Compositions for use in the treatment of Chlamydia

Abstract

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

Description

[Technical Field]

[0001] The present invention is in the field of treating and preventing Chlamydia infections. In particular, the present invention relates to antigens and antigen combinations that can be used as vaccines to immunize against Chlamydia species infections. Vaccines can be delivered as nucleic acids (e.g., mRNA) encoding the antigenic proteins or as recombinant protein antigens. [Background technology]

[0002] The family Chlamydiae is a group of intracellular bacterial pathogens implicated in a variety of human infections, including sexually transmitted diseases and ocular infections (trachoma) caused by Chlamydia trachomatis. The genus Chlamydia further includes the species Chlamydia abortus, Chlamydia pneumoniae, Chlamydia psittaci, Chlamydia pecorum, Chlamydia felis, and Chlamydia caviae.

[0003] Chlamydia trachomatis (C. trachomatis) causes a range of pathological conditions in humans and comprises three biovars that are further subdivided into serovars. 1 Trachoma biovars serotypes AC (A, B, Ba, C) can cause chlamydial conjunctivitis or trachoma, a disease that can lead to blindness. 2 Genital tract serotypes DK (D, E, F, G, H, I, J, and K) cause disease in the genital tract. 3 The biotypes of lymphogranuloma saphenous vein, serotypes L1-3 (L1, L2, L3), cause invasive urogenital and anorectal infections and have become particularly relevant in HIV-infected men who have sex with men.4

[0004] C. trachomatis (serotypes D-K) is the most common bacterial agent of sexually transmitted disease. 5 In 2020, the WHO estimated 129 million new C. trachomatis infections. These are of public health concern, particularly because untreated infections are often asymptomatic or minimally symptomatic but contribute to the transmission of the pathogen. Furthermore, if left untreated, infections can cause, among other conditions, salpingitis, endometritis, pelvic inflammatory disease (PID), ectopic pregnancy, and tubal factor infertility, and may increase the risk of transmitting or acquiring HIV and developing cervical cancer. 6 Although C. trachomatis infections can be effectively controlled with antibiotic therapy, high case prevalence and often delayed diagnosis of asymptomatic cases make C. trachomatis a leading cause of female infertility worldwide. 7 There is a need to develop an effective C. trachomatis vaccine as a sustainable strategy to control C. trachomatis infection rates. Summary of the Invention [Problem to be solved by the invention]

[0005] The present inventors have provided Chlamydia species antigens and antigen combinations that can be used to immunize against Chlamydia species.

[0006] In particular, we have found that antigens derived from the native major outer membrane protein (MOMP) polypeptide of Chlamydia trachomatis, as well as C. trachomatis antigens other than MOMP, when delivered by mRNA encoding the relevant antigen, elicit robust T cell responses, particularly the induction of IFN-γ-producing CD4+ T cells, and / or B cell (i.e., antibody) responses. T cell responses, in particular, are thought to be important for protective immunity. [Means for solving the problem]

[0007] Thus, the present invention provides modified MOMP polypeptides, chimeric MOMP VD polypeptides and CT443, CT584, CT600 and CT812 Chlamydia species polypeptides, and nucleic acids comprising nucleotide sequences encoding such polypeptides.

[0008] The polypeptide antigens described herein can be delivered by, or in the form of, a nucleic acid (eg, mRNA) comprising a nucleotide sequence encoding said polypeptide.

[0009] MOMP MOMP is an integral membrane porin protein found in Chlamydia species of bacteria. 8 This constitutes approximately 60% of the protein mass of the Chlamydia trachomatis membrane. 9 , containing multiple B & T cell epitopes. 10 In C. trachomatis infection, MOMP is the immunodominant antigen. The native MOMP polypeptide of Chlamydia species contains five conserved domains separated by four surface-exposed variable domains (VD1, VD2, VD3, and VD4). 11、12Thus, in native MOMP polypeptides of Chlamydia species, VD1, VD2, VD3, and VD4 are surface-exposed loops. Within Chlamydia species (e.g., C. trachomatis), native MOMP polypeptides differ in their VD sequences across different serotypes. 13、14 Within each serovar of C. trachomatis, the native MOMP polypeptide sequence is highly conserved with greater than 98% sequence identity. Between serovars of C. trachomatis, the protein sequence is highly conserved, with the exception of the surface-exposed VD. Neutralizing antibody epitopes have been mapped to the VD. 15、16、17、18、19

[0010] The present inventors recognized that IFN-γ-producing CD4+ T cells are important for protective immunity. Indeed, HIV+ women are at increased risk of chlamydial reinfection, and cellular IFN-γ production has been shown to strongly correlate with protection. 20 , CD4+ T cells appear to be important 21 There is also evidence that C. trachomatis antibodies (i.e., B cell-mediated responses) alone do not prevent reinfection, and that the presence of anti-C. trachomatis IgG may further increase the risk of incident infection. 22 Anti-C. trachomatis antibodies have been shown to accelerate clearance of primary C. trachomatis antigenic stimulation in mice in the presence of CD4+ T cells, but not in the absence of CD4+ T cells. 23

[0011] As used herein, a "native Chlamydia species MOMP polypeptide" encompasses the mature form of a full-length native MOMP polypeptide of a Chlamydia species, including its native signal peptide sequence and a full-length native MOMP polypeptide of a Chlamydia species, but not including Chlamydia species that contain the native signal peptide sequence. Preferably, the Chlamydia species is referred to herein as Chlamydia trachomatis. The native C. trachomatis MOMP polypeptide can be one of serotypes A-C, D-K, or L1-L3, e.g., serotypes D-K. In some embodiments, the native C. trachomatis MOMP polypeptide is one of serotypes D-G. In some preferred embodiments, the native C. trachomatis MOMP polypeptide is of serotype E. Exemplary sequences of native Chlamydia sp. MOMP polypeptides, including the native signal peptide sequence, are provided by SEQ ID NOs: 1-4.

[0012] The amino acid sequence of a native C. trachomatis MOMP polypeptide of serogroup E is provided in SEQ ID NO:2. [ka]

[0013] The native signal peptide sequence corresponds to residues 1-22 in the amino acid sequence of MOMP from C. trachomatis serovar E (SEQ ID NO: 2) and is shown underlined. Residues 23-393 of SEQ ID NO: 2 correspond to the mature form of the full-length native MOMP polypeptide from C. trachomatis serovar E, lacking the native signal peptide sequence. The VD1-VD4 sequence of the MOMP polypeptide from C. trachomatis serovar E is shown above in bold and underlined text.

[0014] The naturally occurring Chlamydia species MOMP polypeptide is (i) a native MOMP polypeptide of serovar D of C. trachomatis (SEQ ID NO: 1) or its mature form (residues 23 to 393 of SEQ ID NO: 1); (ii) a native MOMP polypeptide of serovar F of C. trachomatis (SEQ ID NO: 3) or its mature form (residues 23-395 of SEQ ID NO: 3); or (iii) the native MOMP polypeptide of serovar G of C. trachomatis (SEQ ID NO: 4) or its mature form (residues 23-395 of SEQ ID NO: 4) It may comprise the sequence:

[0015] Native Chlamydia species MOMP polypeptides contain five conserved domains separated by four variable surface-exposed domains (VD1, VD2, VD3, and VD4). For example, in the native C. trachomatis serovar E MOMP polypeptide, VD1 (SEQ ID NO: 9) corresponds to amino acid residues 86-105 of SEQ ID NO: 2, VD2 (SEQ ID NO: 10) corresponds to amino acid residues 161-181 of SEQ ID NO: 2, VD3 (SEQ ID NO: 11) corresponds to amino acid residues 245-260 of SEQ ID NO: 2, and VD4 (SEQ ID NO: 12) corresponds to amino acid residues 309-337 of SEQ ID NO: 2. The conserved domains correspond to amino acid residues 23-85 (first conserved domain), 106-160 (second conserved domain), 182-244 (third conserved domain), 261-308 (fourth conserved domain), and 338-393 (fifth conserved domain). For serotype E, VD4 (SEQ ID NO: 12) corresponds to positions 309-337 of SEQ ID NO: 2, and the fifth conserved domain corresponds to positions 338-393 of SEQ ID NO: 2, or VD4 (SEQ ID NO: 883) corresponds to positions 309-338 of SEQ ID NO: 2, and the fifth conserved domain corresponds to positions 339-393 of SEQ ID NO: 2.

[0016] For C. trachomatis serotypes D, F, and G, the residues corresponding to VD1, VD2, VD3, and VD4 and the five conserved domains are shown in Table 1. For serotype D, VD4 (SEQ ID NO:8) corresponds to positions 309-337 of SEQ ID NO:1, and the fifth conserved domain corresponds to positions 338-393 of SEQ ID NO:1, or VD4 (SEQ ID NO:882) corresponds to positions 309-338 of SEQ ID NO:1, and the fifth conserved domain corresponds to positions 339-393 of SEQ ID NO:1. For serotype F, VD4 (SEQ ID NO:16) corresponds to positions 310-339 of SEQ ID NO:3, and the fifth conserved domain corresponds to positions 340-395 of SEQ ID NO:3, or VD4 (SEQ ID NO:884) corresponds to positions 310-340 of SEQ ID NO:3, and the fifth conserved domain corresponds to positions 341-395 of SEQ ID NO:3. For serotype G, VD4 (SEQ ID NO: 20) corresponds to positions 310 to 339 of SEQ ID NO: 4, and the fifth conserved domain corresponds to positions 340 to 395 of SEQ ID NO: 4, or VD4 (SEQ ID NO: 885) corresponds to positions 310 to 340 of SEQ ID NO: 4, and the fifth conserved domain corresponds to positions 341 to 395 of SEQ ID NO: 4.

[0017] [Table 1]

[0018] [Table 2]

[0019] Native C. trachomatis MOMP polypeptides of serotypes A-C, H-K, or L1-L3, or other native Chlamydia species MOMP polypeptides, contain VD1-VD4 and conserved domains at positions corresponding to the residue numbering described for native MOMP polypeptides of C. trachomatis serotypes D-G, e.g., serotype E.

[0020] The sequences of other naturally occurring Chlamydia species MOMP polypeptides, including those of serotypes A-C, H-K, or L1-L3 C. trachomatis MOMP polypeptides, are well known and available from public databases. For example, the amino acid sequences of naturally occurring Chlamydia species MOMP polypeptides are available in the Uniprot (https: / / www.uniprot.org / ) and NCBI (https: / / www.ncbi.nlm.nih.gov / ) databases. Nucleic acid sequences encoding naturally occurring Chlamydia species MOMP polypeptides can be obtained from the NCBI (https: / / www.ncbi.nlm.nih.gov / ) database. The locations of the VD and conserved domains within other naturally occurring Chlamydia species MOMP polypeptides are well known and can, in any event, be determined, for example, by aligning the sequences of C. trachomatis serovars D, E, F, and G with the sequences of other naturally occurring Chlamydia species MOMP polypeptides and searching for regions of high sequence conservation, for example, to identify conserved domains.

[0021] Modified MOMP Polypeptides The present inventors have generated modified MOMP polypeptides containing non-native loop sequences in place of the native Chlamydia species MOMP VD sequence. The inventors recognized that such modified MOMP polypeptides could be used to elicit protective immune responses against Chlamydia species infections. In particular, the inventors demonstrated that such modified MOMP polypeptides induce T cell responses, including CD4+ T cells, such as IFNγ-producing CD4+ T cells. In particular, IFNγ-producing CD4+ T cells are believed to be important for protection against Chlamydia species infections. Thus, elimination of the native VD sequence may reduce B cell-mediated (e.g., antibody) responses (e.g., B cell epitopes in the VD) while prioritizing T cell responses against MOMP, for example, sequences in the conserved domains that are conserved across serotypes. The inventors have shown that modified MOMP polypeptides can induce cross-serotype T cell immune responses, i.e., T cell immune responses that are cross-reactive against two or more serotypes of Chlamydia species, e.g., serotypes D-G and J of C. trachomatis.

[0022] Thus, the inventors have demonstrated that these modified MOMP polypeptides are suitable vaccine candidates that can be used as stand-alone antigens or in combination with other Chlamydia species capable of eliciting B cell-mediated (e.g., antibody) and / or T cell-mediated responses in a subject to promote long-lasting memory T cell responses and protective immunity against infection, for example, in combination with one or more chimeric MOMP VD polypeptides provided herein and / or one or more Chlamydia species, CT443, CT584, CT600, or CT812 polypeptides provided herein. The modified MOMP polypeptides described herein can be delivered by nucleic acids comprising a nucleotide sequence encoding the modified MOMP polypeptide.

[0023] Thus, in one aspect, the invention provides a nucleic acid comprising a nucleotide sequence encoding a modified MOMP polypeptide, the modified MOMP polypeptide having an amino acid sequence comprising two or more conserved domain sequences of a naturally occurring Chlamydia species MOMP polypeptide and a non-native loop sequence between the conserved domain sequences. In a further aspect, the invention provides a modified MOMP polypeptide having an amino acid sequence comprising two or more conserved domain sequences of a naturally occurring Chlamydia species MOMP polypeptide and a non-native loop sequence between the conserved domain sequences.

[0024] In a preferred embodiment, the modified MOMP polypeptide does not include a naturally occurring Chlamydia species MOMP variable domain between two or more conserved domain sequences. Thus, in the modified MOMP polypeptide, a non-native loop sequence may replace the naturally occurring Chlamydia species MOMP polypeptide VD sequence. Thus, the modified MOMP polypeptide may exclude the naturally occurring Chlamydia species MOMP VD sequence between two or more conserved domain sequences and may include a non-native loop sequence between two or more conserved domain sequences.

[0025] By replacing the native Chlamydia species MOMP VD sequence with a non-native loop, B cell epitopes (e.g., antibody epitopes) found in the native Chlamydia species MOMP VD are removed. Removal of these B cell epitopes can reduce B cell-mediated responses to antigens in a subject (e.g., to B cell epitopes in the native MOMP VD). This can advantageously increase T cell responses to MOMP in a subject, for example, to sequences in the MOMP conserved domain that share a high degree of identity among MOMP serotypes. Thus, the modified MOMP polypeptides of the present invention can elicit a cross-serotype T cell immune response in a subject, i.e., a T cell immune response that is cross-reactive to two or more serotypes of Chlamydia species (e.g., cross-reactive to two or more serotypes of C. trachomatis (e.g., serotypes D-G and J)). Thus, the modified MOMP polypeptides of the present invention may be capable of enhancing T cell-mediated responses (e.g., compared to the corresponding native MOMP polypeptide). The modified MOMP polypeptides of the present invention may be capable of enhancing CD4+ T cell-mediated responses (e.g., an IFNγ+CD4+ T cell response, such as an IFNγ+IL2+TNFα+CD4+ T cell response), for example, compared to the corresponding native MOMP polypeptide. The modified MOMP polypeptides of the present invention may be capable of inducing a T cell response (e.g., an antigen-specific T cell response) in a subject. The T cell response may be immunodominant. Thus, the modified MOMP polypeptides of the present invention may be capable of eliciting a T cell population responsive to native Chlamydia species MOMP polypeptide in a subject. In some embodiments, the T cell response is a CD4+ T cell response (e.g., an IFNγ+CD4+ T cell response). In some embodiments, the T cell response is an IFNγ+IL2+TNFα+CD4+ T cell response.

[0026] A "non-native loop sequence" refers to a sequence that is not native to any Chlamydia species MOMP VD (VD1, VD2, VD3, or VD4) of any serotype. In some embodiments, the non-native loop sequence is no more than 40%, no more than 35%, no more than 30%, no more than 25%, no more than 20%, no more than 15%, no more than 10%, or no more than 5% identical to any native Chlamydia species MOMP VD (VD1, VD2, VD3, or VD4) sequence of any serotype. In some embodiments, the non-native loop sequence is no more than 40%, no more than 35%, no more than 30%, no more than 25%, no more than 20%, no more than 15%, no more than 10%, or no more than 5% identical to the MOMP VD sequence (VD1, VD2, VD3, or VD4) of serotype E of C. trachomatis (SEQ ID NOS: 9-12).

[0027] The non-native loop sequence may allow the conserved domain sequences in the modified MOMP polypeptide to form a beta-barrel structure, for example, maintaining the beta-barrel structure of the conserved domains of a native Chlamydia species MOMP polypeptide. Thus, in the modified MOMP polypeptides of the present invention, two or more conserved domain sequences are connected by a non-native loop sequence such that the conserved domain sequences form a beta-barrel structure (e.g., composed of antiparallel beta strands). The beta-barrel structure may be as predicted in silico (e.g., using Alphafold2 (Deepmind) software).

[0028] In silico modeling can be used to generate a 3D structure of a native MOMP protein, which contains a beta barrel with VD1-VD4 sequences as loops connecting the individual beta strands. The VD1-VD4 loops are located on the same side of the barrel structure (the surface exposed in the native MOMP polypeptide). Non-native loop sequences can be mapped onto the VD loop in the in silico model, and suitable non-native loop sequences can be determined by those skilled in the art. Suitable non-native loop sequences include, for example, peptide sequences that can span the distance between the ends of the conserved domains adjacent to the native VD as modeled in silico. The distance can correspond, for example, to the distance in angstroms determined by the in silico 3D model between the last conserved domain residue before the VD sequence and the first conserved domain residue after the VD sequence.

[0029] The non-natural loop sequence may exclude sequence motifs found in the subject (e.g., human) proteome, for example, sequence motifs that are 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 amino acids in length. The excluded subject proteome (e.g., human proteome) sequence motifs are typically 8 amino acids or longer in length. This helps minimize undesired cross-reactivity due to homology between the antigen and self-proteins. The non-native loop sequence can be 3 to 30 amino acids in length, for example, 4 to 20 amino acids in length. In some embodiments, the non-native loop sequence that replaces VD1 can have a sequence according to SEQ ID NO: 462 or 466 (e.g., SEQ ID NO: 462). In some embodiments, the non-native loop sequence that replaces VD2 can have a sequence according to SEQ ID NO: 463 or 467 (e.g., SEQ ID NO: 463). In some embodiments, the non-native loop sequence that replaces VD3 can have a sequence according to SEQ ID NO: 464 or 468 (e.g., SEQ ID NO: 464). In some embodiments, the non-native loop sequence that replaces VD4 can have a sequence according to SEQ ID NO: 465 or 469 (e.g., SEQ ID NO: 465). Typically, the modified MOMP polypeptide can include four non-native loop sequences according to SEQ ID NOs: 462-465 in place of VD1, VD2, VD3, and VD4, respectively. The modified MOMP polypeptide may alternatively include four non-native loop sequences according to SEQ ID NOs: 466-469 in place of VD1, VD2, VD3, and VD4, respectively.

[0030] A "conserved domain sequence MOMP polypeptide of a native Chlamydia species" refers to the sequence of a conserved domain of a MOMP polypeptide of any serovar of Chlamydia species, or a variant thereof. Variants include sequences having 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 (preferably at least 95% identity) to a conserved domain of a MOMP polypeptide of any serovar of Chlamydia species. In some embodiments, the conserved domain sequence of the modified MOMP polypeptide 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 (at least 95% identical) to a conserved domain of a native MOMP polypeptide of any serovar Chlamydia species (e.g., a conserved domain defined in Table 1). Variants also include truncated forms of native conserved domains of Chlamydia species MOMP polypeptides, e.g., the conserved domain sequence in the modified MOMP polypeptide lacks up to 3, 5, 8, 10, 15, 20, 25, or 30 (e.g., up to 3 or 5, e.g., up to 3) amino acids of the native Chlamydia species MOMP conserved domain sequence. In some embodiments, the conserved domain sequence of the modified MOMP polypeptide lacks up to 3 or 5 amino acids of the native Chlamydia species MOMP conserved domain sequence. The conserved domain boundaries of C. trachomatis serovars D, E, F, and G are shown in Table 1.

[0031] In some embodiments, the modified MOMP polypeptide comprises two, three, four, or five conserved domains of a native Chlamydia species MOMP polypeptide or a variant thereof. In some embodiments, the modified MOMP polypeptide comprises five conserved domain sequences of a native Chlamydia species MOMP polypeptide or a variant thereof. In some embodiments, the modified MOMP polypeptide comprises all five full-length conserved domains of a native Chlamydia species MOMP polypeptide. The conserved domains of the modified MOMP polypeptide can collectively have at least 95% sequence identity to the conserved domains of a native MOMP polypeptide (e.g., serotype E MOMP). In some embodiments, the conserved domain sequences of the modified MOMP polypeptide collectively have at least 90% or at least 95% sequence identity (e.g., at least 95%) to the conserved domains of a native MOMP polypeptide (e.g., a conserved domain defined in Table 1).

[0032] The conserved domain of the modified MOMP polypeptide can include a conserved domain sequence of MOMP from a serovar of C. trachomatis (e.g., serovars D-K, e.g., serovars D-G). In some embodiments, the conserved domain of the modified MOMP polypeptide can include a conserved domain sequence of MOMP from serovar E of C. trachomatis.

[0033] A modified MOMP polypeptide may contain a non-native loop sequence between each of the conserved domain sequences. Thus, a modified MOMP polypeptide may contain one, two, three, or four non-native loop sequences. One or more non-native loop sequences may replace one, two, three, or four of the corresponding native VD loops. A native VD may be retained between any remaining conserved domains. A modified MOMP polypeptide may have the five conserved domain sequences of a native Chlamydia species MOMP polypeptide. In some embodiments, a modified MOMP polypeptide does not contain a native Chlamydia species MOMP VD between any of the conserved domain sequences. In some embodiments, a modified MOMP polypeptide contains four non-native loop sequences between the conserved domain sequences and does not contain a native Chlamydia species MOMP VD. In some embodiments, a modified MOMP polypeptide does not contain a native Chlamydia species MOMP VD (or a fragment of at least 5, 6, or 7 consecutive amino acids thereof).

[0034] In some embodiments, the modified MOMP polypeptide comprises (1) all five conserved domain sequences of a Chlamydia species MOMP polypeptide of any serotype of C. trachomatis, and (2) four non-native loop sequences, wherein the non-native loop sequences are located between each of the conserved domain sequences, and the modified MOMP polypeptide does not contain a native Chlamydia species MOMP variable domain between any of the conserved domain sequences, and further wherein the non-native loop sequences are 3-30 amino acids in length and are 40% or less identical to any native Chlamydia species MOMP VD (VD1, VD2, VD3, or VD4) sequence of any serotype.

[0035] The modified MOMP polypeptide may have the formula (e.g., N-terminal to C-terminal): C1-L1-C2 (Formula I) and C1 and C2 are two conserved domain sequences of native Chlamydia species MOMP polypeptides, and L1 is a non-native loop sequence.

[0036] In some embodiments, the modified MOMP polypeptide has the formula (e.g., from N-terminus to C-terminus): C1-L1-C2-L2-C3 (Formula II) and C1, C2 and C3 are three conserved domain sequences of native Chlamydia species MOMP polypeptides, and L1 and L2 are non-native loop sequences.

[0037] In some embodiments, the modified MOMP polypeptide has the formula (e.g., from N-terminus to C-terminus): C1-L1-C2-L2-C3-L3-C4 (Formula III) and C1, C2, C3 and C4 are the four conserved domain sequences of native Chlamydia species MOMP polypeptides, and L1, L2 and L3 are non-native loop sequences.

[0038] In some embodiments, the modified MOMP polypeptide has the formula (e.g., from N-terminus to C-terminus): C1-L1-C2-L2-C3-L3-C4-L4-C5 (Formula IV) and C1, C2, C3, C4 and C5 are the five conserved domain sequences of native Chlamydia species MOMP polypeptides, and L1, L2, L3 and L4 are non-native loop sequences.

[0039] The conserved domain sequences and non-native loop sequences may be as described herein.

[0040] In some embodiments, C1-C5 correspond to the first, second, third, fourth, and fifth conserved domains, respectively, of a native Chlamydia trachomatis MOMP polypeptide (e.g., as defined in Table 1).

[0041] Thus, in some embodiments, a nucleic acid of the invention comprises a nucleotide sequence that encodes a modified MOMP polypeptide that comprises or consists of the sequence shown in Formula I, II, III or IV, preferably IV.

[0042] In some embodiments, the modified MOMP polypeptide comprises a sequence according to any one of SEQ ID NOs: 486-489 (e.g., SEQ ID NO: 486), or a sequence having 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 modified MOMP polypeptide comprises a sequence having at least 90% identity to SEQ ID NOs: 486-489 (e.g., SEQ ID NO: 486). In some embodiments, the modified MOMP polypeptide comprises a sequence having at least 95% identity to SEQ ID NOs: 486-489 (e.g., SEQ ID NO: 486).

[0043] In some embodiments, the nucleic acid comprising a nucleotide sequence encoding a modified MOMP polypeptide comprises the nucleotide sequence set forth in any one of SEQ ID NOs:551-566 (e.g., SEQ ID NO:551), 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% (e.g., at least 75%) identity thereto. Typically, the nucleic acid comprises a nucleotide sequence according to SEQ ID NO: 551, 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% (e.g., at least 75%) identity thereto. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding a secretory signal peptide sequence described herein, e.g., the secretory signal peptide sequence set forth in SEQ ID NO: 187.

[0044] In one embodiment, the nucleic acid of the invention comprises the following structural element: (1) 5' cap; (2) a 5' untranslated region (5'UTR) having a nucleic acid sequence according to SEQ ID NO: 838; (3) a protein coding region having a nucleic acid sequence according to SEQ ID NO: 213, optionally followed by a stop codon (e.g., TGA); (4) a 3' untranslated region (3'UTR) having a nucleic acid sequence according to SEQ ID NO: 839; and (5) Poly(A) tail It is an mRNA comprising or consisting of (e.g. consisting of)

[0045] In one embodiment, the nucleic acid of the invention comprises the following structural element: (1) a 5' cap having the following structure: [ka] (2) a 5' untranslated region (5'UTR) having a nucleic acid sequence according to SEQ ID NO: 838; (3) a protein coding region having a nucleic acid sequence according to SEQ ID NO: 213; (4) a 3' untranslated region (3'UTR) having a nucleic acid sequence according to SEQ ID NO: 839; and (5) Poly(A) tail It is an mRNA comprising or consisting of (e.g. consisting of)

[0046] In some embodiments, the 3' end of (1) is directly linked to the 5' end of (2) via a 3' to 5' phosphodiester bond, the 3' end of (2) is directly linked to the 5' end of (3) via a 3' to 5' phosphodiester bond, the 3' end of (3) is directly linked to the 5' end of (4) via a 3' to 5' phosphodiester bond, or the 3' end of (4) is directly linked to the 5' end of (5) via a 3' to 5' phosphodiester bond. In some embodiments, the mRNA is chemically modified, and the chemical modification includes N1-methylpseudouridines in place of all uridines. The mRNA can be encapsulated in LNPs.

[0047] Chimeric MOMP VD polypeptide The present inventors have demonstrated that chimeric Chlamydia species MOMP VD polypeptides containing VD sequences from different C. trachomatis serovars elicit strong antibody responses. In particular, the present inventors showed that mRNAs encoding chimeric MOMP VD polypeptides that combine VD sequences from serovars that exhibit lower levels of VD sequence identity compared to other serovars successfully elicited strong antibody (IgG) responses. Chimeric MOMP VD polypeptides containing only one VD domain from a particular serovar were able to elicit robust IgG responses against that serovar. The level of the IgG antibody response obtained using mRNA encoding the chimeric MOMP VD polypeptide was higher than that induced by immunization with mRNA encoding a polypeptide combining the four VD4 sequences of serotypes D, E, F, and G (based on the polypeptide described in Anja W. Olsen, et al., "Protection Against Chlamydia trachomatis Infection and Upper Genital Tract Pathological Changes by Vaccine-Promoted Neutralizing Antibodies Directed to the VD4 of the Major Outer Membrane Protein," The Journal of Infectious Diseases, Volume 212, Issue 6, 2015, Pages 978-989). Furthermore, the level of the IgG antibody response obtained using mRNA encoding the chimeric MOMP VD polypeptide was higher than that induced by immunization with mRNA encoding the native MOMP protein. The inventors also demonstrated that the chimeric Chlamydia species MOMP VD polypeptides of the present invention elicited strong antibody (e.g., IgG) responses against C. trachomatis elementary bodies (EBs). The elicited antibodies were cross-reactive with EBs from different serotypes of C. trachomatis.The inventors also demonstrated that the Chlamydia spp. MOMP VD polypeptides of the present invention elicited T cell responses, including CD4+ and CD8+ T cells, including IFNγ-producing CD4+ and CD8+ T cells, demonstrating that the chimeric MOMP VD polypeptides of the present invention are suitable vaccine candidates.

[0048] Thus, in another aspect, the present invention provides a nucleic acid comprising a nucleotide sequence encoding a chimeric Chlamydia species MOMP variable domain, wherein the chimeric MOMP VD polypeptide comprises an amino acid sequence comprising two or more Chlamydia species MOMP VD sequences of different serotypes of Chlamydia species. In a further aspect, the present invention provides a chimeric Chlamydia species MOMP VD polypeptide, wherein the chimeric MOMP VD polypeptide comprises an amino acid sequence comprising two or more Chlamydia species MOMP VD sequences of different serotypes of Chlamydia species, wherein native MOMP VD sequences are highly variable across Chlamydia species serotypes and comprise epitopes that elicit a B-cell (i.e., antibody) immune response. The chimeric MOMP VD polypeptides of the present invention can elicit a B-cell (i.e., antibody) response, such as an antigen-specific antibody response, in a subject. The antibody response can be an IgG response. The induced antibodies can bind to the C. trachomatis substrate. The terms "chimeric MOMP VD construct," "chimeric Chlamydia species MOMP VD polypeptide," and "chimeric MOMP VD polypeptide" are used interchangeably herein.

[0049] Thus, chimeric MOMP VD polypeptides of the invention, comprising two or more MOMP VD sequences of different Chlamydia species serotypes, can elicit a B cell (i.e., antibody) response, e.g., a cross-serotype response, against two or more serotypes of C. trachomatis. Thus, chimeric VD polypeptides of the invention can elicit a B cell (antibody) response against two or more serotypes of Chlamydia species. The response can be a protective antibody response.

[0050] The present inventors compared MOMP VD sequences of C. trachomatis and found different levels of sequence variation among serotypes. While the MOMP VD sequences of some serotypes were identical or shared a high level of sequence identity (e.g., at least 70%), other serotypes had a greater degree of sequence variation. For example, the VD1 sequences of serotypes D and E shared a high level of sequence identity, and the VD1 sequences of serotypes F and G were identical. However, there was a much greater level of variation between the VD1 sequences of (i) serotype D or E and (ii) serotype F or G, with the VD2 sequences of serotypes D and E sharing a high level of sequence identity, and similarly, the VD2 sequences of serotypes F and G sharing a high level of sequence identity. However, there is a much greater level of variation between the VD2 sequences of (i) serotypes D or E and (ii) serotypes F or G; the VD3 sequences of serotypes D and F are identical, and the VD3 sequences of serotypes F and G have a high level of sequence identity. The VD3 sequence of serotype E has a lower percentage of sequence identity compared to the VD3 sequences of serotypes D / F compared to serotype G. However, the inventors have found that the VD3 sequence of serotype E contains amino acid motifs that are homologous to sequences found in the human proteome. In the case of VD4, the sequences of serotypes D and E have a high level of sequence identity, and similarly, the sequences of serotypes F and G have a high level of sequence identity. However, there is a much higher level of sequence variation between the VD4 sequences of (i) serotypes D or E and (ii) serotypes F or G.

[0051] Thus, a chimeric MOMP VD polypeptide of the invention can comprise two MOMP VD1 sequences of different serotypes of Chlamydia species, where the two VD1 sequences have lower sequence identity to each other than to the VD1 sequences of other serotypes of Chlamydia species. In some embodiments, in the chimeric MOMP VD polypeptide, the two MOMP VD1 sequences of different serotypes of Chlamydia species are 70%, 69%, 68%, 67%, 66%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25% or less identical (e.g., 50% or less identical) to each other. The serotypes can be selected from C. trachomatis serotypes D through K, e.g., D, E, F, and G.

[0052] The chimeric MOMP VD polypeptides of the invention can comprise two MOMP VD2 sequences of different serotypes of Chlamydia species, where the two VD2 sequences have lower sequence identity to each other than to the VD2 sequences of other serotypes of Chlamydia species. In some embodiments, in the chimeric MOMP VD polypeptide, the two MOMP VD2 sequences of different serotypes of Chlamydia species are 80%, 79%, 78%, 77%, 76%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25% or less identical (e.g., 50% or less) to each other. The serotypes can be selected from C. trachomatis serotypes D through K, e.g., D, E, F, and G.

[0053] The chimeric MOMP VD polypeptides of the present invention can comprise two MOMP VD3 sequences of different serotypes of Chlamydia species, where the two VD3 sequences have lower sequence identity with each other than with the VD3 sequences of other serotypes of Chlamydia species. In some embodiments, in the chimeric MOMP VD polypeptide, the two MOMP VD3 sequences of different serotypes of Chlamydia species are 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25% or less identical (e.g., 70% or less) to each other. In other embodiments, the chimeric MOMP comprises a single MOMP VD3 domain. The serotype may be selected from C. trachomatis serotypes DK, such as D, E, F and G.

[0054] Chimeric MOMP VD polypeptides of the invention can include two MOMP VD4 sequences of different serotypes of Chlamydia species, where the two VD4 sequences have lower sequence identity with each other than with the VD4 sequences of other serotypes of Chlamydia species. In some embodiments, in a chimeric MOMP VD polypeptide, the two MOMP VD4 sequences of different serotypes of Chlamydia species can be 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25% or less identical (e.g., 60% or less identical) to each other. The serotype can be selected from C. trachomatis serotypes D through K, e.g., D, E, F, and G.

[0055] In preferred embodiments, the MOMP VD sequence in the chimeric MOMP VD polypeptide excludes sequence motifs found in the subject (e.g., human) proteome, e.g., sequence motifs that are 8 or more amino acids, 9 or more amino acids, 10 or more amino acids, 11 or more amino acids, 12 or more amino acids, 13 or more amino acids, 14 or more amino acids, or 15 or more amino acids (e.g., 8 or more) in length. The excluded subject (e.g., human) proteome sequence motifs are typically 8 or more amino acids in length. As explained above, this helps minimize undesired cross-reactivity.

[0056] In a preferred embodiment, a chimeric MOMP VD polypeptide comprises a conserved domain sequence portion of a naturally occurring Chlamydia species MOMP polypeptide flanking each of two or more MOMP VD sequences.

[0057] A chimeric MOMP VD polypeptide can comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 MOMP VD sequences. In some embodiments, a chimeric MOMP VD polypeptide comprises 7 MOMP VD sequences.

[0058] The Chlamydia species can be C. trachomatis and can be a serovar DK.

[0059] A chimeric MOMP VD polypeptide can comprise MOMP VDs of two, three, four, five, six, seven, or eight different serotypes, e.g., C. trachomatis serotypes D through K. In some embodiments, a chimeric MOMP VD polypeptide comprises MOMP VDs of four different serotypes, e.g., C. trachomatis serotypes D through G.

[0060] The chimeric MOMP VD polypeptide can comprise (i) two MOMP VD1 sequences of different serotypes of Chlamydia species; and / or (ii) two MOMP VD2 sequences of different serotypes of Chlamydia species; and / or (iii) one MOMP VD3 sequence; and / or (iv) two MOMP VD4 sequences of different serotypes of Chlamydia species. In other embodiments, there are two MOMP VD3 sequences of different serotypes of Chlamydia species. The different serotypes can be selected from serotypes D, E, F, or G of C. trachomatis.

[0061] Thus, a chimeric MOMP VD polypeptide of the invention can comprise a MOMP VD sequence of one of C. trachomatis serovars D or E and a MOMP VD sequence of one of C. trachomatis serovars F or G. In some embodiments, a chimeric MOMP VD polypeptide comprises a MOMP VD1 sequence of C. trachomatis serovars D or E and a MOMP VD1 sequence of C. trachomatis serovars F or G (e.g., a MOMP VD1 sequence of C. trachomatis serovar E and a MOMP VD1 sequence of C. trachomatis serovar G). In some embodiments, a chimeric MOMP VD polypeptide comprises a MOMP VD2 sequence of C. trachomatis serovar D or E and a MOMP VD2 sequence of C. trachomatis serovar F or G (e.g., a MOMP VD2 sequence of C. trachomatis serovar D and a MOMP VD2 sequence of C. trachomatis serovar G). In some embodiments, a chimeric MOMP VD polypeptide comprises a MOMP VD3 sequence of serovar D, F, or G of C. trachomatis (e.g., serovar F). In some embodiments, the chimeric MOMP VD polypeptide comprises a MOMP VD4 sequence of C. trachomatis serovar D or E and a MOMP VD4 sequence of C. trachomatis serovar F or G (e.g., a MOMP VD4 sequence of C. trachomatis serovar D and a MOMP VD4 sequence of C. trachomatis serovar F).

[0062] Chimeric MOMP VD polypeptides of the invention can comprise two, three, or four MOMP VD sequences of C. trachomatis serovars D or E and two, three, or four MOMP VD sequences of C. trachomatis serovars F or G. In some embodiments, a chimeric MOMP VD polypeptide comprises, for each of MOMP VD1, VD2, VD3, and VD4, one MOMP VD sequence of C. trachomatis serovars D or E and one MOMP VD sequence of C. trachomatis serovars F or G. In preferred embodiments, a chimeric MOMP VD polypeptide comprises only one MOMP VD3 sequence corresponding to the VD3 of serovars D, F, or G of C. trachomatis (e.g., serovar F).

[0063] The chimeric MOMP VD polypeptide may comprise at least one (e.g., 1, 2, 3, or 4) of (i) to (iv): (i) a MOMP VD1 sequence from serotype D and a MOMP VD1 sequence from serotype F, or a MOMP VD1 sequence from serotype E and a MOMP VD1 sequence from serotype F; and / or (ii) a MOMP VD2 sequence from serotype E and a MOMP VD2 sequence from serotype F, or a MOMP VD2 sequence from serotype D and a MOMP VD2 sequence from serotype G; and / or (iii) a MOMP VD3 sequence from serotype G, or a MOMP VD3 sequence from serotype F; and / or (iv) a MOMP VD4 sequence from serotype E and a MOMP VD4 sequence from serotype G, or a MOMP VD4 sequence from serotype D and a MOMP VD4 sequence from serotype F, wherein serotypes D, E, F, or G are those of C. trachomatis. In some embodiments, the chimeric MOMP VD polypeptide comprises four of (i) through (iv). In some embodiments, the chimeric MOMP VD polypeptide comprises (i) a MOMP VD1 sequence from serovar E and a MOMP VD1 sequence from serovar G; (ii) a MOMP VD2 sequence from serovar D and a MOMP VD2 sequence from serovar G; and (iii) a MOMP VD3 sequence from serovar F; and (iv) a MOMP VD4 sequence from serovar D and a MOMP VD4 sequence from serovar F, wherein serovars D, E, F, or G are of C. trachomatis. The MOMP VD1 sequence from serovar F of C. trachomatis is the same as the MOMP VD1 sequence from serovar G of C. trachomatis.

[0064] In some embodiments, the chimeric MOMP VD polypeptide comprises (i) a MOMP VD1 sequence from serotype D and a MOMP VD1 sequence from serotype F, a MOMP VD2 sequence from serotype E and a MOMP VD2 sequence from serotype F, a MOMP VD3 sequence from serotype G, a MOMP VD4 sequence from serotype E and a MOMP VD4 sequence from serotype G; or (ii) a MOMP VD1 sequence from serotype E and a MOMP VD1 sequence from serotype F, a MOMP VD2 sequence from serotype D and a MOMP VD2 sequence from serotype G, a MOMP VD3 sequence from serotype F, a MOMP VD4 sequence from serotype D and a MOMP VD4 sequence from serotype F, wherein serotypes D, E, F, or G can comprise C. trachomatis. In some embodiments, the chimeric MOMP VD polypeptide comprises a MOMP VD sequence as in (i). Typically, the chimeric MOMP VD polypeptide comprises a MOMP VD sequence such as (ii).

[0065] Typically, the chimeric MOMP VD polypeptide contains a single amino acid substitution at one or more (e.g., all) positions corresponding to the N-glycosylation sites of a native Chlamydia species MOMP polypeptide, e.g., in one or more MOMP VD domains.

[0066] The chimeric MOMP VD polypeptides described herein can include a conserved domain sequence portion that is part of a conserved domain sequence of a native Chlamydia species MOMP polypeptide that is adjacent to the VD in that native Chlamydia species MOMP polypeptide. The conserved domain sequence that is adjacent to the VD in that native Chlamydia species MOMP polypeptide can be directly adjacent to the VD sequence.

[0067] Each conserved domain sequence portion in the chimeric MOMP VD polypeptide can include 3 to 30 amino acid residues (e.g., between 4 and 20) of a conserved domain sequence of a native Chlamydia species MOMP polypeptide, with 3 to 30 (e.g., 4 to 20) amino acid residues immediately adjacent to the VD sequence of that native Chlamydia species MOMP polypeptide.

[0068] The chimeric MOMP VD polypeptides described herein have the following formula: (Ax-VDx-Bx)y-(Aw-VDw-Bw)z, wherein VDx comprises a VD sequence of VD1, VD2, VD3 or VD4 of a native C. trachomatis serovar yMOMP polypeptide; Ax comprises 3 to 30 (e.g., 4 to 20) amino acid residues immediately preceding VDx of a native Chlamydia MOMP polypeptide sequence of serotype y; Bx comprises 3 to 30 (e.g., between 4 and 20) amino acid residues immediately following VDx of a native Chlamydia MOMP polypeptide sequence of serotype y; VDw comprises the VD sequence of VD1, VD2, VD3, or VD4 of a native C. trachomatis serovar z MOMP polypeptide; Aw comprises 3 to 30 (e.g., between 4 and 20) amino acid residues immediately preceding VDw of a native Chlamydia MOMP polypeptide sequence of serotype z; Bw comprises 3 to 30 (e.g., between 4 and 20) amino acid residues immediately following VDw of a native Chlamydia MOMP polypeptide sequence of serotype z; x and w are independently selected from 1 to 4 and represent a MOMP VD selected from VD1, VD2, VD3, or VD4; and y and z are serotypes of C. trachomatis (eg, selected from serotypes D, E, F, or G).

[0069] In some embodiments, the chimeric MOMP VD polypeptide comprises a sequence according to any one of SEQ ID NOs: 490-505 (e.g., SEQ ID NO: 495 or 503 (e.g., SEQ ID NO: 503)), or a sequence having at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 chimeric MOMP VD polypeptide comprises a sequence having at least 90% identity to any one of SEQ ID NOs: 490-505 (e.g., SEQ ID NO: 495 or 503 (e.g., SEQ ID NO: 503). In some embodiments, the chimeric MOMP VD polypeptide comprises a sequence having at least 95% identity to any one of SEQ ID NOs: 490-505 (e.g., SEQ ID NO: 495 or 503 (e.g., SEQ ID NO: 503).

[0070] In some embodiments, a nucleic acid comprising a nucleotide sequence encoding a chimeric MOMP VD polypeptide comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 567-630 (e.g., SEQ ID NO: 617), 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, a nucleic acid comprising a nucleotide sequence encoding a chimeric MOMP VD polypeptide comprises a nucleotide sequence at least 85% identical to any one of SEQ ID NOs: 567-630 (e.g., SEQ ID NO: 617). In some embodiments, the nucleic acid comprises a nucleotide sequence encoding a secretory signal peptide sequence described herein, e.g., the secretory signal peptide sequence set forth in SEQ ID NO: 187. For example, the nucleic acid may comprise a sequence according to SEQ ID NO: 870.

[0071] In one embodiment, the nucleic acid comprises the following structural elements: (1) 5' cap; (2) a 5' untranslated region (5'UTR) having a nucleic acid sequence according to SEQ ID NO: 838; (3) a protein coding region having a nucleic acid sequence according to SEQ ID NO: 870, optionally followed by a stop codon (e.g., TGA); (4) a 3' untranslated region (3'UTR) having a nucleic acid sequence according to SEQ ID NO: 839; and (5) Poly(A) tail It is an mRNA comprising or consisting of (e.g., consisting of).

[0072] In one embodiment, the nucleic acid comprises the following structural elements: (1) a 5' cap having the following structure: [ka] (2) a 5' untranslated region (5'UTR) having a nucleic acid sequence according to SEQ ID NO: 838; (3) a protein coding region having a nucleic acid sequence according to SEQ ID NO: 870; (4) a 3' untranslated region (3'UTR) having a nucleic acid sequence according to SEQ ID NO: 839; and (5) Poly(A) tail It is an mRNA comprising or consisting of (e.g., consisting of).

[0073] In some embodiments, the 3' end of (1) is directly linked to the 5' end of (2) via a 3' to 5' phosphodiester bond, the 3' end of (2) is directly linked to the 5' end of (3) via a 3' to 5' phosphodiester bond, the 3' end of (3) is directly linked to the 5' end of (4) via a 3' to 5' phosphodiester bond, or the 3' end of (4) is directly linked to the 5' end of (5) via a 3' to 5' phosphodiester bond. In some embodiments, the mRNA is chemically modified, and the chemical modification includes N1-methylpseudouridines in place of all uridines. The mRNA can be encapsulated in LNPs.

[0074] In alternative embodiments, the chimeric MOMP VD polypeptide comprises three MOMP VD4 sequences selected from (i) the MOMP VD4 sequence of C. trachomatis serovar D; (ii) the MOMP VD4 sequence of C. trachomatis serovar E; (iii) the MOMP VD4 sequence of C. trachomatis serovar F; or (iv) the MOMP VD4 sequence of C. trachomatis serovar G. In some embodiments, the chimeric MOMP VD polypeptide comprises all four VD4 domains (i)-(iv). In some embodiments, the chimeric MOMP VD polypeptide comprises a sequence according to any one of SEQ ID NOs: 506, 841, 842, or 843, or a sequence having at least 90% or at least 95% identity thereto.

[0075] Chlamydia species antigens other than MOMP The present inventors have identified Chlamydia species antigens other than MOMP ("non-MOMP antigens") that can be used in the present invention. Non-MOMP antigens or nucleic acids encoding them can be provided or used in combination with MOMP antigens, including the MOMP antigens (and nucleic acids encoding them) described herein. These non-MOMP antigens include the Chlamydia species polypeptides CT443, CT584, CT600, and CT812. These antigens are highly abundant outer membrane proteins and are highly conserved across serotypes of Chlamydia species, e.g., C. trachomatis. Thus, the use of any one or more of these antigens can provide a cross-serotype immune response, i.e., an immune response that is cross-reactive against two or more serotypes of Chlamydia species (e.g., cross-reactive against two or more serotypes of C. trachomatis). The present inventors have demonstrated that the C. trachomatis polypeptides CT443, CT584, CT600, and CT812 elicit T cell responses when delivered as mRNA expressing the relevant antigen or in the form of recombinant protein. The present inventors have also shown that the C. trachomatis polypeptides CT443, CT584, or CT600, as well as at least mRNA encoding recombinant CT443, CT584, or CT600 proteins, induce robust antigen-specific antibody responses.

[0076] CT443 The present inventors have demonstrated that both mRNA encoding C. trachomatis CT443 polypeptide and recombinant C. trachomatis CT443 protein induce IFNγ-producing T cells. Furthermore, C. trachomatis CT443 polypeptide induced robust IgG responses when delivered as antigen-expressing mRNA or in the form of recombinant protein. C. trachomatis CT443 polypeptide delivered as mRNA was shown to induce strong antibody (e.g., IgG) responses against C. trachomatis elementary bodies (EBs). The induced antibodies were cross-reactive with EBs from different serotypes of C. trachomatis. The present inventors have demonstrated that the C. trachomatis CT443 polypeptide of the present invention is a suitable vaccine candidate.

[0077] In one aspect, the present invention provides a nucleic acid comprising a nucleotide sequence encoding a Chlamydia spp. CT443 polypeptide. In another aspect, the present invention provides a polypeptide comprising the amino acid sequence of a Chlamydia spp. CT443 polypeptide. The Chlamydia spp. CT443 polypeptide for use in the present invention can elicit an antibody (e.g., IgG) response (e.g., an antigen-specific antibody response) in a subject. The elicited antibody can bind to a C. trachomatis substrate. The Chlamydia spp. CT443 polypeptide of the present invention can elicit an antibody (IgG) response against two or more serotypes of C. trachomatis, e.g., a cross-serotype response. Thus, the Chlamydia spp. CT443 polypeptide of the present invention can elicit a B cell (antibody) response against two or more serotypes of Chlamydia spp. The response can be a protective antibody response.

[0078] Chlamydia spp. CT443 polypeptides for use in the invention may induce a T cell response (e.g., an antigen-specific T cell response) in a subject. In some embodiments, Chlamydia spp. CT443 polypeptides for use in the invention induce IFNγ-producing T cells, such as IFNγ-producing CD4+ T cells and / or IFNγ-producing CD8+ T cells in a subject.

[0079] CT443 (also called outer membrane protein B (omcB or OMPB)) is an abundant outer membrane protein. It has been suggested that CT443 is an adhesin that facilitates the interaction of C. trachomatis elementary bodies with host cells. 24 CT443, particularly its C-terminus, has been shown to be immunogenic during human infection. 25、26 It has also been reported to be a protective antigen in a mouse model. 27、28

[0080] A "Chlamydia spp. CT443 polypeptide" includes the mature form of a full-length native Chlamydia spp. CT443 polypeptide, without its native signal peptide sequence, and immunogenic variants thereof. Immunogenic variants of native Chlamydia spp. CT443 polypeptides are capable of eliciting an immune response (e.g., an antigen-specific immune response), such as a T cell response and / or an antibody response, in a subject. Immunogenic variants of native Chlamydia spp. CT443 polypeptides include immunogenic fragments of native Chlamydia spp. CT443 polypeptides. Immunogenic CT443 fragments include fragments of naturally occurring Chlamydia spp. CT443 polypeptides that are 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, at least 425, at least 450, at least 475, or at least 500 amino acids in length. Typically, the CT443 polypeptides of the invention are full-length Chlamydia spp. CT443 polypeptides. In some embodiments, immunogenic variants exclude sequence motifs found in a subject (e.g., human) proteome, e.g., sequence motifs that are 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) amino acids in length. The excluded proteome sequence motifs of interest are typically eight amino acids or longer in length, which, as explained above, helps minimize undesired cross-reactivity.

[0081] Exemplary Chlamydia species CT443 polypeptide sequences include the Chlamydia trachomatis CT443 polypeptide sequence (see SEQ ID NO: 507, which lacks the native signal peptide sequence of the Chlamydia trachomatis CT443 polypeptide).

[0082] In some embodiments, the Chlamydia spp. CT443 polypeptide comprises the sequence of any one of SEQ ID NOs:507-508 (e.g., SEQ ID NO:507), or a sequence having 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 Chlamydia spp. CT443 polypeptide comprises a sequence having at least 90% identity thereto. In some embodiments, the Chlamydia spp. CT443 polypeptide comprises a sequence having at least 95% identity thereto.

[0083] In some embodiments, a nucleic acid comprising a nucleotide sequence encoding a Chlamydia sp. CT443 polypeptide comprises a nucleotide sequence according to any one of SEQ ID NOs: 707-710 (e.g., SEQ ID NO: 707), 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 at least 85% identical to any one of SEQ ID NOs: 707-710 (e.g., SEQ ID NO: 707). In some embodiments, the nucleic acid comprises a nucleotide sequence encoding a secretory signal peptide sequence described herein, e.g., the secretory signal peptide sequence set forth in SEQ ID NO: 187. For example, the nucleic acid may comprise a sequence according to SEQ ID NO: 369.

[0084] In one embodiment, the nucleic acid comprises the following structural elements: (1) 5' Cap: (2) a 5' untranslated region (5'UTR) having a nucleic acid sequence according to SEQ ID NO: 838; (3) a protein coding region having a nucleic acid sequence according to SEQ ID NO: 369, optionally followed by a stop codon (e.g., TGA); (4) a 3' untranslated region (3'UTR) having a nucleic acid sequence according to SEQ ID NO: 839; and (5) Poly(A) tail It is an mRNA comprising or consisting of (e.g., consisting of).

[0085] In one embodiment, the nucleic acid comprises the following structural elements: (1) a 5' cap having the following structure: [ka] (2) a 5' untranslated region (5'UTR) having a nucleic acid sequence according to SEQ ID NO: 838; (3) a protein coding region having a nucleic acid sequence according to SEQ ID NO: 369; (4) a 3' untranslated region (3'UTR) having a nucleic acid sequence according to SEQ ID NO: 839; and (5) Poly(A) tail It is an mRNA comprising or consisting of (e.g., consisting of).

[0086] In some embodiments, the 3' end of (1) is directly linked to the 5' end of (2) via a 3' to 5' phosphodiester bond, the 3' end of (2) is directly linked to the 5' end of (3) via a 3' to 5' phosphodiester bond, the 3' end of (3) is directly linked to the 5' end of (4) via a 3' to 5' phosphodiester bond, or the 3' end of (4) is directly linked to the 5' end of (5) via a 3' to 5' phosphodiester bond. In some embodiments, the mRNA is chemically modified, and the chemical modification includes N1-methylpseudouridines in place of all uridines. The mRNA can be encapsulated in LNPs.

[0087] CT584 The present inventors have demonstrated that mRNA encoding C. trachomatis CT584 polypeptide and recombinant C. trachomatis CT584 protein induce IFNγ-producing T cells. Furthermore, C. trachomatis CT584 polypeptide was shown to induce robust IgG responses when delivered as antigen-expressing mRNA or in the form of a recombinant protein. C. trachomatis CT584 polypeptide delivered as mRNA was shown to induce strong antibody (e.g., IgG) responses against C. trachomatis elementary bodies (EBs). The induced antibodies were cross-reactive with EBs of different serotypes of C. trachomatis. The present inventors have demonstrated that the C. trachomatis CT584 polypeptide of the present invention is a suitable vaccine candidate.

[0088] In one aspect, the present invention provides a nucleic acid comprising a nucleotide sequence encoding a Chlamydia spp. CT584 polypeptide. In another aspect, the present invention provides a polypeptide comprising the amino acid sequence of a Chlamydia spp. CT584 polypeptide. The Chlamydia spp. CT584 polypeptide for use in the present invention can elicit an antibody (e.g., IgG) response (e.g., an antigen-specific antibody response) in a subject. The elicited antibody can bind to a C. trachomatis substrate. The Chlamydia spp. CT584 polypeptide of the present invention can elicit an antibody (IgG) response against two or more serotypes of C. trachomatis, e.g., a cross-serotype response. Thus, the Chlamydia spp. CT584 polypeptide of the present invention can elicit a B cell (antibody) response against two or more serotypes of Chlamydia spp. The response can be a protective antibody response.

[0089] Chlamydia spp. CT584 polypeptides for use in the invention may induce a T cell response (e.g., an antigen-specific T cell response) in a subject. In some embodiments, Chlamydia spp. CT584 polypeptides for use in the invention induce IFNγ-producing T cells, such as IFNγ-producing CD4+ T cells and / or IFNγ-producing CD8+ T cells in a subject.

[0090] CT584 is a putative tip protein in the type III secretion system (based on computational structure prediction and homology search studies for C. trachomatis). 29 It has high sequence homology with proteins from other Chlamydiaceae species, but shares no homology with proteins from other bacterial genera. 30 It is also universally conserved among all C. trachomatis serovars, making CT584 a promising target antigen for generating cross-serotype immune responses.

[0091] "Chlamydia spp. CT584 polypeptide" includes full-length native CT584 polypeptides of Chlamydia spp. and immunogenic variants thereof. Immunogenic variants of Chlamydia spp. CT584 polypeptides are capable of eliciting an immune response in a subject, such as a T cell response and / or an antibody response (e.g., an antigen-specific T cell and / or antibody response). Immunogenic variants of CT584 include immunogenic fragments of native Chlamydia spp. CT584 polypeptides. Immunogenic CT584 fragments include fragments of native Chlamydia spp. CT584 polypeptides that are at least 50, at least 75, at least 100, at least 125, at least 150, or at least 175 amino acids in length. Typically, the CT584 polypeptides of the invention are full-length Chlamydia spp. CT584 polypeptides. In embodiments, immunogenic variants exclude sequence motifs found in the subject (e.g., human) proteome, e.g., sequence motifs 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 amino acids in length. The excluded subject proteome (e.g., human proteome) sequence motifs are typically 8 or more amino acids in length. As explained above, this helps minimize undesired cross-reactivity.

[0092] Exemplary Chlamydia spp. CT584 polypeptide sequences include the Chlamydia trachomatis CT584 polypeptide sequence (see SEQ ID NO: 509). Typically, Chlamydia spp. CT584 polypeptides of the invention contain a single amino acid substitution at a position corresponding to residue 11 of SEQ ID NO: 509 (e.g., an N to Q substitution).

[0093] In some embodiments, the Chlamydia spp. CT584 polypeptide comprises the sequence of any one of SEQ ID NOs:509-512 (e.g., SEQ ID NO:510), or a sequence having 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 Chlamydia spp. CT584 polypeptide comprises a sequence having at least 90% identity thereto. In some embodiments, the Chlamydia spp. CT584 polypeptide comprises a sequence having at least 95% identity thereto.

[0094] In some embodiments, a nucleic acid comprising a nucleotide sequence encoding a Chlamydia sp. CT584 polypeptide comprises a nucleotide sequence according to any one of SEQ ID NOs:711-718 (e.g., SEQ ID NO:715), 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 at least 85% identical to any one of SEQ ID NOs:711-718 (e.g., SEQ ID NO:715). In some embodiments, the nucleic acid comprises a nucleotide sequence encoding a secretory signal peptide sequence described herein, e.g., the secretory signal peptide sequence set forth in SEQ ID NO:187. For example, the nucleic acid may comprise a sequence according to SEQ ID NO:377.

[0095] In one embodiment, the nucleic acid comprises the following structural elements: (1) 5' Cap: (2) a 5' untranslated region (5'UTR) having a nucleic acid sequence according to SEQ ID NO: 838; (3) a protein coding region having a nucleic acid sequence according to SEQ ID NO: 377, optionally followed by a stop codon (e.g., TGA); (4) a 3' untranslated region (3'UTR) having a nucleic acid sequence according to SEQ ID NO: 839; and (5) Poly(A) tail It is an mRNA comprising or consisting of (e.g., consisting of).

[0096] In one embodiment, the nucleic acid comprises the following structural elements: (1) a 5' cap having the following structure: [ka] (2) a 5' untranslated region (5'UTR) having a nucleic acid sequence according to SEQ ID NO: 838; (3) a protein coding region having a nucleic acid sequence according to SEQ ID NO: 377; (4) a 3' untranslated region (3'UTR) having a nucleic acid sequence according to SEQ ID NO: 839; and (5) Poly(A) tail It is an mRNA comprising or consisting of (e.g., consisting of).

[0097] In some embodiments, the 3' end of (1) is directly linked to the 5' end of (2) via a 3' to 5' phosphodiester bond, the 3' end of (2) is directly linked to the 5' end of (3) via a 3' to 5' phosphodiester bond, the 3' end of (3) is directly linked to the 5' end of (4) via a 3' to 5' phosphodiester bond, or the 3' end of (4) is directly linked to the 5' end of (5) via a 3' to 5' phosphodiester bond. In some embodiments, the mRNA is chemically modified, and the chemical modification includes N1-methylpseudouridines in place of all uridines. The mRNA can be encapsulated in LNPs.

[0098] CT600 The present inventors have demonstrated that the C. trachomatis polypeptide CT600 elicits robust IgG responses when delivered as antigen-expressing mRNA or in the form of recombinant protein. The present inventors have also shown that mRNA encoding the C. trachomatis CT600 polypeptide and recombinant C. trachomatis CT600 protein can induce IFNγ-producing T cells.

[0099] In one aspect, the present invention provides a nucleic acid comprising a nucleotide sequence encoding a Chlamydia spp. CT600 polypeptide. In another aspect, the present invention provides a polypeptide comprising the amino acid sequence of a Chlamydia spp. CT600 polypeptide. The Chlamydia spp. CT600 polypeptide for use in the present invention can induce an antibody (e.g., IgG) response (e.g., an antigen-specific antibody response) in a subject. C. trachomatis CT600 polypeptide delivered as mRNA has been shown to induce a strong antibody (e.g., IgG) response against C. trachomatis elementary bodies (EBs). The induced antibodies were cross-reactive with EBs of different serotypes of C. trachomatis. The inventors have demonstrated that the C. trachomatis C600 polypeptide of the present invention is a suitable vaccine candidate.

[0100] Chlamydia spp. CT600 polypeptides for use in the invention may induce a T cell response (e.g., an antigen-specific T cell response) in a subject. In some embodiments, Chlamydia spp. CT600 polypeptides for use in the invention induce IFNγ-producing T cells, such as IFNγ-producing CD4+ T cells and / or IFNγ-producing CD8+ T cells in a subject.

[0101] CT600 is a Chlamydia species peptidoglycan-associated lipoprotein (Pal) that is thought to form a bridge between the peptidoglycan and the outer membrane.

[0102] A "Chlamydia spp. CT600 polypeptide" includes the mature form of a full-length native Chlamydia spp. CT600 polypeptide, without its native signal peptide sequence. Immunogenic variants of native Chlamydia spp. CT600 polypeptides are capable of eliciting an immune response (e.g., an antigen-specific immune response), such as a T cell response and / or an antibody response, in a subject. Immunogenic variants of native Chlamydia spp. CT600 polypeptides include immunogenic fragments of native Chlamydia spp. CT600 polypeptides. Immunogenic CT600 fragments include fragments of native Chlamydia spp. CT600 polypeptides that are at least 50, at least 75, at least 100, at least 125, or at least 150 amino acids in length. Preferred immunogenic fragments of Chlamydia spp. CT600 polypeptides include fragments lacking the N-terminal cysteine-rich region of the Chlamydia spp. CT600 polypeptide (e.g., fragments lacking the 13 amino acids immediately following the native signal peptide of the Chlamydia spp. CT600 polypeptide). In embodiments, immunogenic fragments exclude sequence motifs found in a subject (e.g., human) proteome, e.g., sequence motifs 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 amino acids in length. Excluded subject (e.g., human) proteome sequence motifs are typically 8 or more amino acids in length. As explained above, this helps minimize undesirable cross-reactivity.

[0103] Exemplary Chlamydia species CT600 polypeptide sequences include the Chlamydia trachomatis CT600 polypeptide sequence of SEQ ID NO: 513, which lacks the native signal peptide sequence of the Chlamydia trachomatis CT600 polypeptide and the 13 amino acid long region immediately following the signal peptide.

[0104] In some embodiments, the Chlamydia spp. CT600 polypeptide comprises the sequence of SEQ ID NO: 513 or 514, or a sequence having 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 Chlamydia spp. CT600 polypeptide comprises a sequence having at least 90% identity to any one of SEQ ID NOs: 513-514. In some embodiments, the Chlamydia spp. CT600 polypeptide comprises a sequence having at least 95% identity thereto.

[0105] In some embodiments, a nucleic acid comprising a nucleotide sequence encoding a Chlamydia spp. CT600 polypeptide comprises a nucleotide sequence according to any one of SEQ ID NOs: 719-722, 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 at least 85% identical to any one of SEQ ID NOs: 719-722.

[0106] CT812 The present inventors have demonstrated that C. trachomatis CT812 polypeptide and mRNA encoding recombinant C. trachomatis CT812 protein induce IFNγ-producing T cells.

[0107] In one aspect, the invention provides a nucleic acid comprising a nucleotide sequence encoding a Chlamydia spp. CT812 polypeptide. In another aspect, the invention provides a polypeptide comprising the amino acid sequence of a Chlamydia spp. CT812 polypeptide. A Chlamydia spp. CT812 polypeptide for use in the invention may elicit an antibody (e.g., IgG) response (e.g., an antigen-specific antibody response) in a subject. A Chlamydia spp. CT812 polypeptide for use in the invention may elicit a T cell response (e.g., an antigen-specific T cell response) in a subject. In some embodiments, a Chlamydia spp. CT812 polypeptide for use in the invention elicits IFNγ-producing T cells, such as IFNγ-producing CD4+ T cells and / or IFNγ-producing CD8+ T cells in a subject.

[0108] CT812 (also called polymorphic membrane protein D (or PmpD)) is an autotransporter protein anchored in the outer membrane of Chlamydia species bacteria. CT812 is a proposed adhesin protein. Mature CT812 contains a passenger domain and a beta-barrel domain. 32 Immunization with recombinant CT812 protein has been shown to be protective against C. trachomatis serovar D in mice. 31

[0109] As used herein, "Chlamydia spp. CT812 polypeptide" includes the mature form of the full-length native CT812 polypeptide of Chlamydia spp., without its native signal peptide sequence, and immunogenic variants thereof. Immunogenic variants of Chlamydia spp. CT812 polypeptides are capable of eliciting an immune response (e.g., an antigen-specific immune response), such as a T cell response and / or an antibody response, in a subject. Immunogenic variants of native Chlamydia spp. CT812 polypeptides include immunogenic fragments of native Chlamydia spp. CT812 polypeptides. Immunogenic CT812 fragments include fragments of naturally occurring Chlamydia spp. CT812 polypeptides that are at least 50, at least 75, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, at least 650, at least 700, at least 750, at least 800, at least 850, at least 900, at least 950, at least 1000, at least 1050, at least 1100, or at least 1150 amino acids in length. In some embodiments, immunogenic fragments of Chlamydia spp. CT812 polypeptides comprise a fragment of the amino acid sequence of SEQ ID NO: 515. In some embodiments, an immunogenic fragment can be a fragment of the passenger domain, e.g., a fragment comprising residues 52-1003 of SEQ ID NO: 515 or residues 52-1179 of SEQ ID NO: 515. In some embodiments, the immunogenic fragment comprises an amino acid sequence according to SEQ ID NO: 516 or 518. In some embodiments, the fragment of the passenger domain can be at least 600, at least 650, at least 700, at least 750, at least 800, at least 850, at least 900, at least 950, at least 1000, at least 1050, at least 1100, or at least 1150 amino acids in length.In embodiments, the immunogenic fragment excludes sequence motifs found in the subject (e.g., human) proteome, e.g., sequence motifs 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 amino acids in length. The excluded subject proteome (e.g., human proteome) sequence motifs are typically 8 or more amino acids in length. As explained above, this helps minimize undesired cross-reactivity.

[0110] Exemplary Chlamydia spp. CT812 polypeptide sequences include the Chlamydia trachomatis CT812 polypeptide sequence of SEQ ID NO: 515, which lacks the native signal peptide sequence of the Chlamydia trachomatis CT812 polypeptide. Chlamydia spp. CT812 polypeptide sequences also include SEQ ID NOs: 516 and 518.

[0111] In some embodiments, the Chlamydia spp. CT812 polypeptide comprises the sequence of any one of SEQ ID NOs: 515-535, or a sequence having 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 Chlamydia spp. CT812 polypeptide comprises a sequence having at least 90% identity thereto. In some embodiments, the Chlamydia spp. CT812 polypeptide comprises a sequence having at least 95% identity thereto.

[0112] In some embodiments, a nucleic acid comprising a nucleotide sequence encoding a Chlamydia sp. CT812 polypeptide comprises a nucleotide sequence according to any one of SEQ ID NOs: 723-762, 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 at least 85% identical to any one of SEQ ID NOs: 723-762.

[0113] Modified MOMP polypeptides, chimeric MOMP VD polypeptides, and variants of non-MOMP antigenic polypeptides The sequences of the modified MOMP polypeptides, chimeric Chlamydia sp. MOMP VD polypeptides, Chlamydia sp. CT443 polypeptides, Chlamydia sp. CT584 polypeptides, Chlamydia sp. CT600 polypeptides and / or Chlamydia sp. CT812 polypeptides described herein may contain one or more mutations or modifications.

[0114] In some embodiments, the modified MOMP polypeptides, chimeric Chlamydia spp. MOMP VD polypeptides, Chlamydia spp. CT443 polypeptides, Chlamydia spp. CT584 polypeptides, Chlamydia spp. CT600 polypeptides, and / or Chlamydia spp. CT812 polypeptides described herein may contain one or more conservative amino acid substitutions.

[0115] A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art, and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, if an amino acid in a polypeptide is replaced with another amino acid from the same side chain family, the substitution is considered conservative. In some embodiments, amino acid chains can be conservatively replaced with structurally similar chains that differ in the order and / or composition of side chain family members.

[0116] Mutation of cysteine ​​residues One or more cysteine ​​residues in the polypeptides described herein may be mutated by a single amino acid substitution, for example, to a serine residue. Cysteine ​​residues are involved in disulfide bridge formation, and therefore cysteine ​​mutations may limit multimerization of the polypeptide.

[0117] In some embodiments, the modified MOMP polypeptides described herein contain a single amino acid substitution at one or more (e.g., all) positions corresponding to cysteine ​​residues in the conserved domain sequence of a naturally occurring Chlamydia species MOMP polypeptide. In some embodiments, the chimeric MOMP VD polypeptides described herein can contain a single amino acid substitution at one or more (e.g., all) positions corresponding to cysteine ​​residues in the conserved domain sequence of a naturally occurring Chlamydia species MOMP polypeptide. The conserved domain of a naturally occurring C. trachomatis serovar E MOMP polypeptide contains Cys residues at positions C48, C51, C55, C124, C137, C204, C206, C229, and / or C357 of SEQ ID NO:2. The conserved domain of native MOMP polypeptides of Chlamydia species may contain cysteine ​​residues at positions corresponding to those shown for the native MOMP polypeptide of serovar E of C. trachomatis.

[0118] In some embodiments, the Chlamydia spp. CT443 polypeptides described herein comprise a single amino acid substitution at one or more (e.g., all) positions corresponding to cysteine ​​residues in a naturally occurring Chlamydia spp. CT443 polypeptide. In some embodiments, the Chlamydia spp. CT443 polypeptide comprises one or more single amino acid substitutions at positions corresponding to C57, C100, C111, C156, C157, C164, C166, C175, C181, C191, C194, C252, C268, C285, C299, C341, C380, C383, C386, C389, C405, C410, C414, and / or C424 of SEQ ID NO:507. In some embodiments, a Chlamydia spp. CT443 polypeptide comprises one or more single amino acid substitutions at positions corresponding to C57, C100, C111, C156, C157, C164, C166, C175, C181, C191, C194, C252, C268, C285, C299, C341, C380, C386, C405, C410, C414 and / or C424 of SEQ ID NO: 507. Typically, however, the Chlamydia spp. CT443 polypeptides described herein do not comprise a single amino acid substitution at any position corresponding to a cysteine ​​residue in a naturally occurring Chlamydia spp. CT443 polypeptide.

[0119] In some embodiments, the Chlamydia spp. CT584 polypeptides described herein comprise a single amino acid substitution at one or more (e.g., all) positions corresponding to cysteine ​​residues in a naturally occurring Chlamydia spp. CT584 polypeptide. In some embodiments, the Chlamydia spp. CT584 polypeptide comprises one or more single amino acid substitutions at positions corresponding to C44, C114, and / or C138 of SEQ ID NO:509.

[0120] In some embodiments, the Chlamydia spp. CT600 polypeptides described herein comprise a single amino acid substitution at one or more (e.g., all) positions corresponding to cysteine ​​residues in a naturally occurring Chlamydia spp. CT600 polypeptide. In some embodiments, the Chlamydia spp. CT600 polypeptide comprises one or more (e.g., all) single amino acid substitutions at positions corresponding to C1, C5, C10, and / or C13 of SEQ ID NO:844.

[0121] In some embodiments, the Chlamydia spp. CT812 polypeptides described herein comprise a single amino acid substitution at one or more (e.g., all) positions corresponding to cysteine ​​residues in a naturally occurring Chlamydia spp. CT812 polypeptide. In some embodiments, the Chlamydia spp. CT812 polypeptide comprises one or more (e.g., all) single amino acid substitutions at positions corresponding to C1, C96, C160, C169, C179, C187, C233, C291, C300, C331, C348, C365, C401, C432, C560, C570, C571, C641, C665, C756, C838, C859, C963, C980, and / or C1477 of SEQ ID NO:515. In some embodiments, the Chlamydia sp. CT812 polypeptide comprises one or more (e.g., all) single amino acid substitutions at positions corresponding to C96, C169, C291, C300, C331, C348, C365, C401, C432, C560, C570, C571, C756, C838, C859, C963 and / or C980 of SEQ ID NO: 515.

[0122] Glycosylation site mutations Glycosylation can occur in eukaryotic cells but not in prokaryotic cells. As used herein, "glycosylation" refers to the addition of sugar units to proteins. In particular, N-linked glycosylation is the attachment of a glycan to the amide nitrogen of an asparagine (Asn; N) residue in a protein. Glycosylation can occur at any asparagine residue in a protein that is accessible to and recognized by a glycosylation enzyme after protein translation, most commonly at accessible asparagines that are part of an NXS / T motif, where the second amino acid residue following the 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. The attachment process results in a glycosylated protein. This glycan can be a polysaccharide. Non-human glycosylation patterns can make polypeptides undesirably reactogenic when used to elicit antibodies. Furthermore, glycosylation of a normally unglycosylated polypeptide (such as the polypeptides described herein) can alter its immunogenicity. For example, glycosylation can mask important immunogenic epitopes within a protein. Thus, to reduce or eliminate glycosylation, either asparagine or serine / threonine residues can be modified, for example, by substitution with another amino acid.

[0123] In certain embodiments, one or more of the modified MOMP polypeptides, chimeric Chlamydia sp. MOMP VD polypeptides, Chlamydia sp. CT443 polypeptides, Chlamydia sp. CT584 polypeptides, Chlamydia sp. CT600 polypeptides, and / or Chlamydia sp. CT812 polypeptides described herein comprise at least one variant glycosylation site, preferably at least one variant N-linked glycosylation site and / or at least one O-linked glycosylation site. In some embodiments, one or more N-glycosylation sites are removed from one or more of the modified MOMP polypeptides, chimeric Chlamydia spp. MOMP VD polypeptides, Chlamydia spp. CT443 polypeptides, Chlamydia spp. CT584 polypeptides, Chlamydia spp. CT600 polypeptides, and / or Chlamydia spp. CT812 polypeptides described herein. Removal of N-glycosylation sites can reduce glycosylation of the polypeptide. Removal of glycosylation sites can reduce glycosylation of the polypeptide. In some embodiments, one or more of the modified MOMP polypeptides, chimeric Chlamydia spp. MOMP VD polypeptides, Chlamydia spp. CT443 polypeptides, Chlamydia spp. CT584 polypeptides, Chlamydia spp. CT600 polypeptides, and / or Chlamydia spp. CT812 polypeptides described herein have reduced glycosylation compared to the respective native polypeptides. Removal of N-glycosylation sites can eliminate N-glycosylation of the polypeptide. Removal of O-glycosylation sites can eliminate O-glycosylation of the polypeptide.

[0124] In certain embodiments, the modification comprises the substitution of one or more of the N, S, and T amino acids in the NXS / T sequence motif, where X corresponds to any amino acid. In some embodiments, the modification comprises the substitution of one or more serine (Ser) or threonine (Thr) residues in the protein. In some embodiments, the N, S, or T amino acid is replaced with a conservative amino acid substitution. Typically, the N amino acid can be replaced with a Q, S, K, or A amino acid.

[0125] In some embodiments, the chimeric MOMP VD polypeptides described herein contain a single amino acid substitution at one or more (e.g., all) positions corresponding to N-glycosylation sites of the MOMP polypeptide in a naturally occurring Chlamydia species MOMP polypeptide. The N-glycosylation sites in a naturally occurring Chlamydia species MOMP polypeptide can be present in one or more MOMP VD domains. The N-glycosylation sites can be present in serotype E MOMP VD1 (position 9 of SEQ ID NO:9), serotype F MOMP VD1 (position 11 of SEQ ID NO:13), serotype G MOMP VD1 (position 11 of SEQ ID NO:17), serotype E MOMP VD2 (positions 6 and / or 17 of SEQ ID NO:10), serotype F MOMP VD2 (positions 4 and / or 21 of SEQ ID NO:14), or serotype D MOMP VD2 (position 17 of SEQ ID NO:6). MOMP VD2 of serotype G (position 4 and / or 21 of SEQ ID NO: 18), MOMP VD4 of serotype E (position 14 of SEQ ID NO: 12); MOMP VD4 of serotype G (position 14 of SEQ ID NO: 20); MOMP VD4 of serotype D (position 14 of SEQ ID NO: 8) and / or MOMP VD4 of serotype F (position 14 of SEQ ID NO: 16), where serotypes D to G are those of C. trachomatis. In some embodiments, the chimeric MOMP VD polypeptide comprises (i) a MOMP VD1 sequence from serotype E and a MOMP VD1 sequence from serotype G; (ii) a MOMP VD2 sequence from serotype D and a MOMP VD2 sequence from serotype G; and (iii) a MOMP VD3 sequence from serotype F; (iv) a MOMP VD4 sequence from serotype D and a MOMP VD4 sequence from serotype F, where serotypes D, E, F, or G are from C. trachomatis.Typically, the chimeric MOMP VD polypeptide contains a single amino acid substitution at each of the amino acid residues corresponding to position 9 of SEQ ID NO:9 (e.g., an N to A substitution), position 11 of SEQ ID NO:17 (e.g., a T to A substitution), position 17 of SEQ ID NO:6 (e.g., an S to A substitution), positions 4 and 21 of SEQ ID NO:18 (e.g., an N to A and an S to A substitution, respectively), position 14 of SEQ ID NO:8 (e.g., a T to A substitution), and position 14 of SEQ ID NO:16 (e.g., a T to A substitution).

[0126] In some embodiments, the Chlamydia spp. CT443 polypeptides described herein comprise a single amino acid substitution at one or more (e.g., all) positions corresponding to N-glycosylation sites in a naturally occurring Chlamydia spp. CT443 polypeptide. In some embodiments, the Chlamydia spp. CT443 polypeptide comprises one or more (e.g., all) single amino acid substitutions at positions corresponding to residues 18, 29, and 435 of SEQ ID NO:507.

[0127] In some embodiments, the Chlamydia spp. CT584 polypeptides described herein comprise a single amino acid substitution at one or more (e.g., all) positions corresponding to N-glycosylation sites in a naturally occurring Chlamydia spp. CT584 polypeptide. Typically, the Chlamydia spp. CT584 polypeptide comprises a single amino acid substitution (e.g., an N to Q substitution) at a position corresponding to residue 11 of SEQ ID NO:509.

[0128] In some embodiments, the Chlamydia spp. CT600 polypeptides described herein comprise a single amino acid substitution at one or more (e.g., all) positions corresponding to N-glycosylation sites in a naturally occurring Chlamydia spp. CT600 polypeptide. In some embodiments, the Chlamydia spp. CT600 polypeptide comprises one or two single amino acid substitutions at positions corresponding to residues 48 and 65 of SEQ ID NO:513.

[0129] In some embodiments, the Chlamydia spp. CT812 polypeptides described herein contain a single amino acid substitution at one or more (e.g., all) positions corresponding to the N-glycosylation sites of a native Chlamydia spp. CT812 polypeptide.

[0130] Secretory signal peptide sequence The modified MOMP polypeptides, chimeric Chlamydia species MOMP VD polypeptides, Chlamydia species CT443 polypeptides, Chlamydia species CT584 polypeptides, Chlamydia species CT600 polypeptides, and / or Chlamydia species CT812 polypeptides described herein can include a secretory signal peptide sequence. The secretory signal peptide can be cleaved during post-translational processing of Chlamydia species. The polypeptides described herein. Thus, the mature form of a Chlamydia species polypeptide may not include a secretory signal peptide sequence. However, a nucleotide sequence encoding a secretory signal peptide sequence can be present in a nucleic acid described herein encoding a Chlamydia species polypeptide described herein.

[0131] The modified MOMP polypeptides, chimeric Chlamydia sp. MOMP VD polypeptides, Chlamydia sp. CT443 polypeptides, Chlamydia sp. CT584 polypeptides, Chlamydia sp. CT600 polypeptides, and / or Chlamydia sp. CT812 polypeptides described herein can include the secretory signal peptide sequence of the respective native Chlamydia sp. polypeptide, which can be advantageous when the Chlamydia sp. polypeptide is expressed in prokaryotic cells as a recombinant protein.

[0132] In some embodiments, the modified MOMP polypeptides, chimeric Chlamydia spp. MOMP VD polypeptides, Chlamydia spp. CT443 polypeptides, Chlamydia spp. CT584 polypeptides, Chlamydia spp. CT600 polypeptides, and / or Chlamydia spp. CT812 polypeptides described herein can include a viral or eukaryotic (e.g., human) secretory signal peptide (SS) sequence. The use of a viral or eukaryotic secretory signal peptide sequence linked to a polypeptide described herein can provide numerous advantages to immunogenic compositions. Polypeptides of the invention that include an SS sequence, particularly when expressed from mRNA in eukaryotic cells, can have increased extracellular expression compared to polypeptides that do not include an SS sequence. Increased extracellular expression can promote greater immunogenicity and, by extension, better vaccine efficacy.

[0133] Viral SS sequences can be found in publicly accessible databases (e.g., the NCBI or UniProt databases) that contain annotated viral polypeptide sequences and identify experimentally verified SS start and end positions.

[0134] In certain embodiments, the SS sequence and the location of the SS sequence cleavage site for a given known input polypeptide 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 evaluated based on a cumulative rank score that considers the likelihood of detecting standard features of a signal sequence (SS likelihood score) and the likelihood of cleavage at the cleavage site (cleavage probability score). In certain embodiments, a viral secretory 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, as determined using SignalP 6.0. In some embodiments, the viral secretory 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, as determined using SignalP 6.0.

[0135] In certain embodiments, the SS sequence is a viral SS sequence. In certain embodiments, the viral secretory signal peptide sequence is derived from a viral sequence of a virus that can infect humans. The phrases "influenza," "SARS CoV-2," "varicella-zoster virus (VZV)," "measles," "rubella," "rabies," "Ebola," and "smallpox" preceding the phrase "secretory signal peptide sequence" indicate that the secretory signal peptide is derived from the virus corresponding to the name.

[0136] In certain embodiments, the viral secretory signal peptide is derived from a viral sequence selected from the group consisting of influenza secretory signal peptide sequence, SARS CoV-2 secretory signal peptide sequence, varicella zoster virus (VZV) secretory signal peptide sequence, measles secretory signal peptide sequence, rubella secretory signal peptide sequence, mumps secretory signal peptide sequence, Ebola secretory signal peptide sequence, rabies secretory signal peptide sequence, and smallpox secretory signal peptide sequence. These particular signal peptides are derived from viral sequences in viruses that have been administered to humans as vaccines (attenuated, inactivated, or mRNA) and have demonstrated strong safety profiles.

[0137] In certain embodiments, the viral secretory signal peptide is selected from the group consisting of influenza hemagglutinin (HA) secretory signal peptide sequence, SARS CoV-2 spike secretory signal peptide sequence, VZV gB secretory signal peptide sequence, VZV gE secretory signal peptide sequence, VZV gI secretory signal peptide sequence, VZV gK secretory signal peptide sequence, measles F-protein secretory signal peptide sequence, rubella E1 protein secretory signal peptide sequence, rubella E2 protein secretory signal peptide sequence, mumps F-protein secretory signal peptide sequence, Ebola GP protein secretory signal peptide sequence, rabies virus glycoprotein (rabies G) secretory signal peptide sequence, and smallpox 6 kDa IC protein secretory signal peptide sequence.

[0138] In certain embodiments, the viral secretory signal peptide comprises an HA secretory signal peptide sequence from influenza A or influenza B, preferably an HA secretory signal peptide sequence from influenza A.

[0139] Exemplary viral secretory signal peptide amino acid sequences of the present disclosure are shown below in Table 2. Exemplary viral secretory signal peptide amino acid sequences from influenza A or B of the present disclosure are shown below in Table 2.1.

[0140] [Table 3]

[0141] [Table 4]

[0142] [Table 5]

[0143] [Table 6]

[0144] [Table 7]

[0145] [Table 8]

[0146] The secretory signal peptide sequence can be positioned at the N-terminus or C-terminus (eg, at the N-terminus) of the polypeptides described herein.

[0147] In certain embodiments, the SS amino acid sequence is encoded by a codon-optimized polynucleotide sequence.

[0148] In certain embodiments, the viral secretory signal peptide is derived from a viral sequence in a virus capable of infecting humans.

[0149] In certain embodiments, the viral secretory signal peptide is derived from a viral sequence selected from the group consisting of influenza secretory signal peptide sequences and non-influenza secretory signal peptide sequences selected from the group consisting of SARS CoV-2 secretory signal peptide sequences, varicella zoster virus (VZV) secretory signal peptide sequences, measles secretory signal peptide sequences, rubella secretory signal peptide sequences, mumps secretory signal peptide sequences, Ebola secretory signal peptide sequences, smallpox secretory signal peptide sequences, and rabies secretory signal peptide sequences.

[0150] In certain embodiments, the viral secretory signal peptide is selected from the group consisting of influenza hemagglutinin (HA) secretory signal peptide sequence, SARS CoV-2 spike secretory signal peptide sequence, VZV gB secretory signal peptide sequence, VZV gE secretory signal peptide sequence, VZV gI secretory signal peptide sequence, VZV gK secretory signal peptide sequence, measles F-protein secretory signal peptide sequence, rubella E1 protein secretory signal peptide sequence, rubella E2 protein secretory signal peptide sequence, mumps F-protein secretory signal peptide sequence, Ebola GP protein secretory signal peptide sequence, smallpox 6 kDa IC protein secretory signal peptide sequence, and rabies G protein secretory signal peptide sequence, preferably the viral secretory signal peptide comprises an HA secretory signal peptide sequence from influenza A or influenza B, more preferably an HA secretory signal peptide sequence from influenza A.

[0151] In certain embodiments, the HA secretory signal peptide sequence comprises the amino acid sequence MKX1X2LX3VX4LX5TFX6X7X8X9A (SEQ ID NO: 856), where Xi 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, and X7 is selected from T and A. X8 is selected from A and T; and X9 is selected from N and Y.

[0152] In certain embodiments, the HA secretory signal peptide sequence comprises an amino acid sequence selected from MKAKLLVLLCTFTATYA (SEQ ID NO: 187), MKAILVVLLYTFTTANA (SEQ ID NO: 846), MKVKLLVLLCTFTATYA (SEQ ID NO: 847), MKAILVVLLYTFATANA (SEQ ID NO: 188), and MKAILVVMLYTFTTANA (SEQ ID NO: 848).

[0153] In a specific embodiment, the HA secretory signal peptide sequence comprises the amino acid sequence MKX1IIALSX2ILCLVFX3 (SEQ ID NO: 857), wherein X1 is selected from T and A. X2 is selected from Y, N, C, and H, and X3 is selected from T and A.

[0154] In certain embodiments, the HA secretory signal peptide sequence comprises an amino acid sequence selected from MKTIIALSYILCLVFT (SEQ ID NO: 849), MKTIIALSYILCLVFA (SEQ ID NO: 193), MKTIIALSNILCLVFA (SEQ ID NO: 850), MKAIALSNILCLVFA (SEQ ID NO: 851), MKTIIALSCILCLVFA (SEQ ID NO: 852), and MKTIIALSHILCLVFA (SEQ ID NO: 853).

[0155] In a specific embodiment, the HA secretory signal peptide sequence comprises the amino acid sequence MKAIIVLLMVVTSX1A (SEQ ID NO: 858), and X1 is selected from S and N.

[0156] In a specific embodiment, the HA secretory signal peptide sequence comprises the amino acid sequence MX1AIIVLLMVVTSNA (SEQ ID NO: 859), wherein X1 is selected from K and E.

[0157] In certain embodiments, the HA secretory signal peptide sequence comprises an amino acid sequence selected from MKAIIVLLMVVTSNA (SEQ ID NO: 194), MKAIIVLLMVVTSSA (SEQ ID NO: 854), and MEAIIVLLMVVTSNA (SEQ ID NO: 855).

[0158] In certain embodiments, the viral secretory signal peptide is MKAKLLVLLCTFTATYA (SEQ ID NO: 187); MKAILVVLLYTFATANA (SEQ ID NO: 188); MKTIIALSYILCLVFA (SEQ ID NO: 193); MKAIIVLLMVVTSNA (SEQ ID NO: 194); MFVFLVLLPLVS (SEQ ID NO: 195); MFLLTTKRTMFVFLVLLPLVS (SEQ ID NO: 196) MSPCGYYSKWRNRDRPEYRRNLRFRRFFSSIHPNAAAGSGFNGPGVFITSVTGVWLCFLCIFSMFVTAVVS (SEQ ID NO: 803); MGTVNKPVVGVLMGFGIITGTLRITNPVRA (SEQ ID NO: 804); M The amino acid sequence includes an amino acid sequence selected from the group consisting of FLIQCLISAVIFYIQVTNA (SEQ ID NO: 805); MQALGIKTEHFIIMCLLSGHA (SEQ ID NO: 806); MGLKVNVSAIFMAVLLTLQTPTG (SEQ ID NO: 807); MGAAAALTAVVLQGYNPPAYG (SEQ ID NO: 808); MGAPQAFLAGLLLAAVAVGTARA (SEQ ID NO: 809); MKVFLVTCLGFAVFSSSVC (SEQ ID NO: 810); MGVTGILQLPRDRFKRTSFFLWVIILFQRTFS (SEQ ID NO: 811); MRSLIIFLFPSIIYS (SEQ ID NO: 812); and MVPQALLFVPLLVFPLCFG (SEQ ID NO: 845).

[0159] In certain embodiments, the viral secretory signal peptide comprises the amino acid sequence MKAKLLVLLCTFTATYA (SEQ ID NO: 187).

[0160] In certain embodiments, the viral secretory signal peptide is located at the N-terminus of the antigenic prokaryotic polypeptide. In certain embodiments, the viral secretory signal peptide is positioned at the N-terminus of the polypeptide disclosed herein.

[0161] In certain embodiments, the viral secretory signal peptide is located at the C-terminus of the antigenic prokaryotic polypeptide. In certain embodiments, the viral secretory signal peptide is positioned at the C-terminus of the polypeptide disclosed herein.

[0162] In certain embodiments, the viral secretory signal peptide is attached to the antigenic prokaryotic polypeptide with a linker. In certain embodiments, the viral secretory signal peptide is attached to the polypeptide disclosed herein by a linker.

[0163] Heterologous transmembrane domain (TMB) The chimeric Chlamydia spp. MOMP VD polypeptides, Chlamydia spp. CT443 polypeptides, Chlamydia spp. CT584 polypeptides, Chlamydia spp. CT600 polypeptides, and / or Chlamydia spp. CT812 polypeptides described herein can comprise a heterologous transmembrane domain. The inclusion of a TMB sequence can be advantageous because it localizes the antigen to the cell membrane. This can reduce intracellular localization of the antigen and further promote greater immunogenicity compared to antigens lacking the TMB sequence. The chimeric Chlamydia spp. MOMP VD polypeptides, Chlamydia spp. CT443 polypeptides, Chlamydia spp. CT584 polypeptides, Chlamydia spp. CT600 polypeptides, and / or Chlamydia spp. CT812 polypeptides comprising a heterologous transmembrane domain can also comprise a secretory signal peptide sequence. Typically, nucleic acids described herein encoding chimeric Chlamydia spp. MOMP VD polypeptides containing heterologous transmembrane domains also contain a nucleotide sequence encoding a secretory signal peptide sequence. Typically, nucleic acids described herein encoding Chlamydia spp. CT443 polypeptides containing heterologous transmembrane domains also contain a nucleotide sequence encoding a secretory signal peptide sequence. Typically, nucleic acids described herein encoding Chlamydia spp. CT584 polypeptides containing heterologous transmembrane domains also contain a nucleotide sequence encoding a secretory signal peptide sequence. Typically, nucleic acids described herein encoding Chlamydia spp. CT600 polypeptides containing heterologous transmembrane domains also contain a nucleotide sequence encoding a secretory signal peptide sequence. Typically, nucleic acids described herein encoding Chlamydia spp. CT812 polypeptides containing heterologous transmembrane domains also contain a nucleotide sequence encoding a secretory signal peptide sequence.

[0164] The TMB can be derived from any known TMB in the art, including, but not limited to, TMB from eukaryotic transmembrane proteins (e.g., mammalian transmembrane proteins such as human transmembrane proteins), TMB from prokaryotic transmembrane proteins, and TMB from viral transmembrane proteins. The TMB can be further identified through an in silico prediction algorithm, for example, in the TMHMM prediction method described in 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 its entirety. Some features of TMB are described in Albers et al. (Chapter 2 - cell membrane structures and functions. Basic Neurochemistry eighth edition. Pages 26-39, 2012). The TMB is typically, but not exclusively, composed primarily of nonpolar (hydrophobic) amino acid residues and may cross the lipid bilayer once or several times. Those skilled in the art are familiar with 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.

[0165] TMB typically comprises alpha helices, each containing 18-21 amino acids, sufficient to span the lipid bilayer. Thus, in certain embodiments, the transmembrane domain comprises one or more alpha helices. In certain embodiments, the TMB (a) comprises or consists of 15-50 amino acid residues, preferably 15-30 amino acid residues, and more preferably 18-25 amino acid residues; and / or (b) comprises 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) comprises at least one alpha helix.

[0166] In certain embodiments, the transmembrane domain is derived from an integral membrane protein, as further defined herein; in Albers et al., an "integral membrane protein" (also known as an integral membrane protein) is a membrane protein that is permanently bound to a lipid membrane. In certain embodiments, the transmembrane domain is derived from an integral polytopic protein. An integral polytopic protein spans the entire membrane. In certain embodiments, the transmembrane domain is derived from a single-pass (trans) membrane protein, more specifically, for example, a type I or type II bilayer membrane protein. Single-pass membrane proteins cross the membrane only once (i.e., bilayer membrane proteins), while multipass membrane proteins cross the membrane several times, interwoven and interwoven. Single-pass transmembrane proteins can be classified as type I, which are oriented with their carboxyl termini pointing toward the cytosol, or type II, which have their amino termini pointing toward the cytosol. In certain embodiments, the transmembrane domain is derived from an integral monotopic protein. Integral monotonic proteins are those that associate with only one side of the membrane and do not span the lipid bilayer completely.

[0167] In certain embodiments, the heterologous transmembrane domain is derived from a non-human sequence.

[0168] In certain embodiments, the heterologous transmembrane domain is derived from a viral sequence. The phrases "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 was derived from the virus corresponding to that name.

[0169] In certain embodiments, the heterologous transmembrane domain is derived from a viral transmembrane domain sequence selected from the group consisting of influenza transmembrane domain sequence, SARS CoV-2 transmembrane domain sequence, varicella zoster virus (VZV) transmembrane domain sequence, measles transmembrane domain sequence, rubella transmembrane domain sequence, mumps transmembrane domain sequence, rabies transmembrane domain sequence, and Ebola transmembrane domain sequence. These particular transmembrane domains are derived from viral sequences in viruses that have been administered to humans as vaccines (attenuated, inactivated, or mRNA) and have demonstrated strong safety profiles.

[0170] In certain embodiments, the heterologous transmembrane domain is selected from the group consisting of an influenza hemagglutinin (HA) transmembrane domain sequence, a SARS CoV-2 spike transmembrane domain sequence, a VZV gB transmembrane domain sequence, a VZV gE transmembrane domain sequence, a VZV gI transmembrane domain sequence, a VZV gK transmembrane domain sequence, a measles F protein transmembrane domain sequence, a rubella E1 protein transmembrane domain sequence, a rubella E2 protein domain sequence, a mumps F protein transmembrane domain sequence, a rabies virus glycoprotein (rabies G) transmembrane domain sequence, and an Ebola GP protein transmembrane domain sequence.

[0171] In certain embodiments, the heterologous transmembrane domain comprises an HA transmembrane domain sequence from influenza A or influenza B, preferably influenza A.

[0172] Exemplary viral transmembrane domain amino acid sequences of the present disclosure are shown in Table 3 below.

[0173] [Table 9]

[0174] [Table 10]

[0175] In certain embodiments, the heterologous TMB sequence is positioned at the N-terminus or C-terminus (eg, the C-terminus) of a polypeptide described herein.

[0176] In certain embodiments, the TMB amino acid sequence is encoded by a codon-optimized polynucleotide sequence.

[0177] In some embodiments, the chimeric MOMP VD polypeptide comprises a heterologous transmembrane domain as described herein. In some embodiments, the heterologous transmembrane domain does not comprise the sequence of SEQ ID NO: 189. In some embodiments, the heterologous transmembrane domain comprises the sequence of SEQ ID NO: 190. Typically, the chimeric MOMP VD polypeptide does not comprise a heterologous transmembrane domain.

[0178] In alternative embodiments, one or more of the chimeric MOMP VD polypeptides, Chlamydia spp. CT443 polypeptides, Chlamydia spp. CT584 polypeptides, Chlamydia spp. CT600 polypeptides, and / or Chlamydia spp. CT812 polypeptides described herein do not comprise a heterologous transmembrane domain. Such polypeptides may comprise a secretory signal peptide sequence. In further embodiments, the polypeptides of the invention are secreted. In some embodiments, the chimeric MOMP VD polypeptides described herein are secreted polypeptides. In some embodiments, the Chlamydia spp. CT443 polypeptides described herein are secreted polypeptides. In some embodiments, the Chlamydia spp. CT584 polypeptides described herein are secreted polypeptides. In some embodiments, the Chlamydia spp. CT600 polypeptides described herein are secreted polypeptides. In some embodiments, the Chlamydia spp. CT812 polypeptides described herein are secreted polypeptides. In a preferred embodiment, the modified MOMP polypeptides described herein are secreted polypeptides. The secreted polypeptides described herein include a secretory signal peptide sequence.

[0179] In certain embodiments, the TMB (a) comprises or consists of 15 to 50 amino acid residues, preferably 15 to 30 amino acid residues, more preferably 18 to 25 amino acid residues; and / or (b) comprises 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) comprises at least one alpha helix.

[0180] In certain embodiments, the TMB is derived from an integral membrane protein, preferably from a single-pass membrane protein, more preferably from a bitopic membrane protein, even more preferably from a type I bitopic membrane protein.

[0181] In certain embodiments, the TMB is derived from a non-human sequence.

[0182] In certain embodiments, the TMB is derived from a viral sequence.

[0183] In certain embodiments, the TMB is derived from a viral transmembrane domain sequence selected from the group consisting of an influenza transmembrane domain sequence and a non-influenza transmembrane domain sequence selected from the group consisting of 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.

[0184] In certain embodiments, the TMB is selected from the group consisting of an influenza hemagglutinin (HA) transmembrane domain sequence, a SARS CoV-2 spike transmembrane domain sequence, a VZV gB transmembrane domain sequence, a VZV gE transmembrane domain sequence, a VZV gI transmembrane domain sequence, a VZV gK transmembrane domain sequence, a measles F protein transmembrane domain sequence, a rubella E1 protein transmembrane domain sequence, a rubella E2 protein transmembrane domain sequence, a mumps F protein transmembrane domain sequence, an Ebola GP protein transmembrane domain sequence, and a rabies G protein transmembrane domain sequence; preferably, the TMB comprises an HA transmembrane domain sequence from influenza A or influenza B, more preferably an HA transmembrane domain sequence from influenza A.

[0185] In certain embodiments, TMB is selected from the group consisting of ILAIYSTVASSLVLLVSLGAISF (SEQ ID NO: 813); ILAIYSTVASSLVLVVSLGAISF (SEQ ID NO: 814); ILWISFAISCFLLCVVLLGFI (SEQ ID NO: 815); STAASSLAVTLMLAIFIVYMV (SEQ ID NO: 816); WYIWLGFIAGLIAIVMVTIML (SEQ ID NO: 817); FGALAVGLLVLAGLVAAFFAY (SEQ ID NO: 818); AAWTGGLAAVVLLCLVIFLIC (SEQ ID NO: 819); IIIPIVASVMILTAMVI VIVI (SEQ ID NO: 820); YFWCVQLKMIFFAWFVYGMYL (SEQ ID NO: 821); IVYILIAVCLGGLIGIPALIC (SEQ ID NO: 822); LDHAFAAFVLLVPWVLIFMVC (SEQ ID NO: 823); WWQLTLGAICALLLAGLLACC (SEQ ID NO: 824); IVAALVLSILSIIISLLFCCW (SEQ ID NO: 825); WIPAGIGVTGVIIAVIALFCI (SEQ ID NO: 826); and VLLSAGALTALMLIIFLMTCW (SEQ ID NO: 860).

[0186] In certain embodiments, the TMB comprises the amino acid sequence of ILAIYSTVASSLVLLVSLGAISF (SEQ ID NO: 813).

[0187] In certain embodiments, TMB is attached to a polypeptide described herein using a linker.

[0188] In certain embodiments, TMB is positioned at the N-terminus of a polypeptide described herein.

[0189] In certain embodiments, TMB is positioned at the C-terminus of a polypeptide described herein.

[0190] Linker In certain embodiments of the present disclosure, the secretory signal peptide (SS) sequence or transmembrane domain (TMB) is fused directly to a polypeptide described herein (i.e., there is no linker, e.g., an amino acid linker, connecting the SS sequence or TMB to the polypeptide described herein).

[0191] In other embodiments, the SS sequence and TMB of the present disclosure are optionally linked to the polypeptides described herein using a linker. In certain embodiments, the linker is an amino acid linker. In certain embodiments, the amino acid linker is 1 to 10 amino acids in length (e.g., the amino acid linker has a length of 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, or 10 amino acids).

[0192] Illustrative examples of linkers include glycine polymers (Gly)n, where n is at least 1, 2, 3, 4, 5, 6, 7, or 8; glycine-serine polymers (GlySer)n, where n is at least 1, 2, 3, 4, 5, 6, 7, or 8; glycine-alanine polymers; alanine-serine polymers; and other flexible linkers known in the art.

[0193] Glycine and glycine-serine polymers are relatively unstructured and flexible and therefore can function as neutral tethers between the SS sequence and / or TMB and the polypeptides described herein. In certain embodiments, the linker is SGS or GSG.

[0194] Other exemplary linkers include, but are not limited to, the following amino acid sequences: GGG; DGGGS (SEQ ID NO: 827); TGEKP (SEQ ID NO: 828) (Liu et al. Proc. Natl. Acad. Sci. 94:5525-5530, 1997); GGRR (SEQ ID NO: 829); (GGGGS)n (SEQ ID NO: 830), where n=1, 2, 3, 4, or 5 (Kim et al. Proc. Natl. Acad. Sci. 93:1156-1160, 1996); EGKSSGSGSESKVD (SEQ ID NO: 831) (Chaudhary et al. Proc. Natl. Acad. Sci. 87:1066-1070, 1990); KESGSVSSEQLAQFRSLD (SEQ ID NO: 832) (Bird et al. al. Science. 242:423-426. 1988), GGRRGGGS (SEQ ID NO: 833); LQRDGERP (SEQ ID NO: 834); LRQKDGGGSERP (SEQ ID NO: 835); and GSTSGSGKPGSGEGSTKG (SEQ ID NO: 836) (Cooper et al. Blood. 101(4):1637-1644. 2003). Preferred linkers are shorter, for example, consisting of 3, 4, or 5 amino acids.

[0195] Further examples of linkers are provided in Chen et al. (Adv Drug Deliv Rev. 65(10):1357-1369. 2013), which is incorporated herein by reference.

[0196] composition 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 disclosure can 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. An "immunogenic composition" refers to a composition comprising a nucleic acid or protein that, when administered to a subject, elicits an immune response, e.g., an antigen-specific immune response. The response can be a T cell immune response or an antibody response. The compositions of the present invention can be vaccine compositions. The immunogenic composition (e.g., vaccine composition) can elicit protective immunity (e.g., a T cell and / or a B cell (i.e., antibody) response) against Chlamydia species infection. The T cell response can include a CD4+ T cell response and / or a CD8+ T cell response, e.g., IFNγ-producing CD4+ T cells and / or IFNγ-producing CD8+ T cells. In some embodiments, the T cell response includes CD4+ T cells, such as IFNγ-producing CD4+ T cells.

[0197] As used herein, "protective immunity" or "protective immune response" refers to the induction of immunity or an immune response exhibited by a subject against an infectious agent (e.g., a chlamydia pathogen) that prevents or ameliorates infection or reduces at least one symptom thereof. Specifically, the induction of protective immunity or a protective immune response by administration of a composition of the invention is evidenced by the elimination or reduction of the presence of one or more symptoms of chlamydia infection and / or the expansion of a chlamydia-responsive memory T cell population. As used herein, the term "immune response" refers to both humoral and cell-mediated immune responses. Preferably, protective immunity provided by the invention is characterized by protective immunological memory against the pathogen (e.g., a protective memory T cell response). Protective immunity may be characterized by an effective pathogen-responsive (e.g., chlamydia-responsive) memory T cell population. In a preferred embodiment, treatment with the compositions of the invention described herein provides protective immunity against reinfection with a chlamydia pathogen. Protective immunity can be sterilizing immunity (i.e., complete protective immunity), whereby a protected subject can elicit an immune response that completely clears the infection. The chlamydia antigens described herein (e.g., chimeric MOMP VD polypeptides of the invention and Chlamydia spp. CT443 and CT584 polypeptides of the invention) can bind to Chlamydia spp. (e.g., C. trachomatis) entities, e.g., entities of two or more serotypes.

[0198] Nucleic acid composition In one aspect, the invention provides a composition comprising a nucleic acid described herein that comprises a nucleotide sequence encoding a modified MOMP polypeptide described herein.

[0199] In another aspect, the invention provides a composition comprising a nucleic acid described herein that comprises a nucleotide sequence encoding a chimeric MOMP VD polypeptide described herein.

[0200] In another aspect, the invention provides a composition comprising a nucleic acid described herein, which comprises a nucleotide sequence encoding a Chlamydia sp. CT443 polypeptide described herein.

[0201] In another aspect, the invention provides a composition comprising a nucleic acid described herein, which comprises a nucleotide sequence encoding a Chlamydia sp. CT584 polypeptide described herein.

[0202] In another aspect, the invention provides a composition comprising a nucleic acid described herein that comprises a nucleotide sequence encoding a Chlamydia sp. CT600 polypeptide described herein.

[0203] In another aspect, the invention provides a composition comprising a nucleic acid described herein, which comprises a nucleotide sequence encoding a Chlamydia sp. CT812 polypeptide described herein.

[0204] In a further aspect, the invention provides compositions comprising one, two, three, or four of: (a) a nucleic acid described herein comprising a nucleotide sequence encoding a Chlamydia spp. CT443 polypeptide described herein; (b) a nucleic acid described herein comprising a nucleotide sequence encoding a Chlamydia spp. CT584 polypeptide described herein; (c) a nucleic acid described herein comprising a nucleotide sequence encoding a Chlamydia spp. CT600 polypeptide described herein; and / or (d) a nucleic acid described herein comprising a nucleotide sequence encoding a Chlamydia spp. CT812 polypeptide described herein.

[0205] The compositions of the invention can include the combinations described herein of the nucleic acids described herein (e.g., they can be formulated in the same composition). The combinations described herein of the nucleic acids described herein can alternatively be two or more separate compositions (e.g., as a combination of compositions for simultaneous, separate, or sequential administration (e.g., in therapeutic uses described herein)).

[0206] In some embodiments, a composition may comprise (a) a nucleic acid described herein comprising a nucleotide sequence encoding a modified MOMP polypeptide described herein, (b) a nucleic acid described herein comprising a nucleotide sequence encoding a chimeric MOMP VD polypeptide described herein, and (c) a nucleic acid described herein comprising a nucleotide sequence encoding a Chlamydia sp. CT443 polypeptide described herein.

[0207] In some embodiments, a composition may comprise (a) a nucleic acid described herein comprising a nucleotide sequence encoding a modified MOMP polypeptide described herein, (b) a nucleic acid described herein comprising a nucleotide sequence encoding a chimeric MOMP VD polypeptide described herein, and (c) a nucleic acid described herein comprising a nucleotide sequence encoding a Chlamydia sp. CT584 polypeptide described herein.

[0208] Typically, the compositions may include (a) a nucleic acid described herein that includes a nucleotide sequence encoding a modified MOMP polypeptide described herein, (b) a nucleic acid described herein that includes a nucleotide sequence encoding a chimeric MOMP VD polypeptide described herein, (c) a nucleic acid described herein that includes a nucleotide sequence encoding a Chlamydia sp. CT443 polypeptide described herein, and (d) a nucleic acid described herein that includes a nucleotide sequence encoding a Chlamydia sp. CT584 polypeptide described herein.

[0209] Compositions of the present disclosure comprising one or more nucleic acids of the present disclosure can also comprise one or more additional components, such as a small molecule immunostimulant (e.g., a TLR agonist). Compositions of the present disclosure can also comprise a delivery system for the nucleic acids (e.g., RNA) described herein, such as a liposome, an oil-in-water emulsion, or a microparticle. In some embodiments, the composition comprises a lipid nanoparticle (LNP). In certain embodiments, the composition comprises a nucleic acid molecule of the present invention encapsulated within an LNP.

[0210] Polypeptide Composition In one aspect, the invention provides compositions comprising a modified MOMP polypeptide described herein.

[0211] In another aspect, the invention provides compositions comprising a chimeric MOMP VD polypeptide as described herein.

[0212] In another aspect, the invention provides compositions comprising a Chlamydia sp. CT443 polypeptide described herein.

[0213] In another aspect, the present invention provides compositions comprising a Chlamydia sp. CT584 polypeptide described herein.

[0214] In another aspect, the invention provides compositions comprising a Chlamydia sp. CT600 polypeptide described herein.

[0215] In another aspect, the invention provides compositions comprising a Chlamydia sp. CT812 polypeptide described herein.

[0216] In a further aspect, the present invention provides compositions comprising one, two, three, or four of: (a) a polypeptide comprising the amino acid sequence of a Chlamydia spp. CT443 polypeptide described herein; (b) a polypeptide comprising the amino acid sequence of a Chlamydia spp. CT584 polypeptide described herein; (c) a polypeptide comprising the amino acid sequence of a Chlamydia spp. CT600 polypeptide described herein; and / or (d) a polypeptide comprising the amino acid sequence of a Chlamydia spp. CT812 polypeptide described herein.

[0217] The compositions of the invention may include the herein described combinations of polypeptides described herein (e.g., they may be formulated in the same composition). The herein described combinations of polypeptides described herein may alternatively be two or more separate compositions (e.g., as a combination of compositions for simultaneous, separate, or sequential administration (e.g., in therapeutic uses described herein)).

[0218] In some embodiments, a composition may comprise (i) a modified MOMP polypeptide described herein, (ii) a chimeric MOMP VD polypeptide described herein, and (iii) a Chlamydia sp. CT443 polypeptide described herein.

[0219] In some embodiments, a composition may comprise (i) a modified MOMP polypeptide described herein, (ii) a chimeric MOMP VD polypeptide described herein, and (iii) a Chlamydia sp. CT584 polypeptide described herein.

[0220] Typically, the composition may include (i) a modified MOMP polypeptide described herein, (ii) a chimeric MOMP VD polypeptide described herein, (iii) a Chlamydia sp. CT443 polypeptide described herein, and (iv) a Chlamydia sp. CT584 polypeptide described herein.

[0221] Compositions of the present disclosure comprising one or more polypeptides of the present disclosure may include an adjuvant. As used herein, "adjuvant" refers to a substance or vehicle that enhances the immune response to an antigen. Adjuvants can include, but are not limited to, suspensions of minerals (e.g., alum, aluminum hydroxide, or phosphate) to which antigens are adsorbed; water-in-oil or oil-in-water emulsions in which antigen solutions are emulsified in mineral oil or water (e.g., Freund's incomplete adjuvant). Killed mycobacteria may be included to further enhance antigenicity (e.g., Freund's complete adjuvant). Adjuvants can include squalene-based oil-in-water emulsion adjuvants (e.g., AF03, described in, e.g., WO 2007006939 and U.S. Pat. No. 8,703,095; AS03, described in, e.g., WO 1995017209 and WO 1995017210, and U.S. Pat. Nos. 6,623,739, 7,029,678, and 7,510,698; and MF59, described in, e.g., WO 1990014837, and U.S. Pat. Nos. 6,299,884 and 6,451,325). Immunostimulatory oligonucleotides (e.g., CpG motifs) can also be used as adjuvants (see, e.g., U.S. Patent Nos. 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 can also include biological molecules such as Toll-like receptor (TLR) agonists (e.g., AS01, e.g., as described in WO2007068907 and U.S. Pat. Nos. 10,039,823 and 10,143,745; SPA14, e.g., as described in WO2022090359; and LEQ, e.g., as described in WO2023056089) and costimulatory molecules.In some embodiments, the adjuvant is AF03 (a squalene-based oil-in-water emulsion adjuvant). In some embodiments, the adjuvant is capable of inducing a Th1 response.

[0222] Nucleic acid combinations The present invention provides combinations comprising two or more nucleic acids of the present invention.

[0223] Thus, in one aspect, the invention provides a nucleic acid as described herein comprising a nucleotide sequence encoding a modified MOMP polypeptide as described herein, and (i) a nucleic acid described herein comprising a nucleotide sequence encoding a chimeric MOMP VD polypeptide described herein; and / or (ii) (a) a nucleic acid described herein comprising a nucleotide sequence encoding a Chlamydia sp. CT443 polypeptide described herein; (b) a nucleic acid described herein comprising a nucleotide sequence encoding a Chlamydia sp. CT584 polypeptide described herein; (c) a nucleic acid described herein comprising a nucleotide sequence encoding a Chlamydia sp. CT600 polypeptide described herein; and / or (d) a nucleic acid described herein comprising a nucleotide sequence encoding a Chlamydia sp. CT812 polypeptide described herein. Combinations comprising one or more of:

[0224] In some embodiments, the combination includes nucleic acid (a), nucleic acid (b), nucleic acid (c), nucleic acid (d), nucleic acid (a) and (b), nucleic acid (a) and (c), nucleic acid (a) and (d), nucleic acid (b) and (c), nucleic acid (b) and (d), nucleic acid (c) and (d), nucleic acid (a), (b) and (c), nucleic acid (a), (c) and (d), nucleic acid (a), (b) and (d), nucleic acid (b), (c) and (d), or nucleic acid (a), (b), (c) and (d). In some embodiments, the combination includes nucleic acid (a). In some embodiments, the combination includes nucleic acid (b). Typically, the combination includes nucleic acid (a) and nucleic acid (b).

[0225] Any of the nucleic acids (a) to (d) can be located on the same nucleic acid molecule, or the nucleic acids (a) to (d) can be separate nucleic acid molecules.

[0226] Thus, in some embodiments, a combination of the present invention comprises a nucleic acid described herein that comprises a nucleotide sequence encoding a modified MOMP polypeptide described herein and a nucleic acid described herein that comprises a nucleotide sequence encoding a chimeric MOMP VD polypeptide described herein.

[0227] In some embodiments, the combinations of the present invention include a nucleic acid described herein comprising a nucleotide sequence encoding a modified MOMP polypeptide described herein, and one, two, three, or four of: (a) a nucleic acid described herein comprising a nucleotide sequence encoding a Chlamydia spp. CT443 polypeptide described herein; (b) a nucleic acid described herein comprising a nucleotide sequence encoding a Chlamydia spp. CT584 polypeptide described herein; (c) a nucleic acid described herein comprising a nucleotide sequence encoding a Chlamydia spp. CT600 polypeptide described herein; and / or (d) a nucleic acid described herein comprising a nucleotide sequence encoding a Chlamydia spp. CT812 polypeptide described herein. The combinations may include any of the combinations of non-MOMP antigens (a) to (d) above. In some embodiments, the combinations include the nucleic acid of (a). In some embodiments, the combinations include the nucleic acid of (b). Typically, the combinations include the nucleic acid of (a) and the nucleic acid of (b).

[0228] In some embodiments, the combinations of the present invention comprise (i) a nucleic acid described herein comprising a nucleotide sequence encoding a modified MOMP polypeptide described herein; (ii) a nucleic acid described herein comprising a nucleotide sequence encoding a chimeric MOMP VD polypeptide described herein; or (iii) one, two, three, or four of the nucleic acids (a) to (d). The combinations may include any of the non-MOMP antigen combinations (a) to (d) above. In some embodiments, the combinations include the nucleic acid (a). In some embodiments, the combinations include the nucleic acid (b). Typically, the combinations include the nucleic acid (a) and the nucleic acid (b).

[0229] In a further aspect, the present invention provides a combination comprising a nucleic acid described herein comprising a nucleotide sequence encoding a chimeric MOMP VD polypeptide described herein and one, two, three, or four of the nucleic acids (a) through (d). The combination may include any of the non-MOMP antigen combinations (a) through (d) above. In some embodiments, the combination comprises the nucleic acid of (a). In some embodiments, the combination comprises the nucleic acid of (b). Typically, the combination comprises the nucleic acid of (a) and the nucleic acid of (b). In some embodiments, the combination further comprises a nucleic acid described herein comprising a nucleotide sequence encoding a modified MOMP polypeptide described herein.

[0230] In another aspect, the invention provides combinations comprising a nucleic acid described herein comprising a nucleotide sequence encoding a Chlamydia spp. CT443 polypeptide described herein, and one, two, or three of the nucleic acids defined in (b) through (d). The combinations can include any of the non-MOMP antigen combinations of (b), (c), and / or (d) above. Typically, the combinations include the nucleic acid of (b). In some embodiments, the combinations further include a nucleic acid comprising a nucleotide sequence encoding a native Chlamydia spp. MOMP polypeptide or a variant thereof.

[0231] In another aspect, the invention provides a combination comprising a nucleic acid described herein comprising a nucleotide sequence encoding a Chlamydia spp. CT584 polypeptide described herein, and one, two, or three of the nucleic acids defined in (a), (c), or (d). The combination can include any of the non-MOMP antigen combinations of (a), (c), and / or (d) above. Typically, the combination comprises the nucleic acid of (a). In some embodiments, the combination further comprises a nucleic acid comprising a nucleotide sequence encoding a native Chlamydia spp. MOMP polypeptide or a variant thereof.

[0232] In another aspect, the invention provides combinations comprising a nucleic acid described herein comprising a nucleotide sequence encoding a Chlamydia spp. CT600 polypeptide described herein, and one, two, or three of the nucleic acids defined in (a), (b), or (d). The combinations may include any of the non-MOMP antigen combinations of (a), (b), and / or (d) above. In some embodiments, the combinations further comprise a nucleic acid comprising a nucleotide sequence encoding a native Chlamydia spp. MOMP polypeptide or a variant thereof.

[0233] In another aspect, the invention provides combinations comprising a nucleic acid described herein comprising a nucleotide sequence encoding a Chlamydia spp. CT812 polypeptide described herein, and one, two, or three of the nucleic acids defined in (a)-(c). The combinations can include any of the non-MOMP antigen combinations of (a), (b), and / or (c) above. In some embodiments, the combinations further comprise a nucleic acid comprising a nucleotide sequence encoding a native Chlamydia spp. MOMP polypeptide or a variant thereof.

[0234] Thus, the combinations of the present invention can include a native Chlamydia species MOMP polypeptide or a variant thereof, and a nucleic acid comprising a nucleotide sequence encoding one, two, three, or four of the nucleic acids (a) through (d). Variants of native Chlamydia species MOMP polypeptides include MOMP polypeptides containing one or more conservative amino acid substitutions, MOMP polypeptides containing single amino acid substitutions at one or more (e.g., all) positions corresponding to cysteine ​​residues in native Chlamydia species MOMP polypeptides, and MOMP polypeptides containing single amino acid substitutions at one or more (e.g., all) positions corresponding to glycosylation sites, preferably N-glycosylation sites, in native Chlamydia species MOMP polypeptides. Variants of native Chlamydia species MOMP polypeptides also include MOMP polypeptides containing a secretory signal peptide sequence and MOMP polypeptides containing a heterologous transmembrane domain. In some embodiments, a variant of a native Chlamydia species MOMP polypeptide contains a secretory signal peptide sequence. In some embodiments, a variant of a native Chlamydia species MOMP polypeptide comprises a secretory signal peptide sequence and a heterologous transmembrane domain.

[0235] The combinations of nucleic acids disclosed herein include all of the individual nucleic acids in the same composition and all of the individual nucleic acids in one or more separate compositions. In some embodiments, all of the individual nucleic acids in the combinations described herein are in the same composition. In some embodiments, each of the nucleic acids in the combinations described herein is in a separate composition. In some embodiments, two or more nucleic acids in the combinations described herein are in two or more compositions. In some embodiments of the combinations of the present invention, one, two, three, or four of the nucleic acids (a) to (d) above are in one or more compositions. In some embodiments of the combinations of the present invention, two, three, or four of the nucleic acids (a) to (d) above are in two or more separate compositions. Typically, all of the individual nucleic acids are in the same composition.

[0236] Polypeptide Combinations The present invention provides combinations comprising two or more polypeptides of the invention.

[0237] Thus, in one aspect, the present invention provides a modified MOMP polypeptide as described herein, and (i) a chimeric MOMP VD polypeptide described herein; and / or (ii) (a) a polypeptide comprising the amino acid sequence of a Chlamydia species described herein, the CT443 polypeptide; (b) a polypeptide comprising the amino acid sequence of a Chlamydia species described herein, the CT584 polypeptide; (c) a polypeptide comprising the amino acid sequence of a Chlamydia sp. CT600 polypeptide described herein; and / or (d) a polypeptide comprising the amino acid sequence of a Chlamydia sp. CT812 polypeptide described herein. Combinations comprising one or more of:

[0238] In some embodiments, the combination comprises a polypeptide of (a), a polypeptide of (b), a polypeptide of (c), a polypeptide of (d), a polypeptide of (a) and (b), a polypeptide of (a) and (c), a polypeptide of (a) and (d), a polypeptide of (b) and (c), a polypeptide of (b) and (d), a polypeptide of (c) and (d), a polypeptide of (a), (b) and (c), a polypeptide of (a), (c) and (d), a polypeptide of (a), (b) and (d), a polypeptide of (b), (c) and (d), a polypeptide of (a), (b), (c) and (d). In some embodiments, the combination comprises a polypeptide of (a). In some embodiments, the combination comprises a polypeptide of (b). Typically, the combination comprises a polypeptide of (a) and a polypeptide of (b).

[0239] Thus, in some embodiments, a combination of the present invention comprises a modified MOMP polypeptide as described herein and a chimeric MOMP VD polypeptide as described herein.

[0240] In some embodiments, a combination of the present invention comprises a modified MOMP polypeptide described herein and one, two, three, or four of: (a) a polypeptide comprising the amino acid sequence of a Chlamydia spp. CT443 polypeptide described herein; (b) a polypeptide comprising the amino acid sequence of a Chlamydia spp. CT584 polypeptide described herein; (c) a polypeptide comprising the amino acid sequence of a Chlamydia spp. CT600 polypeptide described herein; and / or (d) a polypeptide comprising the amino acid sequence of a Chlamydia spp. CT812 polypeptide described herein. The combination may comprise any of the combinations of non-MOMP antigens (a) to (d) above. In some embodiments, the combination comprises the polypeptide of (a). In some embodiments, the combination comprises the polypeptide of (b). Typically, the combination comprises the polypeptide of (a) and the polypeptide of (b).

[0241] In some embodiments, the combinations of the invention comprise (i) a modified MOMP polypeptide as described herein, (ii) a chimeric MOMP VD polypeptide as described herein, and (iii) one, two, three, or four of the polypeptides in (a) through (d). The combinations may include any of the combinations of non-MOMP antigens (a) through (d) above. In some embodiments, the combinations include the polypeptide of (a). In some embodiments, the combinations include the polypeptide of (b). Typically, the combinations include the polypeptide of (a) and the polypeptide of (b).

[0242] In a further aspect, the present invention provides a combination comprising a chimeric MOMP VD polypeptide described herein and one, two, three, or four of the polypeptides (a) through (d). The combination may include any of the combinations of non-MOMP antigens (a) through (d) above. In some embodiments, the combination includes the polypeptide of (a). In some embodiments, the combination includes the polypeptide of (b). Typically, the combination includes the polypeptide of (a) and the polypeptide of (b). In some embodiments, the composition further comprises a modified MOMP polypeptide described herein.

[0243] In another aspect, the present invention provides a combination comprising a polypeptide comprising the amino acid sequence of a Chlamydia spp. CT443 polypeptide described herein and one, two, or three of the polypeptides defined in (b) through (d). The combination may include any of the non-MOMP antigen combinations of (b), (c), and / or (d) above. Typically, the combination includes the polypeptide of (b). In some embodiments, the combination further includes a native Chlamydia spp. MOMP polypeptide or a variant thereof.

[0244] In another aspect, the present invention provides a combination comprising a polypeptide comprising the amino acid sequence of a Chlamydia spp. CT584 polypeptide described herein and one, two, or three of the polypeptides defined in (a), (c), or (d). The combination may include any of the non-MOMP antigen combinations of (a), (c), and / or (d) above. Typically, the combination includes the polypeptide of (a). In some embodiments, the combination further includes a native Chlamydia spp. MOMP polypeptide or a variant thereof.

[0245] In another aspect, the invention provides combinations comprising a polypeptide comprising the amino acid sequence of a Chlamydia spp. CT600 polypeptide described herein and one, two, or three of the polypeptides defined in (a), (b), or (d). The combinations may include any of the non-MOMP antigen combinations of (a), (b), and / or (d) above. In some embodiments, the combinations further comprise a native Chlamydia spp. MOMP polypeptide or a variant thereof.

[0246] In another aspect, the invention provides combinations comprising a polypeptide comprising the amino acid sequence of a Chlamydia spp. CT812 polypeptide described herein and one, two, or three of the polypeptides defined in (a)-(c). The combinations may include any of the non-MOMP antigen combinations of (a), (b), and / or (c) above. In some embodiments, the combinations further comprise a native Chlamydia spp. MOMP polypeptide or a variant thereof.

[0247] Thus, a combination of the present invention may comprise a native Chlamydia species MOMP polypeptide or a variant thereof, and one, two, three, or four of the polypeptides in (a) through (d). Variants of native Chlamydia species MOMP polypeptides include MOMP polypeptides containing one or more conservative amino acid substitutions, MOMP polypeptides containing single amino acid substitutions at one or more (e.g., all) positions corresponding to cysteine ​​residues in native Chlamydia species MOMP polypeptides, and MOMP polypeptides containing single amino acid substitutions at one or more (e.g., all) positions corresponding to glycosylation sites, preferably N-glycosylation sites, in native Chlamydia species MOMP polypeptides. Variants of native Chlamydia species MOMP polypeptides also include MOMP polypeptides containing secretory signal peptide sequences and MOMP polypeptides containing heterologous transmembrane domains. In some embodiments, variants of native Chlamydia species MOMP polypeptides contain secretory signal peptide sequences. In some embodiments, a variant of a native Chlamydia species MOMP polypeptide comprises a secretory signal peptide sequence and a heterologous transmembrane domain.

[0248] The polypeptide combinations disclosed herein include all of the individual polypeptides in the same composition and all of the individual polypeptides in one or more separate compositions. In some embodiments, all of the individual polypeptides in the combinations described herein are in the same composition. In some embodiments, each of the polypeptides in the combinations described herein is in a separate composition. In some embodiments, two or more polypeptides in the combinations described herein are in two or more compositions. In some embodiments of the combinations of the invention, one, two, three, or four of the polypeptides (a) to (d) above are in one or more compositions. In some embodiments of the combinations of the invention, two, three, or four of the polypeptides (a) to (d) above are in two or more separate compositions. Typically, all of the individual polypeptides are in the same composition.

[0249] LNP In certain embodiments, a composition of the invention (e.g., a composition comprising a nucleic acid of the invention) further comprises a lipid nanoparticle (LNP). In certain embodiments, the nucleic acid of the invention is encapsulated in an LNP.

[0250] LNPs of the present disclosure can include lipids from four categories: (i) ionizable lipids (e.g., cationic lipids), (ii) PEGylated lipids, (iii) cholesterol-based lipids, and (iv) helper lipids.

[0251] A. Ionized lipids Ionizable lipids facilitate encapsulation of mRNA and can be cationic lipids, which provide a positively charged environment at low pH to facilitate efficient encapsulation of negatively charged mRNA drug substances.

[0252] In some embodiments, the cationic lipid is OF-02: [ka] OF-02 is a non-degradable structural analog of OF-Deg-Lin. While OF-Deg-Lin contains a degradable ester linkage connecting the diketopiperazine core and the doubly unsaturated tail, OF-02 contains a non-degradable 1,2-amino-alcohol linkage connecting the same diketopiperazine core and the doubly unsaturated tail (Fenton et al., Adv Mater. (2016) 28:2939; U.S. Patent No. 10,201,618). The exemplary LNP formulation herein, Lipid A, contains OF-02.

[0253] 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]

[0254] An exemplary LNP formulation herein, lipid B, contains cKK-E10.

[0255] In some embodiments, the cationic lipid is GL-HEPES-E3-E10-DS-3-E18-1 (2-(4-(2-(3-(bis((Z)-2-hydroxyoctadec-9-en-1-yl)amino)propyl)disulfanayl(ethyl)piperazin-1-yl)ethyl 4-(bis(2-hydroxydecyl)amino)butanoate), which is a HEPES-based disulfide cationic lipid with a piperazine core and has formula III: [ka]

[0256] An exemplary LNP formulation herein, Lipid C, contains GL-HEPES-E3-E10-DS-3-E18-1. Lipid C has the same composition as Lipid A or Lipid B, but differs in the cationic lipid.

[0257] In some embodiments, the cationic lipid is GL-HEPES-E3-E12-DS-4-E10 (2-(4-(2-((3-(bis(2-hydroxydecyl)amino)butyl)disulfanayl)ethyl)piperazin-1-yl)ethyl 4-(bis(2-hydroxydodecyl)amino)butanoate), which is a HEPES-based disulfide cationic lipid with a piperazine core and has formula IV: [ka]

[0258] An exemplary LNP formulation herein, Lipid D, contains GL-HEPES-E3-E12-DS-4-E10. Lipid D has the same composition as Lipid A or Lipid B, with the exception of the cationic lipid.

[0259] In some embodiments, the cationic lipid is GL-HEPES-E3-E12-DS-3-E14 (2-(4-(2-((3-(bis(2-hydroxytetradecyl)amino)propyl)disulfanayl)ethyl)piperazin-1-yl)ethyl 4-(bis(2-hydroxydodecyl)amino)butanoate), which is a HEPES-based disulfide cationic lipid with a piperazine core and has formula V: [ka]

[0260] An exemplary LNP formulation herein, Lipid E, contains GL-HEPES-E3-E12-DS-3-E14. Lipid E ​​has the same composition as Lipid A or Lipid B, but differs in the cationic lipid.

[0261] 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 shown in Scheme 1.

[0262] Scheme 1: General synthetic scheme for lipids of formula (III), (IV) and (V) [ka] In some embodiments, the cationic lipid is MC3, having formula VI: [ka]

[0263] 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]

[0264] In some embodiments, the cationic lipid is ALC-0315 [(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate), which has Formula VIII: [ka]

[0265] In some embodiments, the cationic lipid is cOrn-EE1 and has formula IX: [ka]

[0266] In some embodiments, the cationic lipid is ATX-126 (4,4'-[[[[3-(dimethylamino)propyl]thio]carbonyl]imino]bis-butanoic acid, 1,1'-bis(1-heptyloctyl) ester), having formula X. [ka]

[0267] In some embodiments, the cationic lipid is 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-Heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102); [(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); [3-(dimethylamino)-2-[(Z)-octadec-9-enoyl]oxypropyl](Z)-octadec-9-enoate noate (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]phenanthren-3-yl]N-[2-(dimethylamino)ethyl]carbamate (DC -Chol; tetrakis(8-methylnonyl)3,3',3'',3'''-(((methylazanediyl)bis(propane-3,1diyl))bis(azanetriyl))tetrapropionic acid (306Oi10); decyl(2-(dioctylammonio)ethyl)phosphate (9A1P9); ethyl 5,5-di((Z)-heptadec-8-en-1-yl)-1-(3-(pyrrolidin-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)azanediyl)dipropionate (BAME-O16B); 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200); 3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione (cKK-E12); Hexa(octa) N-3-yl)9,9',9'',9''',9'''',9'''''-((((benzene-1,3,5-tricarbonyl)iris(azanediyl))tris(propane-3,1-diyl))tris(azanetriyl))hexanoate (FTT5);(((3,6-dioxopiperazine-2,5-diyl)bis(butane-4,1-diyl))bis(azanetriyl))tetrakis(ethane-2,1-diyl) (9Z,9'Z,9''Z,9''Z,12Z,12'Z,12''Z,12''Z)-tetrakis(octadeca-9,12-dienoate) (OF-Deg-Lin);TT3;N; 1 ,N 3 ,N 5 -Tris(3-(didodecylamino)propyl)benzene-1,3,5-tricarboxamide; N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]benzamide (MVL5); Heptadecan-9-yl 8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octano ate (Lipid 5); 4,4'-[[[[3-(dimethylamino)propyl]thio]carbonyl]imino]bis-butanoic acid, 1,1'-bis(1-heptyloctyl) ester (ATX-126) GL-HEPES-E3-E10-DS-3-E18-1; GL-HEPES-E3-E12-DS-4-E10; GL-HEPES-E3-E12-DS-3-E14, IM-001; and combinations thereof.

[0268] In some embodiments, the cationic lipid is IM-001, represented by Formula XI (EP23306049.0): [ka]

[0269] An exemplary LNP formulation herein, Lipid G, contains IM-001. Lipid G has the same composition as Lipid A or Lipid B, except for the cationic lipid.

[0270] The cationic lipid IM-001(XI) can be synthesized according to the general procedure described in Scheme 2.

[0271] Scheme 2: General synthesis scheme for lipids of formula (XI) [ka] Scheme 2 can be carried out as described in Example 14.

[0272] In some embodiments, the cationic lipid is biodegradable.

[0273] In some embodiments, the cationic lipid is not biodegradable.

[0274] In some embodiments, the cationic lipid is cleavable.

[0275] In some embodiments, the cationic lipid is not cleavable.

[0276] Cationic lipids are described in further detail in Dong et al. (PNAS. 111(11):3955-60. 2014); Fenton et al. (Adv Mater. 28:2939. 2016); U.S. Pat. No. 9,512,073; and U.S. Pat. No. 10,201,618, each of which is incorporated herein by reference.

[0277] B. PEGylated lipids PEGylated lipid components provide control over nanoparticle size and stability. The addition of such components can prevent complex aggregation, extend circulation life, and provide a means to enhance delivery of lipid-nucleic acid pharmaceutical compositions to target tissues (Klibanov et al., FEBS Letters 268(1):235-71990). These components can be selected to be rapidly exchanged from the pharmaceutical composition in vivo (see, for example, U.S. Patent No. 5,885,613).

[0278] Contemplated PEGylated lipids include C6-C8 PEG-ceramides, such as derivatized ceramides (e.g., N-octanoyl-sphingosine-1-[succinyl(methoxypolyethylene glycol)] (C8 PEG ceramide)). 20 (e.g., 8, C 10 , C 12 , C 14 , C 16 or C 18 Examples of PEGylated lipids include, but are not limited to, polyethylene glycols (PEGs) of up to 5 kDa in length covalently attached to lipids having alkyl chains of up to 5 kDa in length. In some embodiments, the PEGylated lipid is 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-dialkyloxypropylcarbamate; 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); and combinations thereof.

[0279] In certain embodiments, the PEG has a high molecular weight, e.g., 2000-2400 g / mol. In certain embodiments, the PEG is PEG2000 (or PEG-2K). In certain embodiments, the PEGylated lipid herein is 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.

[0280] C. Cholesterol-based lipids The cholesterol component provides stability to the lipid bilayer structure within the nanoparticle. In some embodiments, the LNP comprises one or more cholesterol-based lipids. Suitable cholesterol-based lipids include, for example, DC-Choi (N,N-dimethyl-N-ethylcarboxamidocholesterol), 1,4-bis(3-N-oleylamino-propyl)piperazine (Gao et al., Biochem Biophys Res Comm. (1991) 179:280; Wolf et al., Biochem Biophys Res Comm. (1991) 179:280), and 1,4-bis(3-N-oleylamino-propyl)piperazine (Gao et al., Biochem Biophys Res Comm. (1991) 179:280). al., BioTechniques (1997) 23:139; U.S. Pat. No. 5,744,335), imidazole cholesterol ester ("ICE"; WO 2011 / 068810), sitosterol (22,23-dihydrostigmasterol), β-sitosterol, sitostanol, fucosterol, stigmasterol (stigmasta-5,22-dien-3-ol), ergosterol; desmosterol (3β-hydroxy-5,24-cholestadiene); lanosterol (8,24-lanostadien-3b-ol); 7-dehydrocholesterol (Δ5,7-cholesterol); dihydrolanosterol (24, 25-dihydrolanosterol); zymosterol (5α-cholesta-8,24-dien-3β-ol); lanosterol (5α-cholest-7-en-3β-ol); diosgenin ((3β,25R)-spirost-5-en-3-ol); campesterol (campest-5-en-3β-ol); campestanol (5a-campestan-3b-ol); 24-methylenecholesterol (5,24(28)-cholestadien-24-methylene-3β-ol); cholesteryl margarate (cholest-5-en-3β-ylheptadecanoate); cholesteryl oleate; cholesteryl stearate and other modified forms of cholesterol. In some embodiments, the cholesterol-based lipid used in the LNP is cholesterol.

[0281] D. Helper lipids Helper lipids improve the structural stability of LNPs and aid in endosomal escape, which improves uptake and release of mRNA drug payloads. In some embodiments, the helper lipids are zwitterionic lipids with fusogenic properties to improve uptake and release of drug payloads. Examples of helper lipids are 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-dielideyl-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).

[0282] Other exemplary helper lipids are dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), phosphatidylserine, sphingolipids, sphingomyelin, ceramide, cerebroside, ganglioside, 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), or combinations thereof. In certain embodiments, the helper lipid is DOPE. In certain embodiments, the helper lipid is DSPC.

[0283] In various embodiments, the LNPs comprise (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, or IM-001, (ii) DMG-PEG2000; (iii) cholesterol; and (iv) DOPE.

[0284] In other embodiments, the LNP comprises (i) SM-102; (ii) DMG-PEG2000; (iii) cholesterol; and (iv) DSPC.

[0285] In yet other embodiments, the LNP comprises: (i) ALC-0315; (ii) ALC-0159; (iii) cholesterol; and (iv) DSPC.

[0286] In yet other embodiments, the LNP comprises (i) ATX-126, (ii) DMG-PEG2000, (iii) cholesterol, and (iv) DSPC.

[0287] E. Molar ratio of lipid components The molar ratios of the above components are important to the effectiveness of LNPs in delivering mRNA. The molar ratios of cationic lipid, PEGylated lipid, cholesterol-based lipid, and helper lipid are provided as A:B:C:D (where A+B+C+D=100%). In some embodiments, the molar ratio of cationic lipid in the LNP to total lipid (i.e., A) is 35-55%, e.g., 35-50% (e.g., 38-42%, e.g., 40% or 45-50%). In some embodiments, the molar ratio of PEGylated lipid component to total lipid (i.e., B) is 0.25-2.75% (e.g., 1-2%, e.g., 1.5%). In some embodiments, the molar ratio of cholesterol-based lipid to total lipid (i.e., C) is 20-50% (e.g., 27-30%, e.g., 28.5% or 38-43%). In some embodiments, the molar ratio of helper lipid to total lipid (i.e., D) is 5-35% (e.g., 28-32%, such as 30%, or 8-12%, such as 10%). In some embodiments, the (PEGylated lipid + cholesterol) components have the same molar amount as the helper lipid. In some embodiments, the LNPs have a molar ratio of cationic lipid to helper lipid greater than 1.

[0288] In certain embodiments, the LNPs of the present disclosure are cationic lipid in a molar ratio of 35% to 55% or 40% to 50% (e.g., 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, or 55% cationic lipid); polyethylene glycol (PEG)-conjugated (PEGylated) lipids at a molar ratio of 0.25% to 2.75% or 1.00% to 2.00% (e.g., 0.25%, 0.50%, 0.75%, 1.00%, 1.25%, 1.50%, 1.75%, 2.00%, 2.25%, 2.50%, or 2.75% PEGylated lipid); Cholesterol-based lipids in a molar ratio of 20% to 50%, 25% to 45%, or 28.5% to 43% (e.g., 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% cholesterol-based lipids); and A molar ratio of helper lipid of 5% to 35%, 8% to 30%, or 10% to 30% (e.g., 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% molar ratio of helper lipid), All molar ratios are relative to the total lipid content of the LNPs.

[0289] In certain embodiments, the LNP comprises a 40% molar ratio of cationic lipid; a 1.5% molar ratio of PEGylated lipid; a 28.5% molar ratio of cholesterol-based lipid; and a 30% molar ratio of helper lipid.

[0290] In certain embodiments, the LNPs of the present disclosure comprise a 45-50% molar ratio of cationic lipid; a 1.5-1.7% molar ratio of PEGylated lipid; a 38-43% molar ratio of cholesterol-based lipid; and a 9-10% molar ratio of helper lipid.

[0291] In certain embodiments, the PEGylated lipid is dimyristoyl-PEG2000 (DMG-PEG2000).

[0292] In various embodiments, the cholesterol-based lipid is cholesterol.

[0293] In some embodiments, the helper lipid is 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE).

[0294] In certain embodiments, the LNP comprises OF-02 at a molar ratio of 35% to 55%; DMG-PEG2000 at a molar ratio of 0.25% to 2.75%; cholesterol at a molar ratio of 20% to 50%; and DOPE at a molar ratio of 5% to 35%.

[0295] In certain embodiments, the LNP comprises cKK-E10 at a molar ratio of 35% to 55%; DMG-PEG2000 at a molar ratio of 0.25% to 2.75%; cholesterol at a molar ratio of 20% to 50%; and DOPE at a molar ratio of 5% to 35%.

[0296] In certain embodiments, the LNPs comprise 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%.

[0297] In certain embodiments, the LNPs comprise 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%.

[0298] In certain embodiments, the LNPs comprise 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%.

[0299] In certain embodiments, the LNPs comprise SM-102 at a molar ratio of 35% to 55%; DMG-PEG2000 at a molar ratio of 0.25% to 2.75%; cholesterol at a molar ratio of 20% to 50%; and DSPC at a molar ratio of 5% to 35%.

[0300] In certain embodiments, the LNPs comprise ALC-0315 at a molar ratio of 35% to 55%; ALC-0159 at a molar ratio of 0.25% to 2.75%; cholesterol at a molar ratio of 20% to 50%; and DSPC at a molar ratio of 5% to 35%.

[0301] In certain embodiments, the LNPs comprise a 40% molar ratio of OF-02, a 1.5% molar ratio of DMG-PEG2000, a 28.5% molar ratio of cholesterol, and a 30% molar ratio of DOPE. This LNP formulation is referred to herein as "Lipid A."

[0302] In certain embodiments, the LNPs comprise 40% molar cKK-E10, 1.5% molar DMG-PEG2000, 28.5% molar cholesterol, and 30% molar DOPE. This LNP formulation is referred to herein as "Lipid B."

[0303] In certain embodiments, the LNPs comprise 40% GL-HEPES-E3-E10-DS-3-E18-1, 1.5% DMG-PEG2000, 28.5% cholesterol, and 30% DOPE. This LNP formulation is referred to herein as "Lipid C."

[0304] In certain embodiments, the LNPs comprise GL-HEPES-E3-E12-DS-4-E10 (40% molar ratio; 1.5% molar ratio DMG-PEG2000; 28.5% molar ratio cholesterol; and 30% molar ratio DOPE; this LNP formulation is referred to herein as "Lipid D."

[0305] In certain embodiments, the LNP comprises a 40% molar ratio of GL-HEPES-E3-E12-DS-3-E14; a 1.5% molar ratio of DMG-PEG2000; a 28.5% molar ratio of cholesterol; and a 30% molar ratio of DOPE; this LNP formulation is referred to herein as "Lipid E."

[0306] In certain embodiments, the LNPs comprise DLin-MC3-DMA (MC3) at a molar ratio of 50%, DMG-PEG2000 at a molar ratio of 1.5%, cholesterol at a molar ratio of 38.5%, and DSPC at a molar ratio of 10%. This LNP formulation is referred to herein as "Lipid F."

[0307] In certain embodiments, the LNP comprises 40% molar IM-001, 1.5% molar DMG-PEG2000, 28.5% molar cholesterol, and 30% molar DOPE; this LNP formulation is referred to herein as "Lipid G."

[0308] In certain embodiments, the LNPs comprise a 50% molar ratio of 9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102); a 10% molar ratio of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); a 38.5% molar ratio of cholesterol; and a 1.5% molar ratio of 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000).

[0309] In certain embodiments, the LNPs comprise a 46.3% molar ratio of (4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); a 9.4% molar ratio of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); a 42.7% molar ratio of cholesterol; and a 1.6% molar ratio of 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159).

[0310] In certain embodiments, the LNPs comprise a 47.4% molar ratio of (4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); a 10% molar ratio of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); a 40.9% molar ratio of cholesterol; and a 1.7% molar ratio of 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159).

[0311] In certain embodiments, the LNPs comprise 4,4'-[[[[3-(dimethylamino)propyl]thio]carbonyl]imino]bis-butanoic acid, 1,1'-bis(1-heptyloctyl) ester (ATX-126) at a molar ratio of 50%, DMG-PEG2000 at a molar ratio of 1.5%, cholesterol at a molar ratio of 38.5%, and DSPC at a molar ratio of 10%.

[0312] In some embodiments, the LNP formulation is as defined for "lipid A," "lipid B," "lipid D," or lipid G (e.g., lipid D or lipid G).

[0313] To calculate the actual amount of each lipid to be included in the LNP formulation, the molar amount of the cationic lipid is first determined 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 to be transported by the LNP). Next, the molar amounts of each of the other lipids are calculated based on the molar amount of the cationic lipid and the selected molar ratio. These molar amounts are then converted to weight using the molecular weight of each lipid.

[0314] F. Nucleic Acids in LNP The LNP compositions described herein can include nucleic acids (eg, mRNA) of the invention.

[0315] Optionally, LNPs can be multivalent. In some embodiments, LNPs can carry nucleic acids, such as mRNAs, encoding two or more polypeptides of the invention, e.g., 2, 3, 4, 5, 6, 7, or 8 polypeptides. For example, LNPs can carry multiple nucleic acids (e.g., mRNAs) of the invention, each encoding a different polypeptide of the invention. Or, LNPs have polycistronic mRNAs that can be translated into two or more polypeptides of the invention (e.g., each antigen-encoding sequence is separated by a nucleotide linker encoding a self-cleaving peptide, such as a 2A peptide). LNPs carrying different nucleic acids (e.g., mRNAs) typically contain (encapsulate) multiple copies of each nucleic acid. For example, LNPs carrying or encapsulating two different nucleic acids typically encapsulate multiple copies of each of the two different nucleic acids.

[0316] In some embodiments, two or more (e.g., 2, 3, or 4) nucleic acids (e.g., mRNAs) described herein encoding different polypeptides described herein are co-encapsulated in the same LNP. For example, an LNP described herein may co-encapsulate ((a) a nucleic acid described herein comprising a nucleotide sequence encoding a modified MOMP polypeptide described herein, (b) a nucleic acid described herein comprising a nucleotide sequence encoding a chimeric MOMP VD polypeptide described herein, and (c) a nucleic acid described herein comprising a nucleotide sequence encoding a Chlamydia spp. CT443 polypeptide described herein. In some embodiments, an LNP described herein may co-encapsulate ((a) a nucleic acid described herein comprising a nucleotide sequence encoding a modified MOMP polypeptide described herein, (b) a nucleic acid described herein comprising a nucleotide sequence encoding a chimeric MOMP VD polypeptide described herein, and (c) a nucleic acid described herein comprising a nucleotide sequence encoding a Chlamydia spp. CT584 polypeptide described herein. In some embodiments, an LNP described herein may co-encapsulate (a) a nucleic acid described herein comprising a nucleotide sequence encoding a modified MOMP polypeptide described herein, (b) a nucleic acid described herein comprising a nucleotide sequence encoding a chimeric MOMP VD polypeptide described herein, and (c) a nucleic acid described herein comprising a nucleotide sequence encoding a Chlamydia spp. CT584 polypeptide described herein. (c) a nucleic acid described herein comprising a nucleotide sequence encoding a Chlamydia spp. CT443 polypeptide described herein; and (d) a nucleic acid described herein comprising a nucleotide sequence encoding a Chlamydia spp. CT584 polypeptide described herein.

[0317] Any two nucleic acids (e.g., two mRNAs) described herein may be present in a weight ratio of 1:1. Any three nucleic acids (e.g., three mRNAs) described herein may be present in a weight ratio of 1:1:1. Any four nucleic acids (e.g., four mRNAs) described herein may be present in a weight ratio of 1:1:1:1.

[0318] In some embodiments, the LNP is as described herein (eg, lipid D or lipid G).

[0319] Alternatively, any two or more (eg, four) nucleic acids (eg, mRNAs) encoding different polypeptides described herein are encapsulated in separate LNPs.

[0320] In some embodiments, a single LNP formulation can include multiple types of LNPs (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more), each type carrying a different nucleic acid (e.g., mRNA).

[0321] When the nucleic acid is mRNA, the mRNA can be unmodified (i.e., containing only natural ribonucleotides A, U, C, and / or G linked by phosphodiester bonds) or chemically modified (e.g., containing nucleotide analogs such as pseudouridine (e.g., N-1-methylpseudouridine), 2'-fluororibonucleotides, and 2'-methoxyribonucleotides, and / or phosphorothioate linkages). The mRNA molecule can include a 5' cap and a poly-A tail.

[0322] G. Buffers and Other Components To stabilize the nucleic acid and / or LNP (e.g., to extend the shelf life of a vaccine product), facilitate administration of the LNP pharmaceutical composition, and / or enhance in vivo expression of the nucleic acid, the nucleic acid and / or LNP can be formulated in combination with one or more carriers, targeting ligands, stabilizing reagents (e.g., preservatives and antioxidants), and / or other pharmaceutically acceptable excipients. Examples of such excipients are parabens, thimerosal, thiomersal, chlorobutanol, benzalkonium chloride, and chelating agents (e.g., EDTA).

[0323] The LNP compositions of the present disclosure can be provided in a frozen liquid form or a lyophilized form. A variety of cryoprotectants can be used, including, but not limited to, sucrose, trehalose, glucose, mannitol, mannose, dextrose, and the like. The cryoprotectant can comprise 5-30% (w / v) of the LNP composition. In some embodiments, the LNP composition includes trehalose, for example, 5-30% (e.g., 10%) (w / v). When formulated with a cryoprotectant, the LNP composition can be frozen (or lyophilized and cryopreserved) at -20°C to -80°C.

[0324] The LNP composition can be provided to the patient in an aqueous buffer solution (thawed if previously frozen, or reconstituted in aqueous buffer solution at the bedside if previously lyophilized). The buffer is preferably isotonic and suitable for, e.g., intramuscular or intradermal injection. In some embodiments, the buffer solution is phosphate buffered saline (PBS).

[0325] nucleic acid The nucleic acids of the present invention can be RNA or DNA. The nucleic acids of the present invention can be single-stranded or double-stranded. In certain embodiments, the nucleic acid is RNA, such as mRNA.

[0326] mRNA In some embodiments, the nucleic acid of the invention is messenger RNA (mRNA). mRNA may be modified or unmodified. mRNA may contain one or more coding and non-coding regions. A coding region is alternatively referred to as an open reading frame (ORF). Non-coding regions in mRNA include the 5' cap, 5' untranslated region (UTR), 3' UTR, and polyA tail. mRNA can be purified from natural sources, produced using recombinant expression systems (e.g., in vitro transcription), and optionally purified or chemically synthesized.

[0327] In certain embodiments, the mRNA comprises an ORF encoding an antigen of interest. In certain embodiments, the RNA (e.g., mRNA) further comprises at least one 5' UTR, 3' UTR, poly(A) tail, and / or 5' cap. In some embodiments, the mRNA comprises: (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) is directly linked to the 5' end of (ii) via a 3' to 5' phosphodiester bond; the 3' end of (ii) is directly linked to the 5' end of (iii) via a 3' to 5' phosphodiester bond; the 3' end of (iii) is directly linked to the 5' end of (iv) via a 3' to 5' phosphodiester bond; or the 3' end of (iv) is directly linked to the 5' end of (v) via a 3' to 5' phosphodiester bond.

[0328] In certain embodiments, the mRNA comprises at least one, at least two, at least three, or more stop codons, wherein the stop codons may be selected from UAA, UGA, and UAG, and wherein the at least two, at least three, or more stop codons may be the same or different. Typically, at least one stop codon comprises UAA or UGA (e.g., UAA). Typically, the 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 from UAA and UGA (e.g., UGAUAA), in particular. Typically, the at least three stop codons comprise UAA, UGA, and UAG (e.g., UGAUAAUAG).

[0329] The mRNA sequence is presented in the 5' to 3' direction unless otherwise indicated.

[0330] 5' Cap The 5' cap on an mRNA may confer resistance to nucleases found in most eukaryotic cells and may promote translation efficiency. Several types of 5' caps are known: 7-methylguanosine cap ("m 7 The nucleotide sequence of the nucleotide sequence of the transcribed nucleotide (also referred to as "Cap-G" or "Cap-0") contains a guanosine linked to the first transcribed nucleotide through a 5'-5'-triphosphate bond.

[0331] A 5' cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5' nucleotide, leaving two terminal phosphates; then, guanosine triphosphate (GTP) is added to the terminal phosphate via a guanylyltransferase, generating a 5'5'5 triphosphate linkage; and then, the 7-nitrogen of guanine is methylated by a 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. Additional cap structures are described in U.S. Patent Application Publication Nos. 2016 / 0032356 and 2018 / 0125989, which are incorporated herein by reference.

[0332] 5'-Capping of polynucleotides can be simultaneously completed during in vitro transcription reactions using the following chemical RNA cap analogs 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). 5'-Capping of modified RNAs can be completed post-transcriptionally using vaccinia virus capping enzyme to generate the Cap 0 structure: m7G(5')ppp(5')G. Cap 1 structure can be generated using both vaccinia virus capping enzyme and 2'-O-methyltransferase to generate m7G(5')ppp(5')G-2'-O-methyl. 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-methyltransferase. 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-methyltransferase.

[0333] In certain embodiments, the mRNA of the invention comprises 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.

[0334] In certain embodiments, the mRNA of the invention comprises the following 5' cap: [ka]

[0335] Untranslated Regions (UTRs) In some embodiments, mRNAs of the present invention comprise 5' and / or 3' untranslated regions (UTRs). In mRNAs, the 5' UTR begins at the transcription initiation site and continues up to, but not including, the start codon. The 3' UTR begins immediately after the stop codon and continues to the transcription termination signal.

[0336] In some embodiments, the mRNAs disclosed herein may comprise a 5' UTR that contains one or more elements that affect mRNA stability or translation. In some embodiments, the 5' UTR may be about 10 to 5,000 nucleotides in length. In some embodiments, the 5' UTR may be about 50 to 500 nucleotides in length. In some embodiments, the 5' UTR may be at least about 10 nucleotides in length, about 20 nucleotides in length, about 30 nucleotides in length, about 40 nucleotides in length, about 50 nucleotides in length, about 100 nucleotides in length, about 150 nucleotides in length, about 200 nucleotides in length, about 250 nucleotides in length, about 300 nucleotides in length, about 350 nucleotides in length, about 400 nucleotides in length, about 450 nucleotides in length, about 500 nucleotides in length, about 550 nucleotides in length, about 600 nucleotides in length, or about The length is 650 nucleotides, about 700 nucleotides, about 750 nucleotides, about 800 nucleotides, about 850 nucleotides, about 900 nucleotides, about 950 nucleotides, about 1,000 nucleotides, about 1,500 nucleotides, about 2,000 nucleotides, about 2,500 nucleotides, about 3,000 nucleotides, about 3,500 nucleotides, about 4,000 nucleotides, about 4,500 nucleotides, or about 5,000 nucleotides.

[0337] In some embodiments, the mRNAs disclosed herein may include a 3' UTR that includes one or more of a polyadenylation signal, a binding site for a protein that affects the stability of the mRNA's location in the cell, or one or more binding sites for an miRNA. In some embodiments, the 3' UTR may be 50-5,000 or more nucleotides in length. In some embodiments, the 3' UTR may be 50-1,000 nucleotides in length or longer. In some embodiments, the 3'UTR is at least about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, or 5,000 nucleotides in length.

[0338] In some embodiments, the mRNAs disclosed herein may include a 5' or 3' UTR that is derived from a gene that is distinct from the gene encoded by the mRNA transcript (i.e., the UTR is a heterologous UTR).

[0339] In certain embodiments, the 5' and / or 3' UTR sequences may be derived from stable mRNAs (e.g., globin, actin, GAPDH, tubulin, histones, or citric acid cycle enzymes) to enhance mRNA stability. For example, the 5' UTR sequence may include a subsequence of the CMV immediate early 1 (IE1) gene or a fragment thereof to improve nuclease resistance and / or improve the half-life of the mRNA. It is also contemplated that a sequence encoding human growth hormone (hGH) or a fragment thereof may be included in the 3' end or untranslated region of the mRNA. Generally, these modifications improve the stability and / or pharmacokinetic properties (e.g., half-life) of the mRNA relative to its unmodified counterpart, including, for example, modifications made to improve such mRNA resistance to in vivo nuclease digestion.

[0340] Exemplary 5'UTRs include sequences from the CMV immediate early 1 (IE1) gene (U.S. Patent Application Publication Nos. 2014 / 0206753 and 2015 / 0157565, each of which is incorporated herein by reference) or the sequence GGGAUCCUACC (SEQ ID NO: 837) (U.S. Patent Application Publication No. 2016 / 0151409, incorporated herein by reference).

[0341] In various embodiments, the 5'UTR can 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 a 5'TOP motif (oligopyrimidine tract) (e.g., U.S. Patent Application Publication Nos. 2017 / 0029847, 2016 / 0304883, 2016 / 0235864, and 2016 / 0166710, each of which is incorporated herein by reference).

[0342] In certain embodiments, the 5'UTR is derived from the ribosomal protein large 32 (L32) gene (US Patent Application Publication No. 2017 / 0029847, supra).

[0343] In certain embodiments, the 5'UTR is derived from the 5'UTR of the hydroxysteroid (17-b) dehydrogenase 4 gene (HSD17B4) (US Patent Application Publication No. 2016 / 0166710, supra).

[0344] In certain embodiments, the 5'UTR is derived from the 5'UTR of the ATP5A1 gene (US Patent Application Publication No. 2016 / 0166710, supra).

[0345] In some embodiments, an internal ribosome entry site (IRES) is used in place of the 5'UTR.

[0346] In some embodiments, the 5'UTR comprises the nucleic acid sequence set forth in SEQ ID NO: 838 and reproduced below: [ka] .

[0347] In some embodiments, the 3'UTR comprises the nucleic acid sequence set forth in SEQ ID NO: 839 and reproduced below: CGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUC (SEQ ID NO: 839).

[0348] The 5'UTR and 3'UTR are described in further detail in WO 2012 / 075040, which is incorporated herein by reference.

[0349] Polyadenylation tail As used herein, the terms "poly(A) sequence," "poly(A) tail," and "poly(A) region" refer to a sequence of adenosine nucleotides at the 3' end of an mRNA molecule. The poly(A) tail may confer stability to the mRNA and protect it from exonuclease degradation. The poly(A) tail may enhance translation. In some embodiments, the poly(A) tail is essentially homopolymeric. For example, a poly(A) tail of 100 adenosine nucleotides may have a length of essentially 100 nucleotides. In certain embodiments, the poly(A) tail may be interrupted by at least one nucleotide that is 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 of more than 100 nucleotides (comprising 100 adenosine nucleotides and at least one nucleotide or stretch of nucleotides that is different from adenosine nucleotides). In certain embodiments, the poly(A) tail comprises the sequence AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 840).

[0350] As used herein, "poly(A) tail" typically relates to RNA. However, in the context of the present disclosure, the term also relates to the corresponding sequence in a DNA molecule (e.g., a "poly(T) sequence").

[0351] The poly(A) tail can comprise 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 can be at least about 10, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or 500 adenosine nucleotides. In some embodiments, the poly(A) tail comprises at least 50, at least 75, or at least 100 adenosine nucleotides.

[0352] In some embodiments where the nucleic acid is RNA, the poly(A) tail of the nucleic acid is obtained from a 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 being transcribed from a DNA template. In various embodiments, the poly(A) tail is generated by enzymatic polyadenylation of the RNA (after in vitro transcription of the RNA) using a commercially available polyadenylation kit and corresponding protocol, or alternatively by using immobilized poly(A) polymerase, for example, using the methods and procedures described in WO 2016 / 174271.

[0353] The nucleic acid may include a poly(A) tail obtained by enzymatic polyadenylation, with the majority of the nucleic acid molecule comprising from about 100 (+ / -20) to about 500 (+ / -50) or about 250 (+ / -20) adenosine nucleotides.

[0354] In some embodiments, the nucleic acid may comprise a poly(A) tail derived from a template DNA, and may additionally comprise at least one additional poly(A) tail generated by enzymatic polyadenylation, e.g., as described in WO 2016 / 091391.

[0355] In certain embodiments, the nucleic acid comprises at least one polyadenylation signal.

[0356] In various embodiments, the nucleic acid can include at least one poly(C) sequence.

[0357] As used herein, the term "poly(C) sequence" is intended to refer to a sequence of up to about 200 cytosine nucleotides. In some embodiments, the poly(C) sequence contains 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 contains about 30 cytosine nucleotides.

[0358] chemical modification The mRNA disclosed herein may be modified or unmodified. Typically, the mRNA includes at least one chemical modification. In some embodiments, the mRNA disclosed herein may contain one or more modifications that typically improve the stability of the RNA. Exemplary modifications can include backbone modifications, sugar modifications, or base modifications. In some embodiments, the disclosed mRNA can 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 mRNAs may contain modified nucleotide analogs or derivatives of purines and pyrimidines, such as 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-(carboxymethylaminomethyl) ... hydroxymethyl)-uracil, 5-fluoro-uracil, 5-bromo-uracil, 5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil, 5-methyl-uracil, N-uracil-5-oxyacetic acid methyl ester, 5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio-uracil, 5'-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil, queosine, β-D-mannosyl-queosine, phosphoramidate, phosphorothioate, peptide nucleotide, methylphosphonate, 7-deazaguanosine, 5-methylcytosine, and inosine.

[0359] In some embodiments, the disclosed mRNAs can comprise at least one chemical modification including, but not limited to, pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-l-methyl-l-deaza-pseudouridine, 2-thio-l-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-l-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine.

[0360] In some embodiments, the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, 5-methylcytosine, 5-methoxyuridine, and combinations thereof.

[0361] In some embodiments, the chemical modification comprises N1-methylpseudouridine. Typically, the chemical modification comprises N1-methylpseudouridine. Typically, the chemical modification comprises N1-methylpseudouridine in place of all uridines, i.e., 100% of the U residues are N1-methylpseudouridine.

[0362] 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.

[0363] 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.

[0364] The preparation of such analogs is described, for example, in U.S. Pat. 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.

[0365] mRNA synthesis The mRNA disclosed herein can be synthesized according to any of a variety of methods. For example, mRNA according to the present disclosure can be synthesized via in vitro transcription (IVT). Some methods for in vitro transcription are described, for example, in Geall et al. (2013) Semin. Immunol. 25(2):152-159; 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 that may contain DTT and magnesium ions, an appropriate RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNase I, pyrophosphatase, and / or RNase inhibitor. 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 can be considered impurities or contaminants that can be purified or removed to provide clean and / or homogeneous mRNA suitable for therapeutic use. While mRNA provided from an in vitro transcription reaction may be desirable in some embodiments, other sources of mRNA can be used in accordance with the present disclosure, including wild-type mRNA produced from bacteria, fungi, plants, and / or animals.

[0366] Processes for Producing the LNP Compositions of the Invention The LNPs can be prepared by various techniques currently known in the art. For example, multilamellar vesicles (MLVs) can be prepared according to conventional techniques, for example, by dissolving the lipids in a suitable solvent, depositing the selected lipids on the inner wall of a suitable container or vessel, and then evaporating the solvent to leave a thin film on the inside of the vessel, or by spray drying. MLVs can then be formed by adding an aqueous phase to the vessel with a vortex motion. Unilamellar vesicles (ULVs) can then be formed by homogenizing, sonicating, or extruding the multilamellar vesicles. In addition, unilamellar vesicles can be formed by detergent removal techniques.

[0367] Various methods are described in U.S. Patent Application Publication Nos. 2011 / 0244026, 2016 / 0038432, 2018 / 0153822, 2018 / 0125989, and PCT / US Patent Application Publication No. 2020 / 043223 (filed July 23, 2020) and can be used to implement the present disclosure. One exemplary process involves encapsulating mRNA by mixing the mRNA with a mixture of lipids without first preforming the lipids into lipid nanoparticles, as described in U.S. Patent Application Publication No. 2016 / 0038432. Another exemplary process involves encapsulating mRNA by mixing preformed LNPs with the mRNA, as described in U.S. Patent Application Publication No. 2018 / 0153822.

[0368] In some embodiments, the process for preparing mRNA-loaded LNPs comprises heating one or more solutions to a temperature above ambient temperature, the one or more solutions being a solution containing preformed lipid nanoparticles, a solution containing mRNA, and a mixed solution containing LNP-encapsulated mRNA. In some embodiments, the process comprises heating one or both of the mRNA solution and the preformed LNP solution prior to the mixing step. In some embodiments, the process comprises heating one or more of the preformed LNP-containing solution, the mRNA-containing solution, and the LNP-encapsulated mRNA solution during the mixing step. In some embodiments, the process comprises heating the LNP-encapsulated mRNA after the mixing step. In some embodiments, the temperature to which one or more of the solutions is heated is greater than or equal to about 30°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, or 70°C. In some embodiments, the temperature to which one or more of the solutions is heated ranges from 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.

[0369] Various methods can be used to prepare an mRNA solution suitable for the present disclosure. In some embodiments, mRNA can be directly dissolved in a buffer solution as described herein. In some embodiments, an mRNA solution can be prepared by mixing an mRNA stock solution with a buffer solution before mixing with a lipid solution for encapsulation. In some embodiments, an mRNA solution can be prepared by mixing an mRNA stock solution with a buffer solution immediately before mixing with a lipid solution for encapsulation. In some embodiments, a suitable mRNA stock solution can contain mRNA in water or a buffer at a concentration 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, 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 more.

[0370] In some embodiments, the mRNA stock solution is mixed with the 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 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 15x, or 20x greater than the rate of the mRNA stock solution. In some embodiments, the buffer solution is mixed at a flow rate in the range of about 100-6000 ml / min (e.g., about 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 about 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 greater.

[0371] In some embodiments, the mRNA stock solution is mixed at a flow rate ranging from about 10 to 600 ml / min (e.g., about 5 to 50 ml / min, about 10 to 30 ml / min, about 30 to 60 ml / min, about 60 to 120 ml / min, about 120 to 240 ml / min, about 240 to 360 ml / min, about 360 to 480 ml / min, or about 480 to 600 ml / min). In some embodiments, the mRNA stock solution is mixed at a flow rate of about 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 greater.

[0372] The process of incorporating desired mRNA into lipid nanoparticles is called "loading". Exemplary methods are described in Lasic et al., FEBS Lett. (1992) 312:255-8. The nucleic acid incorporated into LNPs can be completely 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", and the nucleic acid is completely or substantially contained within the internal space of the lipid nanoparticle.

[0373] Suitable LNPs can be made in various sizes.In some embodiments, the size reduction of lipid nanoparticles is associated with more efficient delivery of mRNA.Selection of appropriate LNP size can take into account the target cell or tissue site and the application for which lipid nanoparticles are made.

[0374] Various methods known in the art are available for sizing lipid nanoparticle populations. A preferred method herein utilizes a Zetasizer Nano ZS (Malvern Panalytical) to measure LNP particle size. In one protocol, 10 μl of LNP sample is mixed with 990 μl of 10% trehalose. This solution is placed in a cuvette and then loaded into the Zetasizer. The z-average diameter (nm) or cumulant average is considered to be the average size of the LNPs in the sample. The Zetasizer machine can also be used to measure the polydispersity index (PDI) using dynamic light scattering (DLS) and cumulant analysis of the autocorrelation function. The average LNP diameter can be reduced by sonication of the formed LNPs. Intermittent sonication cycles can be alternating with quasi-elastic light scattering (QELS) evaluation to guide efficient lipid nanoparticle synthesis.

[0375] In some embodiments, the majority of the purified LNPs, i.e., greater than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the LNPs, 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). In some embodiments, substantially all (e.g., greater 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).

[0376] 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.

[0377] In some embodiments, greater than about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the LNPs in the composition have a size 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), making them particularly suitable for pulmonary delivery via nebulization.

[0378] In some embodiments, the dispersity, or molecular size heterogeneity measure (PDI), of the LNPs in the pharmaceutical compositions provided by the present disclosure 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 about 0.08. The PDI can be measured by a Zetasizer machine, as described above.

[0379] In some embodiments, greater than about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the purified LNPs in the pharmaceutical compositions provided herein encapsulate mRNA within each individual particle. In some embodiments, substantially all (e.g., greater than 80% or 90%) of the purified lipid nanoparticles in the pharmaceutical composition encapsulate mRNA within each individual particle. In some embodiments, the lipid nanoparticles have an encapsulation efficiency of 50% to 99%; or greater than about 60, 65, 70, 75, 80, 85, 90, 92, 95, 98, or 99%. Typically, lipid nanoparticles for use herein have an encapsulation efficiency of at least 90% (e.g., at least 91, 92, 93, 94, or 95%).

[0380] 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 of greater than 1, about 1, about 2, about 3, about 4, about 5, about 6, about 7, or about 8. In further embodiments, exemplary LNPs herein have an N / P ratio of 4.

[0381] In some embodiments, a pharmaceutical composition according to the present disclosure contains 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, a pharmaceutical composition contains between about 0.1 μg and 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, or at least about 1000 μg of encapsulated mRNA.

[0382] In some embodiments, mRNA can be produced by chemical synthesis or by in vitro transcription (IVT) of a DNA template. In this process, a DNA template, such as a cDNA or pDNA template, is used to produce mRNA transcripts, and the DNA template is degraded by DNase in the IVT process. The transcripts are purified by depth filtration and tangential flow filtration (TFF). The purified transcripts are further modified by adding a cap and tail, and the modified RNA is again purified by depth filtration and TFF.

[0383] The mRNA is then prepared in an aqueous buffer and mixed with an amphipathic solution containing the lipid components of the LNP. The amphipathic solution for dissolving the four lipid components of the LNP can be an alcohol solution. In some embodiments, the alcohol is ethanol. The aqueous buffer can be, for example, a citrate, phosphate, acetate, or succinate buffer and can have a pH of about 3.0 to 7.0, e.g., about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, or about 6.5. The buffer can contain other components, such as salts (e.g., sodium, potassium, and / or calcium salts). In certain embodiments, the aqueous buffer has 1 mM citrate, 150 mM NaCl, and a pH of 3.5 or 4.5.

[0384] An exemplary process for producing mRNA-LNP compositions involves mixing a buffered mRNA solution with a solution of lipids in ethanol in a controlled, homogeneous manner, with the lipid:mRNA ratio maintained throughout the mixing process. In this exemplary example, the mRNA is present in an aqueous buffer containing citric acid 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., a cationic lipid, a PEGylated lipid, a cholesterol-based lipid, and a helper lipid) is dissolved in ethanol. The aqueous mRNA solution and the ethanolic lipid solution are mixed in a 4:1 volumetric ratio in a "T" mixer equipped with a nearly "pulseless" 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. TFF can be used to concentrate and buffer exchange the nascent LNPs obtained immediately after formation by the T mixing process. The diafiltration process is a continuous operation in which the volume is kept constant by adding an appropriate buffer at the same rate as the permeate flow.

[0385] vector In one aspect, disclosed herein is a vector comprising a nucleic acid disclosed herein. In some embodiments, the mRNA described herein can be cloned into a vector. Vectors include, but are not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors also include expression vectors, replication vectors, probe generation vectors, sequencing vectors, and vectors optimized for in vitro transcription (IVT).

[0386] In certain embodiments, this vector can be used to express mRNA in a host cell. In various embodiments, this vector can be used as a template for IVT. The construction of optimally translated IVT mRNA suitable for therapeutic use is described in detail in Sahin, et al. (2014). Nat. Rev. Drug Discov. 13, 759-780; Weissman (2015). Expert Rev. Vaccines 14, 265-281.

[0387] In some embodiments, the vectors disclosed herein may include at least the following 5' to 3': an RNA polymerase promoter; a polynucleotide sequence encoding a 5' UTR; a polynucleotide sequence encoding an ORF; a polynucleotide sequence encoding a 3' UTR; and a polynucleotide sequence encoding at least one RNA aptamer. In some embodiments, the vectors disclosed herein may include a polynucleotide sequence encoding a poly(A) sequence and / or a polyadenylation signal.

[0388] Various RNA polymerase promoters are known. In some embodiments, the promoter may be a T7 RNA polymerase promoter. Other useful promoters may include, but are not limited to, T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for the T7 promoter, T3 promoter, and SP6 promoter are known.

[0389] Also disclosed herein are host cells (e.g., mammalian cells, e.g., human cells) containing the vectors or nucleic acids disclosed herein. A "host cell" includes an individual cell or cell culture that can be or has been a recipient of exogenous nucleic acid. A host cell includes the progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation and / or alteration. A host cell includes cells transfected or infected in vivo or in vitro with a nucleic acid or vector disclosed herein.

[0390] Vectors can be introduced into target cells using any of a number of different methods, including, but not limited to, electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM830(BTX) (Harvard Instruments, Boston, Mass.) or Gene Pulser II (BioRad, Denver, Colo.), multiporator (Eppendorf, Hamburg, Germany), cationic liposome-mediated transfection using lipofection, polymer encapsulation, peptide-mediated transfection, "gene guns" (e.g., Nishikawa, et al. (2001). Hum Gene Ther. 12(8):861-70), or commercially available biological particle delivery systems such as the TransIT-RNA transfection Kit (Mirus, Madison, Wis.).

[0391] Chemical means for introducing vectors into host cells include colloidal dispersion systems, such as macromolecule 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 a liposome (e.g., an artificial membrane vesicle).

[0392] Regardless of the method used to introduce exogenous nucleic acid into host cells or otherwise expose the cells to the inhibitors of the present disclosure, various assays can be performed to confirm the presence of the mRNA sequence in the host cells.

[0393] Self-replicating, trans-replicating, and non-replicating RNAs 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.

[0394] Self-replicating RNA: Self-replicating (or self-propagating) RNA can be produced, for example, by using replication elements derived from alphaviruses and replacing structural viral proteins with nucleotide sequences encoding a protein of interest (e.g., a Chlamydia species antigen). Self-replicating RNAs are typically positive-strand molecules that can be directly translated after delivery to a cell; this translation then provides an RNA-dependent RNA polymerase that produces both antisense and sense transcripts from the delivered RNA. Thus, the delivered RNA results in the production of multiple daughter RNAs. These daughter RNAs, and collinear subgenomic transcripts, can either be translated to provide in situ expression of the encoded antigen or can be transcribed to provide additional 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 round of transcription is a large amplification of the number of introduced replicon RNAs, so that the encoded antigen becomes the major polypeptide product of the cell.

[0395] One suitable system for achieving self-replication in this manner is the use of alphavirus-based replicons. These replicons are positive-strand (positive-sense) RNAs that, after delivery to a cell, result in the translation of a replicase (or replicase transcriptase). The replicase is translated as a polyprotein that self-cleaves to provide a replication complex that generates genomic copies of the positive-strand delivered RNA. These negative-strand transcripts can themselves be transcribed to provide additional copies of the positive-strand parent RNA and further to provide subgenomic transcripts encoding antigens. Translation of the subgenomic transcripts thus results in in situ expression of the antigen by the infected cell. Suitable alphavirus replicons may use replicases from Sindbis virus, Semliki Forest virus, Eastern equine encephalitis virus, Venezuelan equine encephalitis virus, and the like. Mutant or wild-type viral sequences can be used; for example, the attenuated TC83 mutant of VEEV has been used in the replicon. See the following reference: WO 2005 / 113782, incorporated herein by reference.

[0396] 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) a Chlamydia species antigen. The polymerase may be, for example, an alphavirus replicase comprising one or more of the alphavirus proteins nsP1, nsP2, nsP3, and nsP4. While naturally occurring alphavirus genomes encode structural virion proteins in addition to nonstructural replicase polyproteins, in certain embodiments, the self-replicating RNA molecule does not encode alphavirus structural proteins. Thus, while a self-replicating RNA may result in the production of its own genomic RNA copies in a cell, it does not result in the production of RNA-containing virions. The inability to produce these virions means that, unlike wild-type alphaviruses, the self-replicating RNA molecule cannot persist on its own in an infectious form. The alphavirus structural proteins required for persistence in wild-type viruses are absent from the self-replicating RNA of the present disclosure; their place is taken by a gene encoding the immunogen of interest, such that the subgenomic transcript encodes that immunogen rather than the structural alphavirus virion proteins. Self-replicating RNA is described in further detail in WO2011005799, which is incorporated herein by reference.

[0397] Trans-replicating RNA: Trans-replicating (or trans-amplifying) RNA has elements similar to the self-replicating RNA described above. However, in trans-replicating RNA, two separate RNA molecules are used. One RNA molecule encodes the RNA replicase described above (e.g., an alphavirus replicase), and the second RNA molecule encodes a protein of interest (e.g., a Chlamydia species antigen described herein). The RNA replicase can replicate one or both of the first and second RNA molecules, thereby greatly increasing the copy number of the RNA molecule encoding the protein of interest. Trans-replicating RNA is described in further detail in International Publication No. WO2017162265, which is incorporated herein by reference.

[0398] Non-replicating RNA: Non-replicating (or non-amplifying) RNA is RNA that does not have the ability to replicate itself.

[0399] therapeutic use In another aspect, the present invention provides a polypeptide, nucleic acid, combination, or composition of the present invention for use as a pharmaceutical. The present invention also provides the use of a polypeptide, nucleic acid, combination, or composition of the present invention for manufacturing a medicament. The medicament can be used to treat or prevent a disease described herein. The present invention further provides a method for treating or preventing a disease, comprising administering a polypeptide, nucleic acid, combination, or composition of the present invention to a subject in need thereof. The polypeptide, nucleic acid, combination, or composition of the present invention can be administered, for example, in an amount effective to treat or prevent the disease in the subject. Thus, the polypeptide, nucleic acid, combination, or composition can be administered in an effective amount. Typically, the treatment is prophylactic.

[0400] In another aspect, the present invention provides a polypeptide, nucleic acid, combination, or composition of the present invention for use in treating or preventing a Chlamydia spp. infection in a subject (e.g., a human). The present invention also provides use of a polypeptide, nucleic acid, combination, or composition of the present invention for the manufacture of a medicament for treating or preventing a Chlamydia spp. infection in a subject (e.g., a human). The present invention further provides a method of treating or preventing a Chlamydia spp. infection in a subject (e.g., a human), the method comprising administering a polypeptide, nucleic acid, combination, or composition of the present invention to the subject. The polypeptide, nucleic acid, combination, or composition of the present invention can be administered, for example, in an amount effective to treat or prevent a Chlamydia spp. infection in a subject (i.e., administered in an effective amount). The polypeptide, nucleic acid, combination, or composition can be used to generate an immune response against a Chlamydia spp. infection in a subject (e.g., a human). In a preferred embodiment, the infection is a C. trachomatis infection.

[0401] The invention provides a polypeptide, nucleic acid, combination or composition of the invention for use in the treatment or prevention of trachoma (an eye disease caused by C. trachomatis infection), genital tract infection by Chlamydia trachomatis, lymphogranuloma venereum (a disease caused by C. trachomatis), oropharyngeal infection by C. trachomatis or rectal infection by C. trachomatis in a subject (e.g., a human). The invention also provides use of a polypeptide, nucleic acid, combination or composition of the invention for the manufacture of a medicament for treating or preventing C. trachomatis infection, genital tract infection with Chlamydia trachomatis, lymphogranuloma venereum (a disease caused by C. trachomatis), oropharyngeal infection with C. trachomatis or rectal infection with C. trachomatis in a subject (e.g., a human). The present invention further provides a method for treating or preventing trachoma caused by C. trachomatis infection, genital tract infection by Chlamydia trachomatis, lymphogranuloma venereum caused by C. trachomatis infection, oropharyngeal infection by C. trachomatis, or rectal infection by C. trachomatis in a subject (e.g., a human), the method comprising administering to the subject a polypeptide, nucleic acid, combination, or composition of the present invention.The polypeptides, nucleic acids, combinations, or compositions of the invention can be administered in an amount effective to treat or prevent, for example, trachoma caused by C. trachomatis infection, genital tract infection by Chlamydia trachomatis, venous lymphogranuloma caused by C. trachomatis infection, oropharyngeal infection by C. trachomatis, or rectal infection by C. trachomatis in a subject (i.e., administered in an effective amount). In a preferred embodiment, the infection is a C. trachomatis infection. Trachoma can be caused by infection with C. trachomatis serovars A, B, Ba, or C. trachomatis serovars A, B, Ba, or C. C. lymphogranuloma venereum can be caused by infection with C. trachomatis serovars L1, L2, and L3. Genital tract infection can be caused by serovars D through K. In a preferred embodiment, the Chlamydia species (e.g., C. trachomatis) infection is a genital tract infection.

[0402] The present invention provides a polypeptide, nucleic acid, combination, or composition of the invention for use in a method of providing protective immunity against Chlamydia spp. infection in a subject. The present invention also provides use of a polypeptide, nucleic acid, combination, or composition of the invention for the manufacture of a medicament for use in a method of providing protective immunity against Chlamydia spp. infection in a subject (e.g., a human). The present invention further provides a method of providing protective immunity against Chlamydia spp. infection in a subject (e.g., a human), comprising administering to the subject a polypeptide, nucleic acid, combination, or composition of the invention. The polypeptide, nucleic acid, combination, or composition of the invention can be administered (i.e., administered in an effective amount), for example, in an amount effective to provide protective immunity against Chlamydia spp. infection in the subject. In a preferred embodiment, the infection is a C. trachomatis infection.

[0403] Polypeptides, nucleic acids, combinations, or compositions of the invention may induce a T cell response (e.g., an antigen-specific T cell response) in a subject. Polypeptides, nucleic acids, combinations, or compositions of the invention may induce a CD4+ T cell response and / or a CD8+ T cell response (e.g., a CD4+ T cell response) in a subject. In some embodiments, polypeptides, nucleic acids, combinations, or compositions of the invention may induce IFNγ-producing T cells, such as IFNγ-producing CD4+ T cells and / or IFNγ-producing CD8+ T cells (e.g., IFNγ-producing CD4+ T cells), in a subject. In some embodiments, polypeptides, nucleic acids, combinations, or compositions of the invention may induce CD4+IFNγ+IL2+TNFα+ T cells (i.e., CD4+ T cells that produce IFNγ, IL2, and TNFα). In some embodiments, polypeptides, nucleic acids, combinations, or compositions of the invention may induce CD8+IFNγ+IL2-TNFα+ T cells or CD8+ T cells that produce IFNγ and TNFα. In some embodiments, the polypeptides, nucleic acids, combinations or compositions of the invention may induce a cross-serotype T cell immune response in a subject, i.e., a T cell immune response that is cross-reactive to two or more serotypes of Chlamydia species (e.g., cross-reactive to two or more serotypes of C. trachomatis). Cytokine production by T cells may be measured by intracellular cytokine staining of T cells, e.g., T cells obtained from a subject, or by detecting secreted cytokines (e.g., by ELISA). The polypeptides, nucleic acids, combinations or compositions of the invention may induce a Th1 response (e.g., a Th1 T cell response) in a subject.

[0404] The polypeptides, nucleic acids, combinations, or compositions of the invention can elicit an antibody (e.g., IgG) response in a subject, such as a neutralizing antibody (IgG) response. The antibody can be of any isotype (e.g., IgA, IgG, IgM, i.e., α, γ, or μ heavy chain), but is generally an IgG. Within the IgG isotype, the antibody can be an IgG1, IgG2, IgG3, or IgG4 subclass. The antibody can have a κ or λ light chain. A "neutralizing antibody" is an antibody that neutralizes the biological effect of an antigen of a Chlamydia species in a subject. In some embodiments, the polypeptides, nucleic acids, combinations, or compositions of the invention can elicit a cross-serotype antibody response in a subject, i.e., an antibody response that is cross-reactive against two or more serotypes of Chlamydia species (e.g., cross-reactive against two or more serotypes of C. trachomatis).

[0405] In some embodiments, a polypeptide, nucleic acid, combination or composition of the invention may elicit a T cell response as described herein and / or an antibody response as described herein.

[0406] Administration of a polypeptide, nucleic acid, combination, or composition of the invention to a subject may enable the subject to generate a Chlamydia species antigen-responsive CD4 memory T cell population or a CD8 memory T cell population (e.g., a CD4 T cell memory population) upon exposure to Chlamydia species bacteria or a Chlamydia species antigen. The Chlamydia species-responsive CD4 memory T cell population may confer "sterilizing" immunity against reinfection (i.e., full protective immunity).

[0407] Administration of a polypeptide, nucleic acid, combination, or composition of the invention to a subject may enable the subject to generate a Chlamydia species antigen-responsive memory B cell population upon exposure to Chlamydia species bacteria or Chlamydia species antigens. The Chlamydia species-responsive memory B cell population may confer "sterilizing" immunity against reinfection (i.e., full protective immunity).

[0408] The polypeptides, nucleic acids, combinations or compositions of the invention may be used to induce a primary immune response and / or to enhance an immune response.

[0409] The polypeptides, nucleic acids, combinations or compositions of the invention can be administered to a subject to enable the subject to develop and / or maintain sterilizing immunity against Chlamydia spp. infection. Protective immunity can be provided or enhanced in the immunized subject following Chlamydia spp. infection.

[0410] The polypeptides, nucleic acids, combinations or compositions of the invention may be used in a prime-boost vaccination regimen. Protective immunity against Chlamydia species infections according to the invention may be provided by administering a priming vaccine comprising a polypeptide, nucleic acid, combination or composition of the invention, followed by a booster vaccine. The booster vaccine may be the same as the primer vaccine.

[0411] In certain embodiments, the subject is a vertebrate, e.g., a mammal, e.g., a human or a veterinary mammal (e.g., a cat, dog, horse, cow, sheep, bovine, deer, goat, pig, rodent (e.g., mouse)). In preferred embodiments, the subject is a human. The subject (e.g., a human subject) can be male or female. In some embodiments, the human subject can be between 7 and 50 years of age (e.g., between 9 and 44).

[0412] Mode of administration The polypeptides, nucleic acids, combinations, or compositions of the invention can be administered parenterally (e.g., intramuscularly, intradermally, subcutaneously, intraperitoneally, intravenously, or into the interstitial space of a tissue) or by rectal, oral, vaginal, topical, transdermal, intranasal, sublingual, ocular, otic, pulmonary, or other mucosal administration. In some embodiments, the compositions of the invention are administered intramuscularly. In some embodiments, the compositions of the invention are delivered by mucosal administration.

[0413] In certain embodiments, the polypeptides, nucleic acids, combinations, or compositions (e.g., compositions) of the present invention are provided for use in intramuscular (IM) injection. The polypeptides, nucleic acids, combinations, or compositions (e.g., compositions) can be administered to a subject's thigh or upper arm, for example, in the deltoid muscle of the upper arm. In some embodiments, the polypeptides, nucleic acids, combinations, or compositions (e.g., compositions) are provided in a pre-filled syringe or injector (e.g., single- or multi-chambered). Injection can be via a needle (e.g., a hypodermic needle), although needleless injection can alternatively be used. A typical intramuscular dose is 0.5 ml. In some embodiments, the polypeptides, nucleic acids, combinations, or compositions (e.g., compositions) are provided for use in inhalation and are provided in a pre-filled pump, aerosolizer, or inhaler.

[0414] In certain embodiments, the polypeptides, nucleic acids, combinations, or compositions (e.g., compositions) of the invention are provided for use in cutaneous injection, e.g., in the epidermal, dermal, or subcutaneous tissues of the skin. In some embodiments, the compositions are provided in a device suitable for cutaneous injection, such as a needle (e.g., an epidermal, dermal, or subcutaneous needle), a needle-free device, a microneedle device, or a microprojection array device. Examples of microneedle or microprojection array devices suitable for dermal injection according to the present invention are described in US Patent Application Publication Nos. 20230270842A1, 20220339416A1, 20210085598A1, 20200246450A1, 20220143376A1, 20180264244A1, 20180263641A1, and 20110245776A1.

[0415] The compositions of the present invention can be used to induce systemic and / or mucosal immunity. The dosage regimen may be a single-dose schedule or a multiple-dose schedule. Multiple doses (e.g., two or three) may be used in a primary immunization schedule and / or a booster immunization schedule. The primary dose schedule may be followed by a booster dose schedule. The multiple doses (e.g., two or three) are typically administered at least one week apart (e.g., about two weeks, about three weeks, about four weeks, about six weeks, about eight weeks, about ten weeks, about twelve weeks, about sixteen weeks, etc.) to the subject in need thereof to achieve the desired therapeutic or prophylactic effect. The doses (e.g., the initial dose and the booster dose) may be separated 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.

[0416] The composition of the present invention can be in the form of ready-to-use preparation, for example, the composition of the present invention can be freeze-dried.Such composition can be reconstituted with physiological buffer solution (for example, PBS) immediately before use.The composition of the present invention can be provided in the form of aqueous solution or frozen aqueous solution, and can be directly administered to subjects without reconstitution (after thawing, if previously frozen).

[0417] In some embodiments of compositions comprising an mRNA described herein, a single dose of the composition comprises 1 to 400 μg, e.g., 1 to 50 μg, of an mRNA described herein (e.g., monovalent or multivalent). For example, a single dose may contain, e.g., for intramuscular (IM) injection, about 2.5 μg, about 5 μg, about 7.5 μg, about 10 μg, about 12.5 μg, or about 15 μg of an mRNA described herein.

[0418] In some embodiments, a composition comprises two nucleic acids (e.g., two mRNAs) described herein encoding different polypeptides described herein, wherein the nucleic acids are present in a 1:1 weight ratio. In some embodiments, a composition comprises three nucleic acids (e.g., three mRNAs) described herein encoding different polypeptides described herein, wherein the nucleic acids are present in a 1:1:1 weight ratio. Typically, the LNPs described herein co-encapsulate (a) a nucleic acid described herein comprising a nucleotide sequence encoding a modified MOMP polypeptide described herein, (b) a nucleic acid described herein comprising a nucleotide sequence encoding a chimeric MOMP VD polypeptide described herein, (c) a nucleic acid described herein comprising a nucleotide sequence encoding a Chlamydia spp. CT443 polypeptide described herein, and (d) a nucleic acid described herein comprising a nucleotide sequence encoding a Chlamydia spp. CT584 polypeptide described herein. Typically, a composition comprises four nucleic acids (e.g., four mRNAs) described herein encoding different polypeptides described herein. Typically, the nucleic acids are present in a 1:1:1:1 weight ratio.

[0419] In further embodiments, the compositions of the invention may be provided as multivalent single doses containing multiple (e.g., 2, 3, or 4) types of LNPs, each against a different antigen, with each type of LNP having an mRNA amount of, for example, 2.5 μg, about 5 μg, about 7.5 μg, about 10 μg, about 12.5 μg, or about 15 μg.

[0420] 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 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 nucleic acid combination includes a combination of two or more nucleic acids described herein. The nucleic acids within the combination may be administered simultaneously, separately, or sequentially.

[0421] In some embodiments, a subject is administered one or more polypeptide compositions of the present invention. The polypeptide compositions may include a polypeptide antigen 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 polypeptides described herein. The nucleic acids within the combination may be administered simultaneously, separately, or sequentially.

[0422] In some embodiments, a subject is administered one or more nucleic acid compositions of the invention and one or more polypeptide compositions of the invention. In some embodiments, a subject is administered a nucleic acid composition comprising a nucleotide sequence encoding a modified MOMP polypeptide (and optionally a nucleic acid composition comprising a nucleotide sequence encoding a chimeric MOMP VD polypeptide) and a polypeptide composition comprising one or more polypeptides comprising a polypeptide sequence of a non-MOMP antigen. In some embodiments, a subject is administered two or more polypeptide compositions, each comprising a polypeptide comprising a polypeptide sequence of a non-MOMP antigen. The nucleic acid composition and one or more polypeptide compositions may be administered simultaneously, separately, or sequentially.

[0423] Compositions administered separately or sequentially can be administered within 12 months of each other, within 6 months of each other, or within 1 month (e.g., within 10 days) of each other. Compositions can be administered within 7 days, 3 days, 2 days, or within 24 hours of each other. Concurrent administration can include administering compositions of the invention at the same time. Concurrent administration can include administering compositions of the invention to a patient within 12 hours, 6 hours, 3 hours, 2 hours, or 1 hour of each other, typically within the same visit to a clinical center.

[0424] The present invention also provides kits containing one or more compositions described herein in one or more containers, or one or more compositions 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 kits may include instructions for use.

[0425] Also provided herein are methods for detecting and quantifying antibodies to one or more of the polypeptides described herein in a serum sample. For example, antibodies to MOMP, CT443, and / or CT584 polypeptides can be detected and quantified. Detection and quantification of antibodies to each polypeptide can be performed separately or simultaneously, for example, as a panel in a single multiwell plate. Thus, further provided herein are multiwell plates containing wells coated with each of the recombinant polypeptides MOMP, CT443, and CT584.

[0426] definition The term "comprising" encompasses "including" and "consisting", for example, a composition "comprising" X may consist only of X, or may include something additional, e.g., X+Y.

[0427] The term "about" in reference to a numerical value x is optional and means, for example, x + / - 10%.

[0428] The term "a" or "an" entity refers to one or more of that entity. For example, a "nucleotide sequence" is understood to refer to one or more nucleotide sequences. Thus, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0429] Furthermore, "and / or," when used herein, should be considered a specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or," as used in phrases such as "A and / or B," is intended herein to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or," as used in phrases such as "A, B, and / or C," is intended to include the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B, or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0430] As used herein, the term "Chlamydia species" refers to species of the genus Chlamydia, including the species Chlamydia trachomatis, Chlamydia abortus, Chlamydia pneumoniae, Chlamydia muridarum, Chlamydia psittaci, Chlamydia pecorum, Chlamydia felis, and Chlamydia caviae. In a preferred embodiment of the present invention, the Chlamydia species is C. trachomatis.

[0431] As used herein, the term "effective amount" refers to an amount (e.g., of a nucleic acid, polypeptide, combination, or composition described herein) sufficient to produce beneficial or desired results. An effective amount can be administered in one or more administrations, applications, or dosages, and is not intended to be limited to a particular formulation or route of administration. The term "effective amount" includes, for example, a therapeutically effective amount and / or a prophylactically effective amount. As used herein, the term "effective amount" refers to an amount (e.g., of a nucleic acid, polypeptide, combination, or composition described herein) effective to produce some desired therapeutic or prophylactic effect in the treatment or prevention of an infection, disease, disorder, and / or condition, at a reasonable benefit / risk ratio applicable to any medical treatment.

[0432] The term "element" refers to one of the forms of Chlamydia bacteria. An element can be released from infected cells and transmitted from one subject to another.

[0433] The terms "fragment" or "variant" when referring to a polypeptide of the present disclosure include any polypeptide that retains at least some of the properties of the reference polypeptide (e.g., the polypeptide's specific antigenic properties or the polypeptide's ability to contribute to inducing antibody binding). Polypeptide fragments include N- and / or C-terminal truncated fragments, e.g., C- and N-terminal fragments, and deletion fragments, but do not include naturally occurring full-length polypeptides (or mature polypeptides). Deletion fragments refer to polypeptides in which one or more internal amino acids are deleted from the full-length polypeptide. Polypeptide variants include the above fragments and also polypeptides with altered amino acid sequences resulting from amino acid substitutions, deletions, or insertions. Variants can be natural or non-natural. Non-natural variants can be generated using mutagenesis techniques known in the art. Variant polypeptides can include conservative or non-conservative amino acid substitutions, deletions, or additions. Such mutations (i.e., truncations and / or amino acid substitutions, deletions, or insertions) can occur either at the amino acid level or, accordingly, at the nucleic acid level.

[0434] Identity for a sequence is defined herein as the percentage of nucleic acid or amino acid residues in a candidate sequence that are identical to the reference amino acid sequence, after aligning the sequences and optionally introducing gaps to achieve the maximum percent sequence identity, and not considering conservative substitutions as part of the sequence identity.

[0435] Sequence identity can be determined by standard methods commonly used to compare the similarity of the amino acids of two polypeptides or the nucleic acid positions of two polynucleotides. For example, using a computer program such as BLAST or FASTA, two polypeptides are aligned (along the entire length of one or both sequences, or along a predetermined portion of one or both sequences) for optimal amino acid matching. The program can use a default opening penalty and a default gap penalty, as well as a scoring matrix such as PAM250 [a standard scoring matrix; see Dayhoff et al., Atlas of Protein Sequence and Structure, vol. 5, sup.3 (1978)] in combination with the computer program. The percent identity can be calculated as follows: The total number of identical matches is multiplied by 100, and then divided by the length of the longer sequence in the matched span plus the number of gaps introduced into the shorter sequence to align the two sequences.

[0436] As used herein, the term "kit" refers to a packaged set of related components, e.g., one or more compounds or compositions and one or more associated materials, e.g., solvents, solutions, buffers, instructions, or desiccants.

[0437] As used herein, the terms "linked" or "attached" refer to a first amino acid sequence or nucleotide sequence covalently linked to a second amino acid sequence or nucleotide sequence, respectively (e.g., a secretory signal peptide amino acid sequence and / or a heterologous transmembrane domain amino acid sequence linked to the amino acid sequence of a Chlamydia species polypeptide). The first amino acid or nucleotide sequence can be directly linked to the second amino acid or nucleotide sequence, or an intervening sequence can covalently link the first sequence to the second sequence. The term "linked" not only refers to a first amino acid sequence being fused to the second amino acid sequence at the C-terminus or N-terminus, but also includes the entire first amino acid sequence (or second amino acid sequence) being inserted within any two amino acids of the second amino acid sequence (or first amino acid sequence, respectively). In one embodiment, the first amino acid sequence can be linked to the second amino acid sequence by a peptide bond or a linker. The first nucleotide sequence can be linked to the second nucleotide sequence by a phosphodiester bond or a linker. A linker can be a peptide or polypeptide (in the case of a polypeptide chain), or a nucleotide or nucleotide chain (in the case of a nucleotide chain), or any chemical moiety (in the case of both polypeptide chains and polynucleotide chains). The term "linked" can also be indicated by a hyphen (-).

[0438] As used herein, the term "native" refers to a sequence that occurs in nature. For example, a native Chlamydia species MOMP polypeptide is a naturally occurring Chlamydia species MOMP polypeptide.

[0439] Embodiment The present invention includes at least the following numbered embodiments:

[0440] 1. A nucleic acid comprising a nucleotide sequence encoding a modified major outer membrane protein (MOMP) polypeptide, wherein the modified MOMP polypeptide has an amino acid sequence comprising two or more conserved domain sequences of a native Chlamydia species MOMP polypeptide and a non-native loop sequence between the conserved domain sequences.

[0441] 2. The nucleic acid of embodiment 1, wherein the Chlamydia species is Chlamydia trachomatis.

[0442] 3. The nucleic acid of embodiment 1 or 2, wherein the modified MOMP polypeptide does not contain a naturally occurring Chlamydia species MOMP variable domain between two or more conserved domain sequences.

[0443] 4. The modified MOMP polypeptide comprises the five conserved domain sequences of a native Chlamydia species MOMP polypeptide, and optionally: (i) the modified MOMP polypeptide contains all five full-length conserved domains of the native Chlamydia species MOMP polypeptide; (ii) the conserved domain sequence of the modified MOMP polypeptide is at least 95% identical to a conserved domain of a native MOMP polypeptide of any serovar Chlamydia species (e.g., a conserved domain defined in Table 1); (iii) the conserved domains of the modified MOMP polypeptide collectively have at least 95% sequence identity to the conserved domains of a native MOMP polypeptide (e.g., serotype E MOMP), and / or (iv) the conserved domain sequence of the modified MOMP polypeptide lacks up to 3 or 5 amino acids of the naturally occurring Chlamydia species MOMP conserved domain sequence; The nucleic acid according to any one of embodiments 1 to 3.

[0444] 5. (i) the modified MOMP polypeptide contains a non-native loop sequence between each of the conserved domain sequences; (ii) the non-native loop sequence is 40% or less identical to any native Chlamydia species MOMP VD (VD1, VD2, VD3, or VD4) sequence of any serovar (e.g., C. trachomatis serovar D, E, F, or G); The nucleic acid according to any one of embodiments 1 to 4.

[0445] 6. The nucleic acid of embodiment 4, wherein the modified MOMP polypeptide comprises four non-native loop sequences and does not contain a native Chlamydia species MOMP variable domain between the conserved domain sequences.

[0446] 7. The nucleic acid of any one of embodiments 1-6, wherein the modified MOMP polypeptide does not contain any naturally occurring Chlamydia species MOMP variable domains between any of the conserved domain sequences.

[0447] 8. (i) the non-native loop sequence is 3 to 30 amino acids in length, e.g., 4 to 20 amino acids in length; and / or (ii) two or more conserved domain sequences are connected by a non-native loop sequence such that the conserved domain sequences form a beta-barrel structure (e.g., composed of antiparallel beta strands), optionally as predicted in silico (e.g., using Alphafold2 (Deepmind) software); The nucleic acid according to any one of embodiments 1 to 7.

[0448] 9. (i) the non-native loop sequence replacing VD1 comprises a sequence according to SEQ ID NO: 462 or 466 (e.g., SEQ ID NO: 462); (ii) the non-native loop sequence replacing VD2 comprises a sequence according to SEQ ID NO: 463 or 467 (e.g., SEQ ID NO: 463); (iii) the non-native loop sequence replacing VD3 comprises a sequence according to SEQ ID NO: 464 or 468 (e.g., SEQ ID NO: 464); and / or (iv) the non-native loop sequence replacing VD4 comprises a sequence according to SEQ ID NO: 465 or 469 (e.g., SEQ ID NO: 465); For example, the nucleic acid of any one of embodiments 1 to 8, wherein the modified MOMP polypeptide comprises four non-native loop sequences according to SEQ ID NOs: 462, 463, 464 and 465 in place of VD1, VD2, VD3 and VD4, respectively.

[0449] 10. The nucleic acid of any one of embodiments 1-9, wherein the modified MOMP polypeptide comprises a single amino acid substitution at one or more (e.g., all) positions corresponding to cysteine ​​residues in a conserved domain sequence of a native Chlamydia species MOMP polypeptide, and optionally, the single amino acid substitution is a substitution of a cysteine ​​with a serine.

[0450] 11. The nucleic acid of any one of embodiments 1 to 10, wherein the modified MOMP polypeptide comprises mutations at one or more (e.g., all) positions corresponding to glycosylation sites, optionally N-glycosylation sites, of a native Chlamydia species MOMP polypeptide, and optionally the mutations are single amino acid substitutions.

[0451] 12. The nucleic acid of any one of embodiments 1 to 11, wherein the modified MOMP polypeptide further comprises a secretory signal peptide sequence.

[0452] 13. The nucleic acid of embodiment 12, wherein the secretory signal peptide sequence is a viral secretory signal peptide sequence, optionally selected from the group consisting of influenza hemagglutinin (HA) secretory signal peptide sequence, SARS CoV-2 spike secretory signal peptide sequence, VZV gB secretory signal peptide sequence, VZV gE secretory signal peptide sequence, VZV gI secretory signal peptide sequence, VZV gK secretory signal peptide sequence, measles F-protein secretory signal peptide sequence, rubella E1 protein secretory signal peptide sequence, rubella E2 protein secretory signal peptide sequence, mumps F-protein secretory signal peptide sequence, Ebola GP protein secretory signal peptide sequence, and smallpox 6 kDa IC protein secretory signal peptide sequence, and optionally wherein the secretory signal peptide sequence comprises an amino acid sequence according to one of the SEQ ID NOs in Table 2 or Table 2.1.

[0453] 14. The nucleic acid of embodiment 13, wherein, for example, the secretory signal peptide sequence comprises the secretory signal peptide sequence of the HA protein of an influenza A virus, for example, the secretory signal peptide sequence comprises a sequence according to SEQ ID NO: 187 or SEQ ID NO: 188.

[0454] 15. The nucleic acid of any one of embodiments 1 to 14, wherein the modified MOMP polypeptide comprises a sequence according to any one of SEQ ID NOs: 486 to 489 or a sequence having at least 70% (e.g., at least 90 or 95%) identity thereto, for example, the modified MOMP polypeptide comprises a sequence according to SEQ ID NO: 486 or a sequence having at least 70% (e.g., at least 90 or 95%) identity thereto.

[0455] 16. The nucleic acid according to any one of embodiments 1 to 15, comprising a nucleotide sequence according to any one of SEQ ID NOs: 551 to 566 or a sequence having at least 50% identity thereto, for example, a nucleotide sequence according to SEQ ID NO: 551 or a sequence having at least 50% (e.g., at least 75%) identity thereto.

[0456] 17. The nucleic acid is messenger RNA (mRNA), optionally (i) the mRNA comprises at least one 5' untranslated region (5' UTR), at least one 3' untranslated region (3' UTR), and / or at least one polyadenylation (poly(A)) sequence; (ii) the mRNA is unmodified or comprises at least one chemical modification, optionally the mRNA comprises at least one chemical modification, e.g., the chemical modification comprises N1-methylpseudouridine, and / or (iii) the mRNA is a self-replicating mRNA or a non-replicating mRNA, e.g., a non-replicating mRNA; 17. The nucleic acid according to any one of embodiments 1 to 16.

[0457] 18. mRNA contains the following structural elements: - a 5' cap, for example, the following structure: [ka] Cap with; - a 5' untranslated region (5'UTR) having a nucleic acid sequence according to SEQ ID NO: 838; - a protein coding region having a nucleic acid sequence according to SEQ ID NO: 213; - a 3' untranslated region (3'UTR) having a nucleic acid sequence according to SEQ ID NO: 839; and -Poly A tail 18. The nucleic acid of embodiment 17, comprising or consisting of (e.g. consisting of):

[0458] 19. The mRNA is chemically modified, and the chemical modification comprises or consists of (e.g., consists of) N1-methylpseudouridine in place of any uridine; 19. The nucleic acid of embodiment 18.

[0459] 20. A modified major outer membrane protein (MOMP) polypeptide having an amino acid sequence comprising two or more conserved domain sequences of a native Chlamydia species MOMP polypeptide and a non-native loop sequence between the conserved domain sequences.

[0460] 21. The modified MOMP polypeptide of embodiment 20, wherein the modified MOMP polypeptide does not contain a naturally occurring Chlamydia species MOMP variable domain between two or more conserved domain sequences.

[0461] 22. The modified MOMP polypeptide comprises the five conserved domain sequences of a native Chlamydia species MOMP polypeptide, and optionally (i) the modified MOMP polypeptide comprises all five full-length conserved domains of a native Chlamydia species MOMP polypeptide, and / or (ii) the conserved domains of the modified MOMP polypeptide collectively have at least 95% sequence identity with the conserved domains of a native MOMP polypeptide (e.g., serotype E MOMP). 22. The modified MOMP polypeptide of embodiment 20 or 21.

[0462] 23. The modified MOMP polypeptide of any one of embodiments 20 to 22, wherein the modified MOMP polypeptide comprises a non-native loop sequence between each of the conserved domain sequences.

[0463] 24. The modified MOMP polypeptide of embodiment 22, wherein the modified MOMP polypeptide comprises four non-native loop sequences and does not contain a native Chlamydia species MOMP variable domain between the conserved domain sequences.

[0464] 25. The modified MOMP polypeptide of any one of embodiments 20 to 24, wherein the modified MOMP polypeptide does not contain any naturally occurring Chlamydia species MOMP variable domains between any of the conserved domain sequences.

[0465] 26. The modified MOMP polypeptide according to any one of embodiments 20 to 25, wherein the non-native loop sequence is 3 to 30 amino acids in length, such as 4 to 20 amino acids in length.

[0466] 27. (i) the non-native loop sequence replacing VD1 comprises a sequence according to SEQ ID NO: 462 or 466 (e.g., SEQ ID NO: 462); (ii) the non-native loop sequence replacing VD2 comprises a sequence according to SEQ ID NO: 463 or 467 (e.g., SEQ ID NO: 463); (iii) the non-native loop sequence replacing VD3 comprises a sequence according to SEQ ID NO: 464 or 468 (e.g., SEQ ID NO: 464); and / or (iv) the non-native loop sequence replacing VD4 comprises a sequence according to SEQ ID NO: 465 or 469 (e.g., SEQ ID NO: 465); For example, a modified MOMP polypeptide can include four non-native loop sequences according to SEQ ID NOs: 462-465 in place of VD1, VD2, VD3, and VD4, respectively. 27. A modified MOMP polypeptide according to any one of embodiments 20 to 26.

[0467] 28. The modified MOMP polypeptide of any one of embodiments 20 to 27, wherein the modified MOMP polypeptide comprises a single amino acid substitution at one or more (e.g., all) positions corresponding to cysteine ​​residues in a conserved domain sequence of a native Chlamydia species MOMP polypeptide, and optionally the single amino acid substitution is a substitution of a cysteine ​​with a serine.

[0468] 29. The modified MOMP polypeptide of any one of embodiments 20 to 28, wherein the modified MOMP polypeptide comprises mutations at one or more (e.g., all) positions corresponding to N-glycosylation sites in a native Chlamydia species MOMP polypeptide, and optionally the mutations are single amino acid substitutions.

[0469] 30. The modified MOMP polypeptide of any one of embodiments 20 to 29, wherein the modified MOMP polypeptide comprises a sequence according to any one of SEQ ID NOs: 486 to 489 or a sequence having at least 70% (e.g., at least 90 or 95%) identity thereto, for example, the modified MOMP polypeptide comprises a sequence according to SEQ ID NO: 486 or a sequence having at least 70% (e.g., at least 90 or 95%) identity thereto.

[0470] 31. A composition comprising a nucleic acid according to any one of embodiments 1 to 19, preferably wherein the composition is an immunogenic composition.

[0471] 32. A composition comprising a polypeptide according to any one of embodiments 20 to 30, preferably wherein the composition is an immunogenic composition.

[0472] 33. The composition comprises: (i) a nucleic acid comprising a nucleotide sequence encoding a chimeric Chlamydia spp. MOMP variable domain (VD) polypeptide, wherein the chimeric MOMP VD polypeptide comprises an amino acid sequence comprising two or more Chlamydia spp. MOMP VD sequences of different serotypes of Chlamydia spp.; and / or (ii) One or more of the following (e.g., 1, 2, 3, or 4): (a) a nucleic acid comprising a nucleotide sequence encoding a Chlamydia species CT443 polypeptide; (b) a nucleic acid comprising a nucleotide sequence encoding a Chlamydia sp. CT584 polypeptide; (c) a nucleic acid comprising a nucleotide sequence encoding a Chlamydia species CT600 polypeptide; and (d) a nucleic acid comprising a nucleotide sequence encoding a Chlamydia sp. CT812 polypeptide; 32. The composition of embodiment 31, further comprising:

[0473] 34. The composition comprises: (i) the nucleic acid and (ii) the nucleic acid of (a); (i) a nucleic acid and (ii) a nucleic acid of (b); or (i) nucleic acid, (ii) nucleic acid(a) and (ii) nucleic acid(b); Including, For example, the composition comprises (i) a nucleic acid, (ii) a nucleic acid, and (ii) a nucleic acid. 34. The composition of embodiment 33.

[0474] 35. One or more nucleic acids is mRNA, optionally (i) the mRNA comprises at least one 5' untranslated region (5' UTR), at least one 3' untranslated region (3' UTR), and / or at least one polyadenylation (poly(A)) sequence; (ii) the mRNA is unmodified or comprises at least one chemical modification, optionally the mRNA comprises at least one chemical modification, e.g., the chemical modification comprises N1-methylpseudouridine, and / or (iii) the mRNA is a self-replicating mRNA or a non-replicating mRNA, e.g., a non-replicating mRNA; 35. The composition of embodiment 33 or 34.

[0475] 36. The composition of any one of embodiments 31 or 33-35, further comprising lipid nanoparticles (LNPs), optionally wherein the nucleic acid is encapsulated in the LNPs.

[0476] 37. The composition comprises: (i) a chimeric Chlamydia species MOMP variable domain (VD) polypeptide, wherein the chimeric MOMP VD polypeptide comprises an amino acid sequence that includes two or more Chlamydia species MOMP VD sequences of different serotypes of Chlamydia species; and / or (ii) One or more of the following (e.g., 1, 2, 3, or 4): (a) a polypeptide comprising the amino acid sequence of a Chlamydia species CT443 polypeptide; (b) a polypeptide comprising the amino acid sequence of a Chlamydia species CT584 polypeptide; (c) a polypeptide comprising the amino acid sequence of a Chlamydia species CT600 polypeptide; or (d) a polypeptide comprising the amino acid sequence of the Chlamydia sp. CT812 polypeptide Further comprising: For example, the composition may comprise: (i) the polypeptide and (ii) the polypeptide of (a); or (i) the polypeptide and (ii)(b) the polypeptide; or The polypeptide of (i), the polypeptide of (ii)(a), and the polypeptide of (ii)(b) Including, For example, the composition of embodiment 32, wherein the composition comprises a polypeptide of (i), a polypeptide of (ii)(a), and a polypeptide of (ii)(b).

[0477] 38. The composition of any one of embodiments 33-37, wherein the Chlamydia species is Chlamydia trachomatis.

[0478] 39. The composition of any one of embodiments 33-38, wherein the chimeric MOMP VD polypeptide comprises conserved domain sequence portions of a native Chlamydia species MOMP polypeptide flanking each of two or more MOMP VD sequences.

[0479] 40. The composition of any one of embodiments 33-39, wherein the chimeric MOMP VD polypeptide comprises MOMP VD sequences of four different serotypes, optionally wherein the different serotypes are selected from serotypes D, E, F, or G of C. trachomatis.

[0480] 41. A chimeric MOMP VD polypeptide comprising: (i) two MOMP VD1 sequences of different serotypes of Chlamydia species; and / or (ii) two MOMP VD2 sequences of different serotypes of Chlamydia species; and / or (iii) one MOMP VD3 sequence; and / or (iv) two MOMP VD4 sequences of different serotypes of Chlamydia species; Optionally, for example, the different serotypes are selected from C. trachomatis serotypes D, E, F, or G. 41. The composition of any one of embodiments 33 to 40.

[0481] 42. The composition of any one of embodiments 33 to 40, wherein the chimeric MOMP VD polypeptide comprises one MOMP VD sequence of C. trachomatis serovar D or E and one MOMP VD sequence of C. trachomatis serovar F or G.

[0482] 43. The chimeric MOMP VD polypeptide is at least one of (i) to (iv) (e.g., 4): (i) a MOMP VD1 sequence from serotype D and a MOMP VD1 sequence from serotype F, or a MOMP VD1 sequence from serotype E and a MOMP VD1 sequence from serotype F or G (e.g., a MOMP VD1 sequence of serotype E and a MOMP VD1 sequence of serotype G); and / or (ii) a MOMP VD2 sequence from serotype E and a MOMP VD2 sequence from serotype F, or a MOMP VD2 sequence from serotype D and a MOMP VD2 sequence from serotype G (e.g., a MOMP VD2 sequence of serotype D and a MOMP VD2 sequence of serotype G); and / or (iii) a MOMP VD3 sequence from serotype G or a VD3 sequence from serotype F (e.g., serotype F); and / or (iv) a MOMP VD4 sequence from serotype E and a MOMP VD4 sequence from serotype G, or a MOMP VD4 sequence from serotype D and a MOMP VD4 sequence from serotype F (e.g., a MOMP VD4 sequence from serotype D and a MOMP VD4 sequence from serotype F) Including, The composition of embodiment 41 or 42, wherein the serotype D, E, F or G is of C. trachomatis.

[0483] 44. A chimeric MOMP VD polypeptide comprising: (i) a MOMP VD1 sequence from serotype D and a MOMP VD1 sequence from serotype F, a MOMP VD2 sequence from serotype E and a MOMP VD2 sequence from serotype F, a MOMP VD3 sequence from serotype G, a MOMP VD4 sequence from serotype E and a MOMP VD4 sequence from serotype G; or (ii) the MOMP VD1 sequence from serotype E and the MOMP VD1 sequence from serotype F, the MOMP VD2 sequence from serotype D and the MOMP VD2 sequence from serotype G, the MOMP VD3 sequence from serotype F, the MOMP VD4 sequence from serotype D and the MOMP VD4 sequence from serotype F Including, serotype D, E, F, or G is C. trachomatis; For example, the composition of embodiment 43, wherein the chimeric MOMP VD polypeptide comprises a MOMP VD sequence as set forth in (ii).

[0484] 45. The composition of any one of embodiments 39 to 44, wherein the conserved domain sequence portion is a portion of a conserved domain sequence of a native Chlamydia species MOMP polypeptide that is adjacent to the VD in that native Chlamydia species MOMP polypeptide.

[0485] 46. ​​(b) A composition described in any one of embodiments 39 to 45, wherein each conserved domain sequence portion comprises 3 to 30 amino acid residues of a conserved domain sequence of a native Chlamydia species MOMP polypeptide, and the 3 to 30 amino acid residues are immediately adjacent to the VD sequence of that native Chlamydia species MOMP polypeptide.

[0486] 47. The composition of any one of embodiments 33-36 or 38-46, wherein the composition comprises a nucleic acid comprising a nucleotide sequence encoding a chimeric Chlamydia species MOMP VD polypeptide, wherein the chimeric MOMP VD polypeptide comprises a secretory signal peptide sequence.

[0487] 48. The composition of embodiment 47, wherein the secretory signal peptide sequence is a viral secretory signal peptide sequence, optionally selected from the group consisting of an influenza hemagglutinin (HA) secretory signal peptide sequence, a SARS CoV-2 spike secretory signal peptide sequence, a VZV gB secretory signal peptide sequence, a VZV gE secretory signal peptide sequence, a VZV gI secretory signal peptide sequence, a VZV gK secretory signal peptide sequence, a measles F-protein secretory signal peptide sequence, a rubella E1 protein secretory signal peptide sequence, a rubella E2 protein secretory signal peptide sequence, a mumps F-protein secretory signal peptide sequence, an Ebola GP protein secretory signal peptide sequence, and a smallpox 6 kDa IC protein secretory signal peptide sequence, and optionally wherein the secretory signal peptide sequence comprises an amino acid sequence according to any one of the SEQ ID NOs in Table 2 or Table 2.1.

[0488] 49. The composition of embodiment 48, wherein, for example, the secretory signal peptide sequence comprises the secretory signal peptide sequence of the HA protein of an influenza A virus, for example, the secretory signal peptide sequence comprises a sequence according to SEQ ID NO: 187 or SEQ ID NO: 188.

[0489] 50. The composition of any one of embodiments 33-49, wherein the chimeric MOMP VD polypeptide comprises a single amino acid substitution at one or more (e.g., all) positions corresponding to cysteine ​​residues in the conserved domain sequence of a native Chlamydia species MOMP polypeptide, and optionally the single amino acid substitution is a substitution of a cysteine ​​with a serine.

[0490] 51. The composition of any one of embodiments 33 to 50, wherein the chimeric MOMP VD polypeptide comprises mutations at one or more (e.g., all) positions corresponding to glycosylation sites, optionally N-glycosylation sites, of a native Chlamydia species MOMP polypeptide, and optionally the mutations are single amino acid substitutions.

[0491] 52. The composition of embodiment 51, wherein the chimeric MOMP VD polypeptide comprises a single amino acid substitution at each of the amino acid residues corresponding to position 9 of SEQ ID NO:9 (e.g., an N to A substitution), position 11 of SEQ ID NO:17 (e.g., a T to A substitution), position 17 of SEQ ID NO:6 (e.g., an S to A substitution), positions 4 and 21 of SEQ ID NO:18 (e.g., an N to A and an S to A substitution, respectively), position 14 of SEQ ID NO:8 (e.g., a T to A substitution), and position 14 of SEQ ID NO:16 (e.g., a T to A substitution).

[0492] 53. The composition of any one of embodiments 33-52, wherein the chimeric MOMP VD polypeptide comprises a sequence according to any one of SEQ ID NOs: 490-505, or a sequence having at least 70% (e.g., at least 90 or 95%) identity thereto, e.g., the chimeric MOMP VD polypeptide comprises a sequence according to SEQ ID NO: 503, or a sequence having at least 70% (e.g., at least 90 or 95%) identity thereto.

[0493] 54. A composition described in any one of embodiments 33 to 36 or 38 to 53, wherein the composition comprises a nucleic acid comprising a nucleotide sequence encoding a chimeric Chlamydia species MOMP VD polypeptide, wherein the nucleic acid comprises a nucleotide sequence according to SEQ ID NO: 567 to 630 or a sequence having at least 50% (e.g., at least 75%) identity thereto, for example, the nucleic acid comprises a nucleotide sequence according to SEQ ID NO: 617 or a sequence having at least 50% (e.g., at least 75%) identity thereto.

[0494] 55. The composition of any one of embodiments 33-36 or 38-54, wherein the composition comprises one or more nucleic acids of (ii)(a)-(d), and wherein the Chlamydia spp. CT443 polypeptide, the Chlamydia spp. CT584 polypeptide, the Chlamydia spp. CT600 polypeptide, and the Chlamydia spp. CT812 polypeptide comprise a secretory signal peptide sequence.

[0495] 56. The composition of embodiment 55, wherein the secretory signal peptide sequence is a viral secretory signal peptide sequence, optionally selected from the group consisting of: influenza hemagglutinin (HA) secretory signal peptide sequence, SARS CoV-2 spike secretory signal peptide sequence, VZV gB secretory signal peptide sequence, VZV gE secretory signal peptide sequence, VZV gI secretory signal peptide sequence, VZV gK secretory signal peptide sequence, measles F-protein secretory signal peptide sequence, rubella E1 protein secretory signal peptide sequence, rubella E2 protein secretory signal peptide sequence, mumps F-protein secretory signal peptide sequence, Ebola GP protein secretory signal peptide sequence, and smallpox 6 kDa IC protein secretory signal peptide sequence, and optionally wherein the secretory signal peptide sequence comprises an amino acid sequence according to one of the SEQ ID NOs in Table 2 or Table 2.1.

[0496] 57. The composition of embodiment 56, for example, wherein the secretory signal peptide sequence comprises the secretory signal peptide sequence of the HA protein of an influenza A virus, for example, the secretory signal peptide sequence comprises a sequence according to SEQ ID NO: 187 or SEQ ID NO: 188.

[0497] 58. The composition of any one of embodiments 55 to 57, wherein one or more of the Chlamydia spp. CT443 polypeptide, Chlamydia spp. CT584 polypeptide, Chlamydia spp. CT600 polypeptide and / or Chlamydia spp. CT812 polypeptide comprises a heterologous transmembrane domain.

[0498] 59. The composition of embodiment 58, wherein the transmembrane domain sequence is selected from the group consisting of an influenza hemagglutinin (HA) transmembrane domain sequence, a SARS COV-2 spike transmembrane domain sequence, a VZV gB transmembrane domain sequence, a VZV gE transmembrane domain sequence, a VZV gI transmembrane domain sequence, a VZV gK transmembrane domain sequence, a measles F protein transmembrane domain sequence, a rubella E1 protein transmembrane domain sequence, a rubella E2 protein domain sequence, a mumps F protein transmembrane domain sequence, and an Ebola GP protein transmembrane domain sequence, and optionally wherein the transmembrane domain comprises an amino acid sequence according to one of the SEQ ID NOs in Table 3.

[0499] 60. The composition of embodiment 58 or 59, wherein the transmembrane domain comprises the sequence of the transmembrane domain of the HA protein of an influenza A virus, for example, the transmembrane domain comprises the sequence according to SEQ ID NO: 813 or SEQ ID NO: 814.

[0500] 61. The composition of any one of embodiments 33-60, wherein one or more of the Chlamydia spp. CT443 polypeptide, the Chlamydia spp. CT584 polypeptide, the Chlamydia spp. CT600 polypeptide, or the Chlamydia spp. CT812 polypeptide comprises a single amino acid substitution at one or more (e.g., all) positions corresponding to a cysteine ​​residue in the respective native Chlamydia spp. polypeptide, and optionally the single amino acid substitution is a substitution of a cysteine ​​with a serine.

[0501] 62. The composition of any one of embodiments 33-61, wherein one or more of the Chlamydia spp. CT443 polypeptide, the Chlamydia spp. CT584 polypeptide, the Chlamydia spp. CT600 polypeptide, or the Chlamydia spp. CT812 polypeptide comprises a mutation at one or more (e.g., all) positions corresponding to N-glycosylation sites in the respective native Chlamydia spp. polypeptide, and optionally the mutation is a single amino acid substitution.

[0502] 63. The composition of embodiment 62, wherein the Chlamydia sp. CT584 polypeptide comprises a single amino acid substitution at a position corresponding to residue 11 of SEQ ID NO: 509 (e.g., an N to Q substitution).

[0503] 64. (a) the Chlamydia sp. CT443 polypeptide comprises a sequence according to SEQ ID NOs: 507-508, or a sequence having at least 70% (e.g., at least 90 or 95%) identity thereto, e.g., the Chlamydia sp. CT443 polypeptide comprises a sequence according to SEQ ID NO: 507, or a sequence having at least 70% (e.g., at least 90 or 95%) identity thereto; (b) the Chlamydia sp. CT584 polypeptide comprises a sequence according to any one of SEQ ID NOs: 509-512, or a sequence having at least 70% (e.g., at least 90 or 95%) identity thereto, e.g., the Chlamydia sp. CT584 polypeptide comprises a sequence according to SEQ ID NO: 510, or any one of a sequence having at least 70% (e.g., at least 90 or 95%) identity thereto; (c) the Chlamydia sp. CT600 polypeptide comprises a sequence according to any one of SEQ ID NOs: 513-514 or a sequence having at least 70% (e.g., at least 90 or 95%) identity thereto; and / or (d) the composition of any one of embodiments 33-63, wherein the Chlamydia sp. CT812 polypeptide comprises a sequence according to any one of SEQ ID NOs: 515-535 or a sequence having at least 70% (e.g., at least 90 or 95%) identity thereto.

[0504] 65. The composition comprises: (1) The chimeric MOMP VD polypeptide comprises a sequence according to any one of SEQ ID NOs: 490 to 505 (e.g., SEQ ID NO: 503) or a sequence having at least 70% (e.g., at least 90 or 95%) identity thereto; (2) The nucleic acid comprises a nucleotide sequence encoding a chimeric Chlamydia sp. MOMP VD polypeptide comprising a nucleotide sequence according to SEQ ID NOs: 567-630 (e.g., SEQ ID NO: 617), or a sequence having at least 50% identity thereto; (3) the Chlamydia sp. CT443 polypeptide comprises SEQ ID NO: 507 or a sequence having at least 70% (e.g., at least 90 or 95%) identity thereto; (4) A nucleic acid comprising a nucleotide sequence encoding a Chlamydia sp. CT443 polypeptide comprises a nucleotide sequence according to any one of SEQ ID NOs: 707 to 710 (e.g., SEQ ID NO: 707) or a sequence having at least 50% (e.g., at least 75%) identity thereto; (5) The Chlamydia sp. CT584 polypeptide comprises a sequence according to any one of SEQ ID NOs: 509-512 (e.g., SEQ ID NO: 510), or a sequence having at least 70% (e.g., at least 90 or 95%) identity thereto; (6) A nucleic acid comprising a nucleotide sequence encoding a Chlamydia sp. CT584 polypeptide comprises a nucleotide sequence according to any one of SEQ ID NOs: 711 to 718 (e.g., SEQ ID NO: 715) or a sequence having at least 50% (e.g., at least 75%) identity thereto; (7) the Chlamydia sp. CT600 polypeptide comprises a sequence according to any one of SEQ ID NOs: 513-514 or a sequence having at least 70% (e.g., at least 90 or 95%) identity thereto; and / or (8) A nucleic acid comprising a nucleotide sequence encoding a Chlamydia sp. CT600 polypeptide comprises a nucleotide sequence according to any one of SEQ ID NOs: 719 to 722 or a sequence having at least 50% (e.g., at least 75%) identity thereto; (9) the Chlamydia sp. CT812 polypeptide comprises a sequence according to any one of SEQ ID NOs: 515-535 or a sequence having at least 70% (e.g., at least 90 or 95%) identity thereto; and / or (10) A nucleic acid comprising a nucleotide sequence encoding a Chlamydia sp. CT812 polypeptide comprises a nucleotide sequence according to any one of SEQ ID NOs: 723 to 762 or a sequence having at least 50% (e.g., at least 75%) identity thereto; Depending on the situation, (ii) the nucleic acid of (a) comprises a nucleotide sequence according to SEQ ID NO: 707 or a sequence having at least 50% (at least 75%) identity thereto; and / or (ii) The composition of any one of embodiments 33 to 36 or 38 to 64, wherein the nucleic acid of (b) comprises the nucleotide sequence set forth in SEQ ID NO: 715 or a sequence having at least 50% (at least 75%) identity thereto.

[0505] 66.(ii) The nucleic acid in (a) is mRNA, and the mRNA contains the following structural elements: - a 5' cap, for example, the following structure: [ka] having a 5' cap; - a 5' untranslated region (5'UTR) having a nucleic acid sequence according to SEQ ID NO: 838; - a protein coding region having a nucleic acid sequence according to SEQ ID NO: 369; - a 3' untranslated region (3'UTR) having a nucleic acid sequence according to SEQ ID NO: 839; and -Poly A tail 66. The composition of any one of embodiments 33-36 or 38-65, comprising or consisting of (e.g., consisting of):

[0506] 67.(ii) The nucleic acid in (b) is mRNA, and the mRNA contains the following structural elements: - a 5' cap, for example, the following structure: [ka] having a 5' cap; - a 5' untranslated region (5'UTR) having a nucleic acid sequence according to SEQ ID NO: 838; - a protein coding region having a nucleic acid sequence according to SEQ ID NO: 377; - a 3' untranslated region (3'UTR) having a nucleic acid sequence according to SEQ ID NO: 839; and -Poly A tail 67. The composition of any one of embodiments 33-36 or 38-66, comprising or consisting of (e.g., consisting of):

[0507] 68. The nucleic acid comprising a nucleotide sequence encoding a chimeric Chlamydia sp. MOMP VD polypeptide is an mRNA, wherein the mRNA comprises the following structural elements: - a 5' cap, for example, the following structure: [ka] having a 5' cap; - a 5' untranslated region (5'UTR) having a nucleic acid sequence according to SEQ ID NO: 838; - a protein coding region having a nucleic acid sequence according to SEQ ID NO: 870; - a 3' untranslated region (3'UTR) having a nucleic acid sequence according to SEQ ID NO: 839; and -Poly A tail 55. The composition of embodiment 54, comprising or consisting of (e.g., consisting of):

[0508] 69. The composition comprises: (1) A nucleic acid (e.g., mRNA) according to embodiment 18 or 19, comprising a nucleotide sequence according to SEQ ID NO: 551 or a sequence having at least 50% (e.g., at least 75%) identity thereto (e.g., the nucleic acid is an mRNA according to embodiment 16); a nucleic acid (e.g., mRNA) comprising a nucleotide sequence encoding a chimeric Chlamydia spp. MOMP VD polypeptide, wherein the nucleic acid comprises a nucleotide sequence according to SEQ ID NO: 617, or a sequence having at least 50% (e.g., at least 75%) identity thereto (e.g., the nucleic acid is the mRNA described in embodiment 68); and a nucleic acid (e.g., mRNA) comprising a nucleotide sequence encoding a Chlamydia sp. CT443 polypeptide, wherein the nucleic acid (e.g., mRNA) comprises a nucleotide sequence according to SEQ ID NO: 707, or a sequence having at least 50% (e.g., at least 75%) identity thereto (e.g., the nucleic acid is the mRNA described in embodiment 67); or (2) A nucleic acid (e.g., mRNA) according to embodiment 18 or 19, comprising a nucleotide sequence according to SEQ ID NO: 551 or a sequence having at least 50% (e.g., at least 75%) identity thereto (e.g., the nucleic acid is an mRNA according to embodiment 16); a nucleic acid (e.g., mRNA) comprising a nucleotide sequence encoding a chimeric Chlamydia spp. MOMP VD polypeptide, wherein the nucleic acid comprises a nucleotide sequence according to SEQ ID NO: 617, or a sequence having at least 50% (e.g., at least 75%) identity thereto (e.g., the nucleic acid is the mRNA described in embodiment 68); and a nucleic acid (e.g., mRNA) comprising a nucleotide sequence encoding a chimeric Chlamydia sp. CT584 polypeptide, wherein the nucleic acid (e.g., mRNA) comprises a nucleotide sequence according to SEQ ID NO: 715, or a sequence having at least 50% (e.g., at least 75%) identity thereto (e.g., the nucleic acid is the mRNA described in embodiment 67); or (3) A nucleic acid (e.g., mRNA) according to embodiment 18 or 19, comprising a nucleotide sequence according to SEQ ID NO: 551 or a sequence having at least 50% (e.g., at least 75%) identity thereto (e.g., the nucleic acid is an mRNA according to embodiment 16); a nucleic acid (e.g., mRNA) comprising a nucleotide sequence encoding a chimeric Chlamydia spp. MOMP VD polypeptide, wherein the nucleic acid (e.g., mRNA) comprises a nucleotide sequence according to SEQ ID NO: 617, or a sequence having at least 50% (e.g., at least 75%) identity thereto (e.g., the nucleic acid is the mRNA described in embodiment 68); a nucleic acid (e.g., mRNA) comprising a nucleotide sequence encoding a Chlamydia sp. CT443 polypeptide, wherein the nucleic acid comprises a nucleotide sequence according to SEQ ID NO: 707 or a sequence having at least 50% (e.g., at least 75%) identity thereto (e.g., the nucleic acid is the mRNA described in embodiment 67); and a nucleic acid (e.g., mRNA) comprising a nucleotide sequence encoding a chimeric Chlamydia sp. CT584 polypeptide, wherein the nucleic acid (e.g., mRNA) comprises a nucleotide sequence according to SEQ ID NO: 715, or a sequence having at least 50% (e.g., at least 75%) identity thereto (e.g., the nucleic acid is the mRNA described in embodiment 67); or (4) The nucleic acid (e.g., mRNA) of embodiment 15, wherein the modified MOMP polypeptide comprises a sequence according to SEQ ID NO: 486 or a sequence having at least 70% (e.g., at least 90 or 95%) identity thereto; a nucleic acid (e.g., mRNA) comprising a nucleotide sequence encoding a chimeric Chlamydia spp. MOMP VD polypeptide, wherein the chimeric MOMP VD polypeptide comprises a sequence according to SEQ ID NO: 503, or a sequence having at least 75% (e.g., at least 90 or 95%) identity thereto; and a nucleic acid (e.g., mRNA) comprising a nucleotide sequence encoding a chimeric Chlamydia spp. CT443 polypeptide, wherein the nucleic acid (e.g., mRNA) comprises a sequence according to SEQ ID NO: 507, or a sequence having at least 75% (e.g., at least 90 or 95%) identity thereto, that encodes a Chlamydia spp. CT443 polypeptide; or (5) The nucleic acid (e.g., mRNA) of embodiment 15, wherein the modified MOMP polypeptide comprises a sequence according to SEQ ID NO: 486 or a sequence having at least 70% (e.g., at least 90 or 95%) identity thereto; a nucleic acid (e.g., mRNA) comprising a nucleotide sequence encoding a chimeric Chlamydia spp. MOMP VD polypeptide, wherein the chimeric MOMP VD polypeptide comprises a sequence according to SEQ ID NO: 503, or a sequence having at least 75% (e.g., at least 90 or 95%) identity thereto; and a nucleic acid comprising a nucleotide sequence encoding a Chlamydia spp. CT584 polypeptide, wherein the Chlamydia spp. CT584 polypeptide comprises a sequence according to SEQ ID NO: 510, or a sequence having at least 75% (e.g., at least 90 or 95%) identity thereto; or (6) The nucleic acid (e.g., mRNA) of embodiment 15, wherein the modified MOMP polypeptide comprises a sequence according to SEQ ID NO: 486 or a sequence having at least 70% (e.g., at least 90 or 95%) identity thereto; a nucleic acid (e.g., mRNA) comprising a nucleotide sequence encoding a chimeric Chlamydia spp. MOMP VD polypeptide, wherein the chimeric MOMP VD polypeptide comprises a sequence according to SEQ ID NO: 503, or a sequence having at least 75% (e.g., at least 90 or 95%) identity thereto; a nucleic acid (e.g., mRNA) comprising a nucleotide sequence encoding a chimeric Chlamydia spp. CT443 polypeptide, the nucleic acid (e.g., mRNA) comprising a sequence according to SEQ ID NO: 507, or a sequence having at least 75% (e.g., at least 90 or 95%) identity thereto, that encodes a Chlamydia spp. CT443 polypeptide; and a nucleic acid comprising a nucleotide sequence encoding a Chlamydia spp. CT584 polypeptide, wherein the Chlamydia spp. CT584 polypeptide comprises a sequence according to SEQ ID NO: 510, or a sequence having at least 75% (e.g., at least 90 or 95%) identity thereto; For example, the composition according to any one of embodiments 33 to 36 or 38 to 68, wherein the composition comprises a nucleic acid defined in (3) or a nucleic acid defined in (6).

[0509] 70. The composition comprises: (1) a modified MOMP polypeptide comprising a sequence according to SEQ ID NO: 486, or a sequence having at least 70% (e.g., at least 90 or 95%) identity thereto; a chimeric Chlamydia sp. MOMP VD polypeptide comprising a sequence according to SEQ ID NO: 503, or a sequence having at least 75% (e.g., at least 90 or 95%) identity thereto; and a chimeric Chlamydia sp. CT443 polypeptide comprising a sequence according to SEQ ID NO: 507 or a sequence having at least 75% (e.g., at least 90 or 95%) identity thereto; or (2) a modified MOMP polypeptide comprising a sequence according to SEQ ID NO: 486 or a sequence having at least 70% (e.g., at least 90 or 95%) identity thereto; a chimeric Chlamydia sp. MOMP VD polypeptide comprising a sequence according to SEQ ID NO: 503 or a sequence having at least 75% (e.g., at least 90 or 95%) identity thereto; and a Chlamydia sp. CT584 polypeptide comprising a sequence according to SEQ ID NO: 510 or a sequence having at least 75% (e.g., at least 90 or 95%) identity thereto; or (3) a modified MOMP polypeptide comprising a sequence according to SEQ ID NO: 486 or a sequence having at least 70% (e.g., at least 90 or 95%) identity thereto; a chimeric Chlamydia sp. MOMP VD polypeptide comprising a sequence according to SEQ ID NO: 503 or a sequence having at least 75% (e.g., at least 90 or 95%) identity thereto; a chimeric Chlamydia sp. CT443 polypeptide comprising a sequence according to SEQ ID NO: 507 or a sequence having at least 75% (e.g., at least 90 or 95%) identity thereto; and a Chlamydia sp. CT584 polypeptide comprising a sequence according to SEQ ID NO: 510 or a sequence having at least 75% (e.g., at least 90 or 95%) identity thereto; For example, the composition of any one of embodiments 32 or 37 to 64, wherein the composition comprises a polypeptide as defined in (3).

[0510] 71. The nucleic acid of any one of embodiments 1 to 19, and (i) a nucleic acid comprising a nucleotide sequence encoding a chimeric Chlamydia spp. MOMP variable domain (VD) polypeptide, wherein the chimeric MOMP VD polypeptide comprises an amino acid sequence comprising two or more Chlamydia spp. MOMP VD sequences of different serotypes of Chlamydia spp.; and / or (ii) One or more of the following (e.g., 1, 2, 3, or 4): (a) a nucleic acid comprising a nucleotide sequence encoding a Chlamydia species CT443 polypeptide; (b) a nucleic acid comprising a nucleotide sequence encoding a Chlamydia sp. CT584 polypeptide; (c) a nucleic acid comprising a nucleotide sequence encoding a Chlamydia species CT600 polypeptide; or (d) a nucleic acid comprising a nucleotide sequence encoding a Chlamydia sp. CT812 polypeptide; A combination including:

[0511] 72. The composition comprises: (i) the nucleic acid and (ii) the nucleic acid of (a); (i) a nucleic acid and (ii) a nucleic acid of (b); or (i) nucleic acid, (ii) nucleic acid (a) and (ii) nucleic acid (b) Including, For example, a composition comprising (i) a nucleic acid, (ii) a nucleic acid, and (ii) a nucleic acid, 72. The combination according to embodiment 71, optionally wherein the nucleic acid is as defined in embodiment 69.

[0512] 73. The polypeptide of any one of embodiments 20 to 30, and (i) a chimeric Chlamydia species MOMP variable domain (VD) polypeptide, wherein the chimeric MOMP VD polypeptide comprises an amino acid sequence that includes two or more Chlamydia species MOMP VD sequences of different serotypes of Chlamydia species; and / or (ii) One or more of the following (e.g., 1, 2, 3, or 4): (a) a polypeptide comprising the amino acid sequence of a Chlamydia species CT443 polypeptide; (b) a polypeptide comprising the amino acid sequence of a Chlamydia species CT584 polypeptide; (c) a polypeptide comprising the amino acid sequence of a Chlamydia species CT600 polypeptide; or (d) a polypeptide comprising the amino acid sequence of the Chlamydia sp. CT812 polypeptide combinations, including:

[0513] 74. The composition comprises: (i) the polypeptide and (ii) the polypeptide of (a); (i) the polypeptide and (ii)(b) the polypeptide; or The polypeptide of (i), the polypeptide of (ii)(a), and the polypeptide of (ii)(b) Including, For example, the composition comprises the polypeptide of (i), the polypeptide of (ii)(a), and the polypeptide of (ii)(b), 74. The combination according to embodiment 73, optionally wherein the polypeptide is as defined in embodiment 70.

[0514] 75. The combination of embodiment 71 or 72, wherein the nucleic acids are present in the same composition or in two or more separate compositions.

[0515] 76. The combination of embodiment 73 or 74, wherein the polypeptides are present in the same composition or in two or more separate compositions.

[0516] 77. The composition of embodiment 37, further comprising an adjuvant.

[0517] 78. The LNP comprises at least one cationic lipid, and optionally: (i) the cationic lipid is selected from the group consisting of OF-02, cKK-E10, OF-Deg-Lin, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4-E10, GL-HEPES-E3-E12-DS-3-E14, SM-102, ALC-0315, ATX-126, and IM-001 (e.g., GL-HEPES-E3-E12-DS-4-E10 or IM-001); and / or (ii) the LNP further comprises a polyethylene glycol (PEG)-conjugated (PEGylated) lipid, a cholesterol-based lipid, and a helper lipid, and optionally the (PEGylated) lipid is DMG-PEG2000 or ALC-0159 (e.g., DMG-PEG2000); the cholesterol-based lipid is cholesterol, and / or the helper lipid is DOPE or DSPC, and / or (iii) LNP is (1) 40% molar ratio of OF-02; 1.5% molar ratio of DMG-PEG2000; 28.5% molar ratio of cholesterol; and 30% molar ratio of DOPE; (2) 40% molar ratio of cKK-E10; 1.5% molar ratio of DMG-PEG2000; 28.5% molar ratio of cholesterol and 30% molar ratio of DOPE; (3) 40% molar ratio of GL-HEPES-E3-E10-DS-3-E18-1; 1.5% molar ratio of DMG-PEG2000; 28.5% molar ratio of cholesterol; and 30% molar ratio of DOPE; (4) GL-HEPES-E3-E12-DS-4-E10 (40% molar ratio; 1.5% molar ratio of DMG-PEG2000; 28.5% molar ratio of cholesterol; and 30% molar ratio of DOPE), (5) 40% molar ratio of GL-HEPES-E3-E12-DS-3-E14; 1.5% molar ratio of DMG-PEG2000; 28.5% molar ratio of cholesterol; and 30% molar ratio of DOPE. (6) A mixture containing 50% DLin-MC3-DMA (MC3), 1.5% DMG-PEG2000, 38.5% cholesterol, and 10% DSPC; or (7) A mixture containing 40% molar IM-001, 1.5% molar DMG-PEG2000, 28.5% molar cholesterol, and 30% molar DOPE. (8) A mixture containing 50% molar SM-102, 1.5% molar DMG-PEG2000, 38.5% molar cholesterol, and 10% molar DSPC. (9) ALC-0315 at a molar ratio of 46.3%; ALC-0159 at a molar ratio of -1.6%; cholesterol at a molar ratio of -42.7%; and DSPC at a molar ratio of 9.4%. (10) A 47.4% molar ratio of ALC-0315, a 1.7% molar ratio of ALC-0159, a 40.9% molar ratio of cholesterol, and a 10% molar ratio of DSPC; or (11) A 50% molar ratio of ATX-126, a 1.5% molar ratio of DMG-PEG2000, a 38.5% molar ratio of cholesterol, and a 10% molar ratio of DSPC; For example, the composition of embodiment 36, wherein the LNP is as defined in (1), (2), (4) or (7), for example, wherein the LNP is as defined in (4) or (7).

[0518] 79. A nucleic acid according to any one of embodiments 1 to 19, a modified MOMP polypeptide according to any one of embodiments 20 to 30, a composition according to any one of embodiments 31 to 70, 77 or 78, or a combination according to any one of embodiments 71 to 76, for use as a medicament.

[0519] 80. A nucleic acid according to any one of embodiments 1 to 19, a modified MOMP polypeptide according to any one of embodiments 20 to 30, a composition according to any one of embodiments 31 to 70, 77 or 78, or a combination according to any one of embodiments 71 to 76, for use in treating or preventing a Chlamydia species infection, preferably a C. trachomatis infection.

[0520] 81. The nucleic acid, modified MOMP polypeptide, composition or combination for use according to embodiment 80, wherein the infectious disease is a genital infection.

[0521] 82. A vaccine comprising the nucleic acid of any one of embodiments 1 to 19, the modified MOMP polypeptide of any one of embodiments 20 to 30, the composition of any one of embodiments 31 to 70, 77 or 78, or the combination of any one of embodiments 71 to 76.

[0522] 83. A nucleic acid comprising a nucleotide sequence encoding a chimeric Chlamydia species MOMP variable domain (VD) polypeptide, wherein the chimeric MOMP VD polypeptide comprises an amino acid sequence comprising two or more Chlamydia species MOMP VD sequences of different serotypes of Chlamydia species.

[0523] 84. The nucleic acid of embodiment 83, wherein the Chlamydia species is Chlamydia trachomatis.

[0524] 85. The nucleic acid of embodiment 83 or 84, wherein the chimeric MOMP VD polypeptide comprises conserved domain sequence portions of naturally occurring Chlamydia species MOMP polypeptides flanking each of two or more MOMP VD sequences.

[0525] 86. The nucleic acid of any one of embodiments 83 to 85, wherein the chimeric MOMP VD polypeptide comprises MOMP VD sequences of four different serotypes, optionally wherein the different serotypes are selected from serotypes D, E, F, or G of C. trachomatis.

[0526] 87. A chimeric MOMP VD polypeptide comprising: (i) two MOMP VD1 sequences of different serotypes of Chlamydia species; and / or (ii) two MOMP VD2 sequences of different serotypes of Chlamydia species; and / or (iii) one MOMP VD3 sequence; and / or (iv) Two MOMP VD4 sequences from different serotypes of Chlamydia species Including, 87. The nucleic acid of any one of embodiments 83 to 86, optionally wherein, for example, the different serotypes are selected from serotypes D, E, F or G of C. trachomatis.

[0527] 88. The nucleic acid of any one of embodiments 83 to 87, wherein the chimeric MOMP VD polypeptide comprises one MOMP VD sequence of C. trachomatis serovar D or E and one MOMP VD sequence of C. trachomatis serovar F or G.

[0528] 89. The chimeric MOMP VD polypeptide is at least one of (i) to (iv) (e.g., 4): (i) a MOMP VD1 sequence from serotype D and a MOMP VD1 sequence from serotype F, or a MOMP VD1 sequence from serotype E and a MOMP VD1 sequence from serotype F or G (e.g., a MOMP VD1 sequence of serotype E and a MOMP VD1 sequence of serotype G); and / or (ii) a MOMP VD2 sequence from serotype E and a MOMP VD2 sequence from serotype F, or a MOMP VD2 sequence from serotype D and a MOMP VD2 sequence from serotype G (e.g., a MOMP VD2 sequence of serotype D and a MOMP VD2 sequence of serotype G); and / or (iii) a MOMP VD3 sequence from serotype G or a VD3 sequence from serotype F (e.g., serotype F); and / or (iv) a MOMP VD4 sequence from serotype E and a MOMP VD4 sequence from serotype G, or a MOMP VD4 sequence from serotype D and a MOMP VD4 sequence from serotype F (e.g., a MOMP VD4 sequence from serotype D and a MOMP VD4 sequence from serotype F) Including, The nucleic acid of embodiment 87 or 88, wherein serotype D, E, F or G is of C. trachomatis.

[0529] 90. A chimeric MOMP VD polypeptide comprising: (i) a MOMP VD1 sequence from serotype D and a MOMP VD1 sequence from serotype F, a MOMP VD2 sequence from serotype E and a MOMP VD2 sequence from serotype F, a MOMP VD3 sequence from serotype G, a MOMP VD4 sequence from serotype E and a MOMP VD4 sequence from serotype G; or (ii) the MOMP VD1 sequence from serotype E and the MOMP VD1 sequence from serotype F, the MOMP VD2 sequence from serotype D and the MOMP VD2 sequence from serotype G, the MOMP VD3 sequence from serotype F, the MOMP VD4 sequence from serotype D and the MOMP VD4 sequence from serotype F Including, The nucleic acid of embodiment 89, wherein serotype D, E, F or G is of C. trachomatis.

[0530] 91. The nucleic acid of embodiment 90, wherein the chimeric MOMP VD polypeptide comprises a MOMP VD sequence according to (ii).

[0531] 92. The nucleic acid of any one of embodiments 85-91, wherein the conserved domain sequence portion is a portion of a conserved domain sequence of a native Chlamydia species MOMP polypeptide that is adjacent to the VD in that native Chlamydia species MOMP polypeptide.

[0532] 93. The nucleic acid of any one of embodiments 85-92, wherein each conserved domain sequence portion comprises 3 to 30 amino acid residues of a conserved domain sequence of a naturally occurring Chlamydia species MOMP polypeptide, and the 3 to 30 amino acid residues are immediately adjacent to the VD sequence of that naturally occurring Chlamydia species MOMP polypeptide.

[0533] 94. The nucleic acid of any one of embodiments 83-93, wherein the chimeric Chlamydia species MOMP VD polypeptide comprises a secretory signal peptide sequence.

[0534] 95. The nucleic acid of embodiment 94, wherein the secretory signal peptide sequence is a viral secretory signal peptide sequence, optionally selected from the group consisting of an influenza hemagglutinin (HA) secretory signal peptide sequence, a SARS CoV-2 spike secretory signal peptide sequence, a VZV gB secretory signal peptide sequence, a VZV gE secretory signal peptide sequence, a VZV gI secretory signal peptide sequence, a VZV gK secretory signal peptide sequence, a measles F-protein secretory signal peptide sequence, a rubella E1 protein secretory signal peptide sequence, a rubella E2 protein secretory signal peptide sequence, a mumps F-protein secretory signal peptide sequence, an Ebola GP protein secretory signal peptide sequence, and a smallpox 6 kDa IC protein secretory signal peptide sequence, and optionally wherein the secretory signal peptide sequence comprises an amino acid sequence according to any one of the SEQ ID NOs in Table 2 or Table 2.1.

[0535] 96. The nucleic acid of embodiment 95, wherein, for example, the secretory signal peptide sequence comprises the secretory signal peptide sequence of the HA protein of an influenza A virus, for example, the secretory signal peptide sequence comprises the sequence according to SEQ ID NO: 187 or SEQ ID NO: 188.

[0536] 97. The nucleic acid of any one of embodiments 83-96, wherein the chimeric MOMP VD polypeptide comprises a single amino acid substitution at one or more (e.g., all) positions corresponding to cysteine ​​residues in the conserved domain sequence of a native Chlamydia species MOMP polypeptide, and optionally the single amino acid substitution is a substitution of a cysteine ​​with a serine.

[0537] 98. The nucleic acid of any one of embodiments 83 to 97, wherein the chimeric MOMP VD polypeptide comprises mutations at one or more (e.g., all) positions corresponding to glycosylation sites, optionally N-glycosylation sites, of a native Chlamydia species MOMP polypeptide, and optionally the mutations are single amino acid substitutions.

[0538] 99. The nucleic acid of embodiment 98, wherein the chimeric MOMP VD polypeptide comprises a single amino acid substitution at each of the amino acid residues corresponding to position 9 of SEQ ID NO:9 (e.g., an N to A substitution), position 11 of SEQ ID NO:17 (e.g., a T to A substitution), position 17 of SEQ ID NO:6 (e.g., an S to A substitution), positions 4 and 21 of SEQ ID NO:18 (e.g., an N to A and an S to A substitution, respectively), position 14 of SEQ ID NO:8 (e.g., a T to A substitution), and position 14 of SEQ ID NO:16 (e.g., a T to A substitution).

[0539] 100. The nucleic acid of any one of embodiments 83 to 99, wherein the chimeric MOMP VD polypeptide comprises a sequence according to any one of SEQ ID NOs: 490 to 505, or a sequence having at least 70% (e.g., at least 90 or 95%) identity thereto, e.g., the chimeric MOMP VD polypeptide comprises a sequence according to SEQ ID NO: 503, or a sequence having at least 70% (e.g., at least 90 or 95%) identity thereto.

[0540] 101. The nucleic acid according to any one of embodiments 83 to 100, wherein the nucleic acid comprises a nucleotide sequence according to SEQ ID NO: 567 to 630 or a sequence having at least 50% (e.g., at least 75%) identity thereto, for example, the nucleic acid comprises a nucleotide sequence according to SEQ ID NO: 617 or a sequence having at least 50% (e.g., at least 75%) identity thereto.

[0541] 102. The nucleic acid is messenger RNA (mRNA), optionally (i) the mRNA comprises at least one 5' untranslated region (5' UTR), at least one 3' untranslated region (3' UTR), and / or at least one polyadenylation (poly(A)) sequence; (ii) the mRNA is unmodified or comprises at least one chemical modification, optionally the mRNA comprises at least one chemical modification, e.g., the chemical modification comprises N1-methylpseudouridine, and / or (iii) the mRNA is a self-replicating mRNA or a non-replicating mRNA, e.g., a non-replicating mRNA; The nucleic acid according to any one of embodiments 83 to 101.

[0542] 103. mRNA contains the following structural elements: - a 5' cap, for example, the following structure: [ka] having a 5' cap; - a 5' untranslated region (5'UTR) having a nucleic acid sequence according to SEQ ID NO: 838; - a protein coding region having a nucleic acid sequence according to SEQ ID NO: 870; - a 3' untranslated region (3'UTR) having a nucleic acid sequence according to SEQ ID NO: 839; and -Poly A tail 103. The nucleic acid of embodiment 102, comprising or consisting of (e.g. consisting of):

[0543] 104. The nucleic acid of embodiment 103, wherein the mRNA is chemically modified, and the chemical modification comprises or consists of (e.g. consists of) N1-methylpseudouridine in place of every uridine.

[0544] 105. A chimeric Chlamydia species MOMP variable domain (VD) polypeptide, wherein the chimeric MOMP VD polypeptide comprises an amino acid sequence that includes two or more Chlamydia species MOMP VD sequences of different serotypes of Chlamydia species.

[0545] 106. The chimeric MOMP VD polypeptide of embodiment 105, wherein the Chlamydia species is Chlamydia trachomatis.

[0546] 107. The chimeric MOMP VD polypeptide of embodiment 105 or 106, wherein the chimeric MOMP VD polypeptide comprises conserved domain sequence portions of naturally occurring Chlamydia species MOMP polypeptides flanking each of two or more MOMP VD sequences.

[0547] 108. A chimeric MOMP VD polypeptide according to any one of embodiments 105 to 107, wherein the chimeric MOMP VD polypeptide comprises MOMP VD sequences of four different serotypes, optionally wherein the different serotypes are selected from serotypes D, E, F, or G of C. trachomatis.

[0548] 109. A chimeric MOMP VD polypeptide comprising: (i) two MOMP VD1 sequences of different serotypes of Chlamydia species; and / or (ii) two MOMP VD2 sequences of different serotypes of Chlamydia species; and / or (iii) one MOMP VD3 sequence; and / or (iv) two MOMP VD4 sequences of different serotypes of Chlamydia species; 109. The chimeric MOMP VD polypeptide of any one of embodiments 105-108, optionally wherein, for example, the different serotype is selected from serotypes D, E, F, or G of C. trachomatis.

[0549] 110. A chimeric MOMP VD polypeptide according to any one of embodiments 105 to 109, wherein the chimeric MOMP VD polypeptide comprises one MOMP VD sequence of C. trachomatis serovar D or E and one MOMP VD sequence of C. trachomatis serovar F or G.

[0550] 111. The chimeric MOMP VD polypeptide is at least one of (i) to (iv) (e.g., 4): (i) a MOMP VD1 sequence from serotype D and a MOMP VD1 sequence from serotype F, or a MOMP VD1 sequence from serotype E and a MOMP VD1 sequence from serotype F or G (e.g., a MOMP VD1 sequence of serotype E and a MOMP VD1 sequence of serotype G); and / or (ii) a MOMP VD2 sequence from serotype E and a MOMP VD2 sequence from serotype F, or a MOMP VD2 sequence from serotype D and a MOMP VD2 sequence from serotype G (e.g., a MOMP VD2 sequence of serotype D and a MOMP V...

Claims

1. 1. A composition comprising: (i) a nucleic acid comprising a nucleotide sequence encoding a modified major outer membrane protein (MOMP) polypeptide, wherein the modified MOMP polypeptide has an amino acid sequence comprising two or more conserved domain sequences of a native Chlamydia trachomatis MOMP polypeptide and a non-native loop sequence between the conserved domain sequences; (ii) a nucleic acid comprising a nucleotide sequence encoding a chimeric Chlamydia trachomatis MOMP variable domain (VD) polypeptide, wherein the chimeric MOMP VD polypeptide comprises an amino acid sequence comprising two or more Chlamydia trachomatis MOMP VD sequences of different serovars of Chlamydia trachomatis; (iii) a nucleic acid comprising a nucleotide sequence encoding a Chlamydia trachomatis CT443 polypeptide; and (iv) a nucleic acid comprising a nucleotide sequence encoding a Chlamydia trachomatis CT584 polypeptide. A composition comprising:

2. 1. A nucleic acid comprising a nucleotide sequence encoding a modified major outer membrane protein (MOMP) polypeptide, wherein the modified MOMP polypeptide has an amino acid sequence comprising two or more conserved domain sequences of a native Chlamydia species MOMP polypeptide and a non-native loop sequence between the conserved domain sequences, and optionally wherein the Chlamydia species is Chlamydia trachomatis.

3. A modified major outer membrane protein (MOMP) polypeptide having an amino acid sequence comprising two or more conserved domain sequences of a native Chlamydia species MOMP polypeptide and a non-native loop sequence between said conserved domain sequences, wherein optionally said Chlamydia species is Chlamydia trachomatis.

4. 4. The composition of claim 1, the nucleic acid of claim 2, or the modified MOMP polypeptide of claim 3, wherein the non-native loop sequence is 40% or less identical to any native Chlamydia species MOMP VD (VD1, VD2, VD3, or VD4) sequence of any serovar (e.g., C. trachomatis serovar D, E, F, or G).

5. the modified MOMP polypeptide (1) does not contain a naturally occurring Chlamydia species MOMP variable domain species between the two or more conserved domain sequences; and / or (2) The modified MOMP polypeptide comprises five conserved domain sequences of a native Chlamydia species MOMP polypeptide, and optionally (i) the modified MOMP polypeptide comprises all five full-length conserved domains of a native Chlamydia species MOMP polypeptide, and / or (ii) the conserved domains of the modified MOMP polypeptide collectively share at least 95% sequence identity with the conserved domains of a native MOMP polypeptide (e.g., serotype E MOMP).

10. A composition according to claim 1 or 4, a nucleic acid according to claim 2 or 4, or a modified MOMP polypeptide according to claim 3 or 4.

6. (1) the modified MOMP polypeptide comprises a non-native loop sequence between each of the conserved domain sequences, e.g., the modified MOMP polypeptide comprises four non-native loop sequences and does not comprise any native Chlamydia species MOMP variable domains between the conserved domain sequences; and / or (2) The non-native loop sequence is 3 to 30 amino acids in length, for example, 4 to 20 amino acids in length. A composition according to any one of claims 1, 4 or 5, a nucleic acid according to any one of claims 2, 4 or 5, or a modified MOMP polypeptide according to any one of claims 3 to 5.

7. the modified MOMP polypeptide (1) all five conserved domain sequences of the Chlamydia species MOMP polypeptide of any serovar of C. trachomatis; and (2) four non-native loop sequences, wherein the non-native loop sequences are located between each of the conserved domain sequences, and the modified MOMP polypeptide does not contain any native Chlamydia species MOMP variable domains between any of the conserved domain sequences. Including, and wherein the non-native loop sequence is 3-30 amino acids in length and is 40% or less identical to any native Chlamydia species MOMP VD (VD1, VD2, VD3, or VD4) sequence of any serotype. A composition according to any one of claims 1 or 4 to 6, a nucleic acid according to any one of claims 2 or 4 to 6, or a modified MOMP polypeptide according to any one of claims 3 to 6.

8. (i) the non-native loop sequence replacing VD1 comprises a sequence according to SEQ ID NO: 462 or 466 (e.g., SEQ ID NO: 462); (ii) the non-native loop sequence replacing VD2 comprises a sequence according to SEQ ID NO: 463 or 467 (e.g., SEQ ID NO: 463); (iii) the non-native loop sequence replacing VD3 comprises a sequence according to SEQ ID NO: 464 or 468 (e.g., SEQ ID NO: 464); and / or (iv) the non-native loop sequence replacing VD4 comprises a sequence according to SEQ ID NO: 465 or 469 (e.g., SEQ ID NO: 465); For example, the modified MOMP polypeptide comprises four non-native loop sequences according to SEQ ID NOs: 462, 463, 464 and 465 in place of VD1, VD2, VD3 and VD4, respectively. A composition according to any one of claims 1 or 4 to 7, a nucleic acid according to any one of claims 2 or 4 to 7, or a modified MOMP polypeptide according to any one of claims 3 to 7.

9. 9. The composition of any one of claims 1 or 4 to 8, the nucleic acid of any one of claims 2 or 4 to 8, or the modified MOMP polypeptide of any one of claims 3 to 8, wherein the modified MOMP polypeptide comprises a sequence according to any one of SEQ ID NOs: 486-489 (e.g., SEQ ID NO: 486), or a sequence having at least 70% (e.g., at least 90 or 95%) identity thereto.

10. (1) the modified MOMP polypeptide further comprises a secretory signal peptide sequence, optionally comprising the secretory signal peptide sequence of the HA protein of influenza A virus, e.g., the secretory signal peptide sequence comprises a sequence according to SEQ ID NO: 187 or SEQ ID NO: 188; and / or (2) The nucleic acid comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 551 to 566 (e.g., SEQ ID NO: 551), or a sequence having at least 50% identity thereto; A composition according to any one of claims 1 or 4 to 9 or a nucleic acid according to any one of claims 2 or 4 to 9.

11. the nucleic acid is messenger RNA (mRNA), and optionally (i) the mRNA comprises at least one 5' untranslated region (5'UTR), at least one 3' untranslated region (3'UTR), and / or at least one polyadenylation (poly(A)) sequence; (ii) the mRNA comprises at least one chemical modification, for example, the chemical modification comprises N1-methylpseudouridine, and optionally the chemical modification comprises N1-methylpseudouridine in place of every uridine; and / or (iii) the mRNA is a self-replicating mRNA or a non-replicating mRNA, e.g., a non-replicating mRNA; A composition according to any one of claims 1 or 4 to 10, or a nucleic acid according to any one of claims 2 or 4 to 10.

12. The mRNA comprises the following structural elements: - 5' cap, for example, the following structure: 【Chemistry 1】 a cap having a 5' untranslated region (5'UTR) having the nucleic acid sequence according to SEQ ID NO: 838; - a protein coding region having a nucleic acid sequence according to SEQ ID NO: 213; a 3' untranslated region (3'UTR) having the nucleic acid sequence according to SEQ ID NO: 839; and - Poly A tail comprising or consisting of (e.g. consisting of), optionally, the mRNA is chemically modified, wherein the chemical modification comprises or consists of (e.g., consists of) N1-methylpseudouridine in place of any uridine; The composition or nucleic acid of claim 11.

13. (1) The nucleic acid according to any one of claims 2 or 4 to 12; or (2) The polypeptide according to any one of claims 3 to 9. A composition comprising: Preferably, the composition is an immunogenic composition. composition.

14. The composition is as defined in (1), and the composition is (i) a nucleic acid (e.g., mRNA) comprising a nucleotide sequence encoding a chimeric Chlamydia species MOMP variable domain (VD) polypeptide, wherein the chimeric MOMP VD polypeptide comprises an amino acid sequence comprising two or more Chlamydia species MOMP VD sequences of different serotypes of Chlamydia species; and / or (ii) one or more (e.g., 1, 2, 3, or 4) of the following: (a) a nucleic acid (e.g., mRNA) comprising a nucleotide sequence encoding a Chlamydia sp. CT443 polypeptide; (b) a nucleic acid (e.g., mRNA) comprising a nucleotide sequence encoding a Chlamydia sp. CT584 polypeptide; (c) a nucleic acid (e.g., mRNA) comprising a nucleotide sequence encoding a Chlamydia sp. CT600 polypeptide; and (d) a nucleic acid (e.g., mRNA) comprising a nucleotide sequence encoding a Chlamydia sp. CT812 polypeptide. Further comprising: Optionally, the composition comprises: the nucleic acid of (i) and the nucleic acid of (ii)(a); the nucleic acid of (i) and the nucleic acid of (ii)(b); or the nucleic acid of (i), the nucleic acid of (ii)(a), and the nucleic acid of (ii)(b); Including, For example, the composition comprises the nucleic acid (i), the nucleic acid (ii)(a), and the nucleic acid (ii)(b), The nucleic acid of (i), the nucleic acid of (ii)(a), and the nucleic acid of (ii)(b) The composition of claim 13 comprising:

15. (A) the one or more nucleic acids is mRNA, and optionally (1) The mRNA comprises at least one 5' untranslated region (5'UTR), at least one 3' untranslated region (3'UTR), and / or at least one polyadenylation (poly(A)) sequence; (2) the mRNA comprises at least one chemical modification, for example, the chemical modification comprises N1-methylpseudouridine, and optionally, the chemical modification comprises N1-methylpseudouridine in place of all uridines; and / or (3) the mRNA is a self-replicating mRNA or a non-replicating mRNA, e.g., a non-replicating mRNA, and / or (B) the composition further comprises a lipid nanoparticle (LNP), and optionally the nucleic acid is encapsulated in the LNP; 15. The composition of claim 1 or 14.

16. The composition is as defined in (2), and the composition is (i) a chimeric Chlamydia species MOMP variable domain (VD) polypeptide, wherein the chimeric MOMP VD polypeptide comprises an amino acid sequence that includes two or more Chlamydia species MOMP VD sequences of different serotypes of Chlamydia species; and / or (ii) one or more (e.g., 1, 2, 3, or 4) of the following: (a) a polypeptide comprising the amino acid sequence of a Chlamydia sp. CT443 polypeptide; (b) a polypeptide comprising the amino acid sequence of a Chlamydia sp. CT584 polypeptide; (c) a polypeptide comprising the amino acid sequence of a Chlamydia species CT600 polypeptide; or (d) a polypeptide comprising the amino acid sequence of a Chlamydia sp. CT812 polypeptide. Further comprising: Optionally, the composition comprises: The polypeptide of (i) and the polypeptide of (ii)(a); or The polypeptide of (i) and the polypeptide of (ii)(b); or The polypeptide of (i), the polypeptide of (ii)(a), and the polypeptide of (ii)(b) Including, For example, the composition comprises the polypeptide of (i), the polypeptide of (ii)(a), and the polypeptide of (ii)(b). The composition of claim 13.

17. 17. The composition of any one of claims 1, 4-12, or 14-16, wherein the chimeric MOMP VD polypeptide comprises conserved domain sequence portions of a naturally occurring Chlamydia species MOMP polypeptide flanking each of the two or more MOMP VD sequences, optionally wherein the conserved domain sequence portions are portions of the conserved domain sequence of a naturally occurring Chlamydia species MOMP polypeptide that flank a VD in the naturally occurring Chlamydia species MOMP polypeptide, and further optionally wherein each conserved domain sequence portion comprises 3 to 30 amino acid residues of a conserved domain sequence of a naturally occurring Chlamydia species MOMP polypeptide, wherein the 3 to 30 amino acid residues are immediately adjacent to the VD sequence of the naturally occurring Chlamydia species MOMP polypeptide.

18. the chimeric MOMP VD polypeptide is (1) MOMP VD sequences of four different serotypes, e.g., the different serotypes are serotypes D, E, F, or G of C. trachomatis; (2) (i) two MOMP VD1 sequences of different serotypes of the Chlamydia species; and / or (ii) two MOMP VD2 sequences of different serotypes of said Chlamydia species; and / or (iii) one MOMP VD3 sequence; and / or (iv) two MOMP VD4 sequences of different serotypes of said Chlamydia species; Optionally, the different serotype is selected from C. trachomatis serotypes D, E, F, or G; and / or (3) One MOMP VD sequence of serovar D or E of C. trachomatis and one MOMP VD sequence of serovar F or G of C. trachomatis.

18. The composition of any one of claims 1, 4-12, or 14-17, comprising:

19. The chimeric MOMP VD polypeptide is at least one (e.g., four) of (1) to (4): (1) a MOMP VD1 sequence from serotype D and a MOMP VD1 sequence from serotype F, or a MOMP VD1 sequence from serotype E and a MOMP VD1 sequence from serotype F or G (e.g., a MOMP VD1 sequence from serotype E and a MOMP VD1 sequence from serotype G); and / or (2) a MOMP VD2 sequence from serotype E and a MOMP VD2 sequence from serotype F, or a MOMP VD2 sequence from serotype D and a MOMP VD2 sequence from serotype G (e.g., a MOMP VD2 sequence from serotype D and a MOMP VD2 sequence from serotype G); and / or (3) a MOMP VD3 sequence from serotype G or a VD3 sequence from serotype F (e.g., serotype F); and / or (4) A MOMP VD4 sequence from serotype E and a MOMP VD4 sequence from serotype G, or a MOMP VD4 sequence from serotype D and a MOMP VD4 sequence from serotype F (e.g., a MOMP VD4 sequence from serotype D and a MOMP VD4 sequence from serotype F). Including, the serotypes D, E, F, and G are of C. trachomatis; Optionally, the chimeric MOMP VD polypeptide comprises: (i) a MOMP VD1 sequence from serotype D and a MOMP VD1 sequence from serotype F, a MOMP VD2 sequence from serotype E and a MOMP VD2 sequence from serotype F, a MOMP VD3 sequence from serotype G, a MOMP VD4 sequence from serotype E and a MOMP VD4 sequence from serotype G; or (ii) a MOMP VD1 sequence from serotype E and a MOMP VD1 sequence from serotype F, a MOMP VD2 sequence from serotype D and a MOMP VD2 sequence from serotype G, a MOMP VD3 sequence from serotype F, a MOMP VD4 sequence from serotype D and a MOMP VD4 sequence from serotype F Including, For example, the chimeric MOMP VD polypeptide comprises a MOMP VD sequence according to (ii). The composition of any one of claims 1, 4-12, or 14-18.

20. (1) The chimeric MOMP VD polypeptide comprises a sequence according to any one of SEQ ID NOs: 490-505 (e.g., SEQ ID NO: 503), or a sequence having at least 70% (e.g., at least 90 or 95%) identity thereto; (2) The nucleic acid comprising a nucleotide sequence encoding a chimeric Chlamydia sp. MOMP VD polypeptide comprises a nucleotide sequence according to SEQ ID NO: 567-630 (e.g., SEQ ID NO: 617), or a sequence having at least 50% identity thereto; (3) the Chlamydia sp. CT443 polypeptide comprises a sequence according to SEQ ID NOs: 507-508 (e.g., SEQ ID NO: 507), or a sequence having at least 70% (e.g., at least 90 or 95%) identity thereto; (4) The nucleic acid comprising a nucleotide sequence encoding the Chlamydia sp. CT443 polypeptide comprises a nucleotide sequence according to any one of SEQ ID NOs: 707-710 (e.g., SEQ ID NO: 707), or a sequence having at least 50% (e.g., at least 75%) identity thereto. (5) The Chlamydia sp. CT584 polypeptide comprises a sequence according to any one of SEQ ID NOs: 509-512 (e.g., SEQ ID NO: 510), or a sequence having at least 70% (e.g., at least 90 or 95%) identity thereto. (6) The nucleic acid comprising a nucleotide sequence encoding the Chlamydia sp. CT584 polypeptide comprises a nucleotide sequence according to any one of SEQ ID NOs: 711-718 (e.g., SEQ ID NO: 715), or a sequence having at least 50% (e.g., at least 75%) identity thereto. (7) The Chlamydia sp. CT600 polypeptide comprises a sequence according to any one of SEQ ID NOs: 513-514, or a sequence having at least 70% (e.g., at least 90 or 95%) identity thereto. (8) The nucleic acid comprising a nucleotide sequence encoding the Chlamydia sp. CT600 polypeptide comprises a nucleotide sequence according to any one of SEQ ID NOs: 719-722, or a sequence having at least 50% (e.g., at least 75%) identity thereto. (9) The Chlamydia sp. CT812 polypeptide comprises a sequence according to any one of SEQ ID NOs: 515-535, or a sequence having at least 70% (e.g., at least 90 or 95%) identity thereto; and / or (10) The nucleic acid comprising a nucleotide sequence encoding the Chlamydia sp. CT812 polypeptide comprises a nucleotide sequence according to any one of SEQ ID NOs: 723-762, or a sequence having at least 50% (e.g., at least 75%) identity thereto.

20. The composition of any one of claims 1, 4 to 12, or 14 to 19.

21. (1) The nucleic acid comprising a nucleotide sequence encoding a Chlamydia sp. CT443 polypeptide is mRNA, and the mRNA comprises the following structural elements: - 5' cap, for example, the following structure: 【Chemistry 2】 a 5' cap having a 5' untranslated region (5'UTR) having the nucleic acid sequence according to SEQ ID NO: 838; - a protein coding region having a nucleic acid sequence according to SEQ ID NO: 369; a 3' untranslated region (3'UTR) having the nucleic acid sequence according to SEQ ID NO: 839; and - Poly A tail comprising or consisting of (e.g. consisting of), (2) The nucleic acid comprising a nucleotide sequence encoding a Chlamydia sp. CT584 polypeptide is mRNA, and the mRNA comprises the following structural elements: - 5' cap, for example, the following structure: 【Transformation 3】 a 5' cap having a 5' untranslated region (5'UTR) having the nucleic acid sequence according to SEQ ID NO: 838; - a protein coding region having a nucleic acid sequence according to SEQ ID NO: 377; a 3' untranslated region (3'UTR) having the nucleic acid sequence according to SEQ ID NO: 839; and - Poly A tail and / or comprising or consisting of (e.g. consisting of) (3) The nucleic acid comprising a nucleotide sequence encoding a chimeric Chlamydia sp. MOMP VD polypeptide is an mRNA, and the mRNA comprises the following structural elements: - 5' cap, for example, the following structure: 【Chemistry 4】 a 5' cap having a 5' untranslated region (5'UTR) having the nucleic acid sequence according to SEQ ID NO: 838; - a protein coding region having a nucleic acid sequence according to SEQ ID NO: 870; a 3' untranslated region (3'UTR) having the nucleic acid sequence according to SEQ ID NO: 839; and - Poly A tail comprising or consisting of (e.g. consisting of), The composition of any one of claims 1, 4 to 12, 14, 15 or 17 to 20.

22. The composition comprises: (1) A nucleic acid (e.g., mRNA) according to claim 10 (2), comprising a nucleotide sequence according to SEQ ID NO: 551 or a sequence having at least 50% (e.g., at least 75%) identity thereto (e.g., the nucleic acid is an mRNA according to claim 12); a nucleic acid (e.g., mRNA) comprising a nucleotide sequence encoding the chimeric Chlamydia sp. MOMP VD polypeptide, wherein the nucleic acid comprises a nucleotide sequence according to SEQ ID NO: 617, or a sequence having at least 50% (e.g., at least 75%) identity thereto (e.g., the nucleic acid is the mRNA of claim 21(3)); and a nucleic acid (e.g., mRNA) comprising a nucleotide sequence encoding the Chlamydia sp. CT443 polypeptide, wherein the nucleic acid comprises a nucleotide sequence according to SEQ ID NO: 707, or a sequence having at least 50% (e.g., at least 75%) identity thereto (e.g., the nucleic acid is an mRNA according to claim 21(1)); or (2) A nucleic acid (e.g., mRNA) according to claim 10(2), comprising a nucleotide sequence according to SEQ ID NO: 551, or a sequence having at least 50% (e.g., at least 75%) identity thereto (e.g., the nucleic acid is an mRNA according to claim 12); a nucleic acid (e.g., mRNA) comprising a nucleotide sequence encoding the chimeric Chlamydia sp. MOMP VD polypeptide, wherein the nucleic acid comprises a nucleotide sequence according to SEQ ID NO: 617, or a sequence having at least 50% (e.g., at least 75%) identity thereto (e.g., the nucleic acid is the mRNA of claim 21(3)); and a nucleic acid (e.g., mRNA) comprising a nucleotide sequence encoding the Chlamydia sp. CT584 polypeptide, wherein the nucleic acid comprises a nucleotide sequence according to SEQ ID NO: 715, or a sequence having at least 50% (e.g., at least 75%) identity thereto (e.g., the nucleic acid is an mRNA according to claim 21(2)); or (3) A nucleic acid (e.g., mRNA) according to claim 10(2), comprising a nucleotide sequence according to SEQ ID NO: 551, or a sequence having at least 50% (e.g., at least 75%) identity thereto (e.g., the nucleic acid is an mRNA according to claim 12); a nucleic acid (e.g., mRNA) comprising a nucleotide sequence encoding the chimeric Chlamydia sp. MOMP VD polypeptide, wherein the nucleic acid comprises a nucleotide sequence according to SEQ ID NO: 617, or a sequence having at least 50% (e.g., at least 75%) identity thereto (e.g., the nucleic acid is the mRNA of claim 21(3)); a nucleic acid (e.g., mRNA) comprising a nucleotide sequence encoding the Chlamydia sp. CT443 polypeptide, wherein the nucleic acid comprises a nucleotide sequence according to SEQ ID NO: 707, or a sequence having at least 50% (e.g., at least 75%) identity thereto (e.g., the nucleic acid is the mRNA of claim 21(1)); and a nucleic acid (e.g., mRNA) comprising a nucleotide sequence encoding the Chlamydia sp. CT584 polypeptide, wherein the nucleic acid comprises a nucleotide sequence according to SEQ ID NO: 715, or a sequence having at least 50% (e.g., at least 75%) identity thereto (e.g., the nucleic acid is an mRNA according to claim 21(2)); or (4) The nucleic acid (e.g., mRNA) of claim 9, wherein the modified MOMP polypeptide comprises a sequence according to SEQ ID NO: 486, or a sequence having at least 70% (e.g., at least 90 or 95%) identity thereto; a nucleic acid (e.g., mRNA) comprising a nucleotide sequence encoding the chimeric Chlamydia sp. MOMP VD polypeptide, wherein the chimeric MOMP VD polypeptide comprises a sequence according to SEQ ID NO:503, or a sequence having at least 75% (e.g., at least 90 or 95%) identity thereto; and a nucleic acid (e.g., mRNA) comprising a nucleotide sequence encoding the Chlamydia spp. CT443 polypeptide, wherein the encoding Chlamydia spp. CT443 polypeptide comprises a sequence according to SEQ ID NO:507, or a sequence having at least 75% (e.g., at least 90 or 95%) identity thereto; or (5) The nucleic acid (e.g., mRNA) of claim 9, wherein the modified MOMP polypeptide comprises a sequence according to SEQ ID NO: 486, or a sequence having at least 70% (e.g., at least 90 or 95%) identity thereto; a nucleic acid (e.g., mRNA) comprising a nucleotide sequence encoding the chimeric Chlamydia sp. MOMP VD polypeptide, wherein the chimeric MOMP VD polypeptide comprises a sequence according to SEQ ID NO:503, or a sequence having at least 75% (e.g., at least 90 or 95%) identity thereto; and a nucleic acid comprising a nucleotide sequence encoding the Chlamydia spp. CT584 polypeptide, wherein the Chlamydia spp. CT584 polypeptide comprises a sequence according to SEQ ID NO: 510, or a sequence having at least 75% (e.g., at least 90 or 95%) identity thereto; or (6) The nucleic acid (e.g., mRNA) of claim 9, wherein the modified MOMP polypeptide comprises a sequence according to SEQ ID NO: 486, or a sequence having at least 70% (e.g., at least 90 or 95%) identity thereto; a nucleic acid (e.g., mRNA) comprising a nucleotide sequence encoding the chimeric Chlamydia sp. MOMP VD polypeptide, wherein the chimeric MOMP VD polypeptide comprises a sequence according to SEQ ID NO:503, or a sequence having at least 75% (e.g., at least 90 or 95%) identity thereto; a nucleic acid (e.g., mRNA) comprising a nucleotide sequence encoding the Chlamydia spp. CT443 polypeptide, wherein the encoding Chlamydia spp. CT443 polypeptide comprises a sequence according to SEQ ID NO:507, or a sequence having at least 75% (e.g., at least 90 or 95%) identity thereto; and CT584 polypeptide, wherein the nucleic acid comprises a nucleotide sequence according to SEQ ID NO:510, or a sequence having at least 75% (e.g., at least 90 or 95%) identity thereto. Including, For example, the composition comprises a nucleic acid defined in (3) or a nucleic acid defined in (6). The composition of any one of claims 1, 4 to 12, 14, 15 or 17 to 21.

23. The composition comprises: (1) a modified MOMP polypeptide comprising a sequence according to SEQ ID NO: 486, or a sequence having at least 70% (e.g., at least 90 or 95%) identity thereto; a chimeric Chlamydia sp. MOMP VD polypeptide comprising a sequence according to SEQ ID NO:503, or a sequence having at least 75% (e.g., at least 90 or 95%) identity thereto; and a chimeric Chlamydia sp. CT443 polypeptide comprising a sequence according to SEQ ID NO: 507 or a sequence having at least 75% (e.g., at least 90 or 95%) identity thereto; or (2) a modified MOMP polypeptide comprising a sequence according to SEQ ID NO: 486, or a sequence having at least 70% (e.g., at least 90 or 95%) identity thereto; a chimeric Chlamydia sp. MOMP VD polypeptide comprising a sequence according to SEQ ID NO:503 or a sequence having at least 75% (e.g., at least 90 or 95%) identity thereto; and a Chlamydia sp. CT584 polypeptide comprising a sequence according to SEQ ID NO: 510 or a sequence having at least 75% (e.g., at least 90 or 95%) identity thereto; or (3) a modified MOMP polypeptide comprising a sequence according to SEQ ID NO: 486, or a sequence having at least 70% (e.g., at least 90 or 95%) identity thereto; a chimeric Chlamydia sp. MOMP VD polypeptide comprising a sequence according to SEQ ID NO:503 or a sequence having at least 75% (e.g., at least 90 or 95%) identity thereto; A Chlamydia sp. CT443 polypeptide comprising a sequence according to SEQ ID NO:507 or a sequence having at least 75% (e.g., at least 90 or 95%) identity thereto; and CT584 polypeptide comprising a sequence according to SEQ ID NO:510 or a sequence having at least 75% (e.g., at least 90 or 95%) identity thereto; For example, the composition comprises a polypeptide defined in (3). The composition according to any one of claims 16 to 20.

24. A nucleic acid according to any one of claims 2 or 4 to 12, and (i) a nucleic acid comprising a nucleotide sequence encoding a chimeric Chlamydia species MOMP variable domain (VD) polypeptide, wherein the chimeric MOMP VD polypeptide comprises an amino acid sequence comprising two or more Chlamydia species MOMP VD sequences of different serotypes of Chlamydia species; and / or (ii) one or more (e.g., 1, 2, 3, or 4) of the following: (a) a nucleic acid comprising a nucleotide sequence encoding a Chlamydia sp. CT443 polypeptide; (b) a nucleic acid comprising a nucleotide sequence encoding a Chlamydia sp. CT584 polypeptide; (c) a nucleic acid comprising a nucleotide sequence encoding a Chlamydia sp. CT600 polypeptide; or (d) a nucleic acid comprising a nucleotide sequence encoding a Chlamydia sp. CT812 polypeptide; A combination comprising: Optionally, the combination comprises: the nucleic acid of (i) and the nucleic acid of (ii)(a); the nucleic acid of (i) and the nucleic acid of (ii)(b); or The nucleic acid of (i), the nucleic acid of (ii)(a), and the nucleic acid of (ii)(b) Including, For example, the combination includes the nucleic acid (i), the nucleic acid (ii)(a), and the nucleic acid (ii)(b), Optionally, the nucleic acid is according to claim 22, Further optionally, the nucleic acids are present in the same composition (e.g., in the same composition). combination.

25. A polypeptide according to any one of claims 3 to 9, and (i) a chimeric Chlamydia species MOMP variable domain (VD) polypeptide, wherein the chimeric MOMP VD polypeptide comprises an amino acid sequence that includes two or more Chlamydia species MOMP VD sequences of different serotypes of Chlamydia species; and / or (ii) one or more (e.g., 1, 2, 3, or 4) of the following: (a) a polypeptide comprising the amino acid sequence of a Chlamydia sp. CT443 polypeptide; (b) a polypeptide comprising the amino acid sequence of a Chlamydia sp. CT584 polypeptide; (c) a polypeptide comprising the amino acid sequence of a Chlamydia species CT600 polypeptide; or (d) a polypeptide comprising the amino acid sequence of a Chlamydia sp. CT812 polypeptide. A combination comprising: Optionally, the combination comprises: The polypeptide of (i) and the polypeptide of (ii)(a); The polypeptide of (i) and the polypeptide of (ii)(b); or The polypeptide of (i), the polypeptide of (ii)(a), and the polypeptide of (ii)(b) Including, For example, the combination includes the polypeptide of (i), the polypeptide of (ii)(a), and the polypeptide of (ii)(b), Optionally, the polypeptide is as defined in claim 23, Further optionally, the polypeptides are present in the same composition or in two or more separate compositions (e.g., in the same composition). combination.

26. 1. A nucleic acid comprising a nucleotide sequence encoding a chimeric Chlamydia species MOMP variable domain (VD) polypeptide, wherein the chimeric MOMP VD polypeptide comprises an amino acid sequence comprising two or more Chlamydia species MOMP VD sequences of different serotypes of Chlamydia species, and optionally, the Chlamydia species is Chlamydia trachomatis.

27. 1. A chimeric Chlamydia species MOMP variable domain (VD) polypeptide, wherein the chimeric MOMP VD polypeptide comprises an amino acid sequence that includes two or more Chlamydia species MOMP VD sequences of different serotypes of Chlamydia species, and optionally, the Chlamydia species is Chlamydia trachomatis.

28. (a) the chimeric MOMP VD polypeptide comprises conserved domain sequence portions of naturally occurring Chlamydia species MOMP polypeptides adjacent to each of the two or more MOMP VD sequences; (b) the chimeric MOMP VD polypeptide comprises MOMP VD sequences of four different serotypes, optionally wherein the different serotypes are selected from serotypes D, E, F, or G of C. trachomatis; 28. The nucleic acid of claim 26 or the chimeric MOMP VD polypeptide of claim 27.

29. the chimeric MOMP VD polypeptide is (1) (i) two MOMP VD1 sequences of different serotypes of the Chlamydia species; and / or (ii) two MOMP VD2 sequences of different serotypes of said Chlamydia species; and / or (iii) one MOMP VD3 sequence; and / or (iv) two MOMP VD4 sequences of different serotypes of said Chlamydia species; optionally, for example, the different serotype is selected from C. trachomatis serotypes D, E, F, or G; and / or (2) one MOMP VD sequence of C. trachomatis serovar D or E and one MOMP VD sequence of C. trachomatis serovar F or G 29. The nucleic acid of claim 26 or 28 or the chimeric MOMP VD polypeptide of claim 27 or 28, comprising:

30. The chimeric MOMP VD polypeptide is at least one of (i) to (iv) (e.g., 4): (i) a MOMP VD1 sequence from serotype D and a MOMP VD1 sequence from serotype F, or a MOMP VD1 sequence from serotype E and a MOMP VD1 sequence from serotype F or G (e.g., a MOMP VD1 sequence of serotype E and a MOMP VD1 sequence of serotype G); and / or (ii) a MOMP VD2 sequence from serotype E and a MOMP VD2 sequence from serotype F, or a MOMP VD2 sequence from serotype D and a MOMP VD2 sequence from serotype G (e.g., a MOMP VD2 sequence of serotype D and a MOMP VD2 sequence of serotype G); and / or (iii) a MOMP VD3 sequence from serotype G or a VD3 sequence from serotype F (e.g., serotype F); and / or (iv) a MOMP VD4 sequence from serotype E and a MOMP VD4 sequence from serotype G, or a MOMP VD4 sequence from serotype D and a MOMP VD4 sequence from serotype F (e.g., a MOMP VD4 sequence of serotype D and a MOMP VD4 sequence of serotype F). Including, the serotype D, E, F, or G is C. trachomatis; Optionally, the chimeric MOMP VD polypeptide comprises: (1) a MOMP VD1 sequence from serotype D and a MOMP VD1 sequence from serotype F, a MOMP VD2 sequence from serotype E and a MOMP VD2 sequence from serotype F, a MOMP VD3 sequence from serotype G, a MOMP VD4 sequence from serotype E and a MOMP VD4 sequence from serotype G; or (2) MOMP VD1 sequence from serotype E and MOMP VD1 sequence from serotype F, MOMP VD2 sequence from serotype D and MOMP VD2 sequence from serotype G, MOMP VD3 sequence from serotype F, MOMP VD4 sequence from serotype D and MOMP VD4 sequence from serotype F Including, the serotype D, E, F, or G is C. trachomatis; For example, the chimeric MOMP VD polypeptide comprises the MOMP VD sequence described in (2).

30. The nucleic acid of claim 29 or the chimeric MOMP VD polypeptide of claim 29.

31. (a) the conserved domain sequence portion is a portion of a conserved domain sequence of a native Chlamydia species MOMP polypeptide that is adjacent to the VD in that native Chlamydia species MOMP polypeptide; and / or (b) each conserved domain sequence portion comprises 3 to 30 amino acid residues of a conserved domain sequence of a naturally occurring Chlamydia species MOMP polypeptide, wherein the 3 to 30 amino acid residues are immediately adjacent to the VD sequence of that naturally occurring Chlamydia species MOMP polypeptide; A nucleic acid according to any one of claims 28 to 30 or a chimeric MOMP VD polypeptide according to any one of claims 28 to 30.

32. 32. The nucleic acid of claim 26 or any one of claims 28-31, or the chimeric MOMP VD polypeptide of any one of claims 27-31, wherein the chimeric MOMP VD polypeptide comprises a sequence according to any one of SEQ ID NOs: 490-505, or a sequence having at least 70% (e.g., at least 90 or 95%) identity thereto, e.g., the chimeric MOMP VD polypeptide comprises a sequence according to SEQ ID NO: 503, or a sequence having at least 70% (e.g., at least 90 or 95%) identity thereto.

33. 33. The nucleic acid of any one of claims 26 or 28 to 32, wherein the nucleic acid comprises a nucleotide sequence according to SEQ ID NO: 567 to 630 or a sequence having at least 50% (e.g. at least 75%) identity thereto, such as for example, wherein the nucleic acid comprises a nucleotide sequence according to SEQ ID NO: 617 or a sequence having at least 50% (e.g. at least 75%) identity thereto.

34. the nucleic acid is messenger RNA (mRNA), and optionally (i) the mRNA comprises at least one 5' untranslated region (5'UTR), at least one 3' untranslated region (3'UTR), and / or at least one polyadenylation (poly(A)) sequence; (ii) the mRNA comprises at least one chemical modification, for example, the chemical modification comprises N1-methylpseudouridine, and optionally the chemical modification comprises N1-methylpseudouridine in place of every uridine; and / or (iii) the mRNA is a self-replicating mRNA or a non-replicating mRNA, e.g., a non-replicating mRNA; A nucleic acid according to any one of claims 26 or 28 to 33.

35. The mRNA comprises the following structural elements: - 5' cap, for example, the following structure: 【Transformation 5】 a 5' cap having a 5' untranslated region (5'UTR) having the nucleic acid sequence according to SEQ ID NO: 838; - a protein coding region having a nucleic acid sequence according to SEQ ID NO: 870; a 3' untranslated region (3'UTR) having the nucleic acid sequence according to SEQ ID NO: 839; and - Poly A tail comprising or consisting of (e.g. consisting of), optionally, the mRNA is chemically modified, wherein the chemical modification comprises or consists of (e.g., consists of) N1-methylpseudouridine in place of any uridine; A nucleic acid according to any one of claims 26 or 28 to 34.

36. A composition comprising a nucleic acid according to any one of claims 26 or 28 to 35, wherein said composition is an immunogenic composition.

37. 33. A composition comprising a chimeric MOMP VD polypeptide according to any one of claims 27 to 32, wherein the composition is preferably an immunogenic composition, for example, the composition comprises an adjuvant.

38. The composition comprises: (i) a nucleic acid comprising a nucleotide sequence encoding a modified MOMP polypeptide, wherein the modified MOMP polypeptide has an amino acid sequence comprising two or more conserved domain sequences of a native Chlamydia species MOMP polypeptide and a non-native loop sequence between the conserved domain sequences; and / or (ii) one or more (e.g., 1, 2, 3, or 4) of the following: (a) a nucleic acid comprising a nucleotide sequence encoding a Chlamydia sp. CT443 polypeptide; (b) a nucleic acid comprising a nucleotide sequence encoding a Chlamydia sp. CT584 polypeptide; (c) a nucleic acid comprising a nucleotide sequence encoding a Chlamydia sp. CT600 polypeptide; or (d) a nucleic acid comprising a nucleotide sequence encoding a Chlamydia sp. CT812 polypeptide; Further comprising: Optionally, the composition comprises: the nucleic acid of (i) and the nucleic acid of (ii)(a); the nucleic acid of (i) and the nucleic acid of (ii)(b); or The nucleic acid of (i), the nucleic acid of (ii)(a), and the nucleic acid of (ii)(b) Including, For example, the composition comprises the nucleic acid (i), the nucleic acid (ii)(a), and the nucleic acid (ii)(b).

37. The composition of claim 36.

39. one or more nucleic acids is mRNA, and optionally (i) the mRNA comprises at least one 5' untranslated region (5'UTR), at least one 3' untranslated region (3'UTR), and / or at least one polyadenylation (poly(A)) sequence; (ii) the mRNA comprises at least one chemical modification, for example, the chemical modification comprises N1-methylpseudouridine, and optionally the chemical modification comprises N1-methylpseudouridine in place of every uridine; and / or (iii) the mRNA is a self-replicating mRNA or a non-replicating mRNA, e.g., a non-replicating mRNA; 39. The composition of claim 38.

40. (1) the nucleic acid comprising a nucleotide sequence encoding the modified MOMP polypeptide is as defined in any one of claims 2 or 4 to 12, and / or (2) Any one of the nucleic acid comprising a nucleotide sequence encoding the Chlamydia spp. CT443 polypeptide, the nucleic acid comprising a nucleotide sequence encoding the Chlamydia spp. CT584 polypeptide, the nucleic acid comprising a nucleotide sequence encoding the Chlamydia spp. CT600 polypeptide, or the nucleic acid comprising a nucleotide sequence encoding the Chlamydia spp. CT812 polypeptide is as defined in any one of claims 20-22.

40. The composition of claim 38 or 39.

41. The composition comprises: (1) A nucleic acid (e.g., mRNA) according to claim 10 (2), comprising a nucleotide sequence according to SEQ ID NO: 551 or a sequence having at least 50% (e.g., at least 75%) identity thereto (e.g., the nucleic acid is an mRNA according to claim 12); 34. The nucleic acid (e.g., mRNA) of claim 33, comprising a nucleotide sequence according to SEQ ID NO: 617, or a sequence having at least 50% (e.g., at least 75%) identity thereto (e.g., the nucleic acid is the mRNA of claim 35); and a nucleic acid (e.g., mRNA) comprising a nucleotide sequence encoding the Chlamydia sp. CT443 polypeptide, wherein the nucleic acid comprises a nucleotide sequence according to SEQ ID NO: 707, or a sequence having at least 50% (e.g., at least 75%) identity thereto (e.g., the nucleic acid is an mRNA according to claim 21(1)); or (2) A nucleic acid (e.g., mRNA) according to claim 10(2), comprising a nucleotide sequence according to SEQ ID NO: 551, or a sequence having at least 50% (e.g., at least 75%) identity thereto (e.g., the nucleic acid is an mRNA according to claim 12); 34. The nucleic acid (e.g., mRNA) of claim 33, comprising a nucleotide sequence according to SEQ ID NO: 617, or a sequence having at least 50% (e.g., at least 75%) identity thereto (e.g., the nucleic acid is the mRNA of claim 35); a nucleic acid (e.g., mRNA) comprising a nucleotide sequence encoding the Chlamydia sp. CT584 polypeptide, wherein the nucleic acid comprises a nucleotide sequence according to SEQ ID NO: 715, or a sequence having at least 50% (e.g., at least 75%) identity thereto (e.g., the nucleic acid is an mRNA according to claim 21(2)); or (3) A nucleic acid (e.g., mRNA) according to claim 10(2), comprising a nucleotide sequence according to SEQ ID NO: 551, or a sequence having at least 50% (e.g., at least 75%) identity thereto (e.g., the nucleic acid is an mRNA according to claim 12); 34. A nucleic acid (e.g., mRNA) according to claim 33, comprising a nucleotide sequence according to SEQ ID NO: 617, or a sequence having at least 50% (e.g., at least 75%) identity thereto (e.g., the nucleic acid is an mRNA according to claim 35); a nucleic acid (e.g., mRNA) comprising a nucleotide sequence encoding the Chlamydia sp. CT443 polypeptide, wherein the nucleic acid comprises a nucleotide sequence according to SEQ ID NO: 707, or a sequence having at least 50% (e.g., at least 75%) identity thereto (e.g., the nucleic acid is the mRNA of claim 21(1)); and a nucleic acid (e.g., mRNA) comprising a nucleotide sequence encoding the Chlamydia sp. CT584 polypeptide, wherein the nucleic acid comprises a nucleotide sequence according to SEQ ID NO: 715, or a sequence having at least 50% (e.g., at least 75%) identity thereto (e.g., the nucleic acid is an mRNA according to claim 21(2)); or (4) The nucleic acid (e.g., mRNA) of claim 9, wherein the modified MOMP polypeptide comprises a sequence according to SEQ ID NO: 486, or a sequence having at least 70% (e.g., at least 90 or 95%) identity thereto; 33. The nucleic acid (e.g., mRNA) of claim 32, wherein the chimeric MOMP VD polypeptide comprises a sequence according to SEQ ID NO: 503, or a sequence having at least 75% (e.g., at least 90 or 95%) identity thereto; and a nucleic acid (e.g., mRNA) comprising a nucleotide sequence encoding the Chlamydia spp. CT443 polypeptide, wherein the Chlamydia spp. CT443 polypeptide comprises a sequence according to SEQ ID NO:507, or a sequence having at least 75% (e.g., at least 90 or 95%) identity thereto; or (5) The nucleic acid (e.g., mRNA) of claim 9, wherein the modified MOMP polypeptide comprises a sequence according to SEQ ID NO: 486, or a sequence having at least 70% (e.g., at least 90 or 95%) identity thereto; 33. The nucleic acid (e.g., mRNA) of claim 32, wherein the chimeric MOMP VD polypeptide comprises a sequence according to SEQ ID NO: 503, or a sequence having at least 75% (e.g., at least 90 or 95%) identity thereto; and a nucleic acid comprising a nucleotide sequence encoding the Chlamydia spp. CT584 polypeptide, wherein the Chlamydia spp. CT584 polypeptide comprises a sequence according to SEQ ID NO: 510, or a sequence having at least 75% (e.g., at least 90 or 95%) identity thereto; or (6) The nucleic acid (e.g., mRNA) of claim 9, wherein the modified MOMP polypeptide comprises a sequence according to SEQ ID NO: 486, or a sequence having at least 70% (e.g., at least 90 or 95%) identity thereto; 33. The nucleic acid (e.g., mRNA) of claim 32, wherein the chimeric MOMP VD polypeptide comprises a sequence according to SEQ ID NO: 503, or a sequence having at least 75% (e.g., at least 90 or 95%) identity thereto; a nucleic acid (e.g., mRNA) comprising a nucleotide sequence encoding the Chlamydia spp. CT443 polypeptide, wherein the Chlamydia spp. CT443 polypeptide comprises a sequence according to SEQ ID NO:507, or a sequence having at least 75% (e.g., at least 90 or 95%) identity thereto; and CT584 polypeptide, wherein the nucleic acid comprises a nucleotide sequence according to SEQ ID NO:510, or a sequence having at least 75% (e.g., at least 90 or 95%) identity thereto. Including, For example, the composition contains the nucleic acid defined in (3) or the nucleic acid defined in (6). The composition according to any one of claims 38 to 40.

42. The composition comprises: (i) a modified MOMP polypeptide having an amino acid sequence that includes two or more conserved domain sequences of a native Chlamydia species MOMP polypeptide and a non-native loop sequence between the conserved domain sequences; and / or (ii) one or more (e.g., 1, 2, 3, or 4) of the following: (a) a polypeptide comprising the amino acid sequence of a Chlamydia sp. CT443 polypeptide; (b) a polypeptide comprising the amino acid sequence of a Chlamydia sp. CT584 polypeptide; (c) a polypeptide comprising the amino acid sequence of a Chlamydia species CT600 polypeptide; or (d) a polypeptide comprising the amino acid sequence of a Chlamydia sp. CT812 polypeptide. Further comprising: Optionally, the composition comprises: The polypeptide of (i) and the polypeptide of (ii)(a); or The polypeptide of (i) and the polypeptide of (ii)(b); or The polypeptide of (i), the polypeptide of (ii)(a), and the polypeptide of (ii)(b) Including, For example, the composition comprises the polypeptide of (i), the polypeptide of (ii)(a), and the polypeptide of (ii)(b).

38. The composition of claim 37.

43. (1) the modified MOMP polypeptide is as defined in any one of claims 3 to 9, and / or (2) Any one of the Chlamydia spp. CT443 polypeptide, the Chlamydia spp. CT584 polypeptide, the Chlamydia spp. CT600 polypeptide, or the Chlamydia spp. CT812 polypeptide is as defined in claim 20 or 22.

43. The composition of claim 42.

44. The composition comprises: (1) a modified MOMP polypeptide comprising a sequence according to SEQ ID NO: 486, or a sequence having at least 70% (e.g., at least 90 or 95%) identity thereto; a chimeric Chlamydia sp. MOMP VD polypeptide comprising a sequence according to SEQ ID NO:503, or a sequence having at least 75% (e.g., at least 90 or 95%) identity thereto; and a Chlamydia sp. CT443 polypeptide comprising a sequence according to SEQ ID NO: 507 or a sequence having at least 75% (e.g., at least 90 or 95%) identity thereto; or (2) a modified MOMP polypeptide comprising a sequence according to SEQ ID NO: 486, or a sequence having at least 70% (e.g., at least 90 or 95%) identity thereto; a chimeric Chlamydia sp. MOMP VD polypeptide comprising a sequence according to SEQ ID NO:503 or a sequence having at least 75% (e.g., at least 90 or 95%) identity thereto; and a Chlamydia sp. CT584 polypeptide comprising a sequence according to SEQ ID NO: 510 or a sequence having at least 75% (e.g., at least 90 or 95%) identity thereto; or (3) a modified MOMP polypeptide comprising a sequence according to SEQ ID NO: 486, or a sequence having at least 70% (e.g., at least 90 or 95%) identity thereto; a chimeric Chlamydia sp. MOMP VD polypeptide comprising a sequence according to SEQ ID NO:503 or a sequence having at least 75% (e.g., at least 90 or 95%) identity thereto; A Chlamydia sp. CT443 polypeptide comprising a sequence according to SEQ ID NO:507 or a sequence having at least 75% (e.g., at least 90 or 95%) identity thereto; and CT584 polypeptide comprising a sequence according to SEQ ID NO:510 or a sequence having at least 75% (e.g., at least 90 or 95%) identity thereto. Including, For example, the composition comprises the polypeptide defined in (3).

44. The composition of claim 42 or 43.

45. A nucleic acid according to any one of claims 26 or 28 to 35, and (i) a nucleic acid comprising a nucleotide sequence encoding a modified MOMP polypeptide, wherein the modified MOMP polypeptide has an amino acid sequence comprising two or more conserved domain sequences of a native Chlamydia species MOMP polypeptide and a non-native loop sequence between the conserved domain sequences; and / or (ii) one or more (e.g., 1, 2, 3, or 4) of the following: (a) a nucleic acid comprising a nucleotide sequence encoding a Chlamydia sp. CT443 polypeptide; (b) a nucleic acid comprising a nucleotide sequence encoding a Chlamydia sp. CT584 polypeptide; (c) a nucleic acid comprising a nucleotide sequence encoding a Chlamydia sp. CT600 polypeptide; or (d) a nucleic acid comprising a nucleotide sequence encoding a Chlamydia sp. CT812 polypeptide; A combination comprising: Optionally, the combination comprises: the nucleic acid of (i) and the nucleic acid of (ii)(a); the nucleic acid of (i) and the nucleic acid of (ii)(b); or The nucleic acid of (i), the nucleic acid of (ii)(a), and the nucleic acid of (ii)(b) Including, For example, the combination includes the nucleic acid (i), the nucleic acid (ii)(a), and the nucleic acid (ii)(b), Optionally, the nucleic acid is according to claim 41 , Further optionally, the nucleic acids are present in the same composition (e.g., in the same composition). combination.

46. A polypeptide according to any one of claims 27 to 32, and (i) a modified MOMP polypeptide having an amino acid sequence that includes two or more conserved domain sequences of a native Chlamydia species MOMP polypeptide and a non-native loop sequence between the conserved domain sequences; and / or (ii) one or more (e.g., 1, 2, 3, or 4) of the following: (a) a polypeptide comprising the amino acid sequence of a Chlamydia sp. CT443 polypeptide; (b) a polypeptide comprising the amino acid sequence of a Chlamydia sp. CT584 polypeptide; (c) a polypeptide comprising the amino acid sequence of a Chlamydia species CT600 polypeptide; or (d) a polypeptide comprising the amino acid sequence of a Chlamydia sp. CT812 polypeptide. A combination comprising: Optionally, the combination comprises: The polypeptide of (i) and the polypeptide of (ii)(a); The polypeptide of (i) and the polypeptide of (ii)(b); or The polypeptide of (i), the polypeptide of (ii)(a), and the polypeptide of (ii)(b) Including, For example, the combination includes the polypeptide of (i), the polypeptide of (ii)(a), and the polypeptide of (ii)(b), Optionally, the polypeptide is as defined in claim 44, Further optionally, the polypeptides are present in the same composition or in two or more separate compositions. combination.

47. (i) the modified MOMP polypeptide further comprises a secretory signal peptide sequence; (ii) the chimeric MOMP VD polypeptide comprises a secretory signal peptide sequence, and / or (iii) the Chlamydia spp. CT443 polypeptide, the Chlamydia spp. CT584 polypeptide, the Chlamydia spp. CT600 polypeptide, and the Chlamydia spp. CT812 polypeptide comprise a secretory signal peptide sequence; optionally, the secretory signal peptide sequence is a viral secretory signal peptide sequence, optionally selected from the group consisting of an influenza hemagglutinin (HA) secretory signal peptide sequence, a SARS CoV-2 spike secretory signal peptide sequence, a VZV gB secretory signal peptide sequence, a VZV gE secretory signal peptide sequence, a VZV gI secretory signal peptide sequence, a VZV gK secretory signal peptide sequence, a measles F-protein secretory signal peptide sequence, a rubella E1 protein secretory signal peptide sequence, a rubella E2 protein secretory signal peptide sequence, a mumps F-protein secretory signal peptide sequence, an Ebola GP protein secretory signal peptide sequence, and a smallpox 6 kDa IC protein secretory signal peptide sequence; optionally, the secretory signal peptide sequence comprises an amino acid sequence according to one of the SEQ ID NOs in Table 2 or Table 2.1; For example, the secretory signal peptide sequence comprises the secretory signal peptide sequence of the HA protein of influenza A virus, for example, the secretory signal peptide sequence comprises a sequence according to SEQ ID NO: 187 or SEQ ID NO: 188; A nucleic acid according to any one of claims 2, 4 to 12, 26 or 28 to 35, a composition according to any one of claims 1, 4 to 12, 13 to 15, 17 to 23, 36 to 41, or a combination according to any one of claims 24 or 45.

48. (i) the modified MOMP polypeptide comprises a single amino acid substitution at one or more (e.g., all) positions corresponding to cysteine ​​residues in a conserved domain sequence of a native Chlamydia species MOMP polypeptide, and optionally, the single amino acid substitution is a cysteine ​​to serine substitution; (ii) the chimeric MOMP VD polypeptide comprises a single amino acid substitution at one or more (e.g., all) positions corresponding to cysteine ​​residues in a conserved domain sequence of a naturally occurring Chlamydia species MOMP polypeptide, optionally wherein the single amino acid substitution is a cysteine ​​to a serine; and / or (iii) one or more of the Chlamydia sp. CT443 polypeptide, the Chlamydia sp. CT584 polypeptide, the Chlamydia sp. CT600 polypeptide, or the Chlamydia sp. CT812 polypeptide comprises a single amino acid substitution at one or more (e.g., all) positions corresponding to a cysteine ​​residue in the respective native Chlamydia sp. polypeptide, and optionally the single amino acid substitution is a cysteine ​​to serine. A nucleic acid according to any one of claims 2, 4-12, 26, 28-35 or 47, a modified MOMP polypeptide according to any one of claims 3-9, a chimeric MOMP VD polypeptide according to any one of claims 27-32, a composition according to any one of claims 1, 4-23, 36-44 or 47, or a combination according to any one of claims 24, 25 or 45-47.

49. (i) the modified MOMP polypeptide comprises a mutation at one or more (e.g., all) positions corresponding to glycosylation sites, optionally N-glycosylation sites, of a native Chlamydia species MOMP polypeptide, and optionally the mutation is a single amino acid substitution; (ii) the chimeric MOMP VD polypeptide comprises mutations in a native Chlamydia species MOMP polypeptide at one or more (e.g., all) positions corresponding to glycosylation sites, optionally N-glycosylation sites, and optionally the mutations are single amino acid substitutions, e.g., the chimeric MOMP VD polypeptide comprises a single amino acid substitution at each of the amino acid residues corresponding to position 9 of SEQ ID NO:9 (e.g., an N to A substitution), position 11 of SEQ ID NO:17 (e.g., a T to A substitution), position 17 of SEQ ID NO:6 (e.g., an S to A substitution), positions 4 and 21 of SEQ ID NO:18 (e.g., an N to A substitution and an S to A substitution, respectively), position 14 of SEQ ID NO:8 (e.g., a T to A substitution), and position 14 of SEQ ID NO:16 (e.g., a T to A substitution); and / or (iii) one or more of the Chlamydia sp. CT443 polypeptide, the Chlamydia sp. CT584 polypeptide, the Chlamydia sp. CT600 polypeptide, or the Chlamydia sp. CT812 polypeptide comprises a mutation at one or more (e.g., all) positions corresponding to N-glycosylation sites in the respective native Chlamydia sp. polypeptide, optionally wherein the mutation is a single amino acid substitution, e.g., the Chlamydia sp. CT584 polypeptide comprises a single amino acid substitution (e.g., an N to Q substitution) at a position corresponding to residue 11 of SEQ ID NO:509; A nucleic acid according to any one of claims 2, 4-12, 26, 28-35, 47 or 48, a modified MOMP polypeptide according to any one of claims 3-9 or 48, a chimeric MOMP VD polypeptide according to any one of claims 27-32 or 48, a composition according to any one of claims 1, 4-23, 36-44, 47 or 48, or a combination according to any one of claims 24, 25 or 45-48.

50. CT443 polypeptide, the Chlamydia spp. CT584 polypeptide, the Chlamydia spp. CT600 polypeptide, or the Chlamydia spp. CT812 polypeptide comprise a heterologous transmembrane domain, and optionally the transmembrane domain sequence is selected from the group consisting of an influenza hemagglutinin (HA) transmembrane domain sequence, a SARS CoV-2 spike transmembrane domain sequence, a VZV gB transmembrane domain sequence, a VZV gE transmembrane domain sequence, a VZV gI transmembrane domain sequence, a VZV gD ... 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, and Ebola GP protein transmembrane domain sequence, optionally said transmembrane domain comprises an amino acid sequence according to one of the SEQ ID NOs in Table 3, for example said transmembrane domain comprises the sequence of the transmembrane domain of the HA protein of influenza A virus, optionally said transmembrane domain comprises a sequence according to SEQ ID NO: 813 or SEQ ID NO: 814; A composition according to any one of claims 1, 4 to 23, 36 to 44 or 47 to 49, or a combination according to any one of claims 24, 25 or 45 to 49.

51. The composition of any one of claims 1, 4 to 15, 17 to 23, and 36 to 41, wherein the composition further comprises a lipid nanoparticle (LNP), and optionally the nucleic acid is encapsulated in the LNP.

52. The LNPs comprise at least one cationic lipid, and optionally (i) the cationic lipid is selected from the group consisting of OF-02, cKK-E10, OF-Deg-Lin, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4-E10, GL-HEPES-E3-E12-DS-3-E14, SM-102, ALC-0315, ATX-126, and IM-001 (e.g., GL-HEPES-E3-E12-DS-4-E10 or IM-001); and / or (ii) the LNP further comprises a polyethylene glycol (PEG)-conjugated (PEGylated) lipid, a cholesterol-based lipid, and a helper lipid, and optionally the (PEGylated) lipid is DMG-PEG2000 or ALC-0159 (e.g., DMG-PEG2000); the cholesterol-based lipid is cholesterol, and / or The helper lipid is DOPE or DSPC; 53. The composition of claim 52.

53. The LNP is (i) 40% molar ratio of OF-02; 1.5% molar ratio of DMG-PEG2000; 28.5% molar ratio of cholesterol; and 30% molar ratio of DOPE; (ii) 40% molar ratio of cKK-E10; 1.5% molar ratio of DMG-PEG2000; 28.5% molar ratio of cholesterol and 30% molar ratio of DOPE; (iii) GL-HEPES-E3-E10-DS-3-E18-1 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%. (iv) GL-HEPES-E3-E12-DS-4-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%. (v) GL-HEPES-E3-E12-DS-3-E14 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%. (vi) 50% molar ratio of DLin-MC3-DMA (MC3), 1.5% molar ratio of DMG-PEG2000, 38.5% molar ratio of cholesterol, and 10% molar ratio of DSPC; (vii) 40% molar ratio of IM-001, 1.5% molar ratio of DMG-PEG2000, 28.5% molar ratio of cholesterol, and 30% molar ratio of DOPE; (viii) 50% molar ratio of SM-102, 1.5% molar ratio of DMG-PEG2000, 38.5% molar ratio of cholesterol, and 10% molar ratio of DSPC; (ix) 46.3% molar ratio of ALC-0315, 1.6% molar ratio of ALC-0159, 42.7% molar ratio of cholesterol, and 9.4% molar ratio of DSPC; (x) 47.4% molar ratio of ALC-0315, 1.7% molar ratio of ALC-0159, 40.9% molar ratio of cholesterol, and 10% molar ratio of DSPC, or (xi) 50% molar ratio of ATX-126, 1.5% molar ratio of DMG-PEG2000, 38.5% molar ratio of cholesterol, and 10% molar ratio of DSPC Including, For example, the LNP is as defined in (i), (ii), (iv) or (vii), for example, the LNP is as defined in (iv) or (vii); 53. The composition of claim 51 or 52.

54. 10. A nucleic acid according to any one of claims 2, 4 to 12, 26, 28 to 35 or 47 to 49, a modified MOMP polypeptide according to any one of claims 3 to 9, 48 or 49, a chimeric MOMP VD polypeptide according to any one of claims 27 to 32, 48 or 49, a composition according to any one of claims 1, 4 to 23, 36 to 44 or 47 to 53, or a combination according to any one of claims 24, 25 or 45 to 50 for use as a medicament.

55. 50. The nucleic acid of any one of claims 2, 4-12, 26, 28-35 or 47-49, the modified MOMP polypeptide of any one of claims 3-9, 48 or 49, the chimeric MOMP VD polypeptide of any one of claims 27-32, 48 or 49, the composition of any one of claims 1, 4-23, 36-44 or 47-53, or the combination of any one of claims 24, 25 or 45-50, for use in treating or preventing a Chlamydia species infection, preferably wherein the infection is a C. trachomatis infection, such as a C. trachomatis genital infection.

56. 52. A vaccine comprising a nucleic acid of any one of claims 2, 4-12, 26, 28-35, or 47-49, a modified MOMP polypeptide of any one of claims 3-9, 48, or 49, a chimeric MOMP VD polypeptide of any one of claims 27-32, 48, or 49, a composition of any one of claims 1, 4-23, 36-44, or 47-53, or a combination of any one of claims 24, 25, or 45-50.

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