Pathogen parts and their uses

Abstract

The present disclosure relates generally to Neisserial surface moieties. More particularly, the disclosure relates to oligosaccharides corresponding to Neisserial lipooligosaccharides and chimeric molecules comprising these moieties for eliciting an immune response against Neisserial organisms and / or for treating or inhibiting the development of Neisserial infection.

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Application No. 63 / 161,399, entitled "Pathogen Moieties and Uses Thereof," filed March 15, 2021, the contents of which are incorporated by reference in their entirety herein.

[0002] government funds This invention was made with Government support under Grant Nos. NIAD AI108255 and NAID AI024616 awarded by the National Institute of Allergy and Infectious Diseases. The Government has certain rights in this invention.

[0003] The present disclosure relates generally to Neisseria surface moieties, and more particularly to oligosaccharides corresponding to Neisseria lipooligosaccharides, and chimeric molecules comprising these moieties for eliciting an immune response against Neisserial organisms and / or for treating or inhibiting the development of Neisserial infections. [Background technology]

[0004] Neisseria gonorrhoeae is a host-adapted bacterial pathogen that causes the sexually transmitted disease gonorrhea. Gonococcal infection can be symptomatic or asymptomatic, with up to 80% of infections in females being asymptomatic. Untreated gonorrhea in females can result in pelvic inflammatory disease, adverse pregnancy outcomes, neonatal complications, or infertility. In addition, it can increase the risk of acquiring and transmitting HIV. It is estimated that there are over 106 million gonococcal cases annually worldwide, and the emergence of multidrug-resistant (MDR) strains of N. gonorrhoeae is a major public health problem. Isolates resistant to nearly all antibiotics used to treat gonorrhea have been identified. In the past 100 years, only three potential gonococcal vaccines have been tested in clinical trials, and none of these vaccines have been successful. Thus, there is an urgent unmet need for novel approaches for treatment and vaccine development.

[0005] Lipooligosaccharide (LOS) is one of the major structural components of the outer membrane and is an important virulence factor of N. gonorrhoeae. It plays many important roles in gonococcal pathogenesis, including mediating direct interactions between N. gonorrhoeae and human urethral epithelial cells (Harvey et al., 2000, Mol Microbiol. 36:1059-70, and Song et al., 2000, J Exp Med 191:949-60). Lipid A of N. gonorrhoeae's LOS binds complement component C3, which is required for activation of complement receptor 3 in primary cervical epithelial cells (Edwards et al., 2002, Cell Microbiol 4:585-98). Bacteria such as N. gonorrhoeae can mimic host structures to avoid detection. One aspect of this mimicry is the expression of sialic acid, a sugar structure found in humans, on the bacterial surface. The gonococcal lipooligosaccharide structure can be terminated with N-acetylneuraminic acid (Neu5Ac), which has multiple roles in gonococcal virulence (Apicella et al., 1989, Pediatr Infect Dis J. 8:901-2, and Mandrell et al., 1993, Immunobiology 187:382-402). However, gonococci cannot synthesize cytidine-5'-monophosphate (CMP)-Neu5Ac, the nucleotide sugar precursor required for lipooligosaccharide biosynthesis, and must acquire CMP-Neu5Ac from the host (Mandrell et al., 1993, Immunobiology 187:382-402). In contrast, the closely related pathogen Neisseria meningitidis can synthesize CMP-Neu5Ac, which is also required for the polysaccharide capsule structure of meningococcal serogroups B, C, W, and Y. Both of these pathogenic Neisseria species have a nearly identical (95% amino acid identity) sialyltransferase, Lst, which transfers Neu5Ac from CMP-Neu5Ac to the terminal galactose of LOS, as shown in Figure 1 (Gilbert et al., 1996, J Biol Chem 271:28271-6).The Lst sialyltransferase expressed by pathogenic Neisseria species is homologous to the LsgB sialyltransferase of non-typeable Haemophilus influenzae (NTHi). Summary of the Invention

[0006] The present disclosure is based in part on the discovery that a bacterial-specific sugar, ketodeoxyoctrosonate (KDO), is commonly found as the terminal lipooligosaccharide in place of sialic acid on Neisseria gonorrhoeae and Neisseria meningitidis, and that KDO is added by the sialyltransferase Lst, as shown in Figure 1. This discovery is in keeping with previous findings in which the enzyme that adds sialic acid to LOS in both organisms Lst was previously thought to be outside the bacterial cell (Shell et al., 2002, Infect Immun. 70:3744-51), whereas the donor molecule for adding KDO to the biomolecule, cytidine-monophosphate KDO (CMP-KDP), was thought to be in the cytoplasm of the bacterial cell. This discovery represents an important paradigm shift with respect to vaccine development, as it allows the bacterial KDO carbohydrate to be considered as a target for the development of new strategies to prevent and treat Neisserial infections, including gonococcal and meningococcal infections. It has been proposed that KDO-based vaccines induce immune responses distinct from those elicited during natural infection, and that antibodies targeting this bacterial KDO epitope mediate bactericidal and opsonophagocytic killing, thus providing immune protection against Neisserial infection.

[0007] Thus, disclosed in one aspect is a chimeric molecule comprising, consisting of, or consisting essentially of a carrier and an oligosaccharide comprising an oligosaccharide sequence corresponding to a Neisserial lipooligosaccharide (LOS) (e.g., Neisseria gonorrhoeae or Neisseria meningitidis LOS), the oligosaccharide sequence comprising ketodeoxyoctulosonate (KDO) as a terminal sugar unit. The oligosaccharide sequence suitably comprises KDO linked to a galactose (Gal), typically further linked to N-acetylglucosamine (GlcNAc). In a representative embodiment, KDO is linked to Gal via an α(2→3) linkage and / or Gal is linked to GlcNAc via a β(1→4) linkage. In an illustrative example of this type, the oligosaccharide sequence comprises KDOα(2→3)Gal, or KDOα(2→3)Galβ(1→4)GlcNAc.

[0008] In some embodiments, the oligosaccharide comprises n occurrences of the oligosaccharide sequence, where n is 1 to 4. In a representative example of this type, n is 2. Suitably, each oligosaccharide sequence is linked to each other by a linker.

[0009] The carrier is typically linked to the oligosaccharide by a linker. Suitably the carrier is a protein, peptide, lipid, polymer, dendrimer, virosome, virus-like particle (VLP), outer membrane vesicle or a combination thereof. In certain embodiments, the carrier is a protein carrier, such as, for example, bacterial toxoids, toxins, exotoxins, and non-toxic derivatives thereof, such as, for example, keyhole limpet hemocyanin (KLH), Hepatitis B virus core protein, thyroglobulin, albumin (such as bovine serum albumin (BSA), human serum albumin (HSA), and ovalbumin), pneumococcal surface protein A (PspA), pneumococcal adhesion protein (PsaA), purified protein derivative of tuberculin (PPD); transferrin binding protein, polyamino acids, such as poly(lysine:glutamic acid), tetanus toxoid, tetanus toxin fragment C, diphtheria toxoid, CRM (non-toxic diphtheria toxin mutant), cholera toxoid, Staphylococcus aureus exotoxin or toxoid, Escherichia coli exotoxin or toxoid, Staphylococcus aureus ... coli heat labile enterotoxin, Pseudomonas aeruginosa exotoxin A, and bacterial outer membrane proteins such as Neisseria meningitidis serogroup B outer membrane protein complex (OMPC) and outer membrane class 3 porin (rPorB). In a particular embodiment, the protein carrier is CRM197.

[0010] Another aspect of the present disclosure relates to a composition comprising, consisting of, or consisting essentially of a chimeric molecule as broadly described above and elsewhere herein and a pharma- ceutically acceptable vehicle or adjuvant. The composition may optionally include one or more adjuvants for treating or inhibiting the development of a Neisserial infection. The adjuvants may be selected from antimicrobial agents and immunogens (e.g., proteinaceous immunogens derived from Neisserial antigens such as AniA, MsrAB, MetQ, NhbA, etc.) for eliciting an immune response against a Neisserial organism.

[0011] In another aspect, disclosed herein is a method of inducing an immune response in a subject against a Neisserial organism, the method comprising, consisting of, or consisting essentially of immunizing the subject with a chimeric molecule or composition as broadly described above and elsewhere herein, thereby inducing an immune response in the subject against a Neisserial organism. Preferably, the Neisserial organism is Neisseria gonorrhoeae or Neisseria meningitidis. In some embodiments, the Neisserial organism is resistant to at least one antibacterial agent.

[0012] Yet another aspect of the present disclosure relates to a method for producing an antigen binding molecule that specifically binds to a Neisseria organism, the method comprising: (1) immunizing an animal with a chimeric molecule or composition as broadly described above and elsewhere herein; (2) detecting B cells from the animal that specifically bind to the chimeric molecule (particularly its oligosaccharide component) or the LOS of the Neisseria organism; and (3) isolating the antigen binding molecule expressed by the B cells.

[0013] Also disclosed in another aspect is a method for producing an antigen-binding molecule that specifically binds to a Neisseria organism, the method comprising: (1) screening a library of antigen-binding molecules with a chimeric molecule or composition as broadly described above and elsewhere herein; (2) detecting an antigen-binding molecule that specifically binds to the chimeric molecule (particularly its oligosaccharide component) or to the LOS of the Neisseria organism; and (3) isolating the detected antigen-binding molecule.

[0014] In a related aspect, the present disclosure relates to antigen-binding molecules produced by the methods broadly described above and elsewhere herein, or antigen-binding molecule derivatives having the same epitope binding specificity as the antigen-binding molecules. In representative embodiments, the antigen-binding molecule derivatives are selected from antibody fragments (Fab, Fab', F(ab')2, Fv, etc.), single chain (scFv) and domain antibodies (including, for example, shark and camelid antibodies), as well as fusion proteins comprising antibodies, and any other modified configuration of immunoglobulin molecules comprising an antigen-binding / recognition site.

[0015] In another related aspect, the present disclosure relates to cells (e.g., hybridomas or cell lines) that produce the antigen-binding molecules as broadly described above and elsewhere herein.

[0016] Also disclosed herein are methods for treating or inhibiting the development of a Neisserial infection in a subject, the methods comprising administering to the subject an effective amount of a chimeric molecule, composition or antigen-binding molecule as broadly described above and elsewhere herein. In some embodiments, the chimeric molecule or antigen-binding molecule is administered simultaneously with an adjuvant (e.g., an antibacterial agent and / or an immunogen).

[0017] In yet another aspect, disclosed herein is a method for detecting the presence of a Neisserial organism in a subject, the method comprising contacting a biological sample taken from the subject with an antigen binding molecule that specifically binds to a Neisserial organism, as broadly described above and elsewhere herein, and detecting the presence of a complex comprising a Neisserial organism or a portion thereof (e.g., LOS), thereby detecting the presence of the Neisserial organism in the subject.

[0018] Yet another aspect of the present disclosure relates to a kit for treating or inhibiting the development of a Neisserial infection in a subject, the kit comprising a chimeric molecule, composition, or antigen-binding molecule as broadly described above and elsewhere herein, and, optionally, instructional materials for carrying out the treatment or inhibition.

[0019] In yet another aspect, a kit for detecting the presence of a Neisseria organism in a subject is provided, the kit comprising an antigen-binding molecule as broadly described above and elsewhere herein, and, optionally, instructional materials for carrying out the detection. [Brief description of the drawings]

[0020] [Figure 1]Figure 1 shows the structure of KDO-containing LOS of N. gonorrhoeae strain 1291. MAb 6E4 recognizes only terminal KDO glycans. Lst is a sialyltransferase that can also incorporate KDO onto terminal Gal in the absence of Neu5Ac. LgtB is a galactosyltransferase that transfers the KDO acceptor galactose (Gal) to N-acetylglucosamine (GlcNAc). In the absence of Lst, Gal is the terminal sugar of LOS in N. gonorrhoeae. [Diagram 2] FIG. 2 is a graph and photographic representation showing a whole cell ELISA of 6E4. (A) ELISA of whole cell N. gonorrhoeae WT and lst mutant strains using 6E4 MAb. Lst transfers a terminal sialic acid to the terminal Gal of the LOS of N. meningitidis. Here, the lst mutant showed that Lst also transfers KDO to the terminal Gal of N. gonorrhoeae. (B) Whole cell ELISA of 6E4 was performed to test 20 clinical isolates of N. gonorrhoeae. 1291 and 1291lst were the positive and negative controls in this ELISA. (C) ELISA of whole cell N. meningitidis MC58 WT, siaB, and lst mutant strains using 6E4 MAb. MC58 WT has Neu5Ac as the terminal glycan of the LOS. Without SiaB, N. meningitidis cannot produce CMP-Neu5Ac, so Lst adds KDO to the end of the LOS instead of sialic acid. (D) Incorporation of KDO into gonococcal LOS. Whole cell ELISA of 6E4 showed that the presence of CMP-Neu5Ac in the growth medium could inhibit the incorporation of KDO into terminal Gal. N. gonorrhoeae 1291 and lst mutants were grown in medium supplemented with 0, 30, and 100 μg / mL CMP-Neu5Ac. (E) Silver staining and 6E4 Western blot analysis of purified LOS samples from N. meningitidis MC58, N. gonorrhoeae 1291 wild type, and glycosyltransferase mutant strains. [Diagram 3]Figure 3 is a photograph showing a confocal microscopy study of a cervical swab from a patient with documented gonococcal infection stained with both anti-KDO MAb 6E4 (Texas Red) and anti-LOS MAb 6B4 (FITC). Infected polymorphonuclear leukocytes (PMNs) can be seen with gonococci stained with separate antibodies (red or green) and with antibodies colocalized to the same organism (yellow). This study shows that KDO termination occurs in vivo and in vitro. [Figure 4] Figure 4 is a graphical representation showing the functional activity of Mab 6E4 against N. gonorrhoeae. Survival of N. gonorrhoeae in the presence of 0 to 10 μg / mL Mab 6E4, primary human PMNs, and 10% normal human serum as a complement source is shown. Data represent the mean survival (± 1 standard deviation) for triplicate samples shown as a percentage of bacteria in the absence of antibody (0 μg / mL Mab 6E4 set at 100% represents 5.07 x 103 CFU). One-way ANOVA (F(6,14) = 59.20, P = < .0001) revealed statistically significant differences between groups. Using a two-tailed Student's t-test, statistically significant differences compared to untreated wild type are indicated: *P<0.05; **P≦0.01; ***P≦0.001 (P values: 1.25 μg / mL, 0.012; 2.5 μg / mL, 0.005; 5 μg / mL, 0.001; 10 μg / mL, 0.0003). [Diagram 5] Figure 5 is a schematic showing the chemical synthesis of the KDO spacer. The protected form of KDO, synthesized using the published chemistry, is converted to a glycosyl fluoride derivative and then reacted with the spacer unit using standard Lewis acid catalyzed glycosidation chemistry. All protecting groups introduced to facilitate chemical synthesis are then removed, leaving the final compound ready for conjugation. [Figure 6] 6A is an illustrative and photographic representation showing the coupling of NH2-linked KDO to CRM197. (B) After binding, CRM197 (Coomassie, lane 2) shifts to a higher apparent molecular weight (Coomassie, lane 1) and becomes KDO positive (Western blot with 6E4, lane 1) compared to unbound CRM197. [Figure 7] Figure 7 is a schematic showing the chemical synthesis of KDOα(2,3)Gal spacer. A protected form of KDO, synthesized using published chemistry, is converted to the glycosyl fluoride derivative. Commercially available galactose is converted to the Gal spacer derivative using standard carbohydrate chemistry manipulations. The KDO fluoride and Gal spacer units are combined together using standard Lewis acid catalyzed glycosidation chemistry. All protecting groups introduced to facilitate chemical synthesis are then removed using standard methods to yield the final compound ready for conjugation. [Figure 8] FIG. 8 is a schematic showing the chemical synthesis of the KDOα(2,3)Galβ(1,4)GlcNAc spacer. A protected form of KDO, synthesized using published chemistry, is converted to the fluorinated glycosyl derivative. Commercially available lactulose is converted to the Gal-GlcNAc spacer derivative using standard carbohydrate chemistry manipulations. The KDO fluoride and Gal-GlcNAc spacer units are combined together using standard Lewis acid catalyzed glycosidation chemistry. All protecting groups introduced to facilitate chemical synthesis are then removed using standard methods, yielding the final compound ready for conjugation. [Figure 9] Figure 9 is a schematic diagram showing the chemoenzymatic approach to synthesize KDO (A) disaccharide and (B) trisaccharide antigens. KdsB and Lst in green are the enzymes involved in the synthesis. [Figure 10]FIG. 10. (A) Schematic and schematic representation showing the in vivo biosynthetic pathway of LOS containing KDO antigen in outer membrane vesicles (OMV); inset electron microscopy image showing OMV production typical of E. coli strain with double mutation lacking expression of OmpA and Braun lipoprotein is modified from Sonntag et al. (1978, J. Bacteriol. 136:280-285). Lacto-N-neotetraose (LNnT) is synthesized by LgtE, LgtA and LgtB. Upregulation of Lst and KdsB (orange) increases the modification of terminal KDO to LNnT. The synthesized LOS is transported to the outer membrane. (B) LgtE, LgtA, LgtB and Lst can be co-expressed to produce the same terminal KDO glycan as Ng in E. coli. Terminal KDO is confirmed by ELISA using mAb 6E4. *P<0.05. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] 1.Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. Methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, and preferred methods and materials are described. For the purposes of this disclosure, the following terms are defined below.

[0022] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0023] The term "adjuvant" as used herein refers to a compound that, when used in combination with a particular immunogen (e.g., a chimeric molecule of the present disclosure) in a composition, enhances the resulting immune response, including enhancing or broadening the specificity of one or both of the antibody and cellular immune responses. In the context of the present disclosure, an adjuvant preferably enhances the specific immunogenic effect of an active agent of the present disclosure. The term "adjuvant" is understood to typically not include an agent that confers immunity by itself. An adjuvant helps the immune system to non-specifically enhance an antigen-specific immune response, for example, by promoting the presentation of antigens to the immune system or the induction of a non-specific innate immune response. In addition, an adjuvant can preferably modulate an antigen-specific immune response, for example, by shifting a dominant Th2-based antigen-specific response to a more Th1-based antigen-specific response, or vice versa. Thus, an adjuvant can advantageously modulate cytokine expression / secretion, antigen presentation, type of immune response, and the like.

[0024] The terms "concurrent administration" or "administering concurrently" or "co-administering" and the like refer to administration of a single composition containing two or more agents, or administration of each agent as a separate composition, and / or delivery by separate routes contemporaneously or simultaneously or sequentially within a sufficiently short period of time that is equivalent to the effective results obtained when all such agents are administered as a single composition. "Contemporaneously" means that the agents are administered at substantially the same time, preferably together in the same composition. "Contemporaneously" means that the agents are administered closely in time, for example, one agent is administered before or after another within about one minute to about one day. Any concurrent time is useful. However, when not administered simultaneously, the agents are often administered within about one minute to about eight hours, preferably within about one hour to about four hours. When administered simultaneously, the agents are suitably administered to the same site on the subject. The term "same site" includes the exact location, but may be within about 0.5 centimeters to about 15 centimeters, preferably within about 0.5 centimeters to about 5 centimeters. As used herein, the term "separately" means that the agents are administered at intervals, for example, from about one day to several weeks or months. The agents may be administered in any order. As used herein, the term "sequentially" means that the agents are administered consecutively, for example, at intervals of minutes, hours, days or weeks. Where appropriate, the agents may be administered in regular repeated cycles.

[0025] As used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).

[0026] As used herein, the term "antigen" and its grammatical equivalents (e.g., "antigenic") refer to a compound, composition, or substance that can be specifically bound by a product of specific humoral or cellular immunity, such as an antibody molecule or a T-cell receptor. Antigens can be any type of molecule, including, for example, haptens, simple intermediate metabolites, sugars (e.g., oligosaccharides), lipids, and hormones, as well as macromolecules such as complex carbohydrates (e.g., polysaccharides), phospholipids, and proteins.

[0027] "Antigen-binding molecule" refers to a molecule that has binding affinity for a target antigen. It will be understood that the term extends to immunoglobulins, immunoglobulin fragments and non-immunoglobulin derived protein frameworks that exhibit antigen-binding activity. Exemplary antigen-binding molecules that are useful in the practice of the present disclosure include polyclonal and monoclonal antibodies and fragments thereof (e.g., Fab, Fab', F(ab')2, Fv), single chain (scFv) and domain antibodies (including, e.g., shark and camelid antibodies), as well as fusion proteins comprising antibodies and any other modified configuration of an immunoglobulin molecule that comprises an antigen binding / recognition site. Antibodies include antibodies of any class, such as IgG, IgA, or IgM (or subclasses thereof), and an antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant region of its heavy chain, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the various classes of immunoglobulins are well known. Antigen-binding molecules also include dimeric antibodies, as well as multivalent forms of antibodies. In some embodiments, the antigen-binding molecules are chimeric antibodies, in which a portion of the heavy and / or light chain is identical or homologous to sequences in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to sequences in an antibody derived from another species or belonging to another antibody class or subclass, so long as the desired biological activity is exhibited (see, e.g., U.S. Pat. No. 4,816,567; and Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81:6851-6855), as well as fragments of such antibodies.Also contemplated are humanized antibodies, which are generally produced by transferring complementarity determining regions (CDRs) from heavy and light chains of non-human (e.g. rodent, preferably mouse) immunoglobulins into human variable domains. Then, typical residues of human antibodies are substituted in the framework regions of the non-human counterpart. The use of antibody components derived from humanized antibodies obviates potential problems associated with the immunogenicity of non-human constant regions. General techniques for cloning non-human, particularly mouse, immunoglobulin variable domains are described, for example, by Orlandi et al. (1989, Proc. Natl. Acad. Sci. USA 86:3833). Techniques for producing humanized monoclonal antibodies are described, for example, in Jones et al. (1986, Nature 321:522), Carter et al. (1992, Proc. Natl. Acad. Sci. USA 89:4285), Sandhu (1992, Crit. Rev. Biotech. 12:437), Singer et al. (1993, J. Immun. 150:2844), Sudhir (ed., Antibody Engineering Protocols, Humana Press, Inc. 1995), Kelley ("Engineering Therapeutic Antibodies," in Protein Engineering: Principles and Practice, Cleland et al. (eds.), pp. 399-434 (John Wiley & Sons, Inc. 1996), and Queen et al., U.S. Patent No. 5,693,762 (1997). Humanized antibodies include "primatized" antibodies in which the antigen-binding region of the antibody is derived from an antibody produced by immunizing a macaque monkey with an antigen of interest. Humanized antibodies are also contemplated as antigen-binding molecules.

[0028] The term "antimicrobial agent" as used herein refers to an agent having antimicrobial activity, i.e., the ability to inhibit or reduce and / or kill a microorganism, for example, by at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 90% or more, compared to the absence of the antimicrobial agent. The term "antimicrobial agent" encompasses agents that inhibit or reduce the growth of a microorganism and / or kill a microorganism by directly interacting with the microorganism and / or cells of the host in which the microorganism resides or resides. Non-limiting examples of antimicrobial agents include silver nanoparticles, small molecules, peptides, peptidomimetics, antibodies or fragments thereof, nucleic acids, enzymes (e.g., antimicrobial metalloendopeptidases such as lysostaphin), aptamers, drugs, antibiotics, chemicals, or any entity capable of inhibiting and / or killing the growth of a microorganism. Examples of antimicrobial peptides that may be included in the compositions described herein include, but are not limited to, mefloquine, venturicidin A, antimycin, myxothiazol, stigmatellin, diuron, iodoacetamide, potassium tellurate hydrate, aDL-vinylglycine, N-ethylmaleimide, L-allylglycine, diarylquinoline, betaine aldehyde chloride, acibusin, psicofurain, buthionine sulfoximine, diaminopemelic acid, 4-phospho-D-erythrone hydroxamic acid, motexafingadolinium and / or oxycitrine, or modified versions or analogs thereof. Exemplary antimicrobial agents include antibiotics, antifungals, antiprotozoal agents, antimalarials, antituberculous agents and antivirals, and mixtures of any of these.

[0029] As used herein, the term "specifically binds" refers to a binding reaction that determines the presence of a chimeric polypeptide or complex of the present disclosure in the presence of a heterogeneous population of molecules, including macromolecules such as proteins and other biologics. In certain embodiments, the term "specifically binds" is used interchangeably with the term "specific immune interaction" and the like when referring to an antigen-binding molecule, and refers to a binding reaction that determines the presence of a chimeric polypeptide or complex of the present disclosure in the presence of a heterogeneous population of proteins and other biologics. Under the specified assay conditions, the molecule specifically binds to a chimeric polypeptide or complex of the present disclosure and does not bind in significant amounts to other molecules (e.g., proteins or antigens) present in the sample. In embodiments of antigen-binding molecules, various immunoassay formats can be used to select antigen-binding molecules that specifically immunointeract with a chimeric polypeptide or complex of the present disclosure. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies that specifically immunointeract with a protein. For a description of immunoassay formats and conditions that can be used to determine specific immune reactivity, see Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York.

[0030] As used herein, the term "carrier" refers to a protein, peptide, lipid, polymer, dendrimer, virosome, virus-like particle (VLP), outer membrane vesicle, or combination thereof, that is coupled to an oligosaccharide of the present disclosure to enhance the immunogenicity of the resulting oligosaccharide-carrier chimeric molecule greater than the oligosaccharide alone. Similarly, the term "protein carrier" as used herein refers to a protein or peptide that is operably linked to an oligosaccharide to enhance the immunogenicity of the resulting oligosaccharide-protein carrier chimeric molecule greater than the oligosaccharide alone. For example, when used as a carrier, a protein carrier can act as a T-dependent antigen that can activate and recruit T cells, thereby enhancing T cell-dependent antibody production.

[0031] The term "chimeric" when used in reference to a molecule means that the molecule contains portions derived from, obtained from, isolated from, or based on two or more different origins or sources. In certain embodiments, the chimeric molecules of the present disclosure are immunogenic chimeric molecules for stimulating or eliciting a specific immune response in an animal. In some embodiments, the immune response is protective in that it allows the animal to better resist infection from the organism against which the immunogenic chimeric molecule is directed.

[0032] Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" are understood to mean the inclusion of a described step or element or group of steps or elements, but not the exclusion of any other step or element or group of steps or elements. Thus, use of the term "comprising" and the like indicates that the recited elements are necessary or mandatory, while other elements are optional and may or may not be present. "Consisting of" means including and limited to what follows the phrase "consisting of". Thus, the phrase "consisting of" indicates that the recited elements are necessary or mandatory, and that other elements are not present. "Consisting essentially of" means including any elements recited after the phrase, and is limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the recited elements. Thus, the phrase "consisting essentially of" indicates that the recited elements are necessary or mandatory, while other elements are optional and may or may not be present depending on whether they affect the activity or action of the recited elements.

[0033] As used herein, the terms "linked," "linked," "fused," or "fusion" and their grammatical equivalents in the context of linking two or more elements or components or domains together by any means, including chemical linkage or recombinant means, are used interchangeably. Methods of chemical linkage (e.g., using heterobifunctional crosslinkers) are known in the art and include covalent and / or non-covalent linkages. As used herein, "linked" refers to an oligosaccharide that is chemically linked to a carrier via a linker and / or crosslinker.

[0034] "Effective amount" in the context of treating, inhibiting the progression of, or preventing a condition means the administration of an amount of a drug or composition to an individual in need of such treatment, inhibition, or prevention, either in a single dose or as part of a series, that is effective in preventing the incurrence of symptoms, identifying such symptoms, and / or treating existing symptoms of the condition. The effective amount will vary depending on the health and physical condition of the individual being treated, the individual taxon being treated, the formulation of the composition, an evaluation of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine testing. For example, non-limiting symptoms of gonorrhea infection include pain in the lower abdomen, pelvis, testicles, or vagina, painful urination, abnormal vaginal discharge, penile discharge, or increased vaginal discharge, fever, frequent urgency, irregular menstruation, pus, or sore throat. Typical symptoms of meningococcal infection include fever and chills, fatigue, vomiting, cold hands and feet, severe aches or pains in the muscles, joints, chest, or abdomen, rapid respiratory diarrhea, and, late, a dark purple rash.

[0035] The term "epitope" refers to an antigenic determinant that is specifically bound by an antigen-binding molecule or antigen receptor, such as a B-cell receptor (BCR) or a T-cell receptor (TCR). Epitopes usually consist of surface groupings of molecules such as amino acids and / or sugar side chains and may be linear or have specific three-dimensional structural characteristics, as well as specific charge characteristics. B-cell epitope sites on proteins, oligosaccharides, or other biopolymers may be composed of moieties from different parts of the macromolecule that are brought together by folding. This type of epitope is called a conformational or discontinuous epitope because the site is composed of segments of the polymer that are discontinuous in the linear sequence but continuous in the folded conformation. Epitopes that are composed of a single segment of a biopolymer or other molecule are called continuous or linear epitopes. T-cell epitopes are generally restricted to linear peptides. Antigen-binding molecules that recognize the same epitope can be identified in a simple immunoassay demonstrating the ability of one antigen-binding molecule to block the binding of another antigen-binding molecule to a target antigen.

[0036] As used herein, the terms "inhibit," "inhibits," or "inhibition" (and their grammatical equivalents) do not imply the complete abolition of a disease, but rather encompass any type of prophylactic treatment that reduces the occurrence of a condition, delays the onset of a condition, and / or reduces the conditions associated with a condition after onset.

[0037] As used herein, "immune response" refers to a response by a subject's immune system. For example, the immune response can be to an antigen / immunogen that the subject's immune system recognizes as foreign (e.g., a non-self antigen) or self (e.g., a self antigen that is recognized as foreign). The immune response can be humoral, involving the production of immunoglobulins or antibodies, or can be cellular, involving various types of B and T lymphocytes, dendritic cells, macrophages, antigen-presenting cells, etc., or both. The immune response can also involve the manufacture or synthesis of various effector molecules, such as cytokines. The term "immune response" encompasses immunogenic responses, which cause, activate, elicit, or induce an immune response in a subject against a particular antigen (e.g., an antigen of a pathogenic organism) or organism (e.g., a pathogenic microorganism), as well as immunosuppressive or tolerogenic immune responses, which inhibit, suppress, reduce, or eliminate an immune response, or render the immune system unresponsive, or delay the development or initiation of an immune response against an allergen, or against self-antigens, or against cells, tissues, or organs expressing such antigens.

[0038] Reference herein to "immunointeractive" includes reference to any interaction, reaction, or other form of association between molecules, particularly where one of the molecules is or mimics a component of the immune system.

[0039] As used herein, the term "immunity" refers to protection from disease (e.g., prevention or attenuation (e.g., suppression) of a sign, symptom, or condition of disease) upon exposure to a microorganism (e.g., a pathogen) capable of causing disease. Immunity can be innate (e.g., a non-adaptive (e.g., non-acquired) immune response that exists in the absence of previous exposure to an antigen) and / or acquired / adaptive (e.g., an immune response mediated by B cells and T cells after previous exposure to an antigen (e.g., exhibiting increased specificity and reactivity to an antigen)).

[0040] As used herein, the term "immunogen" refers to a molecule that stimulates a response from the adaptive immune system, which may include responses elicited from groups including antibody responses, cytotoxic T cell responses, T helper responses, and T cell memory responses. Immunogens may stimulate upregulation of immune responses with a resulting inflammatory response, or may result in downregulation or immunosuppression. Typically, immunogens can initiate lymphocyte activation resulting in an antigen-specific immune response.

[0041] As used herein, the term "immunogenic composition" or "immunogenic formulation" refers to a preparation that, when administered to an animal, such as a vertebrate, particularly a mammal, induces an immunogenic immune response, which may include a response that provides at least some level of immunity in the subject to which the immunogenic composition is administered.

[0042] The terms "linker" and "spacer" are used interchangeably herein and refer to a molecule or group of molecules (such as a monomer or polymer) that links two molecules together and often serves to arrange the two molecules in a desired configuration. In some embodiments, a linker can be a covalent bond linking two groups, or a chemical moiety having a chain ranging from 1 to 50 atoms in length, from 2 to 20 atoms in length, from 3 to 30 atoms in length, from 4 to 40 atoms in length, from 10 to 50 atoms in length, from 10 to 30 atoms in length, from 12 to 26 atoms in length, from 14 to 30 atoms in length, or 1 atom in length. For example, a linker or portion of a linker can have a length that is 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 45, or 50 carbon atoms in length, and the linker may be linear, branched, cyclic, or single atom. In some embodiments, the linker can be 10 Angstroms, 11 Angstroms, 12 Angstroms, 13 Angstroms, 14 Angstroms, 15 Angstroms, 16 Angstroms, 17 Angstroms, 18 Angstroms, 19 Angstroms, 20 Angstroms, 22 Angstroms, 23 Angstroms, 24 Angstroms, 25 Angstroms, 26 Angstroms, 27 Angstroms, 28 Angstroms, 29 Angstroms, 30 Angstroms, 32 Angstroms, 34 Angstroms, 36 Angstroms, 38 Angstroms, 40 Angstroms, 45 Angstroms, 50 Angstroms, or any amount of 1 Angstrom units therein. In some embodiments, one, two, three, four, five or more carbon atoms of the linker backbone may be replaced with sulfur, nitrogen or oxygen heteroatoms. The bonds between the backbone atoms may be saturated or unsaturated, and typically no more than one, two or three unsaturated bonds are present in the linker backbone. The linker may include one or more substituents, for example, with alkyl, aryl or alkenyl groups.Linkers can include, but are not limited to, oligo(ethylene glycol); ethers, thioethers, tertiary amines, alkyls that can be linear or branched, such as methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), and the like. The linker backbone can include a cyclic group, such as an aryl, heterocyclic, or cycloalkyl group, where two or more atoms of the cyclic group, such as 2, 3, or 4 atoms, are included in the backbone. In some embodiments, the linker can be at least one sugar unit. The linker can be cleavable or non-cleavable.

[0043] As used herein, the term "moiety" refers to a portion of a molecule, which may be a functional group, set of functional groups, and / or a particular grouping of atoms within a molecule that is responsible for the molecule's characteristic chemical, biological, and / or pharmaceutical properties.

[0044] The terms "monosaccharide unit" and "monosaccharide residue" are used interchangeably herein to refer to the most basic unit of a carbohydrate, and include aldoses (e.g., D-glucose, D-galactose, D-mannose, D-ribose, D-arabinose, L-arabinose, D-xylose, etc.), ketoses (e.g., D-fructose, D-sorbose, D-tagatose, etc.), deoxysugars (e.g., L-rhamnose, L-fucose, etc.), deoxyaminosugars (e.g., N-acetylglucosamine, N-acetylmannosamine, N-acetylgalactosamine, etc.), aldaric acids, uronic acids, ketoaldonic acids (e.g., sialic acid), uronic acids (e.g., KDO), or the like.

[0045] The term "monosaccharide derivative" or "monosaccharide unit derivative" is used herein to refer to derivatives of monosaccharides in which the monosaccharide moiety is substituted with one or more substituents and / or functional groups, non-limiting examples of which include deoxysugars (alcoholic hydroxy group replaced with hydrogen), aminosugars (alcoholic hydroxy group replaced with amino group), thiosugars (alcoholic hydroxy group replaced with thiol, or C=O replaced with C=S, or ring oxygen replaced with sulfur), selenosugars, tellurosugars, azasugars (ring carbon replaced with nitrogen), aminosugars (ring oxygen replaced with nitrogen), phosphanosugars (ring carbon replaced with phosphorus), C-substituted monosaccharides (hydrogen at a non-terminal carbon atom replaced with carbon), unsaturated monosaccharides, alditols (carbonyl group replaced with CHOH group), aldonic acids (aldehyde group replaced with carboxy group), ketoaldonic acids, uronic acids, aldaric acids, etc. It is understood that the monosaccharide units, etc. may be further substituted.

[0046] The term "Neisseria" refers to a large genus of Gram-negative bacteria that colonize the mucosal surfaces of many animals. Of the eleven species that colonize humans, only two are pathogens: Neisseria meningitidis (one of the pathogens responsible for bacterial meningitis) and Neisseria gonorrhoeae (the pathogen responsible for gonorrhea). In certain embodiments, infections targeted by the agents and compositions of the present disclosure include secondary infections that may result from lack of treatment of primary gonococcal infection. Exemplary secondary infections include urethritis, dysuria, epididymitis, pelvic inflammatory disease, cervicitis and endometritis, and systemic gonococcal infections (e.g., manifesting as arthritis, endocarditis, or meningitis). Gonococcal infections may be caused by strains of Neisseria gonorrhoeae that are resistant to at least one known antibacterial agent, such as at least one β-lactam agent.

[0047] As used herein, the term "neutralize" and its grammatical equivalents refer to the ability of an antigen-binding molecule to inhibit the activity of an antigen to which it binds, including the activity of an organism expressing the antigen. The term "neutralize" also includes within its scope the rendering of a pathogenic organism expressing the antigen non-pathogenic. For example, an organism can be neutralized through the binding of an antigen-binding molecule that induces an immune response against the organism (e.g., by phagocytes). An organism can also be neutralized through the binding of an antigen-binding molecule that renders the organism unable to grow, infect a host, or produce toxins.

[0048] The term "oligosaccharide" refers to a molecule that contains two or more, generally from two to ten, typically from two to six, monosaccharide residues (also referred to herein as "monosaccharide units") suitably glycosidically linked (e.g., by α-glycosidic and / or β-glycosidic bonds). Oligosaccharides are considered to have a reducing end and a non-reducing end.

[0049] The term "oligosaccharide sequence" indicates that the monosaccharide residue / residues in the sequence are part of a larger glycoconjugate, may contain other monosaccharide residues in the chain, may be branched, and / or may have substitution (e.g., natural substitution) modifications of the oligosaccharide chain. The oligosaccharide chain is usually attached or otherwise linked to a lipid anchor or protein. In some embodiments, the oligosaccharide sequence is a non-reducing end oligosaccharide sequence, meaning that the oligosaccharide sequence is not linked to another monosaccharide or oligosaccharide structure except from the reducing end of the oligosaccharide sequence. When present as a conjugate or chimeric molecule, the oligosaccharide sequence is suitably attached or otherwise linked from the reducing end of the oligosaccharide sequence, although other linking positions tolerated by the antigen binding molecule or other binding agent can also be used. It should be understood that adjacent monosaccharide units defined in the oligosaccharide sequences of the present disclosure include within their scope alternative glycosidic linkages between them, and that specific monosaccharide units defined in the disclosed oligosaccharide sequences include within their scope derivatives of those monosaccharide units.

[0050] As used herein, the term "operably linked" refers to a juxtaposition where the components so described are in a relationship permitting them to function in their intended manner. For example, "operably linking" a carrier to an oligosaccharide described herein encompasses positioning and / or orientation of the carrier such that the oligosaccharide is presented to the immune system for eliciting an immune response against the oligosaccharide.

[0051] The terms "patient," "subject," "host," or "individual," as used interchangeably herein, refer to any subject for which treatment or prevention is desired, particularly a vertebrate subject, and even more particularly a mammalian subject. Suitable vertebrates within the scope of the present disclosure include primates (e.g., humans, monkeys, and apes, including monkey species such as the genus Macaca (e.g., Macaca fascicularis, and / or cynomolgus monkeys, such as rhesus monkeys (Macaca mulatta)) and baboons (Papio ursinus), as well as marmosets (Callithrix), squirrel monkeys (Saimiri), and tamarins (Saguinus), and chimpanzees (Pan pantheons). The subject of the present invention includes, but is not limited to, any member of the subphylum Chordata, including monkey species such as Neisseria troglodytes, rodents (e.g., mice, rats, guinea pigs), lagomorphs (e.g., rabbits, hares), bovines (e.g., cows), ovines (e.g., sheep), caprines (e.g., goats), porcines (e.g., pigs), equines (e.g., horses), canines (e.g., dogs), felines (e.g., cats), birds (e.g., chickens, turkeys, ducks, geese, canaries, companion birds such as budgerigars), marine mammals (e.g., dolphins, whales), reptiles (e.g., snakes, frogs, lizards), and fish. A preferred subject is a human, particularly a human in which an immune response against a Neisseria organism needs to be elicited. However, it will be understood that the foregoing term does not imply that symptoms are present.

[0052] "Pharmaceutically acceptable vehicle" refers to a solid or liquid filler, diluent or encapsulating substance that can be safely used in local or systemic administration to animals, preferably mammals, including humans. Representative pharmaceutically acceptable vehicles include any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonicity agents, absorption retardants, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, and the like materials and combinations thereof, as known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329). Except where any conventional carrier is incompatible with the active ingredient, its use in the pharmaceutical composition is contemplated. The term "vehicle" typically refers to a natural or synthetic organic or inorganic ingredient with which the active ingredient is combined to facilitate application.

[0053] The term "pharmaceutically acceptable salts", as used herein, is a broad term and should be given its ordinary and customary meaning to those skilled in the art (not limited to any special or customized meaning), and refers to, but is not limited to, derivatives of the disclosed compounds in which the parent compound is modified by converting an existing acid or base moiety into its salt form (e.g., by reacting a free base group with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. Representative acid addition salts include acetate, adipate, arginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxyethanesulfonate, and the like. Representative salts include salts of the following: acid salts, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations, including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. The pharma- ceutically acceptable salts of the present disclosure include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids.The pharma- ceutically acceptable salts of the present disclosure can be synthesized from parent compounds containing a basic or acidic moiety by conventional chemical methods. In general, such salts can be prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of an appropriate base or acid in water or an organic solvent, or in a mixture of the two, generally in non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile. Lists of suitable salts can be found in Remington's Pharmaceutical Sciences, 17.sup.th.ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418; Pharmaceutical Salts: Properties, Selection, and Use, P.H. Stahl and C.G. Wermuth (eds.), Wiley-VCH, 2008; and Berge et al., Journal of Pharmaceutical Science, 66, 1-19 (1977), each of which is incorporated herein by its entirety.

[0054] "Polypeptide," "peptide," "protein," and "proteinaceous molecule" are used interchangeably herein to refer to a molecule that comprises or consists of a polymer of amino acid residues, and to variants and synthetic analogues thereof. Thus, these terms apply to amino acid polymers in which one or more amino acid residues is a synthetic, non-naturally occurring amino acid, e.g., a chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.

[0055] The term "resistant" is intended to refer to a strain of bacteria that has shown insensitivity to one or more known antibacterial drugs. An insensitive strain is one in which the MIC of a given compound or class of compounds for that strain is shifted to a higher number than the corresponding sensitive strain. For example, it may refer to a strain that is insensitive to β-lactam antibiotics, insensitive to one or more fluoroquinolones, and / or insensitive to one or more other antibiotics (i.e., antibiotics other than β-lactams and fluoroquinolones). In certain embodiments, the term "resistant" may refer to one in which the MIC of a given compound or class of compounds for that strain is shifted to a significantly higher number than for the corresponding sensitive strain. A bacterial strain may be said to be resistant to a given antibiotic if it is inhibited in vitro by a concentration of this drug that is associated with a high probability of treatment failure. The bacterial strain may be resistant to one or more fluoroquinolone antibiotics, such as one or more antibiotics selected from levofloxacin, enoxacin, fleroxacin, lomefloxacin, nadifloxacin, norfloxacin, rufloxacin, balofloxacin, grepafloxacin, pazufloxacin, sparfloxacin, temafloxacin, tosufloxacin, besifloxacin, clinafloxacin, garenoxacin, gemifloxacin, gatifloxacin, sitafloxacin, trovafloxacin, prulifloxacin, ciprofloxacin, pefloxacin, moxifloxacin, ofloxacin, delafloxacin, zavofloxacin, abalofloxacin, and finafloxacin.

[0056] As used herein, the term "sugar unit" refers to a single monosaccharide residue / moiety.

[0057] As used herein, the term "terminal sugar unit" refers to a sugar unit that is linked either to no (in the case of a monosaccharide) or to only one adjacent sugar unit (in the case of an oligosaccharide).

[0058] The terms "treat", "treating" or "treatment" (or grammatical equivalents) mean that the severity of a subject's condition is alleviated, at least partially improved or ameliorated, and / or some alleviation, reduction or diminution of at least one clinical symptom is achieved, and / or there is a delay in the progression of the condition.

[0059] Each embodiment described in this specification applies mutatis mutandis to each and every embodiment unless otherwise specified.

[0060] 2. Abbreviation In this application the following abbreviations are used: CFU = colony forming units CMP = cytidine monophosphate CRM197 = a non-toxic mutant of diphtheria toxin EDC = 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Gal = Galactose Glc = glucose GlcNAc = N-acetylglucosamine Hep = Heptose hr.=hour HRP = horseradish peroxidase Ig = immunoglobulin IgG = immunoglobulin G IgG1 = immunoglobulin G1 IgG2a = immunoglobulin G2a IgG2b = immunoglobulin G2b IgG3=immunoglobulin G3 IgG4 = immunoglobulin G4 IgM = immunoglobulin M kDa = kilodaltons KDO = 3-deoxy-D-manno-oct-2-ulosonic acid, ketodeoxyoctonic acid, or ketodeoxyoctulosonate LNnT = lacto-N-neotetraose LOS = Lipooligosaccharide MAb = monoclonal antibody MDR = multidrug resistant MIC = minimum inhibitory concentration Neu5Ac = N-acetylneuraminic acid NCS = N-chlorosuccinimide NHS = N-hydroxysuccinimide NTHi = non-typeable Haemophilus influenzae OMV=outer membrane vesicle PMN=polymorphonuclear leukocytes TMB = 3,3',5,5-tetramethylbenzidine

[0061] 3. Chimeric molecules containing Neisseria oligosaccharides The present disclosure is based, in part, on the determination that Neisserial LOS α-2,3-sialyltransferase (Lst) is present in the bacterial cytoplasm and that, in addition to transferring Neu5Ac from CMP-Neu5Ac to the terminus of the LOS, Lst can transfer KDO to the LOS in place of Neu5Ac in both N. gonorrhoeae and N. meningitidis. In particular, the inventors have found that KDO is expressed as a terminal LOS structure in in vivo samples from infected women and that anti-KDO monoclonal antibody 6E4 can mediate opsonophagocytic killing of N. gonorrhoeae. Thus, it is proposed that KDO expressed on the gonococcal LOS represents a novel oligosaccharide antigen for the development of immunogenic compositions against Neisserial organisms.

[0062] Thus, the present disclosure relates to chimeric molecules for use in eliciting an immune response against Neisserial organisms, such as Neisseria gonorrhoeae or Neisseria meningitidis, and methods of using such chimeric molecules to treat and / or inhibit Neisserial infections, for example, by eliciting an immune response that targets Neisserial oligosaccharide antigens.

[0063] The chimeric molecule comprises a carrier and an oligosaccharide comprising an oligosaccharide sequence corresponding to Neisseria LOS, the oligosaccharide sequence comprising KDO as a terminal sugar unit. In certain embodiments, the chimeric molecule has formula (I):

number

[0064] The oligosaccharide sequence preferably comprises KDO linked to Gal, suitably the Gal is further linked to GlcNAc. In an exemplary embodiment, KDO is linked to Gal via an α(2→3) linkage and / or Gal is linked to GlcNAc via a β(1→4) linkage. In a non-limiting example of this type, the oligosaccharide sequence comprises KDOα(2→3)Gal or KDOα(2→3)Galβ(1→4)GlcNAc.

[0065] Suitable carriers are known in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th ed., Mack Easton, Pa. (1990)) and can include, for example, proteins, peptides, lipids, polymers, dendrimers, virosomes, virus-like particles (VLPs), outer membrane proteins (OMPs), or combinations thereof, which do not themselves exhibit specific antigenic properties but can support an immunogenic response in the host to the oligosaccharides (antigens) of the present disclosure displayed on the surface of the carrier.

[0066] In certain embodiments, the carrier is a protein carrier, including but not limited to bacterial toxins, toxins, exotoxins, and non-toxic derivatives thereof, such as tetanus toxin, tetanus toxin fragment C, diphtheria toxin, CRM (non-toxic diphtheria toxin mutant), such as CRM 197, cholera toxin, Staphylococcus aureus exotoxin or toxin, Escherichia coli heat labile enterotoxin, Pseudomonas aeruginosa exotoxin A, and recombinantly produced, genetically detoxified variants thereof; bacterial outer membrane proteins, such as Neisseria meningitidis serogroup B outer membrane protein complex (OMPC), outer membrane class 3 porin (rPorB), and other porins; keyhole limpet hemocyanin (KLH), core protein of Hepatitis B virus, thyroglobulin, These include albumins such as bovine serum albumin (BSA), human serum albumin (HSA), ovalbumin, etc.; pneumococcal surface protein A (PspA), pneumococcal adhesion protein (PsaA); purified protein derivative of tuberculin (PPD); transferrin binding protein, polyamino acids such as poly(lysine:giutamic acid); peptidyl agonists of TLR-5 (e.g., flagellin from motile bacteria such as Listeria); and derivatives and / or combinations of the above carriers. Preferred carriers for use in humans include tetanus toxoid, CRM197, and OMPC.

[0067] Depending on the type of bond between the linker and the carrier and the structural nature of the carrier and the oligosaccharide, the carrier may exhibit, on average, for example, between 1 and 500, between 1 and 100, between 1 and 20, or between 3 and 9 oligosaccharide units on its surface.

[0068] Methods for conjugating oligosaccharides to carriers, such as carrier proteins, are conventional, and one of skill in the art can use conventional methods to make conjugates according to the present disclosure. Guidance is also available in a variety of disclosures, including, for example, U.S. Patent Nos. 4,356,170; 4,619,828; 5,153,312; 5,422,427; and 5,445,817; as well as various printed and online Pierce Protein Crosslinking Guides and Catalogs (Thermo Fisher, Rockford, IL).

[0069] Following conjugation of the oligosaccharides to the carrier, the chimeric molecule can be purified by a variety of techniques well known to those skilled in the art. One goal of the purification step is to remove unconjugated oligosaccharides from the conjugation reaction product composition. An exemplary method for purification includes ultrafiltration in the presence of ammonium sulfate, as described in U.S. Pat. No. 6,146,902. Alternatively, the chimeric molecule can be purified from unreacted oligosaccharides and carrier by any number of standard techniques, including, for example, size exclusion chromatography, density gradient centrifugation, hydrophobic interaction chromatography, or ammonium sulfate fractionation (see, for example, Anderson et al., 1986. J. Immunol. 137:1181-1186; and Jennings & Lugowski, 1981. J. Immunol. 127:1011-1018). The composition and purity of the chimeric molecule can be determined by GLC-MS and MALDI-TOF spectroscopy.

[0070] In certain embodiments, the oligosaccharide antigens of the present disclosure are conjugated to CRM197, a commercially available protein carrier used in many FDA-approved vaccines. CRM conjugates have the advantage of being easier to synthesize, purify, and characterize than other FDA-approved carriers such as OMPC. Oligosaccharide antigens can be conjugated to CRMs via thiol-bromoacetyl conjugation chemistry. CRM activation can be achieved by reacting lysine side chains with NHS esters of bromoacetic acid, as described in the aforementioned U.S. Patent Publication No. 2007-0134762. CRMs can be functionalized with 10 to 20 bromoacetyl groups (n=10-20) per protein prior to conjugation. Conjugation can be performed at pH=9 to avoid aggregation of CRM. Careful monitoring of pH must be used to ensure complete CRM reaction with NHS-bromoacetic acid while minimizing background hydrolysis of CRM. Activated CRM can be purified by size-exclusion chromatography prior to conjugation. Antigen-CRM conjugates can be synthesized by reacting a thiol-terminated carbohydrate antigen with a bromoacetamide-activated CRM.

[0071] CRM conjugates can be purified via size exclusion chromatography to remove and recover any unreacted carbohydrate. MBTH (specific for GlcNAc residues) and Bradford assays can be used to determine carbohydrate:protein ratios and protein content, respectively, as previously described (see, e.g., Manzi et al., 1995. Curr. Prot. Mol. Biol., section 17.9.1 (Suppl. 32)). In certain embodiments, a minimum oligosaccharide content of about 10% by weight for each conjugate can be produced. Typically, conjugates can contain from about 3 to about 20 antigens per protein carrier.

[0072] In other embodiments, the oligosaccharide antigens may be coupled to one or more supports suitable for the development of diagnostic assays, including ELISA and microarrays. Exemplary supports for use in such assays include bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), biotin, labels, glass slides or gold surfaces. As an example, synthetic carbohydrate antigens may be coupled to BSA by a thiol-maleimide coupling procedure (Figure 5B). Maleimide-BSA contains between 15 and 20 maleimide groups per protein (n=15-20). Thus, oligosaccharide antigens can be coupled to maleimide-functionalized BSA, whereby a 20-fold molar excess of antigen is coupled to commercially available lmject in maleimide coupling buffer (Pierce). TM React with maleimide BSA (Pierce). To avoid hydrolysis of the maleimide group during conjugation, conjugation may be performed at pH=7.2.

[0073] The BSA conjugate can be purified via size exclusion chromatography to remove unreacted carbohydrates and recover. Characterization by phenol-sulfuric acid and Bradford assays can be performed along with MALDI-MS to provide information on the carbohydrate content and valency of the conjugate. In a preferred embodiment, the conjugate comprises a minimum oligosaccharide content of about 10% by weight per BSA conjugate and more than 8 antigen copies per conjugate.

[0074] In a related aspect, the disclosure relates to oligosaccharides comprising an oligosaccharide sequence corresponding to Neisserial LOS, the oligosaccharide sequence comprising KDO as a terminal sugar unit, as broadly described above and elsewhere herein.

[0075] 4. Antigen-binding molecules The oligosaccharides disclosed herein are structural mimetics of Neisserial LOS and, according to the present disclosure, are useful for producing antigen binding molecules that specifically bind to Neisserial organisms, including pathogenic Neisserial organisms such as Neisseria gonorrhoeae and Neisseria meningitidis.

[0076] Those skilled in the art will appreciate the well-developed knowledge based on antigen binding proteins, for example as described in Abbas et al., Cellular and Molecular Immunology, 6th ed., WB Saunders Company (2010) or Murphey et al., Janeway's Immunobiology, 8th ed., Garland Science (2011). In some embodiments, the antigen binding protein that specifically binds to Neisseria LOS is an antibody. Antibodies include intact antibodies and antigen-binding fragments thereof, as described in the definition section. Antibodies may include complete antibody molecules (including polyclonal, monoclonal, chimeric, humanized, or human forms with full-length heavy and / or light chains) or may include antigen-binding fragments thereof. Antibody fragments include F(ab')2, Fab, Fab', Fv, Fc, and Fd fragments, and can be incorporated into single domain antibodies, single chain antibodies, maxibodies, minibodies, intrabodies, bispecific antibodies, triabodies, tetrabodies, v-NAR, and bis-scFv (see, e.g., Hollinger and Hudson, 2005, Nature Biotechnology, 23, 9, 1126-1136). Also included are antibody polypeptides such as those disclosed in U.S. Patent No. 6,703,199, including fibronectin polypeptide monobodies. Other antibody polypeptides are disclosed in U.S. Patent Application Publication No. 2005 / 0238646, which are single chain polypeptides.

[0077] Numerous methods of preparing antibodies against an antigen of interest are known in the art. For example, monoclonal antibodies that specifically bind to Neisseria LOS can be made using conventional hybridoma techniques, often based on the spirit of Kohler, G. et al. (1975, "Continuous Cultures Of Fused Cells Secreting Antibody Of Predefined Specificity", Nature 256:495-497) or modifications thereof. Typically, monoclonal antibodies are developed in non-human species, such as mice. Generally, mice or rats are used for immunization, although other animals can be used. Antibodies can be produced by immunizing mice with an immunogenic amount of an immunogen, in this case a chimeric molecule of the present disclosure. The immunogen can be administered multiple times, at periodic intervals, such as every other week or every week, or in such a way as to maintain the survival of the animal.

[0078] To monitor the antibody response, a small biological sample (e.g., blood) can be obtained from the animal and tested for antibody titers to the immunogen. The spleen and / or several large lymph nodes can be removed and dissociated into single cells. If necessary, the spleen cells can be screened (after removal of non-specifically adherent cells) by applying a cell suspension to a plate or well coated with the antigen. B cells expressing membrane-bound immunoglobulin specific for the antigen will bind to the plate and will not be washed away with the remainder of the suspension. The resulting B cells, or all dissociated spleen cells, can then be fused with myeloma cells (e.g., X63-Ag8.653, and those from the Salk Institute, Cell Distribution Center, San Diego, Calif.). Polyethylene glycol (PEG) can be used to fuse the spleen or lymphocytes with the myeloma cells to form hybridomas. The hybridomas are then cultured in a selection medium (e.g., hypoxanthine, aminopterin, thymidine medium, also known as "HAT medium"). The resulting hybridomas are then plated by limiting dilution and assayed for the production of antibodies that specifically bind the immunogen, e.g., using FACS (fluorescence activated cell sorting) or immunohistochemistry (IHC) screening. Selected monoclonal antibody-secreting hybridomas are then cultured either in vitro (e.g., in tissue culture bottles or hollow fiber reactors) or in vivo (e.g., as ascites in mice).

[0079] As another alternative to cell fusion techniques, Epstein-Barr Virus (EBV) immortalized B cells can be used to produce monoclonal antibodies that specifically bind to Neisseria LOS. Hybridomas are expanded and subcloned as necessary, and supernatants are assayed for anti-immunogen activity by conventional assay procedures (e.g., FACS, IHC, radioimmunoassay, enzyme immunoassay, fluorescent immunoassay, etc.).

[0080] Accordingly, the present disclosure further contemplates a method of producing an antigen binding molecule that specifically binds to a Neisseria organism, the method comprising: (1) immunizing an animal with a chimeric molecule disclosed herein; (2) detecting B cells from the animal that specifically bind to the chimeric molecule, or a Neisseria organism or its LOS; and (3) isolating the antigen binding molecule expressed by the B cells.

[0081] The present disclosure also encompasses antigen-binding molecules produced by such methods, as well as derivatives thereof. Also encompassed are cells, including hybridomas, capable of producing the antigen-binding molecules of the present disclosure, and methods for producing antigen-binding molecules from those cells. In certain embodiments, the antigen-binding molecules produced by the methods and cells of the present disclosure are preferably neutralizing antigen-binding molecules.

[0082] Chimeric and humanized antibodies are also contemplated. In some embodiments, a humanized monoclonal antibody comprises a variable domain of a mouse antibody (or all or part of its antigen-binding site) and a constant domain derived from a human antibody. Alternatively, a humanized antibody fragment may comprise an antigen-binding site of a mouse monoclonal antibody and a variable domain fragment (lacking the antigen-binding site) derived from a human antibody. Procedures for producing engineered monoclonal antibodies include those described in Riechmann et al., 1988, Nature 332:323; Liu et al., 1987, Proc. Nat. Acad. Sci. USA 84:3439; Larrick et al., 1989, Bio / Technology 7:934; and Winter et al., 1993, TIPS 14:139. In one embodiment, the chimeric antibody is a CDR-grafted antibody. Techniques for humanizing antibodies are described in, for example, U.S. Patent Nos. 5,869,619; 5,225,539; 5,821,337; 5,859,205; 6,881,557; Padlan et al., 1995, FASEB J. 9:133-39; Tamura et al., 2000, J. Immunol. 164:1432-41; Zhang, W. et al., Molecular Immunology 42(12):1445-1451, 2005; Hwang W. et al., Methods 36(1):35-42, 2005; Dall'Acqua WF et al., Methods 36(1):43-60, 2005; and Clark, M., Immunology Today 21(8):397-402, 2000.

[0083] The antibody of the present disclosure can also be a fully human monoclonal antibody. Fully human monoclonal antibodies can be produced by any number of techniques familiar to those skilled in the art. Such methods include, but are not limited to, Epstein-Barr Virus (EBV) transformation of human peripheral blood cells (including, for example, B lymphocytes), in vitro immunization of human B cells, fusion of spleen cells from immunized transgenic mice carrying inserted human immunoglobulin genes, isolation from human immunoglobulin V-region phage library, or other procedures known in the art and based on the disclosure herein.

[0084] Procedures have been developed to generate human monoclonal antibodies in non-human animals. For example, mice have been prepared in which one or more endogenous immunoglobulin genes have been inactivated by various means. Human immunoglobulin genes have been introduced into mice to replace the inactivated mouse genes. In this technique, elements of human heavy and light chain loci are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of endogenous heavy and light chain loci (see also Bruggemann et al., 1997, Curr. Opin. Biotechnol. 8:455-58). For example, the human immunoglobulin transgenes can be minigene constructs, or transgenes on yeast artificial chromosomes, which undergo B cell-specific DNA rearrangement and hypermutation in mouse lymphoid tissues.

[0085] The antibodies produced in the animal incorporate human immunoglobulin polypeptide chains encoded by the human genetic material introduced into the animal. In one embodiment, a non-human animal, such as a transgenic mouse, is immunized with a chimeric polypeptide or conjugate immunogen of interest.

[0086] Examples of techniques relating to the production and use of transgenic animals to produce human or partially human antibodies can be found in U.S. Pat. Nos. 5,814,318, 5,569,825, and 5,545,806; Davis et al., Production of human antibodies from transgenic mice in Lo, ed. Antibody Engineering: Methods and Protocols, Humana Press, NJ:191-200 (2003); Kellermann et al., Curr Opin Biotechnol. 2002, 13:593-97; Russell et al., Infect Immun. 2000, 68:1820-26; Gallo et al., Eur J. Immun. 2000, 30:534-40; Davis et al., Cancer Metastasis Rev. 1999, 18:421-25; Green, J Immunol Methods 2002, 13:593-97; 1999,231:11-23, Jakobovits,Advanced Drug Delivery Reviews 1998,31:33-42, Green et al.,J Exp Med. 1998,188:483-95, Jakobovits A,Exp.Opin.Invest.Drugs 1998,7:607-14, Tsuda et al.,Genomics 1997,42:413-21, Mendez et al.,Nat.Genet. 1997,15:146-56, Jakobovits,Curr Biol. 1994,4:761-63, Arbones et al.,Immunity 1994,1:247-60, Green et al.,Nat.Genet. 1994,7:13-21, Jakobovits et al.,Nature 1993,362:255-58, Jakobovits et al., Proc Natl Acad Sci USA 1993,90:2551-55. Chen, J., M. et al., Int. Immunol. 1993,5:647-656, Choi et al., Nature Genetics 1993,4:117-23, Fishwild et al., Nature Biotech.1996,14:845-51, Harding et al., 1995,Annals of the New York Academy of Sciences, Lonberg et al., 1994,Nature 368:856-59, Lonberg, 1994,Transgenic Approaches to Human Monoclonal Antibodies in Handbook of Experimental Pharmacology 113:49-101, Lonberg et al., Int. Rev. Immunol. 1995,13:65-93, Neuberger, Nature Biotech. 1996,14:826, Taylor et al., Nucleic Acids Research 1992,20:6287-95, Taylor et al., Int. Immunol. 1994,6:579-91, Tomizuka et al., Nature Genetics 1997,16:133-43, Tomizuka et al., Proc Natl. Acad Sci USA 2000, 97:722-27, Tuaillon et al., Proc Natl Acad Sci USA 1993, 90:3720-24, and Tuaillon et al., J. Immunol. 1994, 152:2912-20.; Lonberg et al., Nature 1994, 368:856; Taylor et al., Int. Immunol. 1994, 6:579; U.S. Patent No. 5,877,397; Bruggemann et al., Curr. Opin. Biotechnol. 1997 8:455-58; Jakobovits et al., Ann. NY Acad. Sci. 1995, 764:525-35. Additionally, protocols using the XenoMouse® (Abgenix, now Amgen) are described, for example, in US05 / 0118643 and WO05 / 694879, WO98 / 24838, WO00 / 76310, and U.S. Patent No. 7,064,244.

[0087] Alternatively, the chimeric molecules disclosed herein can be used to screen antigen-binding molecules from an antigen-binding molecule library. For example, the chimeric molecules of the present disclosure can be immobilized on a solid support (e.g., silica gel, resin, derivatized plastic film, glass beads, cotton, plastic beads, polystyrene beads, alumina gel, or polysaccharide, magnetic beads) and screened for binding to antigen-binding molecules. Alternatively, antigen-binding molecules can be immobilized on a solid support and screened for binding to the chimeric molecules. Any screening assay, such as panning assay, ELISA, surface plasmon resonance, or other antigen-binding molecule screening assays known in the art, can be used to screen for antigen-binding molecules that bind to the chimeric molecules disclosed herein, particularly their oligosaccharide components. The antigen-binding molecule library to be screened can be a commercially available library, a library generated in vitro, or a library obtained by identifying and cloning or isolating antibodies from individuals infected with Neisseria organisms. In certain embodiments, the antigen-binding molecule library is generated from subjects infected with Neisseria organisms. The antigen-binding molecule library can be generated according to methods known in the art. In certain embodiments, libraries are generated by cloning antibodies and using them in a phage or phagemid display library.

[0088] The present disclosure further encompasses fragments of anti-Neisserial LOS antibodies. Such fragments may consist entirely of antibody-derived sequences or may contain additional sequences. Examples of antigen-binding fragments include Fab, F(ab')2, single chain antibodies, bispecific antibodies, triabodies, tetrabodies, and domain antibodies. Other examples are provided by Lunde et al., Biochem. Soc. Trans. 2002, 30:500-06.

[0089] Single chain antibodies can be formed by linking heavy and light chain variable domain (Fv region) fragments via an amino acid bridge (short peptide linker), resulting in a single polypeptide chain. Such single chain Fvs (scFvs) have been prepared by fusing DNA encoding a peptide linker between DNA encoding two variable domain polypeptides (VL and VH). The resulting polypeptide can fold back on itself to form an antigen-binding monomer or can form multimers (e.g., dimers, trimers, or tetramers) depending on the length of the flexible linker between the two variable domains (Kortt et al., Prot. Eng. 1997, 10:423; Kortt et al., Biomol. Eng. 2001, 18:95-108). By combining different VL- and VH-containing polypeptides, multimeric scFvs that bind different epitopes can be formed (Kriangkum et al., Biomol. Eng. 2001, 18:31-40). Techniques that have been developed for the production of single chain antibodies include those described in U.S. Pat. No. 4,946,778; Bird, Science 1988, 242:423; Huston et al., Proc. Natl. Acad. Sci. USA 1988, 85:5879; Ward et al., Nature 1989, 334:544, de Graaf et al., Methods Mol. Biol. 2002, 178:379-87.

[0090] Antigen-binding fragments derived from antibodies can also be obtained, for example, by proteolytic hydrolysis of antibodies, for example, by pepsin or papain digestion of whole antibodies by conventional methods. By way of example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment designated F(ab')2. This fragment can be further cleaved using a thiol reducing agent to generate a 3.5S Fab' monovalent fragment. Optionally, the cleavage reaction can be carried out using a blocking group for the sulfhydryl groups resulting from cleavage of disulfide bonds. Alternatively, enzymatic cleavage using papain directly produces two monovalent Fab fragments and an Fc fragment. These methods are described, for example, in Goldenberg, U.S. Patent No. 4,331,647; Nisonoff et al., Arch. Biochem. Biophys. 1960, 89:230; Porter, Biochem. J. 1959, 73:119; Edelman et al., in Methods in Enzymology 1:422 (Academic Press 1967); and Andrews, SM and Titus, JA in Current Protocols in Immunology (Coligan JE et al., editors), John Wiley & Sons, New York (2003), pages 2.8.1-2.8.10 and 2.10A.1-2.10A.5. Other methods for cleaving antibodies, such as separating heavy chains to form monovalent light-heavy chain fragments (Fd), further cleavage of the fragments, or other enzymatic, chemical, or genetic techniques, can also be used, so long as the fragments bind to the antigen recognized by the intact antibody.

[0091] Another form of an antibody fragment is a peptide comprising one or more complementarity determining regions (CDRs) of an antibody. CDRs can be obtained by constructing a polynucleotide encoding the CDRs of interest. Such polynucleotides are prepared, for example, by using the polymerase chain reaction to synthesize the variable region using mRNA from an antibody-producing cell as a template (see, for example, Larrick et al., Methods: A Companion to Methods in Enzymology 2:106, 1991; Courtenay-Luck, "Genetic Manipulation of Monoclonal Antibodies", in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al. (eds.), p. 166 (Cambridge University Press 1995); and Ward et al., "Genetic Manipulation and Expression of Antibodies", in Monoclonal Antibodies: Principles and Applications, Birch et al. (eds.), p. 137 (Wiley-Liss, Inc. 1995). Antibody fragments may further comprise at least one variable region domain of an antibody described herein. Thus, for example, the V region domain may be monomeric and may comprise at least 10 -7 It may be a VL and VH domain that are capable of independently binding to an ectodomain polypeptide or complex of interest with an affinity of M or less.

[0092] The variable region domain may be any naturally occurring variable domain or an engineered version thereof. By engineered version is meant a variable region domain created using recombinant DNA engineering techniques. Such engineered versions include, for example, those created from a specific antibody variable region by inserting, deleting, or changing the amino acid sequence of the specific antibody. Particular examples include engineered variable region domains that contain at least one CDR and optionally one or more framework amino acids from a first antibody and the remainder of the variable region domain from a second antibody.

[0093] The variable region domain may be covalently linked at the C-terminal amino acid to at least one other antibody domain or fragment thereof. Thus, for example, a VH domain present in the variable region domain may be linked to an immunoglobulin CH1 domain or fragment thereof. Similarly, a VL domain may be linked to a CK domain or fragment thereof. Thus, for example, an antibody may be a Fab fragment in which the antigen-binding domain contains the relevant VH and VL domains covalently linked at their C-terminus to the CH1 and CK domains, respectively. The CH1 domain may be extended with further amino acids, for example to provide a hinge region or part of a hinge region domain as found in a Fab' fragment, or to provide further domains, such as antibody CH2 and CH3 domains.

[0094] Antigen binding molecules identified by the methods described herein can be tested for neutralizing activity and lack of autoreactivity using biological assays known in the art or described herein, in some embodiments, antigen binding molecules elicited or identified using the chimeric molecules disclosed herein neutralize Neisseria organisms.

[0095] In some embodiments, antigen binding molecules induced or identified using the chimeric molecules disclosed herein can be used to monitor the effectiveness of treatment and / or disease progression.

[0096] Antigen-binding molecules induced or identified using the chimeric molecules can be used in diagnostic immunoassays to detect the presence of Neisserial organisms in biological samples, passive immunotherapy, and the generation of anti-idiotypic antigen-binding molecules. In addition, the ability of the antigen-binding molecules to neutralize Neisserial organisms and the specificity of the antigen-binding molecules for Neisserial LOS can be tested prior to using the antigen-binding molecules in passive immunotherapy.

[0097] Specific binding of the antigen-binding molecule to the chimeric molecule disclosed herein, particularly its oligosaccharide component, and cross-reactivity with other antigens can be assessed by any method known in the art. Immunoassays that can be used to analyze specific binding and cross-reactivity include, but are not limited to, competitive and non-competitive assay systems using techniques such as Western blot, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassay, immunoprecipitation assay, precipitation reaction, gel diffusion precipitation reaction, immunodiffusion assay, agglutination assay, complement fixation assay, immunoradiometric assay, fluorescent immunoassay, protein A immunoassay, to name a few. Such assays are routine and well known in the art (see, for example, Ausubel et al., eds., 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York).

[0098] In some embodiments, the antigen-binding molecules disclosed herein are used in immunodetection methods to bind, purify, remove, quantify, and generally detect Neisserial organisms, such as Neisseria gonorrhoeae or Neisseria meningitidis. Some immunodetection methods include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), immunoradiometric assays, fluoroimmunoassays, chemiluminescent assays, bioluminescent assays, and Western blots. Immunodetection methods also include methods for detecting and quantifying the amount of Neisserial organisms or their associated components (e.g., LOS) in a sample, as well as detecting and quantifying any immune complexes formed during the binding process. In a non-limiting example, a sample suspected of containing Neisserial organisms is obtained from a patient, the sample is contacted with an antigen-binding molecule that specifically binds to the oligosaccharide component of the chimeric molecule disclosed herein, and the amount of immune complexes formed under specific conditions is then detected and quantified. For antigen detection, the biological sample analyzed can be any sample suspected of containing Neisserial organisms, such as tissue sections or specimens, homogenized tissue extracts, biological fluids, including biological fluids and / or tissues associated with infection with Neisserial organisms.

[0099] Contacting a selected biological sample with an antigen-binding molecule under appropriate conditions and for a sufficient time to allow the formation of immune complexes (primary immune complexes) generally involves simply adding the antigen-binding molecule to the sample and incubating the mixture for a sufficient time for the antigen-binding molecule to form immune complexes, i.e., bind to Neisseria organisms or associated components (e.g., their LOS) present in the sample. After this, the sample antigen-binding molecule composition, such as tissue sections, ELISA plates, dot blots or Western blot analyses, is generally washed to remove non-specifically bound antigen-binding molecule species, allowing only the antigen-binding molecules specifically bound within the primary immune complexes to be detected.

[0100] 5. Composition The chimeric molecules and antigen-binding molecules disclosed herein ("active agents") may be included in pharmaceutical compositions (including therapeutic and prophylactic formulations) and are typically combined with one or more pharma- ceutically acceptable vehicles and, optionally, other therapeutic ingredients (e.g., antibacterial agents, immunogens, etc.).

[0101] A pharma- ceutically acceptable vehicle can be used to enhance the delivery of the active agent and / or control the duration of action. A controlled release formulation can be achieved by using a polymer to complex or absorb the active agent and / or adjuvant. Controlled delivery can be achieved by selecting an appropriate polymer (e.g., polyester, polyamino acid, polyvinyl, pyrrolidone, ethylene vinyl acetate, methyl cellulose, carboxymethyl cellulose, or protamine sulfate) and the concentration of the polymer, as well as the method of incorporation to control release. Another possible method to control the duration of action by a controlled release formulation is to incorporate the active agent disclosed herein into particles of polymeric materials such as polyester, polyamino acid, hydrogel, poly(lactic acid) or ethylene vinyl acetate copolymer. Alternatively, instead of incorporating these drugs into polymer particles, these materials can be encapsulated in microcapsules, for example prepared by interfacial polymerization, e.g., hydroxymethylcellulose or gelatin-microcapsules and poly(methyl methacrylate)-microcapsules, respectively, or in colloidal drug delivery systems, such as liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules, or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, supra.

[0102] Adjuvants can be included in the compositions. Exemplary adjuvants and / or adjuvant combinations include aluminum salts such as aluminum phosphate and aluminum hydroxide (alum) (e.g., Alhydrogel TMRIBI is a mixture of three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate, and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene / Tween® 80 emulsion, and any of the three components, MPL, TDM, and CWS, can be used alone or in combination of two or more. Toll-like receptor (TLR) agonists, including, for example, agonists of TLR-1 (e.g., triacyl lipopeptides); agonists of TLR-2 (e.g., peptidoglycan from gram-positive bacteria such as streptococci and staphylococci; lipoteichoic acid); agonists of TLR-3 (e.g., double-stranded RNA and analogs thereof such as poly 1:C); agonists of TLR-4 (e.g., lipopolysaccharide (endotoxin) from gram-negative bacteria such as Salmonella and E. coli); agonists of TLR-5 (e.g., lipopolysaccharide (endotoxin) from gram-negative bacteria such as Salmonella and E. coli); agonists of TLR-6 (e.g., flagellin of motile bacteria such as Listeria monocytogenes and certain lipids (diacyl lipopeptides)); agonists of TLR-7 (single-stranded RNA (ssRNA) genomes of viruses such as influenza, measles, and mumps; and small synthetic antiviral molecules of guanosine bases such as loxoribine, ssRNA, and their analogues); agonists of TLR-8 (e.g., binds ssRNA); agonists of TLR-9 (e.g., unmethylated CpG and its analogues in the DNA of pathogens); agonists of TLR-10 (function undefined) and TLR-11- (e.g., binds proteins expressed by some infectious protozoa (Apicomplexa). Specific Toll-like receptor agonists include monophosphoryl lipid A (MPL.RTM.), 3-de-O-acylated monophosphoryl lipid A (3 D-MPL), OM-174 (E. coli lipid A derivative); OM triacyl lipid A derivative, and OM triacyl lipid A derivative, as well as MPL.RTM.-SE, RC-529 (Dynavax Technologies), AS01 (liposomes+MPL+QS21), AS02 (oil-in-water PL+QS-21), and AS04 (alum+MPL) (GlaxoSmith Kline, Pa.), CpG-oligodeoxynucleotides (ODN) containing immunostimulatory CpG motifs, double-stranded RNA, polyinosinic:polycytidylic acid (Poly I:C), and other oligonucleotides or polynucleotides, optionally encapsulated in liposomes, other MPL or lipid A based formulations and combinations thereof; oil-in-water emulsions, including AS03 (GlaxoSmith Kline, Pa.), MF-59 (microfluidic detergent stabilized squalene oil-in-water emulsion; Novartis), Montanide ISA-51 VG (stabilized water-in-oil emulsion), and Montanide ISA-720 (stabilized water / squalene; Seppic Pharmaceuticals, Fairfield, NJ); cholera toxin B subunit; saponins such as Quil A or QS21, Quillaja Saponaria HPLC purified non-toxic fraction from the bark of Molina (STIMULON.TM. (Antigenics, Inc., Lexington, Mass.), saponin-based adjuvants such as immune stimulating complexes (ISCOMs; structured complexes of saponins and lipids), and the ISCOMATRIX.TM. and AbISCO.RTM.-100 and -300 series of adjuvants (Isconova AB, Uppsala, Sweden); QS21 and 3 D-MPL, and oil-in-water emulsions as disclosed in U.S. Patent No. 2006 / 0073171; stearyl tyrosine (ST) and its amide analogs; virus-like particles (VLPs) and reconstituted influenza virosomes (ILPs); complete Freund's adjuvant (CFA); incomplete Freund's adjuvant (IFA); Escherichia coli heat-labeled enterotoxin (LT); cytokines such as IL-2, IL-12, GM-CSF, Flt3, B7.1, and immune adjuvants such as mast cell (MC) activators such as mast cell activating compound 48 / 80 (C48 / 80); water insoluble inorganic salts; liposomes including those made from DNPC / Chol and DC Chol; micelles; squalene; squalane; muramyl dipeptides such as N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetyl-muramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1',2'-dipalmitoyl-n-glycero-3-hydroxyphosphoryl as found in U.S. Pat. No. 4,606,918; SAF-1 (Syntex); AS05 (GlaxoSmith Kline, Pa.); and combinations thereof.

[0103] In some embodiments, adjuvant efficacy may be enhanced by combining multiple adjuvants as described above, including combining various delivery systems with immune enhancers to form multi-component adjuvants that have the potential to act synergistically to enhance antigen-specific immune responses in vivo. Exemplary immune enhancers include the adjuvants described above, including, for example, MPL and synthetic derivatives, MDP and derivatives, oligonucleotides (CpGetc), ds RNA, alternative pathogen-associated molecular patterns (PAMPs) (E. coli heat-labile enterotoxin; flagellin, saponins (QS-21, etc.), small molecule immune enhancers (SMIPs, e.g., resiquimod [R848]), cytokines, and chemokines.

[0104] The compositions of the present disclosure can be formulated according to known methods for preparing pharma- ceutically useful compositions, whereby these materials or their functional derivatives are mixed with a pharma- ceutically acceptable vehicle (or diluent). Suitable vehicles and their formulations, including other human proteins, such as human serum albumin, are described, for example, in Remington's Pharmaceutical Sciences, supra. To form a pharma- ceutically acceptable composition suitable for effective administration, such compositions contain an effective amount of the above-mentioned compounds together with an appropriate amount of a protein carrier and / or vehicle.

[0105] Typically, the composition may be prepared as an injectable, either as a liquid solution or suspension; or as a solid form suitable for solution or suspension in a liquid vehicle prior to injection. Aqueous compositions for parenteral administration may, for example, comprise a solution of the active agent dissolved or suspended in a pharma- ceutically acceptable vehicle or diluent, preferably a predominantly aqueous vehicle. Pharmaceutically acceptable vehicles or diluents may include water, saline, including neutral saline buffered with phosphate, Tris, glycerol, ethanol, and the like. Aqueous compositions may be formulated as sterile pyrogen-free buffered saline or phosphate-containing solutions, which may or may not contain preservatives. Suitable preservatives include, for example, benzyl alcohol, parabens, thimerosal, chlorobutanol, and benzalkonium chloride. Aqueous solutions are preferably approximately isotonic, and the isotonicity may be adjusted with agents such as sodium tartrate, sodium chloride, propylene glycol, and sodium phosphate. Additionally, auxiliary substances necessary to approximate physiological conditions, including pH adjusting and buffering agents, isotonicity agents, wetting or emulsifying agents, pH buffering substances, etc., including sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, and the like, can be included with the vehicles described herein.

[0106] These compositions can be sterilized by conventional sterilization methods or can be sterilized by filtration. The resulting aqueous solutions can be packaged for use as is or can be lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration. The preparation of such pharmaceutical compositions is within the skill of the art and can be guided by standard reference texts such as Remington's Pharmaceutical Science, supra.

[0107] The composition can be formulated in solid or liquid form for oral delivery.For solid compositions, non-toxic and / or pharmaceutically acceptable solid protein carriers can include, for example, pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, etc.For oral administration, pharmaceutically acceptable non-toxic compositions can be formed by incorporating any of the commonly used excipients, including the protein carriers listed above, with a unit dose of active ingredient, i.e., one or more active agents disclosed herein.

[0108] The concentration of the active agent in the pharmaceutical composition can vary widely, i.e., from less than, typically or at least about 0.1% by weight, to 20% by weight or more, up to 50% by weight or more, and is selected primarily by fluid volume, viscosity, etc., and according to the particular mode of administration selected. A human unit dose form of the active agent is typically contained in a pharmaceutical composition that includes a human unit dose of an acceptable protein carrier, preferably an aqueous protein carrier, and is administered in a volume of fluid known to those skilled in the art to be used in administering such compositions to humans, adjusted according to commonly understood principles for the particular subject being treated. In certain embodiments, the unit dose of the active agent is provided in a suitable volume of aqueous solution, such as 0.1 mL to 3 mL, preferably 0.2 mL to 2 mL.

[0109] Such pharmaceutical compositions can be administered to a subject by various mucosal administration modes, including by oral, rectal, intranasal, pulmonary, or transdermal delivery, or by topical delivery to other surfaces. Optionally, the active agent can be administered by non-mucosal routes, including intramuscular, subcutaneous, intravenous, intraatrial, intraarticular, intraperitoneal, or parenteral routes. Alternatively, the active agent can be administered ex vivo by direct exposure to cells, tissues, or organs derived from the subject. In certain embodiments, the administration route includes parenteral administration (e.g., intradermal, intramuscular, or subcutaneous delivery), ocular administration, oral administration, intranasal administration, pulmonary administration, intravaginal administration, and rectal administration. The formulation of the pharmaceutical composition can include liquid formulations, such as sterile solutions, suspensions, or emulsions, for parenteral, subcutaneous, intradermal, intramuscular, intravenous, or ocular administration (e.g., injection administration). Formulations of pharmaceutical compositions may also include liquid formulations (e.g., topical or ingestible formulations) for ophthalmic, oral, nasal, anal, and vaginal administration, including solutions, suspensions, syrups, or elixirs. Pharmaceutical compositions can be lyophilized prior to delivery and reconstituted prior to administration.

[0110] The pharmaceutical compositions of the present disclosure are typically sterile and stable under the conditions of manufacture, storage and use. Sterile solutions can be prepared by incorporating the required amount of the conjugate in a suitable solvent with one or a combination of ingredients listed herein as needed, followed by sterilization by filtration. In general, dispersions are prepared by incorporating the active agent and / or other biologically active agent into a sterile vehicle containing a basic dispersion medium and the required other ingredients listed herein. In the case of sterile powders, preparation methods include vacuum drying and freeze-drying, which yields a powder of the conjugate and any additional desired ingredients from its solution that has previously been sterile filtered. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.

[0111] 6.Treatment method The compositions of the present disclosure can be administered to a subject infected with or at risk of developing an infection by a Neisseria organism. According to various treatment methods of the present disclosure, the active agent can be delivered to the subject in a manner consistent with conventional methodologies associated with the management of the Neisseria infection for which treatment or prevention is sought. According to the disclosure herein, a prophylactically or therapeutically effective amount of the active agent is administered to a subject in need of such treatment for a time and under conditions sufficient to prevent, inhibit, and / or ameliorate the Neisseria infection or one or more symptoms thereof. For example, non-limiting symptoms of gonococcal infection include pain in the lower abdomen, pelvis, testicles, or vagina, painful urination, abnormal vaginal discharge, penile discharge, or increased vaginal discharge, fever, frequent urinary urgency, irregular menstruation, pus, or sore throat. Representative symptoms of meningococcal infection include fever and chills, fatigue, vomiting, cold hands and feet, severe aches or pains in the muscles, joints, chest, or abdomen, rapid respiratory diarrhea, and a dark purple rash in the later stages.

[0112] When the composition includes a chimeric molecule of the present disclosure as an active agent, the treatment may be given in a single dose schedule, or preferably a repeated dose schedule, where the primary treatment course may be from one to ten separate doses, followed by subsequent doses at subsequent time intervals required to maintain and / or enhance the response, for example, at one to four months, a second dose, and subsequent doses several months later, if necessary. Examples of suitable treatment schedules include: (i) 0, 1 month and 6 months, (ii) 0, 7 days and 1 month, (iii) 0 and 1 month, (iv) 0 and 6 months, or other schedules sufficient to induce the desired response expected to alleviate disease symptoms or reduce the severity of the disease.

[0113] The amount effective to induce an immune response or provide protective immunity will depend on a variety of factors, including the oligosaccharide antigen component of the chimeric molecule, the associated carrier, the inclusion and nature of any adjuvants, the mode of administration, the weight and general health of the patient, and the judgment of the prescribing physician. By way of example, for a primary immunization (i.e., prophylactic administration), a dose of from about 1.0 μg to about 5,000 μg (e.g., 1.0 μg, 2.0 μg, 2.5 μg, 3.0 μg, 4.0 μg, 4.5 μg, 5.0 μg, 7.0 μg, 8.0 μg, 9.0 μg, 10.0 μg, 11.0 μg, 12.0 μg, 13.0 μg, 14.0 μg, 15.0 μg, 16.0 μg, 17.0 μg, 18.0 μg, 19.0 μg, 20.0 μg, 21.0 μg, 22.0 μg, 23.0 μg, 24.0 μg, 25.0 μg, 26.0 μg, 27.0 μg, 28.0 μg, 29.0 μg, 30.0 μg, 31.0 μg, 32.0 μg, 33.0 μg, 34.0 μg, 35.0 μg, 36.0 μg, 37.0 μg, 38.0 μg, 39.0 μg, 40.0 μg, 41.0 μg, 42.0 μg, 43.0 μg, 44.0 μg, 45.0 μg, 46.0 μg, 47.0 μg, 48.0 μg, 49.0 μ The oligosaccharide antigen may be administered in an amount of 5 μg, 10 μg, 12.5 μg, 15 μg, 17.5 μg, 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, 50 μg, 75 μg, 100 μg, 250 μg, 500 μg, 750 μg, 1,000 μg, 1,500 μg, 2,000 μg, 2,500 μg, 3,000 μg, 3,500 μg, 4,000 μg, 4,500 μg, or 5,000 μg. The actual dose administered to a subject is often determined according to an appropriate amount per kg of the subject's body weight. For example, an effective amount may be from about 0.1 μg to about 5 μg / kg body weight.

[0114] Following the primary administration, the patient may be administered a booster regimen over several weeks to months, depending on the patient's response and condition, by measuring specific antibody titers and / or T cell activity in the patient's blood, followed by administration of about 1.0 to about 1,000 of oligosaccharide antigen (e.g., 1.0 μg, 2.0 μg, 2.5 μg, 3.0 μg, 4.0 μg, 4.5 μg, 5.0 μg, 7.5 μg, 10 μg, 12.5 μg, 16.5 μg, 18.5 μg, 20.5 μg, 22.5 μg, 24.5 μg, 26.5 μg, 28.5 μg, 29.5 μg, 30.5 μg, 31.5 μg, 32.5 μg, 33.5 μg, 34.5 μg, 35.5 μg, 36.5 μg, 37.5 μg, 38.5 μg, 39.5 μg, 40.5 μg, 41.5 μg, 42.5 μg, 43.5 μg, 44.5 μg, 45.5 μg, 46.5 μg, 47.5 μg, 48.5 μg, 49.5 μg, 50.5 μg, 51.5 μg, 52.5 μg, 53.5 μg, 54.5 μg, 55.5 μg, 56.5 μg, 57.5 μg, 58.5 μg, 59.5 μg, 60.5 μg, 61.5 μg, 62.5 μg, 63.5 μg, 64.5 μg, 65.5 μg, 66.5 μg, 67. , 15 μg, 17.5 μg, 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, 50 μg, 75 μg, 100 μg, 250 μg, 500 μg, 750 μg, 1,000 μg, 1,500 μg, 2,000 μg, 2,500 μg, 3,500 μg, 4,000 μg, 4,500 μg, or 5,000 μg).

[0115] The active agent may optionally be administered in combination with a supplementary or complementary pharmacologic active agent. For example, the active agent may be administered in combination with an antibacterial agent useful in the treatment of Neisseria infections, representative examples of which include aminoglycosides (e.g., amikacin, gentamicin, kanamycin, neomycin, netilmicin, streptomycin, tobramycin, and paromycin); ansamycins (e.g., geldanamycin and herbimycin); carbacephems (e.g., loracarbef); carbapenems (e.g., ertapenem, doripenem, imipenem / cilastatin, and meropenem); e.g., cefadroxil, cefazolin, cephalothin (c Cephalosporins (1st generation), including efalotin / cefalothin, cephalexin; Cephalosporins (2nd generation), including e.g., cefaclor, cefamandole, cefoxitin, cefprozil, and cefuroxime; Cephalosporins (3rd generation), including e.g., cefixime, cefdinir, cefditoren, cefperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, and cefdinir; Cephalosporins (4th generation), including e.g., cefepime generation); glycopeptides (e.g., vancomycin and teicoplanin); macrolides (e.g., azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, troleandomycin, telithromycin, and spectinomycin); monobactams (e.g., aztreonam); penicillins (e.g., amosillin, ampicillin, azlocillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, nafcillin, penicillin, piperacillin, and ticarcillin); polypeptides (e.g., bacitracin, polymyxin B, and colistin); quinolones (e.g., ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin, ofloxacin, and trovafloxacin);Sulfonamides (e.g., mafenide, prontosil, sulfacetamide, sulfamethizole, sulfanilimide, sulfasalazine, sulfisoxazole, trimethoprim, trimethoprim-sulfamethoxazole (co-trimoxazole, TMP-SMX)); tetracyclines (e.g., tetracycline (demeclocycline, doxycycline, minocycline, oxytetracycline, and tetracycline); aminocoumarins (e.g., novobiocin, albamycin, coumamycin, and clorobiocin); oxazolidinones (e.g., linezolid and AZD2563); lipopeptides (e.g., daptomycin); streptogramins (e.g., quinupristin / dalfopristin); glycylcyclines (e.g., tigecycline; lantibiotics, such as type A lantibiotics (such as nisin, subtilin, epidermin, mutacin II, mutacin I and III) and type B lantibiotics (such as mersacidin, actagardin, and cinnamycin). Other suitable antibiotics useful as adjuncts include one or more antibiotics selected from arsphenamine, chloramphenicol, clindamycin, lincomycin, ethambutol, fosfomycin, fusidic acid, furazolidone, isoniazid, linezolid, metronidazole, mupirocin, nitrofurantoin, platensimycin, pyrazinamide, quinupristin / dalfopristin, rifampin / rifampicin, and tinidazole;

[0116] Thus, the active agents disclosed herein include penicillin, cloxacillin, dicloxacillin, methicillin, nafcillin, oxacillin, ampicillin, amoxicillin, bacampicillin, capreomycin, cycloserine, azlocillin, carbenicillin, mezlocillin, piperacillin, ticarcillin, azithromycin, clarithromycin, clindamycin, erythromycin, lincomycin, demeclocycline, doxycycline, ethambutol, ethionamide, minocycline, oxytetracycline, tetracycline, quinolones, cinoxacin, nalidixic acid, fluoroquinolones (e.g., levofloxacin, moxafloxacin, and gatifloxacin, ciprofloxacin, enoxacin, grepafloxacin), kanamycin, levofloxacin, It may be used in combination with one or more antibiotics selected from lomefloxacin, norfloxacin, ofloxacin, p-aminosalicylic acid, sparfloxacin, trovafloxacin, bacitracin, colistin, polymyxin B, sulfonamides, trimethoprim-sulfamethoxazole, co-amoxiclav, cephalothin, cefuroxime, ceftriaxone, vancomycin, gentamicin, amikacin, metronidazole, chloramphenicol, streptomycin, nitrofurantoin, cotrimoxazole, rifamycin and its derivatives (e.g., rifampicin, rifabutin, rifapentine, etc.), isoniazid, pyrazinamide, kirromycin, thiostrepton, microcosin, fusidic acid, thiolactomycin, and fosmidomycin. Other suitable antimicrobial adjuvants may be selected from those shown in the table below: DU-6859, erythromycin stinplate, oritavancin, telavancin, dalbavancin, ceftobipro medcaril, tebipenem pivoxil, iclaprim, OPT-80, ceftaroline fosamil, RX-3341, cethromycin, TD-1792, EDP-420, RX-1741, MK-2764, nemonoxacin, phlopristin plus linopristin, tomopenem, ramoplanin, linezolid, cefditoren pivoxil, ertapenem, gemifloxacin, daptomycin, telithromycin, and tigecycline.

[0117] In some embodiments, the chimeric molecules of the present disclosure are used in combination with one or more immunogens derived from Neisserial antigens, including, by way of representative examples, AniA, BamA, CssA, CssB, CssC, CtrA, CtrB, CtrC, CtrD, CtrE, CtrF, ExbB, GNA1030, GNA1162, GNA1220, GNA1870 (fHbp), GNA1946, GNA2001, GNA2091, GNA2132 (NHBA), GNA33, GNA992, LctP, LpdA, LptD, MsrAB, NadA, NGO2054, NGO2139 (MetQ), NspA, OpaD, OpcA, PilC1, PilQ, PorA P1, TamA, TBP2, and TbpA. In certain embodiments, the immunogen is selected from AniA, MsrAB, MetQ, and NhbA.

[0118] Typical subjects intended for treatment with the compositions and methods of the present disclosure include humans, as well as non-human primates and other animals. To identify subjects for prevention or treatment with the methods of the present disclosure, acceptable screening methods are used to determine risk factors associated with the targeted or suspected condition (e.g., cough disease) or to determine the current status of an existing disease or condition in the subject, as discussed herein. These screening methods include, for example, conventional work-ups to determine environmental, familial, occupational, and other such risk factors that may be associated with the targeted or suspected disease or condition, as well as diagnostic methods such as various ELISA and other immunoassay methods that are available and well known in the art to detect and / or characterize disease-related markers. These and other routine methods allow clinicians to select patients who require treatment using the methods and pharmaceutical compositions of the present disclosure. In accordance with these methods and principles, conjugates and / or other biologically active agents can be administered in accordance with the teachings herein as an independent prophylactic or therapeutic program, or as a follow-up, adjunct or coordinating therapeutic regimen to other treatments, including surgery, vaccination, immunotherapy, hormonal therapy, cell, tissue or organ transplantation, and the like.

[0119] Administration of the active agents of the present disclosure may be for either prophylactic or therapeutic purposes. When provided prophylactically, the active agent is provided prior to any symptoms. Prophylactic administration of the active agent serves to prevent or ameliorate any subsequent infection. When provided therapeutically, the conjugate is provided at (or shortly thereafter) the onset of symptoms of disease or infection. Thus, the active agents of the present disclosure can be provided prior to anticipated exposure to a Neisseria organism, after exposure or suspected exposure to the bacteria, or after the actual onset of infection to attenuate the anticipated severity, duration, or extent of infection and / or associated disease symptoms.

[0120] Methods of using the active agents, and related compositions and methods of the disclosure, are useful for increasing resistance to and / or preventing, ameliorating, and / or treating infections and diseases caused by Neisserial organisms, in animal hosts, and in other in vitro applications. Compositions containing the chimeric molecules of the disclosure can be used for active immunization for the prevention of infectious diseases, and for the preparation of immune antibodies.

[0121] 7. Kit The present disclosure also includes kits for carrying out the methods of the present disclosure. These reagents and kits may vary and typically contain any one or more of the active agents or compositions disclosed herein for use in the prevention and treatment of Neisseria infections in vertebrate subjects, and optionally include instructions or means for administering them. Kits for diagnostic use are also provided. In some embodiments, these kits include a container or formulation containing one or more of the chimeric molecules described herein. In one example, the components are formulated into a pharmaceutical formulation for delivery to a subject. The chimeric molecules of the present disclosure are optionally contained in a bulk dispenser container or unit or multi-unit dosage form. Optional distribution means can be provided. The packaging material optionally includes a label or instructions indicating for what processing purpose and / or how the active agent packaged therewith can be used. The pharmaceutical composition or components can be provided in any suitable form (e.g., liquid or lyophilized). The kit can further include a solvent for resuspending or dissolving the lyophilized active agent.

[0122] So that the present disclosure can be readily understood and practically practiced, certain preferred embodiments are illustrated by the following non-limiting examples. EXAMPLES

[0123] Example 1 Evidence that KDO is expressed on the surface of Neisseria gonorrhoeae Blast analysis showed that the gonococcal sialyltransferase Lst belongs to glycosyltransferase superfamily 52 and is homologous to the sialyltransferase LsgB in NTHi (27% identity at the amino acid level, e-value = 6e-31). To determine whether N. gonococcus can transport KDO as the terminal monosaccharide of LOS, as well as NTHi, whole-cell ELISA was performed using the LOS terminal KDO-specific monoclonal antibody (MAb) 6E4 on three well-characterized gonococcal strains 1291, MS11, and F62, and their lst knockout mutants. All three wild-type strains were KDO positive, but all lst mutants of these strains did not react with MAb 6E4 (Figure 2A), indicating that Lst is required for KDO transfer to LOS. Consistent with previous studies, 6E4 cannot detect the basal KDO residue common to all LOS structures. Further analysis detected KDO (Figure 2B) in 19 of 20 gonococcal clinical isolates from patients with mucosal or disseminated gonococcal infection. Variable presentation of KDO on the gonococcal LOS could be due to the absence of a terminal galactose acceptor, when LgtA is switched off, or via competition, when another glycosyltransferase, LgtD, undergoes a phase change "on" and transfers GalNAc in competition with Lst / CMP-KDO for the terminal galactose acceptor. Transfer of KDO to the LOS was also examined in N. meningitidis, which, unlike N. gonococci, can synthesize CMP-Neu5Ac via SiaB. ELISA results in Figure 2C show the absence of KDO in N. meningitidis strain wild type and lst mutants. However, an MC58 siaB mutant unable to synthesize CMP-Neu5Ac was recognized by MAb 6E4, indicating the presence of KDO. These data indicate that there is a competition between CMP-Neu5Ac and the CMP-KDO substrate for Lst-mediated transfer of the terminal sugar to LOS and that mutations in N. meningitidis that eliminate CMP-Neu5Ac synthesis are sufficient to allow KDO addition to LOS.To define the terminal KDO structure on LOS, MAb 6E4 Western blots were performed with LOS purified from MC58, 1291, and their LOS glycosyltransferase (lst and lgtB) mutants. LgtB is a β1,4-galactosyltransferase that transfers CMP-Neu5Ac and CMP-KDO acceptor galactose (Gal) to N-acetylglucosamine (GlcNAc). As shown in Figure 2E, purified LOS from 1291 wild type and MC58 siaB mutant were 6E4 positive, whereas 1291 and MC58 lst and lgtB mutants were all 6E4 negative. LOS from MC58 wild type was only slightly 6E4 positive, indicating the presence of a small amount of KDO-incorporated termini on LOS.

[0124] To determine whether incremental addition of CMP-Neu5Ac to the growth medium could be completed with CMP-KDO for Lst addition to the LOS, thereby decreasing MAb 6E4 binding, N. gonorrhoeae strain 1291 was grown in 0 μg / mL, 30 μg / mL, and 100 μg / mL CMP-Neu5Ac and whole-cell ELISA was performed. The results, shown in Figure 2D, show that there was a dose-dependent inhibition of 6E4 MAb binding to whole cells beginning with 30 μg / mL CMP-Neu5Ac and increasing at 100 μg / mL. These data suggest that CMP-Neu5Ac taken up from the medium competes with endogenous CMP-KDO as a substrate for Lst activity.

[0125] Example 2 The KDO antigen is expressed in vivo To examine whether N. gonococci can incorporate KDO as a terminal glycan of LOS during in vivo infection, we performed confocal microscopy using MAb 6E4 on cervical swabs from patients with documented gonococcal infection. Figure 3 shows that in vivo, terminal KDO epitopes are present in N. gonococci on cervical epithelial cells. This suggests that free CMP-Neu5Ac in the cervical environment was not sufficient to inhibit Lst from incorporating KDO into LOS.

[0126] Example 3 MAb 6e4 mediates opsonophagocytic killing by targeting the KDO epitope To examine whether surface-expressed KDO in N. gonococci could be a vaccine target, MAb 6E4 was used in an opsonophagocytic killing assay against N. gonococcus strain 1291. Dose-dependent opsonophagocytic killing was observed from 0.65 to 10 μg / mL MAb 6E4, with 5 μg / mL MAb 6E4 inducing 54% killing and 10 μg / mL inducing 71% killing compared to untreated controls (Figure 4). These data suggest that antibodies targeting KDO can mediate bacterial killing via opsonophagocytic activity and that the LOS-terminal KDO epitope in N. gonococci is a potential vaccine target.

[0127] Example 4 KDO monosaccharides displayed on the carrier protein CRM-197 elicit immune responses in mice KDO spacer synthesis Commercially available mannose is converted to a protected KDO derivative in two steps according to the method described by Feng et al. (Feng et al., 2015, Org Lett. 17(10):2388-91). The protected KDO is then activated to a glycosyl fluoride according to the procedure of L'Heureux et al. (2010, J Org Chem. 75(10):3401-11). Commercially available 5-amino-1-pentanol has a protected amine group according to the method described by Varala et al. (2007, J Iran Chem Soc. 4:370-374). Glycosidation between the activated KDO and the spacer is carried out using the procedure described by Boltje et al. (2012, J Am Chem Soc. 134, 14255-14262). Deprotection then provides the KDO spacer compound for conjugation. FIG. 5 shows the steps of KDO spacer chemical synthesis.

[0128] Characterization of KDO spacers All small molecule chemicals synthesized in this study will be characterized using standard spectroscopic techniques, specifically nuclear magnetic resonance (NMR, both 1H and 13C), mass spectrometry, and infrared spectroscopy. Evidence that the correct glycosidic bond orientation was formed when KDO was conjugated with the spacer was determined using NMR spectroscopy, specifically the chemical shifts of the protons attached to the anomeric carbon and carbon-3, as described by Kong et al. (Kong et al., 2016, Nat Chem. 8:242-249).

[0129] KDO binding to CRM197 and BSA (or other carrier) The chemical structure of KDO conjugation to CRM197 is shown in Figure 6A. CRM197 (or BSA and other carriers) in 0.3 M NaHCO3 (1 mg / mL) was added to NCS KDO conjugate (200 molar equivalents) in 0.3 M Na2CO3, pH 9 to a final volume of 1 mL. The resulting mixture was gently shaken at 20 °C for 18 h. The reaction products were analyzed using SDS-PAGE and Western blot. In the EDC-NHS reaction, CRM197 and NH2-conjugated KDO were incubated in EDC (25 mM) and NHS (25 mM) (GE, EDC-NHS coupling kit) for 30 min at room temperature. The resulting conjugation reaction was purified by desalting columns (Bio-Spin TM The glycoconjugate CRM197 was analyzed and confirmed using SDS-PAGE Western blot of 6E4 and MS as shown in Figure 6B.

[0130] Mouse antigenicity studies Analysis of mouse antigenicity studies was performed as previously published (Jen et al., 2015, Front Immunol. 10:137). To evaluate the immunogenicity of the glycoconjugate constructs based on KDO monosaccharides, 10 mice were each administered Alhydrogel TMMice were immunized with 100 μg of KDO glycoconjugates or natural carrier protein (CRM197) in a 1:1 formulation with adjuvant. Two booster injections were administered on days 21 and 28 after the first injection, and a final bleed was scheduled for day 42. All animals remained healthy throughout the study period, and no adverse events were observed. Terminal bleed sera were analyzed for anti-KDO immune responses via ELISA assay.

[0131] Analysis of immune responses To examine the immunogenicity of CRM-KDO, 10 mice were immunized with Alhydrogel TM Mice were immunized with CRM-KDO in adjuvant. Sera were evaluated by ELISA. Plate wells were coated with 100 ng of BSA-KDO glycoconjugate protein in 100 μL of coating buffer (0.5 M carbonate / bicarbonate buffer, pH 9.6) for 1 h at room temperature. All ELISAs were performed with mouse pre-immune serum or CRM-KDO immune serum and secondary antibody, polyclonal anti-mouse Ig HRP (Dako). Substrate TMB (3,3',5,5-tetramethylbenzidine) solution (Thermofisher Scientific) was used according to the manufacturer's instructions. An equal volume of 1 N hydrochloric acid was added to stop the reaction. Absorbance was read at 450 nm in a TECAN Model Infinite 200 Pro plate reader. The results of the ELISA using BSA-KDO as coating antigen show KDO-specific immune responses in mice immunized with CRM-KDO with titers of more than 25,000, as shown in Table 1. [Table 1]

[0132] Example 5 Synthesis of KDO antigen (I) Chemical synthesis of KDO disaccharide and trisaccharide antigens KDOα(2,3)Gal spacer synthesis Commercially available mannose is converted to a protected KDO derivative in two steps according to the method described by the Feng group (Feng et al., 2015, supra). The protected KDO is then activated to a glycosyl fluoride following the procedure of L'Heureux et al. (2010, supra). Commercially available galactose is converted to a Gal spacer compound using standard carbohydrate chemistry developed in-house. Glycosidation between the activated KDO and the Gal spacer is achieved using a method adapted from the literature (Kong et al., 2016, supra). Deprotection then yields the KDOα(2,3)Gal spacer compound for conjugation. Figure 7 shows the steps of the KDOα(2,3)Gal spacer chemical synthesis.

[0133] KDOα(2,3)Galβ(1,4)GlcNAc spacer synthesis Commercially available mannose is converted to a protected KDO derivative in two steps according to the method described by the Feng group (Feng et al., 2015, supra). The protected KDO is then activated to a glycosyl fluoride according to the procedure of L'Heureux et al. (2010, supra). Commercially available lactulose is converted to LacNAc (Galβ(1,4)GlcNAc) using the method described by Agoston et al. (2016, Tet Lett. 57:2595-2597). By a series of standard carbohydrate protecting group manipulations developed in-house, LacNAc is converted to the Galβ(1,4)GlcNAc spacer compound. Glycosidation between the activated KDO and the Galβ(1,4)GlcNAc spacer is achieved using a method adapted from the literature (Kong et al., 2016, supra). Deprotection then provides the KDOα(2,3)Galβ(1,4)GlcNAc spacer compound for conjugation. Figure 8 shows the steps in the KDOα(2,3)Galβ(1,4)GlcNAc spacer chemical synthesis.

[0134] Characterization of disaccharide and trisaccharide KDO spacers (i.e., post-synthesis) All small molecule chemicals synthesized in this study will be characterized using standard spectroscopic techniques, specifically nuclear magnetic resonance (NMR, both 1H and 13C), mass spectrometry, and infrared spectroscopy. Evidence that the correct glycosidic bond orientation was formed when KDO was conjugated with Gal was determined using NMR spectroscopy, specifically the chemical shifts of the protons attached to the anomeric carbon and carbon-3, as described by Kong et al. (2016, supra).

[0135] Di- and trisaccharide KDO antigen binding to CRM197 and BSA CRM197 (or BSA or other carrier) in 0.3 M NaHCO3 (1 mg / mL) was added to the NCS KDO conjugate (200 molar equivalents) in 0.3 M Na2CO3, pH 9 to a final volume of 1 mL. The resulting mixture was gently shaken at 20°C for 18 hours. The reaction products were analyzed using SDS-PAGE and Western blot. In the EDC-NHS reaction, CRM197 and NH2-conjugated KDO were incubated in EDC (25 mM) and NHS (25 mM) (GE, EDC-NHS coupling kit) for 30 minutes at room temperature. The resulting conjugation reaction was purified by desalting on a desalting column (Bio-Spin TM The mixture was stopped by passage through a 100-ml buffer (Bio-Rad) to remove excess unbound glycans and the buffer was exchanged into PBS. SDS-PAGE Western blots of 6E4 and MS were used to analyze and characterize the glycoconjugate CRM197.

[0136] Mouse antigenicity studies To evaluate the immunogenicity of the KDO di- and trisaccharide glycoconjugate constructs, 10 mice were each administered 25 μg, 50 μg, or 100 μg of KDO glycoconjugates or a natural carrier protein (CRM197) in Alhydrogel TM The animals are immunized 1:1 with adjuvant. Two booster injections are administered on days 21 and 28 after the first injection, with a final bleed scheduled for day 42. All animals remained healthy throughout the study and no adverse events were observed. Terminal bleed sera are analyzed for anti-KDO immune responses via ELISA assay.

[0137] Analysis of immune responses Analysis of immune responses is performed as previously published (Jen et al., 2015, supra). ELISA is used to determine antibody titers and isotypes of sera produced using plates coated with either BSA-KDO antigen or N. gonorrhoeae whole cells. Plate wells are coated with 100 ng of BSA-KDO glycoconjugates and BSA protein in 100 μL of coating buffer (0.5 M carbonate / bicarbonate buffer, pH 9.6) for 1 hour at room temperature or overnight at 4° C. to test KDO-specific immune responses. All ELISAs are performed using mouse pre-immune serum or CRM-KDO immune serum and secondary antibodies, polyclonal anti-mouse Ig HRP (Dako) or IgG1, IgG2a, IgG2b, IgG3, or IgM HRP (Thermofisher Scientific). Substrate TMB (3,3',5,5-tetramethylbenzidine) solution (Thermofisher Scientific) is used according to the manufacturer's instructions. The reaction is stopped by adding an equal volume of 1N hydrochloric acid. The absorbance is read at 450 nm on a TECAN Model Infinite 200 Pro plate reader.

[0138] Example 6 Synthesis of KDO antigen(II): Chemoenzymatic synthesis of KDO disaccharide and trisaccharide antigens. As shown in Figure 9, the Gal or LacNAc derivatives from Example 5 are deprotected with free NCS or NH2 at the end of the linker allowing EDC-NHS coupling with CRM197. After successfully attaching Gal and LacNAc compounds to CRM197 via the linker, the next step of the chemoenzymatic method is to activate KDO in situ to the CMP-KDO derivative using the enzyme KdsB (CMP-KDO synthase from E. coli) based on (Sugai et al., 1995, Bioorg Med Chem. 3(3):313-20), which can then be transferred to the sugar ligand already attached to CRM197 by the glycosyltransferase Lst (sialyltransferase from Neisseria) base on (Gilbert et al., 1996, J Biol Chem. 271:28271-6).

[0139] Example 7 Synthesis of KDO antigen (III) in vivo synthesis of KDO antigen Overexpressing KDO in gonococcal OMVs as a vaccine candidate As shown in Figure 10A, CMP-KDO synthase (KdsB) and CMP-KDO transferase (Lst) are transferred to the gonococcal chromosome on an IPTG-inducible expression construct. The level of terminal KDO expression on LOS is determined by ELISA using MAb 6E4 and MS. Naturally secreted outer membrane vesicles (OMVs) are isolated / purified by two previously described methods: native purification by ultracentrifugation (Semchenko et al., 2017, Infect Immun. 85(2)) and deoxycholic acid (DOC) detergent extraction (Fredriksen et al., 1991, NIPH Ann. 14(2):67-79). These methods result in different OMV compositions and yields, and both are investigated. Native gonococcal OMVs are isolated from 6-h inoculated cultures (OD600 approx. 0.8) by brief centrifugation (5,000×g) followed by filtration of the supernatant (0.22 μm filter). The filtrate is then centrifuged (100,000×g, 1 h, 4° C.) and the pellet containing the OMVs is washed three times with PBS and then solubilized in PBS containing 0.2% SDS. For detergent extraction, the 6-h inoculated cultures are incubated for 30 min at room temperature in 0.1 M Tris-HCl, pH 8.6, containing 10 mM EDTA and 0.5% DOC, then centrifuged (20,000×g; 30 min; 4° C.). The supernatant is ultracentrifuged (125,000×g; 2 h; 4° C.) and the OMV pellet is resuspended in 50 mM Tris-HCl, pH 8.6, 2 mM EDTA, 1.2% DOC, 20% sucrose and a second ultracentrifugation step is performed. The OMVs are then homogenized in 30% sucrose. All purified OMVs are analyzed by SDS-PAGE and protein concentrations are measured using the BCA protein assay.

[0140] A group of 10 BALB / c mice was randomly assigned to receive Alhydrogel on days 0, 21, and 28. TM (aluminum hydroxide, InvivoGen). Terminal bleeds are performed on day 42 according to standard procedures. Control mice are immunized with 5 μg of native OMVs or Alhydrogel TMImmunize mice with PBS sham using 0.1% DMSO. A pre-bleed of each mouse is collected 4 days prior to immunization. The antibody titer and isotype of raised sera are then determined using ELISA using plates coated with either the immunizing antigen or N. gonorrhoeae whole cells.

[0141] To evaluate KDO-OMVs as vaccine candidates, we overexpress KDO in engineered, virulence-reduced, and controlled blebbing E. coli strains. A strain expressing heterologous oligosaccharides with reduced endotoxins and elevated OMVs of E. coli (C600ΔwaaOBΔlppΔmsbBattλ::ParaB-lppΔompA:pGb3) was generated and may be used to deliver carbohydrate vaccine antigens (Paton et al., 2015, Infect Immun. 83(9):3526-33). To enable expression of the terminal KDO tetrasaccharide in E. coli, plasmids pLNnT (LgtE, LgtA and LgtB expression constructs) and pBAD Lst are transformed into this E. coli strain. As shown in FIG. 10A, LgtE, LgtA, LgtB and Lst encode β-1,4-galactosyltransferase, β-1,3-N-acetylglucosaminyltransferase, β-1,4-galactosyltransferase and sialyltransferase, respectively, with different receptor specificities. Whole cell 6E4 ELISA as shown in FIG. 10B and MS was used to confirm KDO expression on LOS in E. coli blebbing strains. Blebbing of this strain is induced by supplementing the growth culture with 0.2% glucose, and OMVs are isolated by centrifugation (3,900×g, 4° C. for 10 min, then again for 15 min to remove all cells). The resulting supernatant is centrifuged at 150,000×g for 3 h at 4° C. The pellet containing OMVs is washed with PBS, followed by a second centrifugation at 120,000 g for 30 min at 4° C. The OMVs in the pellet are resuspended in PBS for analysis by SDS-PAGE and BCA protein assay.

[0142] Mouse antigenicity testing of purified OMVs was performed as previously published (Jen et al., 2015, supra). Groups of 10 BALB / c mice were incubated at days 0, 21, and 28 with Alhydrogel TM (Aluminum hydroxide, InvivoGen). A terminal bleed is taken on day 42 according to standard procedures. Control mice are immunized with 5 μg of OMVs in a 100% aqueous solution of native E. coli OMVs and Alhydrogel TM Immunize mice with PBS sham using 0.1% DMSO. A pre-bleed of each mouse is collected 4 days prior to immunization. ELISA is then used to determine the antibody titer and isotype of serum produced using plates coated with either the immunizing antigen or N. gonorrhoeae whole cells.

[0143] Alternatively, the same terminal KDO tetrasaccharide antigen shown in Figure 10 can be produced in E. coli and then minicells or bacterial ghosts, instead of OMVs, can be prepared to display the KDO carbohydrate vaccine antigen (Paton et al., 2015, Infect Immun. 83(9):3526-33). A further in vivo strategy for heterologous expression of the KDO antigens shown in Figure 10 is to express the appropriate glycosyltransferase to generate the desired KDO antigen that can be conjugated to a carrier protein in vivo using established methods (Ravenscroft et al., 2019, Glycobiology. 29(9):669-680).

[0144] Example 8 Human serum titers against KDO antigen To assess whether normal human serum (NHS) contains anti-KDO antibodies, an ELISA was performed for KDO antigen probing with NHS. 100 ng of purified gonococcal LOS with KDO (wild-type LOS, see structure in Figure 1) and without KDO (Lst mutant LOS, see structure in Figure 1) was used as coating antigen on the ELISA plate. Ten individual NHS samples from Sigma-Aldrich (catalog number S1-M) and a commercially available pooled NHS were used to test for the presence of antibodies targeting KDO. KDO-specific titers for each NHS sample were measured by subtracting the readings of LOS without KDO (Lst mutant LOS) from the readings of LOS with KDO (wild-type LOS). The endpoint was determined by a positive reading above the secondary antibody only control. The ELISA results showed that the human serum had measurable anti-KDO titers, as shown in Table 2. 6E4 was used as a positive control. [Table 2]

[0145] Materials and Methods Bacterial strains and growth conditions Gonococcal strains 1291, 1291lst, MS11, MS11lst, F62, and F62lst (Mubaiwa et al., 2017, Sci Rep 7:5693) were grown on GC agar (Oxoid) or GC broth with 1% IsoVitaleX and meningococcal strains MC58, MC58lst (Mubaiwa et al., 2017, Sci Rep 7:5693) and MC58siaB (Chopra et al., submitted) were cultured on Brain Heart Infusion (BHI) 1% agar and 10% Levinthal basic medium at 37°C and 5% CO2. Twenty clinical isolates of N. gonorrhoeae from disseminated or mucosal gonococcal infections (Power et al., 2007, Infect Immun 75:3202-4) were also studied. In selected experiments, various concentrations of CMP-Neu5Ac were added to the medium, as indicated.

[0146] Lipooligosaccharide (LOS) purification Cells were grown on BHI or GC solid medium. Bacteria were harvested by scraping and suspended in a solution of 60 mM TRIS, 10 mM EDTA, and 2% (wt / vol) sodium dodecyl sulfate, pH 6.8. Proteinase K was added to a final concentration of 50 μg / ml, and samples were incubated overnight at 37°C. After three ethanol precipitations, samples were treated with DNase I and RNase A. Samples were then phenol extracted, ethanol precipitated once more, and centrifuged at 120,000 × g for 75 min. The pellet was resuspended in water, frozen, and then lyophilized.

[0147] whole cell ELISA Bacteria were grown on BHI or GC plates for 16 h. Cells were harvested and resuspended in PBS at an OD600nm of 0.20. Microtiter plate wells were filled with 50 μL of bacterial suspension and dried overnight at room temperature in a laminar flow cabinet. Bacteria in the dried wells were heat killed at 56°C for 1 h, after which the wells were washed and ELISA was performed with MAb 6E4 at a dilution of 1:64. Secondary antibody (Dako P044701 polyclonal anti-mouse Ig HRP) was used at a dilution of 1:2,000. Substrate TMB (3,3',5,5-tetramethylbenzidine) solution (Thermofisher Scientific) was used according to the manufacturer's instructions. The reaction was stopped by adding an equal volume of 1N hydrochloric acid. Absorbance was read at 450 nm in a TECAN Model Infinite 200 Pro plate reader.

[0148] Fluorescent labeling of clinical samples Paraformaldehyde-fixed preserved clinical specimens from cervical swabs taken from patients presenting with gonorrhea were also dried onto glass microscope slides. These samples were incubated simultaneously with MAbs 6E4 (IgG) and 6B4 (IgM). Fluorescent labeling was provided by incubation with goat anti-mouse IgG3 / FITC to label MAb 6E4 and goat anti-mouse IgM / Texas Red (both conjugates from Jackson ImmunoResearch) for MAb 6B4. Samples were counterstained with Draq5 (Cell Signaling Technologies) to label cell nuclei and viewed by confocal microscopy on a Leica STED system.

[0149] Opsonophagocytic killing assay Polymorphonuclear leukocytes (PMN) were prepared using Polymorphprep according to the manufacturer's instructions. TM Neisseria gonorrhoeae 1291 (approximately 1 × 10 3 CFU) were incubated in serial dilutions of MAb 6E4 (from 0 μg to 10 μg) for 15 min at 37°C. PMNs (approximately 1 × 10 5 Cells) and a complement source (10% v / v normal human serum preabsorbed with N. gonorrhoeae) were added and incubated for 90 min at 37°C. Gonococcal viability was measured after plating serial dilutions on GC agar and survival was calculated as a percentage of the no-antibody control. Statistical significance was calculated using one-way analysis of variance (ANOVA) and Student's t-test.

[0150] The disclosures of all patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety.

[0151] Citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the instant application.

[0152] Throughout this specification, the objective is to describe preferred embodiments of the present disclosure, without limiting the disclosure to any one embodiment or particular collection of features. Thus, those skilled in the art will appreciate in light of this disclosure that various modifications and changes can be made in the specific embodiments illustrated without departing from the scope of the present disclosure. All such modifications and changes are intended to be included within the scope of the appended claims.

Claims

1. A chimeric molecule comprising a carrier and an oligosaccharide comprising an oligosaccharide sequence corresponding to Neisseria lipooligosaccharide, wherein the oligosaccharide sequence comprises ketodeoxyoctulosonate (KDO) as a terminal sugar unit, and the oligosaccharide is other than an oligosaccharide consisting of the oligosaccharide sequence KDOα(2→3)Gal, or the oligosaccharide sequence KDOα(2→3)Galβ(1→4)GlcNAc.

2. Formula (I): [0010] is represented by During the ceremony, KDO-OS represents an oligosaccharide sequence containing 2 to 10 monosaccharide units and KDO as a terminal sugar unit; L is an optional linker, Cr is a support; n is 1 or more and 4 or less, m is at least 1, or a pharma- ceutically acceptable salt thereof.

3. The chimeric molecule of claim 1 or claim 2, wherein the carrier is a protein, a peptide, a lipid, a polymer, a dendrimer, a virosome, a virus-like particle (VLP), an outer membrane vesicle or a combination thereof.

4. The chimeric molecule of any one of claims 1 to 3, wherein the carrier is a protein carrier.

5. 5. The chimeric molecule of claim 4, wherein the protein carrier is selected from bacterial toxoids, toxins, exotoxins and their non-toxic derivatives, Hepatitis B virus core protein, thyroglobulin, albumin, pneumococcal surface protein A (PspA), pneumococcal adhesion protein (PsaA), purified protein derivative of tuberculin (PPD), transferrin binding protein, polyamino acids, tetanus toxoid, tetanus toxin fragment C, diphtheria toxoid, cross-reactive material (CRM), cholera toxoid, Staphylococcus aureus exotoxin or toxoid, Escherichia coli heat-labile enterotoxin, Pseudomonas aeruginosa exotoxin A, and bacterial outer membrane proteins.

6. The chimeric molecule of claim 4, wherein the protein carrier is CRM197.

7. The chimeric molecule of any one of claims 1 to 6, wherein the Neisserial lipooligosaccharide is a Neisseria gonorrhoeae or Neisseria meningitidis lipooligosaccharide.

8. A composition comprising the chimeric molecule of any one of claims 1 to 7 and a pharma- ceutically acceptable vehicle or adjuvant.

9. 9. The composition of claim 8, further comprising an adjunct for treating or inhibiting the development of a Neisserial infection.

10. The composition of claim 9, wherein the adjunct is an antibacterial agent.

11. 10. The composition of claim 9, wherein the adjuvant is an adjuvant immunogen for eliciting an immune response against a Neisserial organism.

12. A pharmaceutical composition comprising a chimeric molecule according to any one of claims 1 to 7 or a composition according to any one of claims 8 to 11 for inducing an immune response against a Neisserial organism in a subject.

13. 13. The pharmaceutical composition of claim 12, wherein the Neisserial organism is Neisseria gonorrhoeae or Neisseria meningitidis.

14. 13. The pharmaceutical composition of claim 12, wherein the Neisseria organism is resistant to at least one antibacterial agent.

15. 12. A method for producing an antigen-binding molecule that specifically binds to a Neisseria organism, the method comprising: (1) immunizing an animal with the chimeric molecule of any one of claims 1 to 7, or the composition of any one of claims 8 to 11; (2) detecting B cells from the animal that specifically bind to the chimeric molecule; and (3) isolating the antigen-binding molecule expressed by the B cells.

16. A method for producing an antigen-binding molecule that specifically binds to a Neisseria organism, the method comprising: (1) screening a library of antigen-binding molecules with the chimeric molecule of any one of claims 1 to 7, or the composition of any one of claims 8 to 11; (2) detecting the chimeric molecule, or an antigen-binding molecule that specifically binds to a Neisseria organism or an LOS of a Neisseria organism; and (3) isolating the detected antigen-binding molecule.

17. An antigen-binding molecule, or an antigen-binding molecule derivative having the same epitope-binding specificity as the antigen-binding molecule, produced by the method of claim 15 or 16.

18. The antigen-binding molecule of claim 17, wherein the antigen-binding molecule derivative is selected from antibody fragments, single chain (scFv) and domain antibodies, and fusion proteins comprising antibodies, and any other modified configuration of an immunoglobulin molecule comprising an antigen-binding / recognition site.

19. A cell that produces the antigen-binding molecule of claim 17 or 18.

20. A pharmaceutical composition comprising a chimeric molecule according to any one of claims 1 to 7, a composition according to any one of claims 8 to 11, or an antigen-binding molecule according to claim 17 or claim 18, for treating or inhibiting the development of a Neisserial infection in a subject.

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