Immunogenic proteins for gonorrhea

Abstract

The present invention relates, inter alia, to immunogenic fragments of the Neisseria heparin-binding antigen (NHBA) protein (SEQ ID NO: 1) of Neisseria gonorrhoeae for the prevention and treatment of diseases and conditions associated with Neisseria gonorrhoeae or Neisseria gonorrhoeae or Neisseria meningitidis. In some embodiments, the immunogenic fragment corresponds to the C-terminal fragment of the protein (SEQ ID NO: 2).

Description

[Technical Field]

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

[0002] The continued emergence of multidrug-resistant strains of Neisseria gonorrhoeae poses a major challenge to the control of sexually transmitted gonorrhea (1, 2), and the World Health Organization (3), the Centers for Disease Control (4), and the Australian National Antimicrobial Resistance (AMR) Strategy (5) have prioritized N. gonorrhoeae as an urgent public health threat requiring immediate action. There are an estimated 106 million cases of gonorrhea annually worldwide (6), and infection rates are rising (e.g., over the past 5 years, there has been a 67% increase in cases in the United States (7) and an 80% increase in cases in Australia (8)). The outcome of N. gonorrhoeae infection varies depending on the site of infection and gender (reviewed in 9, 10) and includes asymptomatic and locally symptomatic infections, but if left undiagnosed and / or untreated, gonorrhea can lead to serious sequelae, such as pelvic inflammatory disease, pregnancy and neonatal complications, and infertility. Infection with N. gonorrhoeae also increases the risk of acquiring and transmitting HIV.

[0003] Due to the high prevalence of N. gonorrhoeae, the severe sequelae it can cause, and the increasing difficulty of treating its multidrug-resistant strains, there is an urgent need for the development of a vaccine to prevent infection. However, the development of a gonococcal vaccine faces various challenges, including the high level of phase and antigenic variation in N. gonorrhoeae surface structures and the lack of protective immunity after infection, meaning that there are no established correlates of protection to guide preclinical vaccine studies (reviewed in 9, 11). Summary of the Invention

[0004] Surprisingly, the present inventors have found that administration of a C-terminal fragment of the Neisseria Heparin Binding Antigen (NHBA) protein from Neisseria gonorrhoeae can elicit the production of antibodies with bactericidal and opsonophagocytic killing at levels higher than the full-length protein, particularly in gonococcal strains with relatively lower NHBA expression.

[0005] Thus, in a broad form, the present invention relates to an NHBA protein (such as that set forth in SEQ ID NO: 1, or a fragment, variant, or derivative thereof that is immunogenic and capable of eliciting an immune response against Neisseria gonorrhoeae or Neisseria gonorrhoeae. In a preferred form, the NHBA protein is a C-terminal fragment comprising the amino acid sequence set forth in SEQ ID NO: 2, or a fragment, variant, or derivative thereof.

[0006] A first aspect of the present invention provides an immunogenic fragment of an isolated Neisseria heparin-binding antigen (NHBA) protein of Neisseria gonorrhoeae.

[0007] Suitably, the isolated NHBA protein comprises the amino acid sequence set forth in SEQ ID NO: 1, or a fragment, variant, or derivative thereof.

[0008] In a specific embodiment, the immunogenic fragment comprises a C-terminal fragment of an isolated NHBA protein (such as the amino acid sequence thereof as set forth in SEQ ID NO: 2, or a fragment, variant, or derivative thereof).

[0009] Suitably, a variant or derivative of SEQ ID NO: 2 comprises an amino acid substitution at one or more of residues 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 181 thereof. More particularly, the one or more amino acid substitutions in SEQ ID NO:2 may be 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, R181Q, and any combination thereof.

[0010] In some embodiments, the immunogenic fragment comprises one or more heparin-binding residues and / or one or more active site residues of the isolated NHBA protein, hi other embodiments, the immunogenic fragment does not comprise the heparin-binding residues and / or active site residues of the isolated NHBA protein.

[0011] In a second aspect, the present invention resides in an isolated protein comprising one or more immunogenic fragments according to the first aspect.

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

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

[0014] In a fifth aspect, the present invention resides in a host cell comprising the genetic construct of the fourth aspect.

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

[0016] In a seventh aspect, the present invention provides an antibody or antibody fragment which binds or is raised against the immunogenic fragment of the first aspect or the isolated protein of the second aspect.

[0017] In an eighth aspect, the present invention resides in a composition comprising one or more immunogenic fragments of the first aspect, an isolated protein of the second aspect, an isolated nucleic acid of the third aspect, a genetic construct of the fourth aspect, a host cell of the fifth aspect, and / or an antibody or antibody fragment of the seventh aspect, optionally together with a pharmaceutically acceptable diluent, carrier, or excipient.

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

[0019] In a ninth aspect, the present invention provides a method for raising an immune response in a subject to Neisseria gonorrhoeae and / or Neisseria meningitidis, the method comprising the step of administering to the subject one or more immunogenic fragments of the first aspect; an isolated protein of the second aspect; an isolated nucleic acid of the third aspect; a 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 composition of the eighth aspect, thereby raising an immune response.

[0020] In a tenth aspect, the present invention relates to a method for inducing immunity in a subject against Neisseria gonorrhoeae and / or Neisseria meningitidis, the method comprising the step of administering to the subject one or more immunogenic fragments of the first aspect; an isolated protein of the second aspect; an isolated nucleic acid of the third aspect; a 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 composition of the eighth aspect, thereby inducing immunity in the subject against Neisseria gonorrhoeae and / or Neisseria meningitidis.

[0021] In an eleventh aspect, the present invention resides in a method of treating or preventing a Neisseria gonorrhoeae and / or Neisseria meningitidis infection in a subject, the method comprising the step of administering to the subject one or more immunogenic fragments of the first aspect; an isolated protein of the second aspect; an isolated nucleic acid of the third aspect; a 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 composition of the eighth aspect, thereby preventing or treating a Neisseria gonorrhoeae and / or Neisseria meningitidis infection in the subject.

[0022] In a twelfth aspect, the present invention provides a method for at least partially inhibiting or preventing Neisseria gonorrhoeae and / or Neisseria meningitidis from binding to cells in a subject, the method comprising the step of administering to the subject one or more immunogenic fragments of the first aspect; an isolated protein of the second aspect; an isolated nucleic acid of the third aspect; a 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 composition of the eighth aspect, thereby inhibiting or preventing Neisseria gonorrhoeae from binding to cells of the subject.

[0023] In a thirteenth aspect, the present invention resides in a method for at least partially inhibiting or reducing serum resistance of Neisseria gonorrhoeae and / or Neisseria meningitidis infection in a subject, the method comprising the step of administering to the subject one or more immunogenic fragments of the first aspect; an isolated protein of the second aspect; an isolated nucleic acid of the third aspect; a 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 composition of the eighth aspect, thereby inhibiting or reducing serum resistance of Neisseria gonorrhoeae and / or Neisseria meningitidis infection in the subject.

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

[0025] In a fifteenth aspect, the present invention relates to the use of one or more immunogenic fragments of the first aspect; an isolated protein of the second aspect; an isolated nucleic acid of the third aspect; a 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 composition of the eighth aspect to raise an immune response to Neisseria gonorrhoeae and / or Neisseria meningitidis in a subject; to induce immunity to Neisseria gonorrhoeae and / or Neisseria meningitidis in a subject; to treat or prevent Neisseria gonorrhoeae and / or Neisseria meningitidis infection in a subject; to at least partially inhibit or prevent Neisseria gonorrhoeae and / or Neisseria meningitidis binding to cells in a subject; and / or to inhibit Neisseria gonorrhoeae and / or Neisseria meningitidis binding to cells in a subject; The present invention relates to the use of the compound in the manufacture of a medicament for at least partially inhibiting or reducing serum resistance of a Meningitidis infection in a subject.

[0026] In a sixteenth aspect, the present invention provides a method for eliciting an immune response to Neisseria gonorrhoeae and / or Neisseria meningitidis in a subject; inducing immunity to Neisseria gonorrhoeae and / or Neisseria meningitidis in a subject; treating or preventing Neisseria gonorrhoeae and / or Neisseria meningitidis infection in a subject; at least partially inhibiting or preventing Neisseria gonorrhoeae and / or Neisseria meningitidis binding to cells in a subject; and / or administering to a subject a therapeutically effective amount of Neisseria gonorrhoeae and / or Neisseria meningitidis. the isolated nucleic acid of the third aspect; the genetic construct of the fourth aspect; the host cell of the fifth aspect; the antibody or antibody fragment of the seventh aspect; and / or the composition of the eighth aspect for or when used for at least partially inhibiting or reducing serum resistance of a Meningitidis infection in a subject.

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

[0028] As used herein, the indefinite articles "a" and "an" are used herein to refer to or cover a singular or plural element or feature and should not be taken as meaning or defining "one" or "single" element or feature.

[0029] Unless the context requires otherwise, the terms "comprise," "comprises," and "comprising," or similar terms, are intended to imply a non-exclusive inclusion, such that an enumerated list of elements or features may include not only those expressly or listed elements, but may also include other elements or features not listed or expressly included.

[0030] By "consisting essentially of" in the context of an amino acid sequence (such as an immunogenic fragment) is meant the amino acid sequence recited together with an additional 1, 2, or 3 amino acids at the N- or C-terminus. [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 (upper panel) (SEQ ID NO: 1) and C-terminal fragment (lower 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 were no significant differences 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 is based, at least in part, on the discovery that a C-terminal fragment of NHBA from Neisseria gonorrhoeae demonstrates substantially improved immunogenicity against gonococci. Immunization with the NHBA protein fragment can elicit antibodies that are bactericidal and opsonophagocytic, and can inhibit the adhesion of gonococci to mucosal epithelial cells.

[0034] A broad aspect of the present invention relates to immunogenic fragments of an isolated Neisseria heparin-binding antigen (NHBA) protein of Neisseria gonorrhoeae (such as those comprising the amino acid sequence set forth in SEQ ID NO: 1, or a fragment, variant, or derivative thereof).

[0035] Neisseria gonorrhoeae (also known as gonococci(es)) is a type of Proteobacterium that causes sexually transmitted urogenital gonorrhea and other gonococcal-associated diseases, disorders, and conditions, including pharyngeal gonorrhea, rectal gonorrhea, disseminated gonococcal sepsis, gonococcal septic arthritis, and neonatal gonococcal conjunctivitis. Generally, as used herein, "Neisseria gonorrhoeae" encompasses all strains and serotypes of N. gonorrhoeae identifiable by one of skill in the art, including those described herein. Neisseria gonorrhoeae also encompasses genetic variants of different strains. Whether the target organism is N. gonorrhoeae can be determined by several methods known in the art, including sequencing the 16S ribosomal RNA (rRNA) gene, as described for N. gonorrhoeae in Chakravorty et al. (2007) (incorporated herein by reference).

[0036] The heparin-binding antigen (NHBA, formerly called GNA2132) of Neisseria meningitidis is a component of 4CMenB and exists as the NHBA-GNA1030 fusion protein (14).Nm ) is a surface-exposed lipoprotein that consists of three regions: an N-terminal region (residues 200–250) predicted to be intrinsically disordered and unfolded (15); a central arginine-rich region (16–18) that binds glycans (including heparin, heparin sulfate, and chondroitin sulfate); and a C-terminal region that folds as an antiparallel β-barrel (15, 19, 20). Nm Although the NHBA is relatively well conserved, the N-terminal region contains multiple insertions / deletions among different meningococcal strains (15). Nm induces serum bactericidal antibodies against diverse N. meningitidis strains ( 17 , 21 , 22 ), and these antibodies are also opsonophagocytic ( 23 , 24 ) and can block N. meningitidis adherence to epithelial cells ( 18 ). The gonococcal homolog of NHBA (NHBA Ng ) is highly conserved among N. gonorrhoeae strains (>93% identity) and shares 67% identity to the NHBA-2 peptide variant in 4CMenB (13, 25). Ng have recently shown that NHBA proteins are surface-exposed and recognized by antibodies from people vaccinated with 4CMenB (13). An example of an NHBA protein from N. gonorrhoeae is shown in SEQ ID NO:1.

[0037] For purposes of this invention, by "isolated" is meant material that has been removed from its natural state or that has otherwise been subjected to human manipulation. Isolated material can be substantially or essentially free of components that normally accompany the material in its natural state, or can be manipulated so as to be in an artificial state together with components that normally accompany the material in its natural state. Isolated material can be in native, chemically synthesized, or recombinant form.

[0038] By "protein" is meant an amino acid polymer. As is well understood in the art, the amino acids can be natural or unnatural amino acids, D-amino acids or L-amino acids.

[0039] The term "protein" includes and encompasses "peptides," which are typically used to describe proteins having 50 or fewer amino acids, and "polypeptides," which are typically used to describe proteins having more than 50 amino acids.

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

[0041] Generally, fragments can comprise an amino acid sequence of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 250, 300, 400, or up to 425 amino acids (such as the full-length NHBA protein set forth in SEQ ID NO:1).

[0042] In certain embodiments, an immunogenic fragment of an isolated NHBA protein comprises or consists of 10 to 250 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, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, or up to 185 amino acids of an isolated NHBA protein (such as that set forth in SEQ ID NO: 1 or SEQ ID NO: 2).

[0043] In certain embodiments, the immunogenic fragment comprises an isolated C-terminal fragment of an NHBA protein. As used herein, the term "C-terminal fragment" as applied to an NHBA protein can comprise a contiguous or stretch of amino acids, typically at least about 15, 20, 25, 30, 35, 40, 45, 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 210, located in or contained within the C-terminal domain of the NHBA protein.

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

[0045] In another embodiment, the immunogenic fragment comprises one or more glycan-binding or heparin-binding residues and / or one or more active site residues of the isolated NHBA protein.

[0046] Thus, an immunogenic fragment may comprise some or all of the extracellular or extracellularly exposed domain of an NHBA protein that corresponds to SEQ ID NO:2 and constitutes a fragment of SEQ ID NO:1, or alternatively, an immunogenic fragment may comprise a fragment of this extracellular or extracellularly exposed domain sequence that includes at least one of its glycan-binding and / or active site residues.

[0047] In the context of the present invention, the term "immunogenic" as used herein refers to the ability or potential of a protein to generate or elicit an immune response (such as against N. gonorrhoeae or its molecular components) upon administration of the protein to an animal. It is envisaged that the immune response may be either B-lymphocyte-mediated or T-lymphocyte-mediated, or a combination thereof. Advantageously, by "immunogenic" it is meant, but is not limited to, being capable of eliciting a B-lymphocyte response. "Immunogenic" also means being capable of eliciting a neutralizing antibody response.

[0048] By "eliciting an immune response" is meant generating or stimulating the production or activity of one or more elements of the immune system, including the cellular immune system, antibodies, and / or the innate immune system. Preferably, the one or more elements of the immune system include B lymphocytes, antibodies, and neutrophils. In one embodiment, the immune response is a mucosal immune response.

[0049] As used herein, a protein "variant" shares a definable nucleotide or amino acid sequence relationship with a reference amino acid sequence. For example, the reference amino acid sequence can be the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. A "variant" protein can have one or more amino acids of the reference amino acid sequence deleted or replaced with different amino acids. It is well understood in the art that some amino acids can be substituted or deleted without altering the activity of the immunogenic fragment and / or protein (conservative substitution). Thus, one or more of the other residues of SEQ ID NO: 1 or SEQ ID NO: 2 can be conservatively modified (e.g., by amino acid substitution or deletion) such that the variant substantially retains the immunogenicity of SEQ ID NO: 1 or SEQ ID NO: 2. Preferably, a protein variant shares 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 a reference amino acid sequence (such as SEQ ID NO: 1 or SEQ ID NO: 2).

[0050] It is also contemplated that modification of a "wild-type" or "unmodified" NHBA protein fragment sequence (such as that of SEQ ID NO: 1 or SEQ ID NO: 2) may substantially improve or enhance the immunogenicity of the immunogenic fragment. As an example, an immunogenic fragment may be modified to substantially match or correspond to the NHBA protein sequence of a particular N. gonorrhoeae strain, thereby improving the immunogenicity of the immunogenic fragment to said strain (such as those described herein). Thus, the term "variant" also encompasses isolated proteins or fragments thereof that are disclosed herein or that are produced from or comprise naturally occurring (e.g., allelic) variants, orthologs (e.g., from species other than N. gonorrhoeae, such as N. meningitidis), and synthetic variants (such as those produced in vitro using mutagenesis techniques). Typically, modifications involve the substitution of one or more amino acids of an NHBA protein fragment.

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

[0052] Suitably, the immunogenic fragment comprises one or more amino acid substitutions at residues 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 181 of SEQ ID NO:2, or the amino acid substitutions shown in Figure 18. In certain embodiments, the one or more amino acid substitutions are a valine (V) amino acid at residue 3 (A3V); a 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); a glutamic acid (E) amino acid at residue 20 (G20E); a serine (S) amino acid at residue 50 (P50S); a serine (S) amino acid at residue 57 (R57S); or a valine (V) amino acid at residue 10 (A3V); a methionine (M) amino acid at residue 11 (I5M); a leucine (L) amino acid at residue 12 (P6L); a serine (S) amino acid at residue 13 (P9S); a glutamic acid (E) amino acid at residue 20 (G20E); a serine (S) amino acid at residue 21 (P50S); a serine (S) amino acid at residue 22 (R57S); a serine (S) amino acid at residue 23 (R57S); or a leucine (L) amino acid at residue 24 (R57S); a serine (S) amino acid at residue 25 (R57S); or a leucine (L an alanine (A) amino acid at residue 60 (G60A); a 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 (T71A); a serine (S) amino acid at residue 75 (N75S); an arginine (R) amino acid at residue 76 (G76R); a threonine (T) amino acid at residue 83 (M83T); a serine (S) amino acid at residue 88 (P88S); a cysteine ​​(C) amino acid (Y89C); a threonine (T) amino acid at residue 91 (S91T); an arginine (R) amino acid at residue 92 (G92R); a serine (S) amino acid at residue 93 (G93S); a glycine (G) amino acid at residue 113 (S113G); an asparagine (N) amino acid at residue 135 (T135N); an aspartic acid (D) amino acid at residue 150 (G150D); a valine (V) amino acid at residue 152 (A1 52V); an aspartic acid (D) amino acid at residue 153 (G153D); a threonine (T) amino acid at residue 167 (A167T); a serine (S) amino acid at residue 173 (G173S); an aspartic acid (D) amino acid at residue 177 (G177D); a glutamic acid (E) amino acid at residue 180 (D180E); a glutamine (Q) amino acid at residue 181 (R181Q); and any combination thereof.

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

[0054] Terms generally used herein to describe the sequence relationships between respective proteins and nucleic acids include "comparison window," "sequence identity," "percentage of sequence identity," and "substantial identity." Because each nucleic acid / protein may contain (1) only one or more portions of the complete nucleic acid / protein sequence shared by the nucleic acids / proteins, and (2) one or more portions that are diverse between the nucleic acids / proteins, sequence comparison is typically performed by comparing sequences over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment, typically 6, 9, or 12 contiguous residues, that is compared to a reference sequence. The comparison window may include no more than about 20% additions or deletions (i.e., gaps) compared to the reference sequence for optimal alignment of the respective sequences. Optimal sequence alignment for aligning a comparison window can be performed by computer implementation of algorithms (GAP, BESTFIT, FASTA, and TFASTA, in the Geneworks program by Intelligenetics; Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive, Madison, WI, USA, incorporated herein by reference) or by inspecting and selecting the best alignment (i.e., resulting in the highest percentage of homology over the comparison window) generated by any of a variety of methods. Reference may also be made to the BLAST family of programs disclosed, for example, by Altschul et al., 1997, Nucl. Acids Res. 25 3389 (incorporated herein by reference). A detailed discussion of sequence analysis can be found in Unit 19.3 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al. (John Wiley & Sons Inc NY, 1995-1999).

[0055] The term "sequence identity" is used herein in its broadest sense to include the exact number of nucleotide or amino acid matches, taking into account the degree to which sequences are identical across a comparison window, taking into account appropriate alignment using standard algorithms. Thus, "percentage of sequence identity" is calculated by comparing two optimally aligned sequences across a comparison window, determining the number of positions where the same nucleic acid base (e.g., A, T, C, G, U) occurs in both sequences to obtain the number of identical positions, dividing the number of identical positions by the total number of positions in the comparison window (i.e., window size), and multiplying the result by 100 to obtain the percentage of sequence identity. For example, "sequence identity" can be understood to mean the "percentage of matches" calculated by the DNASIS computer program (version 2.5 for Windows; available from Hitachi Software Engineering Co., Ltd., South San Francisco, California, USA).

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

[0057] As used herein, a "derivative" is a molecule (such as a protein, fragment, or variant thereof) that has been altered, for example, by conjugation or complexation with other chemical moieties, by post-translational modifications (e.g., phosphorylation, acetylation, and the like), glycosylation modifications (e.g., addition, removal, or alteration of glycosylation), lipid addition, and / or incorporation of additional amino acid sequences, as understood in the art. One particular derivative is through conjugation of the immunogenic fragment to diphtheria toxin (DT). This can be facilitated by the addition of a C-terminal cysteine ​​residue.

[0058] The additional amino acid sequence can include a fusion partner amino acid sequence to generate a fusion protein. For example, the fusion partner amino acid sequence can aid in detection and / or purification of the isolated fusion protein. Non-limiting examples include metal-binding (e.g., polyhistidine) fusion partners, maltose-binding protein (MBP), protein A, glutathione S-transferase (GST), fluorescent protein sequences (e.g., GFP), epitope tags (such as myc, FLAG, and hemagglutinin tags).

[0059] Other additional amino acid sequences may be carrier proteins (such as diphtheria toxoid (DT) or fragments thereof), or CRM protein fragments (such as those described in International Publication WO2017 / 070735). In certain embodiments, the immunogenic fragments or isolated proteins described herein are conjugated, coupled, or otherwise linked to a carrier protein.

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

[0061] In this regard, those skilled in the art are referred to Chapter 15 of CURRENT PROTOCOLS IN PROTEIN SCIENCE, Eds. Coligan et al. (John Wiley & Sons NY 1995-2008) for a more extensive methodology regarding the chemical modification of proteins.

[0062] In a related aspect, the invention provides an isolated protein comprising one or more immunogenic fragments of an NHBA of N. gonorrhoeae. Suitably, the isolated protein is not a full-length or wild-type NHBA of N. gonorrhoeae.

[0063] In one particular embodiment, the present invention contemplates an isolated protein described herein that comprises multiple immunogenic fragments (such as in the form of a "polytope" protein). For example, the immunogenic fragments may be present singly or as repeats, including tandem repeat fragments. Heterologous amino acid sequences (e.g., "spacer" amino acids) may also be included between one or more immunogenic fragments present in the isolated protein.

[0064] In still further embodiments, this aspect of the invention provides isolated proteins or peptides consisting of (i) an immunogenic fragment described herein, or a segment, domain, portion, or region thereof (e.g., an epitope or antigenic determinant thereof), and inclusively, fragments, variants, or derivatives thereof; and (ii) optionally, one or more additional amino acid sequences. In this regard, the additional amino acid sequences are preferably, but not limited to, heterologous amino acid sequences that may be present at the N-terminus and / or C-terminus of the recited amino acid sequence of the aforementioned protein.

[0065] The immunogenic fragments and / or isolated proteins described herein, inclusive of fragments, variants, and derivatives thereof, may be produced by any means known in the art, including, but not limited to, chemical synthesis, recombinant DNA techniques, and proteolytic cleavage to produce peptide fragments.

[0066] Chemical synthesis includes solid-phase synthesis and liquid-phase synthesis. Such methods are well known in the art, and reference is made to examples of chemical synthesis techniques provided in Chapter 9 of Synthetic Vaccines Ed. Nicholson (Blackwell Scientific Publications) and Chapter 15 of Current Protocols in Protein Science Eds. Coligan et al. (John Wiley & Sons, Inc. NY USA 1995-2008). In this regard, reference is also made to International Publications WO99 / 02550 and WO97 / 45444.

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

[0068] In another aspect, the present invention contemplates isolated nucleic acids that encode or are complementary to nucleic acid sequences comprising nucleic acid sequences encoding the immunogenic fragments and isolated proteins disclosed herein or complementary nucleotide sequences thereto.

[0069] Nucleotide sequences encoding the isolated immunogenic proteins, isolated immunogenic fragments, variants, derivatives, and polytopes of the present invention can be readily deduced from the complete genomic nucleic acid sequence of NHBA.

[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-stranded or double-stranded DNA and RNA. DNA includes genomic DNA and cDNA. RNA includes mRNA, RNA, RNAi, siRNA, cRNA, and autocatalytic RNA. Nucleic acids can also be DNA-RNA hybrids. Nucleic acids include nucleotide sequences that typically include nucleotides containing A, G, C, T, or U bases. However, nucleotide sequences can include other bases, such as, but not limited to, inosine, methylycytosine, methylinosine, methyladenosine, and / or thiouridine.

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

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

[0074] A "probe" may be a single- or double-stranded oligonucleotide or polynucleotide, suitably labeled, for the purpose of detecting complementary sequences, for example, in Northern or Southern blotting.

[0075] A "primer" is typically a single-stranded oligonucleotide, preferably having 15-50 contiguous nucleotides, that is capable of annealing to a complementary nucleic acid "template" and being extended in a template-dependent manner by the action of a DNA polymerase (such as Taq polymerase, reverse transcriptase, or Sequenase™).

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

[0077] In one embodiment, the nucleic acid variant encodes a variant of an isolated protein or immunogenic fragment of the invention.

[0078] Suitably, nucleic acid variants share 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%, 81%, 82%, 83%, 84%, or 85%, and even more preferably at least 90%, 91%, 92%, 93%, 94%, or 95% nucleotide sequence identity with the isolated nucleic acids of the invention.

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

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

[0081] It will be appreciated by those skilled in the art that the isolated nucleic acids of the present invention can be conveniently prepared using standard protocols, such as those described in Chapters 2 and 3 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Eds. Ausubel et al. John Wiley & Sons NY, 1995-2008).

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

[0083] "Hybridizing" and "hybridization" are used herein to refer to the pairing of at least partially complementary nucleotide sequences to produce a DNA-DNA hybrid, an RNA-RNA hybrid, or a DNA-RNA hybrid. Hybrid sequences containing complementary nucleotide sequences result through base pairing.

[0084] "Stringency," as used herein, refers to the conditions of temperature and ionic strength during hybridization, as well as the presence or absence of particular organic solvents and / or detergents. The higher the stringency, the higher the required level of complementarity between hybridizing nucleotide sequences will be.

[0085] "Stringent conditions" refer to conditions under which only nucleic acids that have a high frequency of complementary bases will hybridize.

[0086] Stringent conditions are well known in the art, such as those described in Chapters 2.9 and 2.10 of Ausubel et al., supra (incorporated herein by reference). One of skill in the art will also recognize that various factors can be manipulated to optimize the specificity of hybridization. Optimization of the stringency of the final wash can be performed to ensure a high degree of hybridization.

[0087] Complementary nucleotide sequences can be identified by blotting techniques, which involve immobilizing nucleotides on a matrix (preferably a synthetic membrane such as nitrocellulose), hybridizing, and typically detecting using a labeled probe or other complementary nucleic acid. Southern blotting is used to identify complementary DNA sequences; Northern blotting is used to identify complementary RNA sequences. Dot blotting and slot blotting can be used to identify complementary DNA / DNA, DNA / RNA, or RNA / RNA polynucleotide sequences. Such techniques are well known to those skilled in the art and are described in Ausubel et al., supra, pp. 2.9.20-2.9.1. According to such methods, Southern blotting involves separating DNA molecules according to size by gel electrophoresis, transferring the size-separated DNA to a synthetic membrane, and hybridizing the membrane-bound DNA to a complementary nucleotide sequence. Alternative blotting steps are used to identify complementary nucleic acids in cDNA or genomic DNA libraries, such as through the process of plaque or colony hybridization. Other exemplary procedures are described in Chapters 8-12 of Sambrook et al., "MOLECULAR CLONING. A Laboratory Manual" (Cold Spring Harbor Press, 1989).

[0088] Methods for detecting labeled nucleic acids hybridized to immobilized nucleic acids are well known to those of skill in the art and include autoradiographic, chemiluminescent, fluorescent, and colorimetric detection.

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

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

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

[0092] Nucleic acid amplification techniques can include certain quantitative and semi-quantitative techniques such as qPCR, real-time PCR, and competitive PCR, as are well known in the art.

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

[0094] Preferably, the genetic construct is in the form of or includes genetic elements of a plasmid, bacteriophage, cosmid, yeast, or bacterial artificial chromosome, as is well understood in the art. The genetic construct may be suitable for the maintenance and propagation of isolated nucleic acids in bacteria or other host cells for manipulation and / or expression of the nucleic acids or encoded proteins of the invention by recombinant DNA techniques.

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

[0096] By "operably linked" it is meant that said additional nucleotide sequence is positioned relative to the nucleic acid of the invention so as to preferably initiate, regulate or otherwise control transcription.

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

[0098] Typically, the one or more regulatory nucleotide sequences may include, but are not limited to, a promoter sequence, a leader or signal sequence, a ribosomal binding site, a transcription start and stop sequence, a translation start and stop sequence, and an enhancer or activator sequence.

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

[0100] The expression construct may also include an additional nucleotide sequence (typically provided by the expression vector) encoding a fusion partner, so that the recombinant allergenic protein of the invention is expressed as a fusion protein as described above.

[0101] In certain embodiments, the genetic construct is suitable for administration to a subject (such as a human). In a preferred form, the genetic construct is suitable for DNA vaccination of a subject (such as a human).

[0102] Preferably, DNA vaccination is via one or more plasmid DNA expression constructs. Plasmids typically contain a viral promoter (such as the SV40, RSV, or CMV promoter). Intron A may be included to improve mRNA stability and thereby increase protein expression. Plasmids may further contain a multiple cloning site, a strong polyadenylation signal / transcription termination signal (such as the bovine growth hormone or rabbit β-globulin polyadenylation sequence). Plasmids may also contain the Mason-Pfizer simian virus cis-acting transcription element (MPV-CTE), with or without HIV rev-enhanced envelope expression. Additional modifications that may improve expression include the insertion of enhancer sequences, synthetic introns, adenovirus tripartite leader (TPL) sequences, and / or modifications to the polyadenylation sequence and / or transcription termination sequence. A non-limiting example of a DNA vaccine plasmid is pVAC, commercially available from Invivogen.

[0103] A useful reference describing DNA vaccinology is DNA Vaccines, Methods and Protocols, Second Edition (Volume 127 of Methods in Molecular Medicine series, Humana Press, 2006).

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

[0105] Suitable host cells for expression can be prokaryotic or eukaryotic. For example, suitable host cells can include, but are not limited to, mammalian cells (e.g., HeLa, HEK293T, Jurkat cells), yeast cells (e.g., Saccharomyces cerevisiae), insect cells (e.g., Sf9, Trichoplusia ni) used with or without a baculovirus expression system, plant cells (e.g., Chlamydomonas reinhardtii, Phaeodactylum tricornutum), or bacterial cells (e.g., E. coli). Introduction of genetic constructs into host cells (whether prokaryotic or eukaryotic) is well known in the art, for example, as described in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al., (John Wiley & Sons, Inc. 1995-2009), particularly Chapters 9 and 16.

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

[0107] Recombinant proteins can be conveniently prepared by one of skill in the art using standard protocols as described, for example, in Sambrook et al., MOLECULAR CLONING. A Laboratory Manual (Cold Spring Harbor Press, 1989), especially Sections 16 and 17; CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. 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), especially Chapters 1, 5, and 6.

[0108] In a further aspect, the present invention provides antibodies or antibody fragments that bind to and / or are raised against the immunogenic fragments and / or isolated proteins described herein.

[0109] Preferably, the antibody or antibody fragment specifically binds to the isolated immunogenic fragment and / or protein.

[0110] In some embodiments, the antibody may reduce, eliminate, inhibit, or suppress binding of NHBAs of N. gonorrhoeae to one or more glycans and / or substrate molecules (such as GAGs, heparin, heparan sulfate, and chondroitin). In other embodiments, the antibody may reduce, eliminate, inhibit, or suppress the ability of N. gonorrhoeae to bind to or adhere to cells (such as epithelial cells in a subject). In further embodiments, the antibody may reduce, eliminate, inhibit, or suppress the ability of N. gonorrhoeae to induce serum resistance in a subject. In certain embodiments, the antibody induces or mediates complement-dependent lysis and / or opsonophagocytic killing of N. gonorrhoeae cells.

[0111] Suitably, the antibody or antibody fragment specifically binds to an isolated immunogenic peptide comprising the amino acid sequence set forth in SEQ ID NO:2 or a variant, fragment, or derivative thereof. In some embodiments, the antibody or antibody fragment binds to the minimal epitope sequence contained within SEQ ID NO:2 with substantially higher affinity than an antibody raised against the full-length NHBA protein. In this context, by "substantially higher affinity" is meant at least 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold higher affinity at a particular concentration of NHBA protein.

[0112] Antibodies and antibody fragments can be polyclonal or monoclonal, native, or recombinant. Antibody fragments can include Fc, Fab, or F(ab)2 fragments, and / or single-chain Fv antibodies (scFvs). Such scFvs can be prepared, for example, according to the methods described in U.S. Pat. No. 5,091,513, European Patent No. 239,400, or the article by Winter & Milstein, 1991, Nature 349:293. Antibodies can also include multivalent recombinant antibody fragments (such as diabodies, triabodies, and / or tetrabodies containing multiple scFvs) and dimerization-activated demibodies (e.g., WO / 2007 / 062466). By way of example, such antibodies can be prepared according to the methods described in Holliger et al., 1993 Proc Natl Acad Sci USA 90 6444; or Kipriyanov, 2009 Methods Mol Biol 562 177. Well-known protocols applicable to antibody production, purification, and use can be found, for example, in Coligan et al., CURRENT PROTOCOLS IN IMMUNOLOGY (John Wiley & Sons NY, 1991-1994) and Chapter 2 of Harlow, E. & Lane, D. Antibodies: A Laboratory Manual, Cold Spring Harbour, Cold Spring Harbour Laboratory, 1988.

[0113] Methods for producing polyclonal antibodies are well known to those skilled in the art. Exemplary protocols that can be used are described, for example, in Coligan et al., CURRENT PROTOCOLS IN IMMUNOLOGY, supra, and Harlow & Lane, 1988, supra. By way of example, polyclonal antibodies can be raised in a production species (such as a horse) against purified or recombinant NHBA protein, or an immunogenic fragment thereof (e.g., SEQ ID NO: 2), and subsequently purified prior to administration.

[0114] Monoclonal antibodies can be produced by immortalizing spleen or other antibody-producing cells from a production species inoculated with one or more of the isolated proteins, fragments, variants, or derivatives of the invention using standard methods, such as those originally described in the paper by Kohler & Milstein, 1975, Nature 256, 495, or more recent modifications, such as those described in Coligan et al., CURRENT PROTOCOLS IN IMMUNOLOGY, supra. In certain embodiments, the monoclonal antibody or fragment thereof can be in recombinant form. This can be particularly advantageous for "humanizing" the monoclonal antibody or fragment if the monoclonal antibody is initially produced by the spleen cells of a non-human mammal.

[0115] The antibodies and antibody fragments of the present invention may be particularly suitable for affinity chromatography purification of the isolated immunogenic fragments and / or proteins described herein (see, for example, the affinity chromatography procedures described in Chapter 9.5 of Coligan et al., CURRENT PROTOCOLS IN IMMUNOLOGY, supra).

[0116] In some embodiments, antibodies or antibody fragments may be administered to a mammal to provide "passive" immunity to gonococcal infection.

[0117] In another embodiment, antibodies or antibody fragments that bind or are generated against the isolated immunogenic fragments and / or proteins of NHBAs described herein can be used to detect NHBAs expressed on the cell surface.

[0118] Certain further aspects and embodiments of the present invention provide compositions and / or methods for preventing, treating, and / or immunizing against gonorrhea-associated diseases, disorders, or conditions in animals (more particularly humans).

[0119] In one such aspect, the invention resides in a composition for preventing or treating a gonorrhea-associated disease, disorder, or condition, which may comprise: (i) one or more immunogenic fragments and / or proteins described herein; (ii) one or more isolated proteins described herein; (iii) one or more isolated nucleic acids described herein; (iv) one or more genetic constructs described herein; and / or (v) one or more antibodies or antibody fragments that bind or are made against an immunogenic fragment or isolated protein (such as those described herein), optionally together with a pharmaceutically acceptable diluent, carrier, or excipient.

[0120] By "pharmaceutically acceptable carrier, diluent, or excipient" is meant a solid or liquid filler, diluent, or encapsulating material that can be safely used in systemic administration. A variety of carriers well known in the art can be used depending on the particular route of administration. These carriers can be selected from the group including sugars, starches, cellulose and its derivatives, malt, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffer solutions, emulsifying substances, isotonic saline and salts (such as mineral acid salts, including hydrochlorides, bromides, and sulfates), organic acids (such as acetates, propionates, and malonates), and pyrogen-free water.

[0121] A useful reference describing pharmaceutically acceptable carriers, diluents, and excipients is Remington's Pharmaceutical Sciences (Mack Publishing Co. NJUSA, 1991), incorporated herein by reference.

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

[0123] As generally used herein, the terms "immunize," "vaccinate," and "vaccine" refer to methods and / or compositions that elicit a protective immune response against N. gonorrhoeae such that subsequent infection with N. gonorrhoeae is at least partially prevented or minimized.

[0124] Thus, such compositions, upon administration to a subject, can be delivered for, but are not limited to, the purpose of generating at least partial immunity and preferably protective immunity to, or to generate an immune response, preferably a protective immune response, to, N. gonorrhoeae bacteria.

[0125] By "protective immunity" is meant a level of immunity in which responsiveness to an antigen(s) leads to rapid binding and / or clearance of said antigen(s), thereby sufficient to at least partially ameliorate or prevent subsequent N. gonorrhoeae infection in an animal (such as a human subject).

[0126] By "protective immune response" is meant a level of immune response sufficient to prevent or reduce the severity, symptoms, aspects, or characteristics of current and / or future N. gonorrhoeae infection in an animal (such as a human subject).

[0127] In another specific embodiment, the immunogenic composition comprises one or more antibodies disclosed herein for passive immunization of a subject.

[0128] Suitable vaccines may be in the form of proteinaceous vaccines, and in particular comprise one or more immunogenic fragments of the NHBA protein of N. gonorrhoeae, or fragments, variants, or derivatives thereof described herein.

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

[0130] Useful carriers are well known in the art and include, for example, thyroglobulin; albumin (such as human serum albumin); toxins, toxoids, or any mutant cross-reactive material (CRM) of toxins from tetanus, diphtheria, pertussis, Pseudomonas, E. coli, Staphylococcus, and Streptococcus; polyamino acids (such as poly(lysine:glutamic acid)); influenza; rotavirus VP6, parvovirus VP1 and VP2; hepatitis B virus core protein; hepatitis B virus recombinant vaccine, and the like. Alternatively, fragments or epitopes of carrier proteins or other immunogenic proteins can be used. For example, T-cell epitopes of bacterial toxins, toxoids, or CRMs can be used. In this regard, reference can be made to U.S. Pat. No. 5,785,973 (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 adjuvants well known in the art. As understood in the art, "adjuvant" means a composition comprised of one or more substances that enhance the immunogenicity and effectiveness of a vaccine composition.

[0132] Preferably, for the purpose of eliciting an immune response, certain immunological agents may be used in combination with or conjugated to the immunogenic fragments or isolated proteins described herein. The term "immunological agent" includes within its scope carriers, delivery agents, immunostimulants, and / or adjuvants well known in the art. As understood in the art, immunostimulants and adjuvants refer to or include one or more substances that enhance the immunogenicity and / or efficacy of a composition.

[0133] Non-limiting examples of suitable adjuvants or immunostimulants include squalane and squalene (or other oils of plant or animal origin); block copolymers; detergents (Tween® 80); Quil® A, mineral oils (such as Drakeol or Marcol), vegetable oils (such as peanut oil); adjuvants derived from Corynebacterium spp. (such as Corynebacterium parvum); adjuvants derived from Propionibacterium spp. (such as Propionibacterium acne); Mycobacterium bovis (Bacillus Calmette-Guerin or BCG); Bordetella pertussis antigens; tetanus toxoid; diphtheria toxoid; surfactants (such as hexadecylamine, octadecylamine, octadecyl amino acid esters, lysolecithin, dimethyldioctadecylammonium bromide, N,N-dicoctadecyl-N',N'-bis(2-hydroxyethyl-propanediamine), methoxyhexadecylglycerol, and Pluronic polyols); polyamines (such as pyran, dextran sulfate, and polyIC carbopol); peptides (such as muramyl dipeptide and derivatives, dimethylglycine, and tuftsin); oil emulsions; and mineral gels (such as aluminum phosphate, aluminum hydroxide, or alum); interleukins (such as interleukin-2 and interleukin-12); monokines (such as interleukin-1); tumor necrosis factors; interferons (such as gamma interferon); combinations (such as saponin, aluminum hydroxide, or Quinoline). Aluminium hydroxide, etc.); liposomes; ISCOM® adjuvants and ISCOMATRIX® adjuvants; mycobacterial cell wall extracts; synthetic glycopeptides (such as muramyl dipeptide or other derivatives); avridine; lipid A derivatives; dextran sulfate; DEAE dextran alone or with aluminum phosphate; carboxypolymethylene (such as Carbopol'EMA); acrylic copolymer emulsions (such as Neocryl A640) (e.g., U.S. Pat. No. 5,047,238); water-in-oil emulsifying agents (such as Montanide ISA 720);Poliovirus, cowpox, or animal poxvirus proteins; or mixtures thereof;

[0134] With regard to subunit vaccines, examples of such vaccines may be formulated with ISCOMs (such as those described in International Publication No. WO 97 / 45444).

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

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

[0137] Alternatively, the vaccine may be in the form of a nucleic acid vaccine, and in particular a DNA vaccine. A useful reference describing DNA 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.

[0138] In some embodiments, the isolated immunogenic proteins and / or fragments of the invention may be used as vaccines in purified form, fused to immunogenic carrier proteins, or expressed by live vaccine delivery systems (including attenuated viruses, virus-like particles, or live attenuated bacteria).

[0139] In other embodiments, compositions and vaccines of the invention may be administered to humans in the form of attenuated or inactivated bacteria that can be induced to express one or more isolated immunogenic proteins or immunogenic fragments of the invention. Non-limiting examples of attenuated bacteria include Salmonella species (e.g., Salmonella enterica var. Typhimurium or Salmonella typhi). Alternatively, other enteric pathogens (such as Shigella species or E. coli) may be used in attenuated form. Attenuated Salmonella strains have been constructed by inactivating genes in the aromatic amino acid biosynthetic pathway (Alderton et al., Avian Diseases 35 435), by introducing mutations into two genes in the aromatic amino acid biosynthetic pathway (such as those described in U.S. Pat. No. 5,770,214), or into other genes such as htrA (such as those described in U.S. Pat. No. 5,980,907), or into genes encoding outer membrane proteins such as ompR (such as those described in U.S. Pat. No. 5,851,519).

[0140] In one embodiment, the antigen composition comprises outer membrane vesicles (OMVs). OMVs are non-replicating spherical nanoparticles naturally occurring in Gram-negative bacteria and composed of proteins, lipids (often LPS), and periplasmic contents. Suitably, OMVs can be prepared from the naturally secreted or detergent-extracted outer membrane of any bacterial species, such as Neisseria species (e.g., Neisseria gonorrhoeae and / or Neisseria meningitidis) or cultured strains of E. coli. OMVs can be obtained by any method known in the art (see, e.g., Gerritzen et al. 2017, Biotech Adv. 35:565-574; Semchenko et al. 2017, Infect Immun 85(2)e00898-16). In certain embodiments, the immunogenic fragments and / or isolated proteins of the present disclosure can be formulated with OMVs for surface exposure, non-surface exposure, binding to OMVs, or non-binding to OMVs (i.e., simple incorporation). The immunogenic fragment and / or isolated protein and OMV can be produced simultaneously by Gram-negative bacteria, such that the OMV is produced with the immunogenic fragment and / or isolated protein loaded on the surface or in the lumen of the OMV. Alternatively, the immunogenic fragment and / or isolated protein can be attached to the OMV after production, such as by covalent attachment using an affinity tag on the antigen that binds to a fusion protein in the OMV (see, e.g., Alves et al., 2015, ACS Appl. Mater. Interfaces, 7(44):24963-24972). Still further, the immunogenic fragment and / or isolated protein can be loaded into the lumen of the OMV after production, or simply mixed with the OMV after production. Exemplary OMVs for use as adjuvants with the immunogenic fragments and / or isolated proteins of the present disclosure include OMVs produced from any Gram-negative bacteria, including but not limited to N. meningitidis, N. gonorrhoeae, E. coli, and P. aeruginosa.It is further contemplated that the bacterial species from which the OMV is derived may be genetically modified for expression or upregulated expression of an NHBA protein, alone or in combination with other Neisseria gonorrhoeae and / or Neisseria meningitidis antigens, within the OMV from which it is derived.

[0141] Expression of a protein, peptide, fragment, or fusion protein containing a transport or immunogenic function can result in the production of the immunogenic protein, peptide, or fragment that is in the cytoplasm, in the cell wall, exposed on the cell surface, or produced in a secreted form.

[0142] In another aspect, the invention relates to a method of raising an immune response in a subject to Neisseria gonorrhoeae and / or Neisseria meningitidis, the method comprising administering to the subject one or more immunogenic fragments described herein; an isolated protein described herein; an isolated nucleic acid described herein; a genetic construct described herein; a host cell described herein; an antibody or antibody fragment described herein; and / or a composition of the previous aspect; thereby raising an immune response.

[0143] Suitably, the method elicits or promotes an immune response in a subject to prevent or prophylactically or therapeutically treat a gonorrhea-associated disease, disorder, or condition in said subject.

[0144] In a related aspect, the present invention provides a method for inducing immunity in a subject against Neisseria gonorrhoeae and / or Neisseria meningitidis, the method comprising the step of administering to the subject one or more immunogenic fragments described herein; an isolated protein described herein; an isolated nucleic acid described herein; a genetic construct described herein; a host cell described herein; an antibody or antibody fragment described herein; and / or a composition described herein, thereby inducing immunity in the subject against Neisseria gonorrhoeae and / or Neisseria meningitidis.

[0145] Preferably, the immune response or immunization to the N. gonorrhoeae bacteria prevents the animal from contracting a gonorrhea-associated disease, disorder, or condition. Additionally, due to the homology observed in the protein sequences (particularly the C-terminal sequences) for NHBA proteins across different Neisseria species, it will be recognized by those skilled in the art that the methods can also be used to immunize animals against additional Neisseria species (such as Neisseria meningitidis).

[0146] In a further aspect, the present invention resides in a method of treating or preventing a Neisseria gonorrhoeae and / or Neisseria meningitidis infection in a subject, the method comprising the step of administering to the subject one or more immunogenic fragments described herein; an isolated protein described herein; an isolated nucleic acid described herein; a genetic construct described herein; a host cell described herein; an antibody or antibody fragment described herein; and / or a composition described herein, thereby preventing or treating a Neisseria gonorrhoeae and / or Neisseria meningitidis infection in the subject.

[0147] Similar to the previous two embodiments, the method can also be used to treat animals for additional Neisseria species, including but not limited to Neisseria meningitidis.

[0148] As used herein, "treating" (or "treat" or "treatment") refers to a therapeutic intervention that ameliorates signs or symptoms of a gonococcal- or meningococcal-associated disease, disorder, or condition after it has begun to develop. The term "ameliorating" refers to any observable beneficial effect of treatment with respect to a gonococcal- or meningococcal-associated disease, disorder, or condition. Treatment need not be absolute to be beneficial to the subject. A beneficial effect may be determined using any method or criteria known to one of skill in the art.

[0149] As used herein, "preventing" (or "prevent" or "prevention") refers to a procedure (such as administering a therapeutically effective amount of a composition comprising one or more immunogenic proteins of the invention and / or fragments, variants, or derivatives thereof) initiated prior to the onset of a symptom, aspect, or feature of a gonococcal- or meningococcal-associated disease, disorder, or condition, such that the symptom, aspect, or feature is prevented or reduced. It is understood that such prevention need not be absolute to be beneficial to the subject. A "prophylactic" treatment is a treatment administered to a subject who does not exhibit symptoms, or who exhibits only early symptoms, of a gonococcal- or meningococcal-associated disease, disorder, or condition, for the purpose of reducing the risk of developing a symptom, aspect, or feature of the gonococcal- or meningococcal-associated disease, disorder, or condition.

[0150] The term "therapeutically effective amount" describes an amount of a specified agent (such as an isolated immunogenic fragment, isolated protein, and antibody or antibody fragment described herein) sufficient to achieve a desired effect in a subject being treated with the agent. For example, this can be the amount of a composition comprising an isolated immunogenic fragment, isolated protein, and / or antibody or antibody fragment described herein necessary to reduce, alleviate, and / or prevent a gonococcal- or meningococcal-associated disease, disorder, or condition (including gonococcal or meningococcal infection). In some embodiments, a "therapeutically effective amount" is sufficient to reduce or eliminate the symptoms of a gonococcal- or meningococcal-associated disease, disorder, or condition. In other embodiments, a "therapeutically effective amount" is an amount sufficient to achieve a desired biological effect (e.g., an amount sufficient to elicit a protective immune response in a subject so as to inhibit or prevent gonococcal and / or meningococcal infection).

[0151] Ideally, a therapeutically effective amount of an agent is an amount sufficient to induce a desired result without causing substantial cytotoxic effects in the subject. An effective amount of an agent useful for reducing, alleviating, and / or preventing a gonococcal- or meningococcal-associated disease, disorder, or condition (such as a gonococcal or meningococcal infection) will depend on the subject being treated, the type and severity of any associated disease, disorder, and / or condition (e.g., the type of gonococcal- or meningococcal-associated disease, disorder, or condition, and / or the strain of N. gonorrhoeae or N. meningitidis), and 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 gonococcal or Neisseria gonorrhoeae infection (including any clinical pathology resulting from such infection with Neisseria gonorrhoeae, such as those described above).

[0153] Additionally, by "meningococcal-associated disease, disorder, or condition" is meant any meningococcal infection or Neisseria meningitidis infection (including any clinical pathology resulting from such infection with Neisseria meningitidis, such as meningitis, rash, septicemia, fever, nausea, vomiting, and diarrhea).

[0154] In yet another aspect, the present invention provides a method for at least partially inhibiting or preventing Neisseria gonorrhoeae and / or Neisseria meningitidis from binding or adhering to cells in a subject, the method comprising the step of administering to the subject one or more immunogenic fragments described herein; an isolated protein described herein; an isolated nucleic acid described herein; a genetic construct described herein; a host cell described herein; an antibody or antibody fragment described herein; and / or a composition described herein, thereby inhibiting or preventing Neisseria gonorrhoeae and / or Neisseria meningitidis from binding to cells of the subject.

[0155] It will be appreciated that bacterial adhesion to host cells is an initial step and a prerequisite for successful colonization of a host mucosal surface. In certain embodiments, the cells are epithelial cells (such as vaginal, cervical, endometrial, pharyngeal, and urethral epithelial cells).

[0156] In yet another aspect, the present invention relates to a method for at least partially inhibiting or reducing serum resistance of Neisseria gonorrhoeae and / or Neisseria meningitidis infection in a subject, the method comprising the step of administering to the subject one or more immunogenic fragments described herein; an isolated protein described herein; an isolated nucleic acid described herein; a genetic construct described herein; a host cell described herein; an antibody or antibody fragment described herein; and / or a composition described herein, thereby inhibiting or reducing serum resistance of Neisseria gonorrhoeae and / or Neisseria meningitidis infection in the subject.

[0157] Neisseria gonorrhoeae is a frequent cause of sexually transmitted infection in humans worldwide. A small percentage of gonococcal infections can lead to a severe, life-threatening complication commonly referred to as disseminated gonococcal infection (DGI). Virulence and resistance to the complement-dependent bactericidal effects of normal human serum (i.e., serum resistance) appear to be strictly correlated for this Gram-negative diplococcal bacterium.

[0158] Preferably, the aforementioned methods of the present invention are performed on an animal (such as a mammal). In one embodiment, the mammal is a human.

[0159] It will be appreciated that compositions for administration in the methods of the five foregoing aspects may comprise, but are not necessarily limited to, one or more immunogenic fragments and / or isolated proteins of the invention, and / or one or more antibodies or antibody fragments of the invention raised against the immunogenic fragments and / or isolated proteins described herein. Thus, in certain embodiments, such compositions may comprise one or more antibodies or one or more antibody fragments capable of binding to or raised against a C-terminal fragment (such as that set forth in SEQ ID NO:2) of an NHBA protein (e.g., SEQ ID NO:1).

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

[0161] Any safe route of administration may be used to provide the compositions of the present invention to a patient, including oral, rectal, parenteral, sublingual, buccal, intravenous, intraarticular, intramuscular, intradermal, subcutaneous, by inhalation, intraocular, intraperitoneal, intracerebroventricular, intravaginal, and transdermal administration.

[0162] Dosage forms include tablets, dispersions, suspensions, injections, solutions, syrups, lozenges, capsules, nasal sprays, suppositories, aerosols, transdermal patches, and the like. These dosage forms may also include injections or implants of controlled-release devices specifically designed for this purpose, or other forms of implants modified to additionally act in this manner. Controlled release of therapeutic agents can be achieved, for example, by coating the same with hydrophobic polymers (including acrylic resins, waxes, higher aliphatic alcohols, polylactic and polyglycolic acids, and certain cellulose derivatives such as hydroxypropylmethylcellulose). In addition, controlled release can be achieved through the use of other polymer matrices, liposomes, and / or microspheres.

[0163] Compositions of the present invention suitable for oral or parenteral administration may be presented as discrete units (such as capsules, sachets, functional foods / feeds, or tablets, each containing a predetermined amount of one or more therapeutic agents of the present invention), as a powder or granules, or as a solution or suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion, or a water-in-oil liquid emulsion. Such compositions may be prepared by any of the methods of pharmacy, but all methods include the step of bringing into association one or more agents described above with a carrier, which constitutes one or more necessary ingredients. Generally, the compositions may be prepared by uniformly and intimately admixing an agent of the present invention with liquid carriers or finely divided solid carriers, or both, and then, if necessary, shaping the product into the desired presentation.

[0164] The compositions can 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, should be sufficient to achieve a beneficial response in the patient over a reasonable period of time. The amount of drug(s) administered can depend on the subject being treated, including age, sex, weight, and their general health condition, factors that will depend on the judgment of a physician.

[0165] In certain embodiments of the aforementioned methods and compositions, the immunogenic fragments or isolated proteins may be administered in combination with additional immunogenic fragments or proteins derived from the NHBA protein, or additional N. gonorrhoeae or N. meningitidis proteins known in the art, such as the surface-expressed MetQ protein (Semchenko et al. 2017), MsrAB, AniA, and one or more of the four antigenic components present in the Bexsero vaccine (GSK Vaccines) (i.e., factor H binding protein (fHbp) from the New Zealand epidemic strain (MeNZB, which provides PorA), Neisseria adhesin A (NadA), Neisseria heparin-binding antigen (NHBA), and outer membrane vesicles). In this regard, the immunogenic fragments or isolated proteins described herein may be included as components of a multi-antigen vaccine for N. gonorrhoeae and / or Neisseria meningitidis. In some embodiments, the immunogenic fragment or isolated protein of the NHBA and the additional immunogenic fragment or protein may be provided as a single chimeric peptide. In this embodiment, the immunogenic fragment or isolated protein described herein may be N-terminal or C-terminal to the additional immunogenic fragment or protein.

[0166] In a final aspect, the present invention provides a method for detecting N. gonorrhoeae and / or Neisseria meningitidis in a biological sample obtained from an animal, the method comprising contacting the biological sample with an antibody or antibody fragment described herein, thereby detecting N. gonorrhoeae and / or Neisseria meningitidis in the biological sample. Suitably, an NHBA protein is detected on the extracellular surface of one or more N. gonorrhoeae and / or N. meningitidis cells in the biological sample.

[0167] In certain embodiments, the biological sample may be a pathology sample, including one or more bodily fluids, cells, tissues, organs, or organ samples taken from an animal, including, but not limited to, blood, plasma, serum, lymphocytes, urine, feces, amniotic fluid, cervical samples, cerebrospinal fluid, tissue biopsy, bone marrow, bronchoalveolar lavage fluid, sputum, and skin.

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

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

[0170] The label may be selected from the group comprising a chromogen, a catalyst, biotin, digoxigenin, an enzyme, a fluorophore, a chemiluminescent molecule, a radioisotope, a drug, a magnetic bead, and / or a direct visual label.

[0171] In the case of direct visual labeling, colloidal metallic or non-metallic particles, dye particles, enzymes or substrates, organic polymers, latex particles, liposomes, or other vesicles containing signal-producing substances, and the like, can be used.

[0172] The fluorophore can be, for example, fluorescein isothiocyanate (FITC), Alexa dyes, tetramethylrhodamine isothiocyanate (TRITL), allophycocyanin (APC), Texas Red, Cy5, Cy3, or Rphycoerythrin (RPE), as is well known in the art.

[0173] The enzyme can be, but is not limited to, horseradish peroxidase (HRP), alkaline phosphatase (AP), β-galactosidase, or glucose oxidase.

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

[0175] It will be further recognized that such methods for detecting N. gonorrhoeae may have potential utility in characterizing the disease progression and / or severity of gonorrhea-associated diseases, disorders, or conditions in animals. Additionally, such methods may be used to select animals for anti-NHBA treatment (such as by so-called "companion diagnostics").

[0176] As generally used herein, the terms "patient," "individual," and "subject" are used in the context of any mammalian recipient of the treatments or compositions disclosed herein. Accordingly, the methods and compositions disclosed herein may have medical and / or veterinary applications. In a preferred form, the mammal is a human. One or more steps of the methods described herein may be performed in vitro.

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

[0178] Example 1 Introduction Several recent advances support the feasibility of developing a gonococcal vaccine. A recent observational study suggested that a vaccine against the closely related bacterium Neisseria meningitidis (outer membrane vesicle (OMV) meningococcal B vaccine MeNZB) has 31% efficacy against infection with N. gonorrhoeae

[12] . A newer four-component meningococcal B vaccine (4CMenB (commercially available as Bexsero) containing MeNZB OMV components plus three recombinant protein antigens) has been shown to induce cross-reactive antibodies to N. gonorrhoeae proteins, including NHBA

[13] .

[0179] In this example, a detailed analysis of the sequence variation and expression of NHBA of N. gonorrhoeae was performed to identify NHBA Ng The level, type, and functional activity of antibodies generated against the protein will be investigated to assess the potential of the full-length protein and a C-terminal fragment of the protein as a gonococcal vaccine candidate.

[0180] result NHBA is highly conserved in N. gonorrhoeae We previously showed that NHBA is conserved in N. gonorrhoeae

[13] , and here we further investigate sequence variants of NHBA in available gonococcal isolates and genome sequences. A BLAST search (Figure 1A) of the 1,281-nucleotide nhba gene from N. gonorrhoeae strain 1291 (which encodes the 427-amino acid NHBA) against gonococcal genomes available in GenBank revealed that nhba was present in all 594 genomes with 94.1–100% nucleic acid identity. A similar BLAST search against the PubMLST database revealed the presence of the nhba gene in 4,424 isolates with 85.1–100% identity. The 1,228 isolates that did not have a match to nhba in this BLAST search also lacked the annotated 16S and porB genes, indicating that incomplete sequences were available for these isolates. This confirms the widespread distribution and high conservation of nhba in a temporally and geographically diverse panel of gonococcal strains collected between 1960 and 2020 from over 60 different countries.

[0181] As of April 6, 2020, there were 42 unique NHBA_peptide variants among the 3,546 N. gonorrhoeae isolates with annotated NHBA proteins in the PubMLST database. These variants share 97.5–100% amino acid identity. There were two predominant NHBA variants present in 70.3% of PubMLST isolates: NHBA-542 (present in 39.7% of strains, including N. gonorrhoeae 1291) and NHBA-475 (present in 30.4% of strains, including N. gonorrhoeae WHO P and WHO X). Overall, one of the 14 major NHBA variants was present in 97.8% of isolates, while the remaining 28 NHBA peptide variants were rare, present in 1–10 isolates (Table 5). Alignment of these 14 most common variants indicates that the N- and C-termini have the highest levels of conservation, with a variable central region located upstream of the arginine-rich region (Figure 1B; variants are arranged in order of decreasing abundance). The phylogenetic relationships of these NHBA variants are shown in Figure 1C, and a panel of strains representative of NHBA diversity was used in subsequent assays. Given the conserved sequence of the C-terminus (Figure 1B), the structure of the meningococcal NHBA C-terminal region has been characterized [15, 19, 20], and the C-terminal region is likely more exposed and accessible to vaccine-induced antibodies, subsequent investigations focused on both recombinant full-length NHBA and a C-terminal NHBA fragment (NHBA-c) (Figure 1A).

[0182] Recombinant full-length NHBA and C-terminal NHBA fragments are immunogenic and induce antibodies that recognize NHBA variants from various gonococcal strains. To investigate the immunogenicity of gonococcal NHBA, sera from mice immunized with recombinant full-length NHBA plus Freund's adjuvant, or the NHBA-c fragment plus Freund's adjuvant or aluminum hydroxide (Alum) were assessed by ELISA and Western blot. Using whole-cell ELISA, we show that both NHBA and NHBA-c mouse sera can detect native NHBA on the surface of N. gonorrhoeae wild-type (WT) and NHBA-complemented (ΔNHBA_C) strains, with significantly reduced titers for the NHBA mutant strain (ΔNHBA) (Table 1). Analysis of NHBA antisera by Western blot against whole-cell lysates of N. gonorrhoeae wild-type and mutant strains confirmed that the antisera specifically recognized NHBA (Figure 2A). Expression of NHBA in a panel of N. gonorrhoeae strains and cross-reactivity of the NHBA antisera were confirmed by Western blot analysis (Figure 2B). NHBA expression varied between strains, and high, intermediate, and low NHBA expressers were used in subsequent assays.

[0183] ELISA with recombinant NHBAs demonstrated the presence of a predominant IgG1 isotype response in mice immunized with NHBAs (Table 1). However, the isotype and subclass ratios differed between the different formulations, with NHBA-Freund having higher levels of IgG3 and lower levels of IgG2a and IgG2b (IgG1 > IgM > IgG3 > IgG2b > IgG2a) than NHBA-c-Freund (IgG1 > IgM = IgG2b > IgG2a > IgG3) and NHBA-c-Alum (IgG1 > IgM > IgG2b > IgG2a > IgG3). Overall, the ELISA and Western results confirm that gonococcal NHBAs are immunogenic and that anti-NHBA antisera can recognize NHBAs on the surface of multiple N. gonorrhoeae strains expressing different NHBA variants.

[0184] NHBA antibodies enhance C3 fragment deposition To investigate whether NHBA antisera enhance activation of the complement cascade, C3 fragment deposition on the surface of N. gonorrhoeae was examined using flow cytometry. NHBA-Freund and NHBA-c-Freund mouse sera and NHBA-specific IgG purified from these sera were tested; all of them bind to N. gonorrhoeae strain 1291, as evidenced by increased mean fluorescence intensity compared to preimmune serum or control-treated bacteria (Figure 3A-B, top panels). Bacteria incubated with either human complement plus whole serum or purified IgG significantly increased C3 fragment deposition compared to the complement-only control (7.1- and 5.2-fold increases, respectively, for NHBA; 4.8- and 4.7-fold increases, respectively, for NHBA-c; Figure 3A-B, bottom panels).

[0185] NHBA antibodies have bactericidal and opsonophagocytic activities The ability of NHBA and NHBA-c antibodies to mediate complement-dependent lysis and opsonophagocytic killing of N. gonorrhoeae was tested using serum bactericidal activity (SBA) and opsonophagocytic killing (OPA) assays, respectively. Five gonococcal strains containing different NHBA variants and varying levels of NHBA expression were tested. For the SBA assay, N. gonorrhoeae was incubated with NHBA or NHBA-c mouse serum, followed by the addition of a human complement activator, and bacterial survival was measured. Both NHBA-Freund's and NHBA-c-Freund's sera elicited serum bactericidal activity in a concentration-dependent manner, with SBA titers ranging from 100 to 1600 (compared to titers of <50 for preimmune sera) (Figure 20A; Table 2). For OPA assays, N. gonorrhoeae were opsonized with NHBA or NHBA-c antibodies and incubated in the presence of human complement. Human PMNs were killed in a dose-dependent manner with OPA titers ranging from 100 to 6,400 (compared to titers of <50 for preimmune sera) (Fig. 20B; Table 2). Serum raised against NHBA formulated with an adjuvant frequently used in human vaccines (Alum) (NHBA-c-Alum) also induced SBA and OPA killing of N. gonorrhoeae, with titers similar to those observed with NHBA-c-Freund sera (Fig. 20A, B; Table 2). Purified NHBA immunoglobulins from mice immunized with NHBA-c-alum mediated concentration-dependent SBA killing (Fig. 20C). Furthermore, no killing was observed with NHBA-c-alum serum depleted of anti-NHBA antibodies (FIG. 22), confirming the specificity of the immune response for NHBA.

[0186] NHBA antibodies reduce NHBA binding to heparin and gonococcal adhesion to host cells To investigate whether NHBA antisera and NHBA-c antisera could inhibit the functional role of NHBA, we performed surface plasmon resonance (SPR)-based competitive binding experiments with recombinant NHBA and its predicted substrate, heparin, in the presence and absence of NHBA antisera. In the absence of antisera, N. gonorrhoeae NHBA binds heparin. Preimmune serum had no effect on the ability of heparin to interact with NHBA, whereas serum from mice immunized with full-length NHBA reduced heparin binding by 85.7% (P = 0.0001) (Figure 4A; Figure 6). However, NHBA-c serum failed to significantly inhibit the interaction between heparin and NHBA (11% reduction in binding; P = 0.1) (Figure 4A; Figure 6).

[0187] Gonococcal NHBAs are surface-exposed

[13] and likely have similar adhesin functions to meningococcal NHBAs

[18] . Therefore, we investigated whether NHBA antisera and NHBA-c antisera could reduce gonococcal adhesion to human cells. In vitro infection assays were performed using N. gonorrhoeae preincubated with antisera and transformed endocervical (tCX) and urethral (tUEC) epithelial cells. NHBA and NHBA-c sera, but not preimmune sera, were able to reduce adhesion to both tCX and tUEC in a concentration-dependent manner compared to no-antibody controls. For example, a 1:20 dilution of NHBA sera reduced gonococcal adhesion to tCX and tUEC cells by 19-fold and 6-fold, respectively. Similarly, a 1:20 dilution of cNHBA antisera reduced bacterial adhesion to 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, there is an increasing need for the identification and characterization of potential vaccine candidates to aid in the development of a gonococcal vaccine. Herein, we characterize the gonococcal NHBA and show that it is conserved among widely distributed, geographically, and temporally diverse N. gonorrhoeae strains, and that antibodies raised against either the full-length NHBA or a C-terminal fragment of the NHBA mediate bactericidal and opsonophagocytic killing. These antibodies also reduce N. gonorrhoeae adhesion to human epithelial cells and inhibit the glycan-binding activity of the NHBA. While there are currently no known correlates of protection for N. gonorrhoeae (reviewed in [9]), the ability of NHBA to elicit antibodies capable of killing N. gonorrhoeae via two conventional immune killing mechanisms and to mediate functional blocking of critical stages in infection supports its potential use in a gonococcal vaccine.

[0189] Gonococcal NHBA is highly conserved, with ≥97.5% amino acid identity among N. gonorrhoeae strains examined to date, with the majority of strains expressing one of a limited number of NHBA variants (e.g., 70.3% express one of two major variants, and 91.3% express one of seven variants). We also show that NHBA expression varies among strains, even among strains expressing the same NHBA variant (e.g., WHO X and WHO P). However, we show that antisera raised against NHBA variant 542 (from N. gonorrhoeae strain 1291) are cross-reactive and capable of killing strains expressing homologous and heterologous NHBA variants, as well as strains with high, intermediate, and low NHBA expression. Although N. gonorrhoeae and N. meningitidis strains contain different predominant NHBA variants

[13] , our findings are consistent with those for N. meningitidis NHBA, in which an antibody immune response to NHBA-2 (found in 4CMenB) is observed for 99.5% (442 strains) of circulating strains in the United States, regardless of NHBA variant

[26] .

[0190] When adjuvanted with either Freund's or aluminum hydroxide, NHBA and NHBA-c fragments were immunogenic in mice. Overall, IgG1-dominant antibody responses were elicited in all cases, although variations in other isotype and subclass patterns were observed with different antigen and adjuvant combinations. Furthermore, NHBA-c elicited reduced total IgG titers compared with full-length NHBA, but elicited similar or higher SBA and OPA titers against most strains in the panel examined. Immunoglobulin isotypes and subclasses are known to differ in their ability to activate complement and mediate bactericidal and opsonophagocytic activity, but this ability varies between antigenic targets. For example, mouse antibodies targeting N. meningitidis PorA antigens have a hierarchy of IgG3>>IgG2b>IgG2a>>IgG1 for serum bactericidal activity and IgG3>IgG2b=IgG2a>>IgG1 for opsonophagocytic activity

[27] . Similarly, murine antibodies against the N. gonorrhoeae antigen MsrA / B, when adjuvanted with Freund's compared with aluminum hydroxide, had higher titers of IgG2a, IgG2b, and IgG3, and MsrA / B-Freund antisera, but not MsrA / B-Alum antisera, mediated SBA and OPA killing of N. gonorrhoeae

[28] . Our data suggest that IgG2a and IgG2b play a dominant role in anti-NHBA SBA- and OPA-mediated killing of N. gonorrhoeae, because higher total levels of these antibodies were elicited by NHBA-c-Freund compared with NHBA-Freund. Furthermore, NHBA-c-Alum elicited IgG2b > IgG2a > IgG3 and mediated SBA and OPA against all five gonorrhoeae strains tested. This differs from MsrA / B-Alum, which did not elicit IgG2a, IgG2b, or IgG3, and anti-MsrA / B-Alum did not mediate killing of N. gonorrhoeae

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

[0191] Overall, the present invention describes several important features of NHBA that support its use as an antigen in gonococcal vaccines and highlights the potential for using the C-terminal fragment of NHBA as an optimized antigen that can be used alone or as a fusion protein with another antigen.

[0192] method Bacterial strains and growth conditions N. gonorrhoeae strains 1291, FA1090, WHO G, WHO P, and WHO X were used in this study. N. gonorrhoeae was grown on GC agar (Oxoid) containing 1% (v / v) IsoVitaleX (Becton Dickinson) at 37°C or 32°C with 5% CO2. The majority of gonococcal populations used in the assay were piliated and expressed opacity protein, as determined by visual inspection of colonies using a phase-contrast microscope.

[0193] Sequence analysis Sequences were aligned using MacVector, and percentages of amino acid identity and similarity were calculated (BLOSUM90, threshold 0). A neighbor-joining phylogenetic tree (best tree, uncorrected ("p")) of NHBA variants was constructed using MacVector. The presence and conservation of nhba and the encoded NHBA protein among gonococcal strains was determined as of September 19, 2019, using the Basic Local Alignment Search Tool program (BLAST) with nhba from N. gonorrhoeae 1291 (GenBank accession EEH61857.1; genome locus tag NGAG_00725) against 594 gonococcal genomes in GenBank and 5652 N. gonorrhoeae isolates in the Neisseria Multi Locus Sequence Typing website (PubMLST; https: / / pubmlst.org / neisseria / ). We used the previously established PubMLST nomenclature for NHBAs (encoded by NEIS2109) and assigned a unique identification number to each unique peptide sequence (e.g., NHBA_peptide 2 [NHBA-2] is in 4CMenB, and NHBA_peptide 542 [NHBA-542] is in N. gonorrhoeae strain 1291).

[0194] Construction of N. gonorrhoeae NHBA mutant strain The N. gonorrhoeae 1291 nhba gene was amplified using the 5'-ATGTTTAAACGCAGTGTGATTGC-3' (SEQ ID NO: 3) and 5'-TCAATCCCGATCTTTTTTGCCGGC-3' (SEQ ID NO: 4) primers and cloned into pGEM-T Easy vector (Promega). The kanamycin resistance gene (pUC4Kan; Amersham Biosciences) was inserted into the center of the nhba open reading frame using inverse PCR with the 5'-ggatccCCGGCCGAGATTCCGCTGATTCC-3' (SEQ ID NO: 5) and 5'-ggatccGCGACCTCCTCGACCGTGCAGAAC-3' (SEQ ID NO: 6) primers into a BamHI restriction site introduced into the center of the nhba open reading frame (the BamHI restriction enzyme site introduced for subcloning the kanamycin resistance gene into the nhba gene is shown in lowercase). The nhba::kan construct was linearized with NcoI and transformed into N. gonorrhoeae 1291 to generate the 1291 nhba::kan strain (ΔNHBA). The complemented strain (ΔNHBA_C) was generated by introducing the intact nhba gene (amplified using the 5'-GGCATATGGCGGAAACAATA-3' (SEQ ID NO: 7) and 5'-TCAATCCCGATCTTTTTTGCCGGC-3' primers (SEQ ID NO: 8)) into the ΔNHBA strain using the complementing plasmid pCTS32

[29] . Successful deletion of the nhba gene and subsequent complementation were confirmed by PCR and Western blot.

[0195] Recombinant Protein Expression Cloning and expression of full-length recombinant NHBA lacking the predicted signal peptide was previously described

[13] . For expression of the C-fragment of NHBA (NHBA-c), E. coli BL21(DE3) was transformed with pET19b plasmid containing cNHBA amplified from N. gonorrhoeae 1291 using primer 5'-ATTActcgagTCGCTTCCGGCCGAGATTCC-3' (SEQ ID NO: 9) and primer 5'-TGAAggatccCGGCATCAACATCAATC-3' (XhoI and BamHI sites are shown in lowercase letters in each primer) (SEQ ID NO: 10). Expression was monitored by OD 600 Cultures with an ΔΨ of 0.4 were induced by the addition of 1 mM IPTG and incubation for 24 h at 20°C. Proteins were purified using TALON affinity resin (Clontech) as previously described

[13] .

[0196] Generation of polyclonal antibodies Groups of five 3-week-old female BALB / c mice (Animal Resources Center, WA, Australia) were subcutaneously immunized with 25 μg of recombinant protein with Freund's adjuvant (Merck) or aluminum hydroxide (Alhydrogel; InvivoGen) on days 0, 21, 28, and 42. Peripheral blood was collected on day 56, and serum was collected via centrifugation. Pre-immune serum was collected from each mouse before immunization. This study was performed in accordance with the recommendations of the Australian Code for the Care and Use of Animals for Scientific Purposes and with approval from the Griffith University Animal Ethics Committee (AEC).

[0197] Polyclonal NHBA antibodies were purified from mouse serum using affinity chromatography with recombinant NHBA. NHBA was coupled to N-hydroxysuccinimidyl-Sepharose® 4 Fast Flow (Merck) using the manufacturer's instructions and incubated with mouse serum diluted 1 / 2 with PBS. Bound antibodies were eluted with 0.1 M glycine buffer (pH 3.0). The eluted sample was buffer exchanged into PBS using an Amicon-Ultra centrifugal spin unit (Merck). Antibody concentrations were determined by BCA (Thermo).

[0198] Enzyme-linked immunosorbent assay (ELISA) ELISAs were performed in triplicate using 96-well MaxiSorp (NUNC) plates coated with 100 ng of purified recombinant protein in 100 μl of coating buffer (0.5 M carbonate / bicarbonate buffer, pH 9.6) for 1 h at room temperature as previously described [13, 30, 31]. The ELISA titer was the highest serum dilution with an absorbance at 450 nm greater than the mean negative (all reagents except the primary antibody) plus three standard deviations.

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

[30] ) (Figure 21), followed by a 30-min incubation at 37°C, 5% CO2. The OPA assay was performed using a complement source and approximately 1 x 10 5The incubation was initiated by the addition of 100 μg of polymorphonuclear leukocytes (PMNs), followed by a 90-minute incubation at 37°C and 5% CO2. Serial dilutions of the contents of each well were plated on GC agar and grown overnight. The titer was the highest antibody dilution that induced greater than 50% killing in the assay. Statistical analysis was performed using one-way analysis of variance (ANOVA) and two-tailed Student's t-test. Each experiment was performed three times with triplicate samples in each experiment.

[0200] Flow cytometry analysis Antibody binding to N. gonorrhoeae and C3 fragment deposits was measured using flow cytometry as previously described

[28] . Briefly, N. gonorrhoeae 1291 (approximately 1 × 10 7 CFU) in HBSS + Bacteria were preincubated with a 1:100 dilution of heat-inactivated mouse serum or 70 μg / mL of purified NHBA antibody in Hank's balanced salt solution (Hank's balanced salt solution containing 0.15 mM CaCl2, 0.5 mM MgCl2, and 1% BSA (w / v)). Antibody-treated bacteria were washed and incubated with a 1:200 dilution of Alexa Fluor 488-conjugated anti-mouse IgG (Thermo) or 5% normal human serum preabsorbed with N. gonorrhoeae for 15 min at 37°C. C3 fragments were then detected by incubating the bacteria with a 1:200 dilution of FITC-conjugated anti-human C3c antibody (BioRad). Data were captured using a CyAn ADP flow cytometer (Beckman Coulter) and analyzed using FlowJo.

[0201] Surface Plasmon Resonance (SPR) SPR competition assays were performed using a Pall Pioneer FE. Competition assays were performed using NextStep injection in the OneStep assay builder, as previously described

[30] . Pre- and post-immune NHBA mouse serum was used as the first injection (A) and heparin as the second injection (B). Binding of heparin (maximum OneStep concentration of 50 μM) to NHBAs was compared with and without serum, as well as with a 1:200 dilution of pre- or post-immune serum. Data were collected using the Pioneer Software package and analyzed using Qdat analysis software. Percent blocking was calculated based on the relative RMax of heparin binding in the presence of serum (injection A = pre- / post-immune serum; B = heparin) minus serum (injection A = pre- / post-immune serum; B = buffer) versus heparin injection without serum (injection A = buffer; B = heparin).

[0202] Epithelial cell adhesion assay Gonococcal adhesion assays were performed as previously described

[31] with the following modifications: Briefly, monolayers of tCX and tUEC cells were first preincubated with serial dilutions of heat-inactivated mouse serum at room temperature for 30 min at approximately 1 × 10 5 CFU were used for infection (10 min at 37°C). Following infection, cell monolayers were washed three times with warm HBSS to remove non-adherent bacteria, and well contents were plated onto GC agar. Results were calculated as the mean CFU from triplicate wells and expressed as the percentage of adherent bacteria compared to the no-antibody control. Statistical analysis was performed by ANOVA and two-tailed Student's t-test. Each experiment was performed in triplicate. [Table 1] [Table 2]

[0203] References 1 Whiley, D. M., Jennison, A., Pearson, J. & Lahra, M. M. Genetic characterisation of Neisseria gonorrhoeae resistant to both ceftriaxone and azithromycin. Lancet Infect Dis 18, 717-718 (2018). 2 ECDC. Extensively drug-resistant (XDR) Neisseria gonorrhoeae in the United Kingdom and Australia. European Centre for Disease Prevention and Control. Stockholm. https: / / ecdc.europa.eu (2018). 3 WHO. Global priority list of antibiotic-resistant bacteria to guide research, discovery, and development of new antibiotics, <http: / / www.who.int / medicines / publications / global-priority-list-antibiotic-resistant-bacteria / en / > (2017). 4 CDC. Antibiotic Resistance Threats in the United States, 2013, <http: / / www.cdc.gov / drugresistance / threat-report-2013 / pdf / ar-threats-2013-508.pdf> 5 DepartmentofHealth. Responding to the threat of antimicrobial resistance: Australia’s First National Antimicrobial Resistance Strategy 2015-2019. (Department of Health, Australian Government, Canberra, 2015). 6 WHO. Global incidence and prevalence of selected curable sexually transmitted infections - 2008. (World Health Organisation, 2012). 7 CDC. Sexually Transmitted Disease Surveillance 2017. https: / / www.cdc.gov / std / stats (2018). 8 KirbyInstitute. HIV, viral hepatitis and sexually transmissible infections in Australia: annual surveillance report 2018. The Kirby Institute, UNSW Sydney. (2018). 9 Edwards, J. L., Jennings, M. P., Apicella, M. A. & Seib, K. L. Is gonococcal disease preventable? The importance of understanding immunity and pathogenesis in vaccine development. Crit Rev Microbiol 42, 928-941 (2016). 10 WHO. Global incidence and prevalence of selected curable sexually transmitted infections - 2008, <http: / / www.who.int / reproductivehealth / publications / rtis / stisestimates / en / > (2012). 11 Rice, P. A., Shafer, W. M., Ram, S. & Jerse, A. E. Neisseria gonorrhoeae: Drug Resistance, Mouse Models, and Vaccine Development. Annu Rev Microbiol 71, 665-686 (2017). 12 Petousis-Harris, H. et al. Effectiveness of a group B outer membrane vesicle meningococcal vaccine against gonorrhoea in New Zealand: a retrospective case-control study. Lancet 390, 1603-1610 (2017). 13 Semchenko, E. A., Tan, A., Borrow, R. & Seib, K. L. The serogroup B meningococcal vaccine Bexsero elicits antibodies to Neisseria gonorrhoeae. Clin Infect Dis 69, 1101-1111 (2018). 14 Serruto, D., Bottomley, M. J., Ram, S., Giuliani, M. M. & Rappuoli, R. The new multicomponent vaccine against meningococcal serogroup B, 4CMenB: immunological, functional and structural characterization 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 Neisseria meningitidis. J Biol Chem 286, 41767-41775 (2011). 16 Mubaiwa, T. D. et al. The Bexsero Neisseria meningitidis serogroup B vaccine antigen NHBA is a high-affinity chondroitin sulfate binding protein. Scientific reports 8, 6512 (2018). 17 Serruto, D. et al. Neisseria meningitidis GNA2132, a heparin-binding protein that induces protective immunity in humans. Proc Natl Acad Sci U S A 107, 3770-3775 (2010). 18 Vacca, I. et al. Neisserial Heparin Binding Antigen (NHBA) Contributes to the Adhesion of Neisseria meningitidis to Human Epithelial Cells. PLoS One 11, e0162878 (2016). 19 Maritan, M. et al. Structures of NHBA elucidate a broadly conserved epitope identified by a vaccine induced antibody. PLoS One 13, e0201922 (2018). 20 Maritan, M. et al. Crystal structures of human Fabs targeting the Bexsero meningococcal vaccine antigen NHBA. Acta Crystallogr F Struct Biol Commun 73, 305-314 (2017). 21 Pizza, M. et al. Identification of vaccine candidates against serogroup B meningococcus by whole-genome sequencing. Science 287, 1816-1820 (2000). 22 Giuliani, M. M. et al. A universal vaccine for serogroup B meningococcus. Proc Natl Acad Sci U S A 103, 10834-10839 (2006). 23 Welsch, J. A. et al. Antibody to genome-derived neisserial antigen 2132, a Neisseria meningitidis candidate vaccine, confers protection against bacteremia in the absence of complement-mediated bactericidal activity. J Infect Dis 188, 1730-1740 (2003). 24 Plested, J. S. & Granoff, D. M. Vaccine-induced opsonophagocytic immunity to Neisseria meningitidis group B. Clin Vaccine Immunol 15, 799-804 (2008). 25 Muzzi, A., Mora, M., Pizza, M., Rappuoli, R. & Donati, C. Conservation of meningococcal antigens in the genus Neisseria. MBio 4, e00163-00113 (2013). 26 Rajam, G. et al. Meningococcal Antigen Typing System (MATS)-Based Neisseria meningitidis Serogroup B Coverage Prediction for the MenB-4C Vaccine in the United States. mSphere 2 (2017). 27 Michaelsen, T. E., Kolberg, J., Aase, A., Herstad, T. K. & Hoiby, E. A. The four mouse IgG isotypes differ extensively in bactericidal and opsonophagocytic activity when reacting with the P1.16 epitope on the outer membrane PorA protein of Neisseria meningitidis. Scand J Immunol 59, 34-39 (2004). 28 Shaughnessy, J. et al. Human Factor H Domains 6 and 7 Fused to IgG1 Fc Are Immunotherapeutic against Neisseria gonorrhoeae. J Immunol 201, 2700-2709 (2018). 29 Steichen, C. T., Shao, J. Q., Ketterer, M. R. & Apicella, M. A. Gonococcal cervicitis: a role for biofilm in pathogenesis. The Journal of infectious diseases 198, 1856-1861 (2008). 30 Jen, F. E. C., Semchenko, E. A., Day, C. J., Seib, K. L. & Jennings, M. P. The Neisseria gonorrhoeae Methionine Sulfoxide Reductase (MsrA / B) Is a Surface Exposed, Immunogenic, Vaccine Candidate. Frontiers 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 Antibodies. Infect Immun 85 (2017).

[0204] Example 2 Heparin-binding antigen (NHBA) of Neisseria gonorrhoeae is involved in microcolony formation and contributes to serum resistance and adherence to epithelial cells Neisseria heparin-binding antigens (NHBAs) are present in the four-component meningococcal serogroup B vaccine (4CMenB, trade name Bexsero) licensed to protect against invasive disease caused by Neisseria meningitidis (which is closely related to N. gonorrhoeae) [9]. The gonococcal homolog of NHBA is surface-exposed in N. gonorrhoeae strains, is highly conserved (>93% identity), shares 67% identity to meningococcal NHBA variant 2 (NHBA-2) present in 4CMenB

[10] , and is recognized by human sera from people vaccinated with 4CMenB

[11] .

[0205] Meningococcal NHBA, most extensively studied in strain MC58 (expressing NHBA-3), are named based on their ability to bind the glycosaminoglycan (GAG) heparin through their arginine-rich region (Arg region). NHBA binding to heparin increases meningococcal resistance to serum

[12] , and their interaction with heparan sulfate mediates binding to epithelial cells

[13] . NHBA binds several other glycans, with the highest affinity observed for chondroitin sulfate

[14] . Meningococcal NHBA are targets of multiple proteases, including human lactoferrin

[12] , kallikrein

[15] , and C3 convertase

[16] , as well as meningococcal NalP

[12] . NalP cleaves NHBA after the arginine-rich region, and it has been speculated that highly pathogenic strains of N. meningitidis expressing NalP release an NHBA fragment that increases vascular permeability

[17] . NHBA-2 also increases expression at lower temperatures (32°C vs. 37°C)

[18] and plays a role in biofilm formation

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

[20] and its NHBA has a truncated Arg region

[10] , indicating that the NHBA may play a different role in N. gonorrhoeae compared to N. meningitidis. In this example, we explore the function of NHBA in N. gonorrhoeae to describe its role in pathogenesis and support its potential use as a gonococcal therapeutic target or vaccine candidate.

[0206] method Sequence analysis NHBA sequences (N. gonorrhoeae 1291 accession EEH61857.1; N. meningitidis MC58 AAF42586.1) were aligned by CLUSTAL in MacVector. The PubMLST nomenclature for NHBA was used to assign an identification number to each unique peptide sequence (e.g., NHBA_peptide 542 [NHBA-542] in N. gonorrhoeae 1291).

[0207] Growth and phenotypic characterization of N. gonorrhoeae N. gonorrhoeae 1291 was cultured on GC agar (Oxoid) or GC broth containing 1% (v / v) IsoVitaleX (Becton Dickinson) at 32°C or 37°C with 5% CO2. Kanamycin (50 μg / mL) and spectinomycin (100 μg / mL) were used for the knockout and complemented strains, respectively. The majority of gonococcal populations were fimbriae and expressed opacity proteins, as determined by phase-contrast microscopy. Growth rate and aggregation experiments were performed in GC broth and measured by optical density at 600 nm (OD ). 600 ) were measured every hour [21, 22]. SDS-PAGE and Western blot analysis also showed similar molecular weights and abundances of major outer membrane proteins, including pilin (slightly less pilin in WT compared to ΔNHBA and ΔNHBA_C; Figure S4B), Por and Opa (Figure S4C), and lipooligosaccharide (LOS) (Figure S4D) in these strains. Where indicated, N. gonorrhoeae was trypsinized for 5 min in 0.25% trypsin for aggregation analysis.

[0208] Construction of N. gonorrhoeae NHBA mutants and recombinant NHBA The nhba gene (NGAG_00725) was amplified from N. gonorrhoeae strain 1291 (primer 5'-ATGTTTAAACGCAGTGTGATTGC-3' (SEQ ID NO: 3); primer 5'-TCAATCCCGATCTTTTTTGCCGGC-3' (SEQ ID NO: 4)) and cloned into pGEM-T Easy (Promega). The kanamycin resistance gene (pUC4Kan; Amersham Biosciences) was inserted into the BamHI site introduced into the center of nhba using inverse PCR (primer 5'-GGATCCCCGGCCGAGATTCCGCTGATTCC-3' (SEQ ID NO: 5); primer 5'-GGATCCGCGACCTCCTCGACCGTGCAGAAC-3' (SEQ ID NO: 6); BamHI site is underlined). The nhba::kan construct was linearized and transformed into N. gonorrhoeae 1291 to generate nhba::kan (ΔNHBA). The complemented strain (ΔNHBA_C) was generated by introducing an intact nhba gene (primer 5'-GGCATATGGCGGAAACAATA-3' (SEQ ID NO: 7); primer 5'-TCAATCCCGATCTTTTTTGCCGGC-3' (SEQ ID NO: 8)) into ΔNHBA using the complementing plasmid pCTS32

[23] . Deletion of the nhba gene and subsequent complementation were confirmed by PCR and Western blot.

[0209] The N. gonorrhoeae 1291 nhba gene was cloned into pET19b in E. coli BL21, and the full-length mature NHBA (without signal sequence) recombinant protein was expressed and purified as previously described

[11] .

[0210] Generation of polyclonal anti-NHBA Groups of five 3-week-old female BALB / c mice (Animal Resources Center, Western Australia) were immunized subcutaneously with 25 μg of recombinant NHBA with Freund's adjuvant (Merck) on days 0, 21, 28, and 42. Peripheral blood was collected on day 56, and serum was collected via centrifugation. This study was approved by the Griffith University Animal Ethics Committee.

[0211] Western blot, ELISA, and flow cytometry Western blot analysis of NHBA expression from N. gonorrhoeae whole-cell lysates (separated using 4–12% Bis-Tris SDS-PAGE (Thermo)) was performed with mouse anti-NHBA as previously described

[24] . Periplasmic proteins were detected using rabbit anti-NGAG_01228 as previously described

[24] .

[0212] ELISA analysis of His-tagged recombinant NHBA binding to whole-cell N. gonorrhoeae was measured using HRP-conjugated His-tag antibody (Thermo) following standard protocols [ 11 , 25 ] 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 The number of CFU was measured by flow cytometry (as previously described ([24, 26]). The bacteria were incubated with anti-NHBA (1:200, 30 min), washed three times with PBS, incubated with Alexa Fluor 488-conjugated anti-mouse IgG (1:200, 1 h; Thermo), washed, and then fixed in formaldehyde (2.5%, 15 min). N. gonorrhoeae (approximately 10 7 CFU) or human tCX and tUEC cells (approximately 5 × 10 5Binding of FITC-labeled gonococcal NHBA (100 μg / mL) to 100 μg / mL IgG cells was measured after 20 min of incubation at 37°C. All samples were analyzed using a CyAn ADP flow cytometer (Beckman Coulter). Data analysis was performed using FlowJo.

[0214] Microscopy Fluorescence microscopy was used to measure the interaction of tCX cells (grown to full confluence on glass coverslips) with N. gonorrhoeae NHBA (100 μg / mL) incubated for 20 min at 37°C. Cells were washed three times to remove unbound proteins and fixed in formaldehyde (2.5%, 15 min). Ng Antibodies were detected using anti-NHBA (1:1000)

[11] and Alexa Fluor 488-conjugated anti-mouse IgG (1:200; Thermo). Cells were counterstained with Alexa Fluor 568 phalloidin (Thermo) and DAPI for nuclei. Glass coverslips were mounted on microscope slides using ProLong Gold Antifade Mountant (Thermo), images were captured with a Nikon A1R confocal microscope, and data were analyzed using NIS-Elements (Nikon).

[0215] Approximately 1×10 6 Gonococcal microcolony formation was investigated using tUEC cells incubated with CFU of N. gonorrhoeae for 5 h at 37°C. Cell monolayers were washed three times (HBSS) to remove non-adherent bacteria and fixed for 30 min in 2% glutaraldehyde and 5% formaldehyde aqueous solution. Scanning electron microscopy was performed as previously described

[27] , and images were captured using a JCM-5000 NeoScope™ (JEOL).

[0216] Glycan linkage analysis Glycan array experiments were performed using recombinant NHBA (1 μg) and the Institute for Glycomics Glycan Array (v3.0) as previously described [14, 28]. Positive binding was assigned to spots with a mean fluorescence >1-fold above background-adjusted (mean + 3 standard deviations of the slide background) in three independent replicates (Student's t-test, p<0.001).

[0217] Surface plasmon resonance (SPR) was performed on recombinant NHBAs immobilized on flow cells 2–4 by amine coupling on a Series S CM5 sensor chip (GE Healthcare) as previously described [14, 25]. Ng The assay was performed using a BIAcore T200 instrument equipped with ethanolamine (100 μg / ml). Flow cell 1 was used as the reference cell and was immobilized with ethanolamine only. Single cycle kinetics was used to measure the affinity of the interaction with glycans (K) performed in a 1:5 dilution series at concentrations from 100 μM to 1 nM. D ) was calculated. Results were analyzed using BIAcore T200 software 2.0.2.

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

[24] , the resistance of N. gonorrhoeae to serum-mediated killing was tested, and approximately 10 4 CFU were incubated at 37°C for 60 min at 10% (v / v) and subsequently plated on GC agar. Bacteria were preincubated with 6 μM heparin for 30 min where indicated. Bacterial survival was calculated as percent CFU (average from triplicate wells) compared to untreated controls.

[0219] Adhesion assay As previously described

[26] , the gonococcal adhesion assay was performed at approximately 10 5CFU were used to perform E6 / E7-transformed primary human endocervical (tCX) and urethral epithelial (tUEC) cells for 1 h. Adhesion-blocking assays were also performed in cells pretreated with recombinant gonococcal NHBA (1–100 μg / mL) or peanut agglutinin lectin (100 μg / mL PNA; a negative control that does not bind to tCX cells

[26] ) and then infected with N. gonorrhoeae for 10 min. Results were reported as the percent adherent bacteria compared to wild-type (mean from three replicate wells) and adhesion blocking calculated as the percent adherent bacteria compared to the untreated control. Adhesion and serum survival assays were performed in triplicate on three separate occasions, and statistical analysis was performed by ANOVA and Student's t-test.

[0220] result Sequence characteristics and expression of Neisseria gonorrhoeae NHBA The predominant NHBA variant expressed by N. gonorrhoeae strains is NHBA-542, which is present in >40% of gonococcal isolates in the PubMLST database (including strain 1291)

[11] . NHBA-542 (referred to herein as NHBA Ng ), which is 426 amino acids long, is a homolog of the well-characterized NHBA-3 (referred to herein as NHBA) from N. meningitidis strain MC58. Nm It contains sequence features similar to those described in the NHBA (referred to as NHBA), including a lipobox motif and a polyglycine stretch in the N-terminus, and an arginine-rich region in the center of the protein (Figure 7A). However, due to insertions / deletions, NHBA Ng and the NHBA Nm There are several differences between NHBA-3 and N. gonorrhoeae (Figure 7B). The N-terminal half of the N. gonorrhoeae nhba gene has a 63 amino acid deletion compared to NHBA-3, which is consistent among the major gonorrhoeae variants (Figure 13). Ng The Arg region of NHBA NmThis region is highly conserved between Nm strains

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

[11] ).

[0221] To facilitate characterization of gonococcal NHBA, a recombinant His-tagged protein (NHBA-542) was produced in E. coli, and polyclonal anti-NHBA antibodies were generated in mice. Additionally, an isogenic mutation of NHBA was generated by inserting a kanamycin resistance cassette into the open reading frame of the nhba gene in N. gonorrhoeae strain 1291 (ΔNHBA), and this mutation was complemented by reintroducing a single copy of the nhba gene in trans into the genome (ΔNHBA_C). Western blot analysis of whole-cell lysates detected a single band at 58–80 kDa with anti-NHBA serum, confirming NHBA expression in the wild-type strain. The complemented strain expressed NHBA at levels similar to those of the wild-type strain, while NHBA expression was undetectable in the mutant strain (Figure 7C; Figure S13). NHBA was also detected on the surface of whole-cell N. gonorrhoeae WT and ΔNHBA_C strains by flow cytometry (Figure 7C). Growth of N. gonorrhoeae strains to mid-logarithmic phase at 32°C and 37°C revealed that gonococcal NHBA expression is temperature regulated, with higher expression observed at lower temperatures (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 ΔNHBA in vitro, N. gonorrhoeae strain 1291 wild-type, ΔNHBA, and ΔNHBA_C strains were grown in GC broth and GC agar. All strains had comparable growth rates and optimal growth levels in terms of optical density, but the ΔNHBA mutant strain had a significantly reduced sedimentation rate compared to the WT and ΔNHBA_C strains (Figure 8A). Furthermore, samples of equal optical density (OD ) were analyzed. 600When 1000-well plates (p < 0.05) were plated onto GC agar, there were approximately threefold higher numbers of viable CFUs for the ΔNHBA strain compared to the WT or ΔNHBA_C strains (Figure 8B). Treatment of these samples with trypsin resulted in equal CFU counts for all three strains (WT and ΔNHBA_C countable CFUs increased 2.6- and 2.4-fold, respectively, while ΔNHBA CFUs were unaffected) (Figure 8B), indicating that the phenotype was due to cell aggregation rather than a defect in cell separation. Gram stain analysis of the three stains ± trypsin treatment confirmed the presence of cell aggregates in the untreated WT and ΔNHBA_C strains (Figure 15A). Furthermore, Western blot analysis indicated that trypsin treatment digested NHBA from the bacterial surface but did not alter periplasmic control proteins (Figure 15B). Following this finding, the volume of the ΔNHBA sample used in subsequent experiments was adjusted to equalize CFU counts. Gonococcal pili and opacity proteins have previously been implicated in the formation of bacterial aggregates, and phase-contrast microscopy confirmed that the WT, ΔNHBA, and ΔNHBA_C strains shared identical colony morphology with respect to pili formation and opacity.

[0223] NHBA Ng To determine whether NHBA interacts directly with the gonococcal surface to promote agglutination, recombinant NHBA Ng Flow cytometry and ELISA were used. Flow cytometry analysis revealed that FITC-labeled recombinant NHBA was expressed in whole cells of N. gonorrhoeae. Ng This was confirmed using a whole cell ELISA, and the binding of recombinant NHBA was demonstrated (Figure 8C). Ng bound N. gonorrhoeae in a concentration-dependent manner (Fig. 8D).

[0224] To further investigate the role of NHBA in bacteria-bacteria interactions and the formation of gonococcal aggregates, we investigated the ability of ΔNHBA to form microcolonies. Unlike the WT or ΔNHBA_C strains, the ΔNHBA strain was not able to form microcolonies on the surface of glass cover slip slides or on human urethral epithelial monolayers after 5 hours of growth (Figure 9). Ng Biofilm assays were also performed to investigate whether the self-association properties of α-NHBA play a role in the establishment of gonococcal biofilms. However, under static conditions for 24–26 h, no differences in biofilm formation were observed among the WT, ΔNHBA, 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 determined using glycan array analysis, with the array displaying 368 structures representative of glycans found in human cells (including isomers and / or glycans with similar structures but differing in chain length, chemical linkage, or spacer size). Ng bound to 39 glycan structures on the array (Figure 16; Table 4), including the GAGs heparin, heparan sulfate, and chondroitin sulfate. Ng also bound to multiple structures containing lacto-N-biose and N-acetyllactosamine core structures (i.e., LNnT), including their sialylated and fucosylated variants (i.e., sLeX), as well as a limited set of N-acetylglucosamine, N-acetylgalactosamine, glucosyl, and mannosyl glycans.

[0226] Glycans and recombinant NHBA Ng To characterize the kinetics of the interaction, SPR analysis was performed using selected GAG (Figure 10A) and non-GAG glycans (Figure 10B) bound on the array. Ng The highest calculated affinity of the -glycan interaction was with heparin (KD 4.4 nM), followed by chondroitin sulfate (K D The GAG ​​structure, which is composed of repeating polysaccharides with various sulfation patterns, is highly heterogeneous. Ng To determine whether NHBA preferentially binds to glycans with specific sulfate configurations, experiments with three types of chondroitin sulfate (A, B, and C) were performed. Ng bound only to chondroitin sulfate C (chondroitin 6-sulfate); concentration-dependent binding was not observed for chondroitin sulfate A (chondroitin 4-sulfate) or chondroitin sulfate B (dermatan sulfate). Ng has a lower affinity (K D It has also been shown that NHBA binds to non-GAG glycans with a glycan-binding capacity of 2.79 μM. Ng is an 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. Ng is sialyl Lewis X(K D Lewis X (K D Concentration-dependent binding was not observed for hyaluronan (non-sulfated GAG) or H-disaccharides, which were not observed for NHBA on the glycan array. Ng was not bound by and was used as a negative control.

[0227] Gonococcal NHBA binds to epithelial cells NHBA Nm NHBA binds to epithelial cells via its Arg domain by interacting with heparan sulfate proteoglycans

[13] . Ng To investigate whether recombinant NHBA also interacts with epithelial cells, confocal microscopy and flow cytometry analysis were performed. NgFor confocal microscopy, recombinant NHBA was used. Ng (rNHBA Ng ) were incubated with human endocervical epithelial (tCX) cells and detected using mouse anti-NHBA primary antibody and Alexa Flour 488 secondary antibody. Ng Binding of tCX cells was observed (white arrow, Figure 11A(I)), and the protein signal was localized on the surface of the cells (white arrow, Figure 11A(II)). Nonspecific binding of the primary antibody (Figure 11A(III)) or secondary antibody (Figure 11A(IV)) to tCX cells was not observed. Analysis by flow cytometry revealed that NHBA Ng It was further confirmed that IL-14 binds to cervical epithelial cells and urethral epithelial cells (Fig. 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 To investigate the functional role of this interaction, serum survival and epithelial cell adhesion assays were performed. Serum survival assays performed with the WT, ΔNHBA, and ΔNHBA_C strains in 10% normal human serum (a sublethal serum concentration for the WT strain) showed that the ΔNHBA strain had approximately 5-fold reduced survival compared to the WT and ΔNHBA_C strains (Figure 12A). Pretreatment of gonococci with heparin before exposure to human serum increased the survival of the ΔNHBA mutant strain to a level comparable to that of the WT and ΔNHBA_C strains. N. gonorrhoeae expresses several proteins (e.g., Opa

[29] ) that interact with heparin, which may lead to the restoration of serum resistance in the ΔNHBA strain.

[0229] NHBA in N. gonorrhoeae infection NgTo investigate the role of NHBA, in vitro infection assays were performed with human cervical epithelial cells and urethral epithelial cells, as well as with the WT, ΔNHBA, and ΔNHBA_C strains. For the epithelial cell infection assays, the ΔNHBA mutation resulted in 11- and 12-fold reduced adhesion of tCX and tUEC cells, respectively, compared to the WT (Figure 12B). Ng Adhesion assays were also performed with cells pretreated with either NHBA or the negative control protein PNA. Ng Gonococcal adhesion to treated cells increased in a concentration-dependent manner (i.e., 100 and 10 μg / mL of NHBA). Ng The binding of PNA to the cells was reduced by 2.5- and 1.7-fold, respectively, whereas treatment of cells with 100 μg / mL of PNA (a negative control that does not bind these cells) had no effect on bacterial adhesion (FIG. 12C).

[0230] Consideration The sexually transmitted disease, gonorrhea, is a growing public health concern due to rising infection rates and increasing antimicrobial resistance. N. gonorrhoeae primarily colonizes mucosal surfaces, and gonococcal transmission, colonization, and pathogenesis are complex, multifactorial processes [reviewed in 30]. An increased understanding of all stages of gonococcal infection is required to aid in the development of new therapeutic and preventive strategies. In this study, we characterized gonococcal NHBA with respect to glycans and the interaction of N. gonorrhoeae with human epithelial cells, revealing their involvement in microcolony formation, resistance to human serum, and adherence to epithelial cells.

[0231] NHBA was first identified in N. meningitidis via reverse vaccinology as part of the development of the meningococcal serogroup B vaccine 4CMenB [31, 32], and its functional role has since been characterized in detail [12-16]. Despite the relatively high level of sequence identity between gonococcal and meningococcal NHBA proteins (approximately 67% identity

[11] ), multiple differences exist between the NHBA sequence and the pathogenesis seen in the two pathogenic Neisseria species, prompting detailed analysis of NHBA in N. gonorrhoeae. NHBA is more conserved in N. gonorrhoeae than in N. meningitidis

[11] , and we confirm herein that the 63-amino acid deletion and truncated Arg region in the N-terminus of N. gonorrhoeae are conserved in all major gonococcal NHBA variants. Therefore, the data presented herein are likely representative of the role of NHBA in most N. gonorrhoeae strains.

[0232] In the glycan array analysis herein, recombinant NHBA Ng The present inventors have demonstrated that NHBA binds to 39 glycans, including multiple GAGs (e.g., heparin, heparan sulfate, and chondroitin sulfate). Nm We previously showed that NHBA interacts with 28 glycans

[14] , and SPR analysis revealed that NHBA Ng and NHBA Nm Although it was confirmed that all proteins bind at least four glycans (heparin, heparan sulfate, chondroitin sulfate, and Glc-6P), important differences between the proteins are Ng But the NHBA Nm The fact that NHBAs have a higher binding affinity for heparin and a lower affinity for chondroitin sulfate and glucose 6-phosphate than NHBAs is probably due to the binding of NHBAs to GAGs. Nm This is due to differences in the Arg region known to be involved in binding of Ng NHBA [12, 13]. Nginteracts with Lewis X and sialyl Lewis X antigens, lacto-N-neotetraose (LNnT) and its sialylated variants on glycan arrays, which are typically found on the surface of host cells, whereas NHBA Nm No. NHBA by NalP Nm Meningococcal cleavage of NHBA releases a C-terminal fragment called C2

[12] , which contains an Arg-rich region and increases vascular permeability

[17] . Additionally, the human proteases lactoferrin and kallikrein cleave NHBA downstream of the Arg region. Nm [12, 15], and serum C3 convertase can cleave the released NHBA C2 fragment, removing the Arg region and counteracting the toxic effect of the protein on cells

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

[20] , so NHBA Ng was not cleaved upstream of the Arg region. Ng is not cleaved by human lactoferrin (data not shown), and cleavage by kallikrein and C3 convertase has not been investigated. However, due to the absence of NalP, NHBA Ng When cleaved by these human enzymes, a functional Arg region would remain attached to the gonococcal surface where it could mediate roles related to glycan binding.

[0233] N. gonorrhoeae ΔNHBA mutant strains exhibit multiple phenotypes compared to WT and ΔNHBA_C strains, including reduced survival in human serum, reduced clumping and microcolony formation, and reduced adherence to cervical and urethral epithelial cells. Although N. gonorrhoeae rarely causes disseminated disease, its ability to resist serum killing has been extensively studied and found to be mediated by factors including porins [30-32], LOS

[33] , and Opa

[28] , and is relevant during mucosal infections because serum and complement factors are present in the genital tract and other mucosal surfaces [34-36]. NHBA NmHeparin also plays a role in serum survival, and the addition of heparin before serum assays results in increased survival of serum-susceptible non-encapsulated parental meningococcal strains, but not ΔNHBA mutants

[12] . Heparin interacts with multiple complement factors

[37] , and NHBA Nm Heparin-mediated recruitment of complement regulatory proteins by ΔNHBA has been proposed to be a mechanism of action for serum resistance

[12] . We found that the NHBA mutant is responsible for the higher level of recruitment of complement regulatory proteins to the surface of the WT strain compared to the ΔNHBA mutant. Ng We propose a similar mechanism of action for NHBA. Ng The NHBA Nm Although it has a higher affinity for heparin than WT, there are additional gonococcal heparin-binding proteins (e.g., Opa

[28] ) that play a role in serum resistance, which explains the relatively high level of survival of WT under the serum conditions tested and the recovery of resistance of the gonococcal ΔNHBA mutant after the addition of heparin to the assay.

[0234] Gonococcal adhesion to mucosal epithelia is a critical first step in the establishment of infection. Following initial adhesion, N. gonorrhoeae colonization depends on the formation of robust bacterial aggregates and microcolonies on the epithelial cell surface [reviewed in 30]. Both initial adhesion and microcolony formation are mediated by gonococcal factors, including type IV pili, opaque (Opa) protein, and lipooligosaccharide (LOS) [23, 42-44]. However, N. gonorrhoeae can form aggregates even in the absence of pili or Opa, suggesting the existence of unknown host factors that promote GC aggregation

[45] . The ΔNHBA mutant exhibited reduced adhesion to endocervical and urethral epithelial cells, and reduced aggregation and microcolony formation compared to the wild type, suggesting that NHBA Ng Furthermore, recombinant NHBA plays an important role in the establishment of gonorrhea. Ng interacts directly with both epithelial and gonococcal cells, and NHBA Ng is able to block adhesion to epithelial cells in a concentration-dependent manner.Nm This is likely the result of NHBA interactions with host glycans on epithelial cells that mediate their interaction

[13] . Gonococcal NHBA was upregulated at 32°C vs. 37°C, consistent with NHBA regulation in N. meningitidis

[18] , which may be particularly relevant at the time of adhesion by these organisms in the pharynx due to the cooler temperature of this niche.

[0235] NHBA Ng NHBA in vector-bacterial interactions and microcolony formation Ng The role of N. meningitidis aggregates may be facilitated by their interaction with LNnT, which is present on the surface of gonococcal cells as part of the LOS

[46] . Gonococcal microcolonies interact with host microvilli, leading to host cytoskeletal rearrangements and cortical plaque formation [47-51]. Microcolonies are also involved in increased antibiotic resistance in N. gonorrhoeae

[45] , and bacterial aggregate formation is promoted after exposure to seminal plasma, which affects bacterial transmission

[52] . N. meningitidis aggregate formation is also important for resistance to shear forces on the cell surface

[53] , which may also be important for N. gonorrhoeae. However, it is interesting to note that meningococcal NHBAs have not been reported to be involved in aggregation to date [13, 54, 55].

[0236] In summary, the role of NHBAs during multiple stages of gonococcal infection and pathogenesis is emphasized. Therefore, targeting NHBA-self and NHBA-host interactions may be valuable therapeutic and vaccine approaches.

[0237] Throughout this specification, the objective has been to describe preferred embodiments of the present invention without limiting the invention to any one embodiment or particular collection of features. Accordingly, in light of this disclosure, it will be recognized by those of skill in the art that various modifications and changes can be made in the specific exemplary embodiments without departing from the scope of the present invention.

[0238] All computer programs, algorithms, patent and scientific literature, and protein and nucleic acid sequences or accession numbers referred to herein are hereby incorporated 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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The presence of complement in human cervical mucus and its possible relevance to infertility in women with complement-dependent sperm-immobilizing antibodies. Fertil Steril 1979;32:61-6. 40. Schumacher GF. Immunology of spermatozoa and cervical mucus. Hum Reprod 1988;3:289-300. 41. Yu H, Munoz EM, Edens RE, Linhardt RJ. Kinetic studies on the interactions of heparin and complement proteins using surface plasmon resonance. Biochim Biophys Acta 2005;1726:168-76. 42. Zollner R, Oldewurtel ER, Kouzel N, Maier B. Phase and antigenic variation govern competition dynamics through positioning in bacterial colonies. Scientific reports 2017;7:12151. 43. Stein DC, LeVan A, Hardy B, Wang LC, Zimmerman L, Song W. Expression of Opacity Proteins Interferes with the Transmigration of Neisseria gonorrhoeae across Polarized Epithelial Cells. PLoS One 2015;10:e0134342. 44. LeVan A, Zimmerman LI, Mahle AC, et al. 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[0240] 55. Serruto D, Spadafina T, Ciucchi L, et al. Neisseria meningitidis GNA2132, a heparin-binding protein that induces protective immunity in humans. Proceedings of the National Academy of Sciences 2010;107:3770-5. The invention as originally claimed in the present application is described below. [Invention 1] An immunogenic fragment of the Neisserial Heparin Binding Antigen (NHBA) protein isolated from Neisseria gonorrhoeae. [Invention 2] The immunogenic fragment of invention 1, wherein the isolated NHBA protein comprises the amino acid sequence set forth in SEQ ID NO: 1, or a fragment, variant, or derivative thereof. [Invention 3] An immunogenic fragment according to Invention 1 or Invention 2, which comprises or is contained in a C-terminal fragment of the isolated NHBA protein. [Invention 4] An immunogenic fragment according to any one of the preceding inventions, comprising, contained within, consisting of or consisting essentially of the amino acid sequence set forth in SEQ ID NO: 2, or a fragment, variant or derivative thereof. [Invention 5] 5. The immunogenic fragment of claim 4, wherein the variant or derivative comprises one or more amino acid substitutions of residues 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 181 of SEQ ID NO:2. [Invention 6] 6. The immunogenic fragment according to invention 5, wherein the one or more amino acid substitutions in SEQ ID NO: 2 are 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, R181Q, and any combination thereof. [Invention 7] 10. An immunogenic fragment according to any one of the preceding inventions, comprising one or more heparin-binding residues and / or one or more active site residues of said isolated NHBA protein. [Invention 8] An isolated protein comprising one or more immunogenic fragments according to any one of inventions 1 to 7. [Invention 9] An isolated nucleic acid comprising a nucleotide sequence encoding the immunogenic fragment according to any one of Inventions 1 to 7 or the isolated protein according to Invention 8, or comprising a nucleotide sequence complementary thereto. [Invention 10] A genetic construct comprising the isolated nucleic acid according to invention 9. [Invention 11] A host cell comprising the gene construct according to invention 10. [Invention 12] A method for producing an isolated immunogenic fragment according to any one of Inventions 1 to 7 or an isolated protein according to Invention 8, the method comprising: (i) culturing a host cell according to Invention 11; and (ii) isolating the immunogenic fragment or protein from the host cell cultured in step (i). [Invention 13] An antibody or antibody fragment which binds to or is raised against an immunogenic fragment according to any one of inventions 1 to 7 or an isolated protein according to invention 8. [Invention 14] A composition comprising one or more immunogenic fragments according to any one of inventions 1 to 7, an isolated protein according to invention 8, an isolated nucleic acid according to invention 9, a genetic construct according to invention 10, a host cell according to invention 11, and / or an antibody or antibody fragment according to invention 13, optionally together with a pharmaceutically acceptable diluent, carrier, or excipient. [Invention 15] 15. The composition according to claim 14, which is an immunogenic composition. [Invention 16] 16. The composition according to claim 15, which is a vaccine. [Invention 17] A method for eliciting an immune response to Neisseria gonorrhoeae and / or Neisseria meningitidis in a subject, the method comprising the step of administering to the subject one or more immunogenic fragments according to any one of Inventions 1 to 7; an isolated protein according to Invention 8; an isolated nucleic acid according to Invention 9; a gene construct according to Invention 10; a host cell according to Invention 11; an antibody or antibody fragment according to Invention 13; and / or a composition according to any one of Inventions 14 to 16, thereby eliciting the immune response. [Invention 18] A method for inducing immunity against Neisseria gonorrhoeae and / or Neisseria meningitidis in a subject, the method comprising the step of administering to the subject one or more immunogenic fragments according to any one of Inventions 1 to 7; an isolated protein according to Invention 8; an isolated nucleic acid according to Invention 9; a gene construct according to Invention 10; a host cell according to Invention 11; an antibody or antibody fragment according to Invention 13; and / or a composition according to any one of Inventions 14 to 16, thereby inducing immunity against the Neisseria gonorrhoeae and / or Neisseria meningitidis in the subject. [Invention 19] A method for treating or preventing an infection caused by Neisseria gonorrhoeae and / or Neisseria meningitidis in a subject, the method comprising the step of administering to the subject one or more immunogenic fragments described in any one of Inventions 1 to 7; an isolated protein described in Invention 8; an isolated nucleic acid described in Invention 9; a gene construct described in Invention 10; a host cell described in Invention 11; an antibody or antibody fragment described in Invention 13; and / or a composition described in any one of Inventions 14 to 16, thereby preventing or treating an infection caused by Neisseria gonorrhoeae and / or Neisseria meningitidis in the subject. [Invention 20] A method for at least partially inhibiting or preventing the binding of Neisseria gonorrhoeae and / or Neisseria meningitidis to cells in a subject, the method comprising the step of administering to the subject one or more immunogenic fragments described in any one of Inventions 1 to 7; an isolated protein described in Invention 8; an isolated nucleic acid described in Invention 9; a genetic construct described in Invention 10; a host cell described in Invention 11; an antibody or antibody fragment described in Invention 13; and / or a composition described in any one of Inventions 14 to 16, thereby inhibiting or preventing the binding of Neisseria gonorrhoeae and / or Neisseria meningitidis to cells of the subject. [Invention 21] A method for at least partially inhibiting or reducing serum resistance of Neisseria gonorrhoeae and / or Neisseria meningitidis infection in a subject, the method comprising the step of administering to the subject one or more immunogenic fragments described in any one of Inventions 1 to 7; an isolated protein described in Invention 8; an isolated nucleic acid described in Invention 9; a gene construct described in Invention 10; a host cell described in Invention 11; an antibody or antibody fragment described in Invention 13; and / or a composition described in any one of Inventions 14 to 16, thereby inhibiting or reducing serum resistance of Neisseria gonorrhoeae and / or Neisseria meningitidis infection in the subject. [Invention 22] A method for detecting Neisseria gonorrhoeae and / or Neisseria meningitidis in a biological sample obtained from a subject, the method comprising the step of contacting the biological sample with the antibody or antibody fragment described in invention 13, thereby detecting N. gonorrhoeae in the biological sample. [Invention 23] Use of one or more immunogenic fragments according to any one of Inventions 1 to 7; an isolated protein according to Invention 8; an isolated nucleic acid according to Invention 9; a gene construct according to Invention 10; a host cell according to Invention 11; an antibody or antibody fragment according to Invention 13; and / or a composition according to any one of Inventions 14 to 16; in the manufacture of a medicament for eliciting an immune response to Neisseria gonorrhoeae and / or Neisseria meningitidis in a subject; inducing immunity to Neisseria gonorrhoeae and / or Neisseria meningitidis in a subject; treating or preventing infection by Neisseria gonorrhoeae and / or Neisseria meningitidis in a subject; at least partially inhibiting or preventing binding of Neisseria gonorrhoeae and / or Neisseria meningitidis to cells in a subject; or at least partially inhibiting or reducing serum resistance of infection by Neisseria gonorrhoeae and / or Neisseria meningitidis in a subject. [Invention 24] one or more immunogenic fragments according to any one of inventions 1 to 7 for, or when used for, eliciting an immune response to Neisseria gonorrhoeae and / or Neisseria meningitidis in a subject; inducing immunity to Neisseria gonorrhoeae and / or Neisseria meningitidis in a subject; treating or preventing infection by Neisseria gonorrhoeae and / or Neisseria meningitidis in a subject; at least partially inhibiting or preventing binding of Neisseria gonorrhoeae and / or Neisseria meningitidis to cells in a subject; or at least partially inhibiting or reducing serum resistance of infection by Neisseria gonorrhoeae and / or Neisseria meningitidis in a subject; an isolated protein according to invention 8; an isolated nucleic acid according to invention 9; a genetic construct according to invention 10; a host cell according to invention 11; an antibody or antibody fragment according to invention 13; and / or a composition according to any one of inventions 14 to 16.

Claims

1. 1. An isolated immunogenic fragment of a Neisserial Heparin Binding Antigen (NHBA) protein from Neisseria gonorrhoeae, comprising: the immunogenic fragment is 165 to 200 amino acids in length; and the immunogenic fragment comprises, is contained within, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO:2, or a variant thereof, wherein the variant shares at least 97% sequence identity with the amino acid sequence set forth in SEQ ID NO:2; Said immunogenic fragment.

2. The immunogenic fragment of claim 1 , wherein the immunogenic fragment is conjugated, coupled, or otherwise linked to a carrier protein.

3. The immunogenic fragment of claim 1 or 2, wherein the immunogenic fragment consists of or essentially consists of the amino acid sequence set forth in SEQ ID NO: 2, or a variant thereof having at least 97% sequence identity with the amino acid sequence set forth in SEQ ID NO:

2.

4. The immunogenic fragment of any one of claims 1 to 3, wherein the variant shares at least 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:

2.

5. An immunogenic fragment described in claim 1 or 2, wherein the immunogenic fragment comprises an amino acid sequence having at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:

2.

6. An immunogenic fragment described in claim 1 or 2, wherein the immunogenic fragment comprises the amino acid sequence shown in sequence number 2.

7. 6. The immunogenic fragment of any one of claims 1 to 5, wherein the variant comprises an amino acid substitution at one or more of residues 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 181 in the amino acid sequence set forth in SEQ ID NO:

2.

8. 8. The immunogenic fragment of claim 7, wherein the amino acid substitutions in the one or more amino acids in the amino acid sequence set forth in SEQ ID NO: 2 are 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, R181Q, and any combination thereof.

9. The immunogenic fragment of any one of claims 1 to 8, comprising one or more heparin-binding residues and / or one or more active site residues of the isolated NHBA protein.

10. An isolated protein comprising a plurality of immunogenic fragments according to any one of claims 1 to 9.

11. An isolated nucleic acid comprising a nucleotide sequence encoding the immunogenic fragment of any one of claims 1 to 9 or the isolated protein of claim 10.

12. A genetic construct comprising the isolated nucleic acid of claim 11.

13. A host cell comprising the genetic construct of claim 12.

14. 13. A method for producing the isolated immunogenic fragment of any one of claims 1 to 9 or the isolated protein of claim 10, the method comprising: (i) culturing the host cell of claim 13; and (ii) isolating the immunogenic fragment or protein from the host cell cultured in step (i).

15. An antibody or antibody fragment which binds or is raised against the immunogenic fragment of any one of claims 1 to 9 or the isolated protein of claim 10.

16. A composition comprising one or more immunogenic fragments according to any one of claims 1 to 9, an isolated protein according to claim 10, or an isolated nucleic acid according to claim 11, optionally together with a pharmaceutically acceptable diluent, carrier, or excipient.

17. 17. The composition of claim 16, which is an immunogenic composition.

18. 18. The composition of claim 17, which is a vaccine.

19. 19. A pharmaceutical composition for raising an immune response to Neisseria gonorrhoeae and / or Neisseria meningitidis in a subject, the pharmaceutical composition comprising one or more immunogenic fragments of any one of claims 1 to 9; an isolated protein of claim 10; an isolated nucleic acid of claim 11; and / or a composition of any one of claims 16 to 18.

20. 19. A pharmaceutical composition for inducing immunity in a subject against Neisseria gonorrhoeae and / or Neisseria meningitidis, the pharmaceutical composition comprising one or more immunogenic fragments of any one of claims 1 to 9; an isolated protein of claim 10; an isolated nucleic acid of claim 11; and / or a composition of any one of claims 16 to 18.

21. 19. A pharmaceutical composition for treating or preventing a Neisseria gonorrhoeae and / or Neisseria meningitidis infection in a subject, the pharmaceutical composition comprising one or more immunogenic fragments of any one of claims 1 to 9; an isolated protein of claim 10; an isolated nucleic acid of claim 11; and / or a composition of any one of claims 16 to 18.

22. 19. A pharmaceutical composition for at least partially inhibiting or preventing binding of Neisseria gonorrhoeae and / or Neisseria meningitidis to cells in a subject, the pharmaceutical composition comprising one or more immunogenic fragments of any one of claims 1 to 9; an isolated protein of claim 10; an isolated nucleic acid of claim 11; and / or a composition of any one of claims 16 to 18.

23. 19. A pharmaceutical composition for at least partially inhibiting or reducing serum resistance of a Neisseria gonorrhoeae and / or Neisseria meningitidis infection in a subject, the pharmaceutical composition comprising one or more immunogenic fragments of any one of claims 1 to 9; an isolated protein of claim 10; an isolated nucleic acid of claim 11; and / or a composition of any one of claims 16 to 18.

24. 16. A method for detecting Neisseria gonorrhoeae and / or Neisseria meningitidis in a biological sample obtained from a subject, the method comprising contacting the biological sample with the antibody or antibody fragment of claim 15, thereby detecting N. gonorrhoeae in the biological sample.

25. Use of one or more immunogenic fragments according to any one of claims 1 to 9; the isolated protein according to claim 10; the isolated nucleic acid according to claim 11; and / or the composition according to any one of claims 16 to 18; to raise an immune response to Neisseria gonorrhoeae and / or Neisseria meningitidis in a subject; to induce immunity to Neisseria gonorrhoeae and / or Neisseria meningitidis in a subject; to treat or prevent infection with Neisseria gonorrhoeae and / or Neisseria meningitidis in a subject; The use in the manufacture of a medicament for at least partially inhibiting or preventing the binding of Neisseria meningitidis to cells in a subject; or at least partially inhibiting or reducing serum resistance of Neisseria gonorrhoeae and / or Neisseria meningitidis infection in a subject.

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