Immunogenic compositions containing lipooligosaccharides and methods of use thereof

Abstract

Immunogenic and vaccine compositions containing a Neisseria gonorrhoeae lipooligosaccharide (LOS), or a fragment thereof, are provided. The LOS may be conjugated to a carrier such as a carrier protein, virus-like particle (VLP), liposome, inorganic gold particle, dendrimer, outer membrane vesicle (OMV), generalized modules for membrane antigens (GMMA), and protein nanocages. Methods of protecting or treating a subject from Neisseria gonorrhoeae infection and methods of manufacturing the compositions are also provided.

Description

[0001] IMMUNOGENIC COMPOSITIONS CONTAINING LIPOOLIGOSACCHARIDES AND METHODS OF USE THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional applications 63 / 687,873, filed August 28, 2024 and 63 / 698,911, filed September 25, 2024. These applications are incorporated herein by reference in their entirety. FIELD OF THE INVENTION The invention generally relates to an immunogenic or vaccine composition comprising lipooligosaccharides derived from Neisseria gonorrhoeae. The compositions are useful for protecting or treating a subject from Neisseria gonorrhoeae infection. BACKGROUND OF THE INVENTION Neisseria gonorrhoeae is a gram-negative bacterium responsible for gonorrhea, a prevalent sexually transmitted infection. The organism's ability to grow, colonize, and evade the host immune system is significantly influenced by its surface-expressed molecules, particularly lipooligosaccharides (LOS). These glycolipids are crucial for both the pathogen's survival and pathogenicity. LOS are complex molecules composed of lipid A, a core oligosaccharide, and an absence of the O-antigen, distinguishing them from lipopolysaccharides (LPS) found in other gram- negative bacteria. The core oligosaccharides are diverse, providing structural variability that aids in immune evasion. This structural plasticity allows Neisseria gonorrhoeae to modulate its LOS composition in response to environmental pressures, enhancing its adaptability. Structurally, LOS in Neisseria gonorrhoeae are composed of a lipid A moiety, a core oligosaccharide, and an oligosaccharide extension. The lipid A region, critical for anchoring the LOS to the bacterial membrane, comprises a disaccharide backbone of glucosamine units linked by β(1→4) glycosidic bonds. These glucosamine residues are further acylated with fatty acids, which contribute to the molecule's hydrophobic nature and play a role in immune evasion by modulating host immune responses. The core oligosaccharide is attached to the lipid A through a 3-deoxy-D-manno- octulosonic acid (Kdo) residue. This core region is typically composed of heptose residues, including L-glycero-D-manno-heptose, which are linked via glycosidic bonds. The arrangement and types of sugars in the core oligosaccharide can vary, contributing to the antigenic diversity of the LOS. This diversity is a key factor in the bacterium's ability to evade host immunity. The oligosaccharide extension, which is less conserved than the core region, often includes hexose sugars such as glucose, galactose, and N-acetylglucosamine. These sugars can be presented in various configurations, further enhancing the structural diversity. The extension may also contain sialic acid residues (the donor molecule for sialic acid is provided by the host), which mimic host cell surface molecules and help the bacterium evade immune detection. The fine atomic structure of LOS is characterized by intricate glycosidic linkages and the presence of specific functional groups that influence the molecule's overall conformation and interactions with host cells. The stereochemistry of these linkages, particularly the anomeric configurations, can affect the recognition and binding to host immune receptors, such as toll-like receptors (TLRs). Understanding the detailed atomic structure of LOS may shed light on the mechanisms of Neisseria gonorrhoeae pathogenicity and open avenues for the development of vaccines and targeted therapeutics aimed at disrupting these critical structures and mitigating infection. LOS is integral to the outer membrane's structural integrity and functionality, crucial for bacterial viability. The molecules contribute to the membrane's permeability barrier, protecting the bacterium from harmful substances, including antibiotics and detergents. By stabilizing the outer membrane, LOS ensures the maintenance of essential cellular processes, facilitating bacterial growth under various conditions. Furthermore, LOS interacts with host cells to modulate the host environment favorably for bacterial colonization. They are involved in adhesion to epithelial cells, a critical step in establishing infection. The specific binding interactions between LOS and host cell receptors facilitate the initial attachment and subsequent invasion of host tissues. One of the most significant roles of LOS in Neisseria gonorrhoeae is its ability to evade the host immune response. The bacterium employs several strategies to achieve this, primarily through the structural modification of LOS. Neisseria gonorrhoeae can alter the expression of its LOS through phase and antigenic variation. This enables the bacterium to switch between different LOS structures, effectively evading antibody-mediated recognition and destruction. By continuously changing the surface- exposed epitopes, the pathogen stays one step ahead of the host immune system's adaptive response. LOS exhibits molecular mimicry by resembling host glycoconjugates, such as blood group antigens and gangliosides. This mimicry confers an advantage by preventing immune recognition as foreign. The host's immune system is less likely to mount an effective response against structures perceived as self, allowing the bacterium to persist and proliferate. The complement system is a crucial component of the innate immune response, targeting pathogens for destruction through opsonization, inflammation, and lysis. Neisseria gonorrhoeae's LOS can bind regulatory proteins such as factor H, inhibiting the complement cascade and thereby preventing complement-mediated lysis. This mechanism of complement resistance is vital for the bacterium's survival in the hostile environment of the human host. LOS also plays a role in modulating the host immune response to facilitate immune evasion further. They can induce the production of pro-inflammatory cytokines, leading to localized inflammation. While this inflammatory response can help establish infection, it also causes tissue damage, which may aid in the spread of the bacterium. Moreover, the interaction between LOS and toll-like receptors (TLRs) on host immune cells can lead to the activation of signaling pathways that modulate the immune response. By influencing these pathways, Neisseria gonorrhoeae can create an environment that is less hostile and more conducive to its persistence. With rising antibiotic resistance in Neisseria gonorrhoeae, developing a vaccine is becoming critical. Improved immunogenic and vaccine compositions useful for preventing or treating Neisseria gonorrhoeae infection are needed. SUMMARY An aspect of the disclosure provides an immunogenic composition comprising a Neisseria gonorrhoeae lipooligosaccharide (LOS), or a fragment thereof, conjugated to a carrier. In some embodiments, the carrier is selected from the group consisting of a carrier protein, virus-like particle (VLP), liposome, inorganic gold particle, dendrimer, outer membrane vesicle (OMV), generalized modules for membrane antigens (GMMA), ferritin nanoparticles, and protein nanocages. In some embodiments, the LOS is conjugated to a carrier protein and has the following formula: wherein R1 is lactose or PEtn; R2 is H or OAc; R3 is H or PEtn; R4 is H or Gal; and X is a carrier. In some embodiments, the carrier is a protein selected from the group consisting of tetanus toxoid, diphtheria toxoid, keyhole limpet hemocyanin, CRM197, and outer membrane protein complex (OMPC). In some embodiments, a linker is present between the LOS and the carrier. In some embodiments, the linker is a polyethylene glycol (PEG) or maleimide-PEG linker. In some embodiments, the composition further comprises one or more additional bacterial antigens, such as PorB, Neisserial Heparin Binding Antigen (NHBA), Neisserial surface protein A (NspA), Multiple transferable resistance E (MtrE), Transferrin binding proteins A and B (TbpA and TbpB), other TonB dependent transporters (e.g., TdfJ), Methionin-binding lipoprotein (MetQ) or Opacity protein (Opa), as well as antigens identified using artificial intelligence such as NGO1549 and NGO0265. Another aspect of the disclosure provides a vaccine composition comprising an immunogenic composition as described herein and a pharmaceutically acceptable carrier. In some embodiments, the vaccine composition further comprises an adjuvant. Another aspect of the disclosure provides a method of protecting or treating a subject from Neisseria gonorrhoeae infection comprising administering to the subject a vaccine composition as described herein. Another aspect of the disclosure provides a method of manufacturing an immunogenic composition, comprising isolating or synthesizing a Neisseria gonorrhoeae LOS or fragment thereof; and conjugating the LOS or fragment thereof to a carrier. In some embodiments, the carrier is a carrier protein and is conjugated to the LOS via reductive amination, carbodiimide coupling with N-hydroxysuccinimide (EDC / NHS coupling), click chemistry, or maleimide-thiol coupling. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Schematic of gonococcal LOS. LOS biosynthesis genes responsible for addition of glycans are indicated in lower case italics. The α1-4-linked Gal may be added if lgtA is phase varied off and lgtC is phase varied on. Figure 2. Schematic of gonococcal LOS after acid hydrolysis and conjugation to a carrier. Figure 3A-C. Immunogenicity and in vivo efficacy of CP2 configured as a tetramer. A. Mice (n=10) were immunized withTMCP2 (50 µg) adjuvanted with GLA-SE (5 µg) at 0, 3 and 6 weeks, and elicited IgG was measured by LOS ELISA. B. Antisera from immunized mice was depleted of mouse IgM and tested for bactericidal activity against Ngo strain MS11 in a complement-dependent bactericidal assay using 10% human complement (normal human serum depleted of IgG and IgM). X-axis, concentration of immune serum; Y-axis, % survival of bacteria at 30 min relative to 0 min. C. Mice immunized with TMCP2 as above or with GLA-SE alone (adjuvant controls) (n=10 per group), treated with estrogens and antibiotics and challenged 2 weeks post-dose 3 with Ng MS11 (4.2 x 107CFU). Vaginas were swabbed daily to enumerate CFUs. Log10CFU vs time (mean [SEM]) is shown. Figure 4. Structure of the 2,3-Di-O-Substituted Heptose Tetrasaccharide and the retrosynthetic analysis. Bn represents a benzyl protecting group. Figure 5. Synthesis of the heptose sugar intermediate 4. Figure 6. Synthesis of the benzyl-2-azido-glucoacetimidate compound 5. Figure 7. Synthesis of the per-O-benzylated lactosyl trichloroacetimidate 2. Figure 8. Synthesis of the tetrasaccharide 1. Figure 9. Synthesis of LOS tridecasaccharide. Figure 10. Synthesis of fragment F. Figure 11. Synthesis of fragment E. Figure 12. Synthesis of fragment D. Figure 13. Synthesis of fragment C. Figure 14. Synthesis of fragment A. Figure 15. Coupling of fragments A and B to provide the tridecasaccharide. DETAILED DESCRIPTION Embodiments of the disclosure provide compositions comprising lipooligosaccharides (LOS) or fragments thereof derived from Neisseria gonorrhea and their use in vaccines. LOS from Neisseria gonorrhoeae has a structure somewhat similar to lipopolysaccharides (LPS) found in other Gram-negative bacteria but is distinct due to the absence of the repeating O-antigen polysaccharide. The general structure is shown in Figure 1 and includes Lipid A, core oligosaccharide, and outer core sugars. Lipid A is the hydrophobic anchor that embeds the LOS into the bacterial outer membrane. The lipid A portion comprises a disaccharide of glucosamine with multiple fatty acid chains, which are important for the endotoxic properties and immune system activation. The core oligosaccharide is composed of a few, e.g. 4-5, sugars, usually: Glc (Glucose), Hep (Heptose), and Kdo (2-keto-3-deoxyoctonate). The core often shows variability and phase variation, allowing the bacterium to evade the host immune system. The outer core sugars include hexoses (e.g., glucose, galactose), N-acetylglucosamine (GlcNAc) and N- acetylgalactosamine (GalNAc). These sugars are highly variable and can mimic human glycosphingolipids, which helps Neisseria gonorrhoeae in immune evasion by molecular mimicry. Unlike traditional LPS, Neisseria gonorrhoeae lacks the long, repeating O-antigen chains found in other Gram-negative bacteria. This is a distinguishing feature of LOS and contributes to its ability to cause inflammation without the strong antigenic response typically associated with LPS. LOS plays a central role in the pathogenicity of N. gonorrhoeae, contributing to immune evasion, inflammation, and bacterial survival. Targeting LOS in a vaccine would neutralize one of the key mechanisms the bacterium uses to cause infection and evade immune defenses. By targeting LOS, a vaccine would disrupt the bacterium’s ability to adhere to host cells, invade tissues, and resist immune clearance, reducing its ability to establish infection. As described herein in detail, the LOS may be isolated from N. gonorrhoeae or chemically synthesized. If isolated, the lipid A component may be removed by mild acid treatment and the LOS may be conjugated to a carrier (Figure 2). For example, the carboxyl residue on the KDO may be modified with an alkyne hydrazide, such as hex-5-ynehydrazide, which can then be coupled to an azide-containing carrier with click chemistry. The carboxyl group on the KDO could also be cross-linked to carrier proteins such as CRM using heterobifunctional cross linkers. The LOS may be isolated from any strain of N. gonorrhoeae including NG-STAR ST-90, ST-58, ST- 61, ST-64, ST-79, ST-91, ST-139, etc. A fragment of a LOS as described herein is an oligosaccharide portion of LOS that retains at least one antigenic epitope of the LOS. Embodiments provide a LOS, or fragment thereof, as described herein conjugated to a carrier. Exemplary carriers include, but are not limited to: carrier proteins (e.g., diphtheria toxoid, tetanus toxoid, CRM197 etc. or protein-based carriers such as albumin carriers), virus-like particles (VLP), ferritin nanoparticles, lipid-based carriers (such as liposomes and lipid nanoparticles), inorganic nanoparticles such as inorganic gold particles, dendrimers, outer membrane vesicles (OMV), generalized modules for membrane antigens (GMMA), protein nanocages, polymer-based carriers (such as polymeric micelles and microspheres), nanocapsules, hydrogels, transferosomes and antibodies. “Conjugation” to OMV and GMMA carriers as described herein refers to integrating LOS in the OMV or GMMA membrane. Some embodiments provide an LOS having the following structure:

[0002] wherein R1 is lactose or PEtn; R2 is H or OAc; R3 is H or PEtn; R4 is H or Gal; and X is the carrier, which may be a protein. The terms above refer to standard abbreviations in the LOS art, for instance Glc refers to Glucose (or D-glucopyranose), KDO refers 3-deoxy-D-manno-oct-2-ulosonic acid, Hep refers to heptose (or L-glycero-D-manno-heptose), GlcNAc to N-acetylglucosamine, Gal to Galactose, PEtn to phosphoethanolamine, OAc to O-acetyl, etc.

[0003] The LOS is a useful vaccine target because it is an essential structure that plays multiple roles in the bacterium’s survival and pathogenicity. The compositions described herein can thus prevent or reduce the risk of infection and reduce the need for antibiotics. The compositions can also limit the spread of antibiotic-resistant strains by reducing the overall incidence of gonorrhea. Conjugating LOS to a carrier or combining it with other bacterial antigens could enhance its ability to induce a protective immune response. For example, the LOS may be combined with proteins like PorB, Neisserial Heparin Binding Antigen (NHBA), Neisserial surface protein A (NspA), Multiple transferable resistance E (MtrE), Transferrin binding proteins A and B (TbpA and TbpB), other TonB dependent transporters (e.g., TdfJ), Methionin-binding lipoprotein (MetQ) or Opacity protein (Opa), as well as antigens identified using artificial intelligence such as NGO1549 and NGO0265 which are other important surface antigens on N. gonorrhoeae. Conjugating LOS to a carrier protein converts the immune response from T-cell independent (short-lived and limited memory) to T-cell dependent (more robust and longer-lasting memory). The protein component provides T-cell epitopes, allowing helper T cells to be activated, which enhances the immune system's ability to generate a stronger response, including antibody production and immunological memory. In addition, the conjugation process also induces a phenomenon called "immune memory." This means that the immune system, upon encountering the pathogen again, can quickly recognize and neutralize it, ensuring long-lasting protection. This approach is particularly beneficial for young children, whose immune systems are still developing, and for elderly individuals, whose immune responses are often weaker. Suitable protein carriers include, but are not limited to, protein D from non-typeable H. influenzae, tetanus toxoid, diphtheria toxoid, keyhole limpet hemocyanin, CRM197, outer membrane protein complex of serogroup B meningococcus (OMPC), or an antibody specific for an N. gonorrhoeae antigen. Methods for conjugation are known in the art. Before LOS can be conjugated to a protein, it typically needs to be activated to create a functional group for attachment. There are several methods to achieve this: Oxidation of Terminal Groups: LOS has hydroxyl groups on the sugar moieties that can be oxidized to form reactive aldehydes or carboxylic acids. For example, periodate oxidation can selectively oxidize vicinal diols in the carbohydrate portion of LOS, creating aldehyde groups that can react with amine groups on proteins. Derivatization: Synthetic LOS can also be designed with specific functional groups such as thiols, amines, or carboxyl groups to facilitate the conjugation. These modifications are introduced during the chemical synthesis of LOS to provide reactive sites for linking to proteins. Reductive Amination: If LOS has been activated to contain aldehyde groups (via oxidation), it can be conjugated to amine groups on the protein through reductive amination. This reaction forms a stable imine or Schiff base intermediate, which is reduced to form a stable linkage. For instance: LOS-CHO+NH2-Protein→LOS-NH-ProteinLOS-CHO+NH2-Protein→LOS-NH-Protein EDC / NHS Coupling: If the LOS has carboxyl groups (as in some modified versions), it can be coupled to the amine groups of the carrier protein using EDC (1-Ethyl-3-(3- dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide) chemistry. This creates a stable amide bond between the LOS and the protein. Click Chemistry: Another modern approach involves "click chemistry", particularly azide- alkyne cycloaddition reactions. The LOS can be functionalized with an azide group, and the carrier protein can be modified to have an alkyne group. These groups form a stable triazole linkage under mild conditions using a copper catalyst (CuAAC). Maleimide-Thiol Coupling: If LOS is modified to contain a thiol group, it can be conjugated to a maleimide group on the protein through a Michael addition reaction. This creates a highly stable thioether bond. In some embodiments, a linker is present between the LOS and the carrier. Linkers can be incorporated to control the stability, flexibility, and spacing between the conjugated components. Properly chosen linkers ensure that the conjugate maintains its immunogenic properties, allows proper protein folding, and avoids steric hindrance. In some embodiments, a flexible linker is used such as a polyethylene glycol (PEG) linker which is hydrophilic, flexible, and biocompatible. In some embodiments, a non-cleavable linker is used. These form permanent bonds between LOS and the carrier, ensuring long-term stability of the conjugate. They are useful when the LOS does not need to be released from the carrier for immunogenicity. In some embodiments, a maleimide-PEG linker is used. Maleimide reacts with thiol (-SH) groups, often found on cysteine residues in proteins, forming a stable thioether bond. PEG provides flexibility and distance between the LOS and the protein. For example, a maleimide-PEG linker can be used to conjugate the LOS to a carrier protein, such as keyhole limpet hemocyanin (KLH), which has free thiol groups. This results in a stable, flexible conjugate that preserves the function of both LOS and the protein. The reaction would proceed as follows: LOS-SH+Protein-Maleimide→LOS-S-Protein+Stable Thioether BondLOS-SH+Protein- Maleimide→LOS-S-Protein+Stable Thioether Bond In this example, the PEG linker ensures that the LOS is displayed in a flexible manner, allowing immune recognition, while the maleimide group provides a stable and efficient bond with the protein. The choice of linker can also affect the immunogenicity of the vaccine conjugate. For example, a long and flexible linker may help expose the LOS more effectively to the immune system, enhancing the response. Conversely, a too-short or rigid linker could hide the LOS from immune cells, reducing the vaccine's efficacy. In some embodiments, the linker has a length of 5- 30 Angstroms, e.g. 10-20 Angstroms. In some embodiments, the LOS compositions have been detoxified. This may be done by known techniques of hydrazine or alkaline hydrolysis chemical treatments which remove acyl chains from the molecule. Embodiments also include methods of making an LOS, immunogenic composition, or vaccine composition as described herein. Methods of isolating or chemically synthesizing an LOS are known in the art, e.g. as described in the Examples. A method of making or manufacturing a composition may include isolating or synthesizing a Neisseria gonorrhea LOS or fragment thereof and conjugating the LOS or fragment thereof to a carrier. Various protecting groups may be utilized in the synthesis of an a LOS. The term “protecting group” refers to any group which, when bound to one or more hydroxyl group(s) or amine group(s) of a compound of the invention, prevents reactions from occurring at these hydroxyl or amine group(s) and which can be removed by chemical or enzymatic steps to reestablish the hydroxyl or amine group(s). The particular removable protecting group employed is determined by the nature of the compounds and chemical processes being utilized. Removable hydroxyl and amino protecting groups include, without limitation, substituents such as allyl, benzyl, acetyl, chloroacetyl, trifluoroacetyl, thiobenzyl, benzylidene, phenacyl, t- butyldimethylsilyl (TBS), dimethylthexylsilyl (TDS) and trialkylsilyls such as triethylsilyl, triisopropylsilyl, trimethylsilyl, tributylsilyl and the like, as well as azido, allyloxycarbonate (Alloc), 9-fluorenylmethoxycarbamate (Fmoc), levulinate (Lev), allyloxycarbonyl, 2,2,2,- trichloroethoxycarbonyl (Troc), phthalimido, and any other group that can be introduced chemically onto a hydroxyl or amino functionality and later selectively removed either by chemical or enzymatic methods in mild conditions compatible with the nature of the product. In a further embodiment, the composition further comprises a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient can include diluents, vehicles, and inert, non- toxic solid or liquid fillers, or encapsulating material that does not react with the active ingredients of the invention. The excipient may be selected from the group consisting of starch, glucose, lactose, sucrose, trehalose, raffinose, stachyose, melezitose, dextran, mannitol, lactitol, palatinit, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, glycine, arginine, lysine, sodium chloride (NaCl), dried skim milk, glycerol, propylene glycol, water, and ethanol. The final amount of the compounds in the formulations may vary. However, in general, the amount in the formulations will be from about 0.01-99%, weight / volume. In yet another embodiment, the composition further comprises an adjuvant. Suitable adjuvants include an aluminum salt such as aluminum hydroxide gel (alum) or aluminum phosphate (preferably aluminum hydroxide) but may also be a salt of calcium (particularly calcium carbonate), iron or zinc, or may be an insoluble suspension of acylated tyrosine, or acylated sugars, cationically or anionically derivatized polysaccharides, or polyphosphazenes. Other adjuvants that may be added comprise a saponin, more preferably QS21 and / or an oil in water emulsion and tocopherol. The compositions of the present disclosure may also contain other components such as, but not limited to, additives, buffers, tonicity agents, bioadhesive polymers, and preservatives. In any of the compositions of this disclosure, the mixtures are preferably formulated at about pH 5 to about pH 8. This pH range may be achieved by the addition of buffers to the composition. It should be appreciated that the compositions of the present disclosure may be buffered by any common buffer system such as phosphate, borate, acetate, citrate, carbonate and borate-polyol complexes, with the pH and osmolality adjusted in accordance with well-known techniques to proper physiological values. Compositions as described herein may be prepared either as liquid solutions or suspensions, or as solid forms such as tablets, pills, granules, capsules, powders, ampoules, and the like. The liquid may be an aqueous liquid. Solid forms suitable for solution in, or suspension in, liquids prior to administration may also be prepared. Example dosage forms include a tablet, dragee, liquid, drop, capsule, caplet, gelcap, etc. Embodiments also include methods of protecting from, reducing the risk of, or treating a subject with Neisseria gonorrhoeae infection comprising administering to the subject a vaccine composition as described herein. The compositions described herein can be administered to a human or a non-human animal including, but not limited to mammals, dogs, horses, cats, rabbits, gerbils, hamsters, rodents, birds, aquatic mammals, cattle, pigs, camelids, and other zoological animals. The compositions described herein are useful for eliciting an immune response. By eliciting an immune response, it is meant that administration of the antigen causes the synthesis of specific antibodies and / or cellular proliferation. The protective response either wholly or partially prevents or arrests the development of symptoms related to N. gonorrhoeae infection, in comparison to a non-vaccinated (e.g. adjuvant alone) control organism, in which disease progression is not prevented. In some embodiments, the composition is administered to a subject in a therapeutically effective amount. By a "therapeutically effective amount" or an “effective amount” is meant a sufficient amount to treat the disease or disorder at a reasonable benefit / risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compositions of the present disclosure will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific active agent employed; and like factors well known in the medical arts. Vaccine preparation is generally described in Vaccine Design ("The subunit and adjuvant approach" (eds Powell M.F. & Newman MJ.) (1995) Plenum Press New York). An immunoprotective dose of vaccines can be administered via the systemic or mucosal route. These administrations may include injection via the intramuscular, intraperitoneal, intradermal or subcutaneous routes; or via mucosal administration to the oral / alimentary (e.g. intra-nasal administration), respiratory, or genitourinary tracts. The LOS quantity in each vaccine dose is selected as an amount which induces an immunoprotective response without significant, adverse side effects in typical vaccinees. Such amount will vary depending upon which specific immunogen is employed and how it is presented. Generally, it is expected that each dose will comprise l-100μg of each LOS of the invention, e.g. 1-50μg or 1 - 25μg. Due to structural similarities between the LOS of N. gonorrhoeae and other bacteria, including Neisseria meningitidis, the compositions described herein may offer cross-protection against multiple pathogens. Thus, the present disclosure also provides broader-spectrum vaccines that protect against multiple sexually transmitted infections or Gram-negative bacteria. Before exemplary embodiments of the present invention are described in greater detail, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described. All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed. It is noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation. As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible. The invention is further described by the following non-limiting examples which further illustrate the invention, and are not intended, nor should they be interpreted to, limit the scope of the invention. EXAMPLE 1. Rationale for targeting the LOS epitope with a vaccine and targeted therapeutics. A study of immunity to urethral reinfection was carried out in male human volunteers and it was found that the men who resisted Neisseria gonorrhoeae (Ng) reinfection were significantly more likely to have had a >4x increase in anti-LOS IgG following initial infection than men who were reinfected. In humans, the LOS epitope elicits a significant antibody response after natural infection or vaccination with outer membrane antigens that contain LOS. Unlike any other Ng LOS epitopes that mimic host glycans (e.g., the lacto-N-neotetraose epitope that resembles host paraglobosides and the Gal-Gal-Glc trisaccharide that resembles the PK-like blood group antigen), the LOS epitopes contain structures that do not resemble any known human glycans – for example the epitopes recognized by antibodies like 2C7. In vitro and in vivo, it has been shown: i) complement-dependent bactericidal activity of mAb 2C7 against all 62 randomly selected minimally passaged isolates from men in Nanjing, China, ii) dose-dependent efficacy of mAb 2C7 in the mouse vaginal colonization model of gonorrhea, iii) a mechanism of action (MOA) of mAb 2C7 in vivo that is complement (C’)-dependent killing through insertion of the membrane attack complex (C5b-9), and iv) efficacy in chlamydia / Ngo co-infected mice. Moreover, though the carbohydrate target of the 2C7 epitope is not itself immunogenic, a peptide mimic (mimotope) of the 2C7 epitope elicits a protective antibody response in mice when formulated as a multi-antigenic peptide (MAP) (Fig.3A-C). Taken together, these data provide compelling evidence for our ability to target the gonococcal LOS epitope with an appropriately formulated vaccine or a targeted therapeutic. These objectives can be effectively achieved with a vaccine comprising bacterial LOS conjugated to a carrier protein that elicits a safe and protective immune response. EXAMPLE 2. Extraction of Lipooligosaccharides. The extraction of lipooligosaccharide (LOS) from Neisseria gonorrhoeae involves a series of meticulous steps, including fermentation and downstream processing. Here, we describe an exemplary detailed methodology employed to isolate LOS efficiently. Fermentation The initial phase of LOS extraction begins with the cultivation of Neisseria gonorrhoeae. This process is carried out in a controlled environment to ensure optimal growth conditions and high yield of bacterial biomass. Culture Preparation • Media Selection: Gonococcal broth or similar enriched media are used, supplemented with necessary nutrients to support the growth of Neisseria gonorrhoeae. • Inoculation: Pure Neisseria gonorrhoeae strains are inoculated into the media under sterile conditions to prevent contamination. Fermentation Conditions • Temperature: Cultures are incubated at 37°C, which is the optimal temperature for Neisseria gonorrhoeae growth. • Aeration: Adequate aeration is provided to ensure the bacteria have sufficient oxygen, typically achieved by shaking the cultures at around 200 rpm. • Duration: Fermentation is allowed to proceed for 18-24 hours, or until the bacterial growth reaches its exponential phase. Downstream Processing Once the bacterial culture reaches the desired growth phase, the downstream processing steps are initiated to extract LOS from the cells. Cell Harvesting • Centrifugation: The bacterial cells are collected by centrifuging the culture at 5000 x g for 10-15 minutes at 4°C. This helps in separating the cells from the culture supernatant. • Washing: The pelleted cells are washed with cold phosphate-buffered saline (PBS) to remove any residual media components. Cell Disruption • Lysis Methods: The washed cells are lysed using methods such as sonication, bead beating, or enzymatic lysis to release the LOS from the bacterial membranes. • Buffer: A lysis buffer containing detergents like SDS or Triton® X-100 may be used to enhance the disruption of the cell membrane and solubilize the LOS. LOS Extraction • Phase Separation: Following cell lysis, LOS is extracted using hot aqueous phenol (e.g. about 65-68°C) or ethanol precipitation. The phenol-water extraction method separates the LOS into the aqueous phase. • Centrifugation: The mixture is centrifuged at high speeds (e.g., 10000 x g) to facilitate the separation of phases. The aqueous phase containing LOS is carefully collected. • Mild acid hydrolysis may be performed after extraction to remove lipid A. Purification • Dialysis: The extracted LOS is dialyzed against distilled water or PBS to remove phenol and other contaminants. • Column Chromatography: Further purification can be achieved using column chromatography techniques, such as size-exclusion or affinity chromatography, to isolate LOS based on its molecular weight and specific binding properties. Characterization and Storage • Characterization: The purified LOS is characterized using analytical techniques such as SDS-PAGE, mass spectrometry, or NMR spectroscopy to confirm its structure and purity. • Storage: The final LOS preparation is aliquoted and stored at -20°C or below, as it remains stable under these conditions for extended periods. The described fermentation and downstream processing methodology ensures a high yield of pure LOS. EXAMPLE 3. Chemical synthesis of LOS: 2,3-Di-O-Substituted Heptose Tetrasaccharide. While traditional methods of obtaining LOS involve fermentation and downstream processing, chemical synthesis presents several compelling advantages. Consistency and Purity Uniformity: One of the primary benefits of chemical synthesis is the ability to produce LOS with a consistent and uniform structure. Naturally derived LOS can exhibit batch-to-batch variability due to differences in bacterial cultures and fermentation conditions. Chemical synthesis mitigates this issue by providing precise control over the molecular structure, ensuring uniformity across all batches. High Purity: Chemical synthesis allows for the production of LOS with high purity, free from contaminants that may be present in naturally derived LOS. The purification process in fermentation can be complex and may not always result in the desired purity levels. In contrast, chemical synthesis techniques can be finely tuned to achieve the highest purity standards, which is crucial for sensitive applications such as vaccine development. Scalability and Efficiency Scalable Production: Chemical synthesis offers scalable production capabilities that can meet the increasing demand for LOS in various applications. Fermentation processes, while effective, may face limitations in scaling up without compromising yield or quality. Chemical synthesis methods can be adapted to large-scale production, ensuring a steady and reliable supply of LOS. Efficiency: The efficiency of chemical synthesis can be optimized to produce LOS in shorter timeframes compared to fermentation. The latter involves prolonged cultivation periods and intricate downstream processing, which can be time-consuming. Chemical synthesis streamlines the production process, enabling faster turnaround times while maintaining high- quality output. Structural Complexity Precision in Complex Structures: Chemical synthesis excels in constructing complex LOS structures that may be challenging to achieve through natural fermentation. For example, specific glycosylation patterns and stereochemistry can be precisely controlled during chemical synthesis, allowing for the creation of LOS with desired biological properties. This level of precision is often difficult to attain with naturally derived LOS due to the inherent variability in bacterial biosynthesis pathways. Innovation and Customization Novel Derivatives: Chemical synthesis opens the door to the creation of novel LOS derivatives that may not be naturally occurring. Researchers can design and synthesize custom LOS molecules with specific modifications to enhance their immunogenicity or therapeutic efficacy. Tailored Functionality: The ability to customize LOS through chemical synthesis allows for the exploration of new functionalities and applications. By manipulating the molecular structure, scientists can tailor LOS to target specific pathogens or elicit desired immune responses. This level of customization is not feasible with naturally derived LOS, which are limited to the structural constraints of the bacterial source. Synthesis of a 2,3-Di-O-Substituted Heptose Tetrasaccharide of Neisseria gonorrhoeae The bacterial polysaccharide tetrasaccharide is composed of mainly four sugars, the central sugar is a heptose connected via 2-O to N-acetylglucosamine and via 3-O to lactose moiety. This tetrasaccharide contains some challenging bonds to construct by glycosylation. The 1,2 cis configurations at positions indicated by A and B in the retrosynthesis scheme is a challenging task to construct in good yield because of the preferred 1,2 trans bond formation selectivity in glycosylation. The retrosynthetic analysis is novel and loosely based on a report by Ishii et. al.1As shown in the retrosynthetic analysis of the tetrasaccharide (Figure 4), synthesis includes the formation of two key 1,2 cis glycosidic bonds. The two key intermediates compound 3 are constructed by glycosylation of the suitably protected heptose sugar 4 and O-trichloroacetimidate glucose intermediate 5. The intermediate compounds 4 and 5 are synthesized from L-lyxose and glucosamine hydrochloride starting materials respectively, which are commercially available. Synthesis of the L-glycero-D-manno-heptose 4 Synthesis of the L-glycero-D-manno-heptose is carried out starting from commercially available sugar D-lyxose, following a reported procedure from the literature1(Figure 5). D-lyxose on treatment with 3-bromopropenyl acetate in the presence of Indium gives the homologated polyol 8 which on purification followed by ozonolysis and acetylation gives the acetylated L- glycero-D-manno-heptose 11 by a reported route by Stanetty et. al. Synthesis of the glucose acetimidate intermediate compound 5 Synthesis of the benzyl 2-azido-2-deoxy-glucosyl trichloroacetimidate compound 5 is carried out from commercially readily available starting material glucosamine hydrochloride (Figure 6). Amine group in glucosamine is converted to azide by reacting with fluorosulfuryl azide reagent in small batches to avoid any hazards of azide formation. The per-O-acetyled compound 13 on thioglycosylation followed by deacetylation, benzyl protection and acetamidate formation gives azido-gluco acetamidate compound 5. Synthesis of the per-O-benzylated lactosyl trichloroacetimidate 2 The per acetylated lactose 16 is subjected to glycosylation with P-methoxy phenol to give p-methoxyphenyl β-lactoside (17). The deacetylation followed by benzyl protection of compound 17 provides compound 18. The anomeric p-methoxyphenol deprotection in 18 using CAN reagent followed by trichloroacetimidate formation provides the intermediate 22(Figure 7). Synthesis of the tetrasaccharide 1 The assembly of the tetrasaccharide 1 starts with the glycosylation of the heptose intermediate 4 and gluco acetimidate compound 5 to give disaccharide compound 19 (Figure 8). The 1,2 cis bond formation in disaccharide 19 is confirmed by 2D characterization. The ether- protecting group deprotection of compound 19 followed by selective O-TES protection of the 3- OH of heptose, acetylation of the 4-OH and 3-O-TES deprotection gives compound 20. Compound 20 is subjected to glycosylation with per-benzylated lactose acetimidate 2 to give a protected tetrasaccharide 21. Azide to NH-acetyl conversion, O-acetyl deprotection followed by hydrogenation provides the final product tetrasaccharide 1. The structural confirmation of the target molecule 1 is done by mass-spec and 2D NMR analysis. EXAMPLE 4. Protocol for Chemical Conjugation of a Lipo-Oligosaccharide (with a Primary Amine) to a Carrier Protein. Materials Required: • Lipo-oligosaccharide: Chemically synthesized, containing a primary amine group • Carrier Protein: E.g. Tetanus toxoid (TT), diphtheria toxoid (DT), or CRM197 Crosslinker: • Glutaraldehyde (for amine-to-amine conjugation) • EDC (1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide) and Sulfo-NHS (N- hydroxysulfosuccinimide) (for carboxyl-to-amine conjugation) Buffer Solutions: • PBS (Phosphate-Buffered Saline), pH 7.4 • MES buffer (2-(N-morpholino)ethanesulfonic acid), pH 6.0 • Dialysis tubing or desalting columns • Rotary evaporator (optional) • UV-Vis Spectrophotometer • Gel Filtration Chromatography system • Sodium cyanoborohydride (optional, for reductive amination) Safety Considerations: • Work in a fume hood when handling glutaraldehyde, EDC, and other reactive chemicals. • Use appropriate personal protective equipment (PPE), including gloves, lab coat, and safety glasses. Conjugation Protocol: 1. Preparation of Carrier Protein Solution: • Dissolve the carrier protein (e.g., tetanus toxoid, diphtheria toxoid, or CRM197) in PBS, pH 7.4, to a concentration of 2-10 mg / mL. If the protein is lyophilized, reconstitute it as per the manufacturer’s instructions. • Place the solution on ice. 2. Preparation of Lipo-Oligosaccharide Solution: • Dissolve the synthesized lipo-oligosaccharide in PBS, pH 7.4, to a concentration of 5-10 mg / mL. Ensure the solution is homogenous by vortexing or gentle heating if necessary. 3. Conjugation Reaction via Glutaraldehyde (Amine-to-Amine Coupling): a. Crosslinker Activation: • Add glutaraldehyde to the carrier protein solution to achieve a final concentration of 0.1% (v / v). This concentration can be adjusted depending on the desired level of conjugation. • Incubate the protein with glutaraldehyde for 30-60 minutes at room temperature with gentle stirring. b. Conjugation: • Slowly add the lipo-oligosaccharide solution to the activated carrier protein solution while stirring. • Continue stirring the mixture at room temperature for 2-4 hours. For optimal conjugation, you may incubate the reaction overnight at 4°C. c. Quenching the Reaction: • Quench the reaction by adding glycine to a final concentration of 20 mM to neutralize any unreacted glutaraldehyde. • Incubate for 30 minutes at room temperature. 4. Conjugation Reaction via EDC / Sulfo-NHS (Carboxyl-to-Amine Coupling): a. Activation of the Lipo-Oligosaccharide: • If the lipo-oligosaccharide has a carboxyl group (from sialic acid, for example), dissolve it in MES buffer, pH 6.0. • Add EDC (10 mM final concentration) and Sulfo-NHS (25 mM final concentration) to the solution. Incubate for 15-30 minutes at room temperature. b. Conjugation: • Add the activated lipo-oligosaccharide to the carrier protein solution in PBS, pH 7.4, while stirring. • Allow the reaction to proceed for 2-4 hours at room temperature or overnight at 4°C. c. Quenching the Reaction: • Quench the reaction by adding glycine (final concentration of 20 mM) and incubate for 30 minutes. 5. Purification of the Conjugate: a. Dialysis: • Dialyze the reaction mixture against PBS, pH 7.4, to remove unreacted lipo- oligosaccharide, crosslinker, and other small molecules. Use a dialysis membrane with an appropriate molecular weight cut-off (MWCO). b. Gel Filtration Chromatography: • Alternatively, purify the conjugate using gel filtration chromatography to separate the conjugated product from unreacted components. c. Concentration: • If necessary, concentrate the purified conjugate using a centrifugal concentrator or rotary evaporator. 6. Characterization of the Conjugate: a. UV-Vis Spectrophotometry: • Measure the absorbance at 280 nm to estimate the protein concentration and at other relevant wavelengths (e.g., 260 nm for nucleic acids, 490-520 nm for certain chromophores) to confirm the presence of the lipo-oligosaccharide. b. SDS-PAGE: • Run an SDS-PAGE to assess the conjugate’s molecular weight and to verify successful conjugation. c. HPLC or Mass Spectrometry: • Use HPLC or mass spectrometry for detailed analysis of the conjugate and to confirm the ratio of lipo-oligosaccharide to protein. Lipo-Oligosaccharide Conjugation to Carrier Protein The chemically synthesized lipo-oligosaccharide, which contains a primary amine group, may be conjugated to a carrier protein (e.g., tetanus toxoid, diphtheria toxoid, or CRM197) using two distinct chemical strategies: glutaraldehyde-mediated amine-to-amine coupling and EDC / Sulfo-NHS-mediated carboxyl-to-amine coupling. For the glutaraldehyde conjugation, the carrier protein is first activated with 0.1% glutaraldehyde in PBS, pH 7.4, for 1 hour at room temperature. The lipo-oligosaccharide is then added to the activated protein solution, and the reaction is incubated at room temperature for 4 hours with gentle stirring. The reaction is quenched by adding glycine (20 mM final concentration) and incubated for an additional 30 minutes. For the EDC / Sulfo-NHS coupling, the lipo-oligosaccharide is activated with 10 mM EDC and 25 mM Sulfo-NHS in MES buffer, pH 6.0, for 30 minutes at room temperature. The activated lipo-oligosaccharide is then added to the carrier protein in PBS, pH 7.4, and the reaction is allowed to proceed for 4 hours at room temperature. The reaction is quenched with glycine as described above. Following conjugation, the reaction mixtures are dialyzed against PBS, pH 7.4, using a dialysis membrane with an appropriate MWCO to remove unreacted reagents. The conjugates are further purified by gel filtration chromatography. The resulting conjugates are characterized by UV-Vis spectrophotometry, SDS-PAGE, and HPLC to confirm successful conjugation and to determine the protein-to-lipo-oligosaccharide ratio. EXAMPLE 5. Retrosynthetic analysis of an LOS tridecasaccharide. Figure 9 shows the synthesis scheme of an LOS tridecasaccharide. As shown in Figure 10, the suitably protected glucose trichloroacetimidate 1 is subjected to glycosylation with heptose sugar 2 and gives the disaccharide fragment F. The fragment E is synthesized by glycosylating galactose trichloroacetamidate sugar 4 with the suitably protected galactose derivative 5 to give the disaccharide compound 6. The disaccharide 6 is then be subjected for glycosylation with sialic acid sugar 7 to provide trisaccharide fragment E (Figure 11). The synthesis of fragment D is achieved by reacting fragment E and F to form the pentasaccharide fragment D (Figure 12). The synthesis of fragment C starts with the glycosylation of suitably protected galactosamine 12 and galactose sugar 13 to give disaccharide 14 (Figure 13). The regioselective benzylidene ring opening of the disaccharide 14 gives the disaccharide with free hydroxyl compound 15. The disaccharide on glycosylation using sialic acid 16 provides the trisaccharide compound 17. The trisaccharide 17 on further glycosylation with 4C-OH free hydroxyl glucosamine 18 leads to the formation of the tetrasaccharide fragment C. The synthesis of fragment A is achieved by coupling of fragments C and D (Figure 14). The suitably protected fragments C and D are reacted together to give fragment A a nonasaccharide. Finally, the coupling of fragment A and fragment B under glycosylation conditions using a suitable Lewis acid catalyst provides the final oligosaccharide tridecasaccharide (Figure 15). EXAMPLE 6. LOS-Protein Conjugation Approaches. A: Oxidation and Reductive Amination (Simple Aldehyde-Amine Linkage) Step 1: Oxidize the terminal sugar residues of LOS to form aldehyde groups. Step 2: React the aldehyde groups with the primary amine groups (typically on lysine residues) of a carrier protein such as tetanus toxoid. Step 3: The reaction forms a Schiff base intermediate, which is then reduced using a mild reducing agent like sodium borohydride, creating a stable covalent bond. B: EDC / NHS Coupling of Carboxylated LOS (Carboxyl-Amine Coupling) Step 1: Synthesize or modify LOS to introduce carboxyl groups. Step 2: Activate these carboxyl groups using EDC / NHS, which converts them into an active ester intermediate. Step 3: React the active ester with amine groups on the carrier protein, such as CRM197, forming a stable amide bond. C: Click Chemistry Conjugation (Azide-Alkyne Cycloaddition) Step 1: Functionalize LOS with an azide group during the chemical synthesis process. Step 2: Modify the carrier protein (e.g., KLH or tetanus toxoid) with an alkyne group. Step 3: Use CuAAC (copper-catalyzed azide-alkyne cycloaddition) to covalently link the LOS to the protein under mild reaction conditions, forming a stable triazole ring. Quality Control and Verification of Conjugation: Once the LOS is conjugated to the protein, it is essential to verify the efficiency of conjugation and ensure the integrity of both the LOS and the carrier protein. This can be achieved using analytical techniques such as: SDS-PAGE: To check the size and conjugation of the protein. Mass Spectrometry: To determine the molecular mass of the conjugated product and confirm the presence of both LOS and the protein. HPLC / SEC (Size-Exclusion Chromatography): To assess the purity and molecular size of the conjugated product. Immunoassays (ELISA): To verify that the conjugated LOS retains its antigenicity and can be recognized by specific antibodies. Advantages of LOS-Protein Conjugates: Enhanced Immunogenicity: The conjugated LOS is more immunogenic than LOS alone because the protein helps stimulate a stronger immune response, particularly by activating helper T cells. Long-Lasting Immunity: T-cell dependent responses generated by protein conjugation lead to memory B cells, which are crucial for long-term protection. Improved Vaccine Design: Conjugation allows the combination of different LOS epitopes with various carrier proteins, creating multivalent vaccines capable of protecting against multiple strains or pathogens. Table 1. Summary of Conjugation Methods: Method Functional Groups Example Reaction Carrier Protein a enhancing vaccine efficacy while ensuring safety and stability for large-scale production. EXAMPLE 7. Evaluation of immunogenicity of LOS conjugate vaccine The following is an example protocol for evaluating immunogenicity of an LOS conjugate vaccine. Any LOS, carrier, and adjuvant as described herein may be evaluated using this protocol. Immunization of mice: CD1 mice are initially immunized with three doses (e.g., 1, 5 and 20 µg) of synthetic tetrasaccharide-CRM197 conjugate adjuvanted with either Alum, AddaVax (similar to MF59; InVivoGen), AddS03 (similar to AS03; InVivoGen), GLA-SE and 3M-052-SE (the latter two from the Access to Advanced Health Institute [AAHI]). Outbred 6 week-old CD1 mice are used because they have a wider antibody repertoire than inbred strains and may more accurately represent the human population. To address sex as a biological variable, each immunization group contains 4 male and 4 female mice. Vaccines (or adjuvant alone control) are given intramuscularly (IM), the preferred route in humans. Vaccine (or adjuvant alone for controls) is administered at 0, 3 and 6 weeks. Sera is collected pre-immunization, and two weeks post-doses 2 and 3. Antibody (total IgG) measurements by ELISA: Antibody levels in sera against the 2C7 epitope is measured using LOS purified from Ng 15253 (expresses 2C7+ve LOS) using the hot water-phenol extraction method. The isogenic ΔlgtG mutant (2C7-ve LOS) is used as a control for specificity. Comparisons of total IgG (as OD405nm measurements) across groups are made by 2- way ANOVA. IgG subclass analysis: We also pool sera from each group and measure IgG subclasses. The ratio [(IgG2a+IgG3)x0.5] / IgG1 is calculated; ratios >1 or <1 indicate Th1 or Th2 responses, respectively. Immunogenicity in BALB / c mice and durability of Ab responses: We repeat immunizations in CD1 mice and in BALB / c mice to ensure replicability and robustness of the data. BALB / c mice are most commonly used in the mouse vaginal colonization model, therefore it is important to ensure good immune responses in this mouse strain in preparation for future protection experiments. Sera is obtained every 4 weeks after the post-dose 3 bleed for up to 6 months to determine the durability of Ab responses. Mouse numbers, LOS ELISA assays and analysis are described above. Serum bactericidal assays (SBAs): Sera that yield the best Ab responses are assayed for their ability to kill Ng FA1090 (expresses the PorB1B major outer membrane protein) and 15253 (PorB1A) in complement-dependent bactericidal assays. Both strains are otherwise highly resistant to killing by normal human serum and therefore provide a stringent test of functional activity of immune Ab. It has been observed that naïve mouse sera contain natural IgM that is bactericidal against Ng. Therefore, IgM in all sera is depleted by passage through anti-mouse IgM agarose. This permits us to isolate the function of immune IgG. Human complement (IgG and IgM depleted human serum) is purchased from Pel-Freez and used at a final concentration of 10% to simulate complement concentrations in human cervico-vaginal secretions. Antisera is incubated with bacteria at 2-fold dilutions starting at 1:2, and the serum dilution that yields ~50% bacterial survival (IC50) is calculated by plotting survival versus serum dilution and non-linear regression (best-fit) (GraphPad® Prism). We also test isogenic lgtG deletion mutants of FA1090 and 15253 to establish that killing is specifically directed at the 2C7 epitope. These IC50 data guide the selection of serum dilutions to measure the bactericidal activity of immune sera against 20 minimally passaged diverse clinical isolates selected from strains collected between 2009-2013 from a clinic in Nanjing, China, which includes multidrug-resistant isolates. Contemporary MDR isolates are also tested. Expected results. We expect a Th1-biased IgG response (predominance of IgG2a and IgG3) to elicit bactericidal activity against diverse Ng isolates, including minimally passaged MDR clinical isolates. References 1. Ishii, K., Esumi, Y., Iwasaki, Y. and Yamasaki, R. (2004), Synthesis of a 2,3-Di-O-substituted Heptose Structure by Regioselective 3-O-Silylation of a 2-O-Substituted Heptose Derivative. Eur. J. Org. Chem., 2004: 1214-1227. 2. Stanetty, C. and Baxendale, I.R. (2015), Large-Scale Synthesis of Crystalline 1,2,3,4,6,7- Hexa-O-acetyl-L-glycero-α-D-manno-heptopyranose. Eur. J. Org. Chem., 2015: 2718-2726. 3. Meng, G., Guo, T., Ma, T. et al. Modular click chemistry libraries for functional screens using a diazotizing reagent. Nature 574, 86–89 (2019). 4. Kazuyuku Ishii, Hiroyuki Kubo, Ryohei Yamasaki, Synthesis of α-lactosyl-(1→3)-l-glycero-α- d-manno-heptopyranoside, a partial oligosaccharide structure expressed within the lipooligosaccharide produced by Neisseria gonorrhoeae strain 15253, Carbohydrate Research, Volume 337, Issue 1, 2002, Pages 11-20, ISSN 0008-6215. While the invention has been described in terms of its preferred embodiments, those skilled in the art will recognize that the invention can be practiced with modification within the spirit and scope of the appended claims. Accordingly, the present invention should not be limited to the embodiments as described above, but should further include all modifications and equivalents thereof within the spirit and scope of the description provided herein.

Claims

CLAIMS 1. An immunogenic composition comprising a Neisseria gonorrhoeae lipooligosaccharide (LOS), or a fragment thereof, conjugated to a carrier.

2. The immunogenic composition of claim 1, wherein the carrier is selected from the group consisting of a carrier protein, virus-like particle (VLP), liposome, inorganic gold particle, dendrimer, outer membrane vesicle (OMV), generalized modules for membrane antigens (GMMA), ferritin nanoparticles, and protein nanocages.

3. The immunogenic composition of claim 1, wherein the carrier is a carrier protein and the LOS or fragment thereof has the following formula:wherein R1 is lactose or PEtn; R2 is H or OAc; R3 is H or PEtn; R4 is H or Gal; and X is the carrier protein.

4. The immunogenic composition of claim 3, wherein the carrier protein is selected from the group consisting of tetanus toxoid, diphtheria toxoid, keyhole limpet hemocyanin, CRM197, and outer membrane protein complex (OMPC).

5. The immunogenic composition of claim 1, wherein a linker is present between the LOS and the carrier.

6. The immunogenic composition of claim 5, wherein the linker is a polyethylene glycol (PEG) or maleimide-PEG linker.

7. The immunogenic composition of claim 1, further comprising one or more additional bacterial antigens.

8. The immunogenic composition of claim 7, wherein the one or more additional bacterial antigens are selected from the group consisting of PorB, Neisserial Heparin Binding Antigen (NHBA), Neisserial surface protein A (NspA), Multiple transferable resistance E (MtrE), Transferrin binding proteins A and B (TbpA and TbpB), TdfJ, Methionine-binding lipoprotein (MetQ), Opacity protein (Opa), ,NGO1549, and NGO0265.

9. A vaccine composition comprising the immunogenic composition of claim 1 and a pharmaceutically acceptable carrier.

10. The vaccine composition of claim 9, further comprising an adjuvant.

11. A method of protecting or treating a subject from Neisseria gonorrhoeae infection comprising administering to the subject the immunogenic composition of claim 1.

12. A method of manufacturing an immunogenic composition, comprising isolating or synthesizing a Neisseria gonorrhoeae LOS or fragment thereof; and conjugating the LOS or fragment thereof to a carrier.

13. The method of claim 12, wherein the carrier is a carrier protein and is conjugated to the LOS via reductive amination, carbodiimide coupling with N-hydroxysuccinimide (EDC / NHS coupling), click chemistry, or maleimide-thiol coupling.

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