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WO2025097030A1 - Particules pseudo-virales présentant des antigènes de neisseria gonorrhoeae et leur utilisation pour l'immunisation contre la gonorrhée - Google Patents

Particules pseudo-virales présentant des antigènes de neisseria gonorrhoeae et leur utilisation pour l'immunisation contre la gonorrhée Download PDF

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WO2025097030A1
WO2025097030A1 PCT/US2024/054230 US2024054230W WO2025097030A1 WO 2025097030 A1 WO2025097030 A1 WO 2025097030A1 US 2024054230 W US2024054230 W US 2024054230W WO 2025097030 A1 WO2025097030 A1 WO 2025097030A1
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slic
seq
immunogenic composition
acid sequence
amino acid
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Aleksandra E. SIKORA
Fabian MARTINEZ
Adam Frederik SANDER BERTELSEN
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Adaptvac
Oregon State University
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Adaptvac
Oregon State University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/095—Neisseria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/22—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Neisseriaceae (F)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/543—Mucosal route intranasal
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/735—Fusion polypeptide containing domain for protein-protein interaction containing a domain for self-assembly, e.g. a viral coat protein (includes phage display)

Definitions

  • gonorrhea is the second most reported notifiable disease in the United States after chlamydia and the number of cases has risen steadily since the historic low in 2009, increasing by 92% (a total of 616,392 reported cases) in 2019 (CDC, 2018 Sexually Transmitted Diseases Surveillance, 2019). Globally, approximately 87 million gonorrhea infections occurred in 2016 but these statistics are underestimated due to frequent asymptomatic infections (Rowley et al., Bull World Health Organ 97(8):548-562P, 2019; Rice et al., Ann Rev Microbiol 71:665-686, 2017).
  • Neisseria gonorrhoeae (Ng), the gram-negative bacterium and etiological agent of gonorrhea, is categorized as a high-priority pathogen for research and development efforts (World Health Organization, Global priority list of antibiotic-resistant bacteria to guide research, discovery, and development of new antibiotics, 2017; Gottling et al., Vaccine 38(28):4362-73, 2020; CDC, Antibiotic resistance threats in the United States, 2019).
  • gonorrhea In addition to high prevalence and antibiotic resistance, the need for developing an effective gonorrhea vaccine is exacerbated by the brunt of gonorrhea, including infertility and its ability to augment transmission and acquisition of HIV (Fleming and Wasserheit, Sex Transm Infect 75(1):3-17, 1999). In women, gonorrhea may lead to pelvic inflammatory disease, miscarriage, preterm birth, and ectopic pregnancies. In males, this STI presents as uncomplicated urethritis but can ascend to the epididymis or testes (Trojian et al., Am Fam Physician 79(7):583-587, 2009).
  • Gonorrhea primarily affects the genitourinary tract, but other mucosal surfaces can be involved and disseminated disease may also occur (Rice, Infect Dis Clin North Am 19(4):853-861, 2005; Barr and Danielsson, Br Med J 1(5747):482-485, 1971; Knapp and Holmes, J Infect Dis 132(2):204-208, 1975; Lochner and Maraqa, Pediatr Drugs 20(6):501-509, 2018; Humbert and Christodoulides, Pathogens 9(1):10, 2019).
  • Neonatal conjunctivitis can be acquired from the infected birth canal, which if left untreated, can result in corneal scarring and blindness (Humbert and Christodoulides, Pathogens 9(1):10, 2019; Mallika et al., Malays Fam Physician 3(2):77-81, 2008; Yeu and Hauswirth, Clin Ophthalmol 14:805-813, 2020).
  • SUMMARY Described herein are immunogenic compositions that display conserved N. gonorrhoeae antigens on the surface of virus-like particles (VLPs), such as VLPs formed by the capsid protein of an RNA bacteriophage.
  • VLPs virus-like particles
  • immunogenic compositions for protection against gonorrhea.
  • immunogenic compositions that include a capsid protein of an RNA bacteriophage fused to a first peptide tag, and a N. gonorrhoeae antigen fused to a second peptide tag.
  • the first peptide tag and the second peptide tag are joined by an isopeptide bond, and the capsid protein and antigen form a virus-like particle (VLP) displaying the antigen.
  • VLP virus-like particle
  • the antigen is selected from the group consisting of surface-exposed lysozyme inhibitor of c-type lysozyme (SliC), methionine binding protein (MetQ), Neisserial adhesin complex protein (ACP), ⁇ -barrel assembly machinery protein E (BamE), ⁇ -barrel assembly machinery protein G (BamG) and anaerobically induced outer membrane protein A (AniA).
  • the RNA bacteriophage is AP205.
  • the first peptide tag is a SpyTag peptide and the second peptide tag is a SpyCatcher peptide; or the first peptide tag is a SpyCatcher peptide and the second peptide tag is a SpyTag peptide.
  • the immunogenic composition further includes a pharmaceutically acceptable carrier and/or an adjuvant (such as CpG oligodeoxynucleotides and/or a squalene-based oil-in-water emulsion). Also provided herein are nucleic acid molecules that encode the immunogenic compositions disclosed herein.
  • the nucleic acid molecule encodes the first peptide tag fused to the capsid 245-110515-02 OSU-22-30 protein of the RNA bacteriophage, the second peptide tag fused to the antigen, or both.
  • vectors that include the disclosed nucleic acid molecules and isolated host cells that contain the nucleic acid molecules and vectors. Methods of eliciting an immune response against N. gonorrhoeae in a subject, and methods of immunizing a subject against N. gonorrhoeae, by administering to the subject an effective amount of an immunogenic composition disclosed herein are also provided.
  • FIGS 1A-1C Design of the SpyTag plasmid system for the Tag/Catcher-AP205 capsid virus-like particle (cVLP) platform for gonorrhea vaccine development.
  • FIG.1A An outline of in silico designed genetic engineering process to develop the SpyTag plasmid system using pET-22b(+), sliC from Ng FA1090, gene blocks carrying SpyTag on the N- or C-terminus (STN and STC, respectively), a linker, a multicloning site, a tobacco etch virus (TEV) protease cleavage site and a 10 ⁇ His Tag.
  • FIG.1B Gibson assembly was used to clone STN and STC gene blocks to pET-22b(+) yielding pET22-STN and pET22-STC that enable fusion of antigens with the SpyTag on either the N- or C-terminus, respectively.
  • the coli PelB signal sequence is also added to promote proper antigen folding in a heterologous host.
  • the sliC gene was cloned into pET22-STN and pET22-STC to create pET22-N-SliC and pET22-C-SliC.
  • the core AP205 cVLP displaying a complementary Catcher (SpyCatcher VLP) is expressed in E. coli and purified.
  • the AP205 cVLP Catcher is mixed in solution with purified N-SliC or C-SliC.
  • FIGS.2A-2D Assembly and quality assessment of the Tag/Catcher SliC-VLP vaccine.
  • E. coli BL21(DE3) carrying pET22-N-SliC and pET22-C-SliC (N and C, respectively) were cultured without (-) and with (+) IPTG.
  • the whole cell extracts were normalized by OD 600 , separated by SDS-PAGE and stained with Colloidal Coomassie.
  • FIGS.3A-3D The N-SliC-VLP vaccine adjuvanted with ADDAVAX markedly induced antibody titers compared to the corresponding vaccine containing monomeric N-SliC.
  • FIG.3A Systemic total IgG titers were examined in pre-immune (P) and ten days after first (1), second (2) and third (3) subcutaneous administration of cVLP, N-SliC, or N-SliC-VLP. All treatments were adjuvanted with ADDAVAX.
  • FIG. 3B Total IgG, IgG1, IgG2a, IgG3 and IgA antibody titers in final sera from mice immunized with cVLP, N- SliC, or N-SliC-VLP.
  • FIG.3C Vaginal total IgG titers were examined in pre-immune (P) and ten days after first (1), second (2) and third (3) subcutaneous administration of cVLP, N-SliC, or N-SliC-VLP.
  • FIG. 3D Total IgG, IgG1, IgG2a, and IgA titers in final vaginal lavages obtained from mice immunized with cVLP, N-SliC, or N-SliC-VLP. Bar graphs represent geometric mean ELISA titers with error bars showing 95% confidence limits. Statistical significance was determined using Kruskal-Wallis with Dunn’s multiple comparison test.
  • FIGS.4A-4C SliC-specific systemic IgG and IgA and vaginal IgG were elicited by subcutaneous immunization with N-SliC-VLP vaccine adjuvanted with ADDAVAX.
  • Female mice were subcutaneously immunized with cVLP, N-SliC, or N-SliC-VLP adjuvanted with ADDAVAX.
  • Purified N-SliC (FIGS.4A and 4B) and whole cell extracts obtained from the isogenic Ng strain FA1090, the ⁇ sliC knockout, the complemented ⁇ sliC/P::sliC, and a panel of geographically, genetically, and temporally diverse Ng isolates were fractionated by SDS-PAGE. Immunoblotting was performed with pooled serum (FIGS.4A and 4C) and vaginal washes (FIG.4B) collected after the third immunization, followed by secondary antibodies against mouse IgG (FIGS.4A and 4C) or IgA (FIGS.4A and 4B).
  • FIGS.5A-5F Anti-SliC antibody titers elicited by N-SliC and N-SliC-VLP subcutaneous and intranasal immunization.
  • Post-immunization d31 and d52 total IgG (FIG.5A), IgG1 (FIG.5B), IgG2a (FIG.5C), IgG3 (FIG.5D) and IgA (FIG.5E) antibody titers in sera from mice immunized with N-SliC- VLP-ADDAVAX (2.5 and 5 ⁇ g/dose), N-SliC-VLP-CpG (2.5, 5, and 10 ⁇ g/dose), N-SliC, VLP- ADDAVAX, cVLP-CpG, or unimmunized (PBS).
  • N-SliC- VLP-ADDAVAX 2.5 and 5 ⁇ g/dose
  • FIG.5F Post-immunization (d32) vaginal IgG and IgA titers were assessed in female mice administered with N-SliC-VLP-ADDAVAX (2.5 and 5 ⁇ g/dose), N- SliC-VLP-CpG (2.5, 5, and 10 ⁇ g/dose), N-SliC, cVLP-ADDAVAX, cVLP-CpG, or PBS. Bar graphs represent geometric mean ELISA titers with error bars showing 95% confidence limits. Statistical significance between data in groups was determined using Kruskal-Wallis with Dunn’s multiple comparison test. For the comparison of two groups, the non-parametric Mann-Whitney U test was carried out.
  • FIGS.6A-6C N-SliC-VLP-ADDAVAX/CpG vaccines elicited SliC-specific systemic and vaginal IgG and IgA after subcutaneous immunization and intranasal boost.
  • Female mice were administered N-SliC- VLP-ADDAVAX (Add; 2.5 and 5 ⁇ g/dose), N-SliC-VLP-CpG (2.5, 5, and 10 ⁇ g/dose), N-SliC, cVLP- ADDAVAX (cVLP-Add), cVLP-CpG, or PBS, as indicated.
  • Purified N-SliC (FIGS.6A and 6B) and whole cell extracts obtained from the isogenic Ng strain FA1090, the ⁇ sliC knockout, the complemented 245-110515-02 OSU-22-30 ⁇ sliC/P::sliC, the 2016 WHO Ng panel and FA6146 were separated by SDS-PAGE. Immunoblotting was performed with murine pooled serum (FIGS.6A and 6C) and vaginal washes (FIG.6B) collected after the second immunization, followed by secondary antibodies against mouse IgG (FIGS.6A and 6C) or IgA (FIGS.6A and 6B).
  • FIGS.7A-7D Assessment of SliC and adhesin complex protein (ACP) activity against human lysozyme.
  • FIGS.7A-7D Assessment of SliC and adhesin complex protein (ACP) activity against human lysozyme.
  • FIGS.7A-7D Assessment of SliC and adhesin complex protein (ACP) activity against human lysozyme.
  • FIGS.7A-7D Assessment of SliC and adhesin complex protein (ACP) activity against human lysozyme.
  • FIGS.7A-7D Assessment of SliC and adhesin complex protein (ACP) activity against human lysozyme.
  • FIGS.7B-7D To examine if immunization with SliC/ACP elicits antigen function blocking antibodies, SliC/ACP were incubated with pooled sera from immunized rabbits (FIGS.7B and 7C), immunized mice (FIGS.7B and 7D), or control groups (1:10 v/v) for 30 minutes and the lysozyme assays were carried out as described above.
  • FIGS.8A-8E Serum antibody titers elicited by intramuscular (IM) immunization with SliC-VLP or SliC-VLP+CpG vaccines.
  • IM intramuscular
  • Serum IgG, IgG1, IgG2a, IgG3 and IgA were assessed on Days 31 and 52 following immunization. Each dot represents antibody titer in an individual mouse.
  • FIGS.9A-9B Vaginal IgG and IgA antibody titers elicited by IM immunizations with SliC-VLP or SliC-VLP+CpG vaccines. Each dot represents antibody titer in an individual mouse.
  • FIGS.10A-10E Serum antibody responses elicited by SliC-VLP and SliC-VLP+CpG vaccines. Mice received one subcutaneous (SC) dose and two intranasal (IN) doses of vaccine.
  • Serum IgG, IgG1, IgG2a, IgG3 and IgA were assessed on Days 31 and 52 after immunization. Each dot represents antibody titer in an individual mouse.
  • FIGS.11A-11B Vaginal SliC-specific IgG and IgA elicited by one SC and two IN immunizations with SliC-VLP or SliC-VLP+CpG vaccines. Each dot represents antibody titer in individual mouse.
  • FIGS.12A-12D Serum and vaginal IgG and IgA elicited by three IM immunizations with ACP alone or ACP-VLP.
  • FIGS.13A-13B Serum and vaginal antibody responses elicited in mice immunized with ACP+CpG or ACP-SliC+CpG vaccines administered IN, or with ACP-VLP or ACP-VLP+SliC-VLP vaccines administered IM.
  • Antibody titers were determined on Days 31, 52 and 75 following immunization. Each dot represents antibody titer in an individual mouse.
  • FIGS.14A-14E Serum antibody responses elicited by immunization with MetQ-CpG or MetQ- VLP vaccines.
  • SEQ ID NO: 1 is an exemplary amino acid sequence of the AP205 capsid protein (GENBANK Accession No. NP_085472.1).
  • SEQ ID NO: 2 is an exemplary amino acid sequence of a SpyTag peptide.
  • AHIVMVDAYKPTK SEQ ID NO: 3 is an exemplary amino acid sequence of a SpyCatcher peptide.
  • SEQ ID NO: 4 is an exemplary amino acid sequence of the N. gonorrhoeae antigen SliC. PEAYDGGGRGYMPPVQNQAGPDDFRAFSCENGLSVRVRNLDGGKIALRLDGRRAVLSSDVAASGE RYTAEHGLFGNGTEWHQKGGEAFFGFTDAYGNSVETSCRAR
  • SEQ ID NO: 5 is an exemplary amino acid sequence of the N. gonorrhoeae antigen MetQ.
  • SEQ ID NO: 7 is an exemplary amino acid sequence of the N. gonorrhoeae antigen BamE.
  • SEQ ID NO: 8 is an exemplary amino acid sequence of the N.
  • SEQ ID NO: 9 is an exemplary amino acid sequence of the N. gonorrhoeae antigen AniA.
  • PAAQAPAETPAASAEAASSAAQATAETPAGELPVIDAVTTHAPEVPPAIDRDYPAKVRVKMETVEK TMKMDDGVEYRYWTFDGDVPGRMIRVREGDTVEVEFSNNPSSTVPHNVDFHAATGQGGGAAATF TAPGRTSTFSFKALQPGLYIYHCAVAPVGMHIANGMYGLILVEPKEGLPKVDKEFYIVQGDFYTKG KKGAQGLQPFDMDKAVAEQPEYVVFNGHVGAIAGDNALKAKAGETVRMYVGNGGPNLVSSFHVI GEIFDKVYVEGGKLINENVQSTIVPAGGSAIVEFKVDIPGNYTLVDHSIFRAFNKGALGQLKVEGAE NPEIMTQKLSDTAYAGSGAASAPAASAPAASAS SEQ ID NO: 10 is a nucleic acid sequence of the SpyTag gene block with linker.
  • SEQ ID NO: 13 is an exemplary nucleic acid sequence encoding SliC-SpyTagN.
  • SEQ ID NO: 17 is an exemplary nucleic acid sequence encoding SliC-SpyCatcherN.
  • SEQ ID NO: 21 is an exemplary nucleic acid sequence encoding ACP-SpyTagN.
  • SEQ ID NO: 25 is an exemplary nucleic acid sequence encoding ACP-SpyCatcherN.
  • SEQ ID NO: 35 is an exemplary nucleic acid sequence encoding BamE-SpyCatcherC.
  • SEQ ID NO: 39 is an exemplary nucleic acid sequence encoding BamG-SpyTagC.
  • SEQ ID NO: 45 is an exemplary nucleic acid sequence encoding MetQ-SpyTagN.
  • SpyTag sequence SEQ ID NO: 61 is the amino acid sequence of SpytagN_BamG_TEV site_10x His.
  • proteomics and bioinformatics were used to identify conserved vaccine antigens (Zielke et al., Mol Cell Proteomics 15(7):2338-2355, 2016; Zielke et al., Mol Cell Proteomics 13(5):1299-1317, 2014; El-Rami et al., Mol Cell Proteomics 18(1):127-50, 2019; Baarda et al., mSphere 6(1):e00977-20, 2021). Thirty-four gonorrhea protein antigens were discovered through proteome-based reverse vaccinology studies and traditional approaches.
  • the gene sliC (locus NEIP0196) has a total of 12 alleles and 22 single nucleotide polymorphisms (SNPs). There are only eight different amino acid sequences with 11 single amino acid polymorphisms distributed in less than 4% of Ng isolates globally (Baarda et al., mSphere 6(1):e00977-20, 2021).
  • Subunit protein vaccines can fail due to low immunogenicity caused by small antigen size, instability, or improper presentation to the immune system (Thrane et al., J Nanobiotechnology 14:30, 2016; Amanna et al., N Engl J Med 357(19):1903-1915, 2007). Moreover, considering the mechanisms Ng uses to evade the human immune system, an effective vaccine may need to induce a stronger/different type of immune response compared to that elicited during infection (Gott Kunststoff Kunststoffet al., Vaccine 38(28):4362-73, 2020; Russell et al., Front Immunol 10:2417, 2019; Vincent and Jerse, Vaccine 37(50):7419-7426, 2019).
  • Subunit vaccines based on virus-like particles can induce potent B cell responses in humans (Schiller and Lowy, Vaccine 36(32 Pt A):4768-73, 2018; Aves et al., Viruses 12(2):185, 2020), which has led to the licensure of several vaccines, including hepatitis B, human papillomavirus (HPV), malaria, and hepatitis E vaccines.
  • a single dose of the HPV vaccine elicited highly durable (potentially lifelong) antibody responses in humans (Mohsen and Bachmann, Cell Mol Immunol 19(9):993-1011, 2022).
  • cVLPs The intrinsic immunogenicity of cVLPs extends to protein antigens, which are displayed at high density in an orderly fashion on the cVLP (Faizan Zarreen Simnani et al., Materials Today 66, 2023:371-408, 2023). This is especially apparent for antigens that are otherwise weak immunogens (Chackerian, Expert Rev Vaccines 6(3):381-390, 2007; Schodel et al., J Exp Med 180(3):1037-1046, 1994).
  • conserved Ng antigens such as SliC
  • conserved Ng antigens such as SliC
  • cVLPs were formulated using the Tag/Catcher AP205 cVLP platform (Thrane et al., J Nanobiotechnology 14:30, 2016; Aves et al., Viruses 12(2):185, 2020; Zakeri et al., Proc Natl Acad Sci USA 109(12):E690-E697, 2012).
  • the Tag/Catcher- AP205 cVLP uses a split-protein based conjugation system, which was developed by the separation of a bacterial pilin protein into a reactive peptide (Tag) and corresponding protein binding partner (Catcher) (Thrane et al., J Nanobiotechnology 14:30, 201; Zakeri et al., Proc Natl Acad Sci USA 109(12):E690-E697, 2012). Upon mixing in solution, the Tag and Catcher rapidly form a spontaneous isopeptide bond. This platform was developed by genetically fusing AP205 capsid to the split-protein Tag or Catcher, thus displaying 180 copies on the cVLP surface.
  • the Tag/Catcher-AP205 has been utilized to display structurally and functionally diverse antigens, ranging in size from small peptides to large proteins (Escolano et al., Nature 570(7762):468-473, 2019).
  • the resultant VLP-displayed antigens induce antibody titers of higher quality, affinity, and avidity (Thrane et al., J Nanobiotechnology 14:30, 2016; Leneghan et al., Sci Rep 7(1):3811, 2017; Palladini et al., Oncoimmunology 7(3):e1408749, 2018; Fougeroux et al., Nat Commun 12(1):324, 2021).
  • VLPs Protein and peptide antigens are frequently displayed on VLPs either through the genetic fusion of epitopes to the self-assembling coat protein or chemical conjugation to the surface of pre-assembled VLPs.
  • These strategies have their drawbacks, including limitation on antigen size, low-density coupling, interference with VLP assembly, and narrow, epitope-specific antibody responses (Aves et al., Viruses 12(2):185, 2020; Chackerian, Expert Rev Vaccines 6(3):381-390, 2007; Leneghan et al., Sci Rep 7(1):3811, 2017).
  • the Tag/Catcher-AP205 cVLP platform comprised of peptide counterparts SpyTag and SpyCatcher that form irreversible spontaneous isopeptide bond may circumvent these challenges (Zakeri et al., Proc Natl Acad Sci USA 109(12):E690-E697, 2012).
  • the Acinetobacter phage AP205 has a unique structure in that both the N- and C- termini are surface exposed and evenly distributed on the assembled VLP, allowing for genetic fusions at both termini while maintaining stable assembly. Additionally, AP205 has intrinsic immunogenicity, a lack of pre-existing immunity in humans, and can be produced in a cost-effective manner in E.
  • the present disclosure uses the Tag/Catcher-AP205 cVLP for delivery of novel gonorrhea antigens (SliC, ACP and MetQ), and tests different vaccine formulations, doses, and immunization routes.
  • novel gonorrhea antigens SliC, ACP and MetQ
  • the studies disclosed herein demonstrate that vaccines containing monomeric N-SliC failed to induce SliC-specific antibodies when administered alone or with ADDAVAX adjuvant via different immunization routes in mice (see FIGS.3-6).
  • the multivalent repetitive and particulate display of N-SliC via the Tag/Catcher-AP205 cVLP significantly potentiated its immunogenicity as shown by increased kinetics of antibody responses, markedly induced antibody titers in ELISA, serum and vaginal SliC-specific IgG and/or IgA, and functional antibodies with serum bactericidal killing assay (SBA) activity (see FIGS.3-6, Table 1).
  • ACP-VLP and MetQ-VLP compositions are also shown to induce strong serum and vaginal antibody responses (FIGS.12, 14 and 15), such as when administered, IM, SC and/or IN.
  • the disclosed immunogenic compositions satisfy an unmet need for an effective vaccine against gonorrhea. II.
  • Acyl carrier protein A highly conserved N. gonorrhoeae protein.
  • An exemplary ACP protein sequence is set forth herein as SEQ ID NO: 6.
  • Anaerobically induced outer membrane protein A A highly conserved N. gonorrhoeae protein.
  • An exemplary AniA protein sequence is set forth herein as SEQ ID NO: 9.
  • ⁇ -barrel assembly machinery E (BamE): A highly conserved N. gonorrhoeae protein.
  • An exemplary BamE protein sequence is set forth herein as SEQ ID NO: 7.
  • ⁇ -barrel assembly machinery G (BamG): A highly conserved N. gonorrhoeae protein.
  • An exemplary BamG protein sequence is set forth herein as SEQ ID NO: 8.
  • Adjuvant A component of an immunogenic composition used to enhance antigenicity.
  • an adjuvant can include a suspension of minerals (alum, aluminum hydroxide, or phosphate) on which antigen is adsorbed; or water-in-oil emulsion, for example, in which antigen solution is emulsified in mineral oil (Freund incomplete adjuvant), sometimes with the inclusion of killed mycobacteria (Freund's complete adjuvant) to further enhance antigenicity (inhibits degradation of antigen and/or causes influx of macrophages).
  • the adjuvant includes CpG oligodeoxynucleotides and/or ADDAVAX, a squalene-based oil-in-water emulsion.
  • Additional adjuvants for use in the disclosed immunogenic compositions can include, for example, the QS21 purified plant extract, Matrix M, AS01, MF59, and ALFQ adjuvants.
  • Adjuvants also include biological molecules (a “biological adjuvant”), such as costimulatory molecules.
  • biological adjuvants include IL-2, RANTES, GM-CSF, TNF- ⁇ , IFN- ⁇ , G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L, 4-1BBL and toll-like receptor (TLR) agonists, such as TLR-9 agonists.
  • TLR toll-like receptor
  • Antigen A compound, composition, or substance that can stimulate the production of antibodies or a T-cell response in an animal, including compositions that are injected or absorbed into an animal.
  • An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous immunogens.
  • the antigen is a conserved N. gonorrhoeae protein, such as SliC, MetQ, ACP, BamE, BamG or AniA.
  • AP205 A single-stranded RNA bacteriophage that infects Acinetobacter bacteria.
  • the AP205 virus particle is formed by the capsid protein.
  • the capsid protein has an amino acid sequence that includes SEQ ID NO: 1.
  • Bacteriophage A virus that infects and replicates in bacteria or archaea.
  • Conservative amino acid substitution Amino acid substitutions in a protein that do not substantially affect or decrease a function of a protein (e.g., a N. gonorrhoeae antigen), such as the ability of the protein to elicit an immune response when administered to a subject.
  • the term conservative variation also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid.
  • Non-conservative substitutions are those that reduce an activity or function of a protein (such as a N.
  • gonorrhoeae antigen such as the ability to elicit an immune response when administered to a subject. For instance, if an amino acid residue is essential for a function of the protein, even an otherwise conservative substitution may disrupt that activity. Thus, a conservative substitution does not alter the basic function of a protein of interest.
  • Degenerate variant A polynucleotide encoding a polypeptide (such as a N. gonorrhoeae antigen) that includes a sequence that is degenerate as a result of the genetic code. There are 20 natural amino acids, 245-110515-02 OSU-22-30 most of which are specified by more than one codon.
  • Effective amount A quantity of a specific substance (such as a vaccine) sufficient to achieve a desired effect in a subject to whom the substance is administered. For instance, this can be the amount necessary to inhibit, prevent or treat a gonorrhea infection, or to measurably alter outward symptoms of the infection.
  • a prophylactically effective amount refers to administration of an agent or immunogenic composition in an amount that inhibits or prevents establishment of an infection by N. gonorrhoeae.
  • a prophylactically effective amount of a disclosed immunogen/immunogenic composition can be the amount of the immunogen or immunogenic composition sufficient to elicit a priming immune response in a subject that can be subsequently boosted with the same or a different immunogen to elicit a protective immune response.
  • a desired response is to elicit an immune response that inhibits or prevents N. gonorrhoeae infection. The N.
  • gonorrhoeae infection need not be completely eliminated or prevented for the composition to be effective.
  • administration of an effective amount of an immunogenic composition disclosed herein can elicit an immune response that decreases the bacterial load, for example, by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination or prevention of detectable N. gonorrhoeae infection), as compared to the number of N. gonorrhoeae-infected cells in the absence of the immunization.
  • Fusion protein A protein generated by expression of a nucleic acid sequence engineered from nucleic acid sequences encoding at least a portion of two different (heterologous) proteins. To create a fusion protein, the nucleic acid sequences must be in the same reading frame and contain to internal stop codons.
  • a fusion protein includes a SpyCatcher or SpyTag peptide fused to the AP205 capsid protein, or includes a SpyTag peptide or a SpyCatcher peptide fused to a N. gonorrhoeae antigen (either directly or via a linker peptide; see FIG.1C).
  • Heterologous Originating from a separate genetic source or species.
  • a promoter can be heterologous to an operably linked nucleic acid sequence.
  • a SpyTag peptide is heterologous to a N. gonorrhoeae antigen.
  • Immune response A response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus.
  • the response is specific for a particular antigen (an “antigen- specific response”), such as a N. gonorrhoeae antigen (e.g., SliC, MetQ, ACP, BamE, BamG or AniA).
  • the immune response is a T cell response, such as a CD4+ response or a CD8+ response.
  • the response is a B cell response, and results in the production of specific antibodies.
  • Primary an immune response refers to treatment of a subject with a “prime” immunogen/immunogenic composition to induce an immune response that is subsequently “boosted” with a boost immunogen/immunogenic composition. Together, the prime and boost immunizations produce the desired immune response in the subject.
  • Immunogenic composition A composition that includes an immunogen, such as a VLP displaying a N.
  • gonorrhoeae antigen e.g., SliC, MetQ, ACP, BamE, BamG or AniA
  • a measurable immune response such as a T cell response and/or B cell response
  • the immunogenic composition can include the protein or nucleic acid molecule in a pharmaceutically acceptable carrier and may also include other agents, such as an adjuvant.
  • Immunize To render a subject protected from infection by a particular infectious agent, such as N. gonorrhoeae. Immunization does not require 100% protection. In some examples, immunization provides at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% protection against infection compared to infection in the absence of immunization.
  • Linker One or more amino acids that serve as a spacer between two polypeptides of a fusion protein.
  • MetQ A highly conserved N. gonorrhoeae protein. An exemplary MetQ protein sequence is set forth herein as SEQ ID NO: 5.
  • Neisseria gonorrhoeae A species of Gram-negative bacteria that is the causative agent of gonorrhoeae.
  • Operably linked A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter such as the CMV promoter, is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.
  • PelB leader sequence A 22-amino acid signal sequence that directs proteins to which it is attached to the bacterial periplasm.
  • the amino acid sequence of an exemplary pelB leader sequence is MKYLLPTAAAGLLLLAAQPAMA (residues 1-22 of SEQ ID NO: 12).
  • Pharmaceutically acceptable carriers The pharmaceutically acceptable carriers of use are conventional. Remington: The Science and Practice of Pharmacy, 22 nd ed., London, UK: Pharmaceutical Press, 2013, describes compositions and formulations suitable for pharmaceutical delivery of the disclosed immunogens. In general, the nature of the carrier will depend on the particular mode of administration being employed.
  • parenteral formulations usually include injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • injectable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like
  • conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.
  • compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, added preservatives (such as non-natural preservatives), and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • the pharmaceutically acceptable carrier is sterile and suitable for parenteral administration to a subject for example, by injection.
  • the active agent and pharmaceutically acceptable carrier are provided in a unit dosage form such as a pill or in a selected quantity in a vial. Unit dosage forms can include one dosage or multiple dosages (for example, in a vial from which metered dosages of the agents can selectively be dispensed).
  • Recombinant A recombinant nucleic acid, vector or virus is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination can be accomplished, for example, by the artificial manipulation of isolated segments of nucleic acids, for example, using genetic engineering techniques.
  • SpyCatcher/SpyTag A system based on an internal isopeptide bond formed in the CnaB2 domain of the Streptococcus pyogenes FbaB protein.
  • SpyCatcher is an immunoglobulin-like domain of about 138 amino acids from the CnaB2 domain containing a reactive lysine and catalytic glutamate and SpyTag is a peptide of about 13 amino acids containing a reactive aspartate. SpyCatcher and SpyTag bind with high affinity and spontaneously form a covalent peptide bond (Zakeri et al., Proc. Natl. Acad. Sci. USA 109:E690-E697, 2012).
  • the SpyCatcher protein has the amino acid sequence set forth herein as SEQ ID NO: 3 and/or the SpyTag peptide has the amino acid sequence set forth herein as SEQ ID NO: 2.
  • Subject Living multicellular vertebrate organisms, a category that includes human and non-human mammals. In some examples, the subject is a human.
  • SliC protein sequence is set forth herein as SEQ ID NO: 4.
  • Unit dosage form A physically discrete unit, such as a capsule, tablet, or solution, that is suitable as a unitary dosage for a patient (such as a human patient), each unit containing a predetermined quantity of one or more active ingredient(s) calculated to produce a therapeutic or prophylactic effect, in association with at least one pharmaceutically acceptable diluent or carrier, or combination thereof.
  • Vaccine A pharmaceutical composition that elicits a prophylactic or therapeutic immune response in a subject. In some cases, the immune response is a protective immune response. Typically, a vaccine elicits an antigen-specific immune response to an antigen of a pathogen, for example N. gonorrhoeae, or to a cellular constituent correlated with a pathological condition.
  • a vaccine may include a polynucleotide (such as a nucleic acid encoding a disclosed antigen), a peptide or polypeptide (such as a disclosed antigen), a virus or virus-like particle, a cell or one or more cellular constituents.
  • a polynucleotide such as a nucleic acid encoding a disclosed antigen
  • a peptide or polypeptide such as a disclosed antigen
  • virus or virus-like particle such as a virus or virus-like particle, a cell or one or more cellular constituents.
  • 245-110515-02 OSU-22-30 An entity containing a nucleic acid molecule (such as a DNA or RNA molecule) bearing a promoter(s) that is operationally linked to the coding sequence of a protein of interest and can express the coding sequence.
  • Non-limiting examples include a naked or packaged (lipid and/or protein) DNA, a naked or packaged RNA, a subcomponent of a virus or bacterium or other microorganism that may be replication- incompetent, or a virus or bacterium or other microorganism that may be replication-competent.
  • a vector is sometimes referred to as a construct.
  • Recombinant DNA vectors are vectors having recombinant DNA.
  • a vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication.
  • a vector can also include one or more selectable marker genes and other genetic elements known in the art.
  • Viral vectors are recombinant nucleic acid vectors having at least some nucleic acid sequences derived from one or more viruses.
  • VLP Virus-like particle
  • VLPs Protein particles made up of one of more viral structural proteins but lacking a viral genome (or other viral nucleic acid molecules). Because VLPs lack a viral genome, they are non-infectious.
  • the VLP is composed of the capsid protein of the bacteriophage AP205.
  • IV. Immunogenic Compositions for Protection Against Gonorrhea Disclosed herein are immunogenic compositions in which conserved N. gonorrhoeae antigens are displayed on the surface of virus-like particles (VLPs), such as VLPs formed by the capsid protein of an RNA bacteriophage. The disclosed immunogenic compositions satisfy an unmet need for an effective vaccine against gonorrhea.
  • immunogenic compositions that include a capsid protein of an RNA bacteriophage fused to a first peptide tag, and a N. gonorrhoeae antigen fused to a second peptide tag, wherein the first peptide tag and the second peptide tag are joined by an isopeptide bond, and the capsid protein and antigen form a virus-like particle (VLP) displaying the antigen.
  • VLP virus-like particle
  • the antigen is a highly conserved N.
  • gonorrhoeae antigen selected from the group consisting of surface-exposed lysozyme inhibitor of c-type lysozyme (SliC), methionine binding protein (MetQ), Neisserial adhesin complex protein (ACP), ⁇ -barrel assembly machinery protein E (BamE), ⁇ -barrel assembly machinery protein G (BamG) and anaerobically induced outer membrane protein A (AniA).
  • the RNA bacteriophage is AP205, which is a bacteriophage of Acinetobacter.
  • the capsid protein of AP205 is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 1.
  • the amino acid sequence of the capsid protein includes or consists of SEQ ID NO: 1.
  • the RNA bacteriophage is bacteriophage Q ⁇ , bacteriophage fr, bacteriophage GA, bacteriophage R17, bacteriophage SP, bacteriophage MS2, bacteriophage M11, bacteriophage MX1, bacteriophage NL95, bacteriophage f2, or bacteriophage PP7 (see, e.g., WO 2009/130261). 245-110515-02 OSU-22-30
  • the first peptide tag is fused to the N-terminus of the capsid protein. In other aspects, the first peptide tag is fused to the C-terminus of the capsid protein.
  • the second peptide tag is fused to the N-terminus of the antigen. In other aspects, the second peptide tag is fused to the C-terminus of the antigen. In some aspects, the first peptide tag includes a SpyTag peptide, and the second peptide tag includes a SpyCatcher peptide. In other aspects, the first peptide tag includes a SpyCatcher peptide, and the second peptide tag includes a SpyTag peptide.
  • the amino acid sequence of the SpyTag peptide is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 2; or the amino acid sequence of the SpyTag peptide includes or consists of SEQ ID NO: 2.
  • the amino acid sequence of the SpyCatcher peptide is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 3; or the amino acid sequence of the SpyCatcher peptide includes or consists of SEQ ID NO: 3.
  • the immunogenic composition further includes a peptide linker between the capsid protein and the first peptide tag and/or a peptide linker between the antigen and the second peptide tag.
  • the sequence of the peptide linker comprises residues 36-41 of SEQ ID NO: 12.
  • the N. gonorrhoeae antigen is SliC.
  • the amino acid sequence of SliC is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 4.
  • the amino acid sequence of SliC includes or consists of SEQ ID NO: 4.
  • the N. gonorrhoeae antigen is MetQ.
  • the amino acid sequence of MetQ is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 5.
  • the amino acid sequence of Met Q includes or consists of SEQ ID NO: 5.
  • the N. gonorrhoeae antigen is ACP.
  • the amino acid sequence of ACP is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 6.
  • the amino acid sequence of ACP includes or consists of SEQ ID NO: 6.
  • the N. gonorrhoeae antigen is BamE.
  • the amino acid sequence of BamE is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 7.
  • the amino acid sequence of BamE includes or consists of SEQ ID NO: 7.
  • the N. gonorrhoeae antigen is BamG.
  • the amino acid sequence of BamG is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 8.
  • the amino acid sequence of BamG includes or consists of SEQ ID NO: 8.
  • the N. gonorrhoeae antigen is AniA.
  • the amino acid sequence of AniA is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or 245-110515-02 OSU-22-30 at least 99% identical to SEQ ID NO: 9.
  • the amino acid sequence of AniA includes or consists of SEQ ID NO: 9.
  • the immunogenic composition further includes a pharmaceutically acceptable carrier.
  • the immunogenic composition further includes an adjuvant. Adjuvants for use with vaccines are well-known and an appropriate adjuvant for use with the disclosed immunogenic compositions can be selected by a skilled person.
  • the adjuvant includes CpG oligodeoxynucleotides and/or a squalene-based oil-in-water emulsion (e.g., ADDAVAX). Also provided herein are nucleic acid molecule(s) that encode an immunogenic composition of the disclosure.
  • the nucleic acid molecule or molecules includes the nucleic acid sequence of any one of SEQ ID NOs: 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57 and 59, or a degenerate variant of any one of SEQ ID NOs: 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57 and 59.
  • the nucleic acid molecule or molecules further include a nucleic acid molecule encoding a capsid protein of an RNA bacteriophage, such as the AP205 capsid protein set forth herein as SEQ ID NO: 1.
  • the nucleic acid molecule or molecules further include the nucleic acid sequence of nucleotides 1-39 of SEQ ID NO: 10 (encoding a SpyTag peptide) or nucleotides 1-342 of SEQ ID NO: 11 (encoding a SpyCatcher peptide).
  • vectors that include a disclosed nucleic acid molecule or molecules.
  • the vector is a plasmid vector, such as the pET vector (see, e.g., Mierendorf et al., Methods Mol Med 13:257-292, 1998). Isolated host cells that include a nucleic acid molecule or vector disclosed herein are also provided.
  • the host cell is a prokaryotic cell, such as an Escherichia coli cell.
  • methods of eliciting an immune response against N. gonorrhoeae in a subject include administering to the subject an effective amount of an immunogenic composition disclosed herein.
  • methods of immunizing a subject against N. gonorrhoeae include administering to the subject an effective amount of an immunogenic composition disclosed herein.
  • the immunogenic composition is administered to the subject subcutaneously, intramuscularly, intranasally, or any combination thereof.
  • the immunogenic composition is administered subcutaneously as a prime dose and administered intranasally as a boost dose.
  • multiple boost doses are administered intranasally, such as two, three or four doses.
  • the subject is a human subject, such as a human subject at risk of infection by N. gonorrhoeae and/or at risk of developing gonorrhea.
  • the disclosed compositions can be administered to a subject by any of the routes normally used for introducing immunogenic compositions (such as vaccines) into a subject.
  • Methods of administration include, but are not limited to, subcutaneous, intranasal, intramuscular, intradermal, intraperitoneal, 245-110515-02 OSU-22-30 parenteral, intravenous, mucosal, vaginal, rectal, inhalation or oral.
  • Parenteral administration such as subcutaneous, intravenous or intramuscular administration, is generally achieved by injection.
  • Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.
  • Injection solutions and suspensions can be prepared from sterile powders, granules, tablets, and the like. Administration can be systemic or local.
  • the immunogenic composition is administered subcutaneously, intranasally, intramuscularly, or any combination thereof.
  • the immunogenic compositions disclosed herein can be administered with at least one pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions of the present disclosure.
  • Pharmaceutically acceptable carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, sesame oil, ethanol, and combinations thereof.
  • the composition can also contain conventional pharmaceutical adjunct materials such as, pharmaceutically acceptable salts to adjust the osmotic pressure, buffers, preservatives and the like.
  • the carrier and composition can be sterile, and the formulation suits the mode of administration.
  • the composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • the composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • the immunogenic compositions provided herein are formulated for mucosal vaccination, such as oral, intranasal, pulmonary, rectal or vaginal administration. In a specific example, this is achieved by intranasal administration.
  • the disclosed compositions can include one or more biodegradable, mucoadhesive polymeric carriers.
  • Polymers such as polylactide-co-glycolide (PLGA), chitosan (for example in the form of chitosan nanoparticles, such as N-trimethyl chitosan (TMC)-based nanoparticles), alginate (such as sodium alginate) and carbopol can be included.
  • the immunogenic composition includes one or more hydrophilic polymers, such as sodium alginate or carbopol.
  • the composition includes carbopol, for example in combination with starch.
  • the composition is formulated as a particulate delivery system used for nasal administration.
  • the composition can include liposomes, immune-stimulating complexes (ISCOMs) and/or polymeric 245-110515-02 OSU-22-30 particles.
  • the compositions can also include one or more lipopeptides of bacterial origin, or their synthetic derivatives, such as Pam3Cys, (Pam2Cys, single/multiple-chain palmitic acids and lipoamino acids (LAAs).
  • the compositions can also include one or more adjuvants, such as one or more of CpG oligodeoxynucleotides (CpG ODN), Flt3 ligand, and monophosphoryl lipid A (MLA).
  • the adjuvant includes a squalene-based oil-in-water emulsion, such as ADDAVAX.
  • the disclosed immunogenic compositions can be administered as a single dose or as multiple doses (for example, boosters).
  • the first administration is followed by a second administration.
  • the second administration can be with the same, or with a different N. gonorrhoeae immunogenic composition than the first composition administered.
  • the second administration is with the same immunogenic composition as the first composition administered.
  • the second administration is with a different composition than the first composition administered.
  • the immunogenic composition is administered subcutaneously as a prime dose and administered intranasally as a boost dose.
  • an immunogenic composition is administered once subcutaneously, followed by three or more boost doses administered intranasally.
  • the immunogenic compositions are administered as multiple doses, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 doses (such as 2-3 doses).
  • the timing between the doses can be at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 6 weeks, at least 8 weeks, at least 12 weeks, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 1 year, at least 2 years or at least 5 years, such as 1-4 weeks, 2-3 weeks, 1-6 months, 2-4 months, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 12 weeks, 1 month, 2 months, 3, months, 4, months, 5 months, 6 months, 1 year, 2 years, 5 years or 10 years, or combinations thereof (such as where there are at least three administrations, wherein the timing between the first and second, and second and third doses, can be the same or different).
  • the immunogenic composition can be provided in unit dosage form for eliciting an immune response in a subject, for example, to prevent N. gonorrhoeae infection in the subject.
  • a unit dosage form contains a suitable single preselected dosage for administration to a subject, or suitable marked or measured multiples of two or more preselected unit dosages, and/or a metering mechanism for administering the unit dose or multiples thereof.
  • the dose administered to a subject in the context of the present disclosure should be sufficient to induce a beneficial therapeutic response in a subject over time, or to inhibit or prevent N. gonorrhoeae infection and/or the development of gonorrhea.
  • the dose required can vary from subject to subject depending on the species, age, weight and general condition of the subject, the severity of the infection being treated, the particular composition being used and its mode of administration. An appropriate dose can be determined by a skilled person. 245-110515-02 OSU-22-30 VI. Methods of Eliciting an Immune Response
  • the disclosed immunogenic compositions can be used in methods of inducing an immune response to N. gonorrhoeae to prevent, inhibit (including inhibiting transmission), and/or treat a N. gonorrhoeae infection.
  • the immunogenic composition is administered intranasally, subcutaneously and/or intramuscularly.
  • the methods can be used either to avoid infection in a N. gonorrhoeae seronegative subject (e.g., by inducing an immune response that protects against N. gonorrhoeae infection), or to treat existing infection in a N. gonorrhoeae seropositive subject.
  • accepted screening methods are employed to determine risk factors associated with a targeted or suspected disease or condition, or to determine the status of an existing disease or condition in a subject.
  • screening methods include, for example, conventional work-ups to determine behavioral, 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 to detect and/or characterize N. gonorrhoeae infection.
  • diagnostic methods such as various ELISA and other immunoassay methods to detect and/or characterize N. gonorrhoeae infection.
  • a composition can be administered according to the teachings herein, or other conventional methods, as an independent prophylaxis or treatment program, or as a follow- up, adjunct or coordinate treatment regimen to other treatments.
  • the effective amount of the immunogenic composition is administered in a single dose.
  • the immunogenic composition is administered as part of a prime-boost immunization protocol. In some examples, the immunogenic composition is administered as both the prime dose and the boost dose. In other examples, the immunogenic composition is administered as the prime dose and a second N. gonorrhoeae vaccine is administered as the boost dose. In yet other examples, the immunogenic composition is administered as the boost dose and a second N. gonorrhoeae vaccine is administered as the prime dose. In certain aspects, combinatorial immunogenic compositions and coordinate immunization protocols employ separate immunogens or formulations, each directed toward eliciting a N. gonorrhoeae immune response, such as an immune response to a conserved N.
  • a suitable immunization regimen includes at least two separate inoculations with one or more immunogenic compositions disclosed herein with a second inoculation being administered more 245-110515-02 OSU-22-30 than about two weeks, about three weeks, or about four weeks, such about three to eight weeks, following the first inoculation.
  • a third inoculation can be administered several months after the second inoculation, and in specific aspects, more than about four months, five months, or six months after the first inoculation, more than about six months to about two years after the first inoculation, or about eight months to about one year after the first inoculation. Periodic inoculations beyond the third are also desirable to enhance the subject's “immune memory.”
  • the adequacy of the vaccination parameters chosen e.g., formulation, dose, regimen and the like, can be determined by taking aliquots of serum from the subject and assaying antibody titers during the course of the immunization program. Alternatively, the T cell populations can be monitored by conventional methods.
  • the clinical condition of the subject can be monitored for the desired effect, e.g., prevention of N. gonorrhoeae infection, improvement in disease state (e.g., reduction in bacterial load), or reduction in transmission frequency. If such monitoring indicates that vaccination is sub-optimal, the subject can be boosted with an additional dose of immunogenic composition, and the vaccination parameters can be modified in a fashion expected to potentiate the immune response.
  • a dose of a disclosed immunogen can be increased or the route of administration can be changed.
  • the boost may be the same immunogen as another boost, or the prime.
  • the prime and the boost can be administered as a single dose or multiple doses, for example, two doses, three doses, four doses, five doses, six doses or more can be administered to a subject over days, weeks or months. Multiple boosts can also be given, such as one to five, or more. Different dosages can be used in a series of sequential inoculations. For example, a relatively large dose in a primary inoculation and then a boost with relatively smaller doses.
  • the immune response against the selected antigenic surface can be elicited by one or more inoculations of a subject.
  • a disclosed immunogenic composition can be administered to the subject simultaneously with the administration of an adjuvant.
  • the immunogen can be administered to the subject after the administration of an adjuvant and within a sufficient amount of time to elicit the immune response. In other aspects, no adjuvant is administered.
  • N. gonorrhoeae infection does not need to be completely inhibited for the methods to be effective. For example, elicitation of an immune response to N. gonorrhoeae can reduce or inhibit N. gonorrhoeae infection by a desired amount, for example, by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination or prevention of detectable N.
  • An immunogenic composition comprising: a capsid protein of an RNA bacteriophage fused to a first peptide tag; and 245-110515-02 OSU-22-30 a Neisseria gonorrhoeae antigen fused to a second peptide tag, wherein the antigen is selected from the group consisting of surface-exposed lysozyme inhibitor of c-type lysozyme (SliC), methionine binding protein (MetQ), Neisserial adhesin complex protein (ACP), ⁇ -barrel assembly machinery protein E (BamE), ⁇ -barrel assembly machinery protein G (BamG) and anaerobically induced outer membrane protein A (AniA), wherein the first peptide tag and the second peptide tag are joined by an isopeptide
  • Aspect 2 The immunogenic composition of aspect 1, wherein the RNA bacteriophage is AP205.
  • Aspect 3 The immunogenic composition of aspect 2, wherein the amino acid sequence of the AP205 capsid protein is at least 90% identical to SEQ ID NO: 1.
  • Aspect 4. The immunogenic composition of aspect 2 or aspect 3, wherein the amino acid sequence of the AP205 capsid protein comprises or consists of SEQ ID NO: 1.
  • Aspect 5. The immunogenic composition of any one of aspects 1-4, wherein the first peptide tag is fused to the N-terminus of the capsid protein.
  • Aspect 6. The immunogenic composition of any one of aspects 1-4, wherein the first peptide tag is fused to the C-terminus of the capsid protein.
  • Aspect 8 The immunogenic composition of any one of aspects 1-6, wherein the second peptide tag is fused to the C-terminus of the antigen.
  • Aspect 9. The immunogenic composition of any one of aspects 1-8, wherein: the first peptide tag comprises a SpyTag peptide, and the second peptide tag comprises a SpyCatcher peptide; or the first peptide tag comprises a SpyCatcher peptide, and the second peptide tag comprises a SpyTag peptide.
  • Aspect 10 The immunogenic composition of aspect 9, wherein the amino acid sequence of the SpyTag peptide comprises SEQ ID NO: 2.
  • Aspect 11 The immunogenic composition of aspect 9 or aspect 10, wherein the amino acid sequence of the SpyCatcher peptide comprises SEQ ID NO: 3.
  • Aspect 12 The immunogenic composition of any one of aspects 1-11, further comprising a peptide linker between the capsid protein and the first peptide tag and/or a peptide linker between the antigen and the second peptide tag.
  • Aspect 13 The immunogenic composition of aspect 12, wherein the sequence of the peptide linker comprises GSGESG (residues 36-41 of SEQ ID NO: 12).
  • Aspect 14 The immunogenic composition of any one of aspects 1-13, wherein the antigen is SliC.
  • Aspect 15 The immunogenic composition of any one of aspects 1-13, wherein the antigen is SliC.
  • Aspect 16 The immunogenic composition of aspect 14 or aspect 15, wherein the amino acid sequence of SliC comprises or consists of SEQ ID NO: 4.
  • Aspect 17 The immunogenic composition of any one of aspects 1-13, wherein the antigen is MetQ, ACP, BamE, BamG or AniA.
  • Aspect 18 is derived from any one of aspects 1-13, wherein the antigen is MetQ, ACP, BamE, BamG or AniA.
  • the amino acid sequence of MetQ is at least 90% identical to SEQ ID NO: 5; the amino acid sequence of ACP is at least 90% identical to SEQ ID NO: 6; the amino acid sequence of BamE is at least 90% identical to SEQ ID NO: 7; the amino acid sequence of BamG is at least 90% identical to SEQ ID NO: 8; or the amino acid sequence of AniA is at least 90% identical to SEQ ID NO: 9.
  • Aspect 22. A nucleic acid molecule or molecules encoding the immunogenic composition of any one of aspects 1-21.
  • the nucleic acid molecule or molecules of aspect 22 comprising the nucleic acid sequence of any one of SEQ ID NOs: 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57 and 59, or a degenerate variant of any one of SEQ ID NOs: 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57 and 59.
  • Aspect 24 The nucleic acid molecule or molecules of aspect 22 or aspect 23, comprising or further comprising a nucleic acid molecule encoding the AP205 capsid protein set forth as SEQ ID NO: 1.
  • Aspect 25 comprising or further comprising a nucleic acid molecule encoding the AP205 capsid protein set forth as SEQ ID NO: 1.
  • Aspect 26 A vector comprising the nucleic acid molecule or molecules of any one of aspects 22-25.
  • Aspect 27 A host cell comprising the nucleic acid molecule or molecules of any one of aspects 22-25 or the vector of aspect 26.
  • Aspect 28 A method of eliciting an immune response against Neisseria gonorrhoeae in a subject, comprising administering to the subject an effective amount of the immunogenic composition of any one of aspects 1-21.
  • Aspect 29 Aspect 29.
  • a method of immunizing a subject against Neisseria gonorrhoeae comprising administering to the subject an effective amount of the immunogenic composition of any one of aspects 1- 21.
  • Aspect 30 The method of aspect 28 or aspect 29, wherein the immunogenic composition is administered subcutaneously, intramuscularly, intranasally, or any combination thereof. 245-110515-02 OSU-22-30
  • Aspect 31 The method of any one of aspects 28-30, wherein the immunogenic composition is administered subcutaneously as a prime dose and administered intranasally as a boost dose.
  • Aspect 32 The method of any one of aspects 28-31, wherein the subject is a human subject.
  • Example 1 Materials and Methods This example describes the materials and experimental procedures for the studies described in Examples 2-5. Bacteria and culture conditions The serum resistant Ng FA1090 (PorB1B) and the Ng 2016 WHO reference strains were used in the studies described herein (Zielke et al., Mol Cell Proteomics 15(7):2338-2355, 2016; Cohen et al., J Infect Dis 169(3):532-537, 1994).
  • the ⁇ sliC knockout and complementation strain ⁇ sliC/P::sliC were constructed previously using the Ng FA1090 (Zielke et al., Mol Cell Proteomics 15(7):2338-2355, 2016). Ng strains were maintained on GC agar (GCB; Difco) with Kellogg’s Supplement I and 12.5 ⁇ M ferric nitrate or on chocolate agar plates, as indicated in the text, in a 5% CO 2 atmosphere at 37oC for 18-20 hours (Wu et al., Infect Immun 77(3):1091-1102, 2009).
  • coli strains were maintained on Luria-Bertani (LB) agar or cultured in LB broth. Media were supplemented with antibiotics in the following concentrations for Ng: kanamycin 40 ⁇ g/mL and streptomycin 100 ⁇ g/mL; and for E. coli: kanamycin 50 ⁇ g/mL and carbenicillin 50 ⁇ g/mL. Genetic manipulations Cloning procedures, oligonucleotides and gene blocks were designed using SnapGene software version 2.8 (GSL Biotech LLC). Primers and gene blocks were synthesized by Integrated DNA Technologies. Q5 High-Fidelity DNA polymerase, DNA ligase and NEBuilder HiFi DNA Assembly Master Mix were obtained from New England Biolabs (NEB).
  • STN SpyTagN
  • STC SpyTagC
  • STN DNA gene block contains a SpyTag sequence (Thrane et al., J Nanobiotechnology 14:30, 2016) followed by a linker, a multi cloning site, a tobacco etch virus (TEV) protease cleavage site and a 10 ⁇ His Tag.
  • TSV tobacco etch virus
  • the STC DNA fragment includes a 10 ⁇ His Tag followed by a TEV protease recognition site, a multi cloning site, a linker and a SpyTag.
  • pET-22b plasmid was amplified using primer pairs listed in Table 1. The PCR products were gel purified and assembled with corresponding gene blocks using NEBuilder HiFi DNA Assembly Master Mix. The sliC gene was amplified with the primers shown in Table 1 using pRSF-SliC as a template (Zielke et al., PLoS Pathog 14(7):e1007081, 2018).
  • the obtained PCR product was digested with NcoI/XhoI and cloned into similarly digested pET22-STN and pET22-STC to yield pET22-N-SliC and pET22-C-SliC, respectively.
  • Table 1 Primer Sequences Primer Sequence SEQ ID NO: ET STN f d TATAGGCATCGACCATTACGATATGGGCGGCCAT 63 Expression and purification of N-SliC and C-SliC Recombinant N-SliC and C-SliC were purified from 3 L cultures of E.
  • coli BL21(DE3) (Studier and Moffatt, J Mol Biol 189(1):113-130, 1986) carrying pET22-N-SliC and pET22-C-SliC, respectively. Bacteria were incubated at 37°C, 210 rpm until OD600 of ⁇ 0.6 was reached. Cultures were then shifted to 18°C for 1 hour and protein expression was induced with 0.1 mM IPTG. After overnight incubation, the cells were pelleted at 5,000 ⁇ g for 15 minutes at 4°C.
  • Bacteria were suspended in binding/lysis buffer (50 mM Na 2 HPO 4 , pH 8, 200 mM NaCl, 25 mM imidazole, 10% glycerol) supplemented with protease inhibitor mini tablets (Pierce) and lysed by passing through a French Press at 1,500 psi. Cellular debris was removed by centrifugation at 8,000 ⁇ g for 15 minutes at 4°C and the obtained supernatant was passage through 0.45 ⁇ m nylon membrane (EZ flow).
  • binding/lysis buffer 50 mM Na 2 HPO 4 , pH 8, 200 mM NaCl, 25 mM imidazole, 10% glycerol
  • the cleared cell lysate was applied to a 5 mL IMAC Nickel column 245-110515-02 OSU-22-30 (BioRad) and the recombinant proteins were purified on an NGC Medium-Pressure Liquid Chromatography System (BioRad) using binding/lysis buffer and elution buffer (50 mM Na 2 HPO 4 , pH 8, 200 mM NaCl, 250 mM imidazole, 10% glycerol). Elution fractions containing either N-SliC or C-SliC were pooled and applied onto a Vivaspin 20 centrifugal concentrator (GE HealthCare).
  • Samples were supplemented with DTT and EDTA to final concentrations of 1 mM and 0.5 mM, respectively.
  • the His-tag was removed by overnight incubation at 4°C with TEV protease in a 1:100 ratio.
  • Dialysis was performed using a 1:4 ratio of binding/lysis buffer by placing TEV-digested samples into snakeskin dialysis tubing (3.5 MWCO) with low stirring overnight at 4°C. Samples were applied to a 5 mL IMAC Nickel column (BioRad) to remove TEV. The removal of His-tag was confirmed by immunoblotting.
  • Protein samples were concentrated as described above and subjected to size exclusion chromatography using a HiLoad 16/600 Superdex 75 pg column (GE HealthCare) with phosphate buffered saline (PBS) as running buffer to isolate N-SliC and C-SliC. Protein purity was confirmed by sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE) and the protein concentration was measured using the BioRad DC Protein Assay. The rSliC was purified as described previously (Zielke et al., PLoS Pathog 14(7):e1007081, 2018).
  • mice were administered subcutaneous vaccines (day 0) containing N-SliC, N-SliC-VLP adjuvanted with ADDAVAX (2.5 or 10 ⁇ g/dose), N-SliC-VLP adjuvanted with CpG ODN Class B (CpG, Invivogen) at 2.5, 5, or 10 ⁇ g/dose followed by intranasal boost on day 21.
  • Control groups received PBS, cVLP mixed with ADDAVAX or CpG (10 ⁇ g/dose).
  • Venous blood was collected on days 31 and 52 and vaginal lavages were collected on day 31 as described (Sikora et al., Vaccine 38(51):8175-8184, 2020).
  • ELISA Enzyme-linked immunosorbent assays
  • Serum or vaginal lavage samples were serially diluted in PBST and added to each well.
  • the wells were washed with PBST and incubated with secondary antisera: anti-mouse total IgG, anti-mouse IgG1, anti-mouse IgG2a, anti-mouse IgG3 and anti-mouse IgA (Southern Biotech) conjugated to horse radish peroxidase (HRP).
  • Wells were washed and reactions were developed using TMB Peroxidase EIA Substrate (BioRad).
  • End-point titers were determined using the average reading of eight wells incubated with secondary but no primary antibody plus 3 and 2 standard deviations as a baseline for serum and vaginal lavages, respectively.
  • Kruskal-Wallis test with Dunn’s multiple comparisons were applied on non-transformed arithmetic data.
  • the non-parametric Mann- Whitney U test was carried out.
  • p values of ⁇ 0.05 were considered statistically significant.
  • Serum bactericidal assays Sera from mice immunized with tested vaccines and control groups, as described in the text, were pooled and heat inactivated for 30 minutes at 56oC (Zielke et al., Mol Cell Proteomics 15(7):2338-2355, 2016; Gulati et al., PLoS Biol 17(6):e3000323, 2019; Gulati et al., mBio 10(6):e02552-19, 2019). Subsequently, the sera were serially diluted in GC supplemented with balanced salt (0.15 mM CaCl2, 1 mM MgCl2) at two-fold dilutions starting from 1:64 to 1:8192.
  • the Ng FA1090 cells (2 ⁇ 10 4 colony forming units (CFU/mL)) were prepared from non-piliated colonies collected from chocolate agar plates.
  • the Ng cells (1 ⁇ 10 3 in 40 ⁇ L) were added to wells containing test sera, mixed by shaking for 15 seconds, and incubated at 37oC and 5% CO 2 atmosphere for 15 minutes before adding 10 ⁇ L of IgG/IgM-depleted normal human serum (NHS) or heat-inactivated normal human serum (HI-NHS) as the complement source (10% v/v). Samples were incubated for 1 hour at 37°C with 5% CO2.
  • Lysozyme activity assay To determine if the addition of SpyTag affects the SliC-mediated inhibition of c-type human lysozyme (HL, Sigma), the EnzChek Lysozyme Assay kit (ThermoFisher) was used as described previously (41). The lysozyme assay was carried out in black flat-bottom 96 well plates. Samples containing 2.5 ⁇ M HL (Sigma) were incubated with increasing concentrations of rSliC-STN (0-5 ⁇ M) in reaction buffer containing 0.1 M sodium phosphate pH 7.5, 0.1 M NaCl, and 2 mM sodium azide for 30 minutes at 37°C. The controls contained HL alone.
  • N- SliC/ACP were incubated with pooled sera from immunized and control groups (1:10 v/v) for 30 minutes and the lysozyme assays were carried out as described above.
  • Membranes were incubated overnight in PBST supplemented with 5% non-fat dry milk, washed with PBST and probed with pooled sera (1:5,000) or vaginal lavages (1:50) from test or control mice followed by probing the immunoblots with goat anti-mouse IgG (BioRad) or IgA (SouthernBiotech) conjugated to HRP as described previously (3). Cross-reacting proteins were detected using ECL Prime (Amersham) and ImageQuant TM LAS 4000 (GE Healthcare). Statistical analysis Statistical analyses were performed with GraphPad Prism 9 as indicated for each experimental procedure.
  • Example 2 Design of the Tag/Catcher AP205 platform for gonorrhea vaccine development
  • gene blocks were designed to carry SpyTag on the N- or C-terminus (STN and STC, respectively), a linker, a multicloning site, a TEV protease cleavage site and a 10 ⁇ His Tag (FIG.1A) and were cloned into a pET22 vector (FIG.1B).
  • This newly engineered pET22-STN and pET22-STC system 245-110515-02 OSU-22-30 enables cloning and production of a selected antigen with the SpyTag placed on either the N- or C-terminus to ensure optimal antigen folding for purification and coupling to the cVLP (FIG.1C).
  • an E. coli PelB signal sequence was also added to promote proper antigen folding in a heterologous host.
  • the SliC antigen was selected from Ng FA1090, a vaccine prototype strain that carries antigen sequence types identical to the most broadly distributed antigen variants (Baarda et al., mSphere 6(1):e00977-20, 2021), and sliC (NGO1063) was cloned into pET22-STN and pET22-STC (FIG.1B). Both SliC fusion proteins with SpyTag positioned on the N- or C-terminus, N-SliC or C-SliC, respectively, were successfully overproduced in E. coli and migrated on the SDS-PAGE according to the predicted molecular weight of ⁇ 15 kDa (FIG. 2A).
  • Coupling of SliC antigen to the cVLP caused a slight increase in the diameter of the particles of 47.7 nm and 36.9 nm for C-SliC-VLP and N-SliC-VLP, respectively, compared to the 20.8 nm of cVLP (FIG.2C).
  • the C-SliC-VLP had a higher polydispersity (28.7%) compared to N-SliC-VLP (11.7%) and cVLP alone (12.1%), indicating that the C-SliC-VLP population is more heterogeneous and that the coupling is not optimal. Due to the higher coupling efficiency, the N-SliC- VLP complexes were selected for further studies.
  • N-SliC- VLP contained intact and monodisperse particles (FIG.2D).
  • STN affects SliC inhibitory activity of human lysozyme
  • titration reactions were performed with increasing concentrations of purified N-SliC in the presence of peptidoglycan (FIG.7A).
  • recombinant SliC Zielke et al., PLoS Pathog 14(7):e1007081, 2018
  • N-SliC inhibited the lytic activity of human lysozyme in a dose-dependent manner, with complete blocking of lysozyme function at concentrations above 1.25 ⁇ M.
  • N-SliC antigen is functional and couples more efficiently to the cVLPs and thus, a vaccine formulation containing N-SliC-VLP was selected for further immunization studies.
  • Example 4 cVLP significantly enhances SliC immunogenicity, serum bactericidal activity and promotes a Th1 response
  • SliC-VLP we immunized mice with N-SliC-VLP, N-SliC, or cVLP using three subcutaneous injections at three-week intervals.
  • the calculated geometric mean of total IgG antibody responses in terminal sera from mice immunized with N-SliC-VLP was 1.6 ⁇ 10 6 compared to 1.7 ⁇ 10 3 and 1.4 ⁇ 10 3 in mice that received N-SliC and cVLP, respectively (FIG.3B). Furthermore, the SliC- VLP vaccine elicited markedly increased IgG1, IgG2a, IgG3 and IgA titers than N-SliC or cVLP (FIG.3B).
  • Immunization with SliC-VLP resulted in boost of IgG2a and IgG1 antibody responses (1.03 ⁇ 10 6 and 1.4 ⁇ 10 5 ; respectively) that were significantly greater than with N-SliC (0.06 ⁇ 10 3 and 2.1 ⁇ 10 3 ; respectively) or cVLP (0.1 ⁇ 10 3 and 0.5 ⁇ 10 3 ; respectively).
  • Serum IgG3 titers were 168- and >10,000-fold higher in N-SliC-VLP-immunized mice than in N-SliC and cVLP groups, respectively (FIG.3B).
  • Mice administered with N-SliC-VLP vaccine had increased vaginal IgG after the first immunization that augmented with each boost, whereas mice that received N-SliC or cVLP had undetectable IgG in the first and second vaginal samples and ⁇ 100-fold lower titers in the terminal vaginal lavages (FIG.3C).
  • Vaginal IgG, IgG1 and IgG2a were greater after the final immunization with N-SliC-VLP vaccine with the titers of 2.2 ⁇ 10 3 , 0.243 ⁇ 10 3 and 0.243 ⁇ 10 3 , respectively, in comparison to N-SliC (0.027 ⁇ 10 3 , 0.027 ⁇ 10 3 and 0.127 ⁇ 10 3 ) and cVLP (0.081 ⁇ 10 3 , not detectable); however, IgA antibody subtypes were not detected (FIGS.3C and 3D).
  • Example 5 Subcutaneous and intranasal administration of SliC-VLP vaccine formulated with ADDAVAX or CpG
  • SC subcutaneous prime
  • IgA intranasal boost
  • the vaccine dose studies showed that the SliC-VLP-CpG (10 ⁇ g/dose) elicited the highest total IgG titers in the final sera with the geometric mean of 1.15 ⁇ 10 7 , which was 4.6-, 1.7-, 22-, and 14.7-fold fold greater than the titers induced by the same vaccine at 2.5 and 5 ⁇ g/dose, and the N-SliC-VLP-ADDAVAX at 2.5 and 10 ⁇ g/dose, respectively (FIG.5A).
  • mice received N-SliC-VLP- ADDAVAX at 2.5- and 5 ⁇ g/dose and N-SliC-VLP-CpG at 2.5-, 5-, and 10 ⁇ g/dose, respectively.
  • N-SliC-VLP-CpG at 10 ⁇ g/dose resulted in the greatest IgG3 antibody titers in comparison to all tested vaccine formulations with the geometric mean of 2.05 ⁇ 10 5 that increased 67-, 162-, and 354-fold in comparison to those in mice administered with 5 ⁇ g of N-SliC-VLP-CpG, 2.5 ⁇ g of N-SliC-VLP-CpG/10 ⁇ g of N-SliC-VLP-ADDAVAX, and 2.5 ⁇ g of N-SliC- VLP- ADDAVAX, respectively (FIG.5D).
  • SliC-VLP-CpG vaccine (10 ⁇ g/dose) induced the most significant increase in total serum IgG and IgG3 titers and functional antibodies with SBA activity.
  • SliC-VLP vaccine administered with CpG as adjuvant This example tests whether adding CpG adjuvant enhances antibody responses in mice immunized with SliC-VLP in comparison to SliC-VLP alone or control groups (PBS or CpG-VLP). Mice received three IM immunizations.
  • Immune responses elicited by all immunizations were assessed in serum and vaginal lavages on days 31 and 52 after first immunization (day 0) from mice that received PBS, CpG-VLP, SliC- VLP, or SliC-VLP+CpG. Both vaccines elicited similar antibody titers for serum IgG, IgG1, IgG2a, IgG3 and IgA. There was a slight increase in IgG3 titers in mice immunized with SliC-VLP+CpG but it was not significantly higher compared to those observed in mice that received SliC-VLP alone.
  • Example 7 Administration of SliC-VLP via subcutaneous (SC) and intranasal (IN) routes This example describes immunization of mice with SliC-VLP, SliC-VLP+CpG, VLP-CpG, or PBS.
  • Example 9 Comparison of four ACP-based vaccines administered via IN or IM routes This examples compares four different vaccines: (1) ACP administered with CpG as adjuvant (ACP+CpG); (2) ACP and SliC administered with CpG as adjuvant (ACP-SliC+CpG); (3) ACP-VLP administered without adjuvant (ACP-VLP); and (4) ACP-VLP and SliC-VLP administered together without adjuvant (ACP-VLP+SliC-VLP). Mice received either three IN administrations of ACP+CpG or ACP-SliC+CpG, or three IM administrations of ACP-VLP or ACP-VLP+SliC-VLP.
  • mice that received CpG or VLP alone were used as controls.
  • Antibody responses elicited by the four different vaccines were compared on Days 31, 52 and 75.
  • ELISA assays plates were coated with either ACP or SliC to assess immune responses elicited by each antigen. The results are shown in FIGS.13A-13B.
  • ACP+CpG delivered IN elicited similar serum total IgG compared to administration of ACP- SliC+CpG, ACP-VLP, and ACP-VLP+SliC-VLP.
  • ACP+CpG induced higher serum IgA compared to the other three vaccines (ACP-SliC+CpG administered IN, and ACP-VLP or ACP-VLP+SliC-VLP delivered IM).
  • ACP is a “weaker” immunogen in inducing vaginal IgG and IgA when compared to SliC delivered in a dual vaccine IN with CpG.
  • the ACP- SliC+CpG vaccine induced statistically higher SliC-specific IgG and IgA in the vaginal tract, whereas VLP- based vaccines administered IM (due to low concertation issues) failed to induce significant ACP- or SliC- specific IgG and IgA.
  • the total SliC-specific IgG antibody response was three magnitudes higher when compared to the dual protein vaccine (ACP-SliC+CpG) or the ACP-VLP+SliC-VLP vaccine.
  • Example 10 Immunization with MetQ and MetQ-VLP This example compares immunization with MetQ protein plus adjuvant (MetQ-CpG) and immunization with MetQ-VLP via SC and IN routes. Mice received one SC administration and three IN administrations of MetQ-CpG or MetQ-VLP, with each dose administered two weeks apart. Antibody responses in immunized mice were assessed on Days 31, 52 and 79 by measuring serum IgG (FIGS.14A-14E) and vaginal IgG and IgA (FIGS.15A-15B). The results showed that presenting MetQ on VLPs resulted in more consistent and higher antibody responses (both serum and vaginal tract antibodies) compared to those assessed in mice administered MetQ-CpG.
  • MetQ-CpG MetQ protein plus adjuvant
  • a 245-110515-02 OSU-22-30 single MetQ-VLP vaccine dose induced much higher total serum IgG, IgG1 and IgG2a antibodies compared to MetQ-CpG (FIGS.14A-14E), and MetQ-VLP elicited significantly higher serum IgA compared to all other groups.
  • additional doses of MetQ-CpG increased the geometric mean of antibodies titers, the spread of antibody area under the curve (AUC) was much higher compared to those observed in mice administered MetQ-VLP.

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Abstract

L'invention concerne des compositions immunogènes qui comprennent des particules pseudo-virales (PPV) affichant des antigènes de N. gonorrhoeae conservés sur leur surface. Les PPV sont constituées de la protéine de capside d'un bactériophage à ARN (tel que l'AP205) et les antigènes sont affichés sur les PPV à l'aide du système SpyTag/SpyCatcher. L'invention concerne également l'utilisation des compositions immunogènes pour la protection contre la gonorrhée.
PCT/US2024/054230 2023-11-02 2024-11-01 Particules pseudo-virales présentant des antigènes de neisseria gonorrhoeae et leur utilisation pour l'immunisation contre la gonorrhée Pending WO2025097030A1 (fr)

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