Genetically detoxified mutant of neisseria and outer membrane vesicle (OMV) vaccine

Abstract

Disclosed are isolated PorA-PorB-RmpM-LpxL1- N. meningitidis and compositions including an effective amount of OMVs produced from these PorA-PorB-RmpM-LpxL1- N. meningitidis. Also disclosed are methods for using these compositions to induce an immune response to Neisseria, such as N. meningitidis and N. gonorrhoeae.

Description

[0001] GENETICALLY DETOXIFIED MUTANT OF NEISSERIA AND OUTER MEMBRANE VESICLE (OMV) VACCINE

[0002] CROSS REFERENCE TO RELATED APPLICATION

[0003] This application claims priority to U.S. Provisional Application No. 63 / 524,512, filed June 30, 2023, which is incorporated by reference in its entirety.

[0004] FIELD OF THE DISCLOSURE

[0005] This relates to immunogenic compositions comprising outer membrane microvesicles (OMVs) from PorA PorB RmpM LpxLl' Neisseria, such asPorA PorB RmpM LpxLI SiaD' Neisseria (N.) meningitidis, that are of use to induce an immune response to Neisseria, including N. meningitidis and N. gonorrhoeae.

[0006] BACKGROUND

[0007] Neisseria is a genus of Gram-negative bacteria that colonize the mucosal surfaces of many animals. There are eleven species that colonize human, of which two, N. meningitidis and N. gonorrhoeae, are pathogenic. N. meningitidis is the causative agent of meningitis and meningococcal septicemia. N. gonorrhoeae is the causative agent of gonorrhea. The genomes of at least ten of the Neisseria species have been completely sequenced.

[0008] Neisseria meningitidis is estimated to cause 1.2 million cases of invasive disease annually, resulting in approximately 135,000 deaths. Characterized by rapid onset and disease progression, infection with N. meningitidis is also associated with high morbidity, with approximately 20% of invasively infected individuals suffering permanent, debilitating sequelae, including brain damage, hearing loss, and necrosis of the limbs, necessitating amputation. The majority of invasive meningococcal disease is caused by six serogroups, A, C, W, Y, X, and B. Vaccines that target the capsular polysaccharides of serogroups A, C, W, Y, and X have been licensed or are currently in the developmental phase. In contrast, the capsular polysaccharide of scrogroup B meningococcus (McnB) closely resembles a sugar moiety present on human cells and is, as a consequence, poorly immunogenic. MenB-specific vaccines, therefore, must necessarily target bacterial subcapsular antigens. A need remains for additional compositions that can be used to induce an immune response to N. meningitidis and N. gonorrhoeae.

[0009] SUMMARY OF THE DISCLOSURE

[0010] Disclosed are isolated PorA PorB RmpM LpxLI . meningitidis (also called APorAAPorBARmpMALpxL) and compositions including an effective amount of OMVs produced from these PorA PorB RmpM LpxLl ? / . meningitidis. Also disclosed are methods for using these compositions to induce an immune response to Neisseria, such as N. meningitidis and N. gonorrhoeae.

[0011] Also disclosed are methods for inducing an immune response to N. gonorrhoeae in a mammalian subject. These methods include administering to the mammalian subject an immunogenic composition comprising an effective amount of OMVs from PorA PorB RmpM LpxL I \-V. meningitidis and a pharmaceutically acceptable carrier, thereby inducing the immune response to N. gonorrhoeae.

[0012] In some aspects, the PorA PorB RmpM LpxLl V. meningitidis is a PorA PorB RmpM LpxLLSiaD- N. meningitidis.

[0013] The foregoing and other features and advantages of the invention will become more apparent from the following detailed description of several aspects which proceeds with reference to the accompanying figures.

[0014] BRIEF DESCRIPTION OF THE FIGURES

[0015] FIG. 1. Diagram of the lipid A structure of N. meningitidis lipooligosaccharide (LOS). The hatched rectangle indicates the lauroyl chain added to the lipid A backbone by LpxLl.

[0016] FIGS. 2A-2B. Diagram of the mechanism of action of the tetR-sacB protein products. FIG. 2A: In the absence of chlortetracycline (cTc), the tetR gene is transcribed and translated to produce the Tet repressor, which binds to the Ptet promoter, preventing binding of RNA polymerase (RNAP). The sacB gene cannot be transcribed, and le vansucrase will not be produced, allowing for growth of bacteria on plates containing sucrose. FIG. 2B: When present, cTc will bind to the Tet repressor, inducing a conformational change that relieves DNA binding, allowing RNAP to bind to the Ptet promoter. The sacB gene can be transcribed and translated, producing levansucrase, which cleaves sucrose into a product that is toxic to N. meningitidis .

[0017] FIGS. 3A-3C. Cloning strategy for generation of markerless mutant strains. (FIG. 3 A) The 5’ and 3’ untranslated regions (UTRs) of the gene to be deleted (e.g., IpxLl) were amplified from MC58 genomic DNA (gDNA) using primer pairs incorporating restriction enzyme sites RE1 / RE2 and RE2 / RE3, respectively. The 5’ and 3’ UTR sequences were then sequentially cloned into pGEM-3Z via restriction enzyme digest, creating plasmid pINT. (FIG. 3B) The positive / negative selection tetR-sacB-nptll cassette was amplified from plasmid pJJ260 using primers bearing RE2 restriction enzyme site sequences. The PCR product was then cloned into pINT between the 5’ and 3’ UTR sequences via digestion, creating plasmid pDEL. (FIG. 3C) Transformation of pDEL into MC58 functioned to replace the native gene (e.g., IpxLl) with the tetR-sacB-nptll cassette, creating a new strain with an IpxLl deletion (MC58 IpxLlwsacB). Transformation of MC58 IpxLl ::sacB with pINT succeeded in removing the tetR-sacB-nptll cassette from the bacterial genome using double homologous recombination, creating strain MC58AL (also called ALpxLl ). The same methodology was applied to delete porA. porB. rmpM, and siaD.

[0018] FIG. 4. Immunoblots to confirm lack of antigen expression. Whole cell lysates of markerless mutant strains were probed in dot blots (left panel) or western blots (bottom right panel) for PorA, PorB, capsule, lipooligosaccharide (LOS), and RmpM expression. No antigen expression was observed in strains deleted for the relevant protein or capsular genes as expected. LOS production was observed in all strains independent of LpxLl deletion, confirming genetic detoxification had no impact on the structural integrity of LOS. A Coomassie-stained gel of whole cell lysates is shown in the top right panel to confirm equivalent loading for the RmpM western blot.

[0019] FIGS. 5A-5B. Genetically-detoxified OMV do not stimulate robust TLR4 responses. OMV obtained from the markerless mutant strains were isolated and tested for induction of TLR4- (FIG. 5A) and TLR2- (FIG. 5B) specific responses; OMV isolated from the parental wild-type (WT) strain and the APorAAPorBARmpM strain engineered using antibiotic resistance markers were also tested as controls. The genetically-detoxified APorAAPorBARmpMALpxLl and APorAAPorBARmpMASiaDALpxLl OMV were significantly reduced in their ability to stimulate TLR4, but not TLR2, responses compared to the other OMV obtained from LpxLl -expressing strains. Purified LPS and Pam3CSK4 were used as positive controls for TLR4 (FIG. 5A) and TLR2 (FIG. 5B) stimulation, respectively, and OMV were isolated by passage of the bacterial wet mass through a French press. * represents a P-value of at least P < 0.05 for WT OMV, the APorAAPorBARmpM OMV, and APorAAPorBARmpMASiaD OMV relative to both APorAAPorBARmpMALpxLl and APorAAPorBARmpMASiaDALpxll OMV by one-way ANOVA with Tukey’s post hoc test.

[0020] FIGS. 6A-6D. Sera from rabbits immunized with markerless mutant APorAAPorBARmpMALpxLl or APorAAPorBARmpM detergent-extracted, detergent-detoxified OMV (dOMV) exhibit comparable killing profiles relative to sera from rabbits immunized with dOMV from the APorAAPorBARmpM strain engineered with antibiotic resistance markers. Sera from rabbits immunized with APorAAPorBARmpM (KM1 and KM2), the markerless mutant APorAAPorBARmpM (KM3 and KM4), or APorAAPorBARmpMALpxLl (KM5 and KM6) dOMV were tested against a panel of 17 meningococcal strains in human complement serum bactericidal assays; sera from alum-immunized rabbits (KM7 and KM8) were also tested as controls. Meningococcal test strains are divided into those with little-to-no background killing with alum control sera (FIGS. 6A-6B) or those that were sensitive to killing with all sera (FIG. 6C). A summary of the number of strains killed by each scrum is depicted in FIG. 6D.

[0021] FIG. 7. Detergent-extracted, genetically-detoxified OMV (eOMV) retain vesicular structure. Representative APorAAPorBARmpM (constructed with antibiotic resistance markers) detergent-extracted, detergent-detoxified OMV (dOMV) and APorAAPorBARmpMASiaDALpxLl eOMV were isolated, fixed on Formvar carbon film copper grids, and negative stained with 2% methylamine tungstate. Transmission electron micrographs demonstrated that detergent detoxification led to a loss of membrane integrity, while genetically-detoxified eOMV retained vesicular structure.

[0022] FIG. 8. Detergent-extracted, genetically-detoxified OMV (eOMV) elicit robust, cross- reactive anti-meningococcal antibodies in rabbits. Sera from rabbits immunized with detergent-extracted, detergent-detoxified OMV (dOMV) or eOMV were tested against a panel of 18 meningococcal strains in human complement serum bactericidal assays; sera from alum-immunized rabbits (KM 19 and KM20) were also tested as controls. The percentage of strains killed by each antiserum is shown at the bottom of the table. Immunizing antigen / adjuvants: KM9 and KM10, wild-type (WT) dOMV; KM11 and KM12, APorAAPorBARmpM (constructed with antibiotic resistance markers) dOMV; KM 13 and KM 14, APorAAPorBARmpMALpxLl dOMV; KM15 and KM16, APorAAPorBARmpMALpxLl eOMV; KM17 and KM 18, APorAAPorBARmpMASiaDALpxLl eOMV; KM 19 and KM20, Alum.

[0023] FIGS. 9A-9C. Immunization with APorAAPorBARmpMASiaDALpxLl detergent-extracted, genetically-detoxified OMV (eOMV) induces a robust, cross- reactive functional antibody response. Sera from mice (n=10 per group) immunized with wild-type (WT) detergent-extracted, detergent-detoxified OMV (dOMV), APorAAPorBARmpM (constructed with antibiotic resistance markers) dOMV, APorAAPorBARmpMALpxLl dOMV, APorAAPorBARmpMALpxLl eOMV, and APorAAPorBARmpMASiaDALpxLl eOMV were tested in human complement serum bactericidal assays against the parental MC58 strain (FIG. 9 A) or six heterologous meningococcal strains (FIGs. 9B-9C); sera from alum-immunized mice were also tested as controls. Sera from APorAAPorBARmpMASiaDALpxLl eOMV -immunized mice exhibited the most consistent and robust killing profile across the strains tested, with higher geometric mean titers (GMTs) compared to all other groups for all strains. *P < 0.05, **P < 0.01, and ***P < 0.001 by one-way ANOVA with Tukey’s post hoc test.

[0024] FIGS. 10A-10C. All meningococcal vaccines elicit robust serum antibody responses.

[0025] Sera from mice (n=10 per group) immunized with wild-type (WT) detergent-extracted, detergent- detoxified OMV (dOMV), APorAAPorBARmpM (constructed with antibiotic resistance markers) dOMV, APorAAPorBARmpMALpxLl dOMV, APorAAPorBARmpMALpxLl detergent-extracted, genetically-detoxified OMV (eOMV), APorAAPorBARmpMASiaDALpxLl eOMV, or alum alone were assessed in ELISAs for (A) IgA, total IgG, and IgGl antibody levels, in addition to (B) IgG2a, IgG2b, and IgG3. Levels were largely comparable among groups, though higher mean titers of IgG2a, IgG2b, and IgG3 were observed in the WT dOMV group relative to other vaccinated groups. Higher wild-type dOMV IgG2a levels contributed to a diminished IgGl / IgG2a ratio (C), where ratios <0.5, >2.0, and between 0.5 and 2.0 are representative of Thl-skewing, Th2-skewing, and a mixed Thl / Th2 response, respectively. ELISA plates were coated with MC58 OMV, prepared via passage of the bacterial wet mass through a French press. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P<0.0001 by one-way ANOVA with Dunn’s multiple comparisons test.

[0026] FIGS. 11A-11B. Immunization with detergent-extracted, genetically-detoxified OMV (eOMV) elicits antibodies to a greater variety of antigens compared to detergent-extracted, detergent- detoxified OMV (dOMV). Whole cell lysates of meningococcal strains were fractionated by SDS-PAGE and transferred to PVDF membranes. Blots were probed with pooled sera (1 : 1,000,000) from mice (n=10) immunized with each of the antigens. Open and closed arrows indicate RmpM and two unidentified immunogenic antigens, respectively.

[0027] FIG. 12. Antibodies from mice immunized with detergent-extracted, genetically-detoxified OMV (eOMV) demonstrate cross-species cross-reactivity. Whole cell lysates from six antigenically diverse N. gonorrhoeae strains were fractionated by SDS-PAGE and transferred to a PVDF membrane. The blot was probed with pooled serum from mice (1: 1,000,000) immunized with APorAAPorBARmpMASiaDALpxLldetergent-extracted, genetically-detoxified OMV (eOMV).

[0028] FIGS. 13A-13B. Meningococcal vaccines elicit mucosal antibody responses. Vaginal lavages from mice immunized with wild-type (WT) detergent-extracted, detergent-detoxified OMV (dOMV), APorAAPorBARmpM (constructed with antibiotic resistance markers) dOMV, APorAAPorBARmpMALpxLl dOMV, APorAAPorBARmpMALpxLl detergent-extracted, genetically- detoxified OMV (eOMV), APorAAPorBARmpMASiaDALpxLl eOMV, and alum alone were assessed in ELISAs for (FIG. 12) IgA, total IgG, and IgGl antibody levels, in addition to (FIG. 12) IgG2a, IgG2b, and IgG3. Mice vaccinated with APorAAPorBARmpMASiaDALpxLl eOMV produced significant levels of meningococcal-specific IgA and IgG antibodies relative to alum controls. ELISA plates were coated with MC58 OMV, prepared via passage of the bacterial wet mass through a French press. n=10 mice per group, except for APorAAPorBARmpMALpxLl eOMV (n=5), in which five mice were excluded due to contamination of lavages with blood. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P<0.0001 by one-way ANOVA with Dunn’s multiple comparisons test.

[0029] FIGS 14A-14B. Meningococcal vaccines elicit mucosal IgA antibodies against a variety of antigens. Whole cell lysates of meningococcal strains were fractionated by SDS-PAGE and transferred to PVDF membranes. Blots were probed with pooled vaginal lavages (1:100) from mice immunized with (FIG. 13A) wild-type (WT) dctcrgcnt-cxtractcd, detergent-detoxified OMV (dOMV), APorAAPorBARmpM (constructed with antibiotic resistance markers) dOMV, and APorAAPorBARmpMALpxLl dOMV, or (FIG. 13B) APorAAPorBARmpMALpxLl detergent-extracted, genetically-detoxified OMV (eOMV), APorAAPorBARmpMASiaDALpxLl eOMV, and alum alone. Binding of IgA to a similar panel of antigens was noted in the WT dOMV, APorAAPorBARmpMALpxLl eOMV, and APorAAPorBARmpMASiaDALpxLl eOMV groups, though the eOMV groups also elicited antibodies to three unique antigens, including two unidentified antigens (open arrows) and antigens previously identified as two species of lipooligosaccharide.

[0030] DETAILED DESCRIPTION OF SEVERAL ASPECTS

[0031] The first vaccines designed to prevent invasive MenB disease consisted of OMV preparations chemically isolated from outbreak strains (Sierra et al., NIPH Ann. 1991;14:195-210; Bjune et al., Lancet. 1991;338:1093-6). OMV are microvesicles of a size of about-100-300 nm, such as 100 to 200 nm, that recapitulate the composition of the bacterial outer membrane and can induce host anti-Neisseria immune responses (van der Pol et al., Biotechnol J. 2015;10:1689-706). Manufacturing methods typically include: (1) culturing of the outbreak isolate in liquid media, (2) centrifugation to separate the bacterial wet mass, (3) incubation of the wet mass with a low concentration of detergent to extract the OMV from the bacterial membrane, (4) successive steps of centrifugation and ultracentrifugation to isolate the detergent-extracted OMV (eOMV), (5) incubation of eOMV with a high concentration of detergent to decrease the toxic levels of lipooligosaccharide (LOS) present on the eOMV surface, and (6) ultracentrifugation to purify the detergent-detoxified OMV (dOMV) (Fredriksen et al., NIPH Ann. 1991;14:67-8). Although meningococcal dOMV vaccines have been shown to be effective at eliciting protection against the specific outbreak strain from which the vaccine is derived, dOMV are less effective at preventing infection with heterologous MenB strains.

[0032] The low cross-protection afforded by MenB dOMV vaccines is believed to be a result of induction of immunodominant antibody responses to the porin protein PorA, which is highly variable among meningococcal strains (Granoff et al., Vaccines. Clin Infect Dis. 2010;50(S2):S54-S65). Dominant antibody responses are also generated against an alternate porin protein, PorB, which is the major protein on the bacterial / dOMV surface. PorB is highly variable, similar to PorA, and anti-PorB antibodies are expected to strongly cross-react only with strains that express the same PorB type as the vaccine strain (Matthias et aL, Mol Microbiol. 2017;105:934-53). Despite immunodominant anti-PorA and anti-PorB responses, clinical studies suggest that antibody responses to other dOMV antigens can cross-react with heterologous strains and may confer some degree of partial protection against infection with other meningococcal strains (Sierra et al. NIPH Ann. 1991;14: 195-210; Caron et al., Lancet Infect Dis. 2011;! 1:455-6). Likewise, recent retrospective case-control and observational cohort studies have suggested that vaccination with MenB dOMV may confer off-target protection to a low percentage of individuals against infection with N. gonorrhoeae ((Petousis-et al., Lancet. 2017;390: 1603-10.Azze et al., Clin Exp Vaccine Res. 2019;8: l 10-5; Raccagni et al., Sex Transm Dis. 2023;50:247-51; Abara et al., Lancet. 2022;22:1021-9; Wang et al., Lancet. 2022;22: 1011-20; Bruxvoort et al., Clin Infect Dis. 2022;76:el341-e9), which is genetically closely related to N. meningitidis .

[0033] A MenB dOMV vaccine strain, AABR, was constructed that was genetically deleted for expression of PorA and PorB, as well as the structural protein RmpM (see PCT Publication No. WO 2019 / 018744, incorporated herein by reference). Sera from AABR dOMV -immunized animals killed a greater number of heterologous strains in human complement scrum bactericidal assays (hSBAs) relative to sera obtained from animals immunized with porin- sufficient wild-type (WT) dOMV (Matthias et al., Vaccine. 2020;38:2396- 405). However, the titers of killing associated with the anti-AABR dOMV sera were overall lower compared to the anti-WT dOMV sera. When tested in a mouse model of vaginal gonococcal infection, both the WT dOMV and the AABR dOMV vaccines enhanced clearance of N. gonorrhoeae from the lower reproductive tract, though consistent clearance was only observed with AABR dOMV -immunized mice in two independent trials (Matthias et al., J Infect Dis. 2022;225:650-60). Both (1) the divergent patterns of killing in hSBAs conducted with anti-WT and anti-AABR dOMV vaccinated sera and (2) the divergent phenotypes of gonococcal clearance in the WT- and AABR dOMV -immunized mice were associated with induction of antibody responses to varying antigens as assessed in western blot analyses. These data suggested that the absence of PorA, PorB, and RmpM from the dOMV functioned to permit enhanced host responses to other surface antigens that are more cross-reactive than the deleted proteins. However, the antibody responses to the AABR dOMV appeared less potent than those of WT dOMV manufactured by comparable methods. Thus, there was a need to enhance this potency. Summary of Terms

[0034] Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Krebs et al. (eds.), Lewin ’s genes XII, published by Jones & Bartlett Learning, 2017; and Meyers et al. (eds.), The Encyclopedia of Cell Biology and Molecular Medicine, published by Wiley-VCH in 16 volumes, 2008; and other similar references.

[0035] As used herein, the singular forms “a,” “an,” and “the,” refer to both the singular as well as plural, unless the context indicates otherwise. For example, the term “an antigen” includes single or plural antigens and can be considered equivalent to the phrase “at least one antigen.” As used herein, the term “comprises” means “includes.” It is further to be understood that any and all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for descriptive purposes, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described below. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. To facilitate review of the various aspects, the following explanations of terms are provided:

[0036] Administration: The introduction of a composition into a subject by a chosen route. Administration can be local or systemic. For example, if the chosen route is intranasal, the composition is administered by introducing the composition into the nasal passages of the subject. Similarly, if the chosen route is intramuscular, the composition is administered by introducing the composition into a muscle of the subject. If the chosen route is oral, the composition is administered by introducing the subject ingesting the composition. Exemplary routes of administration of use in the methods disclosed herein include, but are not limited to, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), sublingual, rectal, transdcrmal (for example, topical), intranasal, vaginal, oral, and inhalation routes. alpha-2, 8-polysialyltransferase (SiaD): A component of the meningococcal serogroup B capsule biosynthesis pathway. When the siaD gene is deleted, the serogroup B capsule cannot be made and the bacteria are, as a result, unencapsulated.

[0037] Amino acid substitution: The replacement of an amino acid in a polypeptide with one or more different amino acids. In the context of a protein sequence, an amino acid substitution is also referred to as a mutation.

[0038] Antibody: An immunoglobulin, antigen-binding fragment, or derivative thereof, that specifically binds and recognizes an analyte (antigen) such as an antigen on OMV of Neisseria meningitidis. The term “antibody” is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity. Nonlimiting examples of antibodies include, for example, intact immunoglobulins and variants and fragments thereof that retain binding affinity for the antigen. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, Flab'T; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments. Antibody fragments include antigen binding fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies (see, e.g., Kontermann and Dubel (Ed), Antibody Engineering, Vols. 1-2, 2ndEd., Springer Press, 2010).

[0039] Control: A reference standard. In some aspects, the control is a negative control sample obtained from a healthy patient. In other aspects, the control is a positive control sample obtained from a patient immunized with an OMV (such as an microvesicle or bleb) of Neisseria meningitidis. In still other aspects, the control is a historical control or standard reference value or range of values (such as a previously tested control sample, such as a group of patients with known prognosis or outcome, or group of samples that represent baseline or normal values). A control can also be from a subject immunized with a composition comprising an effective amount of PorA PorB RmpM'LpxLl’ detergent-extracted, detergent-detoxified OMV (dOMV) or detergent-extracted, genetically-detoxified OMV (eOMV), such as PorA PorB RmpM" LpxLl SiaD- OMV.

[0040] A difference between a test sample and a control can be an increase or conversely a decrease. The difference can be a qualitative difference or a quantitative difference, for example a statistically significant difference. In some examples, a difference is an increase or decrease, relative to a control, of at least about 5%, such as at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 500%, or greater than 500%.

[0041] Degenerate variant: In the context of the present disclosure, a “degenerate variant” refers to a polynucleotide encoding a polypeptide that includes a sequence that is degenerate as a result of the genetic code. There arc 20 natural amino acids, most of which arc specified by more than one codon. Therefore, all degenerate nucleotide sequences encoding a peptide are included as long as the amino acid sequence of the peptide encoded by the nucleotide sequence is unchanged.

[0042] Effective amount: An amount of agent, such as an immunogen, such as a N. meningitidis OMV, that is sufficient to elicit a desired response, such as an immune response in a subject. It is understood that to obtain a protective immune response against an organism of interest can require multiple administrations of a disclosed immunogen, and / or administration of a disclosed immunogen as the “prime” in a prime boost protocol wherein the boost immunogen can be different from the prime immunogen. Accordingly, an effective amount of a disclosed immunogen can be the amount of the immunogen 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.

[0043] In one example, a desired response is to inhibit or reduce or prevent a Neisseria gonorrhoeae or Neisseria meningitidis infection. The Neisseria gonorrhoeae or N. meningitidis infection does not need to be completely eliminated or reduced or prevented for the method to be effective. For example, administration of an effective amount of the agent can decrease the Neisseria gonorrhoeae or N. meningitidis infection (for example, as measured by bacteria number or by number or percentage of subjects infected by Neisseria gonorrhoeae or Neisseria meningitidis) by a desired amount, 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 Neisseria gonorrhoeae or N. meningitidis infection), as compared to a suitable control.

[0044] Epitope: An antigenic determinant. These are particular chemical groups or peptide sequences on a molecule that are antigenic, such that they elicit a specific immune response, for example, an epitope is the region of an antigen to which B and / or T cells respond. An antibody can bind to a particular antigenic epitope, such as an epitope presented on a microvesicle of Neisseria gonorrhoeae or Neisseria meningitidis. Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein.

[0045] Heterologous: Originating from a different genetic source, so that the biological components that are not found together in nature. The components may be host cells, genes, or regulatory regions, such as promoters. Although the heterologous components are not found together in nature, they can function together, as when a promoter heterologous to a gene is operably linked to the gene. Another example is where a Neisseria sequence is heterologous to the sequence of a different Neisseria strain. “Heterologous” as used herein in the context of proteins expressed in two different bacterial strains, e.g., “heterologous PorA.”

[0046] Immune response: A response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus, such as Neisseria. In one aspect, the response is specific for a particular antigen (an “antigen-specific response”). In one aspect, an immune response is a T cell response, such as a CD4+ response or a CD8+ response. In another aspect, the response is a B cell response, and results in the production of specific antibodies. A “protective immune response” is an immune response that confers protection against a disease caused by a member of Neisseria, such as N. meningitidis scrogroups, particularly serogroups A, B, C, X, Y, and W-135, and / or Neisseria gonorrhoeae. A “therapeutic immune response” treats an existing infection with Neisseria. In some aspects, the subject has a N. meningitidis infection, and administration of the immunogenic composition increases clearance of the N. meningitidis . In other aspects, the subject has a Neisseria gonorrhoeae infection, and administration of the immunogenic composition increases clearance of N. gonorrhoeae.

[0047] Immunogen: A compound, composition, or substance (for example, a composition including outer membrane microvesicles from PorA'PorB RmpM'LpxLl’ Neisseria, such PorA PorB'RmpM LpxLTSiaD’ Neisseria) that can elicit an immune response in an animal, including compositions that are injected or absorbed into an animal. Administration of an immunogen to a subject can lead to immunity against a pathogen of interest, such as N. gonorrhoeae and / or N. meningitidis.

[0048] Immunogenic composition: A composition comprising outer membrane microvesicles from PorA‘ PorB RmpM LpxLE Neisseria meningitidis, such as PorA PorB RmpM LpxLl'SiaD' N. meningitidis, that induces a measurable CTL response against Neisseria gonorrhoeae and / or N. meningitidis, or induces a measurable B cell response (such as production of antibodies) against N. gonorrhoeae and / or N. meningitidis, when administered to a subject. For in vivo use, the immunogenic composition will typically include outer membrane microvesicles from PorA PorB’RmpM'LpxLl’ N. meningitidis, such as PorA PorB" RmpM LpxLI SiaD’ N. meningitidis, in a pharmaceutically acceptable carrier and optionally may also include other agents, such as an adjuvant. The phrase “in an effective amount to elicit an immune response” means that there is a detectable difference between an immune response indicator measured before and after administration of a particular immunogenic composition. Immune response indicators include but are not limited to: antibody titer or specificity, as detected by an assay such as enzyme-linked immunosorbent assay (ELISA), bactericidal assay, flow cytometry, immunoprecipitation, Ouchterlony immunodiffusion; binding detection assays of, for example, spot, western blot or antigen arrays; cytotoxicity assays, etc.

[0049] Inhibiting or treating a disease: Inhibiting the full development of a disease or condition, for example, in a subject who is at risk for a disease such as a N. gonorrhoeae and / or N. meningitidis infection. “Treatment” refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop. The term “ameliorating,” with reference to a disease or pathological condition, refers to any observable beneficial effect of the treatment. Inhibiting a disease can include preventing or reducing the risk of the disease, such as preventing or reducing the risk of bacterial infection. The beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in severity of some or all clinical symptoms of the disease, a slower progression of the disease, a reduction in the bacterial load, an improvement in the overall health or well-being of the subject, or by other parameters that are specific to the particular disease. A “prophylactic” treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing pathology.

[0050] Isolated: An “isolated” biological component has been substantially separated or purified away from other biological components, such as other biological components in which the component naturally occurs, such as other chromosomal and extrachromosomal DNA, RNA, membranes, cells and proteins. OMV that have been “isolated” include those purified by standard purification methods. Isolated does not require absolute purity, and can include microvesicles that are at least 50% isolated, such as at least 75%, 80%, 90%, 95%, 98%, 99%, or even 99.9% isolated from other components of the bacteria that product them.

[0051] 2-keto-3-deoxy octulosonic acid (KDO)2-(lauroyl)-lipid IVA acyltransferase (LpxLl): An acyltransferase in Neisseria that functions in lipid A biosynthesis, see FIG. 1. In vivo, LpxLl transfers laurateto(2-keto-3-deoxyoctulosonicacid)2-lipid IVA [(KDOh-lipid IVA ], after which LpxM can add myristate to complete lipid A acylation. The predominant products formed by LpxLl mutants are tetra- or penta- acylated species (van der Ley et al., 2001, Infect. Immun. 69 5981-5990). LpxLl- Neisseria (also called AL or ALpxLl) exhibit diminished toxicity relative to LpxLl -sufficient Neisseria in vitro, with decreased TLR4 stimulation (Steeghs et al., 2008, Infect. Immun. 76 3801-07; Fransen et al., 2009, PLoS Pathog. 5:el000396; Fransen et al., 2010, Infect. Immun. 78 3177-86) and induction of pro-inflammatory cytokine secretion (van der Ley et al., 2001, Infect. Immun. 69 5981-90; Fransen et al., 2009, PLoS Pathog. 5:el000396; Fransen et aL, 2010, Infect. Immun. 78 3177-86; Brouwer et al., 2011, J. Infect. 62 479-483). Patients infected with spontaneous LpxLl' N. meningitidis mutants correspondingly exhibit a milder clinical pathology with decreased inflammation relative to patients infected with wild-type Neisseria (Fransen et al., 2009, PLoS Pathog. 5:el000396). Immunization of animals with OMV isolated from LpxLl- N. meningitidis results in production of robust host immune responses, including elicitation of bactericidal antibodies (Koeberling et al., 2008, J. Infect. Dis. 198 262-70; Koeberling et aL, 2009, Clin. Vacc. Immunol. 16 156-62; Beernink et al., 2019, mBio 10:e01231-19).

[0052] Outer Membrane Microvesicles: Vesicles that are produced from the outer membrane of Neisseria, and include both microvesicles and blebs. Microvesicles are produced by solubilizing Neisseria in a process wherein the outer membranes form vesicles. Blebs are produced by budding from living Neisseria. Methods from the production of outer membrane microvesicles are known in the art, see for example, van de Waterbeemd et al., PLOS One, doi.org / 10.1371 / journal.pone .0065157, May 31, 2013). OMV and blebs can be purified from intact Neisseria, and are generally 100 to 300 nm, such as 100 to 200 nm. “Detergent extracted outer membrane vesicles” or “eOMV” are OMV (microvesicles) that are formed by chemical treatment of Gram-negative or Gram-positive bacteria with a detergent such that portions of the bacterial membrane are extracted and spontaneously re-form in solution. In some aspects, “eOMV” can be isolated by incubation of genetically-detoxified (LpxLl-) N. meningitidis with a low concentration (0.5%) of sodium deoxycholate, followed by purification. “Detergent detoxified outer member vesicles” or “dOMV” are OMV (such as microvesicles or blebs) that are formed by chemical treatment of microvesicles isolated from Gram-negative or Gram-positive bacteria with a detergent such that the majority of the endogenous, toxic lipooligosaccharide or lipopolysaccharide is removed. dOMV can be detergent-extracted eOMV isolated from LpxLl -sufficient or LpxLl -deficient N. meningitidis that have been incubated with a high concentration (5%) of sodium dcoxycholatc, followed by purification.

[0053] Outer Membrane Protein Reduction Modifiable Protein (Rmp)M: A periplasmic protein from N. meningitidis that comprises an N-terminal domain (residues 1-47) and a separate globular C-terminal domain (residues 65-219) responsible for binding to peptidoglycan. The N-terminal fragment of RmpM binds to both the major outer membrane porins, PorA and PorB. Analysis by semi-native SDS-PAGE established that both recombinant full-length RmpM and an N-terminal fragment were sufficient to stabilize the PorA and PorB oligomeric complexes. The meso-diaminopimelate moiety plays a role in peptidoglycan recognition by RmpM. Site-directed mutagenesis showed that two highly conserved residues, Asp 120 and Argl35, play a role in peptidoglycan binding, see Maharjan et al., Microbiology 162: 364-375, 2016. See also Li et aL, PLOS Biology, doi.org / 10.1371 / journaLpone.0090525, March 4, 2014 and GENBANK Accession No. X05105.1, as available on June 30, 2018, incorporated herein by reference. The yield of OMV is higher in a N. meningitidis strain expressing a truncated N-terminal fragment of RmpM (AC-term RmpM) than in a wild-type strain. This strain is also RpmM- as it does not produce functional RmpM protein. Generally, a RmpM" Neisseria (also called “AR”) does not produce functional RmpM protein.

[0054] Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers of use are conventional. Remington’ s Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 19th Edition, 1995, describes compositions and formulations suitable for pharmaceutical delivery of the disclosed immunogens.

[0055] In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise 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. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions (such as immunogenic compositions) to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate. In particular aspects, suitable for administration to a subject the carrier may be sterile, and / or suspended or otherwise contained in a unit dosage form containing one or more measured doses of the composition suitable to induce the desired immune response. It may also be accompanied by medications for its use for treatment purposes. The unit dosage form may be, for example, in a sealed vial that contains sterile contents or a syringe for injection into a subject, or lyophilized for subsequent solubilization and administration or in a solid or controlled release dosage.

[0056] Polypeptide: Any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). “Polypeptide” applies to amino acid polymers including naturally occurring amino acid polymers and non-naturally occurring amino acid polymer as well as in which one or more amino acid residue is a non-natural amino acid, for example, an artificial chemical mimetic of a corresponding naturally occurring amino acid. A “residue” refers to an amino acid or amino acid mimetic incorporated in a polypeptide by an amide bond or amide bond mimetic. A polypeptide has an amino terminal (N-terminal) end and a carboxy terminal (C-terminal) end. “Polypeptide” is used interchangeably with peptide or protein, and is used herein to refer to a polymer of amino acid residues.

[0057] Polysialyltransferase (SiaD): An enzyme involved in the production of the polysialic acid capsule in Neisseria meningitidis. SiaD functions as a polysialyltransferase, forming a-2,8 linkages between the repeating sialic acid monomers that comprise the MenB capsule. Due to its resemblance to a sugar moiety present on human embryonic neural cells (Rougon et al., 1986, J. Cell Biol. 103 2429-37; Nedelec et aL, 1990, J. NeuroimmunoL 2949-56), the MenB capsule is poorly immunogenic (Wyle et aL, 1972, J. Infect. Dis. 126514-22) and does not constitute a viable target for MenB vaccines. More broadly, N. meningitidis capsules constitute major virulence determinants and have been implicated in evasion of host immune responses by enhancing resistance to defensins (Spinosa et al., 2007, Infect. Immun. 75 3594-3603), phagocytosis (Kolb-Maurer et al., 2001, Infect. Immun. 69 6912-22; Unkmeir et al., 2002, Infect. Immun. 70 2454-62), and complement-mediated killing (Hammerschmidt et al., 1994, Mol. Microbiol. 11 885-96; Vogel et al., 1997, Infect. Immun. 65 4022-29; Kahler et al., 1998, Infect. Immun. 66 5939-47). N. meningitidis strains are rendered unencapsulated by insertional inactivation or deletion of the polysialyltransferase (sial)) gene; SiaD- Neisseria may be referred to as AS or ASiaD.

[0058] Porin (Por)A: PorA is a Neisseria porin. PorA monomer topology shows eight extracellular loops (Derrick et al., 1999, Infect. Immun. 67 2406-13; van der Ley et aL, 1991, Infect. Immun. 59 2963) in the protein. The longest loops (1 and 4) are the most variable, hence are referred to as Variable Region 1 (VR1) and Variable Region 2 (VR2). Less variability is seen in loops 5 and 6 (also called semi-variable SVR1 and 2, or variable region 3 and 4, respectively), with essentially no variability in the remaining loops. Loop 3 is predicted to form a “plug” in the pore formed by each subunit of the PorA trimer. Even within VR1 and VR2, most of the variability is confined to residues predicted to form the tip of each loop. Indeed, in both mice and in immunized human volunteers, epitope mapping showed that the majority of the antibody response is directed at the “top” of loops 1 and 4, the region that is variable between strains (van der Voort, et al., 1997, FEMS Immunol. Med. Microbiol. 17 139-48).

[0059] This protein generates an immune response in both patients and asymptomatic carriers, to the extent that it has been used as a marker for strain identification, representing the serosubtype system (McGuinness et al., 1990, J Exp Med. 171 1871-82). PorA has been used in effective and registered vaccine formulations and elicits effective bactericidal antibodies. However, strain-to-strain variability in surface loops results in a variable target, and vaccines are typically PorA type-specific. Efforts have been made to generate multivalent PorA vaccines covering up to six different PorA types (van der Voort et al., 1996, Infect Immun. 64 2745-51). A PorA' Neisseria (also called AA or APorA) does not produce functional PorA protein.

[0060] Porin B (PorB): A 16-pass transmembrane protein from Neisseria that is a porin and forms a 0- barrel structure with eight surface-exposed loops (L1-L8) (Tanabe et al. (2010) Proc Natl Acad Sci USA 107: 6811-6816). Two of these, L2 and L3, are structural and do not vary considerably in amino acid sequence among different McnB strains. The remaining six, LI and L4-L8, undergo antigenic variation; it is the binding of antibodies to these loops that forms the basis for meningococcal serotyping (Frasch et al. (1985) Rev Infect Dis 7: 504-510). A PorB' Neisseria (also called AB or APorB) does not produce functional PorB protein.

[0061] Prime-boost vaccination: An immunotherapy including administration of a first immunogenic composition (the primer vaccine) followed by administration of another immunogenic composition (the booster vaccine) to a subject to induce an immune response. The primer vaccine and / or the booster vaccine are immunogens to which the immune response is directed. The booster vaccine is administered to the subject after the primer vaccine; a suitable time interval between administration of the primer vaccine and the booster vaccine, and examples of such timeframes are disclosed herein. In some aspects, the primer vaccine, the booster vaccine, or both primer vaccine and the booster vaccine additionally include an adjuvant Recombinant: A recombinant nucleic acid molecule is one that has a sequence that is not naturally occurring, for example, includes one or more nucleic acid substitutions, deletions or insertions, and / or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination can be accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques.

[0062] A recombinant protein 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. In several aspects, a recombinant protein is encoded by a heterologous (for example, recombinant) nucleic acid that has been introduced into a host cell, such as a bacterial or eukaryotic cell, or into the genome of a recombinant virus.

[0063] Sequence identity: The similarity between amino acid sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity; the higher the percentage, the more similar the two sequences are. Homologs, orthologs, or variants of a polypeptide will possess a relatively high degree of sequence identity when aligned using standard methods.

[0064] Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS 5: 151-3, 1989; Corpet et al., Nuc. Acids Res. 16: 10881-90, 1988; Huang et al. Computer Appls. In the Biosciences 8, 155-65, 1992; and Pearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J. Mol. Biol. 215:403-10, 1990, presents a detailed consideration of sequence alignment methods and homology calculations.

[0065] Variants of a polypeptide are typically characterized by possession of at least about 75%, for example, at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity counted over the full-length alignment with the amino acid sequence of interest. Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. When less than the entire sequence is being compared for sequence identity, homologs and variants will typically possess at least 80% sequence identity over short windows of 10-20 amino acids, and may possess sequence identities of at least 85% or at least 90% or 95% depending on their similarity to the reference sequence. Methods for determining sequence identity over such short windows are available at the NCBI website on the internet.

[0066] As used herein, reference to “at least 90% identity” (or similar language) refers to “at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity” to a specified reference sequence.

[0067] Serogroup: Classification, such as of Neisseria meningitidis by virtue of immunologically detectable variations in the capsular polysaccharide. About 12 serogroups are known: A, B, C, X, Y, Z, 29- E, W-135, H, I, K, and L. Any one serogroup can encompass multiple serotypes and multiple serosubtypes. A serotype is classification of N. meningitidis strains based on monoclonal antibody-defined antigenic differences in the outer membrane protein porin PorB, or upon VR typing of amino acid sequences deduced from DNA sequencing. A single serotype can be found in multiple serogroups and multiple serosubtypes. “Serosubtype” is classification of Neisseria meningitidis strains based on antibody-defined antigenic variations on the outer membrane protein porin PorA, or upon VR typing of amino acid sequences deduced from DNA sequencing (Sacchi et al., 2000, J. Infect. Dis. 182: 1169; see also the Multi Locus Sequence Typing web site). Most variability between PorA proteins occurs in two (loops I and IV) of eight putative, surface-exposed loops. The variable loops I and IV have been designated VR1 and VR2, respectively. A single serosubtype can be found in multiple serogroups and multiple serotypes.

[0068] Subject: Living multi-cellular vertebrate organisms, a category that includes human and nonhuman mammals. In an example, a subject is a human. In an additional example, a subject is selected that is in need of inhibiting of a Neisseria infection. For example, the subject is either uninfected and at risk for infection, or is infected in need of treatment.

[0069] Under conditions sufficient for: A phrase that is used to describe any environment that permits a desired activity.

[0070] Vaccine: A preparation of immunogenic material capable of stimulating an immune response, administered for the prevention, amelioration, or treatment of infectious or other types of disease. The immunogenic material may include attenuated or killed microorganisms (such as bacteria or viruses), or antigenic proteins, peptides, or DNA derived from them. A vaccine may include a disclosed immunogen, such as OMVs from PorA PorB RmpM LpxLL Neisseria meningitidis, such PorA PorB’RmpM’LpxLTSiaD’ N. meningitidis. Vaccines can elicit both prophylactic (preventative or protective) and therapeutic responses. Methods of administration vary according to the vaccine, but may include inoculation, ingestion, inhalation, or other forms of administration. Vaccines may be administered with an adjuvant to boost the immune response. In one specific, non-limiting example, a vaccine prevents and / or reduces the severity of the symptoms associated with Neisseria infection and / or decreases the bacterial burden compared to a control.

[0071] In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0072] Neisseria Strains and Outer Membrane Microvesicles

[0073] In some aspects, isolated PorA PorB RmpM LpxLL Neisseria meningitidis, such PorA PorB RmpM’ LpxLl SiaD Neisseria meningitidis, are disclosed herein. These N. meningitidis are not naturally occurring, as PorB’ N. meningitidis do not occur in nature. Isolated PorA’PorB’RmpM’LpxLl’ Neisseria meningitidis, such PorA’PorB’RmpM’LpxLl SiaD’ Neisseria meningitidis, are of use in the disclosed methods.

[0074] Generally, PorA PorB RmpM’ N. meningitidis are deficient from the production of PorA, PorB, and RmpM. Thus, these PorA, PorB, and RmpM subcapsular proteins cannot be detected in the surface of these N. meningitidis. Thus, PorA, PorB, and RmpM do not function in these N. meningitidis.

[0075] Similarly, PorA PorB RmpM LpxLL Neisseria meningitidis are deficient from the production of PorA, PorB, RmpM, and LpxLl. Thus, the PorA, PorB, and RmpM subcapsular proteins cannot be detected in the surface of these N. meningitidis. In addition, inactivation of LpxLl prevents addition of a secondary lauroyl chain to the nonreducing end of the glucasomine disaccharide, transforming the native hexa-acylated structure into a penta-acylated structure in the membrane. Thus, PorA, PorB, RmpM, and LpxLl do not function in these N. meningitidis.

[0076] PorA PorB RmpM LpxLTSiaD' N. meningitidis are deficient from the production of PorA, PorB, RmpM, LpxLl, and SiaD. These N. meningitidis are unencapsulated. In some aspects, the function of LpxLl and / or SiaD is decreased by at least 80%, 85%, 90%, 95%, 98%, 99% or is complete absent.

[0077] In some aspects, there is a deletion in the genes encoding PorA, PorB, RmpM , LpxLl, and / or SiaD in these N. meningitidis, so that the corresponding protein is not produced. In other aspects, there is a stop codon inserted in the gene encoding PorA, PorB, RmpM, LpxLl, and / or SiaD, so that the corresponding protein is not produced. In other aspects, the gene encoding PorA, PorB, RmpM, LpxLl, and / or SiaD includes a mutation such that the protein is not functional or immunogenic, e.g., one or more of these proteins is not present on the outer membrane of the N. meningitidis.

[0078] In some aspects, production of the surface proteins PorA, PorB and RpmM, and the function of LpxLl and / or SiaD, is decreased by at least 80%, 85%, 90%, 95%, 98%, 99% or is completely absent. Generally, in the strains of use in the methods disclosed herein, PorA, PorB, and RmpM is not detectable on the bacterial surface. Suitable methods for detecting PorA, PorB, and RmpM on the cell surface include whole cell ELISA using monoclonal and polyclonal protein- specific antibodies. Furthermore, in the strains of use in the methods disclosed herein, the enzymatic function of LpxLl, and optionally SiaD, cannot be detected using an assay. In more aspects, inactivation of LpxLl prevents addition of a secondary lauroyl chain to the nonreducing end of the glucasomine disaccharide, transforming the native hexa-acylated structure into a penta-acylated structure in the membrane. In further aspects, disruption of the capsule identifies a strain as SiaD'. Deletion of SiaD can be indirectly confirmed using an ELISA or immunoblotting (Sec Fig. 4) by using antibodies specific for the McnB capsule. Also deletion of LpxLl was confirmed using the in vitro TLR4 stimulation assay (see FIG. 5).

[0079] Modified strains can be generated by recombination techniques, and by non-recombinant techniques such as, for example, exposure to chemicals, radiation, or other DNA modifying or damaging agent, and the like. Modified strains having a desired protein expression profile, specifically wherein PorA, PorB, and RmpM, are not detectable on the cell surface can be identified through screening. Similarly, modified strains wherein LpxLl is not present can be identified by their lipid profile (see FIG. 1), and the disruption of the capsule identifies strains with a disruption in SiaD.

[0080] The N. meningitidis can be any type. N. meningitidis strains can be divided into serologic groups, serotypes, and subtypes on the basis of reactions with polyclonal (Frasch et al. (1985) Rev Infect Dis 7: 504- 510, C. E. and Chapman, 1973, J. Infect. Dis. Y2.T. 149-154) or monoclonal antibodies that interact with different surface antigens. Serogroup is based on immunologically detectable variations in the capsular polysaccharide. About 12 serogroups (A, B, C, X, Y, Z, 29-E, and W-135) are known. In some aspects, the PorA'PorB'RmpM'LpxLl' N. meningitidis, such as PorA PorB RmpM LpxLTSiaD' A. meningitidis, are serogroup A, B, or C. N. meningitidis also can be divided into clonal groups or subgroups, using various techniques that directly or indirectly characterize the bacterial genome. These techniques include multilocus enzyme electrophoresis (MLEE), based on electrophoretic mobility variation of an enzyme, which reflects the underlying polymorphisms at a particular genetic locus. By characterizing the variants of a number of such proteins, genetic “distance” between two strains can be inferred from the proportion of mismatches. Similarly, clonality between two isolates can be inferred if the two have identical patterns of electrophoretic variants at a number of loci. Multilocus sequence typing (MLST) can also be used to characterize the microorganisms. Using MLST, the genetic distance between two isolates, or clonality, is inferred from the proportion of mismatches in the DNA sequences of 11 housekeeping genes in N. meningitidis strains (Maiden et al., 1998, Proc. Natl. Acad. Sci. USA 95:3140). Any strain can be selected and used to produce a PorA PorB RmpM'LpxLT / . meningitidis, such as a PorA PorB'RmpM LpxLTSiaD' N. meningitidis.

[0081] Isolated N. meningitidis can be transformed to express a heterologous protein. These recombinant N. meningitidis are also of use in the methods disclosed herein, provided they are PorA PorB RmpM LpxLl', such as a PorA PorB'RmpM LpxLTSiaD'. The isolated N. meningitidis can be transformed to express additional antigens such as those exemplified in PCT Publication Nos. WO 99 / 24578, WO 99 / 36544; WO 99 / 57280, WO 00 / 22430, and WO 00 / 66791, as well as antigenic fragments of such proteins.

[0082] In some aspects, OMVs are produced from PorA PorB RmpM LpxLl / V. meningitidis, such as a PorA PorB'RmpM LpxLTSiaD' N. meningitidis . It is disclosed herein that an effective amount of OMVs from a PorA PorB RmpM LpxLl' N. meningitidis, such as a PorA PorB'RmpM LpxLTSiaD' TV. meningitidis, can be used to induce an immune response to Neisseria, such as to N. meningitidis and / or N. gonorrhoeae. Immunogenic compositions can be produced including an effective amount of OMVs, including microvesicles and / or blebs, from a PorA PorB RmpM'LpxLT N. meningitidis, such as a PorA' PorB RmpM'LpxLl SiaD' N. meningitidis, and a pharmaceutically acceptable carrier. In some aspects, OMVs are isolated from PorA PorB'RmpM LpxLl'A / meningitidis, such as a PorA PorB RmpM'LpxLT SiaD' N. meningitidis, that expresses a heterologous protein. These vesicles do not include the PorA, PorB, RmpM, or LpxLl, but include the heterologous protein. These vesicles are produced without, and do not require, detergent detoxification.

[0083] The OMV can be microvesicles, blebs, or a combination thereof. Blebs are budded from living N. meningitidis. Methods for isolating blebs are also known in the art, see for example, Post et al., J. Biological Chem. 280: 38383-38394, 2005, incorporated herein by reference. Microvesicles are produced by solubilizing Neisseria. Methods of production are disclosed, for example, in U.S. Published Patent Application No. 2012 / 0328643, incorporated herein by reference. Methods for the preparation of OMVs also are disclosed in Claassen et al. (Vaccine (1996) 14:1001-1008); Cartwright et al. (Vaccine (1999) 17:2612-2619); Peelers et al. (Vaccine (1996) 14:1009-1015); Fu et al. (Biotechnology NY (1995) 12:170- 74); Davies et al. (J. Immunol. Meth. (1990) 134:215-225); Saunders et al. (Infect. Immun. (1999) 67:113- 119); Draabick et al. (Vaccine (2000) 18: 160-172); Moreno et al. (Infect. Immun. (1985) 47:527-533); Milagres et al. (Infect. Immun. (1994) 62:4419-4424); Naess et al. (Infect. Immun. (1998) 66:959-965]; Rosenqvist et al. (Dev. Biol. Stand. (1998) 92:323-333); Haneberg et al. (Infect. Immunn. (1998) 66:1334- 41); Andersen et al. (Vaccine (1997) 15:1225-34); and Bjune et al. (Lancet (1991) 338:1093-96).

[0084] In some aspects, OMVs are prepared by deoxy cholate extraction. An extraction protocol is disclosed in Fredriksen et al., (1991) NIPH Ann. 14(2):67-79. Additional methods are disclosed, for example, in, Bjune et al. (Lancet (1991) 338(8775): 1093-96), Fredriksen et al. (1991) NIPH Annals 14: 67-79, Pages 818-824 of Pathobiology and immunobiology of Neisseriaceae (eds. Conde-Glez et al.) ISBN 968-6502-13- 0). The OMV (for example, as obtained by deoxy cholate extraction) can be treated to remove certain components. For instance, pyrogens or toxic components may be removed (e.g. LOS).

[0085] In aspects, the OMVs are eOMVs. In some aspects the OMV have been treated with a low concentration (about 0.5%) of sodium deoxycholate.

[0086] In other aspects, the OMV have not been treated with a high concentration (about 5%) of sodium deoxycholate. In further aspects, the OMV are dOMV. In more aspects, the OMV have been treated with a high concentration (about 5%) of sodium deoxy cholate.

[0087] Immunogenic Compositions and Methods of Use

[0088] Immunogenic compositions of use in the disclosed method include outer membrane microvesicles from PorA PorB RmpM LpxLl- N. meningitidis, such as a PorA PorB RmpM LpxLI SiaD' N. meningitidis. The OMV can be microvesicles and / or blebs. The immunogenic compositions of use in the disclosed methods can include a mixture (e.g., microvesicles and blebs), which microvesicles can be from the same or different strains. In another aspect, the immunogenic compositions can comprise a mixture of vesicles from 2, 3, 4, 5 or more strains, where the OMV can be microvesicles, blebs or both.

[0089] Optionally, an immunogenic composition can include an adjuvant. Adjuvants can include a suspension of minerals (alum, aluminum hydroxide, or phosphate) on which antigen is adsorbed; or water- in-oil emulsion in which antigen solution is emulsified in mineral oil (for example, Freund’s incomplete adjuvant), sometimes with the inclusion of killed mycobacteria (Freund’s complete adjuvant) to further enhance antigenicity. Immunostimulatory oligonucleotides (such as those including a CpG motif) can also be used as adjuvants (for example, see U.S. Patent Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116; 6,339,068; 6,406,705; and 6,429,199). Adjuvants also include biological molecules, such as costimulatory molecules. Exemplary biological adjuvants include interleukin (IL)-2, IL-12, RANTES, granulocyte macrophage colony stimulating factor (GM-CSF), tumor necrosis factor (TNF)-a, and interferon (IFN)-y.

[0090] In one aspect, the OMV, such as the microvesicles and / or blebs, can be used with an aluminum hydroxide adjuvant. One protein:adjuvant ratio is 1:67 (wt / wt). However, other ratios can be used, such as 1:20, 1:30, 1:40, 1:50, 1:33, l;60, 1:65, 1:70, 1:75, 1:80 or 1:85).

[0091] Additional adjuvants include Complete Freund's Adjuvant, Incomplete Freund's Adjuvant, Gerbu adjuvant (GMDP; C.C. Biotech Corp.), RIBI fowl adjuvant (MPL; RIBI Immunochemical Research, Inc.), potassium alum, aluminum phosphate, QS21 (Cambridge Biotech), Titer Max adjuvant (CytRx), and Quil A adjuvant. Exogenous lipopolysaccharide (LPS) can also be used as an adjuvant.

[0092] The immunogenic compositions can also include other agents, such as binders. Binders include, but are not limited to, carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, or gelatin; excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, Primogel, corn starch and the like; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin, a flavoring agent such as peppermint, methyl salicylate or orange flavoring, and a coloring agent. The compositions can also include gum arabic, syrup, lanolin, starch, etc., that forms a vehicle for delivery. Included are substances that, in the presence of sufficient liquid, impart to a composition the adhesive quality needed for the preparation of pills or tablets.

[0093] Exemplary pharmaceutically acceptable carriers include liquid carriers (such as water, saline, culture medium, aqueous dextrose, and glycols) and solid carriers (such as carbohydrates exemplified by starch, glucose, lactose, sucrose, and dextrans, anti-oxidants exemplified by ascorbic acid and glutathione, and hydrolyzed proteins). Exemplary diluents include water, physiological saline solution, human serum albumin, oils, polyethylene glycols, glycerine, propylene glycol, or other synthetic solvents. The compositions can also include antibacterial agents such as benzyl alcohol, antioxidants such as ascorbic acid or sodium bisulphite, chelating agents such as ethylene diamine-tetra-acetic acid, buffers such as acetates, citrates or phosphates, and agents for adjusting the osmolarity, such as sodium chloride or dextrose.

[0094] In some aspects, the immunogenic composition can be formulated for administration by injection via the intramuscular, intraperitoneal, intradermal, or subcutaneous routes; or via mucosal administration to the oral / alimentary, respiratory (e.g., intranasal administration), genitourinary tracts. Although the immunogenic composition can be administered as a single dose, components thereof can also be coadministered together at the same time or at different times. In addition to a single route of administration, two or more different routes of administration can be used.

[0095] Immunogenic compositions can be lyophilized or be in aqueous form, e.g., solutions or suspensions. Liquid formulations allow the compositions to be administered directly from their packaged form, without the need for reconstitution in an aqueous medium. Compositions can be presented in vials, or they can be presented in ready-filled syringes. The syringes can be supplied with or without needles. A syringe will include a single dose of the composition, whereas a vial can include a single dose or multiple doses (e.g. 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses). In one aspect, the dose is for use in a human. In a further aspect, the dose is for an adult, adolescent, toddler, infant, or less than one year old human, and can be administered by injection. Kits can include a measured dose for administration to a subject.

[0096] An immunogenic composition can be lyophilized. When an immunogenic composition requires reconstitution, it can be provided in the form of a kit which can comprise two vials, or can 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.

[0097] The OMVs can be used in conjunction with other agents, such as another vaccine or therapeutic agent. In some aspects, the vaccine can be a meningococcal vaccine, such as a conjugate vaccine or a polysaccharide vaccine. In specific non-limiting examples, the vaccine is MPSV4 (MENOMUNE®), MCV4 (MENACTRA®, MENHIBRIX®, MENVEO®, MENQUADFI®) or a serogroup B meningococcal vaccine (TRUMENBA® and BEXSERO®). Additional vaccines are MENCEVAX®, a purified polysaccharide vaccine, such as NmVac4-A / C / Y / W-135, and NIMENRIX®.

[0098] Methods are disclosed herein for inducing an immune response to Neisseria in a mammalian subject using any of the disclosed immunogenic compositions including an effective amount of OMVs (for example, microvesicles and / or blebs) from PorA PorB RmpM LpxLI A'. meningitidis, such as a PorA PorB RmpM’ LpxLTSiaD’ N. meningitidis. The immune response can be a protective immune response or a therapeutic immune response.

[0099] The subject can be a human or veterinary subject. The subject can be an adult or a juvenile subject. In some aspects, the subject is a human of two weeks, one month, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 months, or one year or 15, 18, or 21 months of age, or a child of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 years of age. The subject can be an adult, such as a human subject 18 or more years of age.

[0100] The method can include administering 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 doses of an effective amount of outer membrane OMVs from PorA PorB RmpM LpxLI A meningitidis, such as PorA PorB RmpM LpxLT SiaD- N. meningitidis. In some aspects, a single dose is used. In other aspects, multiple doses are used, such as in a prime boost protocol. Exemplary non-limiting protocols are shown in the examples section. An initial dose and an additional dose can be administered within days, weeks, or months of each other.

[0101] In some aspects, the subject has a N. meningitidis infection, and administration of the immunogenic composition increases clearance of the N. meningitidis. The N. meningitidis in the immunogenic composition can be any serogroup, such as serogroup A, B, or C. The N. meningitidis infection can be of any serogroup, such as serogroup A, B, and C. Optionally, the N. meningitidis in the immunogenic composition and the N. meningitidis infection arc the same serogroup.

[0102] In other aspects, the subject has a N. gonorrhoeae infection, and administration of the immunogenic composition increases clearance of the N. gonorrhoeae. In further aspects, the subject is a healthy subject, and does not have a N. meningitidis or a N. gonorrhoeae infection.

[0103] In some aspects, methods are provided for inducing an immune response to N. gonorrhoeae in a mammalian subject, comprising administering to the mammalian subject an immunogenic composition comprising an effective amount of OMVs, such as blebs and / or microvesicles, from PorA PorB RmpM LpxLT N. meningitidis, such as PorA PorB RmpM'LpxLTSiaD" N. meningitidis, and a pharmaceutically acceptable carrier. The N. meningitidis can be serogroup A, B or C, or any other serogroup (W, Y, etc.). The N. gonorrhoeae can be of any serotype.

[0104] The methods of the invention provide for administration of one or more antigenic compositions to a mammalian subject (e.g., a human) to elicit an immune response. The immune response can be against more than one strain of Neisseria species bacteria, and thus protection against disease caused by such bacteria.

[0105] The disclosed methods can provide for an immunoprotective immune response against a 1, 2, 3, 4, 5 or more strains of N. meningitidis species, where the strains differ in at least one of serogroup, serotype, or serosubtype. See U.S. Published Patent Application No. US 20170065699, incorporated herein by reference, which discloses methods for immunizing against multiple strains, such as 2, 3, 4, 5, or more strains.

[0106] Optionally, the immunogenic composition can include an adjuvant. Suitable adjuvants are disclosed above.

[0107] The immunogenic composition can be administered by any route. This includes via injection for the intramuscular, intraperitoneal, intradermal, or subcutaneous routes; or via mucosal administration to the oral / alimentary, respiratory (e.g., intranasal administration), genitourinary tracts. Although the immunogenic composition can be administered as a single dose, additional doses can be co-administered together at the same time or at different times. In addition to a single route of administration, two or more different routes of administration can be used.

[0108] Administration can be accomplished by single or multiple doses. 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 infection. The dose required will 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 one of ordinary skill in the art using only routine experimentation.

[0109] In some aspects, an immunogenic composition can be administered orally, nasally, nasopharyngeally, parenterally, enterically, gastrically, topically, transdermally, subcutaneously, intramuscularly, in tablet, solid, powdered, liquid, aerosol form, locally or systemically, with or without added excipients. Actual methods for preparing parenterally administrable compositions are described in such publications as Remington: The Science and Practice of Pharmacy, 22nded., London, UK: Pharmaceutical Press, 2013.

[0110] For oral administration, the compositions may need to be protected from digestion. This is typically accomplished either by association of the composition with an agent that renders it resistant to acidic and enzymatic hydrolysis or by packaging the composition in an appropriately resistant carrier. Means of protecting from digestion are well known in the art.

[0111] The immunogenic compositions are administered to a mammalian subject, such as a human, that has or is at risk for acquiring a Neisseria infection, to prevent or at least partially arrest the development of disease and its complications. An amount adequate to accomplish this is defined as an “effective dose” or an “immunogenically effective amount.” Amounts effective for use will depend on, e.g., the antigenic composition, the manner of administration, the weight and general state of health of the subject, and the judgment of the prescribing physician. Single or multiple doses of the antigenic compositions may be administered depending on the dosage and frequency required and tolerated by the mammalian subject, and route of administration. A prime boost strategy can be utilized.

[0112] The amount of OMVs included in the immunogenic composition is sufficient to elicit an immune response, such as a humoral immune response and / or a cellular immune response, in the subject. Amounts for the immunization of the mixture generally range from about 0.001 mg to about 1.0 milligram (mg) per 70 kilogram (kg) patient, more commonly from about 0.001 mg to about 0.2 mg per 70 kg patient. Dosages from 0.001 up to about 10 mg per patient per day may be used, particularly when the OMV are administered to a secluded site and not into the bloodstream, such as into a body cavity or into a lumen of an organ. Substantially higher dosages (e.g. 10 to 100 mg or more) are possible in oral, nasal, or topical administration. The initial administration of the mixture can be followed by booster immunization of the same of different mixture, with at least one booster, such as two boosters.

[0113] In some aspects, administration is initiated prior to the first sign of disease symptoms, or at the first sign of possible or actual exposure to pathogenic Neisseria. Without being bound by theory, immunoprotective antibodies for N. meningitidis and / or N. gonorrhoeae can be generated by immunization with an immunogenic composition.

[0114] Immunoprotective antibodies for N. meningitidis and / or N. gonorrhoeae can be administered to an individual (e.g., a human patient) to provide for passive immunity, either to prevent infection or disease from occurring, or as a therapy to improve the clinical outcome in patients with established disease (e.g. decreased complication rate such as shock, decreased mortality rate, or decreased morbidity, such as deafness). Antibodies administered to a subject that is of a species other than the species in which they are raised are often immunogenic. Thus, for example, murine or porcine antibodies administered to a human often induce an immunologic response against the antibody. The immunogenic properties of the antibody are reduced by altering portions, or all, of the antibody into characteristically human sequences thereby producing chimeric or human antibodies, respectively.

[0115] Chimeric antibodies are immunoglobulin molecules comprising a human and non-human portion. More specifically, the antigen combining region (or variable region) of a humanized chimeric antibody is derived from a non-human source (e.g. murine), and the constant region of the chimeric antibody (which confers biological effector function to the immunoglobulin) is derived from a human source. The chimeric antibody should have the antigen binding specificity of the non-human antibody molecule and the effector function conferred by the human antibody molecule. A large number of methods of generating chimeric antibodies are well known to those of skill in the art (see, e.g., U.S. Pat. Nos. 5,502,167, 5,500,362, 5,491,088, 5,482,856, 5,472,693, 5,354,847, 5,292,867, 5,231,026, 5,204,244, 5,202,238, 5,169,939, 5,081,235, 5,075,431 and 4,975,369). An alternative approach is the generation of humanized antibodies by linking the CDR regions of non-human antibodies to human constant regions by recombinant DNA techniques. See Queen et al., Proc. Natl. Acad. Sci. USA 86: 10029-10033 (1989) and WO 90 / 07861.

[0116] Fully human antibodies are also of use. Human antibodies consist entirely of characteristically human polypeptide sequences. The human antibodies of this invention can be produced by a wide variety of methods (see, e.g., Larrick et al., U.S. Pat. No. 5,001,065). In one aspect, the human antibodies of the present invention are produced initially in trioma cells (descended from three cells, two human and one mouse). Genes encoding the antibodies are then cloned and expressed in other cells, particularly non-human mammalian cells. The general approach for producing human antibodies by trioma technology has been described by Ostberg et al. (1983), Hybridoma 2: 361-367, Ostberg, U.S. Pat. No. 4,634,664, and Engelman et al., U.S. Pat. No. 4,634,666. Triomas have been found to produce antibody more stably than ordinary hybridomas made from human cells.

[0117] Methods for producing and formulating antibodies suitable for administration to a subject (e.g., a human subject) are well known in the art. For example, antibodies can be provided in a pharmaceutical composition comprising an effective amount of an antibody and a pharmaceutical excipient (e.g., saline). The pharmaceutical composition may optionally include other additives (e.g., buffers, stabilizers, preservatives, and the like). An effective amount of antibody is generally an amount effective to provide for protection against Neisserial disease or symptoms for a desired period (e.g., a period of at least about 2 days to 10 days or 1 month to 2 months).

[0118] EXAMPLES

[0119] Manufacturing methods for OMV typically include: (1) culturing of the outbreak isolate in liquid media, (2) centrifugation to separate the bacterial wet mass, (3) incubation of the wet mass with a low concentration of detergent to extract the OMV from the bacterial membrane, (4) successive steps of centrifugation and ultracentrifugation to isolate the detergent-extracted OMV (eOMV), (5) incubation of eOMV with a high concentration of detergent to decrease the toxic levels of lipooligosaccharide (LOS) present on the eOMV surface, and (6) ultracentrifugation to purify the detergent-detoxified OMV (dOMV) (Fredriksen et al., NIPH Ann. 1991;14:67-80). Although meningococcal dOMV vaccines have been shown to be effective at eliciting protection against the specific outbreak strain from which the vaccine is derived, dOMV are less effective at preventing infection with heterologous MenB strains.

[0120] The low cross-protection afforded by MenB dOMV vaccines may be a result of induction of immunodominant antibody responses to the porin protein PorA, which is highly variable among meningococcal strains (Granoff et al., Clin Infect Dis. 2010;50(S2):S54-S65). Dominant antibody responses are also generated against an alternate porin protein, PorB, which is the major protein on the bacterial / dOMV surface. Like PorA, PorB is highly variable, and anti-PorB antibodies are expected to strongly cross-react only with strains that express the same PorB type as the vaccine strain (Matthias et al., Mol Microbiol. 2017;105:934-53). Despite immunodominant anti-PorA and anti-PorB responses, clinical studies suggest that antibody responses to other dOMV antigens can cross-react with heterologous strains and may confer some degree of partial protection against infection (Sierra et al., NIPH Ann. 1991; 14: 195- 210; Caron et al., Lancet Infect Dis. 2011;11:455-63). Likewise, recent retrospective case-control and observational cohort studies have suggested that vaccination with MenB dOMV may confer off-target protection to a low percentage of individuals against infection with N. gonorrhoeae (Petousis-Harris et al., Lancet. 2017;390:1603-10; Azze et al., Clin Exp Vaccine Res. 2019;8:110-5; Raccagni et al., Sex Transm Dis. 2023;50:247-51; Abara et al., Lancet. 2022;22:1021-9; Wang et al., Lancet. 2022;22: 1011-20; Bruxvoort et al., Clin Infect Dis. 2022;76:el341-e9), which is genetically closely related to N. meningitidis.

[0121] A MenB dOMV vaccine strain, AABR, that was genetically deleted for expression of PorA and PorB, as well as the structural protein RmpM, was produced (Matthias et al., Mol Microbiol. 2017;105:934- 53). Sera from AABR dOMV-immunized animals killed a greater number of heterologous strains in human complement serum bactericidal assays (hSBAs) relative to sera obtained from animals immunized with porin-sufficient wild-type (WT) dOMV (Matthias et al., Vaccine. 2020;38:2396-405). However, the titers of killing associated with the anti-AABR dOMV sera were overall lower compared to the anti-WT dOMV sera. When tested in a mouse model of vaginal gonococcal infection, both the WT dOMV and the AABR dOMV vaccines enhanced clearance of N. gonorrhoeae from the lower reproductive tract, though consistent clearance was only observed with AABR dOMV-immunized mice in two independent trials (Matthias et al., J Infect Dis. 2022;225:650-60). Both (1) the divergent patterns of killing in hSBAs conducted with anti-WT and anti-AABR dOMV vaccinated sera and (2) the divergent phenotypes of gonococcal clearance in the WT- and AABR dOMV-immunized mice were associated with induction of antibody responses to varying antigens as assessed in western blot analyses. These data suggested that the absence of PorA, PorB, and RmpM from the dOMV functioned to permit enhanced host responses to other surface antigens that are more cross-reactive than the deleted proteins. However, the antibody responses to the AABR dOMV appeared less potent than those of WT dOMV manufactured by comparable methods.

[0122] Detergent detoxification is a harsh chemical procedure that is necessary to remove sufficient levels of LOS from the OMV surface that would otherwise be toxic to humans upon vaccination. As an unintended consequence of detergent detoxification, immunogenic, cross-reactive proteins that are loosely associated with the bacterial outer membrane, such as FHbp, may also be removed from the OMV, thereby diminishing efficacy of the final vaccine product. LOS toxicity is mediated by binding of lipid A to Tolllike receptor 4 (TLR4), stimulating secretion of pro -inflammatory cytokines that can lead to necrosis and death (Zughaier et al., Infect Immun. 2004;72:371-8). Toxicity of lipid A is in part a function of both the number and the length of acyl chains present on the 0(1— >6) glucasominc disaccharidc backbone. Inactivation of the gene IpxLl prevents addition of a secondary lauroyl chain to the nonreducing end of the glucasominc disaccharide, transforming the native hexa-acylated structure into a penta-acylated structure (FIG. 1). As the penta-acylated structure is less toxic than the hexa-acylated, subsequent detergent detoxification is unnecessary and loosely associated cross-reactive antigens may be retained in the OMV.

[0123] Example 1 Engineering of Genetically-Detoxified Strains

[0124] To generate a genetically-detoxified AABR vaccine strain, markerless deletions of the porA, porB, rmpM, and IpxLl genes were engineered from the parental MenB strain MC58, in which only the genetic sequence from the native genes of interest were deleted from the genome. Benefits of the mutation approach included the lack of requirement of antibiotics for bacterial growth, which are necessary when genetic deletions are achieved by replacing the native gene with an antibiotic resistance marker (such as in the construction of the AABR strain, see PCT Publication No. WO 2019 / 018744, incorporated herein by reference). Correspondingly, subsequent removal of antibiotics from the final vaccine drug product was rendered unnecessary, streamlining the vaccine formulation process.

[0125] For engineering of the mutant strains, plasmid pJJ260 (Johnston, J. Gene 2012;492:325-328) was obtained. This plasmid contained a positive and a negative genetic selection marker. The full selection cassette (~3.5 kb) includes the tetR gene, encoding the Tet repressor, the sacB gene under the control of an inducible Ptetpromoter, and nptll, encoding an aminoglycoside phosphotransferase, which functions as a kanamycin-resistance gene (FIGS. 2A-2B). Kanamycin resistance was used to positively screen for colonies that integrated the full selection cassette and incorporated this cassette into the genome in place of the gene of interest. Subsequent removal of the cassette from the genome was negatively screened for by growth on plates containing sucrose. Under normal growth conditions, the Tet repressor binds to the operator sequence of the Ptetpromoter, inhibiting transcription and translation of the levansucrase encoded by the sacB gene. In contrast, when grown in the presence of chlortetracycline (cTc), cTc will bind to the Tet repressor, causing it to dissociate from the Ptetpromoter, permitting production of levansucrase, which cleaves sucrose into a product (levan) that is toxic to N. meningitidis. Clones that improperly retain the full selection cassette, including that sacB gene, are killed when grown on plates containing sucrose and cTc; clones that have lost the selection cassette from the genome will be able to grow.

[0126] For construction of the present mutants, approximately 1.0 to 1.5 kb of the 5’ and 3’ untranslated regions (UTRs) directly upstream and downstream of the gene to be deleted were PCR amplified from MC58 genomic DNA (gDNA) using primer pairs engineered to express different restriction enzyme cleavage sites. The 5’ and 3’ UTR PCR products were then cloned successively into plasmid pGEM-3Z (Promega) by restriction enzyme digest, followed by ligation, and transformation into Escherichia coli strain DH5a (FIG. 3A). Once proper insertion of the 5’ and 3’ UTRs of the intermediate plasmid, (pINT), was confirmed, the tetR-sacB-nptll selection cassette was amplified using primers engineered to express the same restriction enzyme sites present on the internal ends of the 5' and 3’ UTRs. The selection cassette and pINT were then digested and ligated together, creating the deletion plasmid (pDEL, FIG. 3B). Following transformation into DH5a and confirmation of the specificity of the pDEL sequence, pDEL was transformed into MC58, or the relevant MC58 strain bearing other gene deletions (i.e., the markerless AABR strain, AABRM, FIG. 3C). Growth on BHI plates containing kanamycin was used to screen for replacement of the native gene of interest by double homologous recombination with the tetR-sacB-nptll cassette. The tetR- sacB-nptll replacement strain was then transformed with pINT (FIG. 3C), and removal of the tetR-sacB- nptll cassette was confirmed by growth on BHI plates containing 10% sucrose and 50 pg / ml cTc.

[0127] The approach defined above was used to delete par A, rmpM, porB, and LpxLl in succession from the MC58 genome, creating strain AABRL. The MenB polysaccharide capsule is poorly immunogenic and may function to decrease immune recognition of cross-reactive subcapsular antigens. Thus, it was decided to apply the same technique to subsequently delete the capsular biosynthesis gene siaD from strain AABRL, creating strain AABRSL. As the capsule is a major MenB virulence determinant, deletion of the capsule from AABRSL had the additional benefit of increasing the safety profile of the vaccine strain for manufacturing purposes. To confirm proper gene deletion, whole genome sequencing (WGS) was conducted on strains AABRL and AABRSL. Strains were grown overnight on BHI agar plates, and genomic DNA was isolated using the Wizard Genomic DNA Purification Kit (Promega). Samples were suspended in DNase-free water and were sequenced by synthesis using the Illumina MiSeq system. Analysis of gene sequences was conducted against the reference MC58 genome sequence using the breseq computational method (Deatherage et al., Methods Mol Biol. 2014;1151:165-8). WGS confirmed deletion of porA, porB, rmpM, and IpxLl from strains AABRL and AABRSL; deletion of siaD was only noted in strain AABRSL as expected. To supplement the WGS results, immunoblots were conducted, probing whole cell lysates of AABRL and AABRSL with antibodies specific for PorA (MN14C11.6, NIBSC), PorB (8B5-5-G9, NIBSC), RmpM (Bl, Matthias et al., Vaccine. 2020;38:2396-405), the MenB polysaccharide capsule (SEAM12, NIBSC), and LOS (4BE12C10, NIBSC). PorA, PorB, and RmpM expression was not detected in strains AABRL and AABRSL (FIG. 4). Likewise, no capsule expression was observed in strain AABRSL, though the capsule was retained in strain AABRL. No changes in LOS expression were detectable in any strain using immunoblot, as antibody 4BE12C10 is specific for an epitope of LOS that is not dependent on lipid A acylation.

[0128] In order to ensure that IpxLl was deleted from strains AABRL and AABRSL, the WT and markerless mutant strains were cultured in TSB broth and centrifuged at 5000 rpm to obtain the bacterial wet mass. The wet mass was suspended in water and passed through a French press to lyse the bacterial cells, and OMV were isolated using centrifugation and ultracentrifugation techniques. Increasing concentrations (0.01 - 1000 ng) of the OMV were incubated with the HEK-BLUE HTLR4™ cell line (InvivoGen) as per the manufacturer’s instructions, where production of SEAP activity, as detected by changes in absorbance, functions as a readout of TLR4 stimulation. Both AABRL and AABRSL OMV elicited significantly less SEAP activity than OMV isolated from the WT strain or AABR strains that retained functional LpxLl (FIGS. 5A-5B). No difference in SEAP activity was observed among any OMV when the same OMV types were incubated with a comparable cell line that overexpressed TLR2 (HEK-BLUE HTLR2™, InvivoGen), highlighting the specificity of the IpxLl mutation on TLR4 stimulation.

[0129] Example 2 dOMV Vaccine Production and Immunogenicity Study

[0130] After confirming the markerless mutant strains were deleted for the appropriate genes, comparability of the immune response to the new OMV types vs. the previously characterized AABR dOMV (Matthias et al., Vaccine. 2020;38:2396-405; Matthias et al., J Infect Dis. 2022;225:650-60) was assessed. To permit a direct comparison, dOMV were first generated using the methods defined in (Fredriksen, NIPH Ann. 1991;14:67-80). Bacteria were cultured for 6-8 h in 250 ml Erlenmeyer flasks with shaking (130 rpm) at 37°C, then 150 ml of the original culture was used to inoculate 1.5 L in a 2 L Erlenmeyer flask for ~16 h with shaking (100 rpm) at 37°C. The wet mass was isolated by centrifugation at 5000 rpm and was suspended at a 5:1 v / w ratio in extraction buffer (100 mM Tris-HCl, 10 mM EDTA, 0.5% sodium deoxycholate, pH 8.6). After stirring at room temperature for 30 min, the solution was centrifuged at 13,000 x g and the supernatant transferred to ultracentrifuge tubes; the wet mass was resuspended at 3: 1 v / w ratio in extraction solution and the extraction / centrifugation process was repeated. The total supernatant from both extraction procedures was ultracentrifuged at 40,000 rpm for 4 h, at which time the pellet was resuspended in 1 ml of PBS and incubated with BENZONASE® (MilliporeSigma) for 30 min at 37°C as per the manufacturer’s instructions. The detergent-extracted OMV (eOMV) were then added to 40 ml of detoxification solution (50 mM Tris-HCl, 2 niM EDTA, 5% deoxycholate, 20% sucrose, pH 8.6) and were incubated for ~ 16 h at 4°C. The following day, the now detoxified dOMV were incubated at room temperature with gentle rocking for 1 h, at which time the dOMV were ultracentrifuged at 40,000 rpm for 4 h. The supernatant was decanted and the pellet washed in 20 ml PBS, followed by ultracentrifugation at 40,000 rpm. After 4 h, PBS was decanted and the dOMV resuspended in 1 ml PBS. The concentration and endotoxin levels of the final product were then measured using the THERMO SCIENTIFIC™ PIERCE™ BCA Protein Assay Kit and Chromogenic Endotoxin Quant Kit, respectively.

[0131] To assess serological responses to the vaccines, two New Zealand white rabbits were immunized three times at one-month intervals with the original published AABR dOMV vaccine (made by replacing the native porA, porB, and rmpM genes with antibiotic resistance markers) or dOMV isolated from the new markerless AABR (designated AABRM) and AABRL strains (each admixed with IMJECT™ adjuvant (ThermoFisher) as per the manufacturer’s instructions). Two-weeks post-3rdimmunization, blood samples were collected and sera from each of the samples were tested in hSBAs against the parental MC58 strain and 16 heterologous MenB strains. Sera KM5 and KM6, obtained from the animals immunized with the AABRL dOMV, killed the greatest number of strains tested, 11 of 17 (FIGS. 6A-6D), suggesting that immune responses to dOMV isolated from the new markerless mutant vaccine strains are at least comparable to those isolated from AABR dOMV.

[0132] Example 3 Extended eOMV Study

[0133] In view of the comparability of host immune responses to AABR and AABRL dOMV, the impact of genetic detoxification on elicitation of functional antibody responses was examined. To accomplish this aim, the same methodology described above was utilized in the production of dOMV to generate genetically-detoxified AABRL and AABRSL vaccines, but instead of suspending the eOMV in detoxification solution following BENZONASE® treatment, they were suspended directly into 20 ml of PBS to wash the eOMV product. After ultracentrifugation, the eOMV were resuspended in 2 ml of PBS and were passed through a 0.2 pm filter to remove contaminants. Protein concentration was then quantified using the THERMO SCIENTIFIC™ PIERCE™ BCA Protein Assay Kit, and eOMV were characterized via electron microscopy (EM). EM imaging confirmed retention of the eOMV vesicular structure, which was in stark contrast to the almost complete degradation of the detergent-detoxified AABR dOMV product (FIG. 7).

[0134] Various vaccine formulations including WT dOMV, AABR dOMV, AABRL dOMV, AABRL eOMV, and AABRSL eOMV, were formulated with IMJECT™ adjuvant and two New Zealand white rabbits were immunized according to the protocol described above, except that blood samples were drawn one-month following the final vaccination for serum isolation. Testing of the sera against a panel of 18 MenB strains demonstrated increased cross-reactivity of anti-AABRL and anti-AABRSL eOMV antibodies relative to anti-AABR and anti-AABRL dOMV antibodies (FIG. 8); comparable or improved cross-reactivity was observed when anti-eOMV antibodies were compared to anti-WT dOMV antibodies. In a parallel study, the same dOMV and eOMV preparations were admixed at a 1:1 ratio with 2% ALHYDROGEL® adjuvant (InvivoGen) and were used to immunize ten mice three times at three-week intervals; control mice were immunized with 1% ALHYDROGEL® in PBS alone. Three-weeks post-3rdimmunization, mice were exsanguinated and corresponding serum samples tested in hSBAs against a panel of seven MenB strains. When assayed against the parental MC58 strain, sera from one (AABR dOMV group) to four (WT dOMV group) mice immunized with the various dOMV formulations were bactericidal at titers ranging from reciprocal values of 4 to 8 (FIG. 9A). In contrast, nine of ten serum samples from the AABRL and AABRSL eOMV -immunized mice killed MC58, with a higher geometric mean titer (GMT) observed for the AABRSL eOMV group compared to the others. Higher GMTs were also noted for the AABRSL eOMV group when tested against three heterologous MenB strains (M12885, M14290, and BB1350, FIG. 9B). Similar GMTs were observed for all vaccinated groups when tested against strains M09057, M17-240156, and Cu385 (FIG. 9C).

[0135] In an effort to gain an understanding of the rationale for the enhanced cross-reactivity afforded by vaccination with the eOMV vaccines, specifically the AABRSL eOMV, isotype-specific ELISAs using WT MC58 membrane preparations as a coating antigen were conducted. Similar serum IgG and IgA antibody profiles were observed for all vaccinated groups independent of immunogen (FIGS. 10A-10B). The mean scrum IgGl:IgG2a ratio for each group was also calculated at a value >2.0, suggestive of Th2 skewing (FIG. 10C), though elevated IgG2a levels in the WT dOMV group (FIG. 10B) resulted in a comparatively lower mean which was closer to the threshold associated with a mixed Thl / Th2 response (value between 0.5 and 2.0).

[0136] Despite measurement of similar antibody responses by ELISA, different immunogenic profiles were observed for the vaccine groups when pooled sera from each group were used to probe whole cell lysates of a panel of eleven MenB strains. Immunization with the WT dOMV elicited antibodies against an antigen of approximately 36 kDa (FIG. 11 A), which has been previously identified as RmpM (Matthias et al., Vaccine. 2020;38:2396-405); this band was absent as expected in immunoblots probed with sera from the animals immunized with the RmpM-deficient dOMV and eOMV. Two bands of approximate sizes 28 kDa and 32 kDa were observed in blots probed with pooled anti-AABRL and anti-AABRSL eOMV sera (FIG. 1 IB). The lack of these bands in any of the blots probed with sera from dOMV-immunized animals suggested that (1) detergent detoxification may have resulted in removal of the antigens from the dOMV during detergent treatment and (2) the antigens may exhibit cross-reactivity that contributed to the enhanced functional antibody responses observed in the hSBAs. The cross-reactivity of the anti-AABRSL eOMV antibodies was further demonstrated by their binding of multiple antigens expressed by six different N. gonorrhoeae strains in western blots (FIG. 12), highlighting the potential for cross-species protection afforded by the eOMV vaccines.

[0137] In addition to systemic humoral responses, all OMV vaccines elicited mucosal (i.e., vaginal) antibodies, with elevated total IgG and IgGl observed for the vaccinated groups relative to alum-immunized controls (FIG. 13A). In contrast, levels of IgG2a, IgG2b, and IgG3 varied depending on the vaccine type, with the WT dOMV group producing higher mean titers of all three isotypes relative to the other vaccinated groups (FIG. 13B). Notably, the AABRSL eOMV group alone exhibited significantly higher MC58-specific vaginal IgA levels vs. the alum control group when assessed via ELISA (FIG. 13A). Robust IgA responses were also observed when vaginal lavages from AABRSL eOMV-immunized mice were pooled and used to probe MenB lysates in immunoblots, with qualitatively higher antibody levels detected compared to the anti- WT dOMV pool (FIGS. 14A-14B). Similar banding profiles were observed for the AABRL eOMV group vs. the AABRSL eOMV group (FIG. 14B), suggesting that presentation of antigens in the eOMV enhanced elicitation of anti-MenB IgA responses.

[0138] Thus, deletion of the IpxLl gene from the markerless AABRL and AABRSL mutant vaccine strains permitted isolation of eOMV with sufficiently low toxicity to render detergent detoxification unnecessary. As shown in the western blot analyses, the requirement for only a low concentration of detergent for eOMV extraction permitted retention of multiple antigens that engaged the host immune response, generating antigen-specific antibodies that were either not observed upon immunization with the dOMV vaccines, or were observed at a lower level. These data suggest that (1) multiple antigens were loosely associated with the bacterial surface and were at least partially removed from the dOMV during detergent detoxification, and (2) the eOMV platform permits enhanced presentation of MenB antigens to the host immune system. Enhanced presentation may in part be due to the maintenance of vesicular structure, which was almost completely abrogated in the dOMV vaccines. In addition, complete elimination of the capsular polysaccharide from the MenB surface was shown to be beneficial in the elicitation of cross-reactive hSBA responses, as potency was enhanced for the AABRSL eOMV vs. AABRL eOMV vaccine. In addition to meningococcal antigens, anti-AABRSL eOMV antibodies were also demonstrated to cross-react with gonococcal antigens, highlighting the potential for MenB eOMV vaccines to protect against N. gonorrhoeae infection.

[0139] In view of the many possible aspects to which the principles of our invention may be applied, it should be recognized that illustrated aspects are only examples of the invention and should not be considered a limitation on the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

We claim:

1. An isolated PorA PorB RmpM LpxLl Neisseria (N.) meningitidis.

2. The isolated PorA'PorB'RmpM'LpxLT N. meningitidis of claim 1, wherein the PorA'PorB' RmpM'LpxLl' Neisseria meningitidis is also SiaD-.

3. The isolated PorA PorB RmpM LpxLl' N. meningitidis of claim 1 or claim 2, wherein the N. meningitidis is serogroup A, B or C.

4. An immunogenic composition comprising an effective amount of outer membrane microvesicles from the isolated PorA PorB RmpM LpxLl' N. meningitidis of any one of claims 1-3, and a pharmaceutically acceptable carrier.

5. The immunogenic composition of claim 4, wherein the outer membrane microvesicles are microvesicles, blebs, or a combination thereof.

6. The immunogenic composition of any one of claims 1-5, further comprising an adjuvant.

7. A method of inducing an immune response to Neisseria in a mammalian subject, comprising administering to the mammalian subject the immunogenic composition of any one of claims 4-6, thereby inducing the immune response.

8. The method of claim 7, wherein the immune response is a protective immune response.

9. The method of claim 8, wherein the protective immune response is for an N. meningitidis infection.

10. The method of claim 8, wherein the protective immune response is for an N. gonorrhoeae infection.

11. The method of claim 7, wherein the immune response is a therapeutic response.

12. The method of claim 7, wherein the subject has a N. meningitidis infection, and administration of the immunogenic composition increases clearance of the TV. meningitidis.

13. The method of claim 7, wherein the subject has a N. gonorrhoeae infection, and administration of the immunogenic composition increases clearance of N. gonorrhoeae .

14. The method of claim 7, wherein the mammalian subject is a healthy subject.

15. The method of any one of claims 7-14, wherein the mammalian subject is a human.

16. A method of inducing an immune response to Neisseria gonorrhoeae in a mammalian subject, comprising administering to the mammalian subject an immunogenic composition comprising an effective amount of outer membrane microvesicles from a PorA PorB RmpM LpxLl N. meningitidis and a pharmaceutically acceptable carrier, thereby inducing the immune response to Neisseria gonorrhoeae.

17. The method of claim 16, wherein the PorA PorB" N. meningitidis is SiaD '.

18. The method of claim 16 or 17, wherein the outer membrane microvesicles are micro vesicles, blebs, or a combination thereof.

19. The method of any one of claims 16-18, wherein the N. meningitidis is serogroup A, B or C.

20. The method of any one of claims 16-19, wherein the immunogenic composition further comprises an adjuvant.

21. The method any one of claims 16-20, wherein the immune response is a protective immune response.

22. The method of any one of claims 16-20, wherein the immune response is a therapeutic response.

23. The method of claim 22, wherein the subject has a N. gonorrhoeae infection, and administration of the immunogenic composition increases clearance of the N. gonorrhoeae.

24. The method of any one of claims 16-20, wherein the mammalian subject does not have an infection with N. gonorrhoeae or N. meningitidis.

25. The method of any one of claims 16-24, wherein the mammalian subject is a human.

26. The method of any one of claims 7-25, wherein the method includes antibodies that bind heterologous N. meningitidis strains in the mammalian subject.

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