Immunogenic proteins for gonorrhea

Abstract

Immunogenic peptides for the prevention and treatment of Neisseria gonorrhoeae or gonorrhea-associated diseases and conditions are provided. [Solution] An immunogenic peptide for the prevention and treatment of Neisseria gonorrhoeae or gonorrhea-related diseases and conditions is provided, which is an immunogenic fragment comprising a specific sequence of a Neisserial Heparin Binding Antigen (NHBA) protein of an isolated Neisseria genus of Neisseria gonorrhoeae.

Description

[Technical Field]

[0001] The present invention relates to Neisseria gonorrhoeae or gonorrhea-related diseases and conditions. The present invention relates to immunogenic peptides for the prevention and treatment of various conditions. [Background technology]

[0002] The continued emergence of multidrug-resistant strains of Neisseria gonorrhoeae has led to a rise in gonorrhea It is a major challenge to the control of sexually transmitted diseases (1, 2) and is a key issue facing the World Health Organization. ganization(3), Centers for Disease Control l(4), and Australian National Antimicrobial The Antimicrobial Resistance (AMR) Strategy (5) is a strategy to tackle the urgent need for action. The study prioritized N. gonorrhoeae as an urgent public health threat, with 1 million cases reported annually worldwide. There are an estimated 606 million cases of gonorrhea (6), and infection rates are rising (e.g., For example, over the past five years, there has been a 67% increase in cases in the United States (7) and in Australia (8). There was an 80% increase in the number of cases (8). The outcome of N. gonorrhoeae infection was The incidence varies by site of infection and gender (reviewed in 9, 10), with asymptomatic infection and localized Symptomatic infections include: If left undiagnosed and / or untreated, gonorrhea can It can cause serious sequelae (such as pelvic inflammatory disease, pregnancy and neonatal complications, and infertility). Infection with N. gonorrhoeae can be a risk factor for acquiring and transmitting HIV. It also increases the risk.

[0003] the high prevalence of N. gonorrhoeae and the severe sequelae it can cause; and Due to the increasing difficulty in treating multidrug-resistant strains, there is a need to develop vaccines to prevent infection. However, there are various challenges in developing a gonorrhea vaccine. High levels of phase and antigenic variation in the surface structure of onorrhoeae and protective immunity following infection. the lack of established evidence of protection to guide preclinical vaccine research. (Reviewed in 9, 11). Summary of the Invention

[0004] Surprisingly, the present inventors have found that Neisseria gonorrhoeae Heparin-binding antigens of Neisseria genus (N) from Gonorrhoeae eisserial Heparin Binding Antigen)(NHBA) Administration of C-terminal fragments of proteins results in higher levels of cytotoxicity than the full-length protein, particularly in relatively good cytotoxicity. Antibody production associated with bactericidal and opsonophagocytic killing in gonococcal strains with lower NHBA expression It was found that the production of

[0005] Thus, in its broadest form, the present invention provides a NHBA protein (shown in SEQ ID NO: 1) or immunogenic and not related to Neisseria gonorrhoeae or and its fragments, variants, or derivatives, which are capable of eliciting an immune response against Neisseria gonorrhoeae. In a preferred embodiment, the NHBA protein has the amino acid sequence shown in SEQ ID NO:2. or a fragment, variant, or derivative thereof.

[0006] A first aspect of the present invention is a method for treating Neisseria gonorrhoeae (Neisseria Heparin-binding antigens of isolated Neisseria species from Gonorrhoeae. (NHBA) protein.

[0007] Preferably, the isolated NHBA protein has the amino acid sequence shown in SEQ ID NO: 1, or or a fragment, variant, or derivative thereof.

[0008] In a specific embodiment, the immunogenic fragment comprises the C-terminal fragment of the isolated NHBA protein. Fragment (the amino acid sequence shown in SEQ ID NO: 2, or a fragment, variant, or Derivatives, etc.

[0009] Suitably, the variant or derivative of SEQ ID NO: 2 is selected from residues 3, 5, 6, 9, 20, 50, 57, 60, 61, 69, 71, 75, 76, 83, 88, 89, 91, 92, 9 3, 113, 135, 150, 152, 153, 167, 173, 177, 180, and 181. More particularly, one of SEQ ID NO: 2. or multiple amino acid substitutions, such as A3V, I5M, P6L, P9S, G20E, P50S, R57S, G60A, E61K, A69V, T71A, N75S, G76R, M83T, P88S, Y89C, S91T, G92R, G93S, S113G, T135N, G15 0D, A152V, G153D, A167T, G173S, G177D, D180E, R 181Q, and any combination thereof.

[0010] In some embodiments, the immunogenic fragment is one of the isolated NHBA proteins. or multiple heparin-binding residues and / or one or more active site residues. In other embodiments, the immunogenic fragment is the heparin-binding fragment of the isolated NHBA protein. residues and / or active site residues.

[0011] In a second aspect, the present invention provides a method for the treatment of a leukemia comprising one or more immunogenic fragments according to the first aspect. The present invention relates to an isolated protein containing the nucleotide sequence.

[0012] In a third aspect, the present invention provides an immunogenic fragment of the first aspect or an isolated antibody of the second aspect. a nucleotide sequence encoding the desired protein, or a nucleotide sequence complementary thereto The present invention relates to an isolated nucleic acid comprising a nucleotide sequence.

[0013] In a fourth aspect, the present invention provides a genetic construct comprising the isolated nucleic acid of the third aspect. Provide a project.

[0014] In a fifth aspect, the present invention provides a host cell comprising a genetic construct of the fourth aspect. It exists.

[0015] In a sixth aspect, the present invention provides an isolated immunogenic fragment of the first aspect or an isolated immunogenic fragment of the second aspect. A method for producing an isolated protein comprising: (i) culturing a host cell of the fifth aspect. and (ii) extracting the immunogen from the host cells cultured in step (i). and isolating the functional fragment or protein.

[0016] In a seventh aspect, the present invention provides an immunogenic fragment of the first aspect or an isolated antibody of the second aspect. and providing antibodies or antibody fragments that bind or are raised against the selected proteins. do.

[0017] In an eighth aspect, the present invention provides a method for the treatment of a leukemia comprising administering to a patient a therapeutically effective amount of one or more immunogenic fragments of the first aspect, The isolated protein of the first aspect, the isolated nucleic acid of the third aspect, the genetic construct of the fourth aspect the construct, the host cell of the fifth aspect, and / or the antibody or antibody fragment of the seventh aspect. , optionally together with a pharmaceutically acceptable diluent, carrier, or excipient. do.

[0018] Suitably, the composition is an immunogenic composition (such as a vaccine).

[0019] In a ninth aspect, the present invention provides a method for the treatment of Neisseria gonorrhoeae bacteria and and / or immune response to Neisseria meningitidis A method of eliciting the antibody comprising administering one or more immunogenic fragments of the first aspect; the isolated protein; the isolated nucleic acid of the third aspect; the genetic construct of the fourth aspect. a host cell of the fifth aspect; an antibody or antibody fragment of the seventh aspect; and / or an eighth aspect the composition; and administering the composition to a subject, thereby eliciting an immune response. Provide the law.

[0020] In a tenth aspect, the present invention provides a method for treating Neisseria gonorrhoeae and and / or Neisseria meningitidis immunity a method for inducing a virulence factor comprising administering to said mammal one or more immunogenic fragments of the first aspect; The isolated protein; the isolated nucleic acid of the third aspect; the gene construct of the fourth aspect a host cell of the fifth aspect; an antibody or antibody fragment of the seventh aspect; and / or an antibody of the eighth aspect. a composition similar to that described above; Immunization against E. coli and / or Neisseria meningitidis The method further relates to the method comprising the step of inducing in an elephant.

[0021] In an eleventh aspect, the present invention provides a method for treating Neisseria gonorrhoeae and and / or Neisseria meningitidis infections A method for treating or preventing a disease comprising administering to said patient one or more immunogenic fragments of the first aspect; The isolated protein of the embodiment; the isolated nucleic acid of the third embodiment; the gene construct of the fourth embodiment. a host cell of the fifth aspect; an antibody or antibody fragment of the seventh aspect; and / or administering to a subject the composition of any one of the eighth aspects of the present invention, thereby preventing or preventing Neisseria gonorrhea. hoeae and / or Neisseria meningitidis infections The method comprises the step of preventing or treating in a subject.

[0022] In a twelfth aspect, the present invention provides a method for treating Neisseria gonorrhoeae and and / or Neisseria meningitidis binding to cells A method of at least partially inhibiting or preventing in a subject, comprising the steps of one of the first and second aspects or a plurality of immunogenic fragments; the isolated protein of the second aspect; the isolated protein of the third aspect The nucleic acid; the genetic construct of the fourth aspect; the host cell of the fifth aspect; and the antibody of the seventh aspect. or an antibody fragment; and / or a composition of the eighth aspect; to a subject, thereby Inhibits or inhibits the binding of Eisseria gonorrhoeae to target cells The method further comprises the step of preventing.

[0023] In a thirteenth aspect, the present invention provides a method for treating Neisseria gonorrhoeae and and / or serum resistance in Neisseria meningitidis infections A method for at least partially inhibiting or reducing the a plurality of immunogenic fragments; an isolated protein of the second aspect; an isolated nucleic acid of the third aspect the gene construct of the fourth aspect; the host cell of the fifth aspect; the antibody or or an antibody fragment thereof; and / or a composition of the eighth aspect; to a subject, thereby Neisseria gonorrhoeae and / or Neisseria meni inhibiting or reducing serum resistance of a B. ngitidis infection in a subject. The present invention relates to the above-mentioned method.

[0024] In a fourteenth aspect, the present invention provides a method for detecting Neiss in a biological sample obtained from a subject. Neisseria gonorrhoeae and / or Neisseria meningit A method for detecting idis, comprising: treating a biological sample with an antibody or antibody fragment of the eighth aspect. thereby detecting N. gonorrhoeae and / or or N. meningitidis.

[0025] In a fifteenth aspect, the present invention provides one or more immunogenic fragments of the first aspect; The isolated protein of the second aspect; the isolated nucleic acid of the third aspect; the gene coding sequence of the fourth aspect. a host cell of the fifth aspect; an antibody or antibody fragment of the seventh aspect; and / or the composition of the eighth aspect; the use of Neisseria gonorrhoeae Targeting immune responses to Neisseria meningitidis and / or Neisseria meningitidis caused by Neisseria gonorrhoeae and / or Ne Inducing immunity in subjects against Isseria meningitidis Neisseria gonorrhoeae and / or Neisseria m Treating or preventing infection with Neis eningitidis in a subject; Seria gonorrhoeae and / or Neisseria meningitidis The method at least partially inhibits or prevents binding of Bacillus itidis to cells in a subject. and / or Neisseria gonorrhoeae and / or Serum resistance in Neisseria meningitidis infections was investigated in a small number of subjects. said use in the manufacture of a medicament for at least partially inhibiting or reducing Regarding.

[0026] In a sixteenth aspect, the present invention provides a method for treating Neisseria gonorrhoeae and and / or the immune response to Neisseria meningitidis caused by Neisseria gonorrhoeae and / or Neisseria gonorrhoeae Inducing immunity in a subject against Seria meningitidis; Eisseria gonorrhoeae and / or Neisseria meningitidis Treating or preventing infection with Neisse ingitidis in a subject; Neisseria gonorrhoeae and / or Neisseria meningitidis and at least partially inhibiting or preventing binding of Bacillus idis to cells in a subject. and / or Neisseria gonorrhoeae and / or Ne Serum resistance in Isseria meningitidis infections is low For use in, or for, inhibiting or reducing, even partially When used, one or more immunogenic fragments of the first aspect; the isolated fragment of the second aspect the isolated protein of the third aspect; the isolated nucleic acid of the fourth aspect; the genetic construct of the fourth aspect; a host cell of the fifth aspect; an antibody or antibody fragment of the seventh aspect; and / or a combination of the eighth aspect. It exists in the composition.

[0027] Preferably, the subject of the foregoing embodiments is a human.

[0028] As used herein, the indefinite articles "a" and "an" refer to a singular or plural element. As used herein, "a" or "an" refers to or encompasses an element or feature. should not be considered to mean or define a "single" element or feature. do not have.

[0029] Unless the context otherwise requires, "comprise", "compris es), and the words "comprising" or similar terms An enumerated list of elements or features is a representation of those specified or listed elements. It does not merely include elements, but also includes other elements or features not listed or explicitly stated. As can be seen, a non-exclusive inclusion is intended to be implied.

[0030] By "consisting essentially of" in the context of amino acid sequences (e.g., immunogenic fragments) , an amino acid listed together with an additional 1, 2, or 3 amino acids at the N-terminus or C-terminus An array is meant. [Brief explanation of the drawings]

[0031] [Figure 1] Overview of gonococcal NHBA. (a) Schematic of the NHBA protein from N. gonorrhoeae strain 1291, showing the signal peptide region (open box) and arginine-rich region (Arg; gray box). The recombinant proteins used in the study, mature NHBA (NHBA; lacking the predicted signal peptide) and the C-terminal fragment of NHBA (NHBA-c), are also shown. (b) Alignment of the amino acid sequences of the 14 major NHBA variants of N. gonorrhoeae. Identical amino acids among all variants are shown as dark gray vertical lines, conserved amino acids among the majority of variants are shown as light gray, and mismatches or gaps are shown as white. The number of NHBA peptides is shown on the left, and the percentage of isolates in PubMLST containing this variant is shown on the right. (c) Neighbor-joining phylogenetic tree of the 14 major NHBA variants. The four NHBA variants present in the strains used in this study are underlined. (d) Amino acid alignment of the four NHBA variants present in the strains used in this study. Matches to the consensus sequence (shown in the bottom row) are indicated by dots, the arginine-rich region is indicated by a dashed line, and the NHBA-c fragment is indicated by a line. [Figure 2] Expression of NHBA in a panel of gonococcal strains. Western blot analysis of NHBA expression in (a) N. gonorrhoeae 1291 wild-type (WT), nhba::kan mutant (ΔNHBA), and complemented (ΔNHBA_C) strains, and (b) gonococcal strains used in SBA and OPA assays. Sera used are indicated below the blots. [Figure 3]Antibody binding and complement activation on N. gonorrhoeae measured by fragment deposition. Flow cytometry of antibody binding and antibody-mediated C3 fragment deposition on the surface of N. gonorrhoeae 1291 in the presence of (a) polyclonal antiserum or (b) purified IgG from mice immunized with either NHBA-Freund or NHBA-c-Freund. Values ​​represent the geometric mean fluorescence of antibody binding to N. gonorrhoeae cells and C3 fragment deposition. Controls of secondary antibody only (-ve), preimmune mouse serum (PI), and complement only (C) are included. [Figure 4] Functional blocking activity of NHBA antisera against N. gonorrhoeae. (a) Blocking of NHBA-heparin interaction by α-NHBA antibody. Surface plasmon resonance (SPR) analysis of NHBA-heparin interaction was performed in the absence of serum (0; white) or in the presence of preimmune serum (PI; light gray), α-NHBA serum (medium gray), or α-NHBA-c serum (dark gray). Data represent the average NHBA-heparin binding (+ / - 1 standard deviation) for triplicate samples as the percentage binding in the absence of antibody (no antibody control (white) set at 100%). (b-c) Blocking of N. gonorrhoeae adhesion to epithelial cells. α-NHBA serum and α-NHBA-c serum-treated gonococci significantly reduced adhesion to (b) cervical epithelial cells and (c) urethral epithelial cells at all tested concentrations compared to the untreated control (0; white) (p<0.05, calculated using a two-tailed Student's t-test). Preimmune serum (PI, light gray) did not affect bacterial adhesion (p>0.05). Results are shown as the average percentage of adherent bacteria from triplicate serum-treated samples compared to the no-antibody control (results for the no-antibody control, set at 100%, are 4.33±0.31×103 and 1.37±0.061×103 adherent CFU for cervical and urethral cells, respectively). Error bars represent ±1 standard deviation. Experiments were performed twice with triplicate samples, and representative results are shown. [Figure 5]Expression of NHBA in N. gonorrhoeae. (A) Coomassie-stained SDS-PAGE and (B) Western blot analysis of whole-cell lysates of N. gonorrhoeae 1291 wild-type (WT), nhba::kan mutant (ΔNHBA), and complemented (ΔNHBA_C) strains, probed with the α-NHBA antibody indicated below the blot. The region of the Western blot shown in Figure 2A is boxed. (C) Coomassie-stained SDS-PAGE and (D) Western blot analysis of whole-cell lysates of a panel of N. gonorrhoeae strains. The region of the Western blot shown in Figure 2B is boxed. [Figure 6] Surface plasmon resonance (SPR) analysis of NHBA-heparin interactions. Representative sensorgrams of SPR analysis of recombinant NHBA binding to heparin in the presence of pre-immune serum (heparin and PI), no serum (heparin), or post-immune serum (heparin and α-NHBA) of α-NHBA-Freund. Response units are arbitrary units generated due to mass change on the sensor chip over time. Time is in seconds. [Figure 7]Characteristics and Expression of NHBA Sequences. (A) Schematic diagrams of the Neisseria gonorrhoeae (Ng) strain 1291 NHBA protein and the Neisseria meningitidis (Nm) strain MC58 NHBA protein are shown, with the lipobox motif (gray box) and glycine stretch (black box) indicated at the N-terminus, and the arginine-rich region (white box) indicated in the central portion of the NHBA. The amino acid (aa) length of each protein is indicated on the right. (B) The Ng and Nm NHBA proteins were aligned using ClustalW in MacVector, with identical amino acids indicated as dark gray vertical lines, mismatches indicated as light gray lines, and gaps indicated as white. The amino acid sequences of the arginine-rich region (boxed) and its flanking sequences in the Ng and Nm NHBA proteins are shown, with identical amino acids shaded gray and mismatches indicated in white. The cleavage sites for meningococcal NalP, human lactoferrin (hLf), kallikrein (hKl), and C3 convertase upstream of the Arg-rich region are indicated. (C) Western blot of whole-cell lysates from N. gonorrhoeae 1291 wild-type (WT), NHBA knockout (ΔNHBA), and complemented (ΔNHBA_C) strains grown at 37°C using a polyclonal anti-NHBA antibody. Upregulation of NHBA expression in WT grown at 32°C vs. 37°C is also shown. The periplasmic protein NGAG_01228 is shown as a loading control. (D) Flow cytometry of whole cells from N. gonorrhoeae WT, ΔNHBA, and ΔNHBA_C strains grown at 32°C and 37°C was performed to confirm the expression of NHBA on the cell surface using a polyclonal anti-NHBA antibody. The negative (-ve) control was WT with secondary antibody alone. Values ​​represent geometric mean fluorescence. [Figure 8]Gonococcal NHBA is involved in cell aggregation. (A) Growth and sedimentation curves of N. gonorrhoeae 1291 wild-type (WT), NHBA knockout (ΔNHBA), and complemented (ΔNHBA_C) strains in GC broth, measured by absorbance at 600 nm. (B) N. gonorrhoeae colony-forming units (CFU) per mL of culture at an OD of 1 before and after trypsinization. Countable CFU of the WT and ΔNHBA_C strains increased 2.6- and 2.4-fold after trypsinization (p = 8.1 × 10-5 and 5.1 × 10-4), respectively, whereas CFU of ΔNHBA was unaffected (p = 0.31). *P < 0.05, **P < 0.01, ***P < 0.001 compared to WT for A and B. (C) Flow cytometric analysis of recombinant NHBANg binding to whole-cell N. gonorrhoeae (Ng) (black, Ng only; white, Ng + labeled NHBANg). (D) Whole-cell ELISA titration curve (black line) showing NHBANg binding to N. gonorrhoeae. The antibody-only control curve (dotted line) demonstrates the absence of nonspecific interactions between whole-cell N. gonorrhoeae and His-tag antibody. [Figure 9] Gonococcal NHBAs are involved in microcolony formation. Scanning electron microscopy of N. gonorrhoeae 1291 wild-type (WT), NHBA knockout (ΔNHBA), and complemented (ΔNHBA_C) strains grown for 5 hours on glass slides (top panel) or on human urethral epithelial cell monolayers on glass slides (bottom panel). For the WT and ΔNHBA_C strains, aggregates and microcolonies can be observed, whereas the ΔNHBA strains appear as single colonies or diplococci. Images were captured at 5,000 magnification. The scale bar at the bottom of each box represents 5 μm. [Figure 10]Gonococcal NHBA binds with high affinity to multiple glycans. Surface plasmon resonance (SPR) analysis of NHBANg binding to (A) glycosylaminoglycans (GAGs) and (B) non-GAG glycans. The name, structure, and sulfation pattern (S) of each glycan are shown, along with the dissociation constant (KD) of NHBANg binding to each glycan. *The KD of the NHBANm interaction

[14] is also shown to allow comparison. NHBANm has a higher affinity for β-Glc6P (KD 0.056 + / - 0.025 μM). NB, no concentration-dependent binding. [Figure 11] Gonococcal recombinant NHBA binds to epithelial cells. (A) Confocal fluorescence images of NHBANg binding to endocervical epithelial cells (tCX) captured under 40x magnification. (I) Expanded focus and (II) xyz cross-section of cells. (III) Control images of cells treated with antibody alone (no recombinant NHBA) and (IV) NHBANg and secondary antibody. White arrows indicate proteins localized on the cell surface. (B) Flow cytometric analysis of recombinant NHBANg binding to human endocervical epithelial cells (tCX) and urethral epithelial cells (tUEC). [Figure 12]Gonococcal NHBA contributes to survival in human serum and adherence to human epithelial cells. (A) Survival of N. gonorrhoeae 1291 wild-type (WT), NHBA knockout (ΔNHBA), and complemented (ΔNHBA_C) strains after 60 minutes in 10% (v / v) normal human serum. Data represent the mean percent survival for triplicate samples as a percentage of the inoculum and are shown relative to WT (results for wild-type, set at 100%, are 5.5 × 10 colony-forming units (CFU)). There was no significant difference between WT survival in serum in the absence or presence of heparin. (B) Adhesion of N. gonorrhoeae 1291 wild-type (WT), NHBANg knockout (ΔNHBA), and complemented (ΔNHBA_C) strains to human uterocervical (tCX) and human urethral (tUEC) epithelial cells. (C) Adhesion of N. gonorrhoeae 1291 wild-type (WT) to tCX cells untreated (No Treatment) or pretreated with recombinant NHBANg (1–100 μg / ml) or PNA as a negative control (100 μg / ml). Data represent the mean percent adhesion or invasion for triplicate samples as a percentage of the inoculum and are shown relative to WT (results for WT, set at 100%, were (B) 1.1 × 10 (tCX) and 1.7 × 10 (tUEC), and (C) 6.5 × 10 adherent CFU). Error bars represent + / - 1 standard deviation. *P<0.05, **P<0.01, ***P<0.001 compared to untreated WT using Student's two-tailed t-test. Experiments were performed at least three times, and representative results are shown. [Figure 13-1] Conservation of gonococcal NHBA. Alignment of the eight most common NHBA variants of N. gonorrhoeae (Ng) is shown with the consensus sequence at the top. The N. meningitidis (Nm) NHBA sequence from strain MC58 is also included (NHBA-3). [Figure 13-2] (As mentioned above.) [Figure 13-3] (As mentioned above.) [Figure 13-4] (As mentioned above.) [Figure 14] Expression of NHBA in N. gonorrhoeae. (A) Coomassie-stained SDS-PAGE and Western blot analysis of whole-cell lysates from N. gonorrhoeae 1291 wild-type (WT), nhba::kan mutant (ΔNHBA), and complemented (ΔNHBA_C) strains, probed with α-NHBA and α-NGAG01228 antibodies. The region of the Western blot shown in Figure 1C is boxed. (B) Coomassie-stained SDS-PAGE and Western blot analysis of pilin preparations from N. gonorrhoeae 1291 WT, NHBA, and NHBA_C strains, probed with α-C311 pilin antibody. (C) Coomassie-stained SDS-PAGE of sarkosyl outer membrane protein (OMP) preparations from N. gonorrhoeae 1291 WT, NHBA, and NHBA_C strains, showing the major OMPs, including opacity (Opa) and porin (Por) proteins. (D) Silver-stained SDS-PAGE gel of lipooligosaccharide (LOS) preparations from N. gonorrhoeae 1291 WT, ΔNHBA, and ΔNHBA_C strains. (E) Coomassie-stained SDS-PAGE and Western blot analysis of whole-cell lysates of N. gonorrhoeae 1291 wild-type (WT) strains grown at 32°C and 37°C and probed with α-NHBA. The region of the Western blot shown in Figure 1C is boxed. (F) Flow cytometry of whole cells of N. gonorrhoeae WT, ΔNHBA, and ΔNHBA_C strains grown at 32°C and 37°C with α-NHBA. The negative (-ve) control is WT with secondary antibody only. Values ​​represent geometric mean fluorescence. [Figure 15]Gonococcal NHBA is involved in cell aggregation. (A) Gram staining of N. gonorrhoeae 1291 wild-type (WT), nhba::kan mutant (ΔNHBA), and complemented (ΔNHBA_C) strains without trypsin ("-"; upper panel) or with trypsin treatment ("+"; lower panel). (B) Western blot analysis of trypsin-free (-) and trypsin-treated (+) whole-cell N. gonorrhoeae 1291 WT probed with antibodies to α-NHBA and the periplasmic protein NGAG_01228. [Figure 16] Glycan binding by Neisseria gonorrhoeae. The heat map shows binding by whole-cell N. gonorrhoeae strain 1291 (black bars) to glycans on the array (average of results from three independent experiments). Glycans are clustered into classes based on their terminal sugars. The number and percentage of bound glycans within each class are shown. The complete data set of glycan binding is shown in Table 4. [Figure 17] Amino acid sequences of the full-length (top panel) (SEQ ID NO: 1) and C-terminal fragment (bottom panel) (SEQ ID NO: 2) of the NHBA protein of N. gonorrhoeae 1291. [Figure 18-1] Amino acid sequence variations about the consensus sequence of the C-terminal immunogenic fragment of SEQ ID NO:2 for 41 N. gonorrhoeae NHBA variants. [Figure 18-2] (As mentioned above.) [Figure 19] Immunogenicity of NHBA. ELISA titers of post-immunization serum from each mouse immunized against purified recombinant NHBA with either NHBA-c-Freund or NHBA-c-Alum. Titers for each of five mice are indicated by symbols, and geometric mean titers (GMTs) are indicated by bars. [Figure 20]Serum bactericidal activity (a, c) and opsonophagocytic activity (b) of anti-NHBA antibodies. (a, c) Serum bactericidal activity (SBA) of anti-NHBA sera. Survival of N. gonorrhoeae strain 1291 in the presence of two-fold dilutions of normal human serum and heat-inactivated mouse serum as a source of complement is shown. Sera were either: (a) anti-NHBA-c serum + adjuvant (Freund or alum) compared to no serum (0) and pre-immune (PI) control serum. A "no complement" control (NC) is also shown (bacteria incubated with a 1 / 100 dilution of mouse serum only); or (c) purified anti-NHBA antibodies from NHBA-c-alum serum. (b) Opsonophagocytic activity (OPA) of anti-NHBA sera. Survival of N. gonorrhoeae strain 1291 in the presence of human polymorphonuclear leukocytes (PMNs), normal human serum, and mouse serum is shown as in (a) above. A "no complement" control (NC) (bacteria incubated with a 1 / 400 dilution of mouse serum only) and a "PMN only" control (PMNs) (bacteria incubated with PMNs but without mouse serum and without complement) are shown. For a–c, data represent the average survival for triplicate samples compared to the results obtained with the untreated wild-type strain (0). (Untreated wild-type, set at 100%, represents 2.5 × 10, 1.6 × 10, and 3.5 × 10 colony-forming units for a–c, respectively.) Error bars represent ±1 standard deviation. Survival was compared to untreated wild-type without serum (0) using a two-tailed Student's t-test. *, p<0.05, **, p≦0.01, ***, p≦0.001. Statistical analysis was also performed for (c) using one-way analysis of variance (ANOVA; p<0.0001) and Dunnett's multiple comparison test (p>0.9 for untreated wild-type control (0) vs. 6, 25, or 12.5; p≦0.0001 for 0 vs. 25, 50, or 100 μg / mL). [Figure 21]Survival of Neisseria gonorrhoeae in human serum. Survival of Neisseria gonorrhoeae 1291 wild-type (WT) and NHBA knockout (ΔNHBA) strains in 0-10% (vol / vol) human serum after 30 minutes is shown. The human serum tested was normal human serum preabsorbed with N. gonorrhoeae to remove any antibodies that cross-react with N. gonorrhoeae. This depleted serum was used as a complement source in serum bactericidal activity (SBA) and opsonophagocytic killing (OPA) assays. Data represent the mean survival for triplicate samples compared to the results obtained with the untreated strain (0). (Untreated WT, set at 100%, represents 2.8 × 103 colony-forming units (CFU); untreated ΔNHBA, set at 100%, represents 2.5 × 103 CFU.) Experiments were performed three times, and representative results are shown. A two-tailed Student's t-test was used to compare survival compared to untreated controls. ** *, p≦0.001. There was no significant difference in survival of WT in the % serum tested compared to untreated (0) controls. [Figure 22] Serum bactericidal activity (SBA) of anti-NHBA sera. Survival of N. gonorrhoeae strain 1291 in the presence of two-fold dilutions of normal human serum and heat-inactivated mouse serum as a source of complement is shown. Sera were either anti-NHBA-c serum + Alum or anti-NHBA-c serum depleted of anti-NHBA antibodies + Alum. Data represent the mean survival for triplicate samples compared to the results obtained with the untreated wild-type strain (0) (untreated wild-type, set at 100%, represents 3.3 × 10 colony-forming units). Error bars represent ±1 standard deviation. Survival was compared to untreated wild-type, shown in white (0), using a two-tailed Student's t-test. *, P < 0.05; ***, P ≤ 0.001.

[0032] [Table 1A] DETAILED DESCRIPTION OF THE INVENTION

[0033] The present invention provides a C-terminal fragment of NHBA from Neisseria gonorrhoeae. demonstrated substantially improved immunogenicity against Neisseria gonorrhoeae. Immunization with NHBA protein fragments is bactericidal and opsonophagocytic, It can elicit antibodies that can inhibit the adhesion of gonococci to mucosal epithelial cells.

[0034] A broad aspect of the present invention is an isolated N of Neisseria gonorrhoeae. Immunogenic fragments of the heparin-binding antigen (NHBA) protein of Eisseria genus (SEQ ID NO: containing the amino acid sequence set forth in No. 1, or a fragment, variant, or derivative thereof; etc.)

[0035] Neisseria gonorrhoeae (also known as gonococci(s)) is a leading cause of sexually transmitted urogenital gonorrhea and other gonorrhea-related diseases, disorders, and conditions ( Pharyngeal gonorrhea, rectal gonorrhea, disseminated gonococcal septicemia, gonococcal septic arthritis, and neonatal gonococcal conjunctivitis Generally, the present specification refers to a type of Proteobacteria that causes bacterial infections, such as As used herein, "Neisseria gonorrhoeae" refers to a substance that is Therefore, it includes all strains and serotypes of N. gonorrhoeae that can be identified, and Neisseria gonorrhoeae includes those described in the book. This includes genetic variants of different strains. Whether or not a particular protein is present can be determined by several methods known in the art, including: For example, Chakravorty et al. (2007) (incorporated herein by reference). Examples include sequencing of the ribosomal RNA (rRNA) gene.

[0036] Neisseria meningitidis Heparin-binding The combined antigen (NHBA, formerly called GNA2132) is a component of 4CMenB. It exists as an NHBA-GNA1030 fusion protein (14). A(NHBA Nm ) are surface-exposed lipoproteins that are intrinsically disordered and misfolded. The N-terminal region (residues 200–250) is predicted to be unpopulated (15), and glycans (including heparin, heparin sulfate, and chondroitin sulfate) an arginine-rich region (16–18), and a C-terminus that folds as an antiparallel β-barrel. It consists of three regions: the end regions (15, 19, and 20). Nm is relatively well preserved Although it has been reported that the N-terminal region contains multiple insertions / deletions among different meningococcal strains (15). NHBA Nm induces serum bactericidal antibodies against diverse N. meningitidis strains (17, 21, 22), these antibodies are also opsonophagocytic (23, 24), epithelial It is possible to block the adhesion of N. meningitidis to cells (18) The gonococcal homolog of NHBA (NHBA Ng ) was highly correlated among N. gonorrhoeae strains. Conserved (>93% identity) NHBA-2 peptide variant in 4CMenB The present inventors have identified the NHBA Ng is exposed to the surface Recently, it has been shown that the 4CMenB vaccine can be recognized by antibodies from people exposed to the virus. (13) An example of an NHBA protein from N. gonorrhoeae is shown in SEQ ID NO: It is indicated by 1.

[0037] For purposes of this invention, "isolated" means removed from its natural state or or otherwise subject to human manipulation. Isolated material is material that is in its natural state. It is substantially or essentially free of components that normally accompany the material in its original state. obtained from, or artificially produced in combination with components normally associated with the material in its natural state. Isolated material may be in its native form, in a chemically synthesized form, or in a form that is It may be in recombinant form.

[0038] By "protein" is meant an amino acid polymer. As will be understood, an amino acid may be a natural or unnatural amino acid, a D-amino acid or It may be an L-amino acid.

[0039] The term "protein" is used to describe proteins having 50 or fewer amino acids. describes "peptides" typically used in and proteins with more than 50 amino acids. The term "polypeptide" typically used to describe a polypeptide is intended to encompass and encompass all polypeptides.

[0040] A "fragment" is a segment of a protein that comprises less than 100% of the amino acid sequence of the protein. A ment, domain, part, or region.

[0041] Generally, fragments are 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 250, 300, 400, or 4 The amino acid sequence of up to 25 amino acids (such as the full-length NHBA protein shown in SEQ ID NO: 1) It may include.

[0042] In certain embodiments, the isolated immunogenic fragment of the NHBA protein is between 10 and 2 50 amino acids, more preferably 15 to 190 amino acids, and even more preferably 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 9 0, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140 , 145, 150, 155, 160, 165, 170, 175, 180, or 185 The isolated NHBA protein (shown in SEQ ID NO: 1 or SEQ ID NO: 2) is or consisting of said protein.

[0043] In certain embodiments, the immunogenic fragment comprises the C-terminus of the isolated NHBA protein. As used herein, "C-terminal" as applied to NHBA proteins includes C-terminal fragments. The term "terminal fragment" refers to a fragment located in or corresponding to the C-terminal domain of the NHBA protein. Typically at least about 15, 20, 25, 30, 35, 40, 4 5, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, or may comprise 210 consecutive or stretches of amino acids.

[0044] In one particular embodiment, the immunogenic fragment is the extracellular domain or extracellular domain of the NHBA. or the amino acid sequence set forth in SEQ ID NO: 2, which essentially comprises the domain exposed to It consists of, consists essentially of, or is contained within said sequence.

[0045] In another embodiment, the immunogenic fragment is one or more of the isolated NHBA proteins. The glycan-binding or heparin-binding residues and / or one or more active Contains the functional site residues.

[0046] Thus, the immunogenic fragment corresponds to SEQ ID NO:2 and constitutes a fragment of SEQ ID NO:1, Some of the extracellular or extracellularly exposed domains of BA proteins Alternatively, the immunogenic fragment may comprise only its glycan-binding residues and / or The extracellular domain sequence or the extracellularly exposed sequence contains at least one of the active site residues. It may comprise a fragment of the exposed domain sequence.

[0047] In the context of the present invention, the term "immunogenic" as used herein means When the protein is administered to an animal, it induces an immune response (N. gonorrhea). the ability or potential to produce or induce oeae or its molecular components The immune response may be B-lymphocyte-mediated or T-lymphocyte-mediated, or a combination thereof. Advantageously, by "immunogenic" it is meant to be understood as meaning that the B-link "Immunotoxicity" means, but is not limited to, being capable of eliciting an immune response. "Epidemic-genic" also means capable of eliciting a neutralizing antibody response.

[0048] By "eliciting an immune response" we mean eliciting an immune response that involves the cellular immune system, antibodies, and / or the innate immune system. It means to generate or stimulate the production or activity of one or more elements of the immune system, including Preferably, the one or more components of the immune system include B lymphocytes, antibodies, and In one embodiment, the immune response is a mucosal immune response.

[0049] As used herein, a protein "variant" refers to a protein that has a similar structure to a reference amino acid sequence. share a definable nucleotide or amino acid sequence relationship, e.g., a reference amino acid sequence. The amino acid sequence may be the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. The protein lacks one or more amino acids of the reference amino acid sequence due to the different amino acids. Several amino acids may be deleted or substituted to make the immunogenic fragment and / or protein It is well understood in the art that amino acids can be substituted or deleted without altering the activity of the (conservative substitution). Thus, one of the other residues in SEQ ID NO: 1 or SEQ ID NO: 2 or wherein the variant substantially retains the immunogenicity of SEQ ID NO: 1 or SEQ ID NO: 2. Conservative modifications may be made (e.g., by amino acid substitution or deletion) such that Preferably, the protein variant is at least 70% or 75%, preferably at least 80% or 85%, or more preferably at least 90%, 91%, 92%, 93% %, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the reference It shares the amino acid sequence (SEQ ID NO: 1 or SEQ ID NO: 2, etc.).

[0050] The "wild-type" or "unmodified" NHBA protein fragment sequence (SEQ ID NO: 1 or SEQ ID NO: 2) can substantially improve or enhance the immunogenicity of the immunogenic fragment. By way of example, an immunogenic fragment may be an N. gonorrhoeae fragment of a particular N. gonorrhoeae strain. HBA protein sequence, thereby identifying the strain (referred to herein as The immunogenic fragment may be modified to improve its immunogenicity to other antigens (such as those described in Thus, the term "variant" refers to any of the variants disclosed herein or naturally occurring. existing (e.g., allelic) variants, orthologs (e.g., N. gonorrhoeae ae species (e.g., N. meningitidis), and synthetic variants (abrupt produced in vitro using mutagenesis techniques) or The term also encompasses isolated proteins or fragments thereof comprising the amino acid sequence of any of the above. , the modification encompasses the substitution of one or more amino acids of the NHBA protein fragment.

[0051] Can the variant retain the biological activity of the corresponding wild-type protein (e.g., allelic Gene or strain variants, paralogs, and orthologs (as described in Figure 18) or may lack biological activity compared to the corresponding wild-type protein. or may have substantially reduced biological activity.

[0052] Preferably, the immunogenic fragment comprises residues 3, 5, 6, 9, 20, 50, 57, 6 of SEQ ID NO:2. 0, 61, 69, 71, 75, 76, 83, 88, 89, 91, 92, 93, 113, 1 35, 150, 152, 153, 167, 173, 177, 180, and 181 Contains one or more amino acid substitutions or the amino acid substitutions shown in Figure 18. In embodiments, the one or more amino acid substitutions are a valine at residue 3 of SEQ ID NO:2 (V) amino acid (A3V); methionine (M) amino acid at residue 5 (I5M); a leucine (L) amino acid at residue 6 (P6L); a serine (S) amino acid at residue 9 (P9S); Glutamic acid (E) amino acid at residue 20 (G20E); serine (S) amino acid at residue 50 acid (P50S); serine (S) amino acid at residue 57 (R57S); alanine (A) amino acid at residue 60 (R57S); the lysine (A) amino acid at residue 61 (G60A); the lysine (K) amino acid at residue 61 (E61K); a valine (V) amino acid at residue 69 (A69V); an alanine (A) amino acid at residue 71 (T 71A); serine (S) amino acid at residue 75 (N75S); arginine at residue 76 ( R) amino acid (G76R); threonine (T) amino acid at residue 83 (M83T); residue Serine (S) amino acid at residue 88 (P88S); Cysteine ​​(C) amino acid at residue 89 ( Y89C); threonine (T) amino acid at residue 91 (S91T); arginine (A) amino acid at residue 92 (S91T); a serine (R) amino acid at residue 93 (G93R); a serine (S) amino acid at residue 93 (G93S); A glycine (G) amino acid at residue 113 (S113G); an asparagine (N) at residue 135 ) amino acid (T135N); aspartic acid (D) amino acid at residue 150 (G150D ); a valine (V) amino acid at residue 152 (A152V); an asparagine at residue 153 acid (D) amino acid (G153D); threonine (T) amino acid at residue 167 (A167 T); serine (S) amino acid at residue 173 (G173S); asparagine at residue 177 a carboxylic acid (D) amino acid at residue 177D; a glutamic acid (E) amino acid at residue 180 (D1 80E); the glutamine (Q) amino acid at residue 181 (R181Q); and any combination thereof combinations thereof.

[0053] In one particular embodiment, the variant protein or variant peptide is may contain one or more residues (such as lysine residues) at the N-terminus and / or C-terminus of The lysine residues (e.g., polylysine) can be a linear sequence of lysine residues or can be a lysine These additional lysine residues may be a branched sequence of residues. These additional lysine residues may facilitate increased peptide solubility. It can be done easily.

[0054] Generally, the term "proteins" is used herein to describe the sequence relationships between the respective proteins and nucleic acids. Terms used in this context include "comparison window," "sequence identity," and "percentage of sequence identity." "Percentage of identity" and "Substantial identity" are used to refer to the identity of each nucleic acid / protein. (1) one or more complete nucleic acid / protein sequences shared by the nucleic acid / protein; (2) only multiple parts, and (3) one or more parts that are diverse among nucleic acids / proteins. , so sequence comparison typically involves identifying and comparing local regions of sequence similarity. This is accomplished by comparing sequences over a "comparison window" to determine whether the sequence is a matched match. A "comparison window" is a window of sequences, typically 6, 9, or 12 consecutive sequences, that are compared to a reference sequence. The comparison window is a conceptual segment of residues that is optimally aligned to each sequence. For the test, there should be no more than about 20% additions or deletions (i.e., gaps) compared to the reference sequence. The optimal sequence alignment for aligning the comparison window may include: Computer implementation of the algorithm (Genew by Intelligenetics) orks program;Wisconsin Genetics Software Pa ckage Release 7.0,Genetics Computer Group GAP, BESTFIT, FASTA, and TFASTA in p. 575 Sci ence Drive, Madison, WI, USA, incorporated herein by reference. (incorporated herein) or by any of a variety of selected methods. The inspection performed and the best alignment (i.e., the one with the highest performance over the comparison window) This can be achieved by, for example, Altschul et al. t al., 1997, Nucl. Acids Res. 25 3389 (see The BLAST family of programs disclosed by For a detailed discussion of sequence analysis, see CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds.Ausubel et al.(Jo Wiley & Sons Inc. NY, 1995-1999) Unit 19 .3.

[0055] The term "sequence identity" refers to the degree of sequence identity determined by appropriate alignment using standard algorithms. Taking into account the extent to which the sequences are identical over the comparison window, the exact nucleobase is determined. The term "antibody" is used herein in its broadest sense to include any number of amino acid or peptide matches. Therefore, the "percentage of sequence identity" is calculated by multiplying the 2 over the comparison window. Two optimally aligned sequences are compared and identical nucleobases (e.g., A, T, C, G, U) are identified. ) occurs in both sequences to give the number of matched positions, and Divide the number by the total number of positions in the comparison window (i.e., the window size) and multiply the result by 100. to obtain the percentage of sequence identity. "Oneness" is a program that runs the DNASIS computer program (version 2.0 for Windows). 5;Hitachi Software engineering Co.,Ltd. Available from South San Francisco, California, USA ) may be understood to mean the "match percentage" calculated by

[0056] The present invention also provides derivatives of the immunogenic fragments disclosed herein. The immunogenic fragment comprises the amino acid sequence shown in SEQ ID NO:2.

[0057] As used herein, a "derivative" refers to a compound that is, for example, a compound that is conjugated with another chemical moiety. By cleavage or complexation, post-translational modifications (e.g., ligation) can be achieved, as understood in the art. glycosylation (e.g., addition, removal of glycosylation), or modification), lipidation, and / or incorporation of additional amino acid sequences. A specific derivative is a molecule (such as a protein, a fragment or a variant thereof). This is due to the conjugation of an immunogenic fragment to diphtheria toxin (DT). can be facilitated by the addition of a C-terminal cysteine ​​residue.

[0058] The additional amino acid sequence may comprise a fusion partner amino acid sequence to form a fusion protein. In one example, the fusion partner amino acid sequence may be used to detect and identify the isolated fusion protein. Non-limiting examples include metal-binding (e.g., polyhistidine) fusion proteins. Binding partners: maltose-binding protein (MBP), protein A, glutathione S- transferase (GST), fluorescent protein sequence (e.g., GFP), epitope tag (such as myc, FLAG, and hemagglutinin tags).

[0059] Other additional amino acid sequences may be found in carrier proteins (diphtheria toxoid (DT) or other fragments thereof), or CRM protein fragments (as described in International Publication WO2017 / 070735) In certain embodiments, the immunoglobulins described herein may be used in combination with other immunoglobulins. The immunogenic fragment or isolated protein may be conjugated, coupled, or otherwise packaged with a carrier protein. or otherwise concatenated.

[0060] Other derivatives contemplated by the present invention include modifications to the side chains, peptide or protein Incorporation of unnatural amino acids and / or their derivatives during protein synthesis and cross-linking Uses and conformationally constraining the immunogenic proteins, fragments, and variants of the invention Other methods include, but are not limited to:

[0061] In this regard, those skilled in the art are familiar with a broader range of methodologies for chemically modifying proteins. CURRENT PROTOCOLS IN PROTEIN SCIENCE,E ds.Coligan et al.(John Wiley & Sons NY 1 See Chapter 15 of the 2008 Act.

[0062] In a related aspect, the present invention provides a method for the preparation of a NHBA of N. gonorrhoeae. provides an isolated protein comprising multiple immunogenic fragments. The protein is not the full-length or wild-type NHBA of N. gonorrhoeae. .

[0063] In one particular embodiment, the present invention provides a method for the production of a medicament as defined herein that comprises a plurality of immunogenic fragments. The present invention contemplates isolated proteins (such as in the form of "polytope" proteins) described above. For example, the immunogenic fragments may be present singly or as repeats, including Heterologous amino acid sequences (e.g., "spacer" amino acids) may also be included in the above sequences. It may be included among one or more immunogenic fragments present in the isolated protein.

[0064] In still further embodiments, the invention of this aspect provides: (i) a method for treating a pulmonary arthritis or pulmonary edema as described herein; Immunogenic fragments, or segments, domains, portions, or regions thereof (e.g., epitopes thereof) antigenic determinants), and inclusive fragments, variants, or derivatives thereof; and and (ii) optionally one or more additional amino acid sequences. In this regard, the additional amino acid sequence preferably comprises: The amino acid sequences of the aforementioned proteins may be modified by alterations that may occur at the N-terminus and / or C-terminus. The amino acid sequence may be, but is not limited to, a species amino acid sequence.

[0065] Immunogenic fragments and / or isolated proteins described herein, inclusive The fragments, variants, and derivatives thereof may be prepared by chemical synthesis, recombinant DNA technology, or other methods to produce peptide fragments. These include, but are not limited to, recombinant DNA techniques, and proteolytic cleavage. It may be produced by any means known in the art.

[0066] Chemical synthesis includes solid phase synthesis and liquid phase synthesis. Such methods are well known in the art. However, Synthetic Vaccines Ed. Nicholson (Bla Chapter 9 of the Blackwell Scientific Publications, and and CURRENT PROTOCOLS IN PROTEIN SCIENCE Ed s.Coligan et al.,(John Wiley & Sons, Inc. Examples of chemical synthesis techniques are provided in Chapter 15 of the NY USA 1995-2008 In this respect, reference is made to International Publication No. WO 99 / 02550 and International Publication No. WO 97 / 02550. See also 45444.

[0067] Recombinant proteins can be prepared using methods such as those described in, for example, Sambrook et al., MOLECULAR CLONING.A Laboratory Manual(Cold Spring Harbor Press, 1989), especially sections 16 and 17; URRENT PROTOCOLS IN MOLECULAR BIOLOGY Ed s.Ausubel et al.,(John Wiley & Sons,Inc. NY USA 1995-2008), particularly Chapters 10 and 16; and C URRENT PROTOCOLS IN PROTEIN SCIENCE Eds. Coligan et al.,(John Wiley & Sons, Inc. NY The standard methods described in the IEC 61014-1995-2008, especially in Chapters 1, 5, and 6, These can be conveniently prepared by those skilled in the art using protocols known in the art. Protein preparation involves expression of a nucleic acid encoding the protein in a suitable host cell. .

[0068] In another aspect, the present invention provides the immunogenic fragments and isolated antibodies disclosed herein. a nucleic acid sequence encoding the protein or a nucleic acid sequence complementary thereto Isolated nucleic acids encoding or complementary to the nucleic acid sequences are contemplated.

[0069] Isolated immunogenic proteins, isolated immunogenic fragments, variants, and derivatives of the present invention The nucleotide sequences encoding the polypeptides and polytopes are provided in the complete genomic nucleic acid sequence of NHBA. can be easily deduced from

[0070] This aspect also encompasses fragments, variants, and derivatives of the isolated nucleic acid.

[0071] The term "nucleic acid" as used herein refers to single- or double-stranded DNA. and RNA. DNA includes genomic DNA and cDNA. RNA includes mRNA. A, RNA, RNAi, siRNA, cRNA, and autocatalytic RNA. Nucleic acids are typically made up of A, G, C, T, or includes nucleotide sequences containing nucleotides containing U bases. The sequence is similar to that of other bases (inosine, methylcytosine) methylinosine, methyladenosine, and / or thiouridine, etc.), Not limited to these.

[0072] Thus, in certain embodiments, the isolated nucleic acid is cDNA.

[0073] A "polynucleotide" is a nucleic acid having 80 or more consecutive nucleotides, , an "oligonucleotide" has fewer than 80 contiguous nucleotides.

[0074] A "probe" is a complementary probe, e.g., in a Northern or Southern blot. For the purpose of detecting specific sequences, preferably labeled single-stranded or double-stranded oligonucleotides are used. It may be a peptide or a polynucleotide.

[0075] A "primer" is a single-stranded nucleic acid having preferably 15 to 50 consecutive nucleotides. An oligonucleotide that is capable of annealing to a complementary nucleic acid "template." DNA polymerase (Taq polymerase, reverse transcriptase, or Sequenase) (trademark), etc., in a template-dependent manner.

[0076] In one embodiment, the present invention provides an isolated immunogenic fragment or protein of the present invention. The present invention provides isolated nucleic acid variants encoding the

[0077] In one embodiment, the nucleic acid variant is an isolated protein or immunogen of the invention. The fragment encodes a variant of the nucleotide sequence.

[0078] Suitably, the nucleic acid variants are at least 35%, 40%, 45%, 50%, 55%, 60% or 65%, 66%, 67%, 68%, 69%, preferably at least 70%, 71%, 72%, 73%, 74%, or 75%, more preferably at least 80%, 8 1%, 82%, 83%, 84%, or 85%, and even more preferably at least 9 0%, 91%, 92%, 93%, 94%, or 95% of the clones are identical to the isolated nucleic acids of the present invention. They share nucleotide sequence identity.

[0079] The present invention also contemplates nucleic acids modified through the use of codon sequence redundancy. In the present invention, codon usage is determined to optimize expression of a nucleic acid in a particular organism or cell type. It can be modified as follows.

[0080] The present invention provides modified purines (e.g., inosine, methylinosine) in the isolated nucleic acids of the invention. adenosine, and methyladenosine) and modified pyrimidines (e.g., thiouridine and methylcytosine) The present invention further provides the use of sine.

[0081] CURRENT PROTOCOLS IN MOLECULAR BIOLOGY( Eds.Ausubel et al.John Wiley & Sons NY,1 Standard processes such as those described in Chapters 2 and 3 of the International Standards Act (ISI) 995-2008 The isolated nucleic acids of the present invention can be conveniently prepared using protocols. , as will be well recognized by those skilled in the art.

[0082] In yet another embodiment, the complementary nucleic acid is a nucleic acid of the present invention under high stringency conditions. hybridizes to the nucleic acid of

[0083] "Hybridize" and "hybridization" refer to the formation of DNA-DNA hybrids. , RNA-RNA, or DNA-RNA hybrids. The term "sequence" is used herein to refer to the pairing of nucleotide sequences that are substantially complementary. A hybrid sequence containing the complementary nucleotide sequence is formed through base pairing.

[0084] "Stringency," as used herein, refers to the degree to which a hybridization The temperature and ionic strength conditions during the reaction and the presence of certain organic solvents and / or detergents The higher the stringency, the more hybridizing nucleotides there are. The required level of complementarity between the sequences will be high.

[0085] "Stringent conditions" are those in which only nucleic acids with a high frequency of complementary bases will hybridize. Indicates the conditions for sizing.

[0086] Stringent conditions are described in Ausubel et al., supra (see (incorporated herein by reference) in Chapters 2.9 and 2.10. Those skilled in the art can manipulate various factors to produce hybrids. It will also be appreciated that the specificity of the lysis can be optimized by the final wash sequence. Optimization of the chromatin density can be performed to ensure a high degree of hybridization. do.

[0087] The complementary nucleotide sequence is determined by the presence of nucleotides in the matrix (preferably nitrocellulose). The steps are immobilization on a synthetic membrane (such as a gel), hybridization, and Typically involves a detection step using a labeled probe or other complementary nucleic acid. Southern blotting is a method for identifying complementary DNA sequences. Northern blotting is used to identify complementary RNA sequences. Dot blotting and slot blotting are used to detect complementary DNA / To identify DNA, DNA / RNA, or RNA / RNA polynucleotide sequences Such techniques are well known to those skilled in the art and may be used in conjunction with Ausubel et al., supra. According to this method, the Southern blot The size separation is performed by separating DNA molecules according to their size using gel electrophoresis. The DNA is transferred to a synthetic membrane, and the membrane-bound DNA is hybridized to a complementary nucleotide sequence. An alternative blotting step involves plaque hybridization. cDNA libraries, such as through the process of DNA sequencing or colony hybridization It is used to identify complementary nucleic acids in a DNA or genomic DNA library. Another representative example of the procedure is Sambrook et al., Molecular Cloning ING.A Laboratory Manual(Cold Spring Harb) or Press, 1989), Chapters 8-12.

[0088] Methods for detecting labeled nucleic acids hybridized to immobilized nucleic acids are well known to those of skill in the art. Such methods include autoradiography, chemiluminescence, fluorescence, and colorimetric detection. Examples include:

[0089] Nucleic acids may also be isolated, detected, and / or detected using nucleic acid sequence amplification methods. Recombinant DNA techniques may be performed.

[0090] Suitable nucleic acid amplification techniques, covering both thermal and isothermal methods, are well known to those skilled in the art. Such methods include polymerase chain reaction (PCR); strand displacement amplification (SDA); Repeated circle replication (RCR); nucleic acid sequence-based amplification (NASBA), Q-β replica These include recombinase amplification, recombinase polymerase amplification, and helicase-dependent amplification (RPA). Examples include, but are not limited to:

[0091] As used herein, an "amplification product" refers to a nucleic acid product produced by nucleic acid amplification. Point to something.

[0092] Nucleic acid amplification techniques include qPCR, real-time PCR, and the like, as are well known in the art. Specific quantitative and semi-quantitative techniques such as PCR, mu PCR, and competitive PCR may be mentioned.

[0093] In another aspect, the invention provides (i) an isolated nucleic acid described herein; or or (ii) an isolated nucleic acid comprising a nucleotide sequence complementary thereto, Provide instruction.

[0094] Preferably, the genetic construct is a plant-derived gene, as is well understood in the art. Gene expression vectors on chromosomes, bacteriophages, cosmids, yeast, or bacterial artificial chromosomes. A genetic construct is in the form of or contains the element. the manipulation and / or development of a known nucleic acid or encoded protein by recombinant DNA technology; Suitable for maintenance and propagation of isolated nucleic acids in bacteria or other host cells for expression. obtain.

[0095] For purposes of host cell expression, the genetic construct is an expression construct. Preferably, the expression construct is operable to one or more additional sequences in an expression vector. An "expression vector" is a self-replicating extrachromosomal vector (e.g., a vector encoding a nucleic acid of the present invention) operably linked to a nucleic acid of the present invention. It can be either a vector (such as a plasmid) or a vector that integrates into the host genome.

[0096] By "operably linked" is preferably meant a molecule capable of initiating, regulating, or otherwise controlling transcription. The additional nucleotide sequence may be positioned relative to the nucleic acid of the invention so as to control It is meant.

[0097] The regulatory nucleotide sequences are generally appropriate for the host cell used for expression. Numerous types of appropriate expression vectors and suitable regulatory sequences are known for a variety of host cells. It is well known in the art.

[0098] Typically, the one or more regulatory nucleotide sequences include a promoter sequence, leader or signal sequence, ribosome binding site, transcription initiation sequence and transcription termination sequence ligation sequences, translation initiation and termination sequences, and enhancer or activator sequences. Examples of such sequences include, but are not limited to:

[0099] Constitutive or inducible promoters, as known in the art are contemplated by the present invention.

[0100] The recombinant allergenic protein of the present invention is expressed as a fusion protein as described above. As shown, the expression construct may contain additional nucleotides encoding a fusion partner. The nucleic acid sequence may also include a sequence (typically provided by an expression vector).

[0101] In certain embodiments, the genetic construct is suitable for administration to a subject (such as a human). In a preferred embodiment, the genetic construct is used as a DNA vaccine in a subject (e.g., a human). It is suitable for vaccination.

[0102] Preferably, DNA vaccination involves the use of one or more plasmid DNA expression constructs. Plasmids typically contain viral promoters (SV4 Intron A contains the promoter for stabilizing the mRNA. Plasmids may be included to improve the efficiency and thereby increase protein expression. , multiple cloning site, strong polyadenylation signal / transcription termination signal (bovine The polyadenylation sequence may further comprise a polyadenylation sequence such as that of growth hormone or rabbit β-globulin. Plasmids containing HIV rev. Mas with or without envelope expression increased The on-Pfizer simian virus cis-acting transcription element (MPV-CTE) was Additional modifications that may improve expression include enhancer sequences, synthetic inserts, and the like. ron, insertion of the adenovirus tripartite leader (TPL) sequence, and / or polyadenylation Modifications to the transcription sequence and / or transcription termination sequence of the DNA vaccine plasmid. A limiting example is pVAC, commercially available from Invivogen.

[0103] A useful reference describing DNA vaccinology is DNA Vaccines, Methods s and Protocols,Second Edition(Volume 12 7 of Methods in Molecular Medicine series s, Humana Press, 2006).

[0104] In a further aspect, the present invention provides a nucleic acid molecule or gene described herein. A host cell transformed with the construct is provided.

[0105] Suitable host cells for expression can be prokaryotic or eukaryotic. For example, suitable Host cells include mammalian cells (e.g., HeLa, HEK293T, Jurkat cells) ), yeast cells (e.g., Saccharomyces cerevisiae), baculoviruses Insect cells (e.g., Sf9, Trichopl) used with or without viral expression systems usia ni), plant cells (e.g., Chlamydomonas reinhardt ii, Phaeodactylum tricornutum), or bacterial cells (E. Host cells (prokaryotic or eukaryotic, such as coli) may include, but are not limited to: The introduction of a gene construct into a target organism (even if it is a target organism) can be performed, for example, by using the CURRENT PRO TOCOLS IN MOLECULAR BIOLOGY Eds.Ausubel et al.,(John Wiley & Sons, Inc.1995-2009) and, as described in Chapters 9 and 16 of the is.

[0106] In yet another aspect, the present invention provides an isolated immunogenic antibody as described herein. A method for producing a fragment or isolated protein comprising: (i) transforming a previously (ii) culturing the host cell described above; and (iii) culturing the host cell cultured in step (i). isolating said fragment or protein from the host cell.

[0107] Recombinant proteins can be prepared using methods such as those described in, for example, Sambrook et al., MOLECULAR CLONING.A Laboratory Manual(Cold Spring Harbor Press, 1989), especially sections 16 and 17; URRENT PROTOCOLS IN MOLECULAR BIOLOGY Ed s.Ausubel et al.,(John Wiley & Sons,Inc. 1995-2009), especially Chapters 10 and 16; and CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds.Coligan et al.,(John Wiley & Sons, Inc.1995-2009 ), particularly in chapters 1, 5, and 6, by those skilled in the art. It can be conveniently prepared by a skilled artisan.

[0108] In a further aspect, the present invention relates to the immunogenic fragments and / or or antibodies that bind to and / or are raised against the isolated protein or Antibody fragments are provided.

[0109] Preferably, said antibody or antibody fragment is a polypeptide of said isolated immunogenic fragment and / or protein. It specifically binds to proteins.

[0110] In some embodiments, the antibody binds to one or more glycans and / or substrate molecules. N.gonorrhea to the children (GAG, heparin, heparan sulfate, and chondroitin, etc.) The binding of oeae to NHBA may be reduced, eliminated, inhibited, or prevented. The antibodies detect the binding or proliferation of N. gonorrhoeae to cells (such as epithelial cells in a subject). In a further embodiment, the ability of an anti- The body reduces, eliminates, or prevents the ability of N. gonorrhoeae to induce serum resistance in a subject. In certain embodiments, the antibody may inhibit or suppress N. gonorrhoeae. Induce or mediate complement-dependent lysis and / or opsonophagocytic killing of eae cells .

[0111] Suitably, the antibody or antibody fragment comprises the amino acid sequence shown in SEQ ID NO: 2 or a variant. specifically binds to an isolated immunogenic peptide comprising the sequence, a fragment, or a derivative thereof In some embodiments, the antibody or antibody fragment is directed against the full-length NHBA protein. The antibody was generated using the smallest epitope contained within SEQ ID NO:2 with substantially higher affinity than the antibody generated using the smallest epitope contained within SEQ ID NO:2. In this context, by "substantially higher affinity" is meant to indicate that the NHB binds to the target polypeptide. At least 2, 3, 4, 5, 6, 7, 8, 9, or 10 at a specific concentration of protein A A 2-fold higher affinity is meant.

[0112] Antibodies and antibody fragments may be polyclonal or monoclonal, native, or recombinant. The antibody fragment may be recombinant. The antibody fragment may include an Fc fragment, an Fab fragment, or an F(ab)2 fragment. Such scFvs may comprise, for example, single chain Fv antibodies (scFvs). US Patent No. 5,091,513, European Patent No. 239,400, or Winter & Milstein, 1991, Nature 349:293, respectively. The antibodies can be prepared according to the methods described. scFvs, diabodies and triabodies and / or tetrabodies, and Also included are dimerization-activated demibodies (e.g., WO / 2007 / 062466). By way of example, such antibodies can be found in the literature as described by Holliger et al., 1993 Proc. atl Acad Sci USA 90 6444; or Kipriyanov,2 According to the method described in Methods Mol Biol 562 177 Well-known protocols applicable to antibody production, purification, and use are available, for example, from C. oligan et al.,CURRENT PROTOCOLS IN IMMUN OLOGY(John Wiley & Sons NY,1991-1994) and H arlow, E. & Lane, D. Antibodies: A Laboratory Manual,Cold Spring Harbor,Cold Spring Harbour Laboratory, 1988, Chapter 2.

[0113] Methods for producing polyclonal antibodies are well known to those skilled in the art. Exemplary methods that can be used include: The protocol is described, for example, in Coligan et al., CURRENT PROTOCOL S IN IMMUNOLOGY, op. cit., and Harlow & Lane, 1988, As an example, polyclonal antibodies can be prepared from purified or recombinant NHBs. A protein, or an immunogenic fragment thereof (e.g., SEQ ID NO: 2), is used in the production species (horse, etc. ) and subsequently purified prior to administration.

[0114] Monoclonal antibodies can be prepared by, for example, Kohler & Milstein, 1975, Na Standard methods as originally described in the paper by ture 256,495, or For example, Colligan et al., CURRENT PROTOCOLS IN I The isolated IgGs of the present invention can be prepared using, for example, recent modifications described in MMUNOLOGY, supra. Inoculated with one or more of the proteins, fragments, variants, or derivatives Antibody antibodies may be produced by immortalization of spleen or other antibody-producing cells derived from the same producing species. In certain embodiments, the monoclonal antibody or fragment thereof may be in recombinant form. This is because monoclonal antibodies are primarily produced by the spleen cells of non-human mammals. It may be particularly advantageous to "humanize" a monoclonal antibody or fragment if .

[0115] The antibodies and antibody fragments of the present invention are the isolated immunogenic fragments and antibodies described herein. and / or may be particularly suitable for affinity chromatography purification of proteins, for example: Coligan et al., CURRENT PROTOCOLS IN IMMU The affinity chromatography procedure described in TECHNOLOGY, supra, Chapter 9.5 It can be referenced.

[0116] In some embodiments, the antibody or antibody fragment provides "passive" immunity to gonorrhea. The compound may be administered to a mammal to provide the compound.

[0117] In another embodiment, an isolated immunogenic fragment of an NHBA described herein Antibodies or antibodies raised against or binding fragments and / or proteins The fragments can be used to detect NHBAs expressed on the cell surface.

[0118] Certain further aspects and embodiments of the present invention relate to the use of steroid hormones in animals (more particularly humans). and a composition for preventing, treating, and / or immunizing against a gonorrhea-associated disease, disorder, or condition. Provides products and / or methods.

[0119] In one such embodiment, the present invention provides a method for preventing or treating a gonorrhea-associated disease, disorder, or condition. The composition for treating the disease comprises (i) one or more of the immunoglobulins described herein. (ii) one or more of the antigenic fragments and / or proteins described herein; (iii) one or more of the isolated proteins described herein; (iv) one or more gene constructs described herein; and / or (v) an immunogenic fragment or isolated protein (as defined herein) one or more antibodies that bind to or are raised against or antibody fragments, optionally together with a pharmaceutically acceptable diluent, carrier, or excipient. It may be included in.

[0120] A "pharmaceutically acceptable carrier, diluent, or excipient" makes the compound safe for systemic administration. This means any solid or liquid filler, diluent, or encapsulating material that may be used in any application. Depending on the particular route of administration, a variety of carriers well known in the art may be used. These carriers include sugar, starch, cellulose and its derivatives, malt, gelatin, tartar, etc. Calcium sulfate, vegetable oil, synthetic oil, polyol, alginic acid, phosphate buffer solution, emulsifier substances, isotonic saline and salts (mineral acid salts including hydrochlorides, bromides, and sulfates), organic acids ( acetate, propionate, and malonate), and pyrogen-free water. may be selected from:

[0121] Useful references describing pharmaceutically acceptable carriers, diluents, and excipients include Rem. ington's Pharmaceutical Sciences(Mack Pu blishing Co. NJUSA, 1991) (hereby incorporated by reference) (This is incorporated herein by reference).

[0122] In one embodiment, the pharmaceutical composition of the present invention is an immunogenic composition. The immunogenic composition is preferably a vaccine.

[0123] As generally used herein, the terms "immunize," "vaccinate," and "vaccinate" refer to The term "cutin" refers to the condition in which subsequent infection with N. gonorrhoeae occurs at least partially. Specifically, a protective immune response to N. gonorrhoeae is prevented or minimized. It refers to a method and / or composition for inducing

[0124] Thus, such compositions, upon administration to a subject, inhibit the growth of N. gonorrhoeae cells. for the purpose of generating at least partial and preferably protective immunity to the bacterium, or The antibodies may be delivered to generate an immune response, preferably a protective immune response, but are not limited to: do not have.

[0125] By "protective immunity" is meant that a response to an antigen(s) is achieved by rapid binding of said antigen(s) and / or leads to elimination and therefore subsequent N. gonorrhoeae in animals (e.g., human subjects). This means a level of immunity sufficient to at least partially ameliorate or prevent infection with E. ae. can be.

[0126] A "protective immune response" may protect animals (e.g., human subjects) from current and / or future N. Preventing or reducing the severity, symptoms, aspects, or characteristics of a gonorrhoeae infection. By this is meant a level of immune response sufficient to induce a response.

[0127] In another specific embodiment, the immunogenic composition is a composition as described herein for passive immunization of a subject. The antibody or antibodies disclosed herein may be one or more of the antibodies disclosed herein.

[0128] Suitable vaccines may be in the form of proteinaceous vaccines, particularly those containing N. gonorrhoeae. one or more immunogenic fragments of the NHBA protein of B. oeae, or The term "antibody" includes fragments, variants, or derivatives thereof as described above.

[0129] The immunogenic compositions and / or vaccines of the present invention may be prepared in an "immunologically acceptable carrier, diluent, It will be appreciated from the above that the term "a substance or excipient" may include "a substance or excipient."

[0130] Useful carriers are well known in the art and include, for example, thyroglobulin; albumin ( human serum albumin, etc.); toxins, toxoids, or tetanus, diphtheria, pertussis, Ps eudomonas, E. coli, Staphylococcus, and Strep Any mutant cross-reacting material (CRM) of toxins from the genus Ptococcus; polyamines Poly(lysine:glutamic acid), etc.; influenza; rotavirus VP6, pal Hepatitis B virus VP1 and VP2; Hepatitis B virus core protein; Hepatitis B virus Alternatively, a carrier protein or other immunogen may be used. Fragments or epitopes of proteins can be used, e.g., bacterial toxins, toxoids. or a T cell epitope of a CRM may be used. No. 85,973, which is incorporated herein by reference.

[0131] "Immunologically acceptable carriers, diluents or excipients" include, within their scope, water, bicarbonate buffer, phosphate buffered saline, or saline, and / or As understood in the art, "ajuva" refers to an adjuvant that is well known in the art. A "vaccine agent" consists of one or more substances that enhance the immunogenicity and efficacy of a vaccine composition. It means a composition made of

[0132] Preferably, for purposes of eliciting an immune response, the specific immunizing agent is one of the agents described herein. in combination with or in conjunction with the immunogenic fragments or isolated proteins described therein. The term "immunizing agent" includes within its scope carriers, delivery agents, , immunostimulants, and / or adjuvants known in the art. As is understood in the art, immunostimulants and adjuvants can improve the immunogenicity of a composition. and / or refers to or includes one or more substances that promote efficacy.

[0133] Non-limiting examples of suitable adjuvants or immunostimulants include squalane and squalene. (or other oils of vegetable or animal origin); block copolymers; detergents ( Tween® 80); Quil® A, mineral oil (Drakeol or Marcol, etc.), vegetable oil (peanut oil, etc.); Corynebacterium spp. (Co Propionib (derived from Rynebacterium parvum, etc.) Ajuva derived from acterium genus (Propionibacterium acne etc.) Mycobacterium bovis (Calmette-Guerin bacillus) or BCG); Bordetella pertussis antigen; tetanus toxoid; Diphtheria toxoid; surfactants (hexadecylamine, octadecylamine, octadecylamine) Tadecyl amino acid ester, lysolecithin, dimethyldioctadecylammonium bromide N,N-dicoctadecyl-N',N'bis(2-hydroxybenzoate) (hydroxyethyl-propanediamine), methoxyhexadecylglycerol, and Pluroni Polyamines (pyran, dextran sulfate, poly Ic carbopol, etc.) ); Peptides (muramyl dipeptide and derivatives, dimethylglycine, tuftsin, etc.); Oils emulsions; and mineral gels (aluminum phosphate, aluminum hydroxide, or mineral gels) alum, etc.); interleukins (interleukin 2 and interleukin 12, etc.); Monokines (interleukin 1, etc.); tumor necrosis factors; interferons (gamma interferons) combination (saponin aluminum hydroxide or Quil A aluminum hydroxide) liposomes; ISCOM® adjuvants and ISCOMATRI® X® adjuvant; mycobacterial cell wall extract; synthetic glycopeptide (Mura) Mildipeptide or other derivatives, etc.); Abridine; Lipid A derivatives; Dextran sulfate Salt; DEAE dextran alone or with aluminum phosphate; Carboxypolymethy Acrylic copolymer emulsion (Neocry) A640, etc.) (e.g., U.S. Patent No. 5,047,238); water-in-oil emulsifying substances (Mo nitanide ISA 720, etc.); poliovirus, cowpox, or animal poxvirus proteins; or mixtures thereof.

[0134] With regard to subunit vaccines, examples of such vaccines include ISCOM (International Publication No. WO 99 / 09999). The compositions may be formulated by methods such as those described in US Pat. No. 7,454,444.

[0135] An example of a vaccine in the form of a water-in-oil formulation is Montanide ISA 720. (such as those described in International Publication WO97 / 45444).

[0136] Any suitable procedure is contemplated for the production of vaccine compositions. Exemplary procedures include: For example, New Generation Vaccines (1997, Levine et al., Marcel Dekker, Inc. New York, Basel , Hong Kong) (incorporated herein by reference). Some examples include:

[0137] Alternatively, the vaccine may be in the form of a nucleic acid vaccine and in particular a DNA vaccine. A useful reference describing vaccinology is DNA Vaccines, Methods and Protocols,Second Edition(Volume 127 of Methods in Molecular Medicine series , Humana Press, 2006), which is incorporated herein by reference. will be done.

[0138] In some embodiments, the isolated immunogenic proteins and / or fragments of the invention The fragments can be used as vaccines in purified form or fused to an immunogenic carrier protein. or a live vaccine delivery system (attenuated virus, virus-like particle, or live attenuated cell It can be expressed by various organisms, including bacteria.

[0139] In other embodiments, the compositions and vaccines of the present invention comprise one or more single Attenuated vectors that can be induced to express isolated immunogenic proteins or immunogenic fragments. They may be administered to humans in the form of bacteria or inactivated bacteria. Non-limiting examples of attenuated bacteria include: Salmonella species (e.g., Salmonella enterica var. Typh Alternatively, other Enteric pathogens (such as Shigella species or E. coli) are used in attenuated forms. Attenuated Salmonella strains can be produced by inactivating genes in the aromatic amino acid biosynthetic pathway. By doing so (Alderton et al., Avian Diseases 35 435), into two genes in the aromatic amino acid biosynthetic pathway (U.S. Patent No. 5, 770,214), or in other genes such as htrA (U.S. Pat. No. 5,627,214). 5,980,907), or outer membrane proteins such as ompR. introducing mutations into the genome (such as those described in U.S. Patent 5,851,519); It was constructed by

[0140] In one embodiment, the antigen composition comprises outer membrane vesicles (OMVs). OMVs are Gram-positive It occurs naturally in bacterial cells and is composed of proteins, lipids (mostly LPS), and periplasmic OMVs are non-replicating spherical nanoparticles consisting of a vesicle. Neisseria species (e.g., Neisseria gonorrhoeae and / or Ne Isseria meningitidis or cultured strains of E. coli OMVs can be prepared from naturally secreted or detergent-extracted outer membranes. It can be obtained by any method known in the art (e.g., Gerritzen t al.2017,Biotech Adv.35:565-574;Semchen ko et al.2017, Infect Immun 85(2)e00898-1 In certain embodiments, the immunogenic fragments and / or isolated proteins of the present disclosure Proteins can be surface-exposed, non-surface-exposed, bound to OMVs, or not bound to OMVs (i.e., The immunogenic fragments and / or isolated fragments may be formulated with OMVs for simple mixing. The isolated proteins and OMVs can be simultaneously produced by Gram-negative bacteria, resulting in The OMVs may contain immunogenic fragments and / or monoclonal antibodies loaded onto the surface or in the lumen of the OMV. Alternatively, immunogenic fragments and / or isolated proteins may be produced. The protein is covalently coupled using an affinity tag on the antigen that binds to the fusion protein in the OMV. The nucleotide sequences can be attached to the OMVs after production, for example by conjugation (see, e.g., Alves et al. l.,2015,ACS Appl.Mater.Interfaces,7(44): (See also 24963-24972.) Still further, immunogenic fragments and / or isolated tags Proteins can be loaded into the lumen of the OMV after it has been produced, or they can be loaded into the lumen of the OMV. After production, the immunogenic fragments and / or monoclonal antibodies of the present disclosure can be simply mixed with the OMVs. Exemplary OMVs for use as adjuvants with isolated proteins include any graft. Negative bacteria (N. meningitidis, N. gonorrhoeae, E. coli i, and P. aeruginosa) The bacterial species from which OMVs are derived may be either alone or in combination with other Neisseria gonorrhoeae antigen and / or Neisseria meningitid expression or expression of the NHBA protein in the OMV from which it is derived in combination with the is antigen It is further contemplated that the nucleotide sequence may be genetically modified for enhanced or upregulated expression. can be.

[0141] Proteins, peptides, fragments, or fusion proteins containing transport or immunogenic functions Protein expression can be in the cytoplasm, the cell wall, exposed on the cell surface, or in a secreted form. This may result in the production of immunogenic proteins, peptides, or fragments produced in the host.

[0142] In another aspect, the present invention provides Neisseria gonorrhoeae and / or or induce an immune response to Neisseria meningitidis in a subject. A method of inducing a leukemia virus comprising administering to a subject a leukemia virus containing one or more immunogenic fragments described herein; The isolated protein described herein; the isolated the nucleic acids described herein; the genetic constructs described herein; a host cell comprising the antibody or antibody fragment described herein; and / or a composition similar to the above; and administering the composition to a subject, thereby eliciting an immune response. Regarding the method.

[0143] Preferably, the method prevents or improves a gonorrhea-associated disease, disorder, or condition in a subject. eliciting or enhancing an immune response in said subject for prophylactic or therapeutic treatment. do.

[0144] In a related aspect, the invention provides Neisseria gonorrhoeae and and / or Neisseria meningitidis immunity A method for inducing one or more immunogenic fragments described herein. an isolated protein as described herein; an isolated the nucleic acid described herein; the genetic construct described herein; a host cell in which the antibody or antibody fragment described herein is to be produced; an antibody or antibody fragment described herein; and / or a host cell in which the antibody or antibody fragment described herein is to be produced; administering to a subject a composition as described in the specification; thereby gonorrhoeae and / or Neisseria meningitidis The method further comprises inducing immunity to the bacteria in the subject.

[0145] Preferably, the immune response or immunity to N. gonorrhoeae bacteria is a disease associated with gonorrhea. In addition, different Neisseria species are The protein sequences (especially the C-terminal sequences) for the NHBA proteins are Due to the homology, the method can be used to identify additional Neisseria species (Neisseria It is understood that the virus can also be used to immunize animals against viruses such as D. meningitidis. will be recognized by the business.

[0146] In a further aspect, the present invention provides Neisseria gonorrhoeae and and / or Neisseria meningitidis infections A method for treating or preventing a disease comprising administering one or more immunogens described herein. a functional fragment; an isolated protein as described herein; a protein as described herein the isolated nucleic acid as described herein; the genetic construct as described herein; a host cell as described herein; an antibody or antibody fragment as described herein; and / or administering to a subject a composition described herein; thereby gonorrhoeae and / or Neisseria meningitidis The method comprises the step of preventing or treating an infection of a subject with Bacillus subtilis. .

[0147] Similar to the previous two embodiments, the method may further comprise the step of detecting additional Neisseria species (Nei (including but not limited to Escherichia coli meningitidis) It may also be used to treat animals.

[0148] As used herein, "treating" (or "treating" or "treatment") "treatment" refers to the treatment of a gonococcal- or meningococcal-related disease, disorder, or condition after symptoms have begun. Refers to a therapeutic intervention that ameliorates signs or symptoms. The term "ameliorating" refers to the treatment of gonorrhea-related or or any observable beneficial treatment effect with respect to a meningococcal-associated disease, disorder, or condition. A treatment does not necessarily have to be beneficial to the subject. can be determined using any method or criteria known to those skilled in the art.

[0149] As used herein, "preventing" (or "preventing" or "prevention") "prevention" means a treatment or treatment for gonorrhea-related or meningitis that prevents or reduces the symptoms, aspects, or characteristics of the disease. Procedures initiated prior to the onset of symptoms, aspects, or characteristics of a fungus-related disease, disorder, or condition ( a therapeutically effective amount of one or more immunogenic proteins of the present invention and / or fragments thereof; Such prevention refers to the administration of a composition containing a compound, variant, or derivative thereof to a subject. It should be understood that a "preventive" treatment need not be absolute to be beneficial. Developing symptoms, aspects, or characteristics of a meningococcal-related or meningococcal-related disease, disorder, or condition for the purpose of reducing the risk of developing a gonorrhea-related or meningococcal-related disease, disorder, or It is a treatment administered to subjects who do not show signs of a disease state or who show only early signs.

[0150] The term "therapeutically effective amount" refers to a defined amount of a drug (such as an isolated drug described herein) administered to a patient. immunogenic fragments, isolated proteins, and antibodies or antibody fragments) by the drug The term "anticoagulant" refers to an amount sufficient to achieve the desired effect in the subject being treated. For example, this may include any gonorrhea-related or meningococcal-related disease, disorder, or condition (gonococcus or meningococcus). and / or to reduce, alleviate, and / or prevent a bacterial infection (including a fungal infection) as described herein. The isolated immunogenic fragments, isolated proteins, and / or antibodies described in In some embodiments, the term "therapeutically effective" may be used to refer to a therapeutically effective amount of a composition comprising an antibody fragment. "amount" reduces or eliminates symptoms of a gonorrhea-associated or meningococcal-associated disease, disorder, or condition. In other embodiments, a "therapeutically effective amount" is sufficient to achieve a desired biological effect. an amount sufficient to achieve an effect (e.g., to inhibit or prevent gonococcal and / or meningococcal infections) is an amount sufficient to elicit a protective immune response in a subject such that the

[0151] Ideally, a therapeutically effective amount of an agent does not cause substantial cytotoxic effects in a subject. in an amount sufficient to induce the desired result. reduce, alleviate, and / or prevent a disease, disorder, or condition (such as gonorrhea or meningococcal infection) An effective amount of an agent useful for preventing or preventing a disease or disorder in a subject being treated, including any associated disease or disorder. and / or the type and severity of the condition (e.g., gonorrhea- or meningococcal-related disease, disability, type of damage or condition, and / or N. gonorrhoeae or N. men The dosage will depend on the strain of B. ingitidis, as well as the mode of administration of the therapeutic composition.

[0152] In the context of the present invention, by "gonococcus-associated disease, disorder, or condition" is meant any gonococcus infection or Neisseria gonorrhoeae infection (such as those described above) , any resulting from such infection with Neisseria gonorrhoeae (including clinical pathology of the disease) is meant.

[0153] Additionally, any meningococcal infection by "meningococcal-related disease, disorder, or condition" or Neisseria meningitidis infection (meningitis, rash, sepsis, Symptoms caused by Neisseria meningitidis include fever, nausea, vomiting, and diarrhea. By "pathological pathology" is meant any clinical pathology resulting from such an infection.

[0154] In yet another aspect, the present invention provides a method for the detection of Neisseria gonorrhoeae bacteria. and / or Neisseria meningitidis binding or adhesion to cells 20. A method of at least partially inhibiting or preventing in a subject the one or more immunogenic fragments as described in the isolated proteins described herein; the isolated nucleic acids described herein; the genetic constructs described herein; the host cells described herein; the antibody or antibody fragment described; and / or the composition described herein; to the subject, thereby infecting the cells of the subject with Neisseria gonorrhoeae. inhibits binding of E. coli and / or Neisseria meningitidis or The method further comprises the step of preventing.

[0155] Bacterial adhesion to host cells is an early step for successful colonization of host mucosal surfaces. It will be appreciated that the conditions and prerequisites are: Cells (vaginal epithelial cells, cervical epithelial cells, endometrial epithelial cells, pharyngeal epithelial cells, and urethral epithelial cells) cells, etc.).

[0156] In yet another aspect, the present invention provides a method for the detection of Neisseria gonorrhoeae bacteria. and / or serum resistance in Neisseria meningitidis infections 20. A method of at least partially inhibiting or reducing the level of a phenotype ... as described herein, comprising: one or more immunogenic fragments thereof; an isolated protein as described herein; a protein; an isolated nucleic acid described herein; a gene described herein a construct; a host cell as described herein; an antibody as described herein administering to a subject an antibody or antibody fragment; and / or a composition described herein; and thereby Neisseria gonorrhoeae and / or Neisseria Inhibits or inhibits serum resistance in Seria meningitidis infections The method further comprises the step of reducing

[0157] Neisseria gonorrhoeae is a common sexually transmitted disease in humans worldwide. A small percentage of gonorrhea cases are commonly referred to as disseminated gonorrhea (DGI). This can lead to serious life-threatening complications. Resistance to the bactericidal effect of erythropoietin (i.e., serum resistance) is strictly Seems to be correlated.

[0158] Preferably, the aforementioned methods of the present invention are carried out in animals (such as mammals). In this case, the mammal is a human.

[0159] The composition for administration in the method of the five preceding embodiments comprises one or more immunoglobulins of the invention. immunogenic fragments and / or isolated proteins, and / or and / or isolated proteins of the present invention. It is recognized that the antibody may include, but is not necessarily limited to, one or more antibodies or antibody fragments. Thus, in certain embodiments, such compositions may be used in combination with NHBAs. It binds to the C-terminal fragment (such as that shown in SEQ ID NO: 2) of a protein (such as SEQ ID NO: 1). one or more antibodies or one or more antibodies obtained or made against It may include body fragments.

[0160] By "administering" or "administration" is meant the introduction of a substance into a subject by a particular chosen route. The introduction of the compositions disclosed herein is meant.

[0161] Any safe route of administration may be used to provide the compositions of the present invention to a patient. For example, oral, rectal, parenteral, sublingual, buccal, intravenous, intra-articular, Intramuscular, intradermal, subcutaneous, inhalation, intraocular, intraperitoneal, intracerebroventricular , intravaginal, and transdermal administration can be used.

[0162] Dosage forms include tablets, dispersions, suspensions, injections, solutions, syrups, lozenges, capsules, etc. These include ointments, nasal sprays, suppositories, aerosols, transdermal patches, and the like. These dosage forms include the injection of controlled release devices specifically designed for this purpose. Other implants or implants, or implants that are modified to additionally act in this manner. The controlled release of therapeutic agents can be achieved by, for example, using hydrophobic polymers (acrylic resins, waxes, etc.). higher aliphatic alcohols, polylactic acid, and polyglycolic acid, and hydroxypropyl (specific cellulose derivatives such as propylmethylcellulose) In addition, controlled release can be achieved by coating other polymer matrices. This can be achieved by the use of co-solvents, liposomes, and / or microspheres.

[0163] Compositions of the present invention suitable for oral or parenteral administration may be prepared in discrete units (predetermined amounts). Capsules, sachets, functional foods / feeds, each containing one or more of the therapeutic agents of the present invention , or tablets, etc.), as powder or granules, or in aqueous, non-aqueous, or water-based liquids. It is presented as a solution or suspension in an oil-based emulsion or a water-in-oil liquid emulsion. Such compositions may be prepared by any of the methods of pharmacy, but All methods involve the use of one or more of the agents described above in a pharmaceutical composition comprising one or more essential ingredients. Generally, the compositions comprise a liquid carrier or a carrier containing an agent of the invention. or finely divided solid carrier, or both, and then, if necessary, the product is mixed to the desired consistency. The composition can be prepared by shaping it into the desired shape.

[0164] The compositions may be administered in a manner compatible with the dosage formulation, and in a pharmaceutically effective amount. The dose administered to a patient, in the context of this specification, is the dose that will be administered to the patient over an appropriate period of time. The amount of drug(s) administered should be sufficient to achieve a beneficial response. Age, sex, weight, and general health and medical condition, factors that may depend on the physician's judgment. The dosage may depend on the subject being treated.

[0165] In certain embodiments of the foregoing methods and compositions, the immunogenic fragment or isolated protein The protein may be a further immunogenic fragment or protein derived from the NHBA protein, or or additional N. gonorrhoeae proteins known in the art or N. meningitidis protein (surface-expressed MetQ protein (Semche nko et al. 2017), MsrAB, AniA, and Bexsero vaccines One of the four antigenic components present in (GSK Vaccines) or Multiple (i.e., the New Zealand epidemic strain (MeNZB), which provides PorA Factor H binding protein (fHbp), Neisseria adhesin A ( NadA), heparin-binding antigen (NHBA) of Neisseria genus, and outer membrane vesicles) In this regard, the immunogenicity described herein may be administered in combination with The fragment or isolated protein may be N. gonorrhoeae and / or Neis Included as a component of a multi-antigen vaccine for Seria meningitidis In some embodiments, an immunogenic fragment or isolated protein of an NHBA may be used. The protein and the additional immunogenic fragment or protein are provided as a single chimeric peptide. In this embodiment, the immunogenic fragment or isolated The protein may be further immunogenic fragmented or N- or C-terminal to the protein. It is possible.

[0166] In a final aspect, the present invention provides a method for detecting N. gon in a biological sample obtained from an animal. orrhoeae and / or Neisseria meningitidis The method comprises treating a biological sample with an antibody or antibody fragment described herein. and thereby detecting N. gonorrhoeae and / or the method further comprising detecting Neisseria meningitidis. Preferably, the NHBA protein is present in one or more N. gonorrhoeae cells and / or N. meningitidis cells Detected on the outer surface.

[0167] In certain embodiments, the biological sample is one or more samples taken from the animal. The sample may be a pathology sample, including any bodily fluid, cell, tissue, organ, or organ sample. Non-limiting examples include blood, plasma, serum, lymphocytes, urine, feces, amniotic fluid, cervical samples, These include cerebrospinal fluid, tissue biopsy, bone marrow, bronchoalveolar lavage fluid, sputum, and skin.

[0168] Preferably, N. gonorrhoeae and / or N. meningitidis Detection involves forming a detectable complex between the antibody or antibody fragment and the NHBA protein. The complex so formed can be prepared by any method known in the art. The present invention can be detected by any technique, assay, or means, including immunoblotting, immunohistochemistry, immunocytochemistry, immunoassay ... Cytochemistry, immunofluorescence, immunoprecipitation, ELISA, flow cytometry, magnetic bead separation, and biosensor-based detection systems (such as surface plasmon resonance). Not limited.

[0169] To facilitate detection, the antibody may be directly labeled or a labeled secondary antibody may be used. Additionally, small molecules can be directly labeled.

[0170] Labels include chromogens, catalysts, biotin, digoxigenin, enzymes, fluorophores, and chemicals. Groups including luminescent molecules, radioisotopes, drugs, magnetic beads, and / or direct visual labels may be selected from:

[0171] In the case of direct visual marking, colloidal metallic or non-metallic particles, dyes Particles, enzymes or substrates, organic polymers, latex particles, liposomes, or signals Other vesicles containing the product, and the like, may be used.

[0172] Fluorophores can be used as known in the art, for example, fluorescein isothiocyanate. cyanate (FITC), Alexa dyes, tetramethylrhodamine isothiocyanate (TRITL), allophycocyanin (APC), Texas Red, Cy5, Cy3 , or R phycoerythrin (RPE).

[0173] The enzymes used were horseradish peroxidase (HRP), alkaline phosphatase (A The enzyme may be, but is not limited to, β-galactosidase, β-galactosidase, or glucose oxidase. Not determined.

[0174] In some embodiments, the detection method is a "high throughput" diagnostic test, or This may be accomplished by a commercial pathology laboratory procedure or a procedure performed in a hospital.

[0175] Characterizing disease progression and / or severity of gonorrhea-associated diseases, disorders, or conditions in animals Such a detection method for N. gonorrhoeae may be useful in identifying It will be further recognized that such methods may have therapeutic properties. used to select animals for treatment (e.g., by so-called "companion diagnostics") obtain.

[0176] As generally used herein, the terms "patient," "individual," and "subject" in the context of any mammalian recipient of the treatments or compositions disclosed herein. Thus, the methods and compositions disclosed herein are useful in In a preferred embodiment, the mammal is a human. One or more steps of the methods described herein may be performed in vitro. It can be carried out.

[0177] In order that the present invention may be fully understood and effectively practiced, reference is made to the following non-limiting examples. do. [Example]

[0178] Example 1 Introduction Several recent advances support the feasibility of developing a gonorrhea vaccine. against the closely related bacterium Neisseria meningitidis The vaccine (Meningococcal B vaccine MeNZB in outer membrane vesicles (OMV)) is effective against N. gonorrhoeae. It has been suggested that it has a 31% efficacy against infections caused by E. hoeae

[12] . A new four-component meningococcal B vaccine (MeNZB OMV component + 3 recombinant 4CMenB (commercially available as Bexsero), which contains a protein antigen, Eliciting cross-reactive antibodies to N. gonorrhoeae proteins, including HBA It has been shown to lead to

[0179] In this example, the sequence variation and expression details of NHBA of N. gonorrhoeae are described. Conducting a detailed analysis, the NHBA Ng The level, type, and functional activity of antibodies raised against The full-length protein and the C-terminal fragment of the protein were investigated as candidates for a gonorrhea vaccine. Evaluate competence.

[0180] result NHBA is highly conserved in N. gonorrhoeae The present inventors have shown that NHBA is conserved in N. gonorrhoeae. As previously shown

[13] , the inventors herein have used available gonorrhea isolates and genome sequences. To further investigate sequence variants of NHBA in the sequences available in GenBank 1281 nucleotides from N. gonorrhoeae strain 1291 against the gonococcal genome blast by the nhba gene (which encodes the 427 amino acid NHBA) The n search (Fig. 1A) showed that nhba was present in all 594 genomes, with a range of 94.1 to 10 0% nucleic acid identity. Similarity blastn searches revealed the presence of the nhba gene in 4,424 isolates, with 85. The BLAST search revealed 1-100% identity. The 1,228 isolates that did not have a match to a had annotated 16S and porB genes were also missing, indicating that incomplete sequences were available for these isolates. This resulted in a time-series database collected from over 60 different countries between 1960 and 2020. and nhba was widely distributed and highly conserved in a panel of geographically diverse gonococcal strains. It is confirmed that this will be done.

[0181] As of April 6, 2020, the PubMLST database contained NH 42 unique isolates among 3,546 N. gonorrhoeae isolates with BA proteins There are NHBA_ peptide variants. These variants account for 97.5-100% of all Two dominant sequences present in 70.3% of PubMLST isolates share amino acid identity. There are several prevalent NHBA variants, which are present in 39.7% of NHBA-542 strains. , N. gonorrhoeae 1291) and NHBA-475 (strain 30 0.4% and includes N. gonorrhoeae WHO P and WHO X. Overall, one of the 14 major NHBA variants was present in 9 of the isolates. While present in 7.8% of cases, the remaining 28 NHBA peptide variants are rare. , present in 1–10 isolates (Table 5). From the alignment, the N- and C-termini have the highest level of conservation, with the arginine-rich region The variable central region upstream of the (Arranged in decreasing order). The phylogenetic relationships of these NHBA variants are shown in Figure 1. A panel of strains representative of NHBA diversity was used in subsequent assays. The C-terminal sequence is conserved (Fig. 1B). The C-terminal region is likely to be more exposed. Given its high potency and accessibility to vaccine-induced antibodies, subsequent investigations will focus on recombinant The results were concentrated in both the full-length NHBA and the C-terminal NHBA fragment (NHBA-c) (Fig. 1A).

[0182] Recombinant full-length NHBA and C-terminal NHBA fragments were immunogenic and expressed in various gonococcal strains. Induction of antibodies that recognize NHBA variants To investigate the immunogenicity of gonococcal NHBA, recombinant full-length NHBA + Freund's antibody was used. adjuvant, or NHBA-c fragment plus Freund's adjuvant or hydroxypropyl ammonium Sera from mice immunized with Alum were analyzed by ELISA and Western blot. Using whole cell ELISA, we were able to assess the activity of NHBA and Both mouse sera from NHBA-c were positive for N. gonorrhoeae wild-type (WT) and N. Native NHBA on the surface of the HBA-complemented (ΔNHBA_C) strain could be detected, and NHBA The mutant strain (ΔNHBA) showed significantly reduced titers (Table 1). Western blots on whole cell lysates of A. gonorrhoeae wild-type and mutants Analysis of the NHBA antiserum by confirmed that the antiserum specifically recognizes NHBA. (Fig. 2A). Expression of NHBA in a panel of N. gonorrhoeae strains and NHBA The cross-reactivity of the antisera was confirmed by Western blot analysis (Fig. 2B). Expression varied between strains, with high, intermediate, and low NHBA expressors being identified in subsequent assays. It was used.

[0183] ELISA with recombinant NHBA demonstrated significant activity in mice immunized with NHBA. The presence of an IgG1 isotype response was demonstrated (Table 1). The ratio of subclasses varied between different formulations, with NHBA-c-Freund (IgG1 > IgM =IgG2b>IgG2a>IgG3) and NHBA-c-Alum(IgG1>IgM >IgG2b>IgG2a>IgG3), NHBA-Freund showed higher levels IgG3 and lower levels of IgG2a and IgG2b (IgG1>IgM>Ig Overall, the results of ELISA and Western The results showed that gonococcal NHBAs are immunogenic and that anti-NHBA antisera were effective against different NHBA barriers. The results showed that the NHBA can be recognized on the surface of multiple N. gonorrhoeae strains expressing the NHBA. It is confirmed that:

[0184] NHBA antibodies enhance C3 fragment deposition To investigate whether NHBA antisera enhance activation of the complement cascade, C3 fragment deposition onto the surface of .gonorrhoeae was analyzed using flow cytometry. The mouse serum of NHBA-Freund and NHBA-c-Freund were investigated. Mouse sera and purified NHBA-specific IgG from these sera were tested and their All were evidenced by an increase in mean fluorescence intensity compared to pre-immune serum or control-treated bacteria. As shown in Figure 3A-B, the 1291-like structure binds to N. gonorrhoeae strain 1291 (top panel). ) Bacteria incubated with human complement plus either whole serum or purified IgG significantly increased C3 fragment deposition compared to the complement-only control (for NHBA, for NHBA-c, 7.1-fold and 5.2-fold increases, respectively; for NHBA-c, 4.8-fold and 4. a 7-fold increase; bottom panels of Figure 3A-B).

[0185] NHBA antibodies have bactericidal and opsonophagocytic activities NHBA antibody and NHBA-c antibody inhibit complement-dependent lysis of N. gonorrhoeae The ability of the antibodies to mediate opsonophagocytic and opsonophagocytic killing was assessed by serum bactericidal activity (SBA) assay, respectively. Different NHBAs were tested using the opsonophagocytic killing (OPA) assay. Five N. gonorrhoeae strains containing variants and with varying levels of NHBA expression were tested. For the A assay, N. gonorrhoeae was incubated with NHBA mouse serum or NHB Ac mouse serum was incubated, followed by the addition of a human complement activator. Survival was measured. NHBA-Freund's serum and NHBA-c-Freund's serum Both elicited serum bactericidal activity in a concentration-dependent manner, with SBA titers ranging from 100 to 1600. The titers of the pre-immune sera were <50 (compared to titers of <50 for the pre-immune sera) (Fig. 20A; Table 2). OPA assay Therefore, N. gonorrhoeae was isolated using NHBA antibody or NHBA-c antibody. When psonized and incubated in the presence of human complement, human PMNs showed a dose-dependent The mice were killed in a controlled manner, with OPA titers ranging from 100 to 6,400 (pre-immune serum titers <50) (Figure 20B; Table 2). Anti-NHBA (NHBA-c-Alum) formulated with Bant (Alum) The serum also induced SBA and OPA killing of N. gonorrhoeae and NHB titers similar to those observed with Ac-Freund's serum. (Fig. 20A, B; Table 2). The purified NHBA immunoglobulin mediated concentration-dependent killing of SBA (Figure 20C). No killing was observed in NHBA-c-alum serum, which was depleted of anti-NHBA antibodies ( Figure 22), confirming the specificity of the immune response for NHBA.

[0186] NHBA antibodies reduce NHBA binding to heparin and gonococcal adhesion to host cells We investigated whether NHBA antiserum and NHBA-c antiserum can inhibit the functional role of NHBA. To investigate whether the NHBA-antiserum is effective, recombinant NHBA and and its predicted substrate, heparin, and surface plasmon resonance (SPR)-based competitive binding In the absence of antiserum, N. gonorrhoeae NHBA binds heparin. Pre-pandemic serum had no effect on the ability of heparin to interact with NHBAs, but Serum from mice immunized with long NHBA reduced heparin binding by 85.7% ( P = 0.0001) (Figure 4A; Figure 6). However, NHBA-c serum was significantly higher than heparin. failed to significantly inhibit the interaction between NHBA (11% reduction in binding; P = 0.1) (Fig. 4A; Fig. 6).

[0187] Gonococcal NHBA is surface-exposed

[13] and has similar adhesin functions to meningococcal NHBA. Therefore, NHBA antiserum and NHBA-c antiserum are likely to have We investigated whether the α-glucanase inhibitor could reduce the adhesion of N. gonorrhoeae to human cells. , N. gonorrhoeae preincubated with antiserum and transformed This was performed using cultured endocervical (tCX) and urethral (tUEC) epithelial cells. BA and NHBA-c sera inhibited tCX and tCX in a concentration-dependent manner compared to no antibody controls. It is possible to reduce adhesion to both tUECs and IgG, but not pre-immune serum. For example, a 1:20 dilution of NHBA serum inhibited N. gonorrhoeae adherence on tCX and tUEC cells. Similarly, a 1:20 dilution of cNHBA antiserum reduced , reducing bacterial adhesion in tCX and tUEC cells by 8-fold and 5-fold, respectively ( Figure 4B-C).

[0188] Consideration In light of the threat of antimicrobial-resistant N. gonorrhoeae, the development of a gonococcal vaccine There is a need for the identification and characterization of potential vaccine candidates to aid in their development. The gonococcal NHBA is characterized herein and is widely distributed. Conservation among geographically and temporally diverse N. gonorrhoeae strains and antibodies raised against either full-length NHBA or a C-terminal fragment of NHBA. These antibodies have been shown to mediate both bactericidal and opsonophagocytic killing of human epithelial cells. Reduces adhesion of N. gonorrhoeae to cytoplasm and inhibits the glycan-binding activity of NHBA The known correlates of protection for N. gonorrhoeae are currently Although there is no evidence that NHBAs can kill HIV-1-associated viruses via two conventional immune killing mechanisms (reviewed in [9]), elicits antibodies capable of killing N. gonorrhoeae and inhibiting infection The ability of these proteins to mediate functional blocking of a critical stage in the process of gonorrhea has led to their use in gonorrhea vaccines. I support the possibility of this.

[0189] Gonococcal NHBA is highly conserved and N. gonorrhoeae strains investigated to date The majority of strains share a limited number of NHBA variants. express one of the two major variants (e.g., 70.3% express one of the two major variants) (91.3% express one of the seven variants, and 91.3% express one of the seven variants). , NHBA expression varies between strains, and strains expressing the same NHBA variant (e.g., WHO However, the present inventors have also shown that there is a significant variation even between NHB A variant 542 (from N. gonorrhoeae strain 1291) If the antisera are cross-reactive and express homologous and heterologous NHBA variants, These results show that it is possible to kill strains with high, intermediate, and low NHBA expression. N. gonorrhoeae and N. meningitidis strains have different predominant NH Although it contains BA variants

[13] , our findings suggest that N. meningitid Consistent with findings about NHBA in is, NHBA-2 (found in 4CMenB) The antibody immune response to 99% of strains circulating in the United States, regardless of the NHBA variant. This is observed in 5% (442 strains)

[26] .

[0190] Adjuvanted with either Freund's or aluminum hydroxide When the NHBA fragment and the NHBA-c fragment were administered, they were immunogenic in mice. As a result, IgG1-dominant antibody responses were elicited in all cases, whereas other isotypes were not. Variation in class and subclass patterns is observed for different antigen and adjuvant combinations. Furthermore, NHBA-c reduced the total IgG titer compared to full-length NHBA. However, similar or higher SBA potency was observed against most strains in the panel studied. The immunoglobulin isotype and subclass were determined by complement cleavage. are known to differ in their ability to activate and mediate bactericidal and opsonophagocytic activities. However, this ability varies between antigenic targets. Murine antibodies targeting the rA antigen showed a serum bactericidal activity of IgG3>>IgG2b> IgG2a>>IgG1 and IgG3>IgG2b=I for opsonophagocytic activity The hierarchy is gG2a>>IgG1

[27] . Mouse antibodies against native MsrA / B were given adjuvant effect by Freund. When used in combination with aluminum hydroxide, it produced higher titers of IgG2a, IgG2b, and and IgG3, and the MsrA / B-Freund's antiserum was mediated SBA and OPA killing of E. ae., but MsrA / B-Alum antiserum did not.

[28] . Our data show that anti-NHBA SB of N. gonorrhoeae The dominant role of IgG2a and IgG2b in A- and OPA-mediated killing This suggests that higher total levels of these antibodies may be associated with NHBA-Freund's In comparison, it was induced by NHBA-c-Freund. Ac-Alum elicited IgG2b>IgG2a>IgG3, and all the antibodies tested SBA and OPA were mediated against five gonococcal strains. Unlike MsrA / B-Alum, which did not elicit IgG3 or IgG2b, anti-MsrA / B-Alum did not mediate killing of N. gonorrhoeae

[28] . This difference in levels and function may be antigen-specific or may be due to differences in different studies. This may be related to the different immunization doses and schedules used in the NHBA, 0, 21 , and 25 μg vs MsrA / B on day 28, 5 μg vs MsrA / B on days 0, 21, 28, and 42 g).

[0191] Overall, the present invention provides evidence supporting the use of NHBA as an antigen in gonorrhea vaccines. It describes several important features and can be used alone or as a fusion protein with another antigen. This highlights the possibility of using the C-terminal fragment of NHBA as an optimized antigen for immunization. do.

[0192] method Bacterial strains and growth conditions N.gonorrhoeae strains 1291, FA1090, WHO G, WHO P, and and WHO X were used in this study. N. gonorrhoeae was cultured in 5% C O2 at 37 or 32°C, with 1% (v / v) IsoVitaleX (Becto The assay was performed on GC agar (Oxoid) containing 100% ethanol (N Dickinson). The majority of gonococcal populations used in this study were identified by visual inspection of colonies using a phase-contrast microscope. As determined by the above, the cells were piliated and expressed opacity protein.

[0193] Sequence analysis The sequences were aligned using MacVector and the amino acid identity and similarity were calculated. The percentage was calculated (BLOSUM90, threshold 0). The phylogenetic tree (best tree, uncorrected ('p')) was generated using MacVecto The presence of nhba and the encoded NHBA protein among gonococcal strains and Conservation was performed using 594 gonococcal genomes and Neisseria Multigenome in GenBank. Locus Sequence Typing website (PubMLST; http 5652 N. gono in For the N. gonorrhoeae isolate, N. gonorrhoeae 1291 (GenBan k accession EEH61857.1; genomic locus tag NGAG_00725) Basic Local Alignment Search T The sequence was determined as of September 19, 2019, using the BLAST program. The PubMLST nomenclature previously established for NHBA (converted by NEIS2109) (loaded) and assign a unique identification number to each unique peptide sequence (e.g. NHBA_peptide 2 [NHBA-2] is in 4CMenB, and NHBA_peptide 5 42 [NHBA-542] in N. gonorrhoeae strain 1291).

[0194] Construction of N. gonorrhoeae NHBA mutant strain The N. gonorrhoeae 1291 nhba gene was cloned using the 5′-ATGTTTAA ACGCAGTGTGATTGC-3' (SEQ ID NO: 3) primer and 5'-TCAAT CCCGATCTTTTTTGCCGGC-3' (SEQ ID NO: 4) primer was used The DNA was amplified and cloned into the pGEM-T Easy vector (Promega). namycin resistance gene (pUC4Kan; Amersham Biosciences) 5'-ggatccCCGGCCGAGATTCCGCTGATTCC-3' (sequence Number 5) Primer and 5'-ggatccGCGACCTCCTCGACCGTGCA Using inverse PCR with the GAAC-3' (SEQ ID NO: 6) primer, The fragment was inserted into a BamHI restriction site introduced into the center of the open reading frame ( Introduced for subcloning of the kanamycin resistance gene into the nhba gene (The BamHI restriction enzyme site is shown in lowercase.) The nhba::kan construct was cloned into Nc It was linearized with oI and transformed into N. gonorrhoeae 1291. The 91 nhba::kan strain (ΔNHBA) was generated using the complementing plasmid pCTS32. Using the intact nhba gene (5'-GGCATATGGCGGAAACAA TA-3' (SEQ ID NO: 7) primer and 5'-TCAATCCCGATCTTTTTT GCCGGC-3' primer (SEQ ID NO: 8) was used to amplify the ΔNHBA strain. The complemented strain (ΔNHBA_C) was generated by introducing the nhb Successful deletion of the a gene and subsequent complementation were confirmed by PCR and Western blot. did.

[0195] Recombinant Protein Expression Cloning and expression of full-length recombinant NHBA lacking the predicted signal peptide For expression of the C-fragment of NHBA (NHBA-c), the platelet was used. timer 5'-ATTActcgagTCGCTTCCGGCCGAGATTCC-3'( SEQ ID NO: 9) and primer 5'-TGAAggatccCGGCATCAACATCA ATC-3' (XhoI site and BamHI site are shown in lowercase in each primer) (SEQ ID NO: 10) was amplified from N. gonorrhoeae 1291 E. coli BL21 (DE3 ) was transformed. Expression was measured by OD 600 Addition of 1 mM IPTG to a culture with a .DELTA. of 0.4 and induced by incubation at 20°C for 24 hours, as previously described.

[13] , proteins were purified using TALON affinity resin (Clontech). Ta.

[0196] Generation of polyclonal antibodies Five 3-week-old female BALB / c mice (Animal Resources Center) were cultured. ter, WA, Australia) with 25 μg of recombinant protein. Reund's adjuvant (Merck) or aluminum hydroxide (Alhydrog The mice were subcutaneously immunized with IgG1 (InvivoGen) on days 0, 21, 28, and 42. Blood was collected on day 56 and serum was collected via centrifugation. Pre-immune serum was collected from each This work was carried out in accordance with the Australian Code for the Prevention of Violence against Humans (AVC). Care and Use of Animals for Scientific Purposes and Griffith University An This was carried out with approval from the American Ethics Committee (AEC).

[0197] Polyclonal NHBA antibodies were purified by affinity chromatography with recombinant NHBA. NHBA was purified from mouse serum using the N-hydroxybenzoates (NHB-1) and N-hydroxybenzoates (NHB-2) using the manufacturer's instructions. Thiosuccinimidyl-Sepharose® 4 Fast Flow (Mer ck) and incubated with mouse serum diluted 1 / 2 in PBS. The bound antibody was eluted with 0.1 M glycine buffer (pH 3.0). The eluted sample was then loaded onto an Amicon-Ultra centrifugal spin column. The buffer was exchanged into PBS using a BCA unit (Merck). (Thermo).

[0198] Enzyme-linked immunosorbent assay (ELISA) 100 μl of coating buffer as previously described [13, 30, 31]. 100 ng of purified recombinant TNF-α in 0.5 M carbonate / bicarbonate buffer, pH 9.6 96-well MaxiSorp (NUNC) plate coated with protein for 1 hour at room temperature ELISA was performed in triplicate using plates. ELISA titers were average negative ( The highest absorbance at 450 nm is greater than +3 standard deviations (all reagents except primary antibody). This is a low serum dilution.

[0199] Serum bactericidal activity (SBA) assay and opsonophagocytic killing (OPA) assay SBA and OPA assays were performed as previously described [30, 31]. Briefly, about 1x10 3 Colony forming units (CFU) of N. gonorrhoeae eae were incubated with heat-inactivated (56°C, 60 min) anti-NHBA mouse serum or pre-immunized mice The SBA assay was performed in serial dilutions of serum at 37°C for 15 minutes. Complement source (10% (v / v) normal human preabsorbed with N. gonorrhoeae Serum

[30] ) was added (Figure 21), followed by incubation at 37°C and 5% CO2 for 30 minutes. The OPA assay was initiated by injecting a complement source and approximately 1 x 10 5 of Polymorphonuclear leukocytes (PMNs) were added, followed by incubation at 37°C and 5% CO for 90 minutes. Serial dilutions of the contents of each well were plated on GC agar. Plated and grown overnight. Titers were determined as those that induced greater than 50% killing in the assay. Statistical analysis was performed using one-way analysis of variance (ANOVA) and St Each experiment was performed in triplicate. This was carried out three times in a simple manner.

[0200] Flow cytometry analysis As previously described

[28] , flow cytometry was used to detect N. gonorrhoeae. Antibody binding to N. rhoeae and C3 fragment deposits was measured. hoeae 1291 (approx. 1 x 10 7 CFU) in HBSS + (0.15 mM CaCl Hanks' balanced salts containing 2 and 0.5 mM MgCl2 and 1% BSA (w / v) 1:100 dilution of heat-inactivated mouse serum or 70 μg / mL purified NHB in PBS (solution) Antibody-treated bacteria were washed and pre-incubated with Alexa Flu A antibody. or 488-conjugated anti-mouse IgG (Thermo) at a 1:200 dilution, was added to 5% normal human serum preabsorbed with N. gonorrhoeae for 15 min at 37°C. After incubation for 1 hour, the C3 fragments were incubated with FITC-conjugated anti-human C3 c Detected by incubation of bacteria with a 1:200 dilution of antibody (BioRad) The data were analyzed using a CyAn ADP flow cytometer (Beckman Coulter). ter) and analyzed using FlowJo.

[0201] Surface Plasmon Resonance (SPR) SPR competition assays were performed using a Pall Pioneer FE. As described

[30] , the NextStep interface in the OneStep Assay Builder A competition assay was performed using injection. Serum was used as the first injection (A) and heparin as the second injection (B). (B) The binding of heparin (maximum OneStep concentration of 50 μM) to NHBA. The cases were compared with and without serum and with a 1:200 dilution of pre- or post-immune serum. Data were collected using the Pioneer Software package and compared. The percentage blocking was calculated using the dat analysis software. Heparin injection (injection A = buffer; B = heparin) vs. blood The supernatant was subtracted (injection A = pre-immune / post-immune serum; B = buffer), serum Binding of heparin in the presence of (injection A = pre-immune / post-immune serum; B = heparin The calculation was based on the relative RMax of the α- and β-paramone (Palin).

[0202] Epithelial cell adhesion assay The gonococcal adhesion assay was performed as previously described

[31] with the following modifications. For purification, monolayers of tCX and tUEC cells were incubated with serial dilutions of heat-inactivated mouse serum. Approximately 1 x 10 cells were first pre-incubated at room temperature for 30 minutes. 5 Infected by CFU Following infection, the cell monolayer was washed three times with warmed HBSS. Non-adherent bacteria were removed by centrifugation, and the well contents were plated onto GC agar. Adherent bacteria were calculated as the average CFU from triplicate wells and compared to the no antibody control. Statistical analysis was performed using ANOVA and two-tailed Student's t-test. Each experiment was performed three times. [Table 1] [Table 2]

[0203] References 1 Whiley, D.M., Jennison, A., Pearson , J. & Lahra, M.M. tion of Neisseria gonorrhoeae resistance to both ceftriaxone and azithromycin. ncet Infect Dis 18, 717-718 (2018). 2 ECDC. Extensively drug-resistant (XD R) Neisseria gonorrhoeae in the United K ingdom and Australia. European Centre fo r Disease Prevention and Control. Stockh olm. https: / / ecdc.europa.eu (2018). 3 WHO. Global priority list of antibio tic-resistant bacteria to guide research , discovery, and development of new anti biotics, <http: / / www.who.int / medicines / p ublications / global-priority-list-antibio tic-resistant-bacteria / en / > (2017). 4 CDC. Antibiotic Resistance Threats i n the United States, 2013, <http: / / www.c dc.gov / drugresistance / threat-report-2013 / pdf / ar-threats-2013-508.pdf> 5 DepartmentofHealth. Responding to th e threat of antimicrobial resistance: Au stralia’s First National Antimicrobial R esistance Strategy 2015-2019. (Departmen t of Health, Australian Government, Canb erra, 2015). 6 WHO. Global incidence and prevalence of selected curable sexually transmitte d infections - 2008. (World Health Organ isation, 2012). 7 CDC. Sexually Transmitted Disease Su rveillance 2017. https: / / www.cdc.gov / std / stats (2018). 8 KirbyInstitute. HIV, viral hepatitis and sexually transmissible infections i n Australia: annual surveillance report 2018. The Kirby Institute, UNSW Sydney. (2018). 9 Edwards, J. L., Jennings, M. P., Api cella, M. A. & Seib, K. L. Is gonococcal disease preventable? The importance of understanding immunity and pathogenesis in vaccine development. Crit Rev Microbi ol 42, 928-941 (2016). 10 WHO. Global incidence and prevalence of selected curable sexually transmitte d infections - 2008, <http: / / www.who.int / reproductivehealth / publications / rtis / st isestimates / en / > (2012). 11 Rice, P. A., Shafer, W. M., Ram, S. & Jerse, A. E. Neisseria gonorrhoeae: Dr ug Resistance, Mouse Models, and Vaccine Development. Annu Rev Microbiol 71, 665 -686 (2017). 12 Petousis-Harris, H. et al. Effective ness of a group B outer membrane vesicle meningococcal vaccine against gonorrhoe a in New Zealand: a retrospective case-c ontrol study. Lancet 390, 1603-1610 (201 7). 13 Semchenko, E. A., Tan, A., Borrow, R . & Seib, K. L. The serogroup B meningoc occal vaccine Bexsero elicits antibodies to Neisseria gonorrhoeae. Clin Infect D is 69, 1101-1111 (2018). 14 Serruto, D., Bottomley, M. J., Ram, S., Giuliani, M. M. & Rappuoli, R. The n ew multicomponent vaccine against mening ococcal serogroup B, 4CMenB: immunologic al, functional and structural characteri zation of the antigens. Vaccine 30 Suppl 2, B87-97 (2012). 15 Esposito, V. et al. Structure of the C-terminal domain of Neisseria heparin binding antigen (NHBA), one of the main antigens of a novel vaccine against Neis seria meningitidis. J Biol Chem 286, 417 67-41775 (2011). 16 Mubaiwa, T. D. et al. The Bexsero Ne isseria meningitidis serogroup B vaccine antigen NHBA is a high-affinity chondro itin sulfate binding protein. Scientific reports 8, 6512 (2018). 17 Serruto, D. et al. Neisseria meningi tidis GNA2132, a heparin-binding protein that induces protective immunity in hum ans. Proc Natl Acad Sci U S A 107, 3770- 3775 (2010). 18 Vacca, I. et al. Neisserial Heparin Binding Antigen (NHBA) Contributes to th e Adhesion of Neisseria meningitidis to Human Epithelial Cells. PLoS One 11, e01 62878 (2016). 19 Maritan, M. et al. Structures of NHB A elucidate a broadly conserved epitope identified by a vaccine induced antibody . PLoS One 13, e0201922 (2018). 20 Maritan, M. et al. Crystal structure s of human Fabs targeting the Bexsero me ningococcal vaccine antigen NHBA. Acta C rystallogr F Struct Biol Commun 73, 305- 314 (2017). 21 Pizza, M. et al. Identification of v accine candidates against serogroup B me ningococcus by whole-genome sequencing. Science 287, 1816-1820 (2000). 22 Giuliani, M. M. et al. A universal v accine for serogroup B meningococcus. Pr oc Natl Acad Sci U S A 103, 10834-10839 (2006). 23 Welsch, J. A. et al. Antibody to gen ome-derived neisserial antigen 2132, a N eisseria meningitidis candidate vaccine, confers protection against bacteremia i n the absence of complement-mediated bac tericidal activity. J Infect Dis 188, 17 30-1740 (2003). 24 Plested, J. S. & Granoff, D. M. Vacc ine-induced opsonophagocytic immunity to Neisseria meningitidis group B. Clin Va ccine Immunol 15, 799-804 (2008). 25 Muzzi, A., Mora, M., Pizza, M., Rapp uoli, R. & Donati, C. Conservation of me ningococcal antigens in the genus Neisse ria. MBio 4, e00163-00113 (2013). 26 Rajam, G. et al. Meningococcal Antig en Typing System (MATS)-Based Neisseria meningitidis Serogroup B Coverage Predic tion for the MenB-4C Vaccine in the Unit ed States. mSphere 2 (2017). 27 Michaelsen, T. E., Kolberg, J., Aase , A., Herstad, T. K. & Hoiby, E. A. The four mouse IgG isotypes differ extensive ly in bactericidal and opsonophagocytic activity when reacting with the P1.16 ep itope on the outer membrane PorA protein of Neisseria meningitidis. Scand J Immu nol 59, 34-39 (2004). 28 Shaughnessy, J. et al. Human Factor H Domains 6 and 7 Fused to IgG1 Fc Are I mmunotherapeutic against Neisseria gonor rhoeae. J Immunol 201, 2700-2709 (2018). 29 Steichen, C. T., Shao, J. Q., Ketter er, M. R. & Apicella, M. A. Gonococcal c ervicitis: a role for biofilm in pathoge nesis. The Journal of infectious disease s 198, 1856-1861 (2008). 30 Jen, F. E. C., Semchenko, E. A., Day , C.J., Seib, K.L. & Jennings, M.P.T. he Neisseria gonorrhoeae Methionine Sulf Oxide Reductase (MsrA / B) Is a Surface Ex posed, Immunogenic, Vaccine Candidate. rontiers in Immunology 10 (2019). 31 Semchenko, EA, Day, CJ & Seib, KL MetQ of Neisseria gonorrhoeae Is a Surface-Expressed Antigen That Elicits Bactericidal and Functional Blocking An tibodies. Infect Immun 85 (2017).

[0204] Example 2 Heparin-binding antigens (NHBA) of Neisseria gonorrhoeae are involved in microcolony formation. contributes to serum resistance and adhesion to epithelial cells Heparin-binding antigens (NHBA) of Neisseria species are expressed in Neisseria me ningitidis (which is closely related to N. gonorrhoeae) A four-component meningococcal serum licensed to protect against invasive disease caused by It is present in the group B vaccine (4CMenB, trade name Bexsero) [9]. The gonococcal homolog is surface-exposed and highly conserved in N. gonorrhoeae strains. (>93% identity), meningococcal NHBA variant 2 (NH BA-2)

[10] and those vaccinated with 4CMenB. It is recognized by human serum from various sources

[11] .

[0205] Meningococcal NHBA has been most extensively studied in strain MC58 (expressing NHBA-3). It has been investigated that glycosaminoglycans (GAGs) bind to arginine-rich regions (Arg regions). Named based on their ability to bind heparin, NHBA binding to heparin is a key factor in determining serum

[12] and its interaction with heparan sulfate increases the resistance of Neisseria meningitidis to hepatitis B virus, which binds to epithelial cells. NHBA mediates the binding of multiple other glycans, with the highest affinity binding occurring at the core. androitin sulfate

[14] . Meningococcal NHBA is a type of NHBA that inhibits the growth of human lactoferrin.

[12] , kallikrein

[15] , and C3 convertase

[16] , and meningococcal Na NalP is a target of several proteases, including NalP

[12] . High virulence of N. meningitidis expressing NalP after cleavage of NHBA It was speculated that the strain releases NHBA fragments that increase vascular permeability

[17] . BA-2 expression was also increased at lower temperatures (32°C vs. 37°C)

[18] , suggesting that bio It plays a role in film formation

[19] . The gonococcal NHBA has not yet been characterized. However, N. gonorrhoeae does not express NalP

[20] , and its NHBA is shortened. The NHBA has a specific Arg region

[10] , and the NHBA is This indicates that it may play a different role in N. gonorrhoeae compared to other In the Examples, its role in pathogenesis is described and it is proposed as a gonococcal therapeutic target or vaccine candidate. To support its potential use, NHB in N. gonorrhoeae Investigate the function of A.

[0206] method Sequence analysis NHBA sequence ( N. gonorrhoeae 1291 accession EEH6185 7.1; N. meningitidis MC58 AAF42586.1) on Mac Alignment was performed using CLUSTAL in Vector. Using the MLST nomenclature, each unique peptide sequence is assigned an identification number (e.g., N HBA_peptide 542 [NHBA-542] is N. gonorrhoeae 1291 inside).

[0207] Growth and phenotypic characterization of N. gonorrhoeae N. gonorrhoeae 1291 was incubated at 32 or 37°C with 5% CO2. , containing 1% (v / v) IsoVitaleX (Becton Dickinson) Cultures were grown on GC agar (Oxoid) or GC broth. Kanamycin (50 μg / m L) and spectinomycin (100 μg / mL) were added to the knockout and complemented strains. The majority of gonococcal populations were determined by phase contrast microscopy. The growth rate and aggregation experiments were performed in GC broth. The optical density at 600 nm (OD 600 ) were measured every hour [21, 22]. SDS-PAGE and Western blot analysis revealed that the pili in these strains Slightly less pilin was detected in WT compared to ΔNHBA and ΔNHBA_C; 14B), major outer membrane proteins including Por and Opa (Fig. 14C), and The similar molecular weight and abundance of pooligosaccharide (LOS) (Figure 14D) was also shown. In this case, N. gonorrhoeae was incubated in 0.25% trypsin for aggregation analysis. Trypsinized for 5 minutes.

[0208] Construction of N. gonorrhoeae NHBA mutants and recombinant NHBA The nhba gene (NGAG_00725) was cloned into N. gonorrhoeae strain 1291 Amplified from (primer 5'-ATGTTTAAACGCAGTGTGATTGC-3' (SEQ ID NO: 3); primer 5'-TCAATCCCGATCTTTTTTGCCGGC -3' (SEQ ID NO: 4)), cloned into pGEM-T Easy (Promega). The kanamycin resistance gene (pUC4Kan; Amersham Biosciences) was inserted into the vector. nces) was used for inverse PCR (primer 5'-GGATCCCCGGCCGAGA TTCCGCTGATTCC-3' (SEQ ID NO: 5); primer 5'-GGATCCGC GACCTCCTCGACCGTGCAGAAC-3' (SEQ ID NO: 6); BamHI site (underlined) into the BamHI site introduced into the center of nhba. The nhba::kan construct was linearized and transformed into N. gonorrhoeae 12 The complementing plasmid was transformed into 91 to generate nhba::kan (ΔNHBA). Use pCTS32 to amplify the intact nhba gene (primer 5'-GGCAT ATGGCGGAAACAATA-3' (SEQ ID NO: 7); primer 5'-TCAATC CCGATCTTTTTTGCCGGC-3' (SEQ ID NO: 8)) into ΔNHBA The complemented strain (ΔNHBA_C) was generated by inducing the nhba gene

[23] . The deletion and subsequent complementation of the offspring were confirmed by PCR and Western blot.

[0209] The N. gonorrhoeae 1291 nhba gene was transfected into E. coli BL21 The full-length mature N The HBA (without signal sequence) recombinant protein was expressed and purified.

[0210] Generation of polyclonal anti-NHBA Five 3-week-old female BALB / c mice (Animal Resources Center) were cultured. ter, Western Australia) group was treated with 25 μg of recombinant NHBA. with Freund's adjuvant (Merck) on days 0, 21, 28, and 42. The mice were immunized subcutaneously in the eye. Peripheral blood was collected on day 56, and serum was collected via centrifugation. The research was supported by the Griffith University Animal Ethics Co. Approved by mmittee.

[0211] Western blot, ELISA, and flow cytometry N from N. gonorrhoeae whole cell lysates as previously described

[24] . Western blot analysis of HBA expression (4–12% Bis-Tris SDS-PAGE (Th The isolation was performed using mouse anti-NHBA antibodies.

[24] , rabbit anti-NGAG_01228 was used to detect periplasmic proteins. was detected.

[0212] ELISA of His-tagged recombinant NHBA binding to whole-cell N. gonorrhoeae Analysis was performed using HRP-conjugated His-tag ELISAs according to standard protocols [11, 25]. Antibodies (Thermo) were used and measured after 30 min of incubation at room temperature.

[0213] NHBA expression on the surface of N. gonorrhoeae was measured by measuring the expression of the bacteria (approximately 10 8 CFU) The IgG was measured by flow cytometry (as previously described ([24, 26]) The bacteria were incubated with anti-NHBA (1:200, 30 min) and then PBS The cells were washed three times with Alexa Fluor 488-conjugated anti-mouse IgG (1 :200, 1 hour; Thermo Scientific), washed, and then Fixed in aldehyde (2.5%, 15 min). e(about 10 7 CFU) or human tCX and tUEC cells (approximately 5 × 10 5 cells) Binding of FITC-labeled gonococcal NHBA (100 μg / mL) was confirmed by incubation at 37°C for 20 min. All samples were measured using a CyAn ADP flow cytometer. (Beckman Coulter). Data analysis was performed using FlowJo This was accomplished using

[0214] Microscopy Using fluorescence microscopy, tCX cells (at full confluence on glass coverslips) were cultured. and N. gonorrhoeae NHBA (100 μg) incubated at 37°C for 20 min. The interaction of the ATP (mg / mL) was measured. The cells were washed three times to remove unbound proteins. The tissue was fixed in formaldehyde (2.5%, 15 min). Ng The anti-NHBA ( 1:1000)

[11] and Alexa Fluor 488-conjugated anti-mouse Cells were detected using Alexa Flu IgG (1:200; Thermo Scientific). Nuclei were counterstained with 568 phalloidin (Thermo) and DAPI. Mount the coverslip on ProLong Gold Antifade Mountant. (Thermo) mounted onto microscope slides and images were taken with a Nikon A1 R confocal microscope and data were analyzed using NIS-Elements (Nikon). analyzed.

[0215] Approximately 1×10 6 Incubate with CFU of N.gonorrhoeae at 37°C for 5 hours The tUEC cells were used to investigate gonococcal microcolony formation. Wash twice with HBSS to remove non-adherent bacteria, then add 2% glutaraldehyde and 5% phosphate buffer. The cells were fixed in aqueous methylaldehyde for 30 min and then analyzed by scanning electron microscopy as previously described

[27] . The CT scan was performed and images were taken using a JCM-5000 NeoScope™ (JEOL). Image captured.

[0216] Glycan linkage analysis Glycan array experiments were performed using recombinant NHBA ( 1 μg) and Institute for Glycomics glycan array (v3. Positive binding was determined by background adjusted counts in three independent replicates. A slide with a mean fluorescence >1x greater than the mean of the slide background (mean + 3 standard deviations) were assigned to pots (Student's t-test, p<0.001).

[0217] Surface plasmon resonance (SPR) was performed as previously described [14, 25]. for amine coupling on a S CM5 sensor chip (GE Healthcare) Therefore, the recombinant NHBA immobilized on flow cells 2 to 4 Ng (100μg / ml) The assay was performed using a BIAcore T200 instrument equipped with flow cell 1 as the reference cell. The ethanolamine was immobilized using single-cycle kinetics. Interactions with glycans were performed in a 1:5 dilution series at concentrations from 100 μM to 1 nM. Affinity for (K D The results were analyzed using BIAcore T200 software 2.0. .2 was used for the analysis.

[0218] Normal human serum (NHS) viability assay As previously described

[24] , N. gonorrhoeae against serum-mediated killing. e resistance test, approximately 10 4 CFU were incubated at 37°C for 60 min with 10% (v / v) Bacteria were cultured and subsequently plated on GC agar where indicated. Preincubation with 6 μM heparin for 30 min. Bacterial survival was assessed using untreated controls. The results were calculated as the percent CFU (average from three replicate wells) compared to the control.

[0219] Adhesion assay As previously described

[26] , the gonococcal adhesion assay was performed at approximately 10 5 CFU, E6 / E 7 Transformed primary human uterine cervical (tCX) cells and urethral epithelial (tUEC) cells for 1 hour Adhesion-blocking assays were performed using recombinant N. gonorrhoeae NHBA (1–100 μg / m L) or peanut agglutinin lectin (PNA; 100 μg / ml; tCX cells and The control was pretreated with a non-binding negative control

[26] , followed by N. gonorrhoeae. The same was performed on cells infected for 10 min. Results are expressed as percentages compared to wild type. Adherent bacteria (average from three replicate wells) and adherent bacteria compared to untreated controls Adhesion blocking was calculated as a percentage and reported. Adhesion and serum viability assays The results were performed in triplicate on three separate occasions, and statistical analysis was performed using ANOVA and Student's method. This was performed by t-test.

[0220] result Sequence characteristics and expression of Neisseria gonorrhoeae NHBA The major NHBA variant expressed by N. gonorrhoeae strains is NHB A-542, and >40% of N. gonorrhoeae isolates in the PubMLST database (strain 1291 NHBA-542 (referred to herein as NHBA-542) is present in the Ng ), which is 426 amino acids long, was isolated from N. meningitidis strain MC58. The well-characterized NHBA-3 (referred to herein as NHBA) Nm (called It contains sequence features (a lipobox motif and polynucleotides in the N-terminus) similar to those described in These include the glycine stretch, as well as the arginine-rich region in the center of the protein. However, due to insertions / deletions, the NHBA Ng and N.H. BA Nm There are several differences between the N-terminal half of the gonococcal nhba gene and N It has a 63 amino acid deletion compared to HBA-3, which is consistent among the major gonococcal variants. (Figure 13) Ng The Arg region of NHBA Nm It is shortened compared to This region is highly conserved between Nm strains

[12] and the major Ng NHBA variants. (Figure 13; the variant shown is present in 94% of 3068 isolates

[0011] ).

[0221] To facilitate the characterization of gonococcal NHBs, recombinant His-tagged proteins (NHBs) were synthesized. A-542) was produced in E. coli and polyclonal anti-NHBA antibodies were generated in mice. In addition, isogenic mutations of NHBA were generated in N. gonorrhoeae. Kanamycin insertion into the open reading frame of the nhba gene in E. coli strain 1291 This mutation was generated by the insertion of a cytochrome P454 (C1) resistance cassette (ΔNHBA) into the genome. Complemented by reintroducing a single copy of the nhba gene in trans (ΔNHBA C) Western blot analysis of whole cell lysates revealed that anti-NHBA serum inhibited 58–8 A single band was detected at 0 kDa, confirming the expression of NHBA in the wild-type strain. The transformed strain expressed NHBA at levels similar to the wild type, while NHBA expression was significantly higher in the mutant strains. NHBA was not detected in the different strains (Fig. 7C; Fig. 13). rhoeae WT and ΔNHBA_C strains by flow cytometry. N. gonorrhoeae reached mid-logarithmic growth phase at 32°C and 37°C (Fig. 7C). From the growth of the e strain, higher expression was observed at lower temperatures, and the expression of N. gonorrhoeae NHBA was controlled by temperature. It was revealed that the nodes were endothelial cells (Fig. 7B and C, Fig. 14).

[0222] Neisseria gonorrhoeae NHBA is involved in cell aggregation and microcolony formation NHBA in proliferation Ng To investigate the role of N. gonorrh in vitro, oeae strain 1291 wild type, ΔNHBA strain, and ΔNHBA_C strain were cultured in GC broth and G C agar. All strains had similar growth rates and optimal growth in terms of optical density. The ΔNHBA mutant strain had a significantly higher level of β-glucanase activity than the WT and ΔNHBA_C strains. The samples were analyzed using a 200-well platelet-free PBS containing 100% PEG-400 HCl and 100% PEG-400 HCl. Pull (OD 600 When the WT strain or Δ Approximately three times higher numbers of viable cells were detected for the ΔNHBA strain compared to the NHBA_C strain. Treatment of these samples with trypsin resulted in the production of CFU in all three The CFU counts for the two strains were equal (WT and ΔNHBA_C). The CFU of ΔNHBA increased by 2.6 and 2.4 times, respectively. (unaffected) (Fig. 8B), suggesting that the phenotype is due to cell aggregation rather than a defect in cell separation. From the Gram stain analysis of three stains ± trypsin treatment, the untreated WT strain and The presence of cell aggregates in the ΔNHBA_C strain was confirmed (Fig. 15A). From the tandem blot analysis, trypsin treatment digested NHBA from the bacterial surface but not from the periphery. It was shown that the plasma control protein was not altered (Figure 15B). The volume of the ΔNHBA sample used in subsequent experiments was adjusted to equalize the number of CFU. Gonococcal pili and opacity proteins have previously been shown to play a role in the formation of bacterial aggregates. As suggested by phase contrast microscopy, the WT, ΔNHBA, and ΔNHBA_C strains were fimbriated. It was confirmed that all the colonies shared the same colony morphology in terms of growth and opacity.

[0223] NHBANg To determine whether gonococci directly interact with the surface of N. gonorrhoeae to promote agglutination, To achieve this, recombinant NHBA Ng Flow cytometry and ELISA were used. Flow cytometry analysis revealed FITC labeling of whole-cell N. gonorrhoeae. Recombinant NHBA Ng This was confirmed by whole cell ELISA. The recombinant NHBA was confirmed using Ng N. gonorrhoeae in a concentration-dependent manner were combined (Figure 8D).

[0224] To further investigate the role of NHBA in bacteria-bacteria interactions and the formation of gonococcal aggregates, To investigate the ability of ΔNHBA to form microcolonies, we investigated the ability of the WT strain or ΔNHBA. Unlike the _C strain, the ΔNHBA strain adhered to the glass coverslip after 5 hours of growth. It was not possible to form microcolonies on the surface of the urethra or on a monolayer of human urethral epithelium. (Figure 9). NHBA Ng The self-association properties of α-glucan play a role in the establishment of gonococcal biofilms. Biofilm assays were also performed to investigate whether the Under static conditions for ~26 hours, the difference in biofilm formation was not significant between the WT and ΔNHBA strains. , and ΔNHBA_C strains (data not shown).

[0225] NHBA Ng binds to multiple glycans with high affinity The glycan binding profile of gonococcal NHBA was compared to that of glycans representative of those found on human cells. The array presents 368 structures (with similar structures but with different chain lengths, chemical linkages, etc.) or glycans with different spacer sizes, including isomers and / or glycans Determined using array analysis. Ng GAGs heparin, heparan sulfate, and On the array containing chondroitin sulfate (Figure 16; Table 4), 39 glycan structures were bound. NHBA Ng The lacto-N-biose core structure and the N-acetyllactosamine core structure structures (i.e., LNnT) (as well as their sialylated and fucosylated variants (i.e., sLeX), and N-acetylglucosamine, N-acetylgalactosamine It also binds to multiple structures containing a limited set of glycosyl, glucosyl, and mannosyl glycans. It met.

[0226] Glycans and recombinant NHBA Ng To characterize the kinetics of the interaction, Selected GAG (Figure 10A) and non-GAG glycans (Figure 10B) were used to conjugate the glycans. SPR analysis was performed using all tested NHBAs. Ng -glycan interactions The highest calculated affinity was for heparin (K D 4.4 nM), followed by chondroitin sulfate (K D The results were due to the presence of 73 nM of ATP. The ATP was composed of repeating polysaccharides with various sulfation patterns. The GAG ​​structures that are produced are highly heterogeneous. Ng However, it has a specific sulfation configuration. To determine whether they preferentially bind to the selected glycans, three types of chondroitin Experiments with tin sulfate (A, B, and C) were performed. Ng Chondroitin sulfate C (chondroitin 6-sulfate), and the concentration-dependent binding was (chondroitin 4-sulfate) or chondroitin sulfate B (dermatan sulfate) However, the NHBA Ng has a lower affinity (K D 2 It has also been shown that NHBA binds to non-GAG glycans at 0.79 μM. Ng teeth, α2-6 sialylated pentasaccharide (LSTc) (K D 0.24 μM) and its isomer LSTb (K D 2.26 μM) and LNnT (K D 4.89 μM) than the non-sialylated variant Furthermore, NHBA binds with high affinity. Ng is sialyl Lewis X(K D 3.65μ Lewis X (K) rather than M D 0.68 μM) Concentration-dependent binding was observed for hyaluronan (non-sulfated GAG) or H-disaccharides. These are the NHBAs on the glycan array. Ng was not bound by and was used as a negative control. was used.

[0227] Gonococcal NHBA binds to epithelial cells NHBA Nm The Arg region of the β-heparan sulfate proteoglycan is activated by its interaction with the β-heparan sulfate proteoglycan. It binds to epithelial cells via NHBA

[13] . Ng whether they also interact with epithelial cells Confocal microscopy and flow cytometry analysis were performed to investigate the recombinant NHBA N g For confocal microscopy, the following was performed: Recombinant NHBA Ng (rNHBA Ng ) with human endocervical epithelial (tCX) cells. Incubation with mouse anti-NHBA primary antibody and Alexa Flour 488 secondary antibody The following antibodies were used to detect NHBA in tCX cells: Ng Binding of 11A(I)), the protein signal was localized to the surface of the cells (white arrows, FIG. 11 A(II)). tCX cells were stained with primary (Figure 11A(III)) or secondary antibodies (Figure 11A(II)). A IV) non-specific binding was not observed. NHBA Ng It was further confirmed that β-glucan binds to cervical epithelial cells and urethral epithelial cells ( Figure 11B).

[0228] NHBA contributes to serum survival and adherence to epithelial cells of Neisseria gonorrhoeae NHBA of glycans, gonococcal cells, and human epithelial cells Ng Investigating the functional role of interactions To investigate the effect of WT serum on the survival and epithelial cell adhesion assays, we performed serum viability and epithelial cell adhesion assays using 10% of normal human serum (WT). The WT, ΔNHBA, and ΔNHBA_C strains were treated with 1000-kJ / mL serum at a sublethal concentration for each strain. From the serum survival assay performed by the present inventors, the ΔNHBA strain was found to be significantly different from the WT and ΔNHBA_C strains. Exposure to human serum demonstrated approximately a five-fold reduction in survival compared to control (Fig. 12A). Pretreatment of N. gonorrhoeae with heparin before exposure significantly improved the survival of the ΔNHBA mutant strain compared with the WT and ΔNHB strains. The levels of N. gonorrhoeae increased to levels comparable to those of strain A_C. They express several proteins (e.g., Opa

[29] ) that interact with ΔNH This may lead to the restoration of serum resistance in the BA strain.

[0229] NHBA in N. gonorrhoeae infection Ng To investigate the role of Cervical epithelial cells and urethral epithelial cells of the WT strain, ΔNHBA strain, and ΔNHBA In vitro infection assays using the C strain were performed. The ΔNHBA mutation increased the expression of tCX cells and tUEC cells by 1.5% compared to WT. There was a 1-fold and 12-fold reduced adhesion (Figure 12B). Ng or negative pair Adhesion assays were also performed with cells pretreated with either control protein PNA. HBA Ng Adhesion of N. gonorrhoeae to treated cells occurred in a concentration-dependent manner (i.e., at 100 and 10 μg / m L's NHBA Ng At 100 μg / mL, the reduction was 2.5-fold and 1.7-fold, respectively. Treatment of cells with PNA (a negative control that does not bind these cells) showed no effect on bacterial adhesion. No results were obtained (Fig. 12C).

[0230] Consideration The sexually transmitted disease gonorrhea is a public health concern due to rising infection rates and increasing antimicrobial resistance. N. gonorrhoeae primarily colonizes mucosal surfaces. The transmission, colonization, and pathogenesis of gonorrhea are complex multifactorial processes. [Reviewed in 30]. Increased understanding of all stages of gonorrhea has led to new treatments and This study is required to support the development of strategies for the prevention and treatment of glycans and Ng characterize gonococcal NHBA with respect to its interaction with human epithelial cells, Revealed involvement in microcolony formation, tolerance to human serum, and adhesion to epithelial cells do.

[0231] The NHBA is developing a reverse vaccine as part of the development of the meningococcal serogroup B vaccine, 4CMenB. It was first identified in N. meningitidis through cutinology [ 31 , 32 ]. Its functional role has since been characterized in detail [12-16]. There is a relatively high level of sequence identity (approximately 67%) between the protein and the meningococcal NHBA protein. Despite the 99.5% identity

[11] ), the NHBA sequence and two pathogenic Neisseria species There are several differences between the pathology seen in N. gonorrhoeae and that in N. gonorrhoeae, details of the NHBA are unclear. This prompted a detailed analysis. NHBA was more likely to be N. gonorrhoeae than N. meningitidis. hoeae

[11] , and herein, N. gonorrhoe A 63 amino acid deletion and a truncated Arg region in the N-terminus of ae are responsible for all major gonococcal NH It is confirmed that the nucleotide sequence is conserved in the BA variants. The data presented indicate the role of NHBA in most N. gonorrhoeae strains. Probably representative.

[0232] In the glycan array analysis herein, recombinant NHBA Ng is a set of multiple GAGs ( It was bound to 39 glycans, including valine, heparan sulfate, and chondroitin sulfate. The inventors are NHBA Nm previously showed that it interacts with 28 glycans

[14] . ,From SPR analysis, NHBA Ng and NHBA Nm have at least four glycans in common Confirmed to bind to (heparin, heparan sulfate, chondroitin sulfate, Glc-6P) However, important differences between the proteins were Ng But the NHBA Nm Rather than Hepari Higher binding affinity for chondroitin sulfate and glucose 6-phosphate This is probably because NHBs have a lower affinity for GAGs. A Nm This is due to differences in the Arg region known to be involved in the binding of Furthermore, Ng NHBA Ng Le, which can typically be found on the surface of the host cell Lewis X and sialyl Lewis X antigens, lacto-N-neotetraose (LNnT) and its sialylated variants on the glycan array, but NHBA Nm No. NHBA by NalP Nm The meningococcal cleavage of It releases a C-terminal fragment called C2

[12] , which increases vascular permeability

[17] . Additionally, the human proteases lactoferrin and kallikrein contain N-terminal amino acids downstream of the Arg region. HBA Nm [12, 15], and serum C3 convertase converts the released NHBA C2 fragments. The fragment was cut off, removing the Arg region and negating the toxic effects of the protein on cells.

[16] N. gonorrhoeae does not have the nalP gene.

[20] So, the NHBA Ng was not cleaved upstream of the Arg region. Ng is human lactate It is not cleaved by ferrin (data not shown) but by kallikrein and C3 convertase However, due to the absence of NaIP, NHBA Ng but Even when cleaved by these human enzymes, the functional Arg region remains associated with glycan binding. It may remain bound to the gonococcal surface where it may mediate a role.

[0233] The N. gonorrhoeae ΔNHBA mutant strain exhibited multiple phenotypes compared with the WT and ΔNHBA_C strains. The phenotypes include reduced survival in human serum, reduced aggregation and microcolony formation. and reduced adhesion to cervical and urethral epithelial cells. rrhoeae rarely causes disseminated disease but has the ability to resist serum killing have been extensively studied and include porins [30-32], LOS

[33] , and Opa

[28] . It has been found that the reproductive system is mediated by factors including serum factors and complement factors. It is present in the tract and other mucosal surfaces, making it relevant during mucosal infections [34-36]. BA Nm The addition of heparin before serum assays also contributes to serum-sensitive noncapsules. This results in increased survival of the cellularized parent meningococcal strain, but not the ΔNHBA mutant strain. Heparin interacts with multiple complement factors

[37] and NHBA Nm Complement regulation by Heparin-mediated mobilization of proteins has been proposed to be a mechanism for serum resistance

[0012] . We found that the WT strain expresses less complement regulatory proteins on its surface than the ΔNHBA mutant. NHBA involved in mobilizing higher levels of quality Ng We propose a similar mechanism of action for Even though the NHBA Ng The NHBA Nm It has a higher affinity for heparin than However, the relatively high level of survival of WT under the serum conditions tested and the accessibility to the assay Role in serum resistance, explaining the recovery of resistance of the gonococcal ΔNHBA mutant after the addition of valine There are additional gonococcal heparin-binding proteins (e.g., Opa

[28] ) that play a role.

[0234] Adhesion of gonococcal bacteria to the mucosal epithelium is a crucial first step in the establishment of infection and is the initial stage of adhesion. Following colonization, N. gonorrhoeae colonization occurs through the steady bacterial adhesion on the epithelial cell surface. The initial adhesion and microcolony formation depend on the formation of aggregates and microcolonies [reviewed in 30]. Both chromosomal colonization and chromosomal colonization are due to the presence of type IV pili, opaque (Opa) protein, and lipooligonucleotides. It is mediated by gonococcal factors, including the loss of sugar (LOS) [23, 42-44]. However, N. gonorrhoeae can agglutinate even in the absence of fimbriae or Opa. This suggests the existence of unknown host factors that promote GC aggregation

[45] . The BA mutations exhibited reduced adhesion to cervical epithelial cells and urethral epithelial cells compared to the wild type. and showed a reduction in aggregation and microcolony formation, thus NHBA Ng However, gonorrhea Furthermore, recombinant NHBA plays an important role in the establishment of Ng is above Direct interaction with both epithelial and gonococcal cells, NHBA Ng in a concentration-dependent manner to epithelial cells It is possible to block adhesion to Hec1B cells and CHO-K1 cells. NHBA with Cells Nm NHBA interactions with host glycans on epithelial cells mediate their interaction. This is likely the result of the use of N. gonorrhoeae

[13] . N. gonorrhoeae NHBA was upregulated at 32°C vs. 37°C. This is consistent with NHBA regulation in N. meningitidis and is due to the lower temperature of this niche at the time of adhesion by these organisms in the pharynx. This may be particularly relevant.

[0235] NHBA Ng NHBA in vector-bacterial interactions and microcolony formation Ng The role of LNnT is to interact with LNnT, which is present on the surface of gonococcal cells as part of LOS. This can be facilitated by the interaction of gonococcal microcolonies with host microvilli and the proliferation of host cells. This leads to the reorganization of the cytoskeleton and the formation of cortical plaques [47-51]. It is also involved in increasing biologic resistance

[45] , and the formation of bacterial aggregates is enhanced after exposure to seminal plasma. This may affect bacterial transmission

[52] . The formation of septa is also important for resistance to shear forces on the cell surface

[53] , which is why N. gon orrhoeae. However, meningococcal NHBA is currently It is interesting to note that no other protein has been reported to be involved in aggregation [13, 54 , 55].

[0236] In summary, the role of NHBA during multiple stages of gonococcal infection and pathogenesis is emphasized. Therefore, targeting NHBA-self and NHBA-host interactions may be a beneficial therapeutic approach. This could be a prophylactic and vaccine approach.

[0237] Throughout this specification, the intention is to limit the invention to any one embodiment or particular set of features. Therefore, the present invention has been described in detail without departing from the spirit and scope of the present invention. In light of the teachings herein, various modifications may be made in the specific exemplary embodiments without departing from the scope of the present invention. It will be recognized by those skilled in the art that modifications and variations can be made.

[0238] All computer programs, algorithms, and other The patent and scientific literature, as well as protein and nucleic acid sequences or accession numbers, are available by reference. No. 6,299,499, filed on Oct. 1, 2003, which is incorporated herein by reference. [Table 3] [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6] [Table 4-7] [Table 4-8] [Table 4-9] [Table 4-10] [Table 5]

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[0240] 55. Serruto D, Spadafina T, Ciucchi L, e t al. Neisseria meningitidis GNA2132, a heparin-binding protein that induces pro tective immunity in humans. Proceedings of the National Academy of Sciences 2010 ;107:3770-5.

Claims

1. An immunogenic fragment of a Neisseria genus heparin-binding antigen (NHBA) protein derived from Neisseria gonorrhoeae, wherein the immunogenic fragment is between 182 and 200 amino acids in length, comprises, consists of, or is essentially composed of, an amino acid sequence having at least 97% sequence identity to the amino acid sequence of Sequence ID No. 2 over the length of Sequence ID No. 2, and the immunogenic fragment is conjugated, coupled, or otherwise linked to a heterogeneous amino acid sequence.

2. The immunogenic fragment according to claim 1, wherein the immunogenic fragment comprises an amino acid sequence having at least 97% sequence identity with respect to the amino acid sequence of SEQ ID NO: 2 over the length of SEQ ID NO:

2.

3. The immunogenic fragment according to claim 2, wherein the immunogenic fragment comprises the amino acid sequence shown in SEQ ID NO:

2.

4. The immunogenic fragment according to claim 1, wherein the immunogenic fragment comprises, or is essentially, an amino acid sequence having at least 97% sequence identity with respect to the amino acid sequence of SEQ ID NO: 2 over the length of SEQ ID NO:

2.

5. The immunogenic fragment according to claim 4, wherein the immunogenic fragment consists of or is essentially derived from the amino acid sequence shown in SEQ ID NO:

2.

6. An immunogenic fragment of a Neisseria genus heparin-binding antigen (NHBA) protein derived from Neisseria gonorrhoeae, wherein the immunogenic fragment has a length between 165 and 181 amino acids, the immunogenic fragment has at least 97% sequence identity with respect to the amino acid sequence of SEQ ID NO: 2 over the length of the immunogenic fragment, and the immunogenic fragment is conjugated, coupled, or otherwise linked to a heterologous amino acid sequence.

7. The immunogenic fragment according to claim 6, wherein the immunogenic fragment has 100% sequence identity with SEQ ID NO: 2 over the length of the immunogenic fragment.

8. The immunogenic fragment according to any one of claims 1, 2, 4, and 6, wherein the immunogenic fragment comprises at least one amino acid substitution at positions 3, 5, 6, 9, 20, 50, 57, 60, 61, 69, 71, 75, 76, 83, 88, 89, 91, 92, 93, 113, 135, 150, 152, 153, 167, 173, 177, 180, and / or 181 of SEQ ID NO:

2.

9. The immunogenic fragment according to claim 8, wherein the at least one amino acid substitution is selected from the group consisting of A3V, I5M, P6L, P9S, G20E, P50S, R57S, G60A, E61K, A69V, T71A, N75S, G76R, M83T, P88S, Y89C, S91T, G92R, G93S, S113G, T135N, G150D, A152V, G153D, A167T, G173S, G177D, D180E, and R181Q of SEQ ID NO: 2, and any combination thereof.

10. The immunogenic fragment according to any one of claims 1 to 9, comprising one or more heparin-binding residues and / or one or more active site residues of the isolated NHBA protein.

11. A protein comprising the immunogenicity fragment described in any one of claims 1 to 10 and the heterogeneous amino acid sequence.

12. An isolated nucleic acid encoding an immunogenic fragment according to any one of claims 1 to 10 or a protein according to claim 11.

13. A gene construct comprising the isolated nucleic acid described in Claim 12.

14. A host cell comprising the gene construct described in claim 13 or the isolated nucleic acid described in claim 12.

15. A method for producing an immunogenic fragment according to any one of claims 1 to 10 or a protein according to claim 11, comprising: (i) culturing a host cell according to claim 14 in a culture medium; and (ii) isolating the immunogenic fragment or protein from the culture medium.

16. An antibody or antibody fragment that is conjugated to or prepared for an immunogenic fragment according to any one of claims 1 to 10 or a protein according to claim 11.

17. A composition comprising an immunogenic fragment according to any one of claims 1 to 10, wherein the composition optionally comprises a pharmaceutically acceptable diluent, carrier, or excipient.

18. The composition according to claim 17, which is an immunogenic composition.

19. The composition according to claim 18, which is a vaccine.

20. A composition comprising the protein according to claim 11, the isolated nucleic acid according to claim 12, the gene construct according to claim 13, the host cell according to claim 14, or the antibody or antibody fragment according to claim 16, wherein the composition optionally comprises a pharmaceutically acceptable diluent, carrier, or excipient, and further optionally the composition is an immunogenic composition and / or vaccine.

21. A pharmaceutical composition for inducing an immune response to Neisseria gonorrhoeae and / or Neisseria meningitidis in a target, wherein the pharmaceutical composition comprises an immunogenic fragment according to any one of claims 1 to 10, a protein according to claim 11, or a composition according to any one of claims 17 to 19.

22. A pharmaceutical composition for inducing immunity against Neisseria gonorrhoeae and / or Neisseria meningitidis in a subject, wherein the pharmaceutical composition comprises an immunogenic fragment according to any one of claims 1 to 10, a protein according to claim 11, or a composition according to any one of claims 17 to 19.

23. A pharmaceutical composition for treating or preventing infections caused by Neisseria gonorrhoeae and / or Neisseria meningitidis, wherein the pharmaceutical composition comprises an immunogenic fragment according to any one of claims 1 to 10, a protein according to claim 11, or a composition according to any one of claims 17 to 19.

24. A pharmaceutical composition for at least partially inhibiting or preventing Neisseria gonorrhoeae and / or Neisseria meningitidis from binding to a cell, wherein the pharmaceutical composition comprises an immunogenic fragment according to any one of claims 1 to 10, a protein according to claim 11, or a composition according to any one of claims 17 to 19.

25. A pharmaceutical composition for at least partially inhibiting or reducing serological resistance to infections caused by Neisseria gonorrhoeae and / or Neisseria meningitidis, wherein the pharmaceutical composition comprises an immunogenic fragment according to any one of claims 1 to 10, a protein according to claim 11, or a composition according to any one of claims 17 to 19.

26. In a subject, (i) for eliciting an immune response to Neisseria gonorrhoeae and / or Neisseria meningitidis, (ii) for inducing immunity against Neisseria gonorrhoeae and / or Neisseria meningitidis, (iii) for treating or preventing infection with Neisseria gonorrhoeae and / or Neisseria meningitidis, (iv) for at least partially inhibiting or preventing Neisseria gonorrhoeae and / or Neisseria meningitidis from binding to cells, or (v) Neisseria A pharmaceutical composition for at least partially inhibiting or reducing serological resistance to infections caused by Bacteria gonorrhoeae and / or Neisseria meningitidis, wherein the pharmaceutical composition comprises an isolated nucleic acid according to claim 12, a gene construct according to claim 13, a host cell according to claim 14, or an antibody or antibody fragment according to claim 16.

27. ​​A method for detecting Neisseria gonorrhoeae and / or Neisseria meningitidis in a biological sample obtained from a subject, comprising the step of contacting the biological sample with the antibody or antibody fragment described in claim 16 to detect Neisseria gonorrhoeae and / or Neisseria meningitidis in the biological sample.

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