Immunogenic compositions containing lipooligosaccharide sialyltransferase and methods of use thereof

Abstract

Immunogenic compositions containing a lipo-oligosaccharide sialyltransferase (LST) protein or antigenic fragment thereof and an adjuvant are provided. Further immunogenic compositions contain a lipid nanoparticle and a nucleic acid encoding LST protein or an antigenic fragment thereof enclosed within the lipid nanoparticle. Vaccine compositions and methods of protecting or treating a subject from a bacterial infection are also provided.

Description

[0001] IMMUNOGENIC COMPOSITIONS CONTAINING LIPOOLIGOSACCHARIDE SIALYLTRANSFERASE AND METHODS OF USE THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional application 63 / 760,712, filed February 20, 2025. This application is incorporated herein by reference in its entirety.

[0003] SEQUENCE LISTING

[0004] This document incorporates by reference an electronic sequence listing xml file, which was electronically submitted along with this document. The xml file is named 15660007TA_seqlisting, is 35507 bytes, and was created on February 10, 2026.

[0005] FIELD OF THE INVENTION

[0006] The invention generally relates to an immunogenic or vaccine composition comprising lipo-oligosaccharide sialyltransferase or an antigenic fragment thereof. The compositions are useful for protecting or treating a subject from Neisseria gonorrhoeae and other bacterial pathogens.

[0007] BACKGROUND OF THE INVENTION

[0008] Neisseria gonorrhoeae is a gram-negative bacterium responsible for gonorrhea, a prevalent sexually transmitted infection. This organism has developed widespread antimicrobial resistance, necessitating the urgent development of effective vaccines and alternative therapeutic approaches. One of the key virulence factors of Neisseria gonorrhoeae is the lipo-oligosaccharide (LOS) sialyltransferase (LST), which facilitates immune evasion by catalyzing the transfer of sialic acid onto LOS, thereby mimicking host cell surfaces and reducing immune recognition. Prior approaches to gonorrhea vaccines have focused on outer membrane proteins and pili, with limited success. Improved immunogenic and vaccine compositions useful for preventing or treating pathogenic bacterial infections are needed.

[0009] SUMMARY

[0010] An aspect of the disclosure provides an immunogenic composition comprising a lipo-oligosaccharide sialyltransferase (LST) protein or antigenic fragment thereof and an adjuvant. In some embodiments, the LST or antigenic fragment thereof has at least 95% sequence identity to a peptide selected from the group consisting of SEQ ID Nos 1-5 and 11-31. In some embodiments, the LST protein or antigenic fragment thereof is conjugated to a carrier protein. In some embodiments, the carrier protein is selected from the group consisting of tetanus toxoid, diphtheria toxoid, keyhole limpet hemocyanin, CRM 197, and outer membrane protein complex (OMPC). In some embodiments, the LST protein or antigenic fragment thereof is conjugated to a vesicle carrier, such as an outer membrane vesicle (OMV). In some embodiments, the composition further comprises a lipooligosaccharide (LOS) or an antigenic fragment thereof. In some embodiments, the LST protein or antigenic fragment thereof and the LOS or antigenic fragment thereof are conjugated to a shared vesicle carrier, such as an OMV. In some embodiments, LST protein, LOS, or antigenic fragments thereof are displayed on the surface of a virus-like particle (VLP).

[0011] In some embodiments, the immunogenic composition further comprises one or more additional bacterial antigens. In some embodiments, 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. In some embodiments, the adjuvant is selected from the group consisting of aluminum hydroxide, monophosphoryl lipid A, CpG, and MF59.

[0012] Another aspect of the disclosure provides an immunogenic composition comprising a lipid nanoparticle and a nucleic acid encoding LST protein or an antigenic fragment thereof enclosed within the lipid nanoparticle. In some embodiments, the nucleic acid has at least 95% sequence identity to a nucleic acid selected from the group consisting of SEQ ID Nos 6-10.

[0013] Another aspect of the disclosure provides a vaccine composition comprising an immunogenic composition as described herein and a pharmaceutically acceptable carrier.

[0014] Another aspect of the disclosure provides a method of protecting or treating a subject from a bacterial infection comprising administering to the subject an immunogenic or vaccine composition as described herein. In some embodiments, the bacterial infection is caused by Neisseria gonorrhoeae, Haemophilus influenzae, Moraxella catarrhalis, or Neisseria meningitidis.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figures 1A-B. Summary of the role of gonococcal LOS sialylation in pathogenesis. A.

[0016] LOS sialylation occurs through LOS sialyltransferase (Lst). B. Mechanisms of host immune evasion by LOS sialic acid (Neu5Ac).

[0017] Figures 2A-B. Decreased Lst expression level impairs gonococcal complement resistance and its ability to colonize mice. A. Low Lst expression levels impair sialylation and complement resistance. N. gonorrhoeae expressing gonococcal Lst from either the gonococcal F621st promoter (Pist-Ng) or the weak meningococcal 1st promoter (Pist-Nm) in 33.3% pooled normal human serum following growth in the absence (0 pg / ml) or presence (0.1 or 1.0 pg / ml) of CMP-Neu5 Ac. The 1st knockout (Alst) is shown as a comparator. Percent survival at 30 min (t30) relative to tO are plotted on the y axis (mean [SEM] of 2 experiments. **, P < 0.01; ***, P < 0.001). B.

[0018] Attenuation of Pist-Nm in the mouse vaginal colonization model. Data representing recovery of Pist-Nm relative to recovery of its isogenic mutant Pist-Ng from estradiol-treated B ALB / c mice coinfected with both strains are expressed as competitive indices (CI); each point represents the CI calculated from an individual mouse. Data representing recovery of Alst relative to that Pist-Ng are also shown. A CI <1.0 indicates a decrease in the ratio of mutant to wild type with respect to that of the inoculum. The ratios of strain Pist-Nm to strain Pist-Ng and of strain Alst to strain Pist-Ng in the inocula were 1 and 0.85, respectively. Horizontal bars represent the geometric mean CI values; open symbols represent mice from whom at least 50 wild-type (Pist-Ng) CFU / ml, but no mutant (Pist-Nm or A 1st) CFU, were recovered. The limit of detection (40 CFU / ml) was used as the number of mutant CFU recovered in these cases. From Lewis LA et al, mBio, 2015.

[0019] Figures 3A-C. V. gonorrhoeae recombinant Lst adjuvanted with monophosphoryl lipid A (MPL) plus Alum decreases the duration and burden of gonococcal vaginal colonization in mice. Six-week-old female BALB / c mice were immunized with recombinant Lst (20 pg / dose) adjuvanted with MPL (20 pg) plus Alum (200 pg) at weeks 0, 2, 4 and 9. Mice in the disestrus phase of the oestrus cycle were treated with Premarin® (Pfizer) and antibiotics to suppress commensal flora, and challenged at week 11 intravaginally with 3.5 x 107CFU N. gonorrhoeae FA1090 (n=7 and 5 in the vaccine and adjuvant control groups, respectively). Vaginal swabs were obtained daily to enumerate CFU. A. Kaplan-Meier curves showing time to clearance (P=0.0009 by Mantel-Cox log-rank test). B. CFU (logio) counts versus time.Comparisons were made by 2-way ANOVA. C. Area under the curve (AUC) analysis for consolidated bacterial burden over time. Pairwise comparisons were by Mann-Whitney nonparametric t test.

[0020] Figures 4A-C. The terminal complement pathway (membrane attack complex, or C5b-9) is required for efficacy of the Lst vaccine. Six-week-old female transgenic (Tg) mice that express the human complement inhibitors Factor H (FH) and C4b-binding protein (C4BP) in a BALB / c background were immunized with 20 pg recombinant Lst adjuvanted with MPL / Alum on weeks 0, 2, 4 and 7. Control animals were given MPL / Alum alone. At week 9, mice in the disestrus phase of the oestrus cycle (n=10 / group) were treated with Premarin® (Pfizer) and antibiotics to suppress commensal flora, and challenged intravaginally with 4.2 x 107CFU N. gonorrhoeae FA 1090 on day 0. Five mice in each group were treated intraperitoneally (IP) with 2 mg of a function-blocking anti-C7 mAb or vehicle (PBS) on days -1, 2 and 5. Vaginal swabs were obtained daily to enumerate CFU. A. Kaplan-Meier curves showing time to clearance. Pairwise comparisons were made with Mantel-Cox log-rank test. The Lst / MPL / Alum group showed significantly faster clearance (P<0.003) compared to all groups. B. CFU (logic) counts versus time. Comparisons with the MPL / Alum group were made by 2-way ANOVA and Dunnett’s multiple comparison test. PcO.OOOl. C. Area under the curve (AUC) analysis for consolidated bacterial burden over time. Pairwise comparisons were by Mann-Whitney nonparametric t test.

[0021] Figure 5. Lst immunization increases the susceptibility of N. gonorrhoeae to complement-dependent killing. Gonococci (FA1090) recovered directly (i.e., without subpassage on artificial media) from the vaginas of BALB / c mice immunized with Lst plus MPL-Alum or MPL-Alum alone on days 1 and 2 were serially diluted and tested for resistance to human complement (pooled normal human serum at a final concentration of 10%). Aliquots of the reaction mixture were plated onto chocolate agar containing vancomycin, colistin, trimethoprim and nystatin to suppress normal flora and selectively permit N. gonorrhoeae growth at 0- and 30-min. Percent survival at 30 min relative to 0 min is shown on the Y-axis. Reaction mixtures that yielded 20 to 100 CFU at 0 min were used to calculate survival.

[0022] Figures 6A-D. Diagram of LST / LOS dual targeting strategy. (A) LST enzyme in outer membrane. (B) Steric blocker antibody (anti-LST) and LOS access block. (C) Functional synergy - masked vs. unmasked LOS. (D) Anti-LOS effector activity and complement- mediated lysis.DETAILED DESCRIPTION

[0023] Embodiments of the disclosure provide compositions comprising lipo-oligosaccharide sialyltransferase (LST) proteins or antigenic fragments thereof and their use in vaccines, e.g. subunit / conjugate vaccines. Figure 1 is a summary of the role of gonococcal lipo-oligosaccharide (LOS) sialylation in pathogenesis. LOS sialylation occurs through LST believed to reside in the outer membrane (Fig. 1 A) (1). CMP-Neu5Ac (or CMP-NANA), the donor molecule for sialic acid, is derived from the host (2). The LOS structure shown represents lacto-N-neotetraose (LNnT; GaL GlcNAc-GaLGlc). Fig. IB shows mechanisms of host immune evasion by LOS sialic acid (Neu5Ac). LOS Neu5Ac inhibits complement activation by reducing IgG binding (3, 4) and by enhancing binding of the complement inhibitor, factor H (FH) (5). Neu5Ac engages sialic acidbinding immunoglobulin-type lectins (Siglecs) on host cell surfaces (6), most of which possess immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and dampen host inflammatory responses. LOS Neu5Ac also confers resistance to cationic antimicrobial peptides (e.g., LL-37) (7).

[0024] LST is a key virulence factor in many bacterial infections, including those caused by Neisseria gonorrhoeae, H. influenzae, M. catarrhalis, etc. LST plays a crucial role in the pathogen's ability to evade the host immune system by mimicking host cell surfaces, thus reducing immune recognition and allowing persistent infection. Further, LST is ubiquitously expressed and highly conserved among different strains of N. gonorrhoeae. The compositions described herein thus provide broad protection against various strains of the bacteria. The disclosed compositions include LST either as a recombinant protein or as a DNA / RNA-based construct to stimulate the immune system to recognize and attack bacteria effectively. This approach not only targets the bacteria directly but also interferes with their mechanism of immune evasion, providing a twofold benefit.

[0025] The LST as described herein may be isolated from a Gram negative bacteria such as the Neisseria, Campylobacter, or Haemophilus families or the LST may be chemically synthesized. In some embodiments, the LST may be isolated from any strain of N. gonorrhoeae including F62, FA1090, FA19, MS 11, NG-STAR ST-90, ST-58, ST-61, ST-64, ST-79, ST-91, ST-139, etc. LSTs may be isolated from other bacterial pathogens including, but not limited to, Neisseria meningitidis, Neisseria lactamica, Haemophilus influenzae, Moraxella catarrhalis,Campylobacter jejuni, Haemophilus ducreyi, Haemophilus parasuis, Helicobacter bizzozeroni , etc.

[0026] Exemplary antigenic peptide sequences include:

[0027] SEQ ID NO:1 — Neisseria gonorrhoeae Lst (strain F62)

[0028] Amino acid sequence MGLKKVCLTVLCLIVFCFGIFYTFDRVNQGERNAVSLLKDKLFNEEGKPVNLIFCYTILQ MKVAERIMAQHPGERFYVVLMSENRNEKYDYYFNQIKDKAERAYFFYLPYGLNKSFNF IPTMAELKVKSMLLPKVKRIYLASLEKVSIAAFLSTYPDAEIKTFDDGTNNLIRESSYLGG EFAVNGAIKRNFARMMVGDWSIAKTRNASDEHYTIFKGLKNIMDDGRRKMTYLPLFDA SELKAGDETGGTVRILLGSPDKEMKEISEKAAKNFNIQYVAPHPRQTYGLSGVTALNSP YVIEDYILREIKKNPHTRYEIYTFFSGAALTMKDFPNVHVYALKPASLPEDYWLKPVYAL FRQADIPILTFDDKN

[0029] SEQ ID NO:2 — Neisseria meningitidis Lst (strain MC58)

[0030] Amino acid sequence MGLKKACLTVLCLIVFCFGIFYTFDRVNQGERNAVSLLKEKLFNEEGEPVNLIFCYTILQ MKVAERIMAQHPGERFYVVLMSENRNEKYDYYFNQIKDKAERAYFFHLPYGLNKSFNF IPTMAELKVKSMLLPKVKRIYLASLEKVSIAAFLSTYPDAEIKTFDDGTGNLIQSSSYLGD EFSVNGTIKRNFARMMIGDWSIAKTRNASDEHYTIFKGLKNIMDDGRRKMTYLPLFDAS ELKTGDETGGTVRILLGSPDKEMKEISEKAAKNFKIQYVAPHPRQTYGLSGVTTLNSPYV IEDYILREIKKNPHTRYEIYTFFSGAALTMKDFPNVHVYALKPASLPEDYWLKPVYALFT QSGIPILTFDDKN

[0031] SEQ ID NO:3 — Neisseria meningitidis Lst (strain 406Y)

[0032] Amino acid sequence MGLKKACLTVLCLIVFCFGIFYTFDRVNHGERNAVSLLKDKLFNEEGEPVNLIFCYTILQ MKVAERIMAQHPGERFYVVLMSENRNEKYDYYFKQIKDKAERAYFFHLPYGLNKSFNF IPTMAELKVKSMLLPKVKRIYLASLEKVSIAAFLSTYPDAEIKTFDDGTGNLIQSSSYLGD EFSVNGTIKRNFARMMIGDWSIAKTRNASDEHYTIFKGLKNIMDDGRRKMTYLPLFDAS ELKAGDETGGTVRILLGSPDKEMKEISEKAAKNFNIQYVAPHPRQTYGLSGVTTLNSPYVIEDYILREIKKNPHTRYEIYTFFSGAALTMKDFPNVHVYALKPASLPEDYWLKPVYALF TQSGIPILTFDDKN

[0033] SEQ ID NO:4 — Neisseria meningitidis Lst (strain 126E)

[0034] Amino add sequence MGLKKACLTVLCLIVFCFGIFYTFDRVNQGERNAVSLLKDKLFNEEGEPVNLIFCYTILQ MKVAERIMAQHPGERFYVVLMSENRNEKYDYYFNQIKDKAEWAYFFHLPYGLNKSFN FIPTMAELKVKAMLLPKVKRIYLASLEKVSIAAFLSTYPDAEIKTFDDGTINLIQSSSYLG DEFSVNGTIKRNFARMMIGDWSIAKTRNASDEHYTIFKGLKNIMDDGRRKMTYLPLFDA SELKAGDETGGTVRILLGSPDKEMKEISEKAAKNFNIQYVAPHPRQTYGLSGVTTLNSPY VIEDYILREIKKNPHTRYEIYTFFSGAALTMKDFPNVHVYALKPASLPEDYWLKPVYALF TQSGIPILTFDDKN

[0035] SEQ ID NO:5 — Neisseria lactamica

[0036] Amino acid sequence MGLKKACLTVLCLIVFCFGIFYTFDRVNQGERNAVSLLKDKLFNEEGEPVNLIFCYTILQ MKVAERIMAQHPGERFYVVLMSENRNEKYDYYFNQIKDKAERAYFFYLPYGLNKSFNF IPTMAELKVKSMLLPKVKRIYLASLEKVSIAAFLSTYPDAEIKTFDDGTNNLIQESSYLGD EFSVNGTIKRNFARMMIGDWSIAKTRNASDEHYTIFKGLKNIMDDGRRKMTYLPLFDAS ELKAGDETGGTVRILLGSPDKEMKEISEKAAKNFKIQYVAPHPRQTYGLSGVTTLNSPY VIEDYILREIKKNPHTRYEIYTFFSGAALTMKDFPNVHVYALKPASLPEDYWLKPVYALF TQS GIPILTFDDKNQS YGKS K

[0037] The corresponding DNA sequences are as follows. For each cDNA sequence presented herein, the invention includes the mRNA equivalent of the cDNA, meaning that the invention includes each cDNA sequence wherein each T is replaced by U.

[0038] SEQ ID NO:6 — Neisseria gonorrhoeae Lst (strain F62)

[0039] Nucleic acid sequence ATGGGGTTGAAAAAAGTCTGTTTGACCGTGTTGTGCCTGATTGTTTTTTGCTTCGGGA TATTTTTATACGTTTGACCGGGTAAATCAGGGGGAAAGGAACGCGGTTTCCCTGCTGAAGGACAAACTCTTCAATGAAGAGGGGAAACCCGTCAATCTGATTTTCTGCTATACC ATATTGCAGATGAAGGTGGCAGAAAGGATTATGGCGCAGCATCCGGGGGAGCGGTT TTATGTGGTGCTGATGTCTGAAAACAGGAATGAAAAATACGATTATTATTTCAATCA GATAAAGGATAAGGCGGAGCGGGCGTATTTTTTCTACCTGCCCTACGGTTTGAACAA ATCGTTTAATTTCATTCCGACGATGGCGGAGCTGAAGGTGAAGTCGATGCTGCTGCC GAAGGTCAAGCGGATTTATTTGGCGAGTTTGGAAAAAGTCAGTATTGCCGCCTTTTT GAGCACTTACCCGGATGCGGAAATCAAAACCTTTGACGACGGCACAAACAACCTGA TACGGGAGAGCAGCTATTTGGGCGGCGAGTTTGCCGTAAACGGGGCGATTAAGCGG AATTTTGCCCGAATGATGGTCGGGGATTGGAGCATCGCCAAAACCCGCAATGCTTCC GACGAGCATTACACGATATTCAAGGGTTTGAAAAACATTATGGATGACGGCCGCCG CAAGATGACTTACCTGCCGCTGTTCGATGCGTCCGAACTGAAGGCGGGGGACGAAA CGGGCGGCACGGTGCGGATACTTTTGGGTTCGCCCGACAAAGAGATGAAGGAAATT TCGGAAAAGGCGGCAAAAAATTTCAACATACAATATGTCGCGCCGCATCCCCGCCA GACCTACGGGCTTTCCGGCGTAACCGCGTTAAATTCGCCCTATGTCATCGAAGACTA TATTTTGCGCGAAATTAAGAAAAACCCGCATACGAGGTATGAAATTTATACCTTTTT CAGCGGTGCGGCGTTGACGATGAAGGATTTTCCCAATGTGCACGTTTACGCATTGAA ACCGGCTTCCCTTCCGGAAGATTATTGGCTCAAGCCCGTTTATGCGCTGTTCCGTCAG GCCGACATTCCGATTTTGACATTTGACGATAAAAATTAA

[0040] SEQ ID NO:7 — Neisseria meningitidis Lst (strain MC58)

[0041] Nucleic acid sequence ATGGGCTTGAAAAAGGCTTGTTTGACCGTGTTGTGTTTGATTGTTTTTTGTTTCGGGA TATTTTATACATTTGACCGGGTAAATCAGGGGGAAAGGAATGCGGTTTCCCTGCTGA AGGAGAAACTTTTCAATGAAGAGGGGGAACCGGTCAATCTGATTTTCTGTTATACCA TATTGCAGATAAAGGTGGCGGAAAGGATTATGGCGCAGCATCCGGGCGAGCGGTTT TATGTGGTGCTGATGTCTGAAAACAGGAATGAAAAATACGATTATTATTTCAATCAG ATAAAGGATAAGGCGGAGCGGGCGTACTTTTTCCACCTGCCCTACGGTTTGAACAAA TCGTTTAATTTCATTCCGACGATGGCGGAGCTGAAGGTAAAGTCGATGCTGCTGCCG AAAGTCAAGCGGATTTATTTGGCAAGTTTGGAAAAAGTCAGCATTGCCGCCTTTTTG AGCACTTACCCGGATGCGGAAATCAAAACCTTTGACGACGGGACAGGCAATTTAAT TCAAAGCAGCAGCTATTTGGGCGATGAGTTTTCTGTAAACGGGACGATCAAGCGGAATTTTGCCCGGATGATGATCGGAGATTGGAGCATCGCCAAAACCCGCAATGCTTCCG ACGAGCATTACACGATATTCAAGGGTTTGAAAAACATTATGGACGACGGCCGCCGC AAGATGACTTACCTGCCGCTGTTCGATGCGTCCGAACTGAAGACGGGGGACGAAAC GGGCGGCACGGTGCGGATACTTTTGGGTTCGCCCGACAAAGAGATGAAGGAAATTT CGGAAAAGGCGGCAAAAAACTTCAAAATACAATATGTCGCGCCGCATCCCCGCCAA ACCTACGGGCTTTCCGGCGTAACCACATTAAATTCGCCCTATGTCATCGAAGACTAT ATTTTGCGCGAGATTAAGAAAAACCGCATACGAGGTATGAAATTTATACCTTTTTCA GCGGCGCGGCGTTGACGATGAAGGATTTTCCCAATGTGCACGTTTACGCATTGAAAC CGGCTTCCCTTCCGGAAGATTATTGGCTCAAGCCGGTGTATGCCCTGTTTACCCAATC CGGCATCCCGATTTTGACATTTGACGATAAAAATTAA

[0042] SEQ ID NO:8 — Neisseria meningitidis Lst (strain 406Y)

[0043] Nucleic acid sequence ATGGGCTTGAAAAAGGCTTGTTTGACCGTGTTGTGTTTGATTGTTTTTTGTTTCGGGA TATTTTATACATTTGACCGGGTAAATCATGGGGAAAGGAATGCGGTTTCCCTGCTGA AGGACAAACTCTTCAATGAAGAGGGGGAACCGGTCAATCTGATTTTCTGCTATACCA TATTGCAGATGAAGGTGGCGGAAAGGATTATGGCGCAGCATCCGGGGGAGCGGTTT TATGTGGTGCTGATGTCTGAAAACAGGAATGAAAAATACGATTATTATTTCAAGCAG ATAAAGGATAAGGCGGAGCGGGCGTATTTTTTCCACCTGCCCTACGGTTTGAACAAA TCGTTTAATTTCATTCCGACGATGGCGGAGCTGAAGGTAAAGTCGATGCTGCTGCCG AAAGTCAAGCGGATTTATTTGGCAAGTTTGGAAAAAGTCAGCATTGCCGCCTTTTTG AGCACTTACCCGGATGCGGAAATCAAAACCTTTGACGACGGGACAGGCAATTTAAT TCAAAGCAGCAGCTATTTGGGCGATGAGTTTTCTGTAAACGGGACGATCAAGCGGA ATTTTGCCCGGATGATGATCGGAGATTGGAGCATCGCCAAAACCCGTAATGCTTCCG ACGAGCATTACACGATATTCAAGGGTTTGAAAAACATTATGGACGACGGCCGCCGC AAGATGACTTACCTGCCGCTGTTCGATGCGTCCGAACTGAAGGCGGGGGACGAAAC GGGCGGCACGGTGCGGATACTTTTGGGTTCGCCCGACAAGGAGATGAAGGAAATTT CGGAAAAGGCGGCAAAAAACTTCAACATACAATATGTCGCACCGCACCCCCGCCAA ACCTACGGGCTTTCCGGCGTAACCACATTAAATTCGCCCTATGTCATCGAAGACTAT ATTTTGCGCGAGATTAAGAAAAACCCGCATACGAGGTATGAAATTTATACCTTTTTC AGCGGCGCGGCGTTGACGATGAAGGATTTTCCCAATGTGCACGTTTACGCATTGAAACCGGCTTCCCTTCCGGAAGATTATTGGCTCAAGCCGGTGTATGCCCTGTTTACCCAAT CCGGCATCCCGATTTTGACATTTGACGATAAAAATTAA

[0044] SEQ ID NO:9 — Neisseria meningitidis Lst (strain 126E)

[0045] Nucleic acid ATGGGCTTGAAAAAGGCTTGTTTGACCGTGTTGTGCCTGATTGTTTTTTGTTTCGGGA TATTTTATACATTTGACCGGGTAAATCAGGGGGAAAGGAATGCGGTTTCCCTGCTGA AGGACAAACTCTTCAATGAAGAGGGGGAACCGGTCAATCTGATTTTCTGCTATACCA TATTGCAGATAAAGGTGGCAGAAAGGATTATGGCGCAGCATCCGGGGGAGCGGTTT TATGTGGTGCTGATGTCTGAAAACAGGAATGAAAAATACGATTATTATTTCAATCAG ATAAAGGATAAGGCGGAGTGWGGCGTATTTTTTCCACCTGCCCTACGGTTTGAACA AATCGTTTAATTTCATTCCGACGATGGCGGAGCTGAAGGTGAAGGCAATGCTGCTGC CGAAGGTCAAGCGGATTTATTTGGCAAGTTTGGAAAAAGTCAGCATTGCCGCCTTTT TGAGCACTTACCCGGATGCGGAAATCAAAACCTTTGACGACGGGACAATCAATTTA ATTCAAAGCAGCAGCTATTTGGGTGATGAGTTTTCTGTAAACGGGACGATCAAGCGG AATTTTGCCCGGATGATGATCGGAGATTGGAGCATCGCCAAAACCCGTAATGCTTCC GACGAGCATTACACGATATTCAAGGGTTTGAAAAACATTATGGACGACGGTCGCCG CAAGATGACTTACCTGCCGCTGTTCGATGCGTCCGAACTGAAGGCGGGGGACGAAA CGGGCGGCACGGTGCGGATACTTTTGGGTTCGCCTGACAAGGAGATGAAGGAAATT TCGGAAAAGGCGGCAAAAAACTTCAACATACAATATGTCGCACCGCACCCCCGCCA AACCTACGGGCTTTCCGGCGTAACCACATTAAATTCGCCCTATGTCATCGAAGACTA TATTTTGCGCGAAATTAAGAAAAACCGCATACGAGGTATGAAATTTATACCTTTTTC AGCGGTGCGGCGTTGACGATGAAGGATTTTCCCAATGTGCACGTTTACGCATTGAAA ACCGGCTTCCCTTCCGGAAGATTATTGGCTCAAGCCGGTGTATGCCCTGTTTACCCA ATCCGGCATCCCGATTTTGACATTTGACGATAAAAATTAA

[0046] SEQ ID NO:10 — Neisseria lactamica

[0047] Nucleotide sequence ATGGGTCTGAAAAAAGCTTGCCTGACCGTTCTGTGCCTGATTGTTTTTTGCTTTGGTA TTTTTACCATTTCAGATCGTGTTAACCAAGGTGAACCGGTTAATCTGGCAGTTAGCCT GCTGAAAGATAAACTGTTTAATGAAGAAGGTGAACCGGTTAATCTGATTTTTTGCTATACCATTCTGCAAATGAAAGTTGCTGAACGTATTATGGCTCAACATCCGGGTGAAAG ATTTTATGTTGTTTTGATGAGCGAAAATCGTAATGAAAAATATGATTATTATTTTAAT CAAATTAAAGATAAAGCTGAACGTGCTTATTTTTTTTATCTGCCGTATGGTCTGAATA AATCCTTTAATTTTATTCCGACCATGGCTGAATTGAAAGTTAAATCGATGCTGCTGCC GAAAGTTAAACGTATTTATCTGGCCTCGCTGGAAAAAGTTAGCATTGCTGCTTTTCT GAGCACCTATCCGGATGCTGAAATTAAAACCTTTGATGATGGTACCAATAATCTGAT TCAAGAAAGCTCGTATCTGGGTGATGAATTTAGCGTTAATGGTACCATTAAACGTAA CTTTGCTCGTATGATGATTGGATGGAGCATTGCTAAAACCCGTAACTATAGCGATGA ACATTATACCATTTCGGTGCTGAAAAAATATTATGGATGATGGTAGACGTAAACGTA TGACCTATCTGCCGCTGTTTGATGCTAGCGAACTGAAAGCTGGTGATGAAACCGGTG GTACCGTTAGAATTCTGCTGGGTAGCCCGGATAAAGAAATGAAAGAAATTTCGGAA AAAGCTGCTAAAAATTTTAAAATTCAATATGTTGCTCCGCATCCGCGTCAAACCTAT GGTCTGAGCGGTGTTACCACCCTGAATAGCCCGTATGTTATTGAAGATTATATTCTG CGTGAAATTAAAAAAAATCCGCATACCAGATATGAAATTTATACCTTTTTTAGCGGT GCTGCTCTGACCATGAAAGATTTTCCGAATGTTCATGTTTATGCTCTGAAACCGGCT AGCCTGCCGGAAGATTATTGGCTGAAACCGGTTTATGCTCTGTTTACCCAATCGGGT ATTCCGATTCTGACCTTTGATGATAAAAATCAATCGTATGGTAAATCGAAATAA

[0048] A fragment of a LST as described herein is a portion of LST that retains at least one antigenic epitope of the LST.

[0049] Preferably, the sequence included in the composition exhibits between about 60-100% sequence identity to a naturally occurring LST sequence or sequence described herein and more preferably the sequences exhibit between about 80-100% sequence identity, e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a naturally occurring polynucleotide or polypeptide LST sequence or a sequence as disclosed herein. In addition, the sequences described herein can be operably linked to each other in any combination. For example, one or more sequences may be expressed from the same promoter and / or from different promoters.

[0050] As used herein, the terms “polypeptide”, “short protein”, “fragment of protein”, “polypeptide or fragment”, “antigenic polypeptide or fragment” and “peptide” are used interchangeably and refer to chains of amino acids comprising between 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15 and 10, or 11, or 12, or 13, or 14, or 15, or 20,or 25, or 30, or 35, or 40, or 45, or 50 or 55 or 60 or 100 or 150 or 200 or 300 or 350 or 400 or 500 amino acids. As used herein, the term “epitope” or “T cell epitope” refers to a sequence of contiguous amino acids contained within a protein antigen that possess a binding affinity to a T cell receptor when presented on the surface of antigen presenting cells. An epitope is antigenspecific but not individual specific. An epitope, a T cell epitope, a polypeptide, a fragment of a polypeptide or a composition comprising a polypeptide or a fragment thereof is “immunogenic” for a specific human individual if it is capable of inducing an immune cell response in that individual. In some embodiments, an “immune response”, “T cell response” or “immunogenic response” are used interchangeably and may further include an antibody response. As used historically, the term “antigen” is used to designate an entity that is bound by an antigen-specific antibody or B-cell antigen receptor.

[0051] As used herein, the term “antigens”, “proteins”, “peptides”, “polypeptides”, “fragments”, or “epitopes” may be used interchangeably. In particular, an “antigen” refers to a molecule containing one or more epitopes (either linear, conformational or both) that will stimulate a host’s immune-system to make a humoral and / or cellular antigen- specific response. The term is used interchangeably with the term “immunogen”. Further, antigenic polypeptide or fragment “derived from” a particular bacterial protein or protein domain refers to a full-length or near full-length bacterial protein or domain, as well as a fragment thereof, or a bacterial protein with internal deletions. Accordingly, the polypeptide may comprise the full-length sequence, fragments, truncated and partial sequences, as well as analogs and precursor forms of the reference molecule. In addition, the term “derived” may refer to construction of a peptide based on the knowledge of a representative protein domain sequence using any one of several suitable means, including, by way of example, isolation or synthesis. Thus, the term includes variations of the specified polypeptide.

[0052] In some embodiments, the antigenic peptides as described herein are 4 to 150 residues in length, e.g. 4 to 100 residues, 4 to 50 residues, or 8 to 30 residues. The antigenic peptides may include consecutive or nonconsecutive sequences from the bacterial peptides. Embodiments include fragments retaining conformational epitopes provided that residues critical for folding and surface exposure are preserved. Exemplary B-cell epitope sequences from N. gonorrhoeae F62 LST are provided in SEQ ID Nos 11-20. Exemplary B-cell epitope sequences from N. meningitidis MC58 LST are provided in SEQ ID Nos 21-25.B-cell epitope peptide sequences from N. gonorrhoeae F62 Lst

[0053] SEQ ID NO: 11 - DRVNQGERNAVS

[0054] SEQ ID NO: 12 - NEKYDYYFNQ

[0055] SEQ ID NO: 13 - GLNK

[0056] SEQ ID NO: 14 - KSMLLP

[0057] SEQ ID NO: 15 - AEIKTFDDGTNNLIRESSYLGGEFAVN

[0058] SEQ ID NO: 16 - SIAKTRNASDEHYTIFKGLKNIMDDGR

[0059] SEQ ID NO: 17 - ASELKAGDETG

[0060] SEQ ID NO: 18 KEMKEI

[0061] SEQ ID NO: 19 - RQTYGLS

[0062] SEQ ID NO:20 - SLPEDYWLK

[0063] B-cell epitope peptide sequences from N. meningitidis MC58 Lst

[0064] SEQ ID NO:21 - DRVNQGERNAVSL

[0065] SEQ ID NO:22 - NEKYDYYFNQIKDKA

[0066] SEQ ID NO:23 - AEIKTFDDGTGNLIQSSSYLGDEFSVNGT

[0067] SEQ ID NO:24 - ASELKTGDETG

[0068] SEQ ID NO:25 - EMKEISE

[0069] The present inventors have also identified mechanistically relevant regions of LST, derived from crystallographic and mechanistic analysis of the homologous Neisseria meningitidis enzyme (PDB 2yk7; Lin et al., Lin, L.Y.-C. et al. 'Structure and Mechanism of the Lipooligosaccharide Sialyltransferase from Neisseria meningitidis.' Journal of Biological Chemistry, 2011). Fragments encompassing these regions or specified residues are included within the present disclosure. In some embodiments, the fragments are associated with LST enzymatic activity, CMP-Neu5Ac binding, or immune evasion.

[0070] Regions Implicated in CMP-Neu5Ac Binding

[0071] The donor substrate CMP-Neu5Ac binds within a composite active site formed at the interface of a domain-swapped homodimer. Key structural features include:i) A C-terminal Rossmann-like domain coordinating the CMP moiety.

[0072] ii) Residues corresponding to His-280 and Glu-300, which interact with the phosphate and ribose groups of CMP.

[0073] iii) Hydrophobic residues (e.g., Pro-281, Ile-299) stabilizing the cytidine base.

[0074] iv) A dimer-interface contribution from the N-terminal swapping module, including residues corresponding to Phe-118 and Glu-124, which help position the sialic acid moiety via direct and water-mediated interactions.

[0075] These observations indicate that donor binding depends on both monomer-intrinsic regions and residues contributed in trans from the adjacent subunit.

[0076] Catalytic and Substrate-Proximal Regions

[0077] Mutagenesis and structural data identify a set of residues forming the rim and interior of the catalytic pocket:

[0078] i) Asp-258: positioned near the anomeric center; mutation abolishes both hydrolysis and transfer activity, supporting a role as a general base or gating residue.

[0079] ii) His-280: positioned to stabilize the leaving group; mutation significantly reduces catalytic efficiency, consistent with a general acid role.

[0080] iii) Arg-282: contributes to donor binding and transition-state stabilization.

[0081] iv) Glu-124 and Phe-118: contribute to positioning of the donor sialic acid and define the entrance to the active site.

[0082] Together, these residues define a catalytic / substrate-proximal region that governs enzymatic activity and substrate access.

[0083] N-terminal Signal Sequence and Membrane-Associated Regions

[0084] The native enzyme is membrane associated and contains an N-terminal transmembrane helix that anchors the catalytic domain to the bacterial outer membrane. Structural studies used a truncated construct lacking this segment, confirming that:

[0085] i) The N-terminal region is responsible for membrane localization rather than catalysis per se.ii) The enzyme contains a hydrophobic tunnel adjacent to the dimer interface that accommodates lipid or detergent molecules, consistent with interaction with lipidated LOS substrates.

[0086] iii) This lipid-binding cavity involves residues within and adjacent to the swapping module (approximately residues 108-121), supporting stable membrane- associated dimerization.

[0087] Thus, embodiments of the disclosure encompass LST or LST fragments which are displayed in membrane contexts (e.g., OMVs or vesicles), enabling antibody access to functionally relevant surfaces.

[0088] Swapping Module and Dimer Interface

[0089] A distinctive feature of Neisseria LST enzymes is a domain- swapped homodimer formed by exchange of N-terminal residues (approximately Pro-49 to Met- 130). This swapping module:

[0090] i) is mediated by a loop-helix-loop region (approximately residues 108-130), ii) contributes residues directly to the composite active site, and

[0091] ii) is implicated in acceptor substrate binding and conformational activation of the enzyme. Because functional activity requires this dimeric architecture, regions within the swapping module and dimer interface represent structurally and mechanistically significant targets.

[0092] Exemplary antigenic fragments encompassing the aforementioned regions include:

[0093] SEQ ID NO: 26 - Domain-swap hinge

[0094] Residues 108-134

[0095] FYVVLMSENRNEKYDYYFNQIKDKAER

[0096] SEQ ID NO: 27 - Composite donor-binding loop

[0097] Residues 113-127

[0098] MSENRNEKYDYYFNQ

[0099] SEQ ID NO: 28 - Catalytic gate loop - Asp258

[0100] Residues 251-265

[0101] GSPDKEMKEISEKAASEQ ID NO: 29 - Donor phosphate I transition-state region

[0102] Residues 273-287

[0103] VAPHPRQTYGLS G VT

[0104] SEQ ID NO: 30 - Acceptor-binding platform

[0105] Residues 165-181

[0106] IKTFDDGTNNLIRESSY

[0107] SEQ ID NO: 31 - C-terminal donor / acceptor surface

[0108] Residues 296-310

[0109] DYILREIKKNPHTRY

[0110] The antigenic peptides described herein may comprise epitopes, i.e. amino acids that bind to an antibody generated in response to such sequence. An epitope for use in the subject invention is not limited to a polypeptide having the exact sequence of the portion of the parent protein from which it is derived. Thus, the term "epitope" encompasses sequences identical to the native sequence, as well as modifications to the native sequence, such as deletions, additions, and substitutions (generally conservative in nature).

[0111] As used herein, the term "conformational epitope" refers to a recombinant epitope having structural features native to the amino acid sequence encoding the epitope within the full-length natural protein. Native structural features include, but are not limited to, glycosylation and three-dimensional structure. The length of the epitope-defining sequence can be subject to wide variations as these epitopes are believed to be formed by the three-dimensional shape of the antigen (e.g„ folding). Thus, amino acids defining the epitope can be relatively few in number, but widely dispersed along the length of the molecule (or even on different molecules in the case of dimers, etc.), being brought into correct epitope conformation via folding. The portions of the antigen between the residues defining the epitope may not be critical to the conformational structure of the epitope. For example, deletion or substitution of these intervening sequences may not affect the conformational epitope provided sequences critical to epitope conformation are maintained (e.g., cysteines involved in disulfide bonding, glycosylation sites, etc.).Embodiments provide an LST, or fragment thereof, as described herein conjugated to a carrier. Exemplary carriers include, but are not limited to: carrier proteins (e.g., tetanus toxoid, diphtheria toxoid, keyhole limpet hemocyanin, CRM 197, outer membrane protein complex (OMPC), etc. or protein-based carriers such as albumin carriers), virus-like particles (VLP) including Hepatitis B Surface Antigen VLP, QP Bacteriophage VLP, Bacteriophage AP205 capsid VLP, etc. ferritin nanoparticles, lipid-based carriers (such as liposomes and lipid nanoparticles), inorganic nanoparticles such as inorganic gold particles, dendrimers, outer membrane vesicles (OMV), extracellular vesicles, dendritic cell-derived small extracellular vesicles, erythrocyte-derived vesicles, generalized modules for membrane antigens (GMMA), protein nanocages, polymer-based carriers (such as polymeric micelles and microspheres), nanocapsules, hydrogels, transferosomes and antibodies.

[0112] Conjugating LST or a fragment thereof to a carrier or combining it with other bacterial antigens could enhance its ability to induce a protective immune response. For example, the LST or fragment 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.

[0113] Conjugating LST or the fragment thereof 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.

[0114] Methods for conjugation are known in the art. In some embodiments, a linker is present between the LST or fragment thereof and the earner. Linkers can be incorporated to control the stability, flexibility, and spacing between the conjugated components. Properly chosen linkersensure 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 LST or fragment thereof and the carrier, ensuring long-term stability of the conjugate. They are useful when the LST or fragment does not need to be released from the carrier for immunogenicity. In some embodiments, a maleimide-PEG linker is used.

[0115] The choice of linker can also affect the immunogenicity of the vaccine conjugate. For example, a long and flexible linker may help expose the LST or fragment more effectively to the immune system, enhancing the response. Conversely, a too-short or rigid linker could hide the LST or fragment 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.

[0116] In some embodiments, a lipooligosaccharide (LOS) is combined with the LST or LST fragment as described herein. LOS is a major surface-exposed virulence determinant that mediates host interaction and is a target of bactericidal antibodies. However, LOS is dynamically modified in vivo by LST-mediated sialylation, which sterically and electrostatically suppresses complement activation. As a result, immune responses directed solely against LOS are functionally attenuated in the presence of active LST.

[0117] The inventive concept underlying dual targeting is the recognition that effective immunological engagement of LOS requires immune-mediated interference with the enzymatic process that modifies and protects LOS. LST is therefore not targeted as an independent antigen, but as a mechanistic control whose disruption restores immune accessibility of LOS epitopes. Because LOS sialylation is required for immune evasion and efficient colonization, immune pressure that reduces LST function constrains the pathogen’s ability to evade LOS-directed antibodies. Alterations that preserve LOS structure but reduce LST activity increase immune susceptibility, while alterations that evade LOS antibodies compromise virulence. This creates a constrained evolutionary landscape that is not produced by targeting LOS or LST in isolation.

[0118] Thus, embodiments of the disclosure provide LST or an antigenic fragment thereof combined with an LOS or antigenic fragment thereof, including native LOS, detoxified LOS, LOS-derived oligosaccharides, or glycan mimics. Such a combination may provide a synergistic bactericidal effect. The LOS antigen may be obtained, for example, from N. gonorrhoeae or fromany other bacterial pathogen as described herein. Exemplary LOS antigens are disclosed in PCT / US2025 / 043665 incorporated herein by reference. The LOS may be conjugated to a carrier, e.g. protein or vesicle carrier as described herein. In some embodiments, the LST antigen to be combined with LOS is selected to elicit antibodies that bind and sterically interfere with functional regions of LST associated with donor binding or catalytic activity as described herein.

[0119] In some embodiments, LST or LST antigenic fragments are presented in proximity to LOS or LOS antigenic fragments within a shared membrane or vesicular carrier, such as outer membrane vesicles (OMVs), VLPs, generalized membrane vesicles (GMMA), bacterial membrane-derived vesicles, or liposomal systems incorporating LOS or LOS mimics.

[0120] With reference to Figures 6A-D, antibodies elicited against LST bind defined functional regions of the enzyme and sterically interfere with its ability to catalyze LOS sialylation. Reduction or prevention of LOS sialylation increases susceptibility of LOS epitopes to complement-mediated bactericidal activity of anti-LOS antibodies. This immune-mediated potentiation is not achieved by immunization against LOS alone, nor by immune recognition of LST that does not affect enzymatic function. The enhanced / synergistic bactericidal effect arises from a mechanistic sequence linking enzyme interference to epitope accessibility.

[0121] In order to increase the immunogenicity of the composition, in some embodiments, the immunogenic compositions comprise one or more adjuvants and / or cytokines. Suitable adjuvants include an aluminum salt such as aluminum hydroxide (alum) or aluminum phosphate, but may also be a salt of calcium, iron or zinc, or may be an insoluble suspension of acylated tyrosine, or acylated sugars, or may be cationically or anionically derivatized saccharides, polyphosphazenes, biodegradable microspheres, monophosphoryl lipid A (MPL), lipid A derivatives (e.g. of reduced toxicity), MF59, 3-0-deacylated MPL [3D-MPL], quit A, Saponin, QS21, Freund's Incomplete Adjuvant (Difco Laboratories, Detroit, Mich.), Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.), AS-2 (Smith-Kline Beecham, Philadelphia, Pa.), CpG oligonucleotides, bioadhesives and mucoadhesives, microparticles, liposomes, polyoxyethylene ether formulations, polyoxyethylene ester formulations, muramyl peptides or imidazoquinolone compounds (e.g. imiquamod and its homologues). Human immunomodulators suitable for use as adjuvants in the disclosure include cytokines such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), granulocyte, macrophage colony stimulating factor (GM-CSF) may also be used as adjuvants. It is expectedthat an adjuvant or cytokine can be added in an amount of about 0.01 mg to about 10 mg per dose, preferably in an amount of about 0.2 mg to about 5 mg per dose. Alternatively, the adjuvant or cytokine may be at a concentration of about 0.01 to 50% by weight, preferably at a concentration of about 2% to 30% by weight.

[0122] In certain aspects, the immunogenic compositions of the disclosure are prepared by physically mixing the adjuvant and / or cytokine with peptides described herein under appropriate sterile conditions in accordance with known techniques to produce the final product. The dose may be determined according to various parameters, especially according to the substance used; the age, weight and condition of the individual to be treated; the route of administration; and the required regimen. The amount of antigen in each dose is selected as an amount which induces an immune response. A physician will be able to determine the required route of administration and dosage for any particular individual. The dose may be provided as a single dose or may be provided as multiple doses, for example taken at regular intervals, for example 2, 3 or 4 doses administered weekly. Typically, peptides, or polynucleotides are typically administered in the range of 1 pg to 1 mg, more typically 1 pg to 10 pg for particle mediated delivery and 1 pg to 1 mg, and more typically 1-150 pg. Generally, it is expected that each dose will comprise 0.01-3 mg of antigen. An optimal amount for a particular vaccine can be ascertained by studies involving observation of immune responses in individuals.

[0123] In further embodiments, the antigens may exist as nucleic acids and may be formulated as a RNA or DNA vaccine for in vivo expression. In some embodiments, DNA vaccines, or gene vaccines, comprise a plasmid with a promoter and appropriate transcription and translation control elements and a nucleic acid sequence encoding one or more polypeptides of the disclosure. In some embodiments, the plasmids also include sequences to enhance, for example, expression levels, intracellular targeting, or proteasomal processing. In some embodiments, DNA vaccines comprise a viral vector containing a nucleic acid sequence encoding one or more polypeptides of the disclosure. In additional aspects, the compositions disclosed herein comprise one or more nucleic acids encoding peptides determined to have immunoreactivity with a biological sample. For example, in some embodiments, the compositions comprise one or more nucleotide sequences encoding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more peptides as described herein. The DNA or gene vaccine also encodes immunomodulatory molecules to manipulate the resulting immune responses, such as enhancing the potency of the vaccine,stimulating the immune system or reducing immunosuppression. Strategies for enhancing the immunogenicity of DNA or gene vaccines include encoding of xenogeneic versions of antigens, fusion of antigens to molecules that activate T cells or trigger associative recognition, priming with DNA vectors followed by boosting with viral vector, and utilization of immunomodulatory molecules. In some embodiments, the DNA vaccine is introduced by a needle, a gene gun, an aerosol injector, with patches, via microneedles, by abrasion, among other forms. In some forms the DNA vaccine is incorporated into a lipid nanoparticle, liposome, or other forms of nanobodies. In some embodiments, the DNA vaccine includes a delivery system selected from the group consisting of a transfection agent; protamine; a protamine liposome; a polysaccharide particle; a cationic nanoemulsion; a cationic polymer; a cationic polymer liposome; a cationic nanoparticle; a cationic lipid and cholesterol nanoparticle; a cationic lipid, cholesterol, and PEG nanoparticle; a dendrimer nanoparticle. In some embodiments, the DNA vaccine is administered by inhalation or ingestion. In some embodiments, the DNA vaccine is introduced into the blood, the thymus, the pancreas, the skin, the muscle, a tumor, or other sites.

[0124] In yet other embodiments, the composition may be prepared as an RNA vaccine. In some embodiments, the RNA is non-replicating mRNA or virally derived, self-amplifying RNA. In some embodiments, the non-replicating mRNA encodes the peptides disclosed herein and contains 5' and 3' untranslated regions (UTRs). In some embodiments, the virally derived, self-amplifying RNA encodes not only the peptides disclosed herein but also the viral replication machinery that enables intracellular RNA amplification and abundant protein expression. In some embodiments, the RNA is directly introduced into the individual. In some embodiments, the RNA is chemically synthesized or transcribed in vitro. In some embodiments, the mRNA is produced from a linear DNA template using a T7, a T3, or a Sp6 phage RNA polymerase, and the resulting product contains an open reading frame that encodes the peptides disclosed herein, flanking UTRs, a 5' cap. and a poly(A) tail. In some embodiments, various versions of 5' caps are added during or after the transcription reaction using a vaccinia virus capping enzyme or by incorporating synthetic cap or anti-reverse cap analogues. In some embodiments, an optimal length of the poly(A) tail is added to mRNA either directly from the encoding DNA template or by using poly(A) polymerase. In some embodiments, the RNA includes signals to enhance stability and translation. In some embodiments, the RNA also includes unnatural nucleotides to increase the half-life or modified nucleosides to change the immuno stimulatory profile.In some embodiments, the RNA is introduced by a needle, a gene gun, an aerosol injector, with patches, via microneedles, by abrasion, among other forms. In some forms the RNA vaccine is incorporated into lipid nanoparticles, liposomes, or other forms of nanobodies that facilitate cellular uptake of RNA and protect it from degradation. In some embodiments, the RNA vaccine includes a delivery system selected from the group consisting of a transfection agent; protamine; a protamine liposome; a polysaccharide particle: a cationic nanoemulsion; a cationic polymer; a cationic polymer liposome; a cationic nanoparticle; a cationic lipid and cholesterol nanoparticle; a cationic lipid, cholesterol, and PEG nanoparticle; a dendrimer nanoparticle; and / or naked mRNA; naked mRNA with in vivo electroporation; protamine-complexed mRNA; mRNA associated with a positively charged oil-in-water cationic nanoemulsion; mRNA associated with a chemically modified dendrimer and complexed with polyethylene glycol (PEG)-lipid; protamine-complexed mRNA in a PEG-lipid nanoparticle; mRNA associated with a cationic polymer such as polyethylenimine (PEI); mRNA associated with a cationic polymer such as PEI and a lipid component; mRNA associated with a polysaccharide (for example, chitosan) particle or gel; mRNA in a cationic lipid nanoparticle (for example, 1,2 dioleoyloxy 3 trimethylammoniumpropane (DOTAP) or dioleoylphosphatidylethanolamine (DOPE) lipids); mRNA complexed with cationic lipids and cholesterol; or mRNA complexed with cationic lipids, cholesterol and PEG-lipid. In some embodiments, the RNA vaccine is administered by inhalation or ingestion. In some embodiments, the RNA is introduced into the blood, the thymus, the pancreas, the skin, the muscle, a tumor, or other sites, and / or by an intradermal, intramuscular, subcutaneous, intranasal, intranodal, intravenous, intrasplenic, intratumoral or other delivery route.

[0125] In some embodiments, the polynucleotide components are naked nucleotide sequences or are in combination with cationic lipids, polymers or targeting systems. They may be delivered by any available technique. For example, the polynucleotide may be introduced by needle injection, preferably intradermally, subcutaneously or intramuscularly. Alternatively, the polynucleotide may be delivered directly across the skin using a delivery device such as particle-mediated gene delivery. The polynucleotide may be administered topically to the skin, or to mucosal surfaces for example by intranasal, oral, or intrarectal administration. Uptake of polynucleotide constructs may be enhanced by several known transfection techniques, for example those including the use of transfection agents. Examples of these agents include cationic agents, for example, calciumphosphate and DEAE-Dextran and lipofectants, for example, lipofectam and transfectam. The dosage of the polynucleotide to be administered can be altered.

[0126] In some embodiments, LST is expressed in attenuated bacterial strains or viral vectors (e.g., adenovirus, MV A).

[0127] Embodiments also include methods of making an immunogenic or vaccine composition as described herein. A method of making or manufacturing a composition may include isolating or synthesizing LST or a fragment thereof, optionally conjugating the LST or fragment thereof to a carrier, and mixing with an adjuvant.

[0128] The immunogenic compositions or vaccines described herein comprise, in addition to one or more peptides, nucleic acids, or vectors, a pharmaceutically acceptable excipient, carrier, diluent, buffer, stabilizer, preservative, adjuvant or other materials well known to those skilled in the art. Such materials are preferably non-toxic and preferably do not interfere with the pharmaceutical activity of the active ingredient(s). 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. “Pharmaceutically acceptable carriers” are typically large, slowly metabolized macromolecules such as proteins, saccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, sucrose (Paoletti et. al., 2001, Vaccine. 19:2118-2126), trehalose (WO 00 / 56365), lactose and lipid aggregates (such as oil droplets or liposomes). Such carriers are well known to those of ordinary skill in the art. 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.

[0129] 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.

[0130] 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.

[0131] In some embodiments, the immunogenic compositions of the disclosure are lyophilized or in aqueous form, i.e„ solutions or suspensions. Liquid formulations of this type allow the compositions to be administered direct from their packaged form, without the need for reconstitution in an aqueous medium, and are thus ideal for injection. In some embodiments, the immunogenic compositions are presented in vials, or they may be presented in ready filled syringes. The syringes may be supplied with or without needles. A syringe will include a single dose, whereas a vial may include a single dose or multiple doses.

[0132] Liquid formulations of the disclosure are also suitable for reconstituting other medicaments from a lyophilized form. Where an immunogenic composition is to be used for such extemporaneous reconstitution, the disclosure provides a kit, which may comprise two vials, or may comprise one ready-filled syringe and one vial, with the contents of the syringe being used to reconstitute the contents of the vial prior to injection.

[0133] In some embodiments, the immunogenic compositions of the disclosure include an antimicrobial, particularly when packaged in a multiple dose format. Antimicrobials may be used, such as 2-phenoxyethanol or parabens (methyl, ethyl, propyl parabens). Any preservative is preferably present at low levels. Preservative may be added exogenously and / or may be a component of the bulk antigens which are mixed to form the composition.

[0134] In some embodiments, the immunogenic compositions are encapsulated in a suitable vehicle either to deliver the peptides into antigen presenting cells or to increase the stability. As will be appreciated by a skilled artisan, a variety of vehicles are suitable for delivering a immunogenic composition of the disclosure. Non-limiting examples of suitable structured fluid delivery systems may include nanoparticles, liposomes, microemulsions, micelles, dendrimers and other phospholipid-containing systems. Methods of incorporating immunogenic compositions into delivery vehicles are known in the art.Embodiments also include methods of protecting from, reducing the risk of, or treating a subject with a bacterial infection comprising administering to the subject an immunogenic or 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.

[0135] 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.

[0136] Beyond gonorrhea, LST and similar sialyltransferases are found in other bacterial pathogens. Thus, the method described herein are useful for protecting or treating a subject from an infection caused by bacteria including, but not limited to, Neisseria gonorrhoeae, Neisseria meningitidis, Neisseria lactamica. Haemophilus influenzae, Moraxella catarrhalis, Campylobacter jejuni, Haemophilus ducreyi. Haemophilus parasuis, Helicobacter bizzozeroni, etc. Thus, the present disclosure provides broader- spectrum vaccines that protect against multiple sexually transmitted infections or Gram-negative bacteria.

[0137] 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 the bacterial infection, in comparison to a nonvaccinated (e.g. adjuvant alone) control organism, in which disease progression is not prevented.

[0138] Other aspects of the disclosure relate to methods of inducing an immune response to at least one bacterial antigen as described above in a subject in need thereof, comprising the steps of: administering to the subject an effective amount of the immunogenic or vaccine composition; allowing a suitable period of time to elapse; and optionally administering at least one additional dose of the immunogenic or vaccine composition. A “suitable period of time” is defined herein as a sufficient time for a subject to produce antibodies against the administered antigens described herein. A sufficient time for a subject to acquire ability to produce antibodies may be days (e.g., 2, 3, 4, 5, 6 or 7 days), weeks (e.g., 1, 2, 3 or 4 weeks), months (e.g., 1, 2, 3, 4, 5, or 6 months), or years (e.g. 1, 2, 3, 4, or 5 years) after a first, second or third dose of the immunogenic composition is administered.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.

[0139] 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. In some embodiments, the compositions of the disclosure are administered in one, or more doses, as well as, by other routes of administration. For example, such other routes include, intracutaneously, intravenously, intravascularly, intraarterially, intraperitnoeally, intrathecally, intratracheally, intracardially, intralobally, intramedullarly, intrapulmonarily, and intravaginally. The LST 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-100pg of each LST of the invention, e.g. l-50pg or 1 - 25pg.

[0140] In some embodiments, the compositions of the disclosure are administered, or the methods and uses for treatment according to the disclosure are performed, alone or in combination with other pharmacological compositions or treatments, for example other antibiotics. In some embodiments, the other therapeutic compositions or treatments are administered eitherT1

[0141] simultaneously or sequentially with (before or after) the composition(s) or treatment of the disclosure.

[0142] 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.

[0143] 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.

[0144] 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.

[0145] 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.

[0146] 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 toserve 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.

[0147] 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.

[0148] 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.

[0149] EXAMPLE 1

[0150] Methods:

[0151] Bacterial strains'. Exchange of the F62AD 1st promoter with the N. meningitidis 126E 1st promoter in strain F62AD Pist-Nm was achieved using a two-step mutagenesis system (8) and plasmids pTOPO / Zprom and pTOPO / Zprom-Erm / Sm. pTOPO Azprom contains (5' to 3') the region upstream of the F621st promoter, the N. meningitidis 126E 1st promoter, and the F621st coding and downstream sequences (9). pTOPOAzprom-Erm / Sm is pTOPOAzprom with a cassette that codes for resistance to Erm and sensitivity to Sm inserted in the HincII site. Strain F62AD was transformed with pTOPOAzprom-Erm / Sm, and Erm-resistant but Sm-sensitive colonies were selected and transformed with pTOPOAzprom. Sm-resistant transformants were selected and screened for sensitivity to Erm (9).

[0152] Serum bactericidal assay: The susceptibility of log-phase gonococci grown in liquid culture with CMP-Neu5Ac (at the concentrations indicated) to complement-mediated killing by PNHS was determined using a serum bactericidal assay. Bacteria from an overnight culture on chocolate agar plates were inoculated into gonococcal liquid media (Morse A + Morse B + Isovitalex) and that did not did not contain CMP-N-acetylneuraminic acid (CMP-NANA) (concentrations indicated on X-axis) to an OD600nm of 0.1. Bacteria were grown for 2-3 hours to the mid-log phase (OD600nm 0.3 - 0.4). Normal human serum was obtained from a healthy human volunteers, pooled and stored at -70 °C till used in bactericidal assays. Briefly, 2000 CFUs ofgonococci were incubated with serum (concentrations specified for each experiment) in a final reaction volume of 150 pl. Aliquots of 25 pl were plated in duplicate at the start of the assay (t o) and after incubating the reaction mixture at 37°C for 30 min (t 30). Survival was calculated as the number of viable colonies at t = 30 min relative to baseline colony counts at t = 0 min, expressed as a percentage.

[0153] Mouse infection experiment'. To determine whether increased expression of 1st provides gonococci with a survival advantage in the mouse model of infection, a competitive infection experiment was performed using a mixture of strains Pist-Ng and Pist-Nm or, as a control, strains Pist-Ngand Alst. Female BALB / c mice (Jackson Laboratories) (10 weeks old) in the diestrus phase of the estrus cycle were treated with water-soluble 170-estradiol (Sigma) (1.5 mg) and antibiotics as described previously (10). In competitive infections, mice were inoculated intravaginally (on day 0) with a suspension of bacteria (total dose, ~8 x 105CFU) containing similar numbers of all strains (strains Pist-Ng [4.68 x 105] and Pist-Nm [4.68 x 105] or strains Pist-Ng [3.92 x 105] and Alst [3.34 x 105CFU]). Mice in each group colonized with N. gonorrhoeae (6 wild-type BALB / c mice / group, and CFU levels were determined daily by plating bacterial dilutions prepared from vaginal swabs onto GC agar supplemented with VCNT inhibitor and 100 pg / ml Sm. The level of detectable infection was 50 CFU per ml (the lowest dilution plated was 20 pl from a 100-pl undiluted sample), and mice from which no CFU were recovered were assigned a value of 40 CFU per ml. The ratio of mutant (strain Pist-Nm or strain Ata) to wild type (strain Pist-Ng) was determined in each mouse daily, using erythromycin resistance to differentiate between strains. Individual colonies (100 colonies per mouse) from each mouse were plated in replicate experiments on a nonselective GC plate and a GC plate containing erythromycin; the latter permits the growth of strain Pist-Ng but not the growth of strain Pist-Nm or strain Alst. Using this method, the level of detection for the wildtype strain relative to the mutant is 0.0099 (<1%). Recovery of the mutant strain relative to the wild-type strain was expressed as a competitive index (CI) as described previously (Wu Jerse). The CI was calculated daily for each mouse by dividing the ratio of the mutant to the wild type in the output by the ratio of the mutant to the wild type in the input as follows: CI = (mutant / wild-type CFU from vaginal cultures on each day) / (mutant / wild-type CFU in the inoculum on day 0). A CI of <1.0 indicates a decrease in the ratio of mutant to wild type compared to the ratio in the inoculum.

[0154] ResultsFigures 2A-B show that replacing the gonococcal 1st promoter with the weaker meningococcal 1st promoter reduces the amount of LOS sialylation such that the meningococcal 1st promoter-bearing mutant is more susceptible to complement and is also more readily cleared from the female genital tract of mice. Thus, even partial blockade of LOS sialylation could significantly attenuate gonococcal virulence.

[0155] EXAMPLE 2

[0156] Methods:

[0157] Immunization and mouse infection'. Six-week-old female BALB / c (The Jackson Laboratory) mice were immunized with recombinant Lst (SEQ ID NO: 1; 20 pg / dose) adjuvanted with monophosphoryl lipid A (MPL) (20 pg) plus Alum (200 pg) intramuscularly (IM) at weeks 0, 2, 4 and 9. Adjuvant control mice received MPL plus Alum alone. Two weeks following dose 4, mice in the diestrus phase of the estrous cycle were with 5 mg estradiol pellets (Innovative Research of America; Cat. No. E-121) on day -2 to prolong the estrus phase of the cycle and promote susceptibility to N. gonorrhoeae infection (10). Antibiotics (vancomycin, colistin, neomycin, trimethoprim, and streptomycin) ineffective against N. gonorrhoeae were used to reduce competitive microflora (10). Mice were challenged intravagin ally with the indicated inoculum ofN. gonorrhoeae. Infection was monitored daily through vaginal swabbing and bacterial enumeration (CFUs).

[0158] Three characteristics of the data were measured: time to clearance, longitudinal trends in mean logio CFU, and the cumulative CFU as AUC. Median time to clearance was estimated using Kaplan-Meier survival curves; times to clearance were compared between groups using the Mantel-Cox log-rank test. The mean AUC (logio CFU) was computed for each mouse to estimate the bacterial burden over time (cumulative infection); the means under the curves were compared between groups using Mann-Whitney’s non-parametric test.

[0159] Results:

[0160] Immunization of mice with recombinant Lst adjuvanted with MPL plus Alum results in significantly accelerated clearance and decreased burden of gonococcal infection in the mouse vaginal colonization model (Figures 3A-C).EXAMPLE 3

[0161] Methods:

[0162] Immunization and mouse infection'. Six-week-old female human Factor H (FH) and C4b-binding protein (C4BP) ‘double’ transgenic mice in a BALB / c background (11) were immunized with recombinant Lst (SEQ ID NO: 1; 20 pg / dose) adjuvanted with MPL (20 pg) plus Alum (200 pg) intramuscularly (IM) at weeks 0, 2, 4 and 7. Adjuvant control mice received MPL plus Alum alone. Two weeks following dose 4, mice in the diestrus phase of the estrous cycle were with 5 mg estradiol pellets (Innovative Research of America; Cat. No. E-121) on day -2 to prolong the estrus phase of the cycle and promote susceptibility to N. gonorrhoeae infection (10). Antibiotics (vancomycin, colistin, neomycin, trimethoprim, and streptomycin) ineffective against N. gonorrhoeae were used to reduce competitive microflora (10). Mice were challenged intravaginally on day 0 with the indicated inoculum of N. gonorrhoeae as previously described (61). Infection was monitored daily through vaginal swabbing and bacterial enumeration (CFUs). To block complement C7, which is necessary for formation of the membrane attack complex, 1 mg of an anti-complement C7 mAb (12) was administered intraperitoneally on days -1, +2 and +5. Three characteristics of the data were measured: time to clearance, longitudinal trends in mean logio CFU, and the cumulative CFU as AUC. Median time to clearance was estimated using Kaplan-Meier survival curves; times to clearance were compared between groups using the Mantel-Cox log-rank test. The mean AUC (logio CFU) was computed for each mouse to estimate the bacterial burden over time (cumulative infection); the means under the curves were compared between groups using Mann-Whitney’s non-parametric test.

[0163] Results:

[0164] Blockade of the terminal complement pathway (i.e., membrane attack complex formation) is necessary for activity of the Lst vaccine (Figures 4A-C). The data unexpectedly show that Lst vaccine efficacy operates through a singular, non-redundant mechanism requiring terminal complement activation and membrane attack complex (MAC) formation. As shown in Figure 4, complete loss of protection upon C7 blockade indicates that, unlike most antibacterial vaccines, Lst-induced immunity does not engage parallel effector pathways such as C3b-mediated opsonophagocytosis. This exclusive reliance on MAC-dependent killing is both unexpected and surprising, representing a mechanistically distinct departure from conventional vaccine-induced antibacterial immunity.EXAMPLE 4

[0165] Methods:

[0166] Six-week-old female BALB / c (The Jackson Laboratory) mice were immunized with recombinant Lst (SEQ ID NO: 1; 20 pg / dose) adjuvanted with MPL (20 jug) plus Alum (200 pg) intramuscularly (IM) at weeks 0, 2, 4 and 9, or with MPL plus Alum alone (adjuvant control mice). Two weeks following dose 4, mice in the diestrus phase of the estrous cycle were with 5 mg estradiol pellets (Innovative Research of America; Cat. No. E- 121) on day -2 to prolong the estrus phase of the cycle and promote susceptibility to A. gonorrhoeae infection (10). Antibiotics (vancomycin, colistin, neomycin, trimethoprim, and streptomycin) ineffective against N. gonorrhoeae were used to reduce competitive microflora (10). Mice were challenged intravaginally with the indicated inoculum of / V. gonorrhoeae. Gonococci (strain FA1090) recovered directly (i.e., without subpassage on artificial media) from the vaginas were serially diluted and tested for resistance to human complement (pooled normal human serum at a final concentration of 10%). Aliquots of the reaction mixture were plated onto chocolate agar containing vancomycin, colistin, trimethoprim and nystatin to suppress normal flora and selectively permit N. gonorrhoeae growth at 0- and 30-min. Percent survival at 30 min relative to 0 min is shown on the Y-axis. Reaction mixtures that yielded 20 to 100 CFU at 0 min were used to calculate survival.

[0167] Results:

[0168] Figure 5 shows that gonococci recovered from the genital tracts of Lst-immunized mice are significantly more susceptible to killing by normal human serum compared to gonococci from mice immunized with adjuvant alone. This suggests that targeting Lst may interfere with gonococcal Lst function, which results in reduced sialylation and increased bacterial susceptibility to complement.

[0169] Discussion:

[0170] The data unexpectedly demonstrate that Lst vaccination elicits bactericidal antibodies despite longstanding controversy regarding whether Lst is accessible to the immune system. The subcellular localization of Lst has been debated for over 20 years. Shell et al. (Infect Immun.

[0171] 2002;70:3744-3751) initially reported Lst as a surface-exposed outer membrane protein based on antibody binding to whole cells, representing what they termed "the first demonstration of thelocalization of a bacterial glycosyltransferase to the outer membrane." However, these same authors noted that anti-Lst antibodies failed to inhibit sialylation of intact cells, suggesting the active site was not accessible. More recently, Jen et al. (mBio. 2021;12:e03666-20) challenged this conclusion with multiple lines of evidence supporting cytoplasmic localization, including: (1) inability of anti-Lst antibodies to bind intact cells without detergent treatment; (2) Lst-mediated transfer of CMP-KDO. which has a very short half-life and is synthesized exclusively in the cytoplasm; and (3) absence of a signal peptide. They concluded that "the conclusion that Lst in the pathogenic Neisseria is an outer membrane protein is not supported by strong evidence."

[0172] Despite this controversy and the weight of evidence suggesting Lst either resides in the cytoplasm or has an inaccessible active site, the present data demonstrate that Lst immunization induces potent bactericidal antibodies that: (1) accelerate bacterial clearance in vivo (Figures 3A-C); (2) require terminal complement pathway activation for efficacy (Figure 4); and (3) functionally inhibit Lst enzymatic activity as evidenced by increased complement susceptibility of bacteria recovered from vaccinated hosts (Figure 5). These results were unexpected because the art taught that antibody-based vaccines cannot target cytoplasmic enzymes, and even proponents of surface localization (Shell et al.) found that antibodies could not access the functional enzyme on intact cells. The successful generation of bactericidal antibodies definitively establishes that immunologically relevant Lst epitopes are surface-accessible and that antibody binding to these epitopes functionally interferes with the enzyme in a manner sufficient to restore complement-mediated bacterial killing. This resolution of the localization controversy through functional vaccine data was not predictable from the conflicting structural and biochemical evidence in the prior art, and represents a surprising validation that Lst presents viable vaccine epitopes despite uncertainty about its precise subcellular distribution.

[0173] REFERENCES

[0174] 1. Shell DM, Chiles L, Judd RC, Seal S, Rest RF. 2002. The Neisseria lipooligosaccharide- specific alpha-2, 3-sialyltransferase is a surface-exposed outer membrane protein. Infect Immun 70:3744-51.

[0175] 2. Nairn CA, Cole JA, Patel PV, Parsons NJ, Fox JE, Smith H. 1988. Cytidine 5'- monophospho-N-acetylneuraminic acid or a related compound is the low Mr factor fromhuman red blood cells which induces gonococcal resistance to killing by human serum. J Gen Microbiol 134:3295-306.

[0176] 3. Elkins C. Carbonetti NH, Varela VA, Stirewalt D, Klapper DG, Sparling PF. 1992.

[0177] Antibodies to N-terminal peptides of gonococcal porin are bactericidal when gonococcal lipopolysaccharide is not sialylated. Mol Microbiol 6:2617-28.

[0178] . Gulati S, Schoenhofen IC, Whitfield DM, Cox AD, Li J, St Michael F, Vinogradov EV, Stupak J, Zheng B, Ohnishi M, Unemo M, Lewis LA, Taylor RE, Landig CS, Diaz S, Reed GW, Varki A, Rice PA, Ram S. 2015. Utilizing CMP-Sialic Acid Analogs to Unravel Neisseria gonorrhoeae Lipooligosaccharide-Mediated Complement Resistance and Design Novel Therapeutics. PLoS Pathogens ll:el005290.

[0179] 5. Ram S, Sharma AK, Simpson SD, Gulati S, McQuillen DP, Pangburn MK, Rice PA.

[0180] 1998. A novel sialic acid binding site on factor H mediates serum resistance of sialylated Neisseria gonorrhoeae. J Exp Med 187:743-752.

[0181] 6. Landig CS, Hazel A, Kellman BP, Fong JJ, Schwarz F, Agarwal S, Varki N, Massari P, Lewis NE, Ram S, Varki A. 2019. Evolution of the exclusively human pathogen Neisseria gonorrhoeae: Human-specific engagement of immunoregulatory Siglecs. Evol Appl 12:337-349.

[0182] . Gulati S, Schoenhofen IC, Lindhout-Djukic T, Lewis LA, Moustafa IY, Saha S, Zheng B, Nowak N, Rice PA, Varki A, Ram S. 2020. Efficacy of Antigonococcal CMP- Nonulosonate Therapeutics Require Cathelicidins. J Infect Dis 222:1641-1650.

[0183] 8. Johnston DM, Cannon JG. 1999. Construction of mutant strains of Neisseria gonorrhoeae lacking new antibiotic resistance markers using a two gene cassette with positive and negative selection. Gene 236:179-84.

[0184] 9. Lewis LA, Gulati S, Burrowes E, Zheng B, Ram S, Rice PA. 2015. alpha-2, 3- Sialyltransferase Expression Level Impacts the Kinetics of Lipooligosaccharide Sialylation, Complement Resistance, and the Ability of Neisseria gonorrhoeae to Colonize the Murine Genital Tract. MB io 6:e02465-14.

[0185] 10. Jerse AE. 1999. Experimental gonococcal genital tract infection and opacity protein expression in estradiol-treated mice. Infect Immun 67:5699-708.

[0186] 11. Shaughnessy J, Chabeda A, Tran Y, Zheng B, Nowak N, Steffens C, DeOliveira RB, Gulati S, Lewis LA, MacLean J, Moss JA, Wycoff KL, Ram S. 2022. An optimizedFactor H-Fc fusion protein against multidrug-resistant Neisseria gonorrhoeae. Front Immunol: 975676.

[0187] 12. Zelek WM, Morgan BP. 2020. Monoclonal Antibodies Capable of Inhibiting Complement Downstream of C5 in Multiple Species. Front Immunol 11:612402.

[0188] 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

1. CLAIMS1. An immunogenic composition comprising a lipo-oligosaccharide sialyltransferase (LST) protein or antigenic fragment thereof and an adjuvant.

2. The immunogenic composition of claim 1, wherein the LST or antigenic fragment thereof has at least 95% sequence identity to a peptide selected from the group consisting of SEQ ID Nos 1-5 and 11-31.

3. The immunogenic composition of claim 1, wherein the LST protein or antigenic fragment thereof is conjugated to a 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, CRM 197, and outer membrane protein complex (OMPC).

5. The immunogenic composition of claim 1, wherein the LST protein or antigenic fragment thereof is conjugated to a vesicle carrier.

6. The immunogenic composition of claim 5, wherein the vesicle carrier is an outer membrane vesicle (OMV).

7. The immunogenic composition of claim 1, wherein the LST or antigenic fragment thereof is displayed on the surface of a virus-like particle (VLP).

8. The immunogenic composition of claim 1, wherein the composition further comprises a lipooligosaccharide (LOS) or an antigenic fragment thereof.

9. The immunogenic composition of claim 8, wherein the LST protein or antigenic fragment thereof and the LOS or antigenic fragment thereof are conjugated to a shared vesicle carrier.lO.The immunogenic composition of claim 9, wherein the shared vesicle carrier is an OMV.

11. The immunogenic composition of claim 8, wherein the LST protein or antigenic fragment thereof and the LOS or antigenic fragment thereof are displayed on the surface of a VLP.

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

13. The immunogenic composition of claim 12, 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.

14. The immunogenic composition of claim 1, wherein the adjuvant is selected from the group consisting of aluminum hydroxide, monophosphoryl lipid A, CpG, and MF59.

15. A vaccine composition comprising the immunogenic composition of any of claims 1-14 and a pharmaceutically acceptable carrier.

16. An immunogenic composition comprising a lipid nanoparticle and a nucleic acid encoding LST protein or an antigenic fragment thereof enclosed within the lipid nanoparticle.

17. The immunogenic composition of claim 16, wherein the nucleic acid has at least 95% sequence identity to a nucleic acid selected from the group consisting of SEQ ID Nos 6-10.

18. A vaccine composition comprising the immunogenic composition of claim 16 and a pharmaceutically acceptable earner.

19. A method of protecting or treating a subject from a bacterial infection comprising administering to the subject the immunogenic composition of claim 1 or claim 16.

20. The method of claim 19, wherein the bacterial infection is caused by Neisseria gonorrhoeae, Haemophilus influenzae, Moraxella catarrhalis, or Neisseria meningitidis.

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