The structure of the chlamydia major outer membrane protein, and applications thereof

Abstract

Provided is the determination of the three-dimensional structure of Chlamydia major outer membrane protein and applications and uses thereof, including for vaccine development, drug development, and diagnostics.

Description

Attorney docket No.00058-089WO1 THE STRUCTURE OF THE CHLAMYDIA MAJOR OUTER MEMBRANE PROTEIN, AND APPLICATIONS THEREOF CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. §119 from Provisional Application Serial No.63 / 708,212, filed October 16, 2024, the disclosure of which is incorporated herein by reference. STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with Government support under Grant Nos: R01AI032248, R01AI030499, U19AI144184, 75N93022C00035 and R35GM145365, awarded by the National Institutes of Health. The Government has certain rights in the invention. INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0003] Accompanying this filing is a Sequence Listing entitled, “00058-089WO1.xml” created on October 16, 2025, and having 58,024 bytes of data, machine formatted on IBM- PC, MS-Windows operating system. The sequence listing is hereby incorporated by reference in its entirety for all purposes. TECHNICAL FIELD

[0004] Provided is the determination of the structure of Chlamydia major outer membrane protein and application and uses thereof, including for vaccine development, drug development, and diagnostics. BACKGROUND

[0005] Chlamydia trachomatis (Ct) is the most common sexually transmitted bacterial infection (STI) 40 worldwide, with ~150 million people infected in 2023. Although Ct STIs are treatable with antibiotics, up to 80% of infected women and 50% of infected men are asymptomatic, leading to undiagnosed cases and continued transmission. In women, acute and chronic Ct infections can cause serious complications, including infertility. In developing nations, Ct ocular infections cause trachoma and preventable blindness. Chlamydia pneumoniae is also a human pathogen that infects the respiratory tract. Zoonotic infections by other chlamydial species, such as Chlamydia psittaci, can be transmitted to humans and threaten animals, e.g. koalas are an endangered species due to Chlamydia pecorum genital infections. The high prevalence and health and environmental burden of chlamydial infections drive vaccines development efforts.Attorney docket No.00058-089WO1 SUMMARY

[0006] Chlamydia trachomatis is the leading cause of preventable blindness and the most common sexually transmitted bacterial infection worldwide, often resulting in infertility. Genitourinary Chlamydia infections are frequently asymptomatic, delaying treatment and increasing transmission rates as well as long-term complications. The Major Outer Membrane Protein (MOMP), the most abundant protein in the chlamydial outer membrane, is a key immunodominant antigen and a promising subunit vaccine candidate. Natively folded MOMP elicits the strongest immune response. To understand MOMP's immunogenicity, cryo-EM structures of native Chlamydia muridarum MOMP from the infectious elementary body (EB) and its complex with a neutralizing monoclonal antibody Fab fragment, mAb-18b, were determined. Native MOMP forms a homotrimer of ten-stranded β-barrels, with an extracellular, tightly folded globular cap formed by immunodominant variable domains (VDs) from all three protomers. In the MOMP-Fab complex, mAb-18b engages three VDs from two protomers. The structural reorganization of these VDs upon Fab binding reveals the molecular basis for neutralizing infectious EBs. These structures will guide subunit vaccine design. MOMP’s topology, cysteine residue positioning, and impermeable structure will prompt a critical reassessment of models and data collected over the past 40 years.

[0007] In a particular embodiment, the disclosure provides an artificial or recombinant construct that expresses a polypeptide sequence for an antigenic head portion of a Chlamydia Major Outer Membrane Protein (MOMP) in its three-dimensional conformation. In another embodiment, the antigenic head portion is from Chlamydia pneumoniae, Chlamydia muridarum, Chlamydia avium, Chlamydia buteonis, Chlamydia caviae, Chlamydia crocodili, Chlamydia felis, Chlamydia gallinacea, Chlamydia poikilotherma, Chlamydia abortus, Chlamydia psittaci, Chlamydia serpentis, Chlamydia suis, Chlamydia pecorum, or Chlamydia trachomatis. In a further embodiment, the antigenic head portion is from Chlamydia trachomatis or Chlamydia pneumoniae. In yet a further embodiment, the antigenic head portion is from a serovar of Chlamydia trachomatis that is selected from A, B / Ba, C, D / Da, E, F, G / Ga, H, I / Ia, J, K, L1, L2, L2a and L3. In a certain embodiment, the Chlamydia trachomatis serovar is selected from A, E, F, D / Da, J, L2, and G / Ga. In another embodiment, the artificial or recombinant construct additionally expresses polypeptide sequences for the β-barrel transmembrane portion and β-barrel extracellular collar of a Chlamydia MOMP. In yet another embodiment, the β-barrel transmembrane portion and β-Attorney docket No.00058-089WO1 barrel extracellular collar is from Chlamydia muridarum. In a further embodiment, the polypeptide sequences for the β-barrel transmembrane portion and / or β-barrel extracellular collar of a Chlamydia MOMP comprises one or more substitutions of non-cysteine amino acids to cysteines to stabilize the Chlamydia MOMP by the formation of disulfide bridges. In yet a further embodiment, the polypeptide sequences for the β-barrel transmembrane portion and / or β-barrel extracellular collar of a Chlamydia MOMP have proline substitutions for non- proline amino acids, wherein the amino acid side chains from the surrounding amino acids have accommodating or favorable interactions with the substituted proline amino acids, and wherein the β-barrel transmembrane portion and / or β-barrel extracellular collar has consistent main chain angles of what is expected for prolines. In a certain embodiment, the polypeptide sequences for the β-barrel and its extracellular collar have been modified or changed so that the expressed polypeptide has increased solubility in aqueous medium while still retaining the antigenic head portion of a Chlamydia MOMP in its three-dimensional conformation. In another embodiment, the artificial or recombinant construct has a polypeptide sequence that is at least 95% identical to SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30. In yet another embodiment, the artificial or recombinant construct has a polypeptide sequence that is at least 98% identical to SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30. In a further embodiment, the artificial or recombinant construct has a polypeptide sequence that is at least 98% identical to SEQ ID NO:28 or SEQ ID NO:29. In yet a further embodiment, the polypeptide sequence for the β-barrel transmembrane portion has been modified or changed so that the β-barrel transmembrane portion has been reduced or truncated. In another embodiment, the artificial or recombinant construct has a polypeptide sequence that is at least 95% identical to SEQ ID NO:30 or SEQ ID NO:31. In yet another embodiment, the artificial or recombinant construct has a polypeptide sequence that is at least 98% identical to SEQ ID NO:30 or SEQ ID NO:31. In a further embodiment, the artificial or recombinant construct additionally expresses a polypeptide sequence for an artificial scaffold that replaces the β-barrel transmembrane portion of a Chlamydia MOMP. In yet a further embodiment, the artificial scaffold is derived from a three-helix bundle, a transmembrane portion of a porin protein, a lipocalin, or a transmembrane portion of a cell adhesion protein. In a certain embodiment, the artificial scaffold is derived from a polypeptide sequence for a lipocalin. In another embodiment, the artificial or recombinant construct has a polypeptideAttorney docket No.00058-089WO1 sequence that is at least 95% identical to SEQ ID NO:33. In yet another embodiment, the artificial or recombinant construct has a polypeptide sequence that is at least 98% identical to SEQ ID NO:33. In a further embodiment, the artificial scaffold is a zinc-finger scaffold that uses zinc-cysteine coordination for stabilization. In yet a further embodiment, the zinc-finger scaffold is based on a C2H2-zinc finger carrying motif. In another embodiment, the C2H2- zinc finger carrying motif is selected from KLF1, KLF8, KLF11, ZEB2, GLI1, IKZFI, and CTCF. In yet another embodiment, the artificial scaffold provides multiple 3-fold axes to create more complex arrangements. In a further embodiment, the multiple 3-fold axes provide for tetrahedral, octahedral or icosahedral symmetries. In yet a further embodiment, the antigenic head portion of a Chlamydia MOMP has been stabilized by domain swapping, wherein the domain swapping can be in the polypeptide sequence for the antigenic head portion of a Chlamydia MOMP, or in some other polypeptide sequence of the artificial or recombinant construct, so long as the antigenic head portion of a Chlamydia MOMP is presented in its three-dimensional conformation. In another embodiment, the artificial or recombinant construct expresses a plurality of polypeptide sequences for multiple antigenic head portions of a Chlamydia MOMP. In yet another embodiment, the multiple antigenic head portions are from different species of Chlamydia MOMPs. In a further embodiment, at least one of the species of Chlamydia is Chlamydia trachomatis or Chlamydia pneumoniae. In yet a further embodiment, the multiple antigenic head portions are from different serovars of Chlamydia trachomatis. In another embodiment, the different serovars of Chlamydia trachomatis are selected from A, E, F, D / Da, J, L2, and G / Ga. In yet another embodiment, the artificial or recombinant construct additionally expresses a polypeptide sequence for a periplasmic loop of a Chlamydia MOMP with / or without the N-terminal stopper segment removed. In a further embodiment, the artificial or recombinant construct additionally expresses another polypeptide sequence for the N-terminal stopper segment extension of the periplasmic loop. In yet a further embodiment, the recombinant or artificial construct further expresses a polypeptide sequence for a T cell epitope.

[0008] In a particular embodiment, the disclosure also provides a vaccine preparation or formulation that comprises the artificial or recombinant construct disclosed herein. In another embodiment, the artificial or recombinant construct comprises polynucleotides that encode the polypeptide sequence for an antigenic head portion of a Chlamydia MOMP in its three- dimensional conformation, and wherein the vaccine preparation or formulation is aAttorney docket No.00058-089WO1 polynucleotide-based vaccine. In yet another embodiment, the polynucleotides comprise DNA, RNA, modified RNA, or hybrids of DNA and RNA. In a further embodiment, liposomes, virus like particles or polymeric nanoparticles are used as a delivery vehicle for the polynucleotides that encode the polypeptide sequence for the antigenic head portion of a Chlamydia MOMP, and wherein the polynucleotides are found within the delivery vehicle as cargo, or on the surface of the delivery vehicle, or both. In yet a further embodiment, the vaccine preparation or formulation further comprises one or more adjuvants. In a certain embodiment, the one or more adjuvants are selected from a CpG oligodeoxynucleotide based adjuvant, ssRNA, an aluminum salt, Montanide ISA 720, CAF01, AS04, MF59, AS01B, and / or CpG 1018.

[0009] In a particular embodiment, the disclosure further provides a vaccine preparation or formulation which comprises an antigenic head portion of a Chlamydia Major Outer Membrane Protein (MOMP) that is selected from Chlamydia pneumoniae, Chlamydia muridarum, Chlamydia avium, Chlamydia buteonis, Chlamydia caviae, Chlamydia crocodili, Chlamydia felis, Chlamydia gallinacea, Chlamydia poikilotherma, Chlamydia abortus, Chlamydia psittaci, Chlamydia serpentis, Chlamydia suis, Chlamydia pecorum, or Chlamydia trachomatis in its three-dimensional conformation. In another embodiment, the antigenic head portion of the Chlamydia MOMP is from Chlamydia trachomatis or Chlamydia pneumoniae. In yet another embodiment, the Chlamydia MOMP is from a Chlamydia trachomatis serovar selected from A, B / Ba, C, D / Da, E, F, G / Ga, H, I / Ia, J, K, L1, L2, L2a and L3. In a further embodiment, the MOMP is from a Chlamydia trachomatis serovar selected from A, E, F, D / Da, J, L2, and G / Ga. In a certain embodiment, liposomes, virus like particles or polymeric nanoparticles are used as a delivery vehicle for the antigenic head portion of the Chlamydia MOMP, and wherein the antigenic head portion of the Chlamydia MOMP is found within the delivery vehicle as cargo, or on the surface of the delivery vehicle, or both. In another embodiment, the vaccine preparation or formulation further comprises one or more adjuvants. In yet another embodiment, the one or more adjuvants are selected from a CpG oligodeoxynucleotide based adjuvant, ssRNA, an aluminum salt, Montanide ISA 720, CAF01, AS04, MF59, AS01B, and / or CpG 1018.

[0010] In a particular embodiment, the disclosure provides a vaccine preparation or formulation which comprises an antigenic head portion of a Chlamydia MOMP expressed from the artificial or recombinant construct of the disclosure. In a further embodiment,Attorney docket No.00058-089WO1 liposomes, virus like particles or polymeric nanoparticles are used as a delivery vehicle for the antigenic head portion of the Chlamydia MOMP, and wherein the antigenic head portion of the Chlamydia MOMP is found within the delivery vehicle as cargo, or on the surface of the delivery vehicle, or both. In yet a further embodiment, the vaccine preparation or formulation further comprises one or more adjuvants. In another embodiment, the one or more adjuvants are selected from a CpG oligodeoxynucleotide based adjuvant, ssRNA, an aluminum salt, Montanide ISA 720, CAF01, AS04, MF59, AS01B, and / or CpG 1018.

[0011] In a particular embodiment, the disclosure provides a method of immunizing a subject to protect against an infection by a Chlamydia bacterium, comprising: administering one or more therapeutically effective doses of the vaccine preparation or formulation disclosed herein to a subject. In another embodiment, multiple doses of the vaccine preparation or formulation are administered to the subject at different time points. In yet another embodiment, the mode of administration is selected from intravenous administration, intraperitoneal administration, intramuscular administration, intracoronary administration, intraarterial administration, subcutaneous administration, transdermal delivery, intratracheal administration, subcutaneous administration, intraarticular administration, intraventricular administration, inhalation, nasal, oral, pulmonary administration, impregnation of a catheter, and direct injection into a tissue.

[0012] In a particular embodiment, the disclosure further provides a method of detecting or monitoring an infection by a Chlamydia bacterium in a subject, comprising: contacting a sample from the subject with a diagnostic agent comprising an antigenic head portion of a Chlamydia MOMP in its three-dimensional conformation, wherein if the subject has antibodies to the antigenic head portion of a Chlamydia MOMP is indicated by detectable change in the diagnostic agent, and wherein if the subject does not have antibodies to the antigenic head portion of a Chlamydia MOMP results in no change in the diagnostic agent.

[0013] In a certain embodiment, the disclosure provides for a composition or a method as substantially described in the disclosure and figures presented herein. DESCRIPTION OF DRAWINGS

[0014] The accompanying drawings, which are incorporated into and constitute a part of this specification, illustrate one or more embodiments of the disclosure and, together with the detailed description, serve to explain the principles and implementations of the invention.Attorney docket No.00058-089WO1

[0015] FIG.1 shows serological variants of Chlamydia trachomatis, with the serovars selected for the studies presented herein: serotypes that are circled (MOMP structure available) and serotypes that are boxed, with EB-neutralizing antibodies targeting MOMP indicated by light gray text.

[0016] FIG.2 demonstrates the isolation and purification of Cm COMC (left) and native Cm MOMP (right). Monomeric and trimeric MOMP migrate anomalously on SDS-PAGE and are resistant to SDS-induced denaturation. Only a combination of SDS and heat fully denatures the protein. This feature provides validation of sample quality. If trimers are not observed at ~60 kDa, it means that the protein is not properly folded. It is expected that this feature will be preserved in any recombinantly produced MOMP. The purity of COMC / MOMP preparations obtained with the protocols presented herein was excellent.

[0017] FIG.3A-G presents a Cryo-EM structure of the Cm EB MOMP trimer. (A) Schematic representation of the MOMP protomer sequence, with VDs labeled. (B) Molecular density map of MOMP at a contour level of 0.2 (arbitrary units dependent on software- specific scaling). (C) The atomic model of the MOMP trimer, colored by chain (gray, light gray, dark gray), with side, periplasmic, and extracellular views and their dimensions labeled. (D) Detailed view of the MOMP protomer. The axis of the barrel is indicated by a grey dashed line. The extracellular domain includes ordered loops (circle 1), two β-strands exposed to the solvent (circle 2), two β-strands buried beneath the extracellular loops (circle 3), three β-strands at the trimerization interface (circle 4), and two β-strands extending above the membrane into the extracellular space at the trimer interface (circle 5). The transmembrane domain consists of a 10-stranded β-barrel with a shear number of 12 (circle 6). The cysteine-rich periplasmic region contains short β-turns and a partially disordered loop (circle 7). (E) Detailed views of selected β-strands along the barrel. Top panel: interior and exterior sides of the extracellular β-sheets at the trimer interface (circles 4 and 5 in panel D). Residues with 963 side chains facing the barrel's interior are mostly non-polar, while those facing the exterior are 964 mostly polar. Bottom panel: interior and exterior sides of the transmembrane β-sheets (circle 6 in panel D). Residues with side chains facing the barrel's interior are mostly polar, while those facing the exterior are mostly non-polar. (F) Arrangement of VDs on the extracellular side (VD1: dark gray; VD2: medium gray; VD3: light gray; VD4: gray). One protomer is highlighted in tube representation, while the other two are shown as ribbon diagrams. Interactions between VDs are highlighted in boxes 1-5.Attorney docket No.00058-089WO1 (G) Surface electrostatic potential of MOMP and charge distribution inside the β-barrel, showing that the interior is polar, with electronegative character in the transmembrane portion of the barrel interior.

[0018] FIG.4 shows the alignment of C. muridarum, C. trachomatis serovars, C. pecorum, C. pneumoniae, and C. psittaci MOMP sequences: C_muridarum / 1-365 (SEQ ID NO:34), Ct_A_Har-13 / 1-374 (SEQ ID NO:35), Ct_B_JAL120 / 1-372 (SEQ ID NO:36), Ct_Ba_Aus28 / 1-372 (SEQ ID NO:37), Ct_C_TW-3 / 1-375 (SEQ ID NO:38), Ct_D_UW-3 / 1- 371 (SEQ ID NO:39), Ct_E_Bour / 1-371 (SEQ ID NO:40), Ct_F_SW4 / 1-373 (SEQ ID NO:41), Ct_G_11074 / 1-373 (SEQ ID NO:42), Ct_H_H_R319754 / 1-375 (SEQ ID NO:43), Ct_la_CS190 / 96 / 1-375 (SEQ ID NO:44), Ct_J_31-98 / 1-375 (SEQ ID NO:45), Ct_K_Soton / 1-375 (SEQ ID NO:46), Ct_L1_440 / 1-371 (SEQ ID NO:47), Ct_L2_434 / 1-372 (SEQ ID NO:48), Ct_L3_404 / 1-375 (SEQ ID NO:49), C_percorum_DBDeUG / 1-367 (SEQ ID NO:50), C_pneumoniae_CM-1 / 1-366 (SEQ ID NO:51), and C_psitacci_6BC / 1-380 (SEQ ID NO:52). The alignment illustrates per-residue conservation of Cm MOMP, the locations of variable domains (VDs), and the TTLNPTIAG (SEQ ID NO: 9) motif in VD4. High to low per-residue conservation is colored from dark gray to white. Conserved cysteine residues are labeled with gray stars.

[0019] FIG.5A-H presents selected Cm EB MOMP cryo-EM data processing results. (A) Motion-corrected micrograph from the MOMP dataset, with particles labeled with white circles. (B) Selected 2D classes from the MOMP reconstruction. (C) Fourier Shell Correlation (FSC) curves for the final MOMP reconstruction. (D) MOMP map colored according to local resolution, ranging from 2.5 Å to 4.5 Å. The left and right panels show a high-contour map (level 0.2), while the middle panel displays a low-contour map (level 0.12) to indicate the location of the detergent lipid bilayer. (E) Motion-corrected micrograph from the MOMP / mAb-18b complex dataset. (F) Selected 2D classes from the MOMP / mAb-18b complex reconstruction. (G) FSC 1079 curves for the final MOMP / mAb-18b complex reconstruction. (H) Local resolution, ranging from 10802.5 Å to 7.0 Å, for the MOMP / mAb- 18b complex map. A low contour map is displayed 1081 in the middle panel to show the location of the detergent lipid bilayer (contour level 0.1).

[0020] FIG.6A-E presents features of the EB MOMP β-barrel interior. (A) External dimensions of the β-barrel. (B) Arrangement of the N-terminal stopper segment (cyan) inside the barrel. (C) Interactions between the residues of the N-terminal stopper segment (cyan)Attorney docket No.00058-089WO1 and the side chains of residues lining the barrel interior (gray) with selected hydrogen bonds labeled. (D) Tunnels inside of the MOMP protomer, calculated using Caver with a probe radius of 0.9 Å. Each tunnel is represented by a different color. (E) Radii of the β-barrel accessible volume mapped along each tunnel.

[0021] FIG.7 presents a topological diagram of the 10-stranded β-barrel model of Cm EB MOMP (SEQ ID NO:1). The diagram was created by calculating the angular and vertical position of residues along an axis running through the β-barrel lumen and translating those positions into X and Y coordinates in R, creating an “unrolled” 2D depiction. Residues are color coded by region (VD1: dark gray, VD2: medium gray, VD3: pale gray, VD4: gray with TTWNPTISG (SEQ ID NO:10) (Ct TTLNPTIAG (SEQ ID NO:11) motif highlighted, transmembrane region: shades of orange (light-medium gray). Aromatic girdles residues are highlighted in light gray. It is suggested that these girdles prevent conformational damage of the protein due to mechanical movements of the membrane. Cysteine residues are highlighted in gray. Secondary structures are represented by shapes (β-strand: square, end of β-strand: triangle, α-helix: oval, loop: circle). The positions of N- and C- termini and cysteine residues outside of the transmembrane region are labeled.

[0022] FIG.8A-O presents molecular density maps (unsharpened) and fits of selected structural segments of the Cm EB MOMP model. The EB MOMP protomers are represented in gray, light gray, and dark gray, with ligands shown in light-medium gray. Hydrogen bond distances are indicated by black dashed line, while hydrophobic and van der Waals interactions are depicted as thicker gray lines. Panel (A) shows the density of the less ordered periplasmic loop. Panel (B) displays the density of a sphingomyelin molecule located at the interface between two MOMP protomers. Panel (C) illustrates the Mg2+binding site and its density map. Panels (D) and (E) provide two views of the lauric acid (DAO) and N- acetylglucosamine (NAG) molecules in their binding sites. Selected amino acid residues from the extracellular sequence segments are shown in panels (C)-(E) and (F)-(K). Selected residues from the transmembrane regions are presented in panels (B) and (L)-(N). The maps of the periplasmic regions are shown in panels (A) and (O).

[0023] FIG.9A-E presents the location of cysteine residues in the EB MOMP structure. (A) Positions of cysteine residues mapped on the trimer and the protomer structures. (B)-(E) Molecular density maps showing the placement of cysteine residues and their lack ofAttorney docket No.00058-089WO1 participation in disulfide bridges. The density maps for C26, C29, C33 are presented in FIG. 8A.

[0024] FIG.10A-G presents a Cryo-EM structure of the Cm EB MOMP / mAb-18b complex. (A) Molecular density map of the MOMP / mAb-18b complex (contour level 0.15). (B) Atomic model of the MOMP / mAb-18b complex, colored by chain (MOMP: gray, light gray, very dark gray; mAb-18b Fab light chain: pale gray, medium gray, very light gray; heavy chain: light blue, black, dark gray). (C) Extracellular view of the MOMP / mAb-18b complex. For clarity, only one unit of the MOMP / mAb-18b complex is shown (MOMP: gray; mAb-18b light chain: medium gray; mAb-18b heavy chain: light gray). (D) Schematic of MOMP / mAb-18b extracellular view showing one unit of mAb-18b Fab interacting with two MOMP protomers. (E) Structural changes in MOMP upon mAb-18b binding. For clarity, the main chain RMSD is mapped onto one MOMP protomer in tube representation, while the other two protomers are displayed as a gray ribbon to illustrate the structural changes within the MOMP trimer. Significant changes are observed at VD1 (dashed box 1) and VD4 (dashed box 2). (F) Superposition of Fab-bound MOMP (gray, light gray and dark gray) and unbound MOMP (gray). One protomer is highlighted in tube representation, with the other two protomers shown as ribbon diagram. Dashed boxes 1 and 2 indicate the regions with the most substantial changes, as in panel E. (G) Top panel: Close-up view of VD1 in MOMP (gray) and in MOMP / mAb-18b complex (medium gray), showing the largest change at residue Asp71, with a Cα distance of ~7.7 Å. Bottom panel: Close-up view of VD4 in MOMP (gray) and in MOMP / mAb-18b complex (medium gray) showing the largest shift at residue T307, with a Cα distance of ~11.7 Å.

[0025] FIG.11 demonstrates the predicted interactions between MOMP, OmcA, and OmcB. A top-down view of a protein complex predicted by AlphaFold 3, consisting of one MOMP trimer (light gray) bound to three copies of OmcA (gray) and OmcB (dark gray). The highly cysteine-rich proteins OmcA and OmcB are predicted to interact and form disulfide bonds with the periplasmic side of the MOMP trimer. OmcB forms an elongated star-shaped trimer, enabling it to form a cross-linked protein mesh in the periplasm.

[0026] FIG.12A-E demonstrates the RMSD between Cm EB MOMP and MOMP / mAb- 18b structures. (A) Secondary structure of EB MOMP with VDs highlighted. (B) The RMSD for main chain atoms (gray circles) is plotted for each MOMP residue. The VDs of MOMP are highlighted in boxes (VD1: medium to dark gray, VD2: dark gray, VD3: light gray, VD4:Attorney docket No.00058-089WO1 gray). The most significant changes are present in VD1 and VD4. The RMSD spike for amino acid residues between positions 20 and 30 is due to the flexibility of the partially disordered periplasmic loop, not due to Fab binding. (C) Density map of the VD1 region in the MOMP / mAb-18b complex. (D) Density map of the VD4 region in the MOMP / mAb-18b complex. (E) Density map of the VD4 region in the MOMP / mAb-18b complex, showing that the D311 position is not compatible with the coordination of the Mg2+cation.

[0027] FIG.13A-I provides for Cm EB MOMP and mAb-18b Fab binding interface. (A) MOMP residues involved in mAb-18b Fab binding are shown as buried surface area mapped onto one MOMP protomer. The other two MOMP protomers are displayed as ribbon diagrams. A surface representation of VDs in the same view is shown in the dashed box as a reference. (B) mAb-18b Fab residues involved in MOMP binding are shown as buried surface area mapped onto one Fab molecule (one light chain and one heavy chain). The other two Fab molecules are displayed as ribbon diagrams. Light chain residues (H36, S37, and A38) are marked with a black dashed line, while heavy chain residues are marked with a gray solid line. (C) Overall binding interface between MOMP and mAb-18b. Left panel: two MOMP protomers (gray and light gray) and one mAb-18b Fab molecule (light chain: dark gray; heavy chain: medium gray) involved in binding. VDs of MOMP and CDRs of mAb- 18b Fab are labeled (light chain CDRs: L1, L2, L3; heavy chain CDRs: H1, H2, H3) (see Table 3). Right panel: epitopes and paratopes are shown as spheres. Dark gray spheres represent MOMP residues in direct contact with mAb-18b (epitopes), while very dark gray spheres are mAb-18b residues in direct contact with MOMP (paratopes). (D)-(I) Detailed view of interactions between MOMP and mAb-102318b through VDs and CDRs (MOMP: gray, light gray; Fab light chain: medium gray; Fab heavy chain: light to medium gray). Main chain atoms are omitted for clarity.

[0028] FIG.14A-D provides structural analysis of species-specific conserved epitopes in VD4: Cm TTWNPTISG (SEQ ID NO:10) and Ct TTLNPTIAG (SEQ ID NO:11). (A) CTH522 extVD4 mapped onto the Cm MOMP (± Fab) structures and its involvement in the N-acetylglucosamine (NAG) and lauric acid (DAO) binding sites. (B) CTH522 extVD4 mapped on the MOMP monomer. (C) CTH522 extVD4 mapped onto the MOMP antigenic cap. MOMP structures with and without Fab are superimposed to highlight Fab-induced changes in the CTH522 extVD4 region. (D) Evolutionary conservation of VDs across all Chlamydia species, as determined by ConSurf. The left panel shows conservation mappedAttorney docket No.00058-089WO1 onto the trimer; the middle panel shows conservation patterns in the antigenic cap, which mediates host cell adhesion; and the right panel shows the locations of the VDs on the antigenic cap in relation to the conservation pattern.

[0029] FIG.15 demonstrates the interactions between the VDs in the antigenic head and the locations of ligand-binding sites. Left panel: extracellular view of VDs and ligand binding sites in MOMP alone structure. Right panel: side view of VDs and ligand binding sites in MOMP (± Fab) structures.

[0030] FIG.16A-C shows that linear B- and T-cell epitopes for Ct MOMP are differently distributed between non-conserved and conserved regions of the Ct MOMP sequence. The log ratio of positive to negative epitope assays for Ct serovar D reported per- residue in the IEDB is mapped onto a homology model of Ct serovar D MOMP. (A) Mapped B-cell epitopes. (B) Mapped T-cell epitopes. (C) The log ratios visualized in panels A and B plotted as a function of sequence position for B-cell (black) and T-cell (dark blue) epitopes. B-cell epitopes are more abundant in the non-conserved VDs (shown in dark gray, very dark gray, light gray, and gray), while T-cell epitopes are more prevalent in the conserved β-barrel, at the trimer interface, and in VD3. This is expected since B-cell epitopes are usually located on the surface exposed regions of pathogens while T-cell epitopes are in antigenic domains that are conformationally stable.

[0031] FIG.17A-B presents cryoEM-SPR data. (A) Fragmented COMC from Ct serovar J. (B) 2D class averages from cryoEM-SPR analysis of this sample. A hexagonal network is clearly visible, including lattice defects.

[0032] FIG.18A-H presents images of compact nucleoids, asymmetric arrays of the T3SS, and intermediate developmental forms. (A) EB with nucleoid marked by a white cross; (B) EB with an asymmetric distribution of T3SS complexes; note the slightly larger size and rearranged mem-branes; (C) Ordered molecular pattern on the EB surface; interparticle distances are consistent with MOMP; (D) Ordered pattern across the outer membrane; the distances and density sizes are consistent with a MOMP trimer; (E) Early EB with a “fluffy” outer membrane, well-separated leaflets of both membranes, and COMC densities located in between; (F) Class averages illustrating the quality of subtomogram averaging; (G) Reconstruction without symmetry (C1); (H) Reconstruction with threefold symmetry (C3).Attorney docket No.00058-089WO1

[0033] FIG.19 demonstrates differences in electrostatic potential (top row), hydrophobicity (bottom row), and surface shape of the antigenic head that interacts with the host cell.

[0034] FIG.20 demonstrates differences in two reconstructions of serovar F, one in the presence of antibody (left) and another without the antibody (right).

[0035] FIG.21 provides exemplary soluble MOMP constructs where the overall fold was preserved, or where an antigenic head was grafted onto the constructs. The antigenic head can maintain its structure in trimeric and circular constructs, as well as when grafted onto modified sequence of human fatty acid binding protein 4 (FABP4), as one of many possible scaffolds.

[0036] FIG.22 presents the polypeptide sequence for the CM_MOMP_01 construct (SEQ ID NO:24), where the sequence for the antigenic head has been highlighted and underlined. The sequence for the antigenic head may be replaced with antigenic head sequences from different Chlamydia species, while the nonantigenic head sequences are not replaced.

[0037] FIG.23 presents the polypeptide sequence for the CM_MOMP_020 construct (SEQ ID NO:25), where the sequence for the antigenic head has been highlighted and underlined. The sequence for the antigenic head may be replaced with antigenic head sequences from different Chlamydia species, while the nonantigenic head sequences are not replaced.

[0038] FIG.24 presents the polypeptide sequence for the CM_MOMP_025 construct (SEQ ID NO:26), where the sequence for the antigenic head has been highlighted and underlined. The sequence for the antigenic head may be replaced with antigenic head sequences from different Chlamydia species, while the nonantigenic head sequences are generally not replaced.

[0039] FIG.25 presents the polypeptide sequence for the CM_MOMP_030-02 construct (SEQ ID NO:27), where the sequence for the antigenic head has been highlighted and underlined. The sequence for the antigenic head may be replaced with antigenic head sequences from different Chlamydia species, while the nonantigenic head sequences are generally not replaced.

[0040] FIG.26 presents the polypeptide sequence for the CM_MOMP_030-00X construct (SEQ ID NO:28), where the sequence for the antigenic head has been highlightedAttorney docket No.00058-089WO1 and underlined. The sequence for the antigenic head may be replaced with antigenic head sequences from different Chlamydia species, while the nonantigenic head sequences are generally not replaced.

[0041] FIG.27 presents the polypeptide sequence for the CM_MOMP_030-00Y construct (SEQ ID NO:29), where the sequence for the antigenic head has been highlighted and underlined. The sequence for the antigenic head may be replaced with antigenic head sequences from different Chlamydia species, while the nonantigenic head sequences are generally not replaced.

[0042] FIG.28 presents the polypeptide sequence for the CM_MOMP_030-3 construct (SEQ ID NO:30), where the sequence for the antigenic head has been highlighted and underlined. The sequence for the antigenic head may be replaced with antigenic head sequences from different Chlamydia species, while the nonantigenic head sequences are generally not replaced.

[0043] FIG.29 presents the polypeptide sequence for the CM_MOMP_HO construct (SEQ ID NO:31), where the sequence for the antigenic head has been highlighted and underlined. The sequence for the antigenic head may be replaced with antigenic head sequences from different Chlamydia species, while the nonantigenic head sequences are generally not replaced.

[0044] FIG.30 presents the polypeptide sequence for the CM_MOMP_CIRC construct (SEQ ID NO:32), where the sequence for the antigenic head has been highlighted and underlined. The sequence for the antigenic head may be replaced with antigenic head sequences from different Chlamydia species, while the nonantigenic head sequences are generally not replaced.

[0045] FIG.31 presents the polypeptide sequence for the CM_MOMP_H20 construct (SEQ ID NO:33), where the sequence for the antigenic head has been highlighted and underlined. The sequence for the antigenic head may be replaced with antigenic head sequences from different Chlamydia species, while the nonantigenic head sequences are generally not replaced. DETAILED DESCRIPTION

[0046] As used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a vaccine" includes a plurality of such vaccines and reference to "theAttorney docket No.00058-089WO1 adjuvant" includes reference to one or more adjuvants and equivalents thereof known to those skilled in the art, and so forth.

[0047] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although many methods and reagents are similar or equivalent to those described herein, the exemplary methods and materials are disclosed herein.

[0048] All publications mentioned herein are incorporated by reference in full for the purpose of describing and disclosing methodologies that might be used in connection with the description herein. The publications are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior disclosure. Moreover, with respect to any term that is presented in one or more publications that is similar to, or identical with, a term that has been expressly defined in this disclosure, the definition of the term as expressly provided in this disclosure will control in all respects.

[0049] An "amino acid sequence" is a polymer of amino acids (a protein, polypeptide, etc.) or a character string representing an amino acid polymer, depending on context. The terms "protein" and "polypeptide" are used interchangeably herein. "Amino acid" is a molecule having the structure wherein a central carbon atom is linked to a hydrogen atom, a carboxylic acid group (the carbon atom of which is referred to herein as a "carboxyl carbon atom"), an amino group (the nitrogen atom of which is referred to herein as an "amino nitrogen atom"), and a side chain group, R. When incorporated into a peptide, polypeptide, or protein, an amino acid loses one or more atoms of its amino acid carboxylic groups in the dehydration reaction that links one amino acid to another. As a result, when incorporated into a protein, an amino acid is referred to as an "amino acid residue."

[0050] A particular amino acid sequence of a given protein (i.e., the polypeptide's "primary structure," when written from the amino-terminus to carboxy-terminus) is determined by the nucleotide sequence of the coding portion of a mRNA, which is in turn specified by genetic information, typically genomic DNA (including organelle DNA, e.g., mitochondrial or chloroplast DNA). Thus, determining the sequence of a gene assists in predicting the primary sequence of a corresponding polypeptide and more particular the role or activity of the polypeptide or proteins encoded by that gene or polynucleotide sequence.Attorney docket No.00058-089WO1

[0051] As used herein, "conservative amino acid substitution" or, simply, "conservative substitution" of a particular sequence refers to the replacement of one amino acid, or series of amino acids, with different amino acids that have similar biochemical properties (e.g., charge, hydrophobicity and size). One of skill will recognize that individual mutations, deletions or additions which alter, add or delete a single amino acid or a percentage of amino acids in an encoded sequence result in "conservative variations" where the alterations result in the deletion of an amino acid, addition of an amino acid, or substitution of an amino acid with a chemically similar amino acid. For purposes of this disclosure a “conservative amino acid substitution” does not significantly affect the stability and / or antigenicity of a Chlamydia polypeptide expressed herein. For example, the engineered polypeptide of the disclosure may comprise conservative amino acid mutations in regions of the sequence that do not impact the binding site for the noncanonical cofactor, e.g., conservative amino acid changes on the surface of the protein. Further, the sequence of a polypeptide disclosed herein can be aligned with polypeptide sequence(s) from other Chlamydia family members that have similar structures and / or catalytic activity in order to identify amino acids that likely do not affect the catalytic activity and / or structural stability of the engineered polypeptide. Moreover, there are many protein modeling programs available, including those specifically recited herein (e.g., Alphafold3, Alphafold2, ColabFold, ParaFold, ESM-2, ESMFold, OpenFold, OmegaFold, UniFold, FastFold, RoseTTA Fold, ManyFold, Alphafold2-Multimer, AF2Complex, MoLPC, PeSTo, TCRdock, AlphaPulldown, Spartan, RosettaDesign, ColabDesign, ProteinMPNN, ESM-IF1, ECNet, ProteinSolver, RFDiffusion, LM-Design, InstructPLM, DiffBindFR, AlphaFill, ProtGPT2, EvoDiff, PoET, and ProtTrans), which can identify conservative amino acid mutations with a high degree of probability / certainty that would not significantly affect the catalytic activity and / or structural stability of an engineered polypeptide disclosed herein (e.g., see Ng et al., Predicting Deleterious Amino Acid Changes Genome Res 11:863-874 (2001)). As such, it is expected that one of skill in the art could reasonably predict that the sequence for a polypeptide disclosed herein can comprise a percentage of conservative amino acid mutations, as is described more fully below, and still have similar or the same stability and / or antigenicity of a Chlamydia polypeptide expressed herein.

[0052] Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, one conservative substitution group includes AlanineAttorney docket No.00058-089WO1 (A), Serine (S), and Threonine (T). Another conservative substitution group includes Aspartic acid (D) and Glutamic acid (E). Another conservative substitution group includes Asparagine (N) and Glutamine (Q). Yet another conservative substitution group includes Arginine (R) and Lysine (K). Another conservative substitution group includes Isoleucine, (I) Leucine (L), Methionine (M), and Valine (V). Another conservative substitution group includes Phenylalanine (F), Tyrosine (Y), and Tryptophan (W).

[0053] Thus, "conservative amino acid mutations" of a polypeptide sequence disclosed herein include mutations of a percentage, typically less than 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, or 40% of the amino acids of the polypeptide sequence, with a conservatively selected amino acid of the same conservative substitution group. Accordingly, a conservatively substituted variation of a polypeptide of the disclosure can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 conservative amino acid substitutions or any range that includes or is in between any two of the foregoing numbers.

[0054] It is envisaged herein that the recombinant or artificial constructs will comprise both conservative and non-conservative amino acid substitutions. For example, for a recombinant or artificial construct of the disclosure expresses a polypeptide sequence for an antigenic head portion of a Chlamydia MOMP folded into its three-dimensional conformation that had resulted from co-evolving contacts in the three-dimensional conformation so that important contacts are preserved, but the amino acids may be substituted with conservative and / or nonconservative amino acid substitutions.

[0055] One of skill in the art will appreciate that many conservative variations of the nucleic acid constructs which are disclosed yield a functionally identical construct. For example, as discussed above, owing to the degeneracy of the genetic code, "silent mutations" (i.e., mutations in a nucleic acid sequence which do not result in an alteration in an encoded polypeptide) are an implied feature of every nucleic acid sequence which encodes an amino acid. Similarly, "conservative amino acid mutations," in one or a few amino acids in an amino acid sequence are substituted with different amino acids with highly similar properties, are also readily identified as being highly similar to a disclosed construct. Such conservative variations of each disclosed sequence are a feature of the polypeptides provided herein.

[0056] As used herein, "conservative variants" are proteins or enzymes in which a given amino acid residue has been changed without altering overall conformation and function ofAttorney docket No.00058-089WO1 the protein, including, but not limited to, replacement of an amino acid with one having similar properties, including polar or non-polar character, size, shape and charge. Amino acids other than those indicated as conserved may differ in a protein or enzyme so that the percent protein or amino acid sequence similarity (or identity) between any two proteins of similar function may vary and can be, for example, at least 30%, at least 50%, at least 70%, at least 80%, or at least 90%, as determined according to an alignment scheme. As referred to herein, "sequence similarity" means the extent to which nucleotide or protein sequences are related. The extent of similarity between two sequences can be based on percent sequence identity and / or conservation. "Sequence identity" herein means the extent to which two nucleotide or amino acid sequences are invariant. "Sequence alignment" means the process of lining up two or more sequences to achieve maximal levels of identity (and, in the case of amino acid sequences, conservation) for the purpose of assessing the degree of similarity. Numerous methods for aligning sequences and assessing similarity / identity are known in the art such as, for example, the Clustal Omega program, wherein similarity is calculated with the modified mBed algorithm, as well as BLASTN, BLASTP, and FASTA programs (Lipman and Pearson, 1985; Pearson and Lipman, 1988). When using all of these programs, the preferred settings are those that results in the highest sequence similarity.

[0057] Non-conservative modifications of a particular polypeptide are those which substitute any amino acid not characterized as a conservative substitution. For example, any substitution which crosses the bounds of the six groups set forth above. These include mutations of basic or acidic amino acids for neutral amino acids, (e.g., Asp, Glu, Asn, or Gln for Val, Ile, Leu or Met), aromatic amino acid for basic or acidic amino acids (e.g., Phe, Tyr or Trp for Asp, Asn, Glu or Gln) or any other substitution not replacing an amino acid with a like amino acid. Basic side chains include lysine (K), arginine (R), histidine (H); acidic side chains include aspartic acid (D), glutamic acid (E); uncharged polar side chains include glycine (G), asparagine(N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), cysteine (C); nonpolar side chains include alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), methionine (M), tryptophan (W); beta-branched side chains include threonine (T), valine (V), isoleucine (I); aromatic side chains include tyrosine (Y), phenylalanine (F), tryptophan (W), and histidine (H).Attorney docket No.00058-089WO1

[0058] A "protein" or "polypeptide", which terms are used interchangeably herein, refers to one or more chains of chemical building blocks called amino acids that are linked together by chemical bonds called peptide bonds.

[0059] A protein has "homology" or is "homologous" to a second protein if the nucleic acid sequence that encodes the protein has a similar sequence to the nucleic acid sequence that encodes the second protein. Alternatively, a protein has homology to a second protein if the two proteins have "similar" amino acid sequences. (Thus, the term "homologous proteins" is defined to mean that the two proteins have similar amino acid sequences).

[0060] As used herein, two proteins (or a region of the proteins) are substantially homologous when the amino acid sequences have at least about 30%, 40%, 50% 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In one embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, typically at least 40%, more typically at least 50%, even more typically at least 60%, and even more typically at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[0061] When "homologous" is used in reference to proteins or peptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In casesAttorney docket No.00058-089WO1 where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of homology may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art (see, e.g., Pearson et al., 1994, hereby incorporated herein by reference).

[0062] Those of skill in the art will recognize that, due to the degenerate nature of the genetic code, a variety of codons differing in their nucleotide sequences can be used to encode a given amino acid. The disclosure includes polynucleotides of any sequence that encode a polypeptide of the disclosure that comprises the same amino acid sequence of said polypeptide.

[0063] The disclosure provides for polynucleotides in the form of recombinant DNA expression vectors or plasmids, as described in more detail elsewhere herein, that encode a Chlamydia MOMP, or a portion thereof, as disclosed herein. For microorganisms, such vectors can either replicate in the cytoplasm of a microorganism or integrate into the DNA of the microorganism. In either case, the vector can be a stable vector (i.e., the vector remains present over many cell divisions, even if only with selective pressure) or a transient vector (i.e., the vector is gradually lost by host microorganisms with increasing numbers of cell divisions). For cellular systems (e.g., mammalian cells), such vectors are able to introduce proper protein folding, post-translational modifications, and product assembly. For expressing heterologous genes in mammalian cells, usually vectors derived from mammalian viruses are used. These include viruses such as Simian Viruses 40 (SV40), polyomavirus, herpesvirus and papovirus. Additionally, plasmids have been designed to be expressed in mammalian cells (e.g., pHEK293 Ultra Expression Vectors, BacMam system, etc.)

[0064] The disclosure provides DNA molecules in isolated (i.e., not pure, but existing in a preparation in an abundance and / or concentration not found in nature) and purified (i.e., substantially free of contaminating materials or substantially free of materials with which the corresponding DNA would be found in nature) form.

[0065] As will be understood by those of skill in the art, it can be advantageous to modify a coding sequence to enhance its expression in a particular host. The genetic code is redundant with 64 possible codons, but most organisms typically use a subset of these codons. The codons that are utilized most often in a species are called optimal codons, and those not utilized very often are classified as rare or low-usage codons. Codons can beAttorney docket No.00058-089WO1 substituted to reflect the preferred codon usage of the host, a process sometimes called "codon optimization" or "controlling for species codon bias."

[0066] Optimized coding sequences containing codons preferred by a particular prokaryotic or eukaryotic host (see also, Murray et al. (1989) Nucl. Acids Res.17:477-508) can be prepared, for example, to increase the rate of translation or to produce recombinant RNA transcripts having desirable properties, such as a longer half-life, as compared with transcripts produced from a non-optimized sequence. Translation stop codons can also be modified to reflect host preference. For example, typical stop codons for S. cerevisiae and mammals are UAA and UGA, respectively. The typical stop codon for monocotyledonous plants is UGA, whereas insects and E. coli commonly use UAA as the stop codon (Dalphin et al. (1996) Nucl. Acids Res.24: 216-218). Methodology for optimizing a nucleotide sequence for expression in a plant is provided, for example, in U.S. Pat. No.6,015,891, and the references cited therein.

[0067] As used herein, “reference sequence" refers to a defined sequence used as a basis for a sequence comparison. A reference sequence may be a subset of a larger sequence, for example, a segment of a full-length gene or polypeptide sequence. Generally, a reference sequence can be at least 20 nucleotides or amino acid residues in length, at least 25 nucleotide or residues in length, at least 50 nucleotides or residues in length, or the full length of the nucleic acid or polypeptide. Since two polynucleotides or polypeptides may each comprise a sequence (i.e., a portion of the complete sequence) that is similar between the two sequences and may further comprise a sequence that is divergent between the two sequences, sequence comparisons between two (or more) polynucleotides or polypeptides are typically performed by comparing sequences of the two polynucleotides or polypeptides over a “comparison window” to identify and compare local regions of sequence similarity.

[0068] As used herein, "sequence identity" means that two polypeptide sequences are substantially identical (i.e., on an amino acid-by-amino acid basis) over a window of comparison. The term "sequence similarity" refers to similar amino acids that share the same biophysical characteristics. The term "percentage of sequence identity" or "percentage of sequence similarity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical residues (or similar residues) occur in both polypeptide sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in theAttorney docket No.00058-089WO1 window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity (or percentage of sequence similarity). Regarding polynucleotide sequences, the terms sequence identity and sequence similarity have comparable meaning as described for protein sequences, with the term "percentage of sequence identity" indicating that two polynucleotide sequences are identical (on a nucleotide-by-nucleotide basis) over a window of comparison. As such, a percentage of polynucleotide sequence identity (or percentage of polynucleotide sequence similarity, e.g., for silent mutations or other mutations, based upon the analysis algorithm) also can be calculated. Maximum correspondence can be determined by using one of the sequence algorithms described herein (or other algorithms available to those of ordinary skill in the art) or by visual inspection. In a particular embodiment, a polypeptide sequence will have about 80%, about 85% or more, about 90% or more, about 95% or more, and about 98% or more, sequence identity to another polypeptide sequence.

[0069] As applied to polypeptides, the term substantial identity or substantial similarity means that two peptide sequences, when optimally aligned, such as by the programs BLAST, GAP or BESTFIT using default gap weights or by visual inspection, share sequence identity or sequence similarity. Similarly, as applied in the context of two nucleic acids, the term substantial identity or substantial similarity means that the two nucleic acid sequences, when optimally aligned, such as by the programs BLAST, GAP or BESTFIT using default gap weights (described elsewhere herein) or by visual inspection, share sequence identity or sequence similarity.

[0070] One example of an algorithm that is suitable for determining percent sequence identity or sequence similarity is the FASTA algorithm, which is described in Pearson, W. R. & Lipman, D. J., (1988) Proc. Natl. Acad. Sci. USA 85:2444. See also, W. R. Pearson, (1996) Methods Enzymology 266:227-258. Preferred parameters used in a FASTA alignment of DNA sequences to calculate percent identity or percent similarity are optimized, BL50 Matrix 15: -5, k-tuple=2; joining penalty=40, optimization=28; gap penalty -12, gap length penalty=-2; and width=16.

[0071] Another example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments to show relationship and percent sequence identity or percent sequence similarity. It also plots a tree or dendrogram showing the clustering relationships used to create the alignment.Attorney docket No.00058-089WO1 PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, (1987) J. Mol. Evol.35:351-360. The method used is similar to the method described by Higgins & Sharp, CABIOS 5:151-153, 1989. The program can align up to 300 sequences, each of a maximum length of 5,000 nucleotides or amino acids. The multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences. This cluster is then aligned to the next most related sequence or cluster of aligned sequences. Two clusters of sequences are aligned by a simple extension of the pairwise alignment of two individual sequences. The final alignment is achieved by a series of progressive, pairwise alignments. The program is run by designating specific sequences and their amino acid or nucleotide coordinates for regions of sequence comparison and by designating the program parameters. Using PILEUP, a reference sequence is compared to other test sequences to determine the percent sequence identity (or percent sequence similarity) relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps. PILEUP can be obtained from the GCG sequence analysis software package, e.g., version 7.0 (Devereaux et al., (1984) Nuc. Acids Res.12:387-395).

[0072] Another example of an algorithm that is suitable for multiple DNA and amino acid sequence alignments is the CLUSTALW program (Thompson, J. D. et al., (1994) Nuc. Acids Res.22:4673-4680). CLUSTALW performs multiple pairwise comparisons between groups of sequences and assembles them into a multiple alignment based on sequence identity. Gap open and Gap extension penalties were 10 and 0.05 respectively. For amino acid alignments, the BLOSUM algorithm can be used as a protein weight matrix (Henikoff and Henikoff, (1992) Proc. Natl. Acad. Sci. USA 89:10915-10919).

[0073] A "vector" generally refers to a polynucleotide that can be propagated and / or transferred between organisms, cells, or cellular components. Vectors include viruses, bacteriophage, pro-viruses, plasmids, phagemids, transposons, and artificial chromosomes such as YACs (yeast artificial chromosomes), BACs (bacterial artificial chromosomes), and PLACs (plant artificial chromosomes), and the like, that are "episomes," that is, that replicate autonomously or can integrate into a chromosome of a host cell. A vector can also be a naked RNA polynucleotide, a naked DNA polynucleotide, a polynucleotide composed of both DNA and RNA within the same strand, a poly-lysine-conjugated DNA or RNA, a peptide- conjugated DNA or RNA, a liposome-conjugated DNA, or the like, that are not episomal inAttorney docket No.00058-089WO1 nature, or it can be an organism which comprises one or more of the above polynucleotide constructs such as an agrobacterium or a bacterium.

[0074] The various components of an expression vector can vary widely, depending on the intended use of the vector and the host cell(s) in which the vector is intended to replicate or drive expression. Expression vector components suitable for the expression of genes and maintenance of vectors in E. coli, yeast, Streptomyces, and other commonly used cells are widely known and commercially available. For example, suitable promoters for inclusion in the expression vectors of the disclosure include those that function in eukaryotic or prokaryotic host organisms. Promoters can comprise regulatory sequences that allow for regulation of expression relative to the growth of the host organism or that cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus. For E. coli and certain other bacterial host cells, promoters derived from genes for biosynthetic enzymes, antibiotic-resistance conferring enzymes, and phage proteins can be used and include, for example, the galactose, lactose (lac), maltose, tryptophan (trp), beta- lactamase (bla), bacteriophage lambda PL, and T5 promoters. In addition, synthetic promoters, such as the tac promoter (U.S. Pat. No.4,551,433, which is incorporated herein by reference in its entirety), can also be used. For E. coli expression vectors, it is useful to include an E. coli origin of replication, such as from pUC, p1P, p1, and pBR.

[0075] Thus, recombinant expression vectors contain at least one expression system, which, in turn, is composed of at least a portion of a gene coding sequences operably linked to a promoter and optionally termination sequences that operate to effect expression of the coding sequence in compatible host cells. The host cells are modified by transformation with the recombinant DNA expression vectors of the disclosure to contain the expression system sequences either as extrachromosomal elements or integrated into the chromosome.

[0076] Chlamydia trachomatis is a Gram-negative, obligate intracellular bacterium that primarily infects the mucous membranes of the human body, causing a variety of diseases. It is best known as the causative agent of chlamydia, the most frequently reported bacterial sexually transmitted infection (STI) worldwide. In addition to genital infections, C. trachomatis can cause eye infection trachoma, which is a leading cause of preventable blindness globally, particularly in developing nations. The bacteria have a unique biphasic life cycle, alternating between an infectious elementary body and a replicative reticulate body. Although often asymptomatic, untreated infections can lead to serious complications,Attorney docket No.00058-089WO1 including infertility, pelvic inflammatory disease, and chronic pain. Due to its asymptomatic nature in many cases, regular screening and early treatment with antibiotics are crucial for controlling its spread.

[0077] Chlamydiae have a unique developmental cycle. Their extracellular infectious forms, known as elementary bodies (EBs), are spore-like, ~300 nm in diameter, highly resistant to osmotic and mechanical stress, oxidized, and exhibit minimal metabolic activity. EBs are endocytosed by the host cell into a cytoplasmic inclusion, where they enlarge into intermediate bodies (IBs) and continue growing until they reach ~1,000 nm, becoming reticulate bodies (RBs). RBs are not infectious, have a flexible outer membrane, are highly susceptible to physical changes, reduced, metabolically active, and replicate. Some RBs divide over 30-72 hours, while others revert to IBs and eventually form EBs. This asynchronous division continues until the inclusion either ruptures the host cell or is extruded, releasing new EBs to infect other cells. Unlike other Gram-negative bacteria, where the peptidoglycan layer provides mechanical support for the cell envelope, in Chlamydiae, the rigidity of the EB envelope is provided by cysteine-rich proteins crosslinked with disulfide bonds, forming the Chlamydial Outer Membrane Complex (COMC). The COMC is reduced during differentiation from the EB to the RB stage, allowing for RB growth and replication.

[0078] MOMP is a key component of the COMC, accounts for about 60% of the EB outer membrane mass and has emerged as the most promising subunit vaccine candidate due to its abundance and ability to induce serovar / serogroup-specific protection. EB MOMP is a homotrimeric integral membrane protein with a ~42 kDa protomer. Each protomer consists of five conserved sequence domains (CDs) interspersed with four variable sequence domains (VDs) that determine the fifteen major C. trachomatis serovars. EB MOMP adheres to host cells, and its extracellularly exposed VDs contain most of the known B-cell epitopes, some of which elicit neutralizing antibodies. MOMP also was shown to function as a porin, diffusing sugars and ATP, with an estimated pore size of ~1-2 nm based on liposome swelling assays. However, MOMP is unlikely to function as a porin in EBs, which are minimally metabolically active and thus do not require passive transfer.

[0079] Native trimeric MOMP has shown superior immune responses and protection compared to peptide-based, denatured, and recombinant MOMP preparations. Preclinical studies of Ct infections primarily use Chlamydia muridarum (Cm; initially called C.Attorney docket No.00058-089WO1 trachomatis mouse pneumonitis biovar), a murine pathogen related to Ct, because Cm vaginal infection in mice mimics acute and long-term sequelae of Ct infections in humans. In contrast, Ct vaginal infections in animal models fail to replicate short and long-term characteristics of Ct human infections. The importance of the native EB MOMP structure for vaccine design has driven 40 years of intense studies on MOMP properties. Researchers have speculated about a possible similarity to the archetypal trimeric b-barrel porin, Escherichia coli OmpF, and hypothetical b-barrel models have been published, all of which had some similarity to OmpF but differed in the number of transmembrane strands (14 vs 16), extent and orientation of the transmembrane strands, location of the cysteines, and even left- or right-handedness of the barrel.

[0080] Provided herein are studies which allowed for the determination of cryogenic electron microscopy single particle reconstruction (cryo-EM SPR) structures of Cm MOMP isolated from EBs in its native trimeric state and in complex with the Fab fragment of a discontinuous neutralizing monoclonal antibody (mAb-18b). The studies presented herein have identified a new family of trimeric β-barrel adhesins and address long-standing questions about MOMP topology and its epitopes’ structure and cellular presentation.

[0081] The structures presented herein reveal a novel trimeric arrangement of 10- stranded β-barrels, with extracellular loops from all protomers forming a compact, magnesium-binding cap that contains immunogenic VDs. This extracellular antigenic head also contains several positively charged cavities, some of which bind ligands, indicating potential sites of interaction with chlamydial and host molecules. The presence of a sphingolipid molecule intercalated between two MOMP protomers suggests that sphingolipid mediated MOMP trimerization may contribute to Chlamydiae’s reliance on host sphingolipids. The experimental EB MOMP structure significantly differs from previous theoretical models proposing 16-stranded or 14-standed β-barrel topologies, indicating the need for a critical reassessment of the experimental data on MOMP gathered over the last 40 years and interpreted in the context of these models. In particular, the EB MOMP structure does not allow for diffusion, suggesting that other MOMP forms might be present during the chlamydial developmental cycle or that other proteins perform this function. The conformational change in trimeric MOMP upon binding the neutralizing antibody mAb-18b provides insights into the structural rearrangements involved in neutralization of infection.Attorney docket No.00058-089WO1

[0082] Most importantly, the structural results presented in the studies herein allow for the development of structure-based vaccines against C. trachomatis infections, including the design of constructs for improved expression, solubility, and stability, enabling the production of recombinant MOMP in its three-dimensional conformation. Deep learning tools can optimize sequences, remove non-essential cysteines, and modify trimer formation features for a more cross-reactive vaccine candidate (see FIG.21). The immunogenic extracellular cap can be grafted onto different scaffolds, e.g., human fatty acid binding protein 4 (FABP4), for enhanced efficacy, with immune refocusing targeting conserved, surface-exposed epitopes while eliminating immunodominant ones (see FIG.21). Several scaffolds decorated with MOMP VDs have shown encouraging results in tests and the structures presented here can be used to design even better candidates.

[0083] The disclosure provides for recombinant or artificial constructs that express or produce the antigenic portion of a Chlamydia MOMP in its three-dimensional conformation as is disclosed herein. In a particular embodiment, the recombinant or artificial construct comprises a vector as disclosed above. In a further embodiment, the vector is an expression vector that expresses a MOMP, or a portion thereof, from a polynucleotide sequence for a MOMP from C. pneumoniae, C. muridarum, C. avium, C. buteonis, C. caviae, C. crocodili, C. felis, C. gallinacea, C. poikilotherma, C. abortus, C. psittaci, C. serpentis, C. suis, C. pecorum, or C. trachomatis. In yet a further embodiment, the expression vector expresses a MOMP, or a portion thereof, from a polynucleotide sequence for a MOMP, or a portion thereof from a C. trachomatis serovar selected from A, B / Ba, C, D / Da, E, F, G / Ga, H, I / Ia, J, K, L1, L2, L2a and L3. It should be noted that the sequences for MOMPs are publicly available from UniProt, GenPept, etc. A listing of Chlamydia MOMP sequences can be found at ebi.ac.uk / interpro / entry / InterPro / IPR000604 / protein / UniProt / #table, which is incorporated herein. In a certain embodiment, the recombinant or artificial constructs will comprise a tightly folded antigenic head exposing regions of MOMP responsible for serovar specificity, inducing immune response, and adhesion to host cell. This antigenic head is formed by polypeptide chains from all three monomers. In an alternate embodiment, the antigenic head is formed by single polypeptide sequence that encodes the VDs of each monomer. In such a case, the single polypeptide sequence produces a polypeptide that folds into the same structure as a Chlamydia MOMP in its three-dimensional conformation as disclosed herein.Attorney docket No.00058-089WO1 Further, said single polypeptide sequence can comprise VDs from different serovars, or VDs from one serovar.

[0084] In a particular embodiment, the artificial constructs take advantage of structurally similar proteins (e.g., porin proteins, cell adhesion proteins, etc.) which can be used as a scaffold or backbone for the production or expression of chimeric proteins which comprise the antigenic portions from MOMP in the native three-dimensional conformation (e.g., the tightly folded antigenic head exposing regions of MOMP). For example, the antigenic portions from MOMP, like the antigenic head, are grafted on to the scaffold or backbone structures from membrane proteins or soluble proteins from different organisms. In additional embodiments, the antigenic portions from MOMP, like the antigenic head, can be made to be accessible or antigenic by selectively mutating some amino acid residues while keeping other amino acid residues unchanged or by adding flexible or stiff polypeptide linkers to attach the antigenic portions from MOMP to the backbone structures. Ideally, in foregoing embodiments, that the antigenic portion of MOMP should be presented in its three- dimensional conformation as disclosed herein, despite the manner of attachment to the backbone or scaffold, or whether optional linkers or mutations are utilized.

[0085] Additionally, the backbone or scaffold can be selected to form larger oligomeric structures e.g., multiple basic trimeric units. In a further embodiment, the backbone or scaffold portion of the artificial or recombinant construct is embedded in a membrane. The type of membrane lipid that can be added when using membrane-embedded scaffolds to maintain stability of the trimerization and dimerization interfaces, e.g. a fatty acid to maintain one of the interfaces and a sphingolipid to support the other, can be modulated by changing both the amino acid facing the lipids and the lipid itself, if lipids are required for the oligomerization.

[0086] In a particular embodiment, the disclosure provides for recombinant or artificial constructs that express or produce the antigenic portion of MOMP from C. trachomatis in its three-dimensional conformation as disclosed herein, where the antigenic portion of MOMP comprises variable domains (VDs) from the same C. trachomatis serovar (e.g., VDs from a C. trachomatis serovar selected from A, B / Ba, C, D / Da, E, F, G / Ga, H, I / Ia, J, K, L1, L2, L2a and L3). In an alternate embodiment, the disclosure provides for recombinant or artificial constructs that express or produce the antigenic portion of MOMP from C. trachomatis in its three-dimensional conformation as disclosed herein, where the antigenic portion of MOMPAttorney docket No.00058-089WO1 comprises variable domains (VDs) from different serovars (e.g., VDs from any combination of C. trachomatis serovar selected from A, B / Ba, C, D / Da, E, F, G / Ga, H, I / Ia, J, K, L1, L2, L2a and L3). With regards to the foregoing embodiment, the antigenic portion from MOMP can be arranged in heterotrimers, where each monomer contains antigenic loops from different serovars, or by mixing several homotrimers, each with a specific serovar antigenic head. In an alternate embodiment, the antigenic head is formed by single polypeptide sequence that encodes the VDs of each monomer. In such a case, the single polypeptide sequence produces a polypeptide that folds into the same structure as a Chlamydia MOMP in its three-dimensional conformation as disclosed herein. Further, said single polypeptide sequence can comprise VDs from different serovars, or VDs from one serovar.

[0087] In a particular embodiment, the disclosure provides for recombinant or artificial constructs comprising polynucleotide(s) the encode at least the antigenic portion of MOMP. In a further embodiment, the polynucleotides lack or have the sequence removed that encodes the periplasmic region of MOMP. As the periplasmic region of MOMP is flexible and not involved in important interactions inducing immune response, it is not necessary for the polynucleotide to comprise such a sequence and is optional. In a further embodiment, the polynucleotide(s) comprise the sequence that encodes MOMP except for the periplasmic region of MOMP.

[0088] The lipid binding in the transmembrane portion of MOMP was found to be important in the studies presented herein for trimerization and dimerization interfaces, including lipid binding at the three-fold axis and between each pair of monomers. For artificial or recombinant constructs that comprise sequences which encode or express the transmembrane portion of MOMP, these sequences can comprise mutations or substitution of amino acids that keep the trimer intact but no longer require the interaction with lipids at trimerization and dimerization interfaces. Examples of such mutations or substitutions can be replacement of amino acids that interact with lipids, with amino acids that have side chains that would generate a similar effect.

[0089] In a certain embodiment, the disclosure provides for recombinant or artificial constructs that expresses a recombinant polypeptide for a MOMP or portion thereof, that is soluble in aqueous solutions, by comprising one or more amino acid substitutions in the polypeptide sequence for the MOMP or a portion thereof. In further embodiment, the recombinant polypeptide for a MOMP or portion thereof comprises one or more amino acidAttorney docket No.00058-089WO1 substitutions that modify the surface of the trimeric beta-barrel assembly so that the expressed polypeptide does not require a membrane to fold into a trimeric beta-barrel structure with a low RMSD but still retains the antigenic head portion of a Chlamydia MOMP in its three-dimensional conformation.

[0090] In another embodiment, the disclosure provides for recombinant or artificial constructs comprising polynucleotide(s) that encode the β-barrel portion of MOMP from Cm MOMP but the antigenic head from C. pneumoniae, C. muridarum, C. avium, C. buteonis, C. caviae, C. crocodili, C. felis, C. gallinacea, C. poikilotherma, C. abortus, C. psittaci, C. serpentis, C. suis, C. pecorum, or C. trachomatis.

[0091] In yet another embodiment, the recombinant or artificial constructs further comprise T cell epitopes, such as universal T cell helper peptides, other MOMP regions, in addition to the antigenic portion of a Chlamydia MOMP in its three-dimensional conformation as is disclosed herein.

[0092] In a particular embodiment, a Chlamydia MOMP, or a portion thereof, in its three-dimensional conformation as disclosed herein is used as an antigen in a vaccine to protect against a Chlamydia infection. The protection induced by MOMP is dependent on its structure. Therefore, the determination of the three-dimensional structure of MOMP disclosed herein is an important step for the successful formulation of a Chlamydia vaccine. MOMP constitutes 60% of the protein mass in the chlamydial elementary body (EB) and is the primary target for surface binding and neutralizing antibodies for a Chlamydia infection. MOMP comprises: (1) variable domains, which determine serotype specificity, and (2) constant domains containing CD4 and CD8 T cell epitopes. Despite MOMP's potential as a subunit vaccine candidate, attempts at inducing protection using denatured MOMP or its derivatives haven not been successful. In another embodiment, a Chlamydia MOMP, or a portion thereof, in its three-dimensional conformation as disclosed herein is used as an antigen in a vaccine to protect against a C. trachomatis infection. A successful C. trachomatis vaccine should induce (a) humoral immune responses including chlamydial- specific serum immunoglobulin (Ig)G and mucosal IgG and IgA responses with neutralizing capabilities and (b) cell-mediated immune (CMI) responses, including upregulation of interferon (IFN)-γ, tumor necrosis factor (TNF)-α and interleukin (IL)-17, and downregulation of IL-4 and IL-10.Attorney docket No.00058-089WO1

[0093] In a certain embodiment, a vaccine formulation or preparation comprises a Chlamydia MOMP, or a portion thereof, having the three-dimensional structure disclosed herein that was expressed or produced from an artificial or recombinant construct disclosed herein. In a further embodiment, liposomes, virus like particles or polymeric nanoparticles are used as a delivery vehicle for Chlamydia MOMP, or a portion thereof, having the three- dimensional structure disclosed herein. In a further embodiment, the Chlamydia MOMP, or a portion thereof, having the three-dimensional structure disclosed herein is found on the surface of the delivery vehicle. In another embodiment, liposomes, virus like particles or polymeric nanoparticles are used as a delivery vehicle for the Chlamydia MOMP, or a portion thereof, or a polynucleotide sequence encoding thereof, is found within the delivery vehicle as cargo, or on the surface of the delivery vehicle, or both.

[0094] In a certain embodiment, the disclosure further provides a vaccine preparation or formulation that comprises the artificial or recombinant construct disclosed herein. In a further embodiment, the artificial or recombinant construct comprises polynucleotides that encode the polypeptide sequence for an antigenic head portion of a Chlamydia MOMP in its three-dimensional conformation, and wherein the vaccine preparation or formulation is a polynucleotide-based vaccine. In yet a further embodiment the polynucleotides comprise DNA, RNA, or hybrids of DNA and RNA.

[0095] In a particular embodiment, a vaccine formulation or preparation comprises the antigenic head from a MOMP Chlamydia, having the three-dimensional structure disclosed herein, that was expressed or produced from an artificial or recombinant construct disclosed herein. With specific regards to the antigenic head of MOMP, the antigenic head contains ligand-binding cavities that can bound with NAG or other sugar derivatives as decoys to prevent the antigen head from binding to host cells. Alternatively, the ligand-binding cavities of the antigen head can be modified or mutated so as to prevent binding by NAG, effectively creating artificial or recombinant constructs that should still induce immune response but would not be able to bind and enter the cell, which might be useful in some applications. For instance, the antigenic head is highly electronegative. The proteoglycan of the host cell is also highly electronegative. These two surfaces need to be balanced for the attachment of the pathogen to the cell. Even simple solutions like modulating pH, adding specific polyions might be an effective strategy for preventing chlamydial ocular infections. Less simple methods would involve development of specific peptides / sugars that would occupy bindingAttorney docket No.00058-089WO1 sites were identified on the antigenic head. The neutralizing antibodies can also be used but would be expensive in this context.

[0096] In additional embodiments, Chlamydia MOMP, or a portion thereof, having the three-dimensional structure disclosed herein is used as an antigen in a vaccine to protect against a Chlamydia infection is used in combination with one or more adjuvants. Adjuvants in immunology are often used to modify or augment the effects of a vaccine by stimulating the immune system to respond to the vaccine more vigorously, and thus providing increased immunity to a particular disease. Adjuvants accomplish this task by mimicking specific sets of evolutionarily conserved molecules, so called pathogen-associated molecular patterns, which include liposomes, lipopolysaccharide, molecular cages for antigens, components of bacterial cell walls, and endocytosed nucleic acids such as RNA, double-stranded RNA, single-stranded DNA, and unmethylated CpG dinucleotide-containing DNA. Because immune systems have evolved to recognize these specific antigenic moieties, the presence of an adjuvant in conjunction with the vaccine can greatly increase the innate immune response to the antigen by augmenting the activities of dendritic cells, lymphocytes, and macrophages by mimicking a natural infection. Examples of adjuvants include, but are not limited to, CpG oligodeoxynucleotide based adjuvants, ssRNA, aluminum salts (e.g., aluminum hydroxide, aluminum phosphate, and aluminum potassium sulfate), Montanide ISA 720, CAF01, AS04, MF59, AS01B, and CpG 1018.

[0097] The disclosure further provides for specified modes of administration for administering the vaccine formulation or preparation disclosed herein. In one embodiment, the disclosure provides for a pharmaceutical composition that comprises a vaccine formulation or preparation disclosed herein and a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier” as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agents from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the composition and is compatible with administration to a subject, for example a human. Such compositions can be specifically formulated for administration via one or more of a number of routes, such as the routes of administration described herein. Supplementary active ingredients also can be incorporated into the compositions. When an agent, formulation orAttorney docket No.00058-089WO1 pharmaceutical composition described herein, is administered to a subject, preferably, a therapeutically effective amount is administered. As used herein, the term “therapeutically effective amount” refers to an amount that results in an improvement or remediation of the condition.

[0098] The disclosure further provides for the use of a vaccine formulation or preparation disclosed herein for vaccinating a subject. Suitable methods of administering a vaccine formulation or preparation described herein to a patient include by any route of in vivo administration that is suitable for delivering such a vaccine formulation or preparation to a patient. Examples of modes of administration include, but are not limited to, intravenous administration, intertumoral administration, intraperitoneal administration, intramuscular administration, intracoronary administration, intraarterial administration (e.g., into a carotid artery), subcutaneous administration, transdermal delivery, intratracheal administration, subcutaneous administration, intraarticular administration, intraventricular administration, inhalation (e.g., aerosol), nasal, oral, pulmonary administration, impregnation of a catheter, and direct injection into a tissue.

[0099] Intravenous, intraperitoneal, and intramuscular administrations can be performed using methods standard in the art. Aerosol (inhalation) delivery can also be performed using methods standard in the art (see, for example, Stribling et al., Proc. Natl. Acad. Sci. USA 189: 11277-11281, 1992, which is incorporated herein by reference in its entirety). Oral delivery can be performed by complexing a vaccine formulation or preparation disclosed herein to a carrier capable of withstanding degradation by digestive enzymes in the gut of an animal. Examples of such carriers include plastic capsules or tablets, such as those known in the art.

[0100] The appropriate dosage and treatment regimen for the vaccine formulation or preparation described herein will vary with respect to the needed vaccination schedule of the subject. In certain cases, only one vaccine formulation or preparation may need to be administered to a subject to bring about effective immunity to a pathogen or noninfectious disease. In other cases, one or more booster shots of the vaccine formulation or preparation disclosed herein may be needed. In such a case, the one or more booster shots may have the same dose of targeted antigen(s) or be of a lower dose. For multiple doses of the vaccine formulation or preparation disclosed herein, they may be administered a week or more apart.Attorney docket No.00058-089WO1

[0101] The disclosure further provides for design or repurpose strain-specific drugs that inhibit essential bacterial processes, such as attachment, invasion, and nutrient transport through nMOMP pores.

[0102] The disclosure further provides for diagnostic methods and / or dry reagent test strips comprising, Chlamydia MOMP, or a portion thereof, having the three-dimensional structure disclosed herein for detecting, monitoring or determining strain-specific Chlamydial infections.

[0103] The disclosure also provides methods enabling cell-delivery by using the epitopes identified on Chlamydia MOMP, or a portion thereof, having the three-dimensional structure disclosed herein, to induce uptake of whatever is attached to the epitope.

[0104] For use in the therapeutic and diagnostic applications described herein, kits and articles of manufacture are also described herein. Such kits can comprise a carrier, package, or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in a method described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers can be formed from a variety of materials such as glass or plastic.

[0105] For example, the container(s) can comprise one or more artificial constructs or diagnostics described herein, optionally in a composition or in combination with another agent as disclosed herein. The container(s) optionally have a sterile access port (for example the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). Such kits optionally comprise an identifying description or label or instructions relating to its use in the methods described herein.

[0106] A kit will typically comprise one or more additional containers, each with one or more of various materials (such as reagents, optionally in concentrated form, and / or devices) desirable from a commercial and user standpoint for use of a compound described herein. Non-limiting examples of such materials include, but are not limited to, buffers, diluents, filters, needles, syringes; carrier, package, container, vial and / or tube labels listing contents and / or instructions for use, and package inserts with instructions for use. A set of instructions will also typically be included.

[0107] A label can be on or associated with the container. A label can be on a container when letters, numbers or other characters forming the label are attached, molded or etchedAttorney docket No.00058-089WO1 into the container itself, a label can be associated with a container when it is present within a receptacle or carrier that also holds the container, e.g., as a package insert. A label can be used to indicate that the contents are to be used for a specific therapeutic application. The label can also indicate directions for use of the contents, such as in the methods described herein. These other therapeutic agents may be used, for example, in the amounts indicated in the Physicians' Desk Reference (PDR) or as otherwise determined by one of ordinary skill in the art.

[0108] The disclosure further provides that the compositions, systems and methods described herein can be further defined by the following aspects (aspects 1 to 62): 1. An artificial or recombinant construct that expresses a polypeptide sequence for an antigenic head portion of a Chlamydia Major Outer Membrane Protein (MOMP) in its three-dimensional conformation. 2. The artificial or recombinant construct of aspect 1, wherein the antigenic head portion is from Chlamydia pneumoniae, Chlamydia muridarum, Chlamydia avium, Chlamydia buteonis, Chlamydia caviae, Chlamydia crocodili, Chlamydia felis, Chlamydia gallinacea, Chlamydia poikilotherma, Chlamydia abortus, Chlamydia psittaci, Chlamydia serpentis, Chlamydia suis, Chlamydia pecorum, or Chlamydia trachomatis. 3. The artificial or recombinant construct of aspect 1 or 2, wherein the antigenic head portion is from Chlamydia trachomatis or Chlamydia pneumoniae. 4. The artificial or recombinant construct of any one of aspects 1 to 3, wherein the antigenic head portion is from a serovar of Chlamydia trachomatis that is selected from A, B / Ba, C, D / Da, E, F, G / Ga, H, I / Ia, J, K, L1, L2, L2a and L3. 5. The artificial or recombinant construct of aspect 4, wherein the Chlamydia trachomatis serovar is selected from A, E, F, D / Da, J, L2, and G / Ga. 6. The artificial or recombinant construct of any one of aspects 1 to 5, wherein the artificial or recombinant construct additionally expresses polypeptide sequences for the β- barrel transmembrane portion and β-barrel extracellular collar of a Chlamydia MOMP. 7. The artificial or recombinant construct of aspect 6, wherein the β-barrel transmembrane portion and β-barrel extracellular collar is from Chlamydia muridarum. 8. The artificial or recombinant construct of aspect 6 or aspect 7, wherein the polypeptide sequences for the β-barrel transmembrane portion and / or β-barrel extracellularAttorney docket No.00058-089WO1 collar of a Chlamydia MOMP comprises one or more substitutions of non-cysteine amino acids to cysteines to stabilize the Chlamydia MOMP by the formation of disulfide bridges. 9. The artificial or recombinant construct of any one of aspects 6 to 8, wherein the polypeptide sequences for the β-barrel transmembrane portion and / or β-barrel extracellular collar of a Chlamydia MOMP have proline substitutions for non-proline amino acids, wherein the amino acid side chains from the surrounding amino acids have accommodating or favorable interactions with the substituted proline amino acids, and wherein the β-barrel transmembrane portion and / or β-barrel extracellular collar has consistent main chain angles of what is expected for prolines. 10. The artificial or recombinant construct of any one of aspects 6 to 9, wherein the polypeptide sequences for the β-barrel and its extracellular collar have been modified or changed so that the expressed polypeptide has increased solubility in aqueous medium while still retaining the antigenic head portion of a Chlamydia MOMP in its three-dimensional conformation. 11. The artificial or recombinant construct of aspect 10, wherein the artificial or recombinant construct has a polypeptide sequence that is at least 95% identical to SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30. 12. The artificial or recombinant construct of aspect 10 or aspect 11, wherein the artificial or recombinant construct has a polypeptide sequence that is at least 98% identical to SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30. 13. The artificial or recombinant construct of any one of aspects 10 to 12, wherein the artificial or recombinant construct has a polypeptide sequence that is at least 98% identical to SEQ ID NO:28 or SEQ ID NO:29. 14. The artificial or recombinant construct of any one of aspects 6 to 13, wherein the polypeptide sequence for the β-barrel transmembrane portion has been modified or changed so that the β-barrel transmembrane portion has been reduced or truncated. 15. The artificial or recombinant construct of aspect 14, wherein the artificial or recombinant construct has a polypeptide sequence that is at least 95% identical to SEQ ID NO:30 or SEQ ID NO:31.Attorney docket No.00058-089WO1 16. The artificial or recombinant construct of aspect 14 or aspect 15, wherein the artificial or recombinant construct has a polypeptide sequence that is at least 98% identical to SEQ ID NO:30 or SEQ ID NO:31. 17. The artificial or recombinant construct of any one of aspects 1 to 5, wherein the artificial or recombinant construct additionally expresses a polypeptide sequence for an artificial scaffold that replaces the β-barrel transmembrane portion of a Chlamydia MOMP. 18. The artificial or recombinant construct of aspect 17, wherein the artificial scaffold is derived from a three-helix bundle, a transmembrane portion of a porin protein, a lipocalin, or a transmembrane portion of a cell adhesion protein. 19. The artificial or recombinant construct of aspect 17 or aspect 18, wherein the artificial scaffold is derived from a polypeptide sequence for a lipocalin. 20. The artificial or recombinant construct of any one of aspects 17 to 19, wherein the artificial or recombinant construct has a polypeptide sequence that is at least 95% identical to SEQ ID NO:33. 21. The artificial or recombinant construct of any one of aspects 17 to 20, wherein the artificial or recombinant construct has a polypeptide sequence that is at least 98% identical to SEQ ID NO:33. 22. The artificial or recombinant construct of aspect 17, wherein the artificial scaffold is a zinc-finger scaffold that uses zinc-cysteine coordination for stabilization. 23. The artificial or recombinant construct of aspect 22, wherein the zinc-finger scaffold is based on a C2H2-zinc finger carrying motif. 24. The artificial or recombinant construct of aspect 22 or aspect 23, wherein the C2H2-zinc finger carrying motif is selected from KLF1, KLF8, KLF11, ZEB2, GLI1, IKZFI, and CTCF. 25. The artificial or recombinant construct of any one of aspects 17 to 24, wherein the artificial scaffold provides multiple 3-fold axes to create more complex arrangements. 26. The artificial or recombinant construct of aspect 25, wherein the multiple 3-fold axes provide for tetrahedral, octahedral or icosahedral symmetries. 27. The artificial or recombinant construct of any one of aspects 1 to 26, wherein the antigenic head portion of a Chlamydia MOMP has been stabilized by domain swapping, wherein the domain swapping can be in the polypeptide sequence for the antigenic head portion of a Chlamydia MOMP, or in some other polypeptide sequence of the artificial orAttorney docket No.00058-089WO1 recombinant construct, so long as the antigenic head portion of a Chlamydia MOMP is presented in its three-dimensional conformation. 28. The artificial or recombinant construct of any one of aspects 1 to 27, wherein the artificial or recombinant construct expresses a plurality of polypeptide sequences for multiple antigenic head portions of a Chlamydia MOMP. 29. The artificial or recombinant construct of aspect 28, wherein the multiple antigenic head portions are from different species of Chlamydia MOMPs. 30. The artificial or recombinant construct of aspect 29, wherein at least one of the species of Chlamydia is Chlamydia trachomatis or Chlamydia pneumoniae. 31. The artificial or recombinant construct of aspect 28, wherein the multiple antigenic head portions are from different serovars of Chlamydia trachomatis. 32. The artificial or recombinant construct of aspect 31, wherein the different serovars of Chlamydia trachomatis are selected from A, E, F, D / Da, J, L2, and G / Ga. 33. The artificial or recombinant construct of any one of aspects 1 to 32, wherein the artificial or recombinant construct additionally expresses a polypeptide sequence for a periplasmic loop of a Chlamydia MOMP with / or without the N-terminal stopper segment removed. 34. The artificial or recombinant construct of aspect 33, wherein the artificial or recombinant construct additionally expresses another polypeptide sequence for the N- terminal stopper segment extension of the periplasmic loop. 35. The artificial or recombinant construct of any one of aspects 1 to 34, wherein the recombinant or artificial construct further expresses a polypeptide sequence for a T cell epitope. 36. A vaccine preparation or formulation that comprises the artificial or recombinant construct of any one of any one of aspects 1 to 35, 37. The vaccine preparation or formulation of aspect 36, wherein the artificial or recombinant construct comprises polynucleotides that encode the polypeptide sequence for an antigenic head portion of a Chlamydia MOMP in its three-dimensional conformation, and wherein the vaccine preparation or formulation is a polynucleotide-based vaccine. 38. The vaccine preparation or formulation of aspect 36 or aspect 37, wherein the polynucleotides comprise DNA, RNA, modified RNA, or hybrids of DNA and RNA.Attorney docket No.00058-089WO1 39. The vaccine preparation or formulation of any one of aspects 36 to 38, wherein liposomes, virus like particles or polymeric nanoparticles are used as a delivery vehicle for the polynucleotides that encode the polypeptide sequence for the antigenic head portion of a Chlamydia MOMP, and wherein the polynucleotides are found within the delivery vehicle as cargo, or on the surface of the delivery vehicle, or both. 40. The vaccine preparation or formulation of any one of aspects 36 to 39, wherein the vaccine preparation or formulation further comprises one or more adjuvants. 41. The vaccine preparation or formulation of aspect 40, wherein the one or more adjuvants are selected from a CpG oligodeoxynucleotide based adjuvant, ssRNA, an aluminum salt, Montanide ISA 720, CAF01, AS04, MF59, AS01B, and / or CpG 1018. 42. A vaccine preparation or formulation which comprises an antigenic head portion of a Chlamydia MOMP expressed from the artificial or recombinant construct of any one of aspects 1 to 35. 43. The vaccine preparation or formulation of aspect 42, wherein liposomes, virus like particles or polymeric nanoparticles are used as a delivery vehicle for the antigenic head portion of the Chlamydia MOMP, and wherein the antigenic head portion of the Chlamydia MOMP is found within the delivery vehicle as cargo, or on the surface of the delivery vehicle, or both. 44. The vaccine preparation or formulation of aspect 42 or aspect 43, wherein the vaccine preparation or formulation further comprises one or more adjuvants. 45. The vaccine preparation or formulation of any one of aspects 42 to 44, wherein the one or more adjuvants are selected from a CpG oligodeoxynucleotide based adjuvant, ssRNA, an aluminum salt, Montanide ISA 720, CAF01, AS04, MF59, AS01B, and / or CpG 1018. 46. A vaccine preparation or formulation which comprises an antigenic head portion of a Chlamydia Major Outer Membrane Protein (MOMP) that is selected from Chlamydia pneumoniae, Chlamydia muridarum, Chlamydia avium, Chlamydia buteonis, Chlamydia caviae, Chlamydia crocodili, Chlamydia felis, Chlamydia gallinacea, Chlamydia poikilotherma, Chlamydia abortus, Chlamydia psittaci, Chlamydia serpentis, Chlamydia suis, Chlamydia pecorum, or Chlamydia trachomatis in its three-dimensional conformation. 47. The vaccine preparation or formulation of aspect 46, wherein the antigenic head portion of the Chlamydia MOMP is from Chlamydia trachomatis or Chlamydia pneumoniae.Attorney docket No.00058-089WO1 48. The vaccine preparation or formulation of aspect 46 or aspect 47, wherein the Chlamydia MOMP is from a Chlamydia trachomatis serovar selected from A, B / Ba, C, D / Da, E, F, G / Ga, H, I / Ia, J, K, L1, L2, L2a and L3. 49. The vaccine preparation or formulation of any one of aspects 46 to 48, wherein the MOMP is from a Chlamydia trachomatis serovar selected from A, E, F, D / Da, J, L2, and G / Ga. 50. The vaccine preparation or formulation of any one of aspects 46 to 49, wherein liposomes, virus like particles or polymeric nanoparticles are used as a delivery vehicle for the antigenic head portion of the Chlamydia MOMP, and wherein the antigenic head portion of the Chlamydia MOMP is found within the delivery vehicle as cargo, or on the surface of the delivery vehicle, or both. 51. The vaccine preparation or formulation of any one of aspects 46 to 50, wherein the vaccine preparation or formulation further comprises one or more adjuvants. 52. The vaccine preparation or formulation of aspect 51, wherein the one or more adjuvants are selected from a CpG oligodeoxynucleotide based adjuvant, ssRNA, an aluminum salt, Montanide ISA 720, CAF01, AS04, MF59, AS01B, and / or CpG 1018. 53. A method of immunizing a subject to protect against an infection by a Chlamydia bacterium, comprising: administering one or more therapeutically effective doses of the vaccine preparation or formulation of any one of aspects 36 to 41 to a subject. 54. The method of aspect 53, wherein multiple doses of the vaccine preparation or formulation are administered to the subject at different time points. 55. The method of aspect 53 or aspect 54, wherein the mode of administration is selected from intravenous administration, intraperitoneal administration, intramuscular administration, intracoronary administration, intraarterial administration, subcutaneous administration, transdermal delivery, intratracheal administration, subcutaneous administration, intraarticular administration, intraventricular administration, inhalation, nasal, oral, pulmonary administration, impregnation of a catheter, and direct injection into a tissue. 56. A method of immunizing a subject to protect against an infection by a Chlamydia bacterium, comprising: administering one or more therapeutically effective doses of the vaccine preparation or formulation of any one of aspects 42 to 45 to a subject.Attorney docket No.00058-089WO1 57. The method of aspect 56, wherein multiple doses of the vaccine preparation or formulation are administered to the subject at different time points. 58. The method of aspect 56 or aspect 57, wherein the mode of administration is selected from intravenous administration, intraperitoneal administration, intramuscular administration, intracoronary administration, intraarterial administration, subcutaneous administration, transdermal delivery, intratracheal administration, subcutaneous administration, intraarticular administration, intraventricular administration, inhalation, nasal, oral, pulmonary administration, impregnation of a catheter, and direct injection into a tissue. 59. A method of immunizing a subject to protect against an infection by a Chlamydia bacterium, comprising: administering one or more therapeutically effective doses of the vaccine preparation or formulation of any one of aspects 46 to 52 to a subject 60. The method of aspect 59, wherein multiple doses of the vaccine preparation or formulation are administered to the subject at different time points. 61. The method of aspect 59 or aspect 60, wherein the mode of administration is selected from intravenous administration, intraperitoneal administration, intramuscular administration, intracoronary administration, intraarterial administration, subcutaneous administration, transdermal delivery, intratracheal administration, subcutaneous administration, intraarticular administration, intraventricular administration, inhalation, nasal, oral, pulmonary administration, impregnation of a catheter, and direct injection into a tissue. 62. A method of detecting or monitoring an infection by a Chlamydia bacterium in a subject, comprising: contacting a sample from the subject with a diagnostic agent comprising an antigenic head portion of a Chlamydia MOMP in its three-dimensional conformation, wherein if the subject has antibodies to the antigenic head portion of a Chlamydia MOMP is indicated by detectable change in the diagnostic agent, and wherein if the subject does not have antibodies to the antigenic head portion of a Chlamydia MOMP results in no change in the diagnostic agent.

[0109] The following examples are intended to illustrate but not limit the disclosure. While they are typical of those that might be used, other procedures known to those skilled in the art may alternatively be used. EXAMPLESAttorney docket No.00058-089WO1

[0110] The invention is illustrated in the following examples, which are provided by way of illustration and are not intended to be limiting.

[0111] Isolation and purification of native Cm EB MOMP. The trimeric native Cm MOMP was extracted and purified as described in Sun et al., J. Bacteriol 189:6222-6235 (2007). Briefly, Cm-infected Hela-229 cell monolayers were collected, centrifuged at 10,000 × g for 30 min at 4 °C (Beckman Coulter, Inc. J2-21 centrifuge; Brea, CA) and washed with PBS (pH 7.4). The pellet was resuspended in 0.02 M Tris (pH 7.4), 1.0 M NaCl, 0.012 M MgCl2, and 1 mM phenylmethylsulfonyl fluoride (PMSF) (Calbiochem, La Jolla, CA) by sonication using a cup horn with iced water (Virsonic 300 Ultrasonic cell disrupter; The VirTis Company, Inc; Gardiner, N.Y.). After adding 25 µg of DNase (Sigma; Aldrich), the suspension was incubated on ice for 2 hours with constant mixing. Following centrifugation, the pellet was resuspended by sonication in 0.2 M sodium phosphate buffer (pH 5.5), containing 1 mM each of EDTA and PMSF, 100 mM DTT (Roche Applied Sciences; Indianapolis, IN), and 2% CHAPS (Anatrace; Maumee, OH), and rotated at 37 °C for 2 h. After ultracentrifugation at 100,000 × g for 1 h at 4 °C (Beckman Coulter Inc. L2 ultracentrifuge), the pellet was resuspended by sonication and extracted again with CHAPS- containing buffer for 1 h at 37 °C. After ultracentrifugation, the pellet was resuspended by sonication and extracted with 2% Anzergent Z3-14 (Anatrace; Maumee, OH) in the same phosphate buffer. Following incubation at 37 °C for 2 h, the sample was centrifuged at 100,000 × g for 1 h at 4 °C, and MOMP was recovered in the supernatant.

[0112] To purify the MOMP trimer, a 1 × 20 cm hydroxyapatite (Bio-Gel ®HTP- hydroxyapatite; Bio-Rad Laboratories Inc., CA) column was used. The column was equilibrated with 0.02 M phosphate buffer (pH 5.5) containing 0.1% Z3-14 and 1 mM each of EDTA, PMSF, and DTT. Elution was performed using a 0.02 M to 0.5 M linear gradient of sodium phosphate buffer containing 0.1% Z3-14 and 1 mM each of EDTA, PMSF and DTT. The peak fraction was collected, concentrated by ultrafiltration (Amicon ®Ultra 15; Merck Millipore, Ltd; Darmstadt, Germany), and the purity of the MOMP trimer was confirmed by SDS-PAGE stained with Coomassie blue (see FIG.2). The concentration of endotoxins as assessed by the Limulus amoebocyte assay (Associates of Cape Cod, Inc. MA), was less than 0.05 EU / mg of protein.

[0113] Production of Cm MOMP monoclonal antibodies and Fab fragments purification. To generate monoclonal antibodies against MOMP, 4- to 6-week-old femaleAttorney docket No.00058-089WO1 BALB / c-H2dmice were inoculated intranasally with 104inclusion-forming units (IFU) of Cm as described in Sun et al. J Bacteriol 189:6222-35 (2007). Six weeks later, the mice were injected intraperitoneally with 107IFU of Cm. Spleen cells from these infected mice were fused with the mouse myeloma cell line S194 / 5XX0.Bu.1, using the method described by Kohler et al., Nature 256:495-497 (1975). Culture fluid from the hybridomas was screened for the presence of antibodies to Cm EB using a microimmunofluorescence assay. Positive hybridomas were selected and cloned by limiting dilution. To produce ascites fluid, 106cloned hybridomas cells were injected intraperitoneally into 6- to 8-week-old female BALB / c mice that have been pre-treated with Pristane (Sigma Chemical Co.; St Louis, MO) two weeks prior. Ascites fluid was collected, centrifuged at 10,000 × g for 30 min at 4 °C, and frozen at -70 °C.

[0114] To map the binding site of the monoclonal antibodies, 25-mer MOMP peptides were chemically synthesized, and an ELISA was performed. Monoclonal antibodies that did not recognize linear peptides were then tested against both heated and non-heated Cm EBs using a fluorescently labeled anti-mouse antibody. This test aimed to identify antibodies that recognize discontinuous epitopes on EB MOMP trimers present in the non-heated samples.

[0115] Fab fragments were generated using Immobilized Papain Agarose Resin (cross- linked 6% agarose; Pierce Biotechnology, Rockford, IL) following the manufacturer’s protocol. Briefly, antibodies purified using Affi-Gel® Protein A MAPS® II Kit (Bio-Rad Laboratories; Hercules, CA) were dialyzed against sample buffer (20 mM sodium phosphate, 10 mM EDTA, pH 7.0) and concentrated to 5 mg / mL in a total volume of 2 mL. Immediately prior to digestion, L-Cysteine-HCl was added to the sample to a final concentration of 20 mM, and the pH was adjusted to 7.0. The Immobilized Papain Agarose Resin (0.5 mL) equilibrated with digestion buffer (20 mM sodium phosphate, EDTA 10 mM, L-Cysteine HCl 20 mM, pH 7.0) was mixed with each antibody’s solution at a 1:2 v / v ratio. Digestion proceeded for about 12 h in a high-speed shaking water bath at 37 °C. After digestion, the papain agarose was separated by centrifugation and washed with 10 mM Tris HCl pH 7.5. Fab fragments were then separated from undigested IgG and Fc fragments using the Affi- Gel® Protein A MAPS® II Kit, according to the manufacturer instructions.

[0116] Cryo-EM grids preparation and data collection. Purified samples were applied to Quantifoil R 1.2 / 1.3300 mesh gold grids, which had been glow discharged for 90 s at 30 mA using a PELCO airglow™ Glow Discharge Cleaning System to create a hydrophilicAttorney docket No.00058-089WO1 surface. The glow-discharged grids were then used to prepare vitrified samples using the Thermo Scientific Vitrobot Mark IV System. A 3 μL aliquot of the purified sample was applied to the glow-discharged grid surface at 4 °C and 100% humidity, followed by blotting for 5.0 to 5.5 s with a blot force of either 18 or 19. The data were acquired with a 300 kV Titan Krios G2 microscope (Thermo Fisher) equipped with a K3 Summit direct electron camera (Gatan) operating in super-resolution mode at a nominal magnification of 81,000×, with a physical pixel size of 1.079 Å. Neither a phase plate nor the objective aperture was used. SerialEM was used for automated data collection in beam-image shift mode, with nine images acquired per stage movement with a defocus range from -1.0 to -3.0 μm and compensation for aberrations induced by beam-image shift. The slit width of the GIF Quantum Energy Filter was set to 25 eV. Movies were dose-fractionated into 100 frames with a total dose of ~80 e− / Å2.2,826 movies were collected for Cm EB MOMP and 3,569 movies for MOMP / mAb-18b complex.

[0117] Cryo-EM image processing and model building. All movies were imported into CryoSPARC, followed by patch motion correction using binning of 2 and patch CTF correction. For the EB MOMP dataset, particles were initially picked using the blob picker and extracted with a box size of 360 pixels. After several rounds of 2D classification, 118,614 clean particles were used for ab initio reconstruction with three classes. Non-uniform refinement with C3 symmetry using 87,699 particles resulted in a 2.96 Å resolution density map (e.g., see FIG.5).

[0118] For the EB MOMP / mAb-18b complex, an initial batch of 25,575 particles was obtained using the blob picker across all micrographs, extracted with box size of 360 pixels, and cleaned through several rounds of 2D classification. Ab initio reconstruction with three classes, using all particles, yielded a single interpretable 3D model of the EB MOMP / mAb- 18b complex. The particle pool was further enriched using the Topaz particle picker. The Topaz model was iteratively trained and optimized on a subset of micrographs, then applied across all micrographs to extract 265,993 particles. After one round of 2D classification, 110,855 particles were retained for further processing. These particles were further filtered through heterogeneous refinement against the three original ab initio classes. A final set of 51,710 particles was used as an input to non-uniform refinement with C3 symmetry, producing a 3.36 Å resolution density map (e.g., see FIG.5). No additional masking was applied beyond cryoSPARC's default dynamic masking, and no sharpening was applied to theAttorney docket No.00058-089WO1 deposited maps. Local resolution maps were generated using the CryoSPARC Local Resolution Estimation job.

[0119] The initial atomic models were generated by docking the AF2 models of EB MOMP and mAb-18b Fab to the maps using MOLREP, implemented in CCPEM. The models were then iteratively rebuilt manually in Coot and refined using Phenix.real_space_refine and Servalcat. Model validation statistics were calculated with MolProbity and Servalcat (see Table 1). The atomic coordinates and maps have been deposited in the Protein Data Bank (PDB: 8V7Y and 8VT1) and the Electron Microscopy Data Bank (EMD-43010 and EMD-43515). Table 1. Cryo-EM data collection, processing, and model refinement statistics Data collection Instrument Titan Krios G2 Detector K3 Summit Energy filter Yes Objective aperture No Nominal magnification 81,000× Data collection mode Beam-Image Shift; 3×3 holes per position Frames per movie 100 Electron dose (e- / A2 / frame) 0.8 Exposure time (s / frame) 0.04 Super-resolution mode Yes Detector pixel size (Å) 1.079 Data pixel size (Å) 0.539 Movies acquired 2,826 for MOMP; 3,569 for MOMP / mAb-18b Reconstruction MOMP MOMP / mAb-18b Molecular weight (kDa) 119 262 Reconstruction symmetry C3 C3 Particles used in refinement 87,699 51,710 Resolution FSC0.143(Å) 2.96 3.36 Refinement MOMPMOMP / mAb-18b Non-hydrogen atoms 9,006 18,531 Protein residues 1,095 2,343 Ligands 18 15 RMSD bond lengths (Å) 0.009 0.009 RMSD bond angles (°) 1.373 1.267 Model-to-map FSC (all) 0.778 0.726 MolProbity score 1.52 (100thpercentile) 1.97 (100thpercentile) Clashscore (all atom) 5.27 (100thpercentile) 11.61 (97thpercentile) Poor rotamers (%) 0 0.6Attorney docket No.00058-089WO1 Ramachandran (%) favored 96.42 94.15 allowed 33 572models were performed with Coot, Pymol and UCSF ChimeraX. The comparisons of three- dimensional structures were conducted using LSQKAB from the CCP4 suite. Shape complementarity was analyzed with the SC, and buried surfaces were calculated and analyzed using the PISA server. Contact patterns were examined using CONTACT, AREAIMOL, and LIGPLOT2+. The Caver 3.0 Pymol plugin was used to search for tunnels, channels and cavities, with the search conducted using a minimum probe radius of 0.9 Å and the starting point near Asn5 inside the b-barrel. The 524 evolutionary conservation was mapped onto the experimental models using Consurf.

[0121] Epitope mapping. B- and T-cell epitopes for Ct serovar D MOMP were downloaded from the Immune Epitope Database (IEDB). Epitopes with less than 100% sequence identity to the Ct serovar D sequence were excluded from the analysis, resulting in 447 B-cell and 22 T-cell epitopes. For each amino acid position, the number of positive and negative B- and T-cell assays reported in the IEDB for epitopes containing that amino acid was counted, and an amino acid score was calculated: log^1 + ^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^ / (1 +^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^).

[0122] The serovar D trimer structure was modeled by AlphaFold-Multimer using the ColabFold v1.5.5 notebook provided by Sergey Ovchinnikov, utilizing the Cm EB MOMP cryo-EM structure as a template.

[0123] Conformational sampling was done using the Colab implementation of AlphaFold2-MULTIMER using MMseqs2, with settings max_msa: 16:32, num_seeds: 8. Note that the AF2 predicted structure for MOMP as stored in the AlphaFold Protein Structure Database and available through Uniprot and PDB is flawed, as it includes the N-terminal signal peptide, which distorts the β-barrel structure.

[0124] Cm EB MOMP forms a unique trimer exposing tightly folded antigenic cap. The structure of Cm EB MOMP was reconstructed using cryo-EM SPR at 2.96 Å global resolution (see Table 1, and FIG.5A-C). The density map reveals well-ordered side chains in both the β-barrel and extracellular domains (see FIG.3B-C, FIG.5D and FIG.6). The periplasmic region is less-ordered (see FIG.5D and FIG.6A). Lipid and detergent moleculesAttorney docket No.00058-089WO1 are visible in the density, marking the EB MOMP transmembrane region (see FIG.5D and FIG.6B-D).

[0125] The EB MOMP protomer is a 10-stranded anti-parallel β-barrel spanning the membrane (~40% of the polypeptide) with an extended extracellular structure (~50% of the polypeptide) and a periplasmic region (~10% of the polypeptide) (see FIG.3C-E, and FIG. 7). The β-barrel strands are angled ~41° to the axis (see FIG.3D, and FIG.7), consistent with a shear number of 12. In the transmembrane region of the β-barrel, hydrophobic residues face the lipid bilayer pointing outward, while polar residues point inward (see FIG.3D-E). Some β-barrel strands extend into the extracellular space, forming a collar-like structure where this inward-outward polarity pattern is reversed, characteristic of globular, soluble proteins (see FIG.3D). The extracellular region contains structured loops, which include the four variable domains VD1-VD4 (see FIG.3D-F, and FIG.7), two antiparallel β-strands in solvent-exposed regions that flank VD1 (see FIG.3D, circle 2), two other short β strands stabilized by VD3 and the extracellular β-barrel collar (see FIG.3D, circle 3 and FIG.7), and five β strands at the trimer interface (see FIG.3D) that extend from the β-barrel to form a β-sheet.

[0126] VDs intra- and inter-protomer interactions stabilize the EB MOMP trimer. VD1 of one protomer (VD11) interacts with VD2 of the same protomer (VD21) and with VD4 of the neighboring protomer (VD42); VD21interacts with VD31, VD41, and VD42; VD31interacts with VD21and VD41; and VD41interacts with VD21, VD31, VD13, VD23, VD42and VD43(see FIG.3F, and FIG.8F-K). These extracellular interactions are primarily mediated by hydrogen bonds (see FIG.8F-I), with fewer non-polar interactions (see FIG. 8J-K). Each protomer contains a magnesium cation octahedrally coordinated by five water molecules and the Oδ1 atom of D311 (CD5) (see FIG.3G and FIG.8C). The water molecules coordinating Mg2+form an H-bond network involving residues from VD11(2,3)and VD42(3,1). The Mg2+binding is consistent with an early observation in C. psittaci, showing that adding cations promoted chlamydial attachment, likely by counteracting repulsive forces between electronegative surfaces of MOMP and host cells. Two other ligands were modeled in the cap region: an N-acetylglucosamine molecule (NAG 403), a common component of chlamydial lipooligosaccharide (LOS), and lauric acid (DAO 404), one of the most abundant fatty acids known to be scavenged by Ct (see FIG.8D-E).Attorney docket No.00058-089WO1

[0127] The transmembrane region of the EB MOMP trimer has fewer direct inter- protomer interactions than the extracellular regions (see FIG.8L-N), with interactions primarily localized at the bottom of the β-barrel between strand 7 of one protomer and strand 10 of another. It was observed elongated densities near 3-fold axis that likely represent trapped lipids mediating trimerization. However, due to possible C3 averaging artifacts, these densities were not modeled. Non-protein densities away from axis could be modeled, taking into account compounds used in purification and the lipid composition of chlamydial and host membranes. Host lipids are expected to bind because EB MOMP was isolated from Cm- infected Hela-229 cells, and Chlamydiae are known to hijack host lipids for growth and development. A sphingomyelin backbone was modeled between two protomers (see FIG. 8B). This molecule forms numerous hydrophobic interactions with the EB MOMP β-barrel, including the side chains of A100 (CD21), I333 (CD51) and A361 (CD52). It spans ~80% of the length of the EB MOMP β-barrel and buries a surface area of 307 Å2 with one protomer and 403 Å2 with another, thus likely playing an important role in stabilizing the trimeric assembly. It was observed additional molecular density near the modeled sphingolipid and on the β-barrel surface, suggesting possible LOS binding. However, local resolution was insufficient to confidently model LOS fragments. Previously postulated EB MOMP N- glycosylation was not observed. Interactions between the EB MOMP protomers and ligands bury ~19,200 Å2, ~31% of the total surface area (~61,500 Å2), confirming the trimer as the functional unit of EB MOMP.

[0128] Each protomer has ~40 periplasm-exposed amino acid residues, including residues 14-34 in the flexible loop, β turns in positions 107-109, 179-181, and 340-342, a fragment of a β-turn at N257 (see FIG.8O), and five out of the eight cysteine residues: C26, C29, C33, C179, and C181 (see FIG.9). C26, C29, and C33, located in the flexible loop, are not modeled with high confidence (see FIG.8A), while C179 and C181 are well ordered (see FIG.9E). No disulfide bridges were observed, as expected for protein isolated in reducing conditions.

[0129] Both the periplasmic and extracellular surfaces have a negative electrostatic potential (see FIG.3G). On the extracellular side, this negative potential is partially neutralized by Mg2+(see FIG.3G). The transmembrane surface has neutral potential with positively charged pockets, indicating putative binding sites for phosphate groups ofAttorney docket No.00058-089WO1 phospholipids, LOS, or sulfate groups of proteoglycans that aid bacterial adhesion to host cells. Inside the β-barrel, the transmembrane region is polar (see FIG.3G).

[0130] The EB MOMP transmembrane β-barrel protomer has canonical features of transmembrane β-barrels including aromatic girdles marking the transmembrane boundaries (see FIG.7) and an alternating pattern of hydrophobic and hydrophilic residues forming a polar interior and hydrophobic exterior (see FIG.3E). However, the trimeric architecture is unique. Other experimental transmembrane 10-stranded β-barrel structures, classified “OmpT-like”, are monomeric multi-functional adhesins and include: OmpT, an outer membrane protease from E. coli, PLA, a plasminogen activator from Yersinia pestis, and OpcA, a proteoglycan-binding adhesin from Neisseria meningitidis. These proteins extend above the membrane, with their extracellular segments conferring activity and substrate specificity. Similarly, EB MOMP extends a significant portion of its β-barrel above the membrane, with the β-barrel interior being occluded by extracellular loops, as in OpcA. Monomers of OpcA (PDB: 2VDF) and EB MOMP align with an RMSD of 3.5 Å for 272 Cαatoms. Unlike monomeric 10-stranded β-barrels, EB MOMP is a trimer, with three β-barrels topped by a globular extracellular cap formed by tightly interacting VDs from all three protomers. This cap has its own hydrophobic core, burying ~10,000 Å2out of the ~22,000 Å2total cap surface. Thus, EB MOMP can be considered the founding member of a new family of trimeric adhesins, with a transmembrane stem formed by three β-barrels and a multimeric cap that mediates cell adhesion.

[0131] Conserved cysteine residues in transmembrane region of EB MOMP. Unlike most membrane β-barrel proteins which lack cysteine residues, MOMP contains eight cysteine residues: C26, C29, C33, C114, C179, C181, C204, and C329 (see FIG.9). Seven of these are highly conserved, while one (C204) is not (see FIG.4 and Table 2). In the EB MOMP structure, C26, C29, C33, C179, and C181 are located in the periplasmic space; C204 in the extracellular space; C114 and C329 in the transmembrane region (see FIG.9, and FIG. 8A).

[0132] Extracellular C204 is well-ordered but too distant from other cysteine residues, including C204 of other monomers, to form disulfide bridges. In non-reducing conditions, all periplasmic cysteine residues likely form intermolecular disulfide bridges with other EB MOMP molecules and COMC components. The protein used in the experiments was isolated under reducing conditions, so intramolecular disulfide bridges C26-C33 and C26-C29,Attorney docket No.00058-089WO1 detected in previous mass spectrometry studies are not observed in the data. The intramolecular disulfide bridge between C179 and C181 is unlikely due to their position in a tight β-turn (see FIG.9E).

[0133] Cysteine residues have not been previously observed in the transmembrane regions of experimentally determined β-barrel structures 72, likely due to their destabilizing effect in these segments. However, C114 and C329 are exposed to the membrane and are evolutionarily conserved (see FIG.9A, C-D; FIG.4; and Table 2), suggesting an important role in EB MOMP function. Structure predictions for the chlamydial polymorphic outer membrane protein (Pmp) family similarly show outward-facing cysteine residues in their transmembrane β-barrels. Like EB MOMP, Pmps are part of COMC and form intermolecular disulfide bridges. Our structure raises the possibility that COMC is stabilized by intramembrane disulfide bridges between its components, in addition to the periplasmic ones. Previous theoretical models of MOMP structure placed cysteine residues in the extracellular space, making it difficult to reconcile MOMP's role in COMC, which is known to involve disulfide bridges in the periplasmic space. The ability of cysteine residues to form and reform disulfide bonds in response to redox changes, along with regulation through both periplasmic and intramembrane disulfide bridges, could provide COMC with the flexibility needed for Chlamydiae to transition between different developmental forms. Table 2. Sequence conservation of the eight cysteine residues in chlamydial MOMP. C26 100% C C29 100% C C33 98% C C114 100% C C179 98% C C181 100% C C204 50% S, 47% C, 1% T, 1% F C329 82% C, 14% T, 2% A The results are based on sequence conservation analysis performed with Consurf using default server settings, which includes all chlamydial MOMP sequences showing significant sequence similarity to Cm but excludes sequences more than 97% identical to the Cm MOMP sequence.

[0134] The EB MOMP structure also serves as a reference for analyzing the EB outer membrane organization. The inner leaflet of the OM in EB is associated with a highly organized, pseudo-crystalline hexagonal array, likely composed of MOMP, small cysteine- rich outer membrane protein OmcA, and large cysteine-rich periplasmic protein OmcB.Attorney docket No.00058-089WO1 AlphaFold3 heteromultimer predictions of the MOMP trimer, OmcA and OmcB, consistently show OmcA and OmcB interacting with the periplasmic side of MOMP and potentially forming multiple disulfide bonds (see FIG.11). OmcB is predicted to form elongated star- shaped trimers, consistent with its putative role in forming a crosslinked periplasmic protein mesh that compensates for the lack of a peptidoglycan layer in the EB periplasm. OmcA is predicted to interact with the MOMP-OmcB interface and may use its N-terminal lipid anchor to tie the periplasmic protein layer to the OM. In the model, MOMP and OmcB form the hexagonal array, while OmcA likely fills gaps in the array through transmembrane interactions, with intra- and inter- molecular disulfide bridges between MOMP and OmcB molecules providing periplasmic support for the array.

[0135] The EB MOMP cannot function as a porin. Some studies have shown that MOMP functions as a porin, diffusing sugars and ATP, with an estimated pore size of ~1-2 nm, based on liposome swelling assays. However, in EB, MOMP is not expected to function as a porin, as passive transport would likely compromise the stability of this almost metabolically inert developmental form. Consistent with these expectations, the EB MOMP structure determined in this study cannot passively transfer the molecular species characterized before.

[0136] The EB MOMP protomer is a 10-stranded, oval-shaped β-barrel, with dimensions of ~23 Å by ~19 Å (see FIG.6A). The first 12 residues at the N-terminus form a partially helical stopper segment that is inserted into the MOMP barrel from the periplasmic side, with a ~2,100 Å2buried surface area between the β-barrel interior and the stopper segment (see FIG.6B-C). This segment is stabilized by 15 H-bonds between atoms of the stopper segment and polar amino acids inside the β-barrel, along with additional internal H-bonds within the stopper segment itself (FIG.6C). Although the stopper segment does not fully occlude the barrel interior, a Caver analysis revealed bottlenecks (FIG.6D-E), which prevent diffusion. None of the tunnels traverse the extracellular tightly folded cap. Although Caver analysis of just the transmembrane β-barrel, with the stopper region and the extracellular domain removed, shows a tunnel with a 4.3 Å bottleneck, which is sufficient to allow the passage of water molecules (2.75 Å in diameter), the diffusion of sugars and ATP observed in planar lipid bilayers and liposome swelling assays with MOMP remains impossible. To accommodate transfer of these molecules, EB MOMP would have to be refolded to another topologically distinct β-barrel. Importantly, the EB MOMP forms a complex with mAb-18b,Attorney docket No.00058-089WO1 an antibody raised against Cm EBs. Thus, the isolated MOMP likely retains its native structure present in EBs and does not undergo major conformational changes that could affect pore size.

[0137] If MOMP functions as a porin in other phases of the chlamydia life cycles, the necessary structural rearrangements likely occur after differentiation from EB to RB, coinciding with the reduction of disulfide bridges in COMC proteins and the volume increase between EB and RB. In RB MOMP, all cysteine residues are reduced. The EB MOMP structures are determined using protein purified under reducing conditions and are incompatible with passive transport, so simply reducing disulfide bridges is insufficient to yield a porin-permissive MOMP conformation.

[0138] A similar challenge of β-barrels being too narrow to support passive transfer, despite porin activity observed in planar lipid bilayers and liposome swelling assays, has been described for other systems. For instance, OprF in Pseudomonas aeruginosa 80 forms a closed 8-stranded β-barrel that supports the OM, but a minor conformation forming wider β- barrels with an estimated pore size of 2 nm has been proposed to explain the transport of large substrates observed in liposome swelling assays. Discussions on the transition to the second putative conformation of OprF invoke refolding influenced by redox potential, with multiple folding intermediates postulated.

[0139] It remains to be determined whether a different structure of chlamydial MOMP exists, whether the experiments detected a contaminating porin instead of MOMP, or whether MOMP misfolding in liposomes affected experimental observations.

[0140] mAb-18b Fab binds a conformational epitope on the EB MOMP trimer. mAb-18b is a mouse monoclonal neutralizing antibody (IgG2b / κ) raised against Cm EB (see Table 3) which recognizes a discontinuous epitope of the trimeric Cm EB MOMP but not Ct EB MOMP. The complex of mAb-18b Fab and EB MOMP (MOMP / mAb-18b) was reconstructed at a global resolution of 3.36 Å (see FIG.10A, FIG.5E-H, and Table 1). The local resolution ranges from 3 to 3.5 Å for EB MOMP and 3.5 to 4 Å for the variable regions of the Fab. The constant regions of the Fab have lower resolution (> 5 Å) due to the flexibility of the antibody elbow region (see FIG.10B-C, and FIG.5H). Table 3. mAb-18b Complementarity-Determining Regions (CDRs) Variable SequenceAttorney docket No.00058-089WO1 Heavy chain QVQLQQSGPELVRPGVSVKISCKGSGYTFTDYSMHWVKQSHAKS (chains B, E, H) LEWIGVISPYSGNTKYNQKFKDKATMTVDKSSSTAFMELARLTSE DSAIFYCARGITTGYYAMDYWGQGTSVTVSS (SEQ ID NO:12) CDR1 (H1) 26-GYTFTDYS-33 (SEQ ID NO:13) CDR2 (H2) 51-ISPYSGNT-58 (SEQ ID NO:14) CDR3 (H3) 97-ARGITTGYYAMDY-109 (SEQ ID NO:15) Light chain DIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQR (chains A, D, G) PGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFILRISRVEAEDVG VYYCMQHLEYPLTFGAGTKLELK (SEQ ID NO:16) CDR1 (L1) 32-KSLLHSNGNTY-42 (SEQ ID NO:17) CDR2 (L2) 60-RMS-62 CDR3 (L3) 99-MQHLEYPLT-107 (SEQ ID NO:18)

[0141] Although the stoichiometry of the complex is 1:1, with three EB MOMP molecules binding three mAb-18b Fab molecules, each Fab interacts with the extracellular VDs of two EB MOMP molecules, inducing significant local structural changes (see FIG. 10C-G, and FIG.12). The RMSD calculated between EB MOMP and the MOMP / mAb-18b structures is 2.0 Å. While the β-barrel segment of the trimer is unchanged upon Fab binding, VD1 (E61 to E82) undergoes dramatic rearrangements, with the largest main chain shift of ~8 Å at D71. VD4 (L284 to T310) is also rearranged, with large main chain shifts in the segment from S301 to I309, with T307 requiring an ~12 Å shift for optimal superposition (see FIG. 10E-G, and FIG.12). The molecular density map for VD1 residues is well resolved, while the density for two VD4 residues in the most rearranged region suggests disorder (see FIG. 12). The most probable conformation was selected for the final model of this region based on molecular density and the pattern of chemical interactions.

[0142] The conformational changes in EB MOMP upon mAb-18b binding also alter the local environment that originally coordinated the Mg2+cation (see FIG.10F and FIG.12E). The Oδ1 of D311 (CD51) rotates away from its coordinating position, while the side chain of I309280 (VD41) moves into the space previously occupied by two water molecules coordinating Mg2+. Although alternative modes of Mg2+binding cannot be entirely ruled out, the dominant conformation is incompatible with Mg2+binding in its original position. In Ct, antibody neutralization efficiency decreases with increased Mg2+concentration. The structures presented here are consistent with this observation, as additional energetic costs would be incurred to induce conformational change of EB MOMP VDs, which are stabilized by Mg2+, during antibody binding and neutralization.

[0143] The interaction interface between mAb-18b Fab and EB MOMP results in a total buried interaction area of ~1,175 Å2 (see FIG.13A-B) and a shape complementarity (Sc) ofAttorney docket No.00058-089WO1 0.62, both values consistent with the expectations for an antibody-protein complex. Each Fab molecule interacts with two MOMP protomers, engaging residues in the complementary- determining regions (CDRs) of both the light (L) and heavy (H) chains (see FIG.13A-I and Table 3). The Fab primarily interacts with one EB MOMP protomer which contributes ~90% of the interface buried area, while the second protomer contributes ~10% of the buried area (see FIG.10C-D and FIG.13C). CDR1 (L1) and CDR3 (L3) of the mAb-18b Fab L chain interact with VD11 and VD21 (see FIG.13C-E) through four H-bonds. Although L2 does not directly participate in binding, R60 from L2 stabilizes Y104 from H3 through π-π interactions, ensuring the optimal orientation of the Y104 side chain for interactions with the Cβ of A145 (VD21). Additionally, interactions between EB MOMP and L1 reduce the solvent-accessible area of VD31. All three CDRs of the Fab H chain interact with EB MOMP (see FIG.13C and F-I). H1 interacts with VD21and VD42, H2 interacts with VD11, VD21, VD41and VD42, while H3 interacts with VD11and VD21. These interactions involve ten H- bonds with the first EB MOMP protomer and one H-bond with the second. Binding is further stabilized by non-bonding interactions at all interfaces. Neutralization with mAb-18b happens most likely through steric hindrance, with bound antibody shielding EB MOMP surfaces from binding to host cells.

[0144] The recognition mode and conformational changes induced by antibody binding in the EB MOMP / mAb-18b complex will enable computational simulations to explore the dynamics of antigenic cap structural changes for Ct serovars.

[0145] VDs, adhesion model and subunit vaccine development. Experiments using Cm in a mouse model clearly demonstrated that the three-dimensional structure of EB MOMP is critical for inducing robust humoral and cellular immune responses. Chlamydial vaccine development models invoke VD-mediated interactions between the surface-exposed EB MOMP and host cell surfaces. These interactions are likely responsible for species / serovar specificity and tissue tropism, although the exact mechanism and molecular targets are uncertain. Based on trypsin digestion experiments, Ct EB MOMP has been described as a non-specific adhesin, interacting with the host cell surface through VD2 and VD4 for serovar B and only VD4 for serovar L2. Researchers have proposed that heparan sulfate-like glycosaminoglycans (GAGs) act as adhesion receptors, but the reported results are inconsistent. A loss-of-function screen in human haploid cells for the Ct L2 serovar revealed that sulfation levels, rather than the amount of heparan sulfate, are critical for EBAttorney docket No.00058-089WO1 attachment, clarifying some of the earlier discrepancies. The Ct A-K serovars infect the epithelia, while the LGV isolates affect the reticuloendothelial system, so the EB MOMP adhesion mechanism may differ between serovars.

[0146] In the Cm EB MOMP structure, VD1, VD2 and VD4 are extracellularly exposed, with 325 VD2 and VD4 interacting with the neutralizing mAb-18b antibody raised against Cm EB. The negatively charged antigenic head of the EB MOMP trimer, formed by VDs, also contains positively charged patches and grooves, including the N-acetylglucosamine- binding site (see FIG.14A, FIG.8D-E and FIG.15), which may be an interaction site for sulfated proteoglycans on host cells. While other proteins contribute to adhesion MOMP’s high concentration on the EB likely makes it important in the early stages of attachment.

[0147] The species-specific constant region TTLNPTIAG (located in VD4), conserved across MOMPs from all Ct serovars, corresponds to 292-TTWNPTISG-300 (SEQ ID NO:10) in the Cm EB MOMP structure (see FIG.14B, FIG.4, and FIG.7; and Table 4). This sequence induces neutralizing antibodies in both species and is solvent-inaccessible in some Ct serovars. It has been proposed as the host cell binding site and is included in the MOMP- based vaccine CTH522, which has completed two Phase I clinical trials. The species-specific constant region does not induce an immune response by itself but requires the flanking regions and repeats to do so. Table 4. Sequences inducing neutralizing antibodies in Cm and sequences of the extended VD4s from Ct serovars used in the CTH522 vaccine. Cm sequence MTTWNPTISGSGI (SEQ ID NO:19) CTH522 Ct vaccine extVD4DNMFTPYIGVKWSRSAFDSDTIRIAQPKSATAIFDTTTLNPTIAGAGDVKTGAE GQLGDTMQIVSLQLN (SEQ ID NO:20) extVD4ENMFTPYIGVKWSRSAFDSDTIRIAQPKSATAIFDTTTLNPTIAGAGDVKASAE GQLGDTMQIVSLQLN (SEQ ID NO:21) extVD4FNMFTPYIGVKWSRSAFDSDTIRIAQPRLVTPVVDITTLNPTIAGSGSVAGANTE FQISDTMQIVSLQLN (SEQ ID NO:22) extVD4GNMFTPYIGVKWSRASFDSNTIRIAQPKLAKPVVDITTLNPTIAGSGSVVAANS EGQISDTMQIVSLQLN (SEQ ID NO:23) The species-specific constant region of VD4 is in bold typeface.Attorney docket No.00058-089WO1

[0148] In both structures, this species-specific nonapeptide is not solvent-accessible but participates in trimerization and contributes to ligand-binding sites occupied by N- acetylglucosamine and lauric acid molecules (see FIG.14). In the EB MOMP structure, residues from this motif also coordinate water molecules around an Mg2+ion (see FIG.14A). A significant structural rearrangement in VD4 upon mAb-18b binding occurs in the segment immediately following the motif (see FIG.10, FIG.14 and FIG.15). It was postulated that during EB adhesion, an appropriate host receptor induces a conformational change in the antigenic cap, larger than that caused by mAb-18b binding, exposing this hidden motif for binding to another host factor. The cap structure shielding the species-specific nonapeptide provides an evolutionary advantage because mutants unable to adhere to host cells cannot replicate and are removed from the population but still divert the host immune response toward the exposed regions of VDs.

[0149] The antigenic cap structure helps explain how antigenic variability arises at the molecular level, including species and serovar specificity, tissue tropism, and adhesion modulation. The cap is formed by three copies of all VDs folded into a single domain, with the most divergent sequences exposed on the surface and at the ligand-binding sites (see FIG. 14D). In this arrangement, even a single amino acid substitution is amplified by the trimeric organization of MOMP leading to coordinated changes in three-dimensional structure, surface charge and complementarity, and conformational dynamics, which together influence adhesion specificity and interactions with the host immune system. Such functional impact of minor sequence variations has been observed for Ct A serotype, where infections with two strains differing by only two residues in the VDs produced distinct neutralizing antibody titers. The role of MOMP as a reservoir of antigenic variability is also supported by the observation that the nonsynonymous mutation rate in cap-forming regions is ~3 times higher than in the rest of the protein.

[0150] mAb-18b binds to the EB MOMP VDs, where predicted and observed B-cell epitopes are located. However, resolving intracellular infections requires T-cell mediated immunity, which is critical for protection against Ct-induced upper genital tract pathology. To assess the relevance of the structure, data from the Immune Epitope Database (IEDB) were mapped for Ct serovar D, which has most published epitope data, onto a Cm MOMP- based homology model of Ct serovar D MOMP. Mapping results show that B-cell epitopes are more frequent in the extracellular antigenic head, while T-cell epitopes are more prevalentAttorney docket No.00058-089WO1 in the conserved parts of EB MOMP, with VD3 harboring both B-cell and T-cell epitopes (see FIG.16). Reconciling the serovar / serogroup specificity of Ct MOMP with the location of T-cell epitopes in the conserved β-strands presents an immunological paradox that requires further studies. The lack of information on chlamydial non-linear B- and T-cell protective epitopes limits our ability to generate synthetic antigens. The identification of the antigenic structure recognized by mAb-18b is the first step towards solving this problem.

[0151] Characterization of the envelope organization of infectious elementary body (EB) in Cm and Ct serovars A, F, and L2 using cryoET. An 11 Å resolution cryoET reconstruction of purified Cm EB was obtained, which demonstrates the feasibility of the near-molecular-resolution structural analysis (see FIG.18). Separate batches of the same preparation were crosslinked with UV light or formaldehyde to render the material non- infectious and to assess potential morphological artifacts before large-scale data collection. Both treatments produced morphologically similar EBs without defects in organizational defects, indicating preserved structural integrity. CryoEM grids were prepared using either a Thermo Fisher Vi-trobot (formaldehyde-treated samples) or a Leica EM GP2 (UV-treated samples). Grids were vitrified in liquid-ethane cooled by liquid nitrogen. Data were collected on a Titan Krios microscope (300 kV, 64,000× nominal magnification, 1.9 Å pixel size) using parallel-beam illumination, a dose rate of 10.2 e⁻ / px / s, and 41 tilts per series. Motion and CTF corrections were performed in RELION 5.0, fol-lowed by tomogram reconstruction in IMOD and denoising with cryoCARE. From 99,523 extracted subtomograms, iterative 3D classification and re-extraction yielded a final dataset of 3,882 subtomo-grams (2× binned, C3 symmetry), achieving 11 Å resolution with a cylindrical mask focused on the outer membrane. The resulting map revealed a well-preserved tri-partite envelope and a quasi- hexagonal lattice of MOMP oligomers, confirming that native EBs remain structurally intact after inactivation and are suitable for in situ subtomogram averaging and molecular mapping of the chlamydial outer mem-brane complex (COMC). Compact nucleoids were observed, asymmetric arrays of the T3SS, and intermediate developmental forms (see FIG.18A-B). During averaging, nucleoid compactness and membrane separation served as criteria for tomogram selection.

[0152] The envelope of chlamydial elementary bodies (EBs) governs infectivity and immune recognition, yet it’s in situ molecular organization remains incompletely defined. EBs are ~300 to 400 nm rigid particles with inner and outer membranes and a Sarkosyl-Attorney docket No.00058-089WO1 insoluble COMC stabilized by extensive disulfide crosslinking that is developmentally regulated and confers mechanical stability. COMC composition is dominated by MOMP (~60%), together with the cysteine-rich proteins OmcA and OmcB (~15% each), which scaffold the envelope and present key antigens to the host. The EB adhesion mechanism is not fully understood, but a consensus model includes: (1) low-affinity reversible tethering to the heparan sulfate (HS)- containing glycocalyx driven by surface proteins, including MOMP and OmcB, with strong serovar dependence (L2≫E), (2) higher-affinity adhesin interactions, likely medicated by OmcB and selected Pmps that cluster re-ceptors, and (3) rapid type III secretion system (T3SS) activation with translocation of early effectors that remodel actin, trigger uptake, and initiate inclusion formation, accompanied by contact-dependent rearrangements of the injectosome. EB-to-RB conversion begins with envelope reorganization: the EB outer membrane is thicker than the RB outer membrane, and intermembrane spacing expands as development proceeds. EB and RB volumes differ by ~25 to 50-fold. These developmental and geometric variables determine what can be successfully averaged during cryoET STA. cryoET has revealed EB polarity with focal T3SS enrichment and host-contact-induced T3SS remodeling. In studies presented herein, non-uniform T3SS distribution was observed, implying that envelope patterning, including local protein packing, could tune entry and early inclusion remodeling. Serovar-linked entry phenotypes further argue for mapping EB envelope organization across multiple serovars. Comparative work shows that Ct L2 is more dependent on heparan-sulfate (HS) interactions than Ct E, which often uses HS-independent routes; OmcB functions as an adhesin with biovar-specific differences in glycosaminoglycan binding, and multiple Pmps also contribute to attachment in serovar dependent manner. Together, these observations reinforce that EB surface organization, and not just composition, varies across biovars selected for these studies (A, F, L2), with likely consequences for tropism and immune recognition. Methodologically, recent advances now make molecular mapping of intact bacterial envelopes realistic: direct electron detection, robust CTF / dose workflows, modern STA, and membrane-aware analysis that registers particle orientation and packing along curved surfaces. In sum, defining EB envelope organization in Cm and in Ct serovars A, F, and L2 establishes the next layer of structural hierarchy needed to interpret molecular determinants of MOMP serospecificity.

[0153] Characterization of MOMP from Ct serovar F (genitourinary strain) using cryoET SPR. The studies presented above were carried out with MOMP from ChlamydiaAttorney docket No.00058-089WO1 muridarum (Cm) and MOMP from serovar A (ocular strain) of Chlamydia trachomatis (Ct). Structures from additional serovars of the Major Outer Membrane Protein (MOMP) were characterized using cryogenic electron microscopy single particle reconstructions (cryoEM SPR). In particular, the structure of MOMP from Ct serovar F (genitourinary strain) was solved. In addition, the same serovar was reconstructed in the presence of a neutralizing antibody that comprised the antigenic cap structure, although the antibody itself was not resolved. The serovar F structure shows, as expected, a nearly identical transmembrane β- barrel trimer, but the antigenic head differs in charge distribution, hydrophobicity, shape, and hydration compared to Cm and serovar A MOMP (see FIG.19). These features dictate how the infectious form of chlamydia interacts with the host cells, because binding is modulated by electrostatics, hydrophobicity, shape complementarity, and solvation of interacting surfaces.

[0154] The structure of serovar F MOMP in complex with neutralizing antibody E-4 shows a weakened density in the cap region, indicating partial opening. This suggests flexibility in variable regions and that trimer stability is maintained through interactions among the barrels and the lower part of the head. Expression constructs need therefore be stabilized either below or above the opening regions (see FIG.20), depending on the intended use, for example a decoy (closed form) can be stabilized by cross-linking the head while for a cell-interacting construct the interactions stabilizing trimeric or trimer-like structures should be permissible to the structure opening. This flexibility supports the possibility that, outside of the EB form, MOMP may exist as a monomer.

[0155] Designing soluble constructs of MOMP that preserves the overall fold or comprises a grafted antigenic head. Several constructs were designed using AlphaFold 3 with or without the use of ProteinMPNN to generate the soluble constructs with the overall fold preserved or comprise a grafted antigenic head on the scaffold. Examples of the constructs are presented in FIG.21, showing that surface solubility can be increased using AI tools without major structural changes in the antigenic head, and that the antigenic head can maintain its structure in trimeric and circular constructs, as well as when grafted onto modified sequence of human fatty acid binding protein 4 (FABP4) as one of several possible scaffolds. Four categories of artificial and recombinant constructs are presented: Category A: The original MOMP sequence (in these examples, Chlamydia muridarum (CM)) is modified. The antigenic head sequence (highlighted and underline sequence in FIGs.22-Attorney docket No.00058-089WO1 28) but the transmembrane (insoluble) region sequence was modified. These constructs, CM_MOMP_01 (SEQ ID NO:24) to CM_MOMP_030-3 (SEQ ID NO:30), become progressively more soluble. However, there is still sequence similarity to the original CM MOMP, even in the modified regions. Category B: A construct, CM_MOMP_HO (SEQ ID NO:31), containing only the antigenic head with the collar (the collar is considered part of the antigenic head) (see FIG.29). Parts of it are connected with GSS-type linkers. The loops in the head can fold into a trimeric structure on their own. Category C: A construct, CM_MOMP-CIRC (SEQ ID NO:32), where three pieces were combined into a single sequence (see FIG.30), again being connected with GSS-type linkers. Category D: A construct, M_MOMP_H20 (SEQ ID NO:33), where the antigenic head was grafted onto an artificial scaffold (see FIG.31), a 10-stranded soluble beta barrel based on FABP4, a lipocalin, where the sequence for FAB4 was modified by replacing its loops, removing some residues, and mutating others. It is envisaged that other types of lipocalins may also be used as artificial scaffolds. Examples of which are presented in Flower et al., Biochim Biophys Acta 1482(1-2):9-24 (2000), which is incorporated herein in full.

[0156] A number of embodiments have been described herein. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of this disclosure. Accordingly, other embodiments are within the scope of the following claims.

Claims

Attorney docket No.00058-089WO1 WHAT IS CLAIMED IS:

1. An artificial or recombinant construct that expresses a polypeptide sequence for an antigenic head portion of a Chlamydia Major Outer Membrane Protein (MOMP) in its three- dimensional conformation.

2. The artificial or recombinant construct of claim 1, wherein the antigenic head portion is from Chlamydia pneumoniae, Chlamydia muridarum, Chlamydia avium, Chlamydia buteonis, Chlamydia caviae, Chlamydia crocodili, Chlamydia felis, Chlamydia gallinacea, Chlamydia poikilotherma, Chlamydia abortus, Chlamydia psittaci, Chlamydia serpentis, Chlamydia suis, Chlamydia pecorum, or Chlamydia trachomatis.

3. The artificial or recombinant construct of claim 2, wherein the antigenic head portion is from Chlamydia trachomatis or Chlamydia pneumoniae.

4. The artificial or recombinant construct of claim 3, wherein the antigenic head portion is from a serovar of Chlamydia trachomatis that is selected from A, B / Ba, C, D / Da, E, F, G / Ga, H, I / Ia, J, K, L1, L2, L2a and L3.

5. The artificial or recombinant construct of claim 4, wherein the Chlamydia trachomatis serovar is selected from A, E, F, D / Da, J, L2, and G / Ga.

6. The artificial or recombinant construct of claim 1, wherein the artificial or recombinant construct additionally expresses polypeptide sequences for the β-barrel transmembrane portion and β-barrel extracellular collar of a Chlamydia MOMP.

7. The artificial or recombinant construct of claim 6, wherein the β-barrel transmembrane portion and β-barrel extracellular collar is from Chlamydia muridarum.

8. The artificial or recombinant construct of claim 6, wherein the polypeptide sequences for the β-barrel transmembrane portion and / or β-barrel extracellular collar of a Chlamydia MOMP comprises one or more substitutions of non-cysteine amino acids to cysteines to stabilize the Chlamydia MOMP by the formation of disulfide bridges.Attorney docket No.00058-089WO1 9. The artificial or recombinant construct of claim 6, wherein the polypeptide sequences for the β-barrel transmembrane portion and / or β-barrel extracellular collar of a Chlamydia MOMP have proline substitutions for non-proline amino acids, wherein the amino acid side chains from the surrounding amino acids have accommodating or favorable interactions with the substituted proline amino acids, and wherein the β-barrel transmembrane portion and / or β-barrel extracellular collar has consistent main chain angles of what is expected for prolines.

10. The artificial or recombinant construct of claim 6, wherein the polypeptide sequences for the β-barrel and its extracellular collar have been modified or changed so that the expressed polypeptide has increased solubility in aqueous medium while still retaining the antigenic head portion of a Chlamydia MOMP in its three-dimensional conformation.

11. The artificial or recombinant construct of claim 10, wherein the artificial or recombinant construct has a polypeptide sequence that is at least 95% identical to SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:

30.

12. The artificial or recombinant construct of claim 10, wherein the artificial or recombinant construct has a polypeptide sequence that is at least 98% identical to SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:

30.

13. The artificial or recombinant construct of claim 10, wherein the artificial or recombinant construct has a polypeptide sequence that is at least 98% identical to SEQ ID NO:28 or SEQ ID NO:

29.

14. The artificial or recombinant construct of claim 6, wherein the polypeptide sequence for the β-barrel transmembrane portion has been modified or changed so that the β-barrel transmembrane portion has been reduced or truncated.

15. The artificial or recombinant construct of claim 14, wherein the artificial or recombinant construct has a polypeptide sequence that is at least 95% identical to SEQ ID NO:30 or SEQ ID NO:31.Attorney docket No.00058-089WO1 16. The artificial or recombinant construct of claim 14, wherein the artificial or recombinant construct has a polypeptide sequence that is at least 98% identical to SEQ ID NO:30 or SEQ ID NO:

31.

17. The artificial or recombinant construct of claim 1, wherein the artificial or recombinant construct additionally expresses a polypeptide sequence for an artificial scaffold that replaces the β-barrel transmembrane portion of a Chlamydia MOMP.

18. The artificial or recombinant construct of claim 17, wherein the artificial scaffold is derived from a three-helix bundle, a transmembrane portion of a porin protein, a lipocalin, or a transmembrane portion of a cell adhesion protein.

19. The artificial or recombinant construct of claim 18, wherein the artificial scaffold is derived from a polypeptide sequence for a lipocalin.

20. The artificial or recombinant construct of claim 19, wherein the artificial or recombinant construct has a polypeptide sequence that is at least 95% identical to SEQ ID NO:

33.

21. The artificial or recombinant construct of claim 19, wherein the artificial or recombinant construct has a polypeptide sequence that is at least 98% identical to SEQ ID NO:

33.

22. The artificial or recombinant construct of claim 17, wherein the artificial scaffold is a zinc-finger scaffold that uses zinc-cysteine coordination for stabilization.

23. The artificial or recombinant construct of claim 22, wherein the zinc-finger scaffold is based on a C2H2-zinc finger carrying motif.

24. The artificial or recombinant construct of claim 23, wherein the C2H2-zinc finger carrying motif is selected from KLF1, KLF8, KLF11, ZEB2, GLI1, IKZFI, and CTCF.Attorney docket No.00058-089WO1 25. The artificial or recombinant construct of claim 17, wherein the artificial scaffold provides multiple 3-fold axes to create more complex arrangements.

26. The artificial or recombinant construct of claim 25, wherein the multiple 3-fold axes provide for tetrahedral, octahedral or icosahedral symmetries.

27. The artificial or recombinant construct of claim 1, wherein the antigenic head portion of a Chlamydia MOMP has been stabilized by domain swapping, wherein the domain swapping can be in the polypeptide sequence for the antigenic head portion of a Chlamydia MOMP, or in some other polypeptide sequence of the artificial or recombinant construct, so long as the antigenic head portion of a Chlamydia MOMP is presented in its three- dimensional conformation.

28. The artificial or recombinant construct of claim 1, wherein the artificial or recombinant construct expresses a plurality of polypeptide sequences for multiple antigenic head portions of a Chlamydia MOMP.

29. The artificial or recombinant construct of claim 28, wherein the multiple antigenic head portions are from different species of Chlamydia MOMPs.

30. The artificial or recombinant construct of claim 29, wherein at least one of the species of Chlamydia is Chlamydia trachomatis or Chlamydia pneumoniae.

31. The artificial or recombinant construct of claim 28, wherein the multiple antigenic head portions are from different serovars of Chlamydia trachomatis.

32. The artificial or recombinant construct of claim 31, wherein the different serovars of Chlamydia trachomatis are selected from A, E, F, D / Da, J, L2, and G / Ga.

33. The artificial or recombinant construct of claim 1, wherein the artificial or recombinant construct additionally expresses a polypeptide sequence for a periplasmic loop of a Chlamydia MOMP with / or without the N-terminal stopper segment removed.Attorney docket No.00058-089WO1 34. The artificial or recombinant construct of claim 33, wherein the artificial or recombinant construct additionally expresses another polypeptide sequence for the N- terminal stopper segment extension of the periplasmic loop.

35. The artificial or recombinant construct of claim 1, wherein the recombinant or artificial construct further expresses a polypeptide sequence for a T cell epitope.

36. A vaccine preparation or formulation that comprises the artificial or recombinant construct of any one of claims 1 to 35.

37. The vaccine preparation or formulation of claim 36, wherein the artificial or recombinant construct comprises polynucleotides that encode the polypeptide sequence for an antigenic head portion of a Chlamydia MOMP in its three-dimensional conformation, and wherein the vaccine preparation or formulation is a polynucleotide-based vaccine.

38. The vaccine preparation or formulation of claim 37, wherein the polynucleotides comprise DNA, RNA, modified RNA, or hybrids of DNA and RNA.

39. The vaccine preparation or formulation of claim 38, wherein liposomes, virus like particles or polymeric nanoparticles are used as a delivery vehicle for the polynucleotides that encode the polypeptide sequence for the antigenic head portion of a Chlamydia MOMP, and wherein the polynucleotides are found within the delivery vehicle as cargo, or on the surface of the delivery vehicle, or both.

40. The vaccine preparation or formulation of claim 36, wherein the vaccine preparation or formulation further comprises one or more adjuvants.

41. The vaccine preparation or formulation of claim 40, wherein the one or more adjuvants are selected from a CpG oligodeoxynucleotide based adjuvant, ssRNA, an aluminum salt, Montanide ISA 720, CAF01, AS04, MF59, AS01B, and / or CpG 1018.Attorney docket No.00058-089WO1 42. A vaccine preparation or formulation which comprises an antigenic head portion of a Chlamydia MOMP expressed from the artificial or recombinant construct of any one of claims 1 to 35.

43. The vaccine preparation or formulation of claim 42, wherein liposomes, virus like particles or polymeric nanoparticles are used as a delivery vehicle for the antigenic head portion of the Chlamydia MOMP, and wherein the antigenic head portion of the Chlamydia MOMP is found within the delivery vehicle as cargo, or on the surface of the delivery vehicle, or both.

44. The vaccine preparation or formulation of claim 42, wherein the vaccine preparation or formulation further comprises one or more adjuvants.

45. The vaccine preparation or formulation of claim 44, wherein the one or more adjuvants are selected from a CpG oligodeoxynucleotide based adjuvant, ssRNA, an aluminum salt, Montanide ISA 720, CAF01, AS04, MF59, AS01B, and / or CpG 1018.

46. A vaccine preparation or formulation which comprises an antigenic head portion of a Chlamydia Major Outer Membrane Protein (MOMP) that is selected from Chlamydia pneumoniae, Chlamydia muridarum, Chlamydia avium, Chlamydia buteonis, Chlamydia caviae, Chlamydia crocodili, Chlamydia felis, Chlamydia gallinacea, Chlamydia poikilotherma, Chlamydia abortus, Chlamydia psittaci, Chlamydia serpentis, Chlamydia suis, Chlamydia pecorum, or Chlamydia trachomatis in its three-dimensional conformation.

47. The vaccine preparation or formulation of claim 46, wherein the antigenic head portion of the Chlamydia MOMP is from Chlamydia trachomatis or Chlamydia pneumoniae.

48. The vaccine preparation or formulation of claim 47, wherein the Chlamydia MOMP is from a Chlamydia trachomatis serovar selected from A, B / Ba, C, D / Da, E, F, G / Ga, H, I / Ia, J, K, L1, L2, L2a and L3.

49. The vaccine preparation or formulation of claim 46, wherein the MOMP is from a Chlamydia trachomatis serovar selected from A, E, F, D / Da, J, L2, and G / Ga.Attorney docket No.00058-089WO1 50. The vaccine preparation or formulation of claim 46, wherein liposomes, virus like particles or polymeric nanoparticles are used as a delivery vehicle for the antigenic head portion of the Chlamydia MOMP, and wherein the antigenic head portion of the Chlamydia MOMP is found within the delivery vehicle as cargo, or on the surface of the delivery vehicle, or both.

51. The vaccine preparation or formulation of claim 46, wherein the vaccine preparation or formulation further comprises one or more adjuvants.

52. The vaccine preparation or formulation of claim 47, wherein the one or more adjuvants are selected from a CpG oligodeoxynucleotide based adjuvant, ssRNA, an aluminum salt, Montanide ISA 720, CAF01, AS04, MF59, AS01B, and / or CpG 1018.

53. A method of immunizing a subject to protect against an infection by a Chlamydia bacterium, comprising: administering one or more therapeutically effective doses of the vaccine preparation or formulation of claim 36 to a subject 54. The method of claim 53, wherein multiple doses of the vaccine preparation or formulation are administered to the subject at different time points.

55. The method of claim 54, wherein the mode of administration is selected from intravenous administration, intraperitoneal administration, intramuscular administration, intracoronary administration, intraarterial administration, subcutaneous administration, transdermal delivery, intratracheal administration, subcutaneous administration, intraarticular administration, intraventricular administration, inhalation, nasal, oral, pulmonary administration, impregnation of a catheter, and direct injection into a tissue.

56. A method of immunizing a subject to protect against an infection by a Chlamydia bacterium, comprising: administering one or more therapeutically effective doses of the vaccine preparation or formulation of claim 42 to a subject.Attorney docket No.00058-089WO1 57. The method of claim 56, wherein multiple doses of the vaccine preparation or formulation are administered to the subject at different time points.

58. The method of claim 56, wherein the mode of administration is selected from intravenous administration, intraperitoneal administration, intramuscular administration, intracoronary administration, intraarterial administration, subcutaneous administration, transdermal delivery, intratracheal administration, subcutaneous administration, intraarticular administration, intraventricular administration, inhalation, nasal, oral, pulmonary administration, impregnation of a catheter, and direct injection into a tissue.

59. A method of immunizing a subject to protect against an infection by a Chlamydia bacterium, comprising: administering one or more therapeutically effective doses of the vaccine preparation or formulation of claim 46 to a subject 60. The method of claim 59, wherein multiple doses of the vaccine preparation or formulation are administered to the subject at different time points.

61. The method of claim 60, wherein the mode of administration is selected from intravenous administration, intraperitoneal administration, intramuscular administration, intracoronary administration, intraarterial administration, subcutaneous administration, transdermal delivery, intratracheal administration, subcutaneous administration, intraarticular administration, intraventricular administration, inhalation, nasal, oral, pulmonary administration, impregnation of a catheter, and direct injection into a tissue.

62. A method of detecting or monitoring an infection by a Chlamydia bacterium in a subject, comprising: contacting a sample from the subject with a diagnostic agent comprising an antigenic head portion of a Chlamydia MOMP in its three-dimensional conformation, wherein if the subject has antibodies to the antigenic head portion of a Chlamydia MOMP is indicated by detectable change in the diagnostic agent, andAttorney docket No.00058-089WO1 wherein if the subject does not have antibodies to the antigenic head portion of a Chlamydia MOMP results in no change in the diagnostic agent.

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