Neisseria outer membrane vesicles and uses thereof

Abstract

The present invention features outer membrane vesicles (OMVs), such as native or detergent extracted OMVs from N. gonorrhoeae. For example, the compositions may include OMVs obtained from two or more different gonococcal strains, or OMVs containing different outer membrane proteins and / or lipooligosaccharides (LOS). The invention also provides for genetically modified gonococci as well as immunogenic compositions containing the outer membrane vesicles.

Description

[0001] PATENT

[0002] ATTORNEY DOCKET NO: 51838-002WO3

[0003] NEISSERIA OUTER MEMBRANE VESICLES AND USES THEREOF

[0004] BACKGROUND OF THE INVENTION

[0005] The Gram-negative bacteria Neisseria gonorrhoeae is a human pathogen that causes the sexually transmitted disease gonorrhoea. Neisseria gonorrhoeae infection is a considerable global health concern with an estimated incidence of more than 106 million cases per year worldwide. Gonorrhoea is the second most reported communicable disease in the US and its prevalence world-wide appears to be increasing. However, because asymptomatic infections are common (occurring in up to 80% of infected females and 40% of infected males) the true prevalence of N. gonorrhoeae is not fully understood. Left untreated, or if undiagnosed, N. gonorrhoeae infection can lead to serious consequences, such as endometritis, pelvic inflammatory disease, urogenital tract abscesses, adverse pregnancy outcomes, neonatal complications (including blindness) and infertility. The control of N. gonorrhoeae is largely based on antibiotic treatment. This approach is compromised by the rapid and continued emergence of antimicrobial resistance. N. gonorrhoeae has developed resistance to many antibiotics that were previously successful in treating the infection.

[0006] In order to circumvent the need for antibiotic treatment, efforts have been focused on the development of a gonococcal vaccine. However, no gonococcal-specific vaccine candidate has yet demonstrated a desired level clinical protection. Thus, a need exists for improved therapeutics to treat and prevent gonorrhea.

[0007] SUMMARY OF THE INVENTION

[0008] Disclosed herein are compositions containing outer membrane vesicles (OMVs), such as native or detergent extracted OMVs from N. gonorrhoeae. For example, the compositions may include OMVs from a plurality (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) of strains of N. gonorrhoeae.

[0009] In one aspect, the disclosure provides a composition that includes OMVs from a plurality (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) of strains of N. gonorrhoeae, wherein the composition includes a first OMV isolated from a first strain of N. gonorrhoeae and a second OMV from a second strain of N. gonorrhoeae that is different from the first strain.

[0010] In some embodiments, the first strain expresses a first major outer membrane protein (PorB) protein and the second strain expresses a second PorB protein having at most 95% (e.g. at most 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 69.6%, 65%, or less) sequence identity to the first PorB protein. In some embodiments, the second PorB protein has at most 75% sequence identity to the first PorB protein. In some embodiments, the first strain expresses a IB allele of PorB protein (PorB. IB) and the second strain expresses a IA allele of PorB protein (PorB.IA).

[0011] In some embodiments, the first strain expresses a first neisserial heparin binding antigen (NHBA) protein, and the second strain expresses a second NHBA protein having at most 99% (e.g. at most 99%, 98%, 97%, 96%, 95%, 94.9%, 94%, 93%, 92%, 91 %, 90%, 85%, 80%, 75%, 70%, 65%, or less) sequence identity to the first NHBA protein.

[0012] In some embodiments, the first strain expresses a first neisserial surface protein A (NspA) protein, and the second strain expresses a second NspA protein having at most 99.5% (e.g. at most PATENT

[0013] ATTORNEY DOCKET NO: 51838-002WO3

[0014] 99.5%, 99.4%, 99%, 98%, 97%, 96%, 95%, 94.9%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, or less) sequence identity to the first NspA protein. In some embodiments, the second NspA protein has at most 95% sequence identity to the first NspA protein.

[0015] In some embodiments, the first strain expresses a first transferrin binding protein B (TbpB) protein, and the second strain expresses a second TbpB protein having at most 95% (e.g. at most 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75.9%, 75%, 70%, 65%, or less) sequence identity to the first TbpB protein. In some embodiments, the second TbpB protein has at most 75% sequence identity to the first TbpB protein.

[0016] In some embodiments, the first strain expresses a first ferric enterobactin transporter (FetA) protein, and the second strain expresses a second FetA protein having at most 98% (e.g. at most 98%, 97.2%, 97%, 96%, 95%, 94.9%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, or less) sequence identity to the first FetA protein. In some embodiments, the second FetA protein has at most 95% sequence identity to the first FetA protein.

[0017] In some embodiments, the first strain expresses a first L-methionine binding lipoprotein (MetQ) protein, and the second strain expresses a second MetQ protein having at most 98% (e.g. at most 98%, 97.2%, 97%, 96%, 95%, 94.9%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, or less) sequence identity to the first MetQ protein. In some embodiments, the second MetQ protein has at most 95% sequence identity to the first MetQ protein.

[0018] In some embodiments, the first strain expresses a first p-barrel assembly machinery A (BamA) protein, and the second strain expresses a second BamA protein having at most 98% (e.g. at most 98%, 97.2%, 97%, 96%, 95%, 94.9%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, or less) sequence identity to the first BamA protein. In some embodiments, the second BamA protein has at most 95% sequence identity to the first BamA protein.

[0019] In some embodiments, the first strain expresses a first translocator assembly module A (TamA) protein, and the second strain expresses a second TamA protein having at most 98% (e.g. at most 98%, 97.2%, 97%, 96%, 95%, 94.9%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, or less) sequence identity to the first TamA protein. In some embodiments, the second TamA protein has at most 95% sequence identity to the first TamA protein.

[0020] In some embodiments, the first strain expresses a first lipopolysaccharide transport protein D (LptD) protein, and the second strain expresses a second LptD protein having at most 98% (e.g. at most 98%, 97.2%, 97%, 96%, 95%, 94.9%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, or less) sequence identity to the first LptD protein. In some embodiments, the second LptD protein has at most 95% sequence identity to the first LptD protein.

[0021] In some embodiments, the first strain expresses a first opacity (Opa) family protein, and the second strain expresses a second Opa family protein having at most 98% (e.g. at most 98%, 97.2%, 97%, 96%, 95%, 94.9%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, or less) sequence identity to the first Opa family protein. In some embodiments, the second Opa family protein has at most 95% sequence identity to the first Opa family protein.

[0022] In some embodiments, the composition further includes a recombinant protein (e.g., a surface protein from an OMV). In some embodiments, an endogenous copy of the recombinant protein is typically PATENT

[0023] ATTORNEY DOCKET NO: 51838-002WO3 expressed on OMVs but exhibits low (e.g., at least 10% less expression, e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, or 99% less expression than a reference surface protein) or undetectable levels of expression or is lost during preparation of the OMV. In some embodiments, the recombinant protein is TbpB. In some embodiments, an endogenous copy of the recombinant protein is typically expressed on OMVs, and the recombinant protein is modified (e.g., a mutant or a biologically active fragment). In some embodiments, the recombinant protein is NHBA. In some embodiments, the recombinant protein is NspA. In some embodiments, the recombinant protein is FetA. In some embodiments, the recombinant protein is MetQ. In some embodiments, the recombinant protein is BamA. In some embodiments, the recombinant protein is TamA. In some embodiments, the recombinant protein is LptD. In some embodiments, the recombinant protein is an Opa protein.

[0024] In some embodiments, the first strain and / or the second strain includes a genetic modification that reduces or abolishes expression and / or function of a lipid A biosynthesis lauroyl acyltransferase (IpxLI ) gene, mRNA, and / or polypeptide. In some embodiments, each of the strains includes a genetic modification that reduces or abolishes expression and / or function of IpxLI . In some embodiments, the genetic modification that reduces or abolishes expression and / or function of IpxLI is a mutation of IpxLI . In some embodiments, the genetic modification that reduces or abolishes expression and / or function of IpxLI suppresses IpxLI in a strain that expresses wild-type IpxLI . In some embodiments, the genetic modification that reduces or abolishes expression and / or function of IpxLI includes a point mutation, a knock-out, or a mutation to a promoter.

[0025] In some embodiments, the first strain and / or the second strain includes a genetic modification that reduces or abolishes expression and / or function of a reduction modifiable protein (rmpM) gene, mRNA, and / or polypeptide. In some embodiments, each of the strains includes a genetic modification that reduces or abolishes expression and / or function of rmpM. In some embodiments, the genetic modification that reduces or abolishes expression and / or function of rmpM is a mutation of rmpM. In some embodiments, the genetic modification that reduces or abolishes expression and / or function of rmpM suppresses rmpM in a strain that expresses wild-type rmpM. In some embodiments, the genetic modification that reduces or abolishes expression and / or function of rmpM includes a point mutation, a knock-out, or a mutation to a promoter.

[0026] In some embodiments, the composition further includes a third OMV from a third strain of N. gonorrhoeae that is different from both the first strain and the second strain.

[0027] In some embodiments, the first strain expresses a first PorB protein, the second strain expresses a second PorB protein, and the third strain expresses a third PorB, wherein the third PorB protein has at most 95% (e.g. at most 95%, 94%, 93%, 92%, 91 %, 90%, 85%, 80%, 75%, 70%, 69.6%, 65%, or less) sequence identity to the first and / or the second PorB protein.

[0028] In some embodiments, the third PorB protein has at most 75% sequence identity to the first and / or the second PorB protein. In some embodiments, the third strain expresses a PorB. IB, and the first strain and / or the second strain expresses a PorB.IA. In some embodiments, the third strain expresses a PorB.IA, and the first strain and / or the second strain expresses a PorB. IB.

[0029] In some embodiments, the first strain expresses a first NHBA protein, the second strain expresses a second NHBA protein, and the third strain expresses a third NHBA protein, wherein the third PATENT

[0030] ATTORNEY DOCKET NO: 51838-002WO3

[0031] NHBA protein has at most 99% (e.g. at most 99%, 98%, 97%, 96%, 95%, 94.9%, 94%, 93%, 92%, 91 %, 90%, 85%, 80%, 75%, 70%, 65%, or less) sequence identity to the first and / or the second NHBA protein.

[0032] In some embodiments, the first strain expresses a first NspA protein, the second strain expresses a second NspA protein, and the third strain expresses a third NspA protein, wherein the third NspA protein has at most 99.5% (e.g. at most 99.5%, 99.4%, 99%, 98%, 97%, 96%, 95%, 94.9%, 94%, 93%, 92%, 91 %, 90%, 85%, 80%, 75%, 70%, 65%, or less) sequence identity to the first and / or the second NspA protein. In some embodiments, the third NspA protein has at most 95% sequence identity to the first and / or the second NspA protein.

[0033] In some embodiments, the first strain expresses a first TbpB protein, the second strain expresses a second TbpB protein, and the third strain expresses a third TbpB, wherein the third TbpB protein has at most 95% (e.g. at most 95%, 94%, 93%, 92%, 91 %, 90%, 85%, 80%, 75.9%, 75%, 70%, 65%, or less) sequence identity to the first and / or the second TbpB protein. In some embodiments, the third TbpB protein has at most 75% sequence identity to the first and / or the second TbpB protein.

[0034] In some embodiments, the first strain expresses a first FetA protein, the second strain expresses a second FetA protein, and the third strain expresses a third FetA, wherein the third FetA protein has at most 98% (e.g. at most 98%, 97.2%, 97%, 96%, 95%, 94.9%, 94%, 93%, 92%, 91 %, 90%, 85%, 80%, 75%, 70%, 65%, or less) sequence identity to the first and / or the second FetA protein. In some embodiments, the third FetA protein has at most 95% sequence identity to the first and / or the second FetA protein.

[0035] In some embodiments, the third strain includes a genetic modification that reduces or abolishes expression and / or function of rmpM. In some embodiments, the genetic modification that reduces or abolishes expression and / or function of rmpM in the third strain is a mutation of rmpM. In some embodiments, the genetic modification that reduces or abolishes expression and / or function of rmpM in the third strain suppresses rmpM in a strain that expresses wild-type rmpM. In some embodiments, the genetic modification that reduces or abolishes expression and / or function of rmpM in the third strain includes a point mutation, a knock-out, or a mutation to a promoter.

[0036] In some embodiments, the third strain includes a genetic modification that reduces or abolishes expression and / or function of IpxLI . In some embodiments, the genetic modification that reduces or abolishes expression and / or function of IpxLI in the third strain is a mutation of IpxLI . In some embodiments, the genetic modification that reduces or abolishes expression and / or function of IpxLI in the third strain suppresses IpxLI in a strain that expresses wild-type IpxLI . In some embodiments, the genetic modification that reduces or abolishes expression and / or function of IpxLI in the third strain includes a point mutation, a knock-out, or a mutation to a promoter.

[0037] In some embodiments, at least one (e.g., one, two, three, or more) OMV was extracted using detergent.

[0038] In some embodiments, all OMVs were extracted using detergent.

[0039] In some embodiments, at least one (e.g., one, two, three, or more) OMV was not extracted using detergent.

[0040] In some embodiments, all OMVs were not extracted using detergent.

[0041] In some embodiments, at least one (e.g., one, two, three, or more) OMV is a native OMV. PATENT

[0042] ATTORNEY DOCKET NO: 51838-002WO3

[0043] In some embodiments, all OMVs are native OMVs.

[0044] In some embodiments, at least one (e.g., one, two, three, or more) OMV was naturally secreted.

[0045] In some embodiments, all OMVs are naturally secreted.

[0046] In some embodiments, the composition further includes an adjuvant. In some embodiments, the adjuvant is alum, a Toll-like receptor 4 (TLR4) agonist, a TLR9 agonist, or saponin.

[0047] In another aspect, the invention provides a pharmaceutical composition including the composition of any one of the aforementioned aspects or embodiments and a pharmaceutically acceptable carrier.

[0048] In another aspect, the invention provides an immunogenic composition that includes the composition of any one of the aforementioned aspects or embodiments or the pharmaceutical composition of the aforementioned embodiment.

[0049] In another aspect, the invention provides a method of treating or preventing a N. gonorrhoeae infection in a subject, the method including administering to the subject a composition, pharmaceutical composition, or immunogenic composition as described herein.

[0050] In another aspect, the invention provides a method of producing an immune response in a subject by administering to the subject a composition, pharmaceutical composition, immunogenic composition as described herein.

[0051] In some embodiments, the method includes administering a plurality (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) of doses of the composition.

[0052] In some embodiments, the method provides protection against N. gonorrhoeae for at least one year (e.g. one, two, three, four, five, ten, fifteen, twenty, or more years).

[0053] In some embodiments, the composition provides resistance to at least 80% (e.g. 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) of genetically characterized N. gonorrhoeae strains.

[0054] In some embodiments, the composition is administered to a population of subjects and reduces the risk of infection of N. gonorrhoeae in the population of subjects by at least 50% (e.g. 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more).

[0055] In another aspect, the invention provides a composition that includes a cell from a first strain of N. gonnorhoeae and a cell from a second strain of N. gonnorhoeae, wherein the second strain is different from the first strain. In some embodiments, the composition further includes a cell from a third strain of N. gonnorhoeae, wherein the third strain is different from both the first strain and the second strain.

[0056] In some embodiments, the composition includes OMVs from a plurality of strains of N. gonorrhoeae.

[0057] In some embodiments, at least one of the OMVs includes a sialic acid moiety.

[0058] In another aspect, the invention provides a composition including a first OMV isolated from a first strain of N. gonorrhoeae that was incubated with sialic acid or a precursor of sialic acid.

[0059] In some embodiments, the first OMV is a native OMV.

[0060] In some embodiments, the composition further includes a second OMV isolated from a second strain of N. gonorrhoeae that was incubated with sialic acid or a precursor of sialic acid.

[0061] In some embodiments, the second OMV is a native OMV. PATENT

[0062] ATTORNEY DOCKET NO: 51838-002WO3

[0063] In some embodiments, the second strain of N. gonorrhoeae includes a genetic modification that reduces or abolishes expression and / or function of a rmpM gene, mRNA, and / or polypeptide.

[0064] In some embodiments, the first strain of N. gonorrhoeae includes a genetic modification that reduces or abolishes expression and / or function of a rmpM gene, mRNA, and / or polypeptide.

[0065] In some embodiments, the composition further includes a third OMV from a third strain of N. gonorrhoeae.

[0066] In some embodiments, the third OMV is a detergent-extracted OMV.

[0067] In some embodiments, the third OMV includes PorB.IA, PorB.IB, translocator assembly module A (TamA), lipopolysaccharide transport protein D (LptD), p-barrel assembly machinery A (BamA), NspA, FetA, or opacity (Opa) proteins.

[0068] In some embodiments, the third OMV includes PorB.IA, PorB.IB, LptD, BamA, or NspA.

[0069] In some embodiments, the third strain of N. gonorrhoeae is different from the first strain of N. gonorrhoeae.

[0070] In some embodiments, the third strain of N. gonorrhoeae is different from the second strain of N. gonorrhoeae.

[0071] In some embodiments, the second strain of N. gonorrhoeae is different from the first strain of N. gonorrhoeae.

[0072] In some embodiments, the third strain of N. gonorrhoeae is the same strain as the first strain of N. gonorrhoeae.

[0073] In some embodiments, the third strain of N. gonorrhoeae is the same strain as the second strain of N. gonorrhoeae.

[0074] In some embodiments, the composition further includes OMVs from a plurality of strains of N. gonorrhoeae.

[0075] In another aspect, the invention provides a composition including OMVs from N. gonorrhoeae, wherein the composition includes a first OMV isolated from a first strain of N. gonorrhoeae and a second OMV from a second strain of N. gonorrhoeae, wherein the first OMV is a native OMV and the second OMV is a detergent-extracted OMV.

[0076] In some embodiments, the first strain of N. gonorrhoeae was incubated with sialic acid or a precursor of sialic acid.

[0077] In some embodiments, the first OMV includes 2HexG+, 3HexG+, 4HexG+, 4HexG-, 5HexG+, and / or 5HexG- lipooligosaccharides (LOS) on a surface of the OMV.

[0078] 2Hex, 3Hex, 4Hex, or 5Hex refer to a structure in which there are two, three, four or five hexose molecules attached to a terminal heptose I (Hep I), respectively. G+ or G- refer to the presence or absence of galactose at the end of oligosaccharide chain of terminal heptose II (Hep II) or that the oligosaccharide chain is truncated to a glucose.

[0079] In some embodiments, expression of NHBA, MetQ, TbpB, and / or Pilin proteins on a surface of the first OMV is reduced following extraction of the first OMV.

[0080] In some embodiments, the second OMV includes PorB.IA, PorB.IB, TamA, LptD, BamA, NspA, FetA, and / or Opa proteins on a surface of the OMV. PATENT

[0081] ATTORNEY DOCKET NO: 51838-002WO3

[0082] In some embodiments, expression of PorB.IA, PorB.IB, TamA, LptD, BamA, NspA, FetA, and / or Opa proteins on a surface of the second OMV is increased following the detergent extraction of the second OMV.

[0083] In some embodiments, expression of NHBA, MetQ, TbpB, and / or Pilin proteins on a surface of the second OMV is reduced following detergent extraction of the second OMV.

[0084] In some embodiments, accumulation of LOS on a surface of the second OMV is significantly reduced following detergent extraction of the second OMV.

[0085] In some embodiments, the second OMV includes PorB.IA, PorB.IB, LptD, BamA, or NspA.

[0086] In some embodiments, the composition further includes a third OMV isolated from a third strain of N. gonorrhoeae that was incubated with sialic acid or a precursor of sialic acid.

[0087] In some embodiments, the third OMV is a native OMV.

[0088] In some embodiments, the third strain of N. gonorrhoeae includes a genetic modification that reduces or abolishes expression and / or function of a rmpM gene, mRNA, and / or polypeptide.

[0089] In some embodiments, the third strain of N. gonorrhoeae is different from the first strain of N. gonorrhoeae.

[0090] In some embodiments, the third strain of N. gonorrhoeae is different from the second strain of N. gonorrhoeae.

[0091] In some embodiments, the second strain of N. gonorrhoeae is different from the first strain of N. gonorrhoeae.

[0092] In some embodiments, the second strain of N. gonorrhoeae is the same strain as the first strain of N. gonorrhoeae.

[0093] In some embodiments, third strain of N. gonorrhoeae is the same strain as the second strain of N. gonorrhoeae.

[0094] In some embodiments, the first strain of N. gonorrhoeae includes a genetic modification that reduces or abolishes expression and / or function of a rmpM gene, mRNA, and / or polypeptide.

[0095] In some embodiments, the composition further includes a recombinant protein.

[0096] In some embodiments, the recombinant protein is NHBA, L-methionine binding lipoprotein (MetQ), BamA, TamA, LptD, TbpB, FetA, NspA, Opa, or Pilin.

[0097] In some embodiments, the recombinant protein is NHBA, MetQ, BamA, TamA, LptD, or TbpB.

[0098] In some embodiments, the recombinant protein is NHBA, MetQ, or TbpB.

[0099] In some embodiments, the recombinant protein is TbpB.

[0100] In some embodiments, the recombinant protein is NHBA.

[0101] In some embodiments, the composition further includes an adjuvant.

[0102] In some embodiments, the adjuvant is alum, a TLR4 agonist, a TLR9 agonist, or saponin.

[0103] In some embodiments, the adjuvant is alum.

[0104] In another aspect, the invention provides a method of making an isolated OMV from a first strain of N. gonorrhoeae, the method including: (a) incubating a N. gonorrhoeae with sialic acid or a precursor of sialic acid; and (b) isolating an OMV from the N. gonorrhoeae.

[0105] In some embodiments, isolating the OMV includes native extraction of the OMV. PATENT

[0106] ATTORNEY DOCKET NO: 51838-002WO3

[0107] In some embodiments, the native extraction includes centrifuging a culture of N. gonorrhoeae and collecting a first supernatant.

[0108] In some embodiments, the culture is centrifuged at about 1000 x g for about 5 minutes.

[0109] In some embodiments, the native extraction further includes centrifuging the first supernatant and collecting a second supernatant.

[0110] In some embodiments, the culture is centrifuged at about 5000 x g for about 10 minutes.

[0111] In some embodiments, the native extraction further includes filtering the second supernatant.

[0112] In some embodiments, the second supernatant is filtered using an about 0.2 pm filter.

[0113] In some embodiments, the native extraction further includes tangential flow filtration, multimodal chromatography, and / or concentration.

[0114] In some embodiments, the N. gonorrhoeae includes a genetic modification that reduces or abolishes expression and / or function of a rmpM gene, mRNA, and / or polypeptide.

[0115] In another aspect, the invention provides an OMV from N. gonorrhoeae produced by the method of one of the preceding embodiments.

[0116] In another aspect, the invention provides an engineered OMV from N. gonorrhoeae including a sialic acid moiety.

[0117] In some embodiments, at least 50% of LOS on a surface of the OMV are sialylated.

[0118] In some embodiments, the sialic acid moiety is on a 2HexG+, 3HexG+, 4HexG+, 4HexG-, 5HexG+, and / or 5HexG- LOS.

[0119] In some embodiments, the N. gonorrhoeae includes a genetic modification that reduces or abolishes expression and / or function of a rmpM gene, mRNA, and / or polypeptide.

[0120] In some embodiments, the OMV is isolated.

[0121] In some embodiments, the OMV is a native OMV.

[0122] In another aspect, the invention provides a method of identifying a first candidate strain of N. gonorrhoeae as suitable for producing an immunogenic OMV, the method including: (a) culturing the first candidate strain of N. gonorrhoeae in the presence of sialic acid or a sialic acid precursor; (b) collecting a first OMV from the N. gonorrhoeae; (c) inoculating a mammal with the OMV; (d) collecting serum from the mammal; (e) performing an assay to determine an activity of the first OMV on a test strain of N. gonorrhoeae; and (f) identifying the candidate strain as suitable for producing an immunogenic OMV if the activity of the isolated first OMV on the test strain exceeds a threshold.

[0123] In some embodiments, the method further includes: (i) culturing a second candidate strain of N. gonorrhoeae in the absence of sialic acid or a sialic acid precursor; and (ii) collecting a second OMV from the second candidate strain of N. gonorrhoeae, wherein the mammal is also inoculated with the second OMV.

[0124] In some embodiments, collecting the second OMV includes isolating the second OMV.

[0125] In some embodiments, isolating the second OMV includes isolating an OMV by a detergent extraction.

[0126] In some embodiments, the method further includes repeating steps (e) and (f) with a plurality of test strains of N. gonorrhoeae.

[0127] In some embodiments, at least 5, 10, 15, or 17 test strains of N. gonorrhoeae are tested. PATENT

[0128] ATTORNEY DOCKET NO: 51838-002WO3

[0129] In some embodiments, the candidate strains are identified as suitable for producing an immunogenic OMV if the activity of the isolated OMVs on the test strain exceeds an activity threshold for a threshold percent of strains.

[0130] In some embodiments, the first candidate strain and the second candidate strain are identified as suitable for producing an immunogenic OMV if the activity of the first OMV and the second OMV on the test strain exceeds an activity threshold for a threshold percent of strains.

[0131] In some embodiments, the threshold percent of strains is at least 50%.

[0132] In some embodiments, the second candidate strain of N. gonorrhoeae includes a genetic modification that reduces or abolishes expression and / or function of a rmpM gene, mRNA, and / or polypeptide.

[0133] In some embodiments, collecting the first OMV includes isolating the first OMV.

[0134] In some embodiments, isolating the first OMV includes isolating a native OMV.

[0135] In some embodiments, the test strain of N. gonorrhoeae was incubated with sialic acid or a sialic acid precursor prior to step (e).

[0136] In some embodiments, the method further includes repeating steps (e) and (f) with a plurality of test strains of N. gonorrhoeae.

[0137] In some embodiments, at least 5, 10, 15, or 17 test strains of N. gonorrhoeae are tested.

[0138] In some embodiments, the first candidate strain is identified as suitable for producing an immunogenic OMV if the activity of the isolated first OMV on the test strain exceeds an activity threshold for a threshold percent of strains.

[0139] In some embodiments, the first candidate strain is identified as suitable for producing an immunogenic OMV if the activity of the isolated first OMV on the test strain exceeds an activity threshold for a threshold percent of strains.

[0140] In some embodiments, the threshold percent of strains is at least 50% (e.g., at least 60%, 70%, 80%, 90%, 95%, 97%, 99%, or more).

[0141] In some embodiments, the assay includes mixing the serum with N. gonorrhoeae of the test strain and complement protein and determining a titer of the serum required to kill a threshold percent of bacteria of the test strain.

[0142] In some embodiments, the candidate strain is identified as suitable for producing an immunogenic OMV if the titer is below a threshold titer.

[0143] In some embodiments, the first candidate strain of N. gonorrhoeae includes a genetic modification that reduces or abolishes expression and / or function of a rmpM gene, mRNA, and / or polypeptide.

[0144] Definitions

[0145] As used herein, “adjuvant” means a compound or substance (or combination of compounds or substances) that, when administered to a subject in conjunction with an antigen or antigens, for example as part of an immunogenic composition, increases or enhances the subject's immune response to the administered antigen or antigens (compared to the immune response obtained in the absence of adjuvant). With respect to the present disclosure an adjuvant administered to subject in conjunction with PATENT

[0146] ATTORNEY DOCKET NO: 51838-002WO3 outer membrane vesicles increases or enhances the subject's immune response to antigen or antigens present in the surface of the OMVs. An adjuvant may be administered to a subject before, in combination with, or after administration of an immunogenic composition (e.g., a vaccine). Examples of chemical compounds used as adjuvants include, but are not limited to, aluminum compounds (e.g., alum, aluminum hydroxide, Alhydrogel), oils, block polymers, immune stimulating complexes, vitamins and minerals (e.g., vitamin E, vitamin A, selenium, and vitamin B12), Quil A (saponins), bacterial and fungal cell wall components (e.g., lipopolysaccarides, lipoproteins, and glycoproteins), hormones, cytokines, and co-stimulatory factors.

[0147] As used herein the term “genetic modification(s)” means any alteration to the constitution, structure, or operation of the genetic material in a cell to provide a specified effect (e.g. decreasing or abolishing expression). The skilled person is aware of numerous means to decrease or abolish gene and / or protein expression in comparison to that of a non-modified (e.g. naturally occurring bacterium) or a bacterium including the wild type gene of interest. The genetic material within a cell relates to either DNA or RNA. As such, the term genetic modification as used herein, means any artificial alteration to the constitution, structure or operation of either gonococcal DNA or RNA such as to decrease and / or abolish expression and / or function of the specified genes. A genetic modification may be, for example, a point mutation to a gene, a knock-out of a gene, or, or a mutation to a promoter As used herein, “genetically modified” with regards to gonococcal bacterium refers to a gonococcus that has had its genetic material artificially altered. Genetically modified gonococcal bacteria do not include wild type gonococcal bacteria. A genetically modified gonococcal bacterium includes for example a gonococcal bacterium into which an exogenous polynucleotide has been introduced. A genetically modified gonococcal bacterium also refers to a bacterium that has been genetically manipulated such that endogenous nucleotides have been altered to include a mutation, such as a deletion, an insertion, a substitution or a combination thereof. For instance, an endogenous coding and / or non-coding region could be deleted or replaced. Such genetic modifications may result either in depleted and / or abolished expression of a polypeptide and / or may result in a polypeptide having a different amino acid sequence than was encoded by the endogenous polynucleotide. Another example of a genetically modified gonococcal bacterium is one having an altered regulatory sequence, such as a promotor, to result in increased or decreased expression of an operably linked endogenous coding region.

[0148] As used herein the term “gene deletion” or “gene knockout” refers to a combination of genetic techniques that has the potential to render a specific gene inoperable or inactive. In some embodiments a gene deletion decreases or abolishes expression of a polypeptide from the gene. In some embodiments both the mRNA and protein are reduced or eliminated. In certain embodiments the expression of gene is substantially decreased or abolished. Substantially decreased means that the expression of a gene is reduced by at least 70%, at least 80%, at least 90%, at least 95% or at least 98% when compared to an endogenous level of expression of a gene. In a certain embodiment the expression of a gene is abolished. Abolished means that, using techniques to monitor the expression of either the mRNA transcribed from a gene, or the expression of protein translated from a particular mRNA, no level of detection is observed. Expression of a gene can be determined by a suitable technique (e.g., by measuring transcript levels by RT / Q-PCR or expressed protein levels by immunoassay e.g. Western PATENT

[0149] ATTORNEY DOCKET NO: 51838-002WO3

[0150] Blot). Such techniques are known to the person skilled in the art. Gene deletion or gene knockout, might include not only deletion of genetic elements but also addition, substitution or modification, such that the gene is inoperable or inactive, i.e. insertion of a genetic sequence may cause mistranslation of the gene, by for example, incorporating an early stop codon, or by causing a missense translation. Genes may for example be deleted by replacement of the gene, or a fragment of the gene, with a different heterologous gene (e.g. an antibiotic resistance gene) for example by homologous recombination.

[0151] By “immunogenic” is meant any substance that is capable of inducing an immune response in a subject.

[0152] As used herein the term “immunogenic composition” relates to a composition of matter suitable for administration to a human or animal subject (e.g., in an experimental or clinical setting) that is capable of eliciting a specific immune response, e.g., against a pathogen, such as Neisseria. As such, an immunogenic composition includes one or more antigens (for example, polypeptide antigens) or antigenic epitopes. An immunogenic composition can also include one or more additional components capable of eliciting or enhancing an immune response, such as an excipient, carrier, and / or adjuvant. In certain instances, immunogenic compositions are administered to elicit an immune response that protects the subject, wholly or partially, against symptoms or conditions induced by a pathogen. In the context of this disclosure, the term immunogenic composition will be understood to encompass compositions that are intended for administration to a subject or population of subjects for the purpose of eliciting a protective pre-exposure immune response against Neisseria or palliative post-exposure immune response against Neisseria.

[0153] As used herein the term “outer membrane vesicle(s)” or “OMV(s)” relates to proteoliposomic vesicles obtained by disruption of, or blebbing from the outer membrane of Gram-negative bacteria, to form vesicles therefrom that retains antigens from the outer membrane. Gram-negative bacteria naturally shed OMVs which are released into the growth medium. Heterologous antigens may be expressed in the Gram-negative bacteria such that they assemble in the membrane that is then released into the culture supernatant. OMVs from such bacteria are representative of the outer membrane and periplasmic bacterial compartments and allow the presentation of membrane proteins in their natural composition and structure. In the broadest sense, OMVs relates to any such proteoliposomic vesicles. However, the term OMVs includes native OMVs (nOMVs), detergent-extracted OMVs (dOMVs), and blebs, which are outer membrane protrusions that remain attached to bacteria prior to release as MVs.

[0154] The terms “pharmaceutically acceptable carrier” and “pharmaceutically acceptable excipient” are used interchangeably and mean a carrier or excipient that is physiologically acceptable to the treated subject while retaining the therapeutic properties of the compound with which it is administered. One exemplary pharmaceutically acceptable carrier substance is physiological saline. Other physiologically acceptable carriers and their formulations are known to those skilled in the art and described, for example, in Remington’s Pharmaceutical Sciences, (20th edition), ed. A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, PA.

[0155] By “pharmaceutical composition” is meant a composition containing a composition as described herein formulated with a pharmaceutically acceptable excipient and manufactured or sold with the approval of a governmental regulatory agency as part of a therapeutic regimen for the treatment or PATENT

[0156] ATTORNEY DOCKET NO: 51838-002WO3 prevention of a disease or event in a mammal. Pharmaceutical compositions can be formulated, for example, for intravenous administration (e.g., as a sterile solution free of particulate emboli and in a solvent system suitable for intravenous use), for oral administration (e.g., a tablet, capsule, caplet, gelcap, or syrup), or any other formulation described herein, e.g., in unit dosage form.

[0157] As used herein, the terms “percent (%) identity” or “percent sequence identity” refers to the percentage of amino acid residues of a candidate sequence, e.g., an OMV surface protein, that are identical to the amino acid residues of a reference sequence, e.g., an OMV surface protein from a second strain, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity (i.e., gaps can be introduced in one or both of the candidate and reference sequences for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). Alignment for purposes of determining percent identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. In some embodiments, the percent amino acid sequence identity of a given candidate sequence to, with, or against a given reference sequence (which can alternatively be phrased as a given candidate sequence that has or includes a certain percent amino acid sequence identity to, with, or against a given reference sequence) is calculated as follows:

[0158] 100 x (fraction of A / B) where A is the number of amino acid residues scored as identical in the alignment of the candidate sequence and the reference sequence, and where B is the total number of amino acid residues in the reference sequence. In some embodiments where the length of the candidate sequence does not equal to the length of the reference sequence, the percent amino acid sequence identity of the candidate sequence to the reference sequence would not equal to the percent amino acid sequence identity of the reference sequence to the candidate sequence.

[0159] As used herein the term “protect” in the context of infection, diseases or conditions caused by Neisseria (most particularly N. gonorrhoeae) means to protect via prophylaxis. Protection may relate to a reduction in the incidence of an infection, disease, or condition caused by Neisseria (in symptomatic and asymptomatic states) leading to the control of the disease and / or to the control of associated reproductive health adverse outcomes caused by Neisseria. Protection may lead to a reduction in the number of clinical visits. The term protect (or protection) may herein be used in relation to protection against the primary infection by Neisseria in terms of prevention of acute diseases (cervicitis and urethritis), reduction of the impact of anti- microbial resistance, gonococcal-related HIV acquisition and long-term reproductive complications occurring as a result of the infection. Protection may be achieved against disease causing gonococcal infections in different anatomical sites (urogenital, anorectal, oropharyngeal). As used herein the term “prevent” (or prevention) means that, as a result of increased protection, diseases or conditions caused by N gonorrhoeae are substantially averted resulting in improved population health outcomes.

[0160] A “subject” as used herein is an animal, preferably a mammal, including humans, non-human primates and non-primate mammals such as members of the rodent genus (including but not limited to PATENT

[0161] ATTORNEY DOCKET NO: 51838-002WO3 mice and rats), the Cavia genus (including but not limited to guinea pigs) and members of the order Lagomorpha (including but not limited to rabbits). As used herein, the subject is most preferably a human.

[0162] By “treating” or “treatment” is meant the medical management of a subject with the intent to cure, ameliorate, stabilize, reduce the likelihood of, or prevent a disease, pathological condition, disorder, or event, by administering a pharmaceutical composition. This term includes active treatment, that is, treatment directed specifically toward the improvement or associated with the cure of a disease, pathological condition, disorder, or event, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, disorder, or event. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, disorder, or event; symptomatic treatment, that is, treatment directed toward constitutional symptoms of the associated disease, pathological condition, disorder, or event; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, disorder, or event, e.g., in a subject who is not yet ill, but who is susceptible to, or otherwise at risk of, a particular disease, pathological condition, disorder, or event; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, disorder, or event.

[0163] By “vaccine,” as used herein, is meant a biological preparation that provides active acquired immunity to a particular infectious disease.

[0164] As used here, the term “sialic acid precursor” refers to a compound that can be incorporated within or converted to a sialic acid moiety or modified form thereof. For example, a sialic acid precursor can be a molecule that is conjugated to or incorporated within a lipooligosaccharide (LOS) to form a sialylated LOS. A sialic acid precursor can be a compound that can undergo one or more chemical reactions (e.g., in the presence of N. gonorrhoeae) to be incorporated within or conjugated to an LOS to form a sialylated LOS. An exemplary sialic acid precursor is cytidine Monophospho-N-Acetylneuraminic acid.

[0165] BRIEF DESCRIPTION OF THE DRAWINGS

[0166] FIG. 1A is a heatmap showing serum bactericidal activity (SBA) against sialylated and non-silylated N. gonorrhoeae. Outer membrane vesicles (OMVs) from 6 different N. gonorrhoeae strains were used to inoculate mice. SBA activity was assessed on a panel of 15 different N. gonorrhoeae strains. Gray dots indicate a test strain that matches the strain used to inoculate a mouse. Ng= / V. gonorrhoeae; PorB 1a=major outer membrane protein IA; PorB 1 b=major outer membrane protein IB.

[0167] FIG. 1 B is a set of graphs showing SBA activity against sialylated and non-silylated N. gonorrhoeae. Outer membrane vesicles (OMVs) from 6 different N. gonorrhoeae strains were used to inoculate mice. SBA activity was assessed on a panel of 15 different N. gonorrhoeae strains, represented as single dots. Dashed purple line indicates threshold for activity. hSBA=human serum bactericidal activity; OMV=outer membrane vesicle; PorB 1a=major outer membrane protein IA; PorB 1 b=major outer membrane protein IB. PATENT

[0168] ATTORNEY DOCKET NO: 51838-002WO3

[0169] FIG. 1C and 1 D are tables showing mouse SBA activity against non-sialylated bacteria (FIG. 10) or non-sialylated and sialylated bacteria (FIG. 1 D) resulting from vaccination of OMVs from 7 different strains. Shading indicates SBA activity over a threshold. OMV=outer membrane vesicle; nOMV=native outer membrane vesicle; CMP-NANA=cytidine Monophospho-N-Acetylneuraminic acid; Alum=alum adjuvant; PorB 1 a=major outer membrane protein IA; PorB 1 b=major outer membrane protein IB.

[0170] FIG. 2 is a table showing SBA activity from nOMVs from N. gonorrhoeae strain WHO-F relative to pre-immune controls. Shading indicates SBA activity over a threshold of a 4-fold increase over pre- immune SBA activity. nOMV=native outer membrane vesicle; PorB 1 a=major outer membrane protein IA; PorB 1 b=major outer membrane protein IB.

[0171] FIG. 3 is a set of tables showing SBA activity against sialylated N. gonorrhoeae bacteria (top) and strain coverage against sialylated N. gonorrhoeae bacteria by SBA from mice inoculated with OMVs from a single N. gonorrhoeae strain or with OMVs from a combination of N. gonorrhoeae strains. hSBA=human serum bactericidal activity; nOMV=native outer membrane vesicle; PorB 1a=major outer membrane protein IA; PorB 1 b=major outer membrane protein IB; CMP-NANA=cytidine Monophospho-N-Acetylneuraminic acid; Alum=aluminum; AS# = animal study #.

[0172] FIGS. 4A and 4B are tables showing SBA activity of serum from rabbits inoculated with Bexsero vaccine or controls against sialylated and non-sialylated N. gonorrhoeae. Shading indicates SBA activity over a threshold of a 4-fold increase over pre-immune SBA activity. CMP-NANA=cytidine Monophospho- N-Acetylneuraminic acid; lgG=immunoglobulin G; PorB 1 a=major outer membrane protein IA; PorB 1 b=major outer membrane protein IB.

[0173] FIG. 5A is a table showing SBA activity of OMVs against non-sialylated N. gonorrhoeae strains that were killed by OMVs from different strains in SBAs. SBA activities are presented as fold increase, relative to alum adjuvant control. Shading indicates SBA activity over a threshold. CMP-NANA=cytidine Monophospho-N-Acetylneuraminic acid; lgG=immunoglobulin G; PorB 1 a=major outer membrane protein IA; PorB 1 b=major outer membrane protein IB; OMV=outer membrane vesicle.

[0174] FIG. 5B is a table showing the number and percent of tested non-sialylated N. gonorrhoeae strains that were killed by OMVs from different strains in SBAs. OMV=outer membrane vesicle; SBA=serum bactericidal activity.

[0175] FIG. 6 is a table summarizing SBA activity of nOMVs against sialylated and non-sialylated N. gonorrhoeae bacteria. nOMV=native outer membrane vesicle; PorB 1a=major outer membrane protein IA; PorB 1 b=major outer membrane protein IB; Alum=alum adjuvant; CMP-NANA=cytidine Monophospho-N-Acetylneuraminic acid.

[0176] FIG. 7 is a set of immunoblots showing accumulation of 2C7 lipooligosaccharide (LOS; top) epitopes and of 4C8 LOS epitopes on different strains of N. gonorrhoeae. PorB-1 A=major outer membrane protein IA; PorB-1 B=major outer membrane protein IB.

[0177] FIGS. 8A and 8B are silver stained Novex Tricine Mini Protein Gel of bacterial pellets and specified OMVs with Proteinase K treatment, of stated strains of N. gonorrhoeae. Sia=sialylated; dOMV=detergent-extracted outer membrane vesicle.

[0178] FIGS. 9A and 9B are immunoblots showing staining of sialylated and non-sialylated WHO-F and WHO-O strains of N. gonorrhoeae, which express 4-HexG+ and 4-HexG-, respectively. Antibodies for PATENT

[0179] ATTORNEY DOCKET NO: 51838-002WO3 staining were antibodies induced by Bexsero (FIG. 9A) and commercially-available anti-2C7 antibodies. Sia=sialylated.

[0180] FIGS. 10A and 10B are graphs showing liquid chromatography-mass spectrometry peaks from a pellet of N. gonorrhoeae of strain FA1090 (FIG. 10A) or of strain FA19 (FIG. 10B).

[0181] FIGS. 11 A and 11 B are tables summarizing predicted LOS structures of different N. gonorrhoeae strains. NT=not tested.

[0182] FIG. 12A is a micrograph showing Coomassie staining of N. gonorrhoeae native OMVs (nOMVs) and detergent-extracted OMVs (dOMVs), and proteins neisserial heparin binding antigen (NHBA) and L-methionine binding lipoprotein (MetQ). Dashed box shows major outer membrane protein (PorB) species.

[0183] FIG. 12B is a set of immunoblots showing staining with antibodies against the stated proteins from N. gonorrhoeae OMVs. nOMV=native outer membrane vesicle; dOMV=detergent-extracted outer membrane vesicle; NHBA=neisserial heparin binding antigen; MetQ=L-methionine binding lipoprotein. Dashed boxes show protein species of interest.

[0184] FIG. 13 is a micrograph showing silver staining of N. gonorrhoeae bacteria or OMVs. nOMV=native outer membrane vesicle; dOMV=detergent-extracted outer membrane vesicle.

[0185] FIG. 14 is a set of micrographs showing Coomassie staining of N. gonorrhoeae native OMVs (nOMVs), detergent-extracted OMVs (dOMVs), sialylated nOMVs (Sia-nOMVs), sialylated dOMVs (Sia-dOMV), and proteins neisserial heparin binding antigen (NHBA) and L-methionine binding lipoprotein (MetQ). Dashed box shows major outer membrane protein (PorB) species. kDa=kilodalton.

[0186] FIG. 15 is a set of micrographs showing silver staining (top) and western blot with anti-2C7 antibodies (bottom) from sialylated and non-sialylated N. gonorrhoeae bacteria and OMVs. Bacteria were treated with phenol-water; OMVs were treated with proteinase Sia-Bacteria=sialylated bacteria, nOMV=native outer membrane vesicle; dOMV=detergent-extracted outer membrane vesicle; Sia-=sialylated.

[0187] FIG. 16 is a set of graphs showing enzyme linked-immunosorbent assay showing binding of anti-2C7 antibodies to N. gonorrhoeae OMVs. nOMV=native outer membrane vesicle; dOMV=detergent-extracted outer membrane vesicle; Sia-=sialy lated; OD=optical density; cone. concentration.

[0188] FIG. 17A is a set of graphs showing enzyme linked-immunosorbent assays showing binding of anti-NHBA, anti-MetQ, and anti-PorBIA antibodies to N. gonorrhoeae OMVs. nOMV=native outer membrane vesicle; dOMV=detergent-extracted outer membrane vesicle; Sia-=sialylated; NHBA=neisserial heparin binding antigen; MetQ=L-methionine binding lipoprotein; PorB1A=major outer membrane protein IA.

[0189] FIG. 17B is a set of immunoblots showing staining with anti-NHBA, anti-MetQ, and anti-PorBIA antibodies of sialylated and non-sialylated N. gonorrhoeae bacteria and OMVs. Dashed boxes show proteins of interest. nOMV=native outer membrane vesicle; dOMV=detergent-extracted outer membrane vesicle; Sia-=sialylated.

[0190] FIG. 18 is a set of graphs showing enzyme linked-immunosorbent assays from N. gonorrhoeae bacteria or N. gonorrhoeae bacteria grown in the presence of CMP-NANA (sialylated bacteria). PATENT

[0191] ATTORNEY DOCKET NO: 51838-002WO3

[0192] NHBA=neisserial heparin binding antigen; MetQ=L-methionine binding lipoprotein; PorB1A=major outer membrane protein IA; CMP-NANA=cytidine Monophospho-N-Acetylneuraminic acid; OD=optical density.

[0193] FIG. 19 is a set of immunoblots showing expression of stated proteins in sialylated and non-sialylated N. gonorrhoeae bacteria and OMVs. kDa=kilodaltons; TamA=translocator assembly module A; BamA= p-barrel assembly machinery A (BamA); FetA=ferric enterobactin transporter; NspA=neisserial surface protein A (NspA) TbpB=transferrin binding protein B.

[0194] FIG. 20 is a table summarizing outer membrane protein presence on N. gonorrhoeae bacteria, nOMVs, and dOMVs. Protein abundance is qualitatively shown with an increasing number of “+” signs corresponding to increased protein abundance as observed by Western Blot. TamA=translocator assembly module A; BamA= p-barrel assembly machinery A (BamA); FetA=ferric enterobactin transporter; NspA=neisserial surface protein A (NspA); TbpB=transferrin binding protein B; NHBA=neisserial heparin binding antigen; MetQ=L-methionine binding lipoprotein; PorB=major outer membrane protein; PorB1A=major outer membrane protein IA; LOS=lipidoligosaccharide; nOMV=native outer membrane vesicle; dOMV=detergent-extracted outer membrane vesicle.

[0195] FIG. 21 A is a table showing SBA activities against test strains of N. gonorrhoeae bacteria cultured in the presence or absence of CMP-NANA. Shading shows SBA titers above a threshold. nOMV=native outer membrane vesicle; dOMV=detergent-extracted outer membrane vesicle; Sia- =sialylated; PorB 1 a=major outer membrane protein IA; PorB 1 b=major outer membrane protein IB; CMP- NANA=cytidine Monophospho-N-Acetylneuraminic acid.

[0196] FIG. 21 B is table showing SBA activities against test strains of sialylated N. gonorrhoeae bacteria. nOMV=native outer membrane vesicle; dOMV=detergent-extracted outer membrane vesicle; Sia-=sialylated.

[0197] FIG. 22 is a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of N. gonorrhoeae strain FA1090 mutants. Dashed boxes show lanes from bacteria, nOMVs, or dOMVs. kDa=kilodalton; WT=wild-type; Alpxll =deletion of lipid A biosynthesis lauroyl acyltransferase; ArmpM=deletion of reduction modifiable protein; nOMV=native outer membrane vesicle; dOMV=detergent-extracted outer membrane vesicle.

[0198] FIG. 23A is a set of graphs showing enzyme linked-immunosorbent assays from N. gonorrhoeae strain FA1090 WT or mutant nOMVs and dOMVs. nOMV=native outer membrane vesicle; dOMV=detergent-extracted outer membrane vesicle; A=deletion of reduction modifiable protein; AA=deletions of reduction modifiable protein and lipid A biosynthesis lauroyl acyltransferase; WT=wild-type; OD=optical density; Cone. concentration.

[0199] FIG. 23B is a set of immunoblots from N. gonorrhoeae showing expression of NHBA (left), MetQ (middle), and PorBIA (right). Boxes show which lanes came from bacteria, nOMVs, and dOMVs. Dashed boxes show protein species of interest. nOMV=native outer membrane vesicle; dOMV=detergent-extracted outer membrane vesicle; ArmpM=deletion of reduction modifiable protein; Alpxll ArmpM=deletions of reduction modifiable protein and lipid A biosynthesis lauroyl acyltransferase; kDa=kilodaltons; NHBA=neisserial heparin binding antigen; MetQ=L-methionine binding lipoprotein; PorB 1 a=major outer membrane protein IA. PATENT

[0200] ATTORNEY DOCKET NO: 51838-002WO3

[0201] FIG. 24 is a set of immunoblots showing expression of stated proteins in wild-type and mutant N. gonorrhoeae bacteria and OMVs. Boxes show which lanes came from bacteria, nOMVs, and dOMVs. Dashed boxes show protein species of interest. nOMV=native outer membrane vesicle; dOMV=detergent-extracted outer membrane vesicle; TamA=translocator assembly module A; BamA= p-barrel assembly machinery A (BamA); FetA=ferric enterobactin transporter; NspA=neisserial surface protein A (NspA); TbpB=transferrin binding protein B; ArmpM=deletion of reduction modifiable protein; AlpxM ArmpM=deletions of reduction modifiable protein and lipid A biosynthesis lauroyl acyltransferase; WT=wild-type.

[0202] FIG. 25A is a table showing SBA activities against sialylated and non-sialylated N. gonorrhoeae bacteria using serum from rabbits inoculated with stated N. gonorrhoeae recombinant proteins. Shading shows SBA titers above a threshold of four-fold greater than pre-immune control. TamA=translocator assembly module A; BamA= p-barrel assembly machinery A (BamA); FetA=ferric enterobactin transporter; NspA=neisserial surface protein A (NspA); TbpB=transferrin binding protein B; PorB1 A=major outer membrane protein IA; PorB1 B=major outer membrane protein IB; NHBA=neisserial heparin binding antigen; MetQ=L-methionine binding lipoprotein; CMP-NANA=cytidine Monophospho-N-Acetylneuraminic acid.

[0203] FIG. 25B is a table showing SBA activities against sialylated and non-sialylated N. gonorrhoeae bacteria using serum from mouse. Shading shows SBA titers above a threshold. MPLA=monophosphoryl lipid A; OMV=outer membrane vesicle; Alum=alum adjuvant; MetQ=L-methionine binding lipoprotein; NHBA= neisserial heparin binding antigen; TbpB=transferrin binding protein B; CMP-NANA=cytidine Monophospho-N-Acetylneuraminic acid.

[0204] FIG. 26A and 26B are sets of graphs showing ultraviolet-visible light spectroscopy (UVA / is; FIG. 26A) and dynamic light scattering (DLS; FIG. 26B) characterization of OMVs of stated strains of N. gonorrhoeae. Z-Avg Dia.=Z- average diameter.

[0205] FIG. 26C is a micrograph of an SDS-PAGE of OMVs from stated strains of N. gonorrhoeae. kDa=kilodaltons.

[0206] FIG. 26D is a table summarizing statistics of OMVs from stated strains of N. gonorrhoeae.

[0207] FIG. 27 is a table summarizing filter options for tangential flow filtration.

[0208] FIG. 28A is a set of graphs showing N. gonorrhoeae bacterial growth in the presence or absence of Antifoam 204. OD600=optical density at 600 nm; W / =with; W / O=without.

[0209] FIGS. 28B and 28C are micrographs showing Coomassie staining (FIG. 28B) and silver staining (FIG. 28C). W / =with; W / O=without; kDa=kilodalton; antifoam 300K= SARTOCON® Slice 50 | 300K; antifoam 500K=Discover12 Hollow Fiber | 500K; antifoam 1 = SARTOCON® Slice 50 | 300K; antifoam 2=Discover12 Hollow Fiber | 500K.

[0210] FIG. 28D is a set of graphs showing properties of nOMVs from N. gonorrhoeae grown in the presence of stated antifoam reagents.

[0211] DETAILED DESCRIPTION OF THE INVENTION

[0212] Neisseria gonorrhoeae is a human pathogen that causes the sexually transmitted disease gonorrhea and can lead to a variety of secondary health complications. Given that many N. gonorrhoeae PATENT

[0213] ATTORNEY DOCKET NO: 51838-002WO3 strains are resistant to antibiotics that were previously successful in treating the infection, immunization with bacterial cellular components remains an attractive mode to prevent and treat gonorrhea infections. The present invention relates to compositions and methods for inducing protective immune responses against Neisseria gonorrhoeae (Ng). The primary immune mechanism for an effective gonococcal vaccine is the induction of bactericidal activity, which is initiated by the binding of antibodies to bacterial surface antigens, thereby facilitating complement activation and / or opsonophagocytic clearance.

[0214] However, N. gonorrhoeae exists in numerous genetically diverse strains, such that immunization against one strain may not be sufficient to protect against a different strain, e.g., that expresses a distinct set of surface antigens. The present invention is premised on the discovery of a number of genetically diverse surface antigens on N. gonorrhoeae outer membranes. By combining antigens from a plurality of genetically diverse gonorrhea strains, the components can provide protection against the widest variety of known circulating gonorrhea strains.

[0215] Thus, the present invention features compositions containing gonorrhea strains, e.g., from a plurality of gonorrhea strains, and outer membrane vesicles (OMVs) derived therefrom that include distinct sets of surface antigens to provide sufficient protection against the highest number of circulating strains.

[0216] Two principal antigenic targets on the outer membrane of Ng have been identified, lipooligosaccharides (LOS) and outer membrane proteins (OMPs). LOS-specific immune responses must accommodate the structural diversity of LOS molecules, which vary in sugar chain length and composition. Importantly, these epitopes may also be modified by sialylation, a process that occurs in vivo when Ng acquires sialic acid from host cells. Sialylation of LOS has been shown to inhibit complement deposition and enhance binding of host factor H, thereby reducing the efficacy of antibody-mediated killing.

[0217] It was observed that sialylation of lipooligosaccharides on outer membranes of N. gonorrhoeae can protect N. gonorrhoeae from antibodies that recognize non-sialylated LOS. However, antibodies raised against sialylated LOS epitopes may target sialylated LOS. In addition, some antibodies to LOS may be less affected by sialylation. Outer membrane vesicles (OMVs) from N. gonorrhoeae present a variety of antigens composed of a variety of epitopes. LOS antigens are diverse in its composition of sugar length, e.g., hexose sugar length having 2Hex, 3Hex, 4Hex, and the like and presence or absence of galactose off Hep II, e.g., G+ or G-..The combinatory presentation of such LOS results in a diversity of antibody responses. Therefore, careful selection of strains for a combination of strains can better represent this LOS diversity, leading superior coverage as vaccine. Biochemically, the bacterial outer membrane is composed of approximately 60% in mass of LOS, and this heavy representation of LOS may skew a host immune response towards LOS. As a consequence, the antibodies to outer membrane proteins, e.g., PorB.IA, PorB.I B, NHBA, NspA, BamA, MetQ, are minuscule after vaccination. Composition of OMPs can be enhanced by stripping of LOS using detergent extraction, which can deplete over 80%of LOS. However, different methods of detergent extracting OMVs lead to differential enrichment and de-enrichment of different types of epitopes (e.g., surface outer membrane proteins vs. LOS). PATENT

[0218] ATTORNEY DOCKET NO: 51838-002WO3

[0219] To address these challenges, the invention also provides immunogenic compositions including native outer membrane vesicles (nOMVs) derived from one or multiple Ng strains. These nOMVs can be selected to encompass a broad spectrum of LOS epitopes, including those modified by sialylation. The composition may further include nOMVs from two, three, or a plurality of Ng strains cultured in the presence of a sialic acid precursor, such as CMP-NANA (cytidine monophosphate-N-acetylneuraminic acid) to ensure sialylation of LOS to allow for expanded coverage of Ng strains.

[0220] Collectively, the compositions and methods described herein provide a rational framework for the development of a broadly protective Ng immunogenic compositions (e.g., vaccines) capable of overcoming the immunomodulatory effects of LOS sialylation and targeting of conserved outer membrane antigens.

[0221] Neisseria gonorrhoeae Strains

[0222] The present invention provides a composition of a plurality of different strains of N. gonorrhoeae. In some embodiments, the compositions described herein include 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different strains of N. gonorrhoeae.

[0223] Gonococcal bacterial strains are known in the art. While it will be understood in the art that an individual strain may differ marginally (for example, between different laboratories) due to natural sequence variation, the skilled person will be aware of methods to determine whether a given gonococcus is of the same or of a different strain. For example, the person skilled in the art may use Neisseria gonorrhea multi-antigen sequence typing (NG-MAST) to determine whether a given gonococcal strain is of the same or of a different strain (see, e.g., Demczuk et al. Journal of clinical microbiology 55.5: 1454-1468, 2017, which is hereby incorporated by reference).

[0224] Outer Membrane Vesicles

[0225] Also featured herein are OMVs from at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) different gonococcal bacterial strains. Outer membrane vesicles are proteoliposomic vesicle obtained by disruption of or blebbing from a gonococcal outer membrane to form vesicles that retain antigens from the outer membrane. OMVs can be prepared by various methods that are known to the person skilled in the art. For example, OMVs can be prepared artificially from bacteria, e.g., using detergent treatment (e.g. with deoxycholate) or by non-detergent extraction. In one embodiment, OMVs may be prepared by centrifugation followed by filtration of culture supernatant and its concentration using Tangential Flow Filtration.

[0226] In one embodiment, the outer membrane vesicles are native outer membrane vesicles, e.g., that are not detergent extracted. In an embodiment, the outer membrane vesicles are obtained via non-detergent extraction. The outer membrane vesicles may be obtained from blebbing or obtained from disruption of the outer membrane, wherein the disruption does not substantially include detergent extraction of the OMV from the outer membrane. Methods for obtaining outer membranes vesicles in the absence of detergent include using techniques such as sonication, homogenization, microfluidization, cavitation, osmotic shock, grinding, French press, blending, etc. Methods using no or low detergent can retain useful antigens as described in PCT Pub. No. WG2004 / 019977, the disclosure of which is hereby PATENT

[0227] ATTORNEY DOCKET NO: 51838-002WO3 incorporated by reference in its entirety. In another embodiment, the outer membrane vesicles may be obtained with detergent extraction.

[0228] In another embodiment, the OMVs are naturally secreted. In one embodiment, OMVs may be prepared by centrifugation. In an embodiment, OMVs are filtered using Tangential Flow Filtration.

[0229] In an embodiment, the outer membrane vesicles are, when administered to a subject, cross-bactericidal. In an embodiment the outer membrane vesicles, when administered to a subject, are able to induce cross-bactericidal antibody titers. In an embodiment, the outer membrane vesicles are, when administered to a subject, cross-bactericidal against heterologous and homologous strain(s) of N. gonorrhoeae.

[0230] In some embodiments, the OMVs have an average diameter of 40 nm to 120 nm (e.g., 60 nm to 80 nm, e.g., 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, or 140 nm), e.g., as determined by electron microscopy or dynamic light scattering. Furthermore, the OMVs may be substantially free from cytoplasmic contamination.

[0231] OMVs are released spontaneously during bacterial growth and can be purified from the culture medium. The purification may involve separating the OMVs from living and / or intact N. gonorrhoeae bacteria e.g. by using low speed centrifugation to pellet cells while leaving blebs in suspension and / or by size-based filtration using a filter, such as a 0.22 pm filter, which allows the blebs to pass through but which does not allow intact bacteria to pass through. Thus, unlike the culture medium, OMV containing compositions can be prepared to be substantially free from whole bacteria, whether living or dead. The size of the blebs means that they can readily be separated from whole bacteria by filtration e.g. via filter sterilization. Although blebs will pass through a standard 0.22 pm filters, these can rapidly become clogged by other material, and so it may be useful to perform sequential steps of filter sterilization through a series of filters of decreasing pore size before using a 0.22 pm filter. Examples of preceding filters would be those with pore size of 0.8 pm, 0.45 pm, etc. In an embodiment, the outer membrane vesicle is purified via filtering through a sterile filter with a pore size of less than 0.5, 0.4 or 0.3 pm.

[0232] A useful process for OMV preparation is described in POT Pub. No. W02005 / 004908, which is hereby incorporated by reference in its entirety, and involves ultrafiltration of crude OMVs rather than high speed centrifugation. The process may involve a step of ultracentrifugation after the ultrafiltration takes place.

[0233] Surface Antigens

[0234] The bacterial cells and OMVs described herein include one or more surface antigens (e.g. proteins) present at the surface of the cells and OMVs. In some embodiments, the bacterial cells and / or OMVs include one or more of the following proteins: major outer membrane protein (PorB), neisserial heparin binding antigen (NHBA), neisserial surface protein A (NspA), transferrin binding protein B (TbpB), ferric enterobactin transporter (FetA), L-methionine binding lipoprotein (MetQ), translocator assembly module A (TamA), lipopolysaccharide transport protein D (LptD) , p-barrel assembly machinery A (BamA), an Opacity (Opa) protein, and Pilin. PATENT

[0235] ATTORNEY DOCKET NO: 51838-002WO3

[0236] PorB is a surface-exposed voltage-gated pore protein that is abundant on gonococcal OMVs and has strong serum bactericidal activity (SBA). In some embodiments, the PorB present in the OMVs from a first strain is at most 99%, at most 98%, at most 97%, at most 96%, at most 95%, at most 94%, at most 93%, at most 92%, at most 91%, at most 90%, at most 85%, at most 80%, at most 75%, at most 70%, at most 69.6%, or at most 65% identical to the PorB present in the OMVs from the second strain. In some embodiments, the PorB present in the OMVs from a third strain is at most 99%, at most 98%, at most 97%, at most 96%, at most 95%, at most 94%, at most 93%, at most 92%, at most 91%, at most 90%, at most 85%, at most 80%, at most 75%, at most 70%, at most 69.6%, or at most 65% identical to the PorB present in the OMVs from the first and / or second strain.

[0237] NHBA is a is a surface-exposed lipoprotein that is abundant on gonococcal OMVs and has strong SBA. In some embodiments, the NHBA present in the OMVs from a first strain is at most 99%, at most 98%, at most 97%, at most 96%, at most 95%, at most 94.9%, at most 94%, at most 93%, at most 92%, at most 91%, at most 90%, at most 85%, at most 80%, at most 75%, at most 70%, or at most 65% identical to the NHBA present in the OMVs from the second strain. In some embodiments, the NHBA present in the OMVs from a third strain is at most 99%, at most 98%, at most 97%, at most 96%, at most 95%, at most 94.9%, at most 94%, at most 93%, at most 92%, at most 91%, at most 90%, at most 85%, at most 80%, at most 75%, at most 70%, or at most 65% identical to the NHBA present in the OMVs from the first and / or second strain.

[0238] NspA is a surface-exposed protein that binds factor H, is abundant on gonococcal OMVs, and has strong SBA. In some embodiments, the NspA present in the OMVs from a first strain is at most 99.4%, at most 99%, at most 98%, at most 97%, at most 96%, at most 95%, at most 94%, at most 93%, at most 92%, at most 91%, at most 90%, at most 85%, at most 80%, at most 75%, at most 70%, or at most 65% identical to the NspA present in the OMVs from the second strain. In some embodiments, the NspA present in the OMVs from a third strain is at most 99.4%, at most 99%, at most 98%, at most 97%, at most 96%, at most 95%, at most 94%, at most 93%, at most 92%, at most 91%, at most 90%, at most 85%, at most 80%, at most 75%, at most 70%, or at most 65% identical to the NspA present in the OMVs from the first and / or second strain.

[0239] TbpB is a surface-exposed lipoprotein that plays a role in iron uptake, is abundant on gonococcal OMVs, and has strong SBA. In some embodiments, the TbpB present in the OMVs from a first strain is at most 99%, at most 98%, at most 97%, at most 96%, at most 95%, at most 94%, at most 93%, at most 92%, at most 91%, at most 90%, at most 85%, at most 80%, at most 75.9%, at most 75%, at most 70%, or at most 65% identical to the TbpB present in the OMVs from the second strain. In some embodiments, the TbpB present in the OMVs from a third strain is at most 99%, at most 98%, at most 97%, at most 96%, at most 95%, at most 94%, at most 93%, at most 92%, at most 91%, at most 90%, at most 85%, at most 80%, at most 75.9%, at most 75%, at most 70%, or at most 65% identical to the TbpB present in the OMVs from the first and / or second strain.

[0240] FetA is a surface-exposed protein associated with iron uptake that is abundant on gonococcal OMVs and has strong SBA. In some embodiments, the FetA present in the OMVs from a first strain is at most 99%, at most 98%, at most 97.2%, at most 97%, at most 96%, at most 95%, at most 94%, at most 93%, at most 92%, at most 91%, at most 90%, at most 85%, at most 80%, at most 75%, at most 70%, or PATENT

[0241] ATTORNEY DOCKET NO: 51838-002WO3 at most 65% identical to the FetA present in the OMVs from the second strain. In some embodiments, the FetA present in the OMVs from a third strain is at most 99%, at most 98%, at most 97.2%, at most 97%, at most 96%, at most 95%, at most 94%, at most 93%, at most 92%, at most 91%, at most 90%, at most 85%, at most 80%, at most 75%, at most 70%, or at most 65% identical to the FetA present in the OMVs from the first and / or second strain.

[0242] MetQ is a surface-exposed protein associated with ATP-binding cassette (ABC) transporters. In some embodiments, the MetQ present in the OMVs from a first strain is at most 99%, at most 98%, at most 97.2%, at most 97%, at most 96%, at most 95%, at most 94%, at most 93%, at most 92%, at most 91%, at most 90%, at most 85%, at most 80%, at most 75%, at most 70%, or at most 65% identical to the MetQ present in the OMVs from the second strain. In some embodiments, the MetQ present in the OMVs from a third strain is at most 99%, at most 98%, at most 97.2%, at most 97%, at most 96%, at most 95%, at most 94%, at most 93%, at most 92%, at most 91%, at most 90%, at most 85%, at most 80%, at most 75%, at most 70%, or at most 65% identical to the MetQ present in the OMVs from the first and / or second strain.

[0243] TamA is a surface-exposed protein associated with secretion of autotransporters. In some embodiments, the TamA present in the OMVs from a first strain is at most 99%, at most 98%, at most 97.2%, at most 97%, at most 96%, at most 95%, at most 94%, at most 93%, at most 92%, at most 91%, at most 90%, at most 85%, at most 80%, at most 75%, at most 70%, or at most 65% identical to the TamA present in the OMVs from the second strain. In some embodiments, the TamA present in the OMVs from a third strain is at most 99%, at most 98%, at most 97.2%, at most 97%, at most 96%, at most 95%, at most 94%, at most 93%, at most 92%, at most 91%, at most 90%, at most 85%, at most 80%, at most 75%, at most 70%, or at most 65% identical to the TamA present in the OMVs from the first and / or second strain.

[0244] LptD is a surface-exposed protein associated with lipopolysaccharide transport. In some embodiments, the LptD present in the OMVs from a first strain is at most 99%, at most 98%, at most 97.2%, at most 97%, at most 96%, at most 95%, at most 94%, at most 93%, at most 92%, at most 91%, at most 90%, at most 85%, at most 80%, at most 75%, at most 70%, or at most 65% identical to the LptD present in the OMVs from the second strain. In some embodiments, the LptD present in the OMVs from a third strain is at most 99%, at most 98%, at most 97.2%, at most 97%, at most 96%, at most 95%, at most 94%, at most 93%, at most 92%, at most 91%, at most 90%, at most 85%, at most 80%, at most 75%, at most 70%, or at most 65% identical to the LptD present in the OMVs from the first and / or second strain.

[0245] BamA is a surface-exposed protein associated with assembly and structure of outer membranes. In some embodiments, the BamA present in the OMVs from a first strain is at most 99%, at most 98%, at most 97.2%, at most 97%, at most 96%, at most 95%, at most 94%, at most 93%, at most 92%, at most 91%, at most 90%, at most 85%, at most 80%, at most 75%, at most 70%, or at most 65% identical to the BamA present in the OMVs from the second strain. In some embodiments, the BamA present in the OMVs from a third strain is at most 99%, at most 98%, at most 97.2%, at most 97%, at most 96%, at most 95%, at most 94%, at most 93%, at most 92%, at most 91%, at most 90%, at most 85%, at most 80%, at most 75%, at most 70%, or at most 65% identical to the BamA present in the OMVs from the first and / or second strain. PATENT

[0246] ATTORNEY DOCKET NO: 51838-002WO3

[0247] Opa proteins are surface-exposed proteins associated with interaction with host cells. In some embodiments, the Opa protein present in the OMVs from a first strain is at most 99%, at most 98%, at most 97.2%, at most 97%, at most 96%, at most 95%, at most 94%, at most 93%, at most 92%, at most 91%, at most 90%, at most 85%, at most 80%, at most 75%, at most 70%, or at most 65% identical to the Opa proteins present in the OMVs from the second strain. In some embodiments, the Opa proteins present in the OMVs from a third strain is at most 99%, at most 98%, at most 97.2%, at most 97%, at most 96%, at most 95%, at most 94%, at most 93%, at most 92%, at most 91%, at most 90%, at most 85%, at most 80%, at most 75%, at most 70%, or at most 65% identical to the Opa proteins present in the OMVs from the first and / or second strain.

[0248] Pilin is a surface-exposed protein associated with interaction with host cells. In some embodiments, the Pilin present in the OMVs from a first strain is at most 99%, at most 98%, at most 97.2%, at most 97%, at most 96%, at most 95%, at most 94%, at most 93%, at most 92%, at most 91%, at most 90%, at most 85%, at most 80%, at most 75%, at most 70%, or at most 65% identical to the Pilin present in the OMVs from the second strain. In some embodiments, the Pilin present in the OMVs from a third strain is at most 99%, at most 98%, at most 97.2%, at most 97%, at most 96%, at most 95%, at most 94%, at most 93%, at most 92%, at most 91%, at most 90%, at most 85%, at most 80%, at most 75%, at most 70%, or at most 65% identical to the Pilin present in the OMVs from the first and / or second strain.

[0249] In some embodiments, the composition further includes a recombinant protein (e.g., a surface protein from an OMV). In some embodiments, an endogenous copy of the recombinant protein is typically expressed on OMVs but exhibits low (e.g., at least 10% less expression, e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, or 99% less expression than a reference surface protein) or undetectable levels of expression or is lost during preparation of the OMV. In some embodiments, an endogenous copy of the recombinant protein is typically expressed on OMVs, and the recombinant protein is modified (e.g., a mutant or a biologically active fragment). In some embodiments, the recombinant protein is NHBA, MetQ, translocator assembly module A (TamA), lipopolysaccharide transport protein D (LptD), p-barrel assembly machinery A (BamA), TbpB, FetA, NspA, Opacity (Opa) proteins, or Pilin. In some embodiments, the recombinant protein is NHBA. In some embodiments, the recombinant protein is MetQ. In some embodiments, the recombinant protein is TamA. In some embodiments, the recombinant protein is LptD. In some embodiments, the recombinant protein is BamA. In some embodiments, the recombinant protein is TbpB. In some embodiments, the recombinant protein is FetA. In some embodiments, the recombinant protein is NspA. In some embodiments, the recombinant protein is an Opa protein. In some embodiments, the recombinant protein is Pilin. In some embodiments, the recombinant protein is NHBA.

[0250] Lipooligosaccharides

[0251] Outer surfaces of N. gonorrhoeae bacteria include lipooligosaccharides (LOS). These LOS may be sialylated or non-sialylated. Sialylation of LOS can protect N. gonorrhoeae from host antibodies by blocking host antibodies from accessing non-sialylated epitopes. OMVs from N. gonorrhoeae can similarly include sialylated and non-sialylated LOS. The present disclosure features compositions that PATENT

[0252] ATTORNEY DOCKET NO: 51838-002WO3 include sialylated OMVs. For example, any of the compositions described herein can include an OMV with a sialic acid moiety.

[0253] In an aspect, the disclosure features a composition including a first OMV isolated from a first strain of N. gonorrhoeae that was incubated with sialic acid or a precursor of sialic acid (e.g., cytidine Monophospho-N-Acetylneuraminic acid (CMP-NANA)). The composition can include a second OMV from a second strains of N. gonorrhoeae that was incubated with sialic acid or a precursor of sialic acid (e.g., CMP-NANA). The composition can include a plurality (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) of OMVs from N. gonorrhoeae that were incubated with sialic acid or a precursor of sialic acid (e.g., CMP-NANA). The plurality of OMVs can be from a plurality of strains of N. gonorrhoeae.

[0254] The OMVs may be native OMVs (i.e., natively extracted OMVs).

[0255] The first strain of N. gonorrhoeae can include a genetic mutation as described herein. The second strain of N. gonorrhoeae can include a genetic mutation as described herein. The plurality of strains of N. gonorrhoeae can include a genetic mutation as described herein. In one embodiment, the strain may be engineered to enhance self “blebing”, e.g., deletion of rmpM gene]. In another embodiment, the strain may be engineered to deplete LpxL1 gene so to “detoxify” LPS associated with secreted OMV. In some embodiment, these and other genetic modifications could be combined for the purpose of production naturally blebbed OMV.

[0256] The composition can further include a third OMV from a third strain of N. gonorrhoeae. The composition may further include an additional plurality of OMVs from an additional plurality of strains of N. gonorrhoeae. The third OMV can be a detergent-extracted OMV. The additional plurality of OMVs can be detergent-extracted OMVs. The third OMV or the additional plurality of OMVs can include PorB.IA, PorB.IB, translocator assembly module A (TamA), lipopolysaccharide transport protein D (LptD), p-barrel assembly machinery A (BamA), NspA, FetA, or opacity (Opa) proteins. The third OMV or the additional plurality of OMVs can include PorB.IA. The third OMV or the additional plurality of OMVs can include PorB.IB. The third OMV or the additional plurality of OMVs can include TamA. The third OMV or the additional plurality of OMVs can include LptD. The third OMV or the additional plurality of OMVs can include BamA. The third OMV or the additional plurality of OMVs can include NspA. The third OMV or the additional plurality of OMVs can include FetA. The third OMV or the additional plurality of OMVs can include Opa proteins.

[0257] The first strain of N. gonorrhoeae, the second strain of N. gonorrhoeae, and the plurality of strains of N. gonorrhoeae can be different from each other. The first strain of N. gonorrhoeae, the second strain of N. gonorrhoeae, and the plurality of strains of N. gonorrhoeae can be different from the third strain of N. gonorrhoeae and the additional plurality of strains of N. gonorrhoeae.

[0258] Native and Detergent-Extracted OMVs

[0259] The disclosure features OMVs from N. gonorrhoeae. OMVs can be natively extracted or detergent extracted. Native extraction of OMVs can more effectively preserve LOS than detergent extraction. Detergent extraction can better preserve some protein antigens than native extraction. Thus there are advantages to including both detergent extracted OMVs and natively extracted OMVs in an immunogenic composition to provide a diverse sets of surface LOS and protein antigens. PATENT

[0260] ATTORNEY DOCKET NO: 51838-002WO3

[0261] In an aspect, the disclosure features a composition including OMVs from N. gonorrhoeae, wherein the composition includes a first OMV isolated from a first strain of N. gonorrhoeae and a second OMV from a second strain of N. gonorrhoeae, wherein the first OMV is a native OMV and the second OMV is a detergent-extracted OMV.

[0262] The first strain can be incubated with sialic acid or a precursor of sialic acid (e.g., CMP-NANA). The first OMV can include 2HexG+, 3HexG+, 4HexG+, 4HexG-, 5HexG+, and / or 5HexG- LOS on a surface of the OMV. Incubation with sialic acid or a precursor of sialic acid can result in sialylated epitope in LOS, which could expand antibody repertoire against N. gonorrhoeae. Thus, the first OMV can include sialylated or non-sialylated 2HexG+, 3HexG+, 4HexG+, 4HexG-, 5HexG+, and / or 5HexG- LOS on a surface of the OMV. Native extraction of the OMV can result in preservation of LOS structures on a surface of the OMV. Native extraction of the OMV can result in reduced levels of NHBA, MetQ, TbpB, and / or Pilin proteins on a surface of the OMV. A second or even third naturally produced OMVs can be added to the composition, e.g., to further expand the antibody repertoire against diverse LOS presentations in N. gonorrhoeae in circulation. The combination of the plural strains can be tested in assays using N. gonorrhoeae that have been cultured with sialic acid.

[0263] OMVs of the second category can be detergent extracted in contrast to natively secreted. They can include, for example, PorB.IA, PorB.IB, TamA, LptD, BamA, NspA, FetA, and / or Opa proteins on a surface of the OMV. Detergent extraction of the OMV can result in preservation of PorB.IA, PorB.IB, TamA, LptD, BamA, NspA, FetA, and / or Opa proteins on a surface of the second OMV. Detergent extraction of the OMV can result in reduced expression of NHBA, MetQ, TbpB, and / or Pilin proteins on a surface of the second OMV. Detergent extraction can result in reduced presentation of LOS on a surface of the second OMV.

[0264] The composition can include a third OMV from a third strain of N. gonorrhoeae or a plurality of OMVs from a plurality of strains of N. gonorrhoeae. The third strain or the plurality of strains can be incubated with sialic acid or a precursor of sialic acid (e.g., CMP-NANA). The third strain or the plurality of strains can be natively extracted.

[0265] The composition can include an additional plurality of OMVs from an additional plurality of strains of N. gonorrhoeae. The additional plurality of OMVs can be detergent-extracted.

[0266] The first strain can include a genetic mutation as described herein. The second strain can include a genetic mutation as described herein. The third strain can include a genetic mutation as described herein. The plurality of strains can include a genetic mutation as described herein. The additional plurality of strains can include a genetic mutation as described herein.

[0267] The first strain of N. gonorrhoeae, the second strain of N. gonorrhoeae, and the plurality of strains of N. gonorrhoeae can be different from each other. The first strain of N. gonorrhoeae, the second strain of N. gonorrhoeae, and the plurality of strains of N. gonorrhoeae can be different from the third strain of N. gonorrhoeae and the additional plurality of strains of N. gonorrhoeae.

[0268] Methods of Making Outer Membrane Vesicles

[0269] The disclosure features a method of making an isolated OMV from a first strain of N. gonorrhoeae. The method includes: (a) incubating a N. gonorrhoeae with sialic acid or a precursor of PATENT

[0270] ATTORNEY DOCKET NO: 51838-002WO3 sialic acid (e.g., CMP-NANA); and (b) isolating an OMV from the N. gonorrhoeae. The OMV may be natively extracted. The native extraction may include centrifuging (e.g., at about 1000 x g for about 5 minutes) a culture of N. gonorrhoeae and collecting a first supernatant, centrifuging the first supernatant (e.g., at about 5000 x g for about 10 minutes) and collecting a second supernatant, and filtering the second supernatant (e.g., with a 0.2 pm filter). The OMV may be purified using tangential flow filtration (e.g., ultrafiltration and / or diafiltration). The OMV may be polished using multimodal chromatography (e.g., with CAPTO™ Core 400 multipmodal chromatography resin). The OMV may be further purified with tangential flow filtration (e.g., ultrafiltration and diafiltration).

[0271] Methods of Identifying Immunogenic N. gonorrhoeae strains

[0272] The disclosure also provides a method of identifying a first candidate strain of N. gonorrhoeae as suitable for producing an immunogenic OMV, the method including: (a) culturing the first candidate strain of N. gonorrhoeae in the presence of sialic acid or a sialic acid precursor (e.g., CMP-NANA); (b) collecting a first OMV from the N. gonorrhoeae; (c) inoculating a mammal (e.g., a mouse or a rabbit) with the OMV; (d) collecting serum from the mammal; (e) performing an assay to determine an activity of the first OMV on a test strain of N. gonorrhoeae, e.g., that has been cultured in the presence of sialic acid; and (f) identifying the candidate strain as suitable for producing an immunogenic OMV if the activity of the isolated first OMV on the test strain exceeds a threshold.

[0273] The method can also include inoculating the mammal with a second OMV from a second candidate strain of N. gonorrhoeae or a plurality of OMVs from a plurality of candidate strains of N. gonorrhoeae. The first OMV can be natively extracted. The second OMV can be detergent extracted. The plurality of OMVs can be natively extracted or detergent extracted.

[0274] The method can include testing at least s (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , or more) test strains of N. gonorrhoeae.

[0275] The assay can be a serum bactericidal assay (SBA). The SBA can be a human SBA (hSBA; i.e., the SBA can include human complement protein).

[0276] The candidate strain or strains can be identified as suitable for producing an immunogenic OMV if the activity of the isolated OMVs on the test strain exceeds an activity threshold for a threshold percent of strains. For example, the activity threshold can be a threshold titer in an SBA (e.g., an at least two-fold (e.g., two-fold, three-fold, four-fold, five-fold, ten-fold, or more) greater titer compared to pre-immune serum). The threshold percent of strains may be at least 50% (e.g., 50%, 60%, 70%, 80%, 9 %, 95%, or more) of tested strains. The candidate strains can include a genetic mutation as described herein.

[0277] Genetic Mutations

[0278] In an aspect, the invention provides compositions containing gonococcal bacteria or OMVs derived therefrom in which the cell contains at least one genetic modification that decreases or abolishes expression and / or function of the IpxLI gene, mRNA, and / or polypeptide. Also provided herein are compositions containing gonococcal bacteria or OMVs derived therefrom in which the cell contains at least one genetic modification that decreases or abolishes expression and / or function of the rmpM gene, mRNA, and / or polypeptide. Gonoccoccal rmpM encodes the outer membrane protein RmpM, which can PATENT

[0279] ATTORNEY DOCKET NO: 51838-002WO3 inhibit serum bactericidal activity of OMVs when anti-RmpM antibodies are generated. Similarly, the IpxLI gene (also referred to as msbB) encodes the polypeptide Lipid A biosynthesis lauroyl acyltransferase (LpxL1). LpxL1 plays a role in lipid A biosynthesis, and its reduction or deletion can reduce lipooligosaccharide endotoxicity. Neisseria organisms genetically modified to provide for decreased or no detectable functional LpxL1 encoded protein produce OMVs with reduced endotoxicity. This may occur because the amount of lipid A acylation and the nature of the acylation are major factors that affect LOS toxicity. Reduction or deletion of one or more of these genes may also provide for improved OMV production yield during manufacture.

[0280] In some embodiments, one or more of the gonococcal strains expresses less than 10%, less than 5%, or less than 1% of the LpxL1 polypeptide compared to the expression of the LpxL1 polypeptide in an unmodified (e.g. wild-type) gonococcal strain.

[0281] In some embodiments, one or more of the gonococcal strains expresses less than 10%, less than 5%, or less than 1% of the RmpM polypeptide compared to the expression of the RmpM polypeptide in an unmodified (e.g. wild-type) gonococcal strain.

[0282] In some embodiments, one or more of the gonococcal strains expresses a de minimum level of the LpxL1 polypeptide compared to the level of the LpxL1 polypeptide in an unmodified (wild-type) gonococcal strain.

[0283] In some embodiments, one or more of the gonococcal strains expresses a de minimus level of the RmpM polypeptide compared to the level of RmpM polypeptide in an unmodified (wild-type) gonococcal strain.

[0284] In some embodiments, one or more of the gonococcal strains does not express the LpxL1 polypeptide and / or the RmpM polypeptide. In some embodiments, the one or more gonococcal strains does not express the LpxL1 and / or RmpM polypeptide at a detectable level as measured for example by immunoassay. In some embodiments, the one or more gonococcal strains does not express the LpxL1 and / or RmpM polypeptide at a detectable level as measured by Western Blot or ELISA.

[0285] In an embodiment, decreased expression refers to one or more gonococcal strains that expresses less IpxLI and / or rmpM mRNA and / or LpxL1 and RmpM protein compared to an unmodified (wild type) gonococcal bacteria or a gonococcal strain including the wild type IpxLI and / or rmpM genes. Expression may be considered decreased when any reduction in mRNA and / or protein expression is observed compared to an unmodified (wild type) gonococcal strain or a gonococcal strain including the wild type IpxLI / rmpM genes. Expression may be considered decreased when an over 5%, over 10%, over 25%, over 50%, over 60%, over 70%, over 80%, over 90%, or over 95% reduction in mRNA and / or protein expression is observed compared to the mRNA and / or protein expression, respectively, in an unmodified (wild-type) gonococcal strain or a gonococcal strain including the wild type IpxLI / rmpM genes. In some embodiments, no IpxLI mRNA and / or protein and / or no rmpM mRNA and / or protein can be detected in the gonococcal bacterium using the technique used by the skilled person to measure expression.

[0286] The level of expression of the IpxLI and / or rmpM genes can be measured using techniques well known to the skilled person, for example using polymerase chain reaction (PCR) based techniques (for example using Q / RT-PCR). The level of expression of the LpxL1 and / or RmpM polypeptides can be PATENT

[0287] ATTORNEY DOCKET NO: 51838-002WO3 measured using techniques well known to the skilled person. For example, the level of expression of both the LpxL1 and / or RmpM polypeptides can be measured using Western Blotting or ELISA. The level of expression of the RmpM polypeptide can be measured using SDS-PAGE and LC / MS-MS.

[0288] The genetic modification(s) may decrease or abolish the expression and / or function of the IpxLI gene mRNA and / or polypeptide. As such, the genetic modification(s) may result in retained expression of the LpxL1 polypeptide but wherein the polypeptide is non-functional. The function of LpxL1 can be determined for example by examining the extent to which the Lipid A component of the outer membrane vesicle lipooligosaccharide is penta-acylated as opposed to being hexa-acylated. If the genetically modified gonococcal bacterium includes a genetic modification that decreases or abolishes the function of the LpxL1 protein, the Lipid A will be penta-acylated (for example it will be at least 80%; at least 90%; 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 is 100% penta-acylated.

[0289] Similarly, the genetic modification(s) may decrease or abolish the expression and / or the function of the rmpM gene mRNA and / or polypeptide. As such, the genetic modification(s) may result in the retention of expression of the RmpM polypeptide but wherein the polypeptide is non-functional. The function of RmpM can be determined for example by examining the extent to which the gonococcus blebs. If the genetically modified gonococcal bacterium includes a genetic modification that decreases or abolishes the function of the RmpM protein the gonococcus may be hyperblebbing or prone to vesiculation compared to a gonococcal bacterium that includes the wild type rmpM gene. Accordingly, in some embodiments, the genetically modified gonococcal bacteria may be tested for whether it produces more OMVs (e.g. whether a strain is hyperblebbing) compared to the same measures from the gonococcal bacterium that includes the wild type rmpM gene, e.g., by comparing the OMV yield obtained from one strain with the OMV yield from another (using the same OMV blebbing protocol). Such methods are disclosed in Maharjan et al. Microbiology, 1620: 364-375, 2016, which is hereby incorporated by reference.

[0290] In some embodiments, one of the gonococcal strains has a genetic modification that decreases or abolishes expression of the IpxLI gene mRNA and / or polypeptide. In some embodiments, one of the gonococcal strains has a genetic modification that decreases or abolishes expression of the rmpM gene mRNA and / or polypeptide. In some embodiments, one of the gonococcal strains has a genetic modification that decreases or abolishes expression of the IpxLI gene mRNA and / or polypeptide, and a genetic modification that decreases or abolishes expression of the rmpM gene mRNA and / or polypeptide. In some embodiments, two of the gonococcal strains has one or more genetic modifications that decreases or abolishes expression of the IpxLI gene mRNA and / or polypeptide, and / or a genetic modification that decreases or abolishes expression of the rmpM gene mRNA and / or polypeptide. In some embodiments, three of the gonococcal strains has one or more genetic modifications that decreases or abolishes expression of the IpxLI gene mRNA and / or polypeptide, and / or a genetic modification that decreases or abolishes expression of the rmpM gene mRNA and / or polypeptide.

[0291] In some embodiments, the genetic modification is a gene deletion. In an embodiment the gene deletion is a result of a sequence addition, substitution or deletion modification within the IpxLI and rmpM loci. In some embodiments, the genetic modification(s) are gene deletions wherein the gene deletion is a PATENT

[0292] ATTORNEY DOCKET NO: 51838-002WO3 result of replacing a portion (or portions) of the IpxLI and / or rmpM genes with heterologous sequences, e.g., a heterologous gene.

[0293] In an embodiment there is provided a genetically modified gonococcal bacterium, including genetic modification(s) that decreases or abolishes expression of the LpxL1 polypeptide and decreases or abolishes expression of the RmpM polypeptide, wherein the genetic modification(s) are gene deletions, thereby resulting in the double-mutant gonococcus (AlpxLI , ArmpM).

[0294] Any suitable technique can be used to delete the endogenous IpxLI and rmpM genes (i.e. to generate a gene knockout). Gene knockouts in gonococci can for example be made by transposon mutagenesis, in vitro genetic engineering to modify genes contained on plasmids or Bacterial Artificial Chromosomes (BACs) and moving the modified construct to the organism of interest, and in vivo homologous recombination. In an embodiment the genes are knocked out by disabling an endogenous promoter, operon or regulatory element that is essential for transcription or translation of the genes. In an embodiment, the genes are deleted using CRISPR-Cas9 technology.

[0295] In an embodiment the endogenous IpxLI and / or rmpM genes are deleted by homologous recombination. Homologous recombination may be performed, for example, as described in PCT Pub. No. W02001 / 009350 or using techniques described in Dillard et al. Current protocols in microbiology Chapter 4, 2011 , which are hereby incorporated by reference. During the process of homologous recombination, the endogenous IpxLI and / or rmpM genes are deleted by either adding a different gene into the coding sequence of the IpxLI and / or rmpM genes or by replacing the gene or fragment thereof with the different gene (e.g., a heterologous gene, or non-functional gene) by recombination. In an embodiment, the heterologous gene is an antibiotic resistance gene.

[0296] In an embodiment, the genetic modification(s) may be to coding and / or non-coding regions. In an embodiment, the genetic modification(s) are to the coding region, non-coding region, or a combination thereof

[0297] In an embodiment, all gonococcal strains as described herein include genetic modifications to IpxLI and to rmpM.

[0298] In an embodiment, two gonococcal strains as described herein include genetic modifications to IpxLI and to rmpM.

[0299] In an embodiment, two gonococcal strains as described herein include genetic modifications to IpxLI and to rmpM and one gonococcal strain includes a genetic modification to rmpM.

[0300] The disclosure provides engineered OMVs. The engineered OMV can include any of the genetic mutation described herein (e.g., a mutation to the rmpM gene). The engineered OMV can include a sialic acid moiety. At least 50% (e.g., 50%, 60%, 70%, 80%, 90%, 95%, or more) of LOS on a surface of the OMV can be sialylated. The sialic acid moiety can be on a 2HexG+, 3HexG+, 4HexG+, 4HexG-, 5HexG+, and / or 5HexG- LOS. The engineered OMV can be isolated. The engineered OMV can be a native OMV.

[0301] Pharmaceutical and Immunogenic Compositions

[0302] The present disclosure provides pharmaceutical and immunogenic compositions containing OMVs for use in treating or preventing gonorrhea. The OMVs described herein can be formulated as PATENT

[0303] ATTORNEY DOCKET NO: 51838-002WO3 pharmaceutical compositions for administration to human subjects in a biologically compatible form suitable for administration in vivo.

[0304] The compositions described herein may be administered to a subject (e.g., a human) in a variety of forms depending on the selected route of administration, as will be understood by those skilled in the art. The compositions described herein may be administered, for example, by any route that allows the composition (e.g., the OMVs) to reach the target cells. The composition may be administered, for example, by oral, parenteral, intrathecal, intracerebroventricular, intraparenchymal, buccal, sublingual, nasal, rectal, patch, pump, or transdermal administration and the pharmaceutical compositions formulated accordingly. Parenteral administration includes intravenous, intraperitoneal, subcutaneous, intramuscular, transepithelial, nasal, intrapulmonary, intrathecal, intracerebroventricular, intraparenchymal, rectal, and topical modes of administration. In one embodiment, the composition administered via aero parenteral administration may be by continuous infusion over a selected period of time.

[0305] A composition described herein may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard- or soft-shell gelatin capsules, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, a composition described herein may be incorporated with an excipient and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, and wafers. A composition described herein may also be administered parenterally. Solutions of a composition described herein can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, DMSO, and mixtures thereof with or without alcohol, and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms. Conventional procedures and ingredients for the selection and preparation of suitable formulations are described, for example, in Remington’s Pharmaceutical Sciences (2012, 22nd ed.) and in The United States Pharmacopeia: The National Formulary (USP 41 NF 36), published in 2018. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that may be easily administered via syringe. Compositions suitable for buccal or sublingual administration include tablets, lozenges, and pastilles, where the active ingredient is formulated with a carrier, such as sugar, acacia, tragacanth, gelatin, and glycerin. Compositions for rectal administration are conveniently in the form of suppositories containing a conventional suppository base, such as cocoa butter.

[0306] The composition described herein may be administered to an animal, e.g., a human, alone or in combination with pharmaceutically acceptable carriers, as noted herein, the proportion of which is determined by the solubility and chemical nature of the composition, chosen route of administration, and standard pharmaceutical practice.

[0307] The dosage of the compositions (e.g., a composition including an OMV) described herein, can vary depending on many factors, such as the pharmacodynamic properties of the OMV, the mode of administration, the age, health, and weight of the recipient, the nature and extent of the symptoms, the frequency of the treatment, and the type of concurrent treatment, if any, and the clearance rate of the PATENT

[0308] ATTORNEY DOCKET NO: 51838-002WO3 composition in the animal to be treated. The compositions described herein may be administered initially in a suitable dosage that may be adjusted as required, depending on the clinical response. In some embodiments, the dosage of a composition (e.g., a composition containing an OMV) is a prophylactically or a therapeutically effective amount. Furthermore, it is understood that all dosages may be continuously given or divided into dosages given per a given time frame. The composition can be administered, for example, every hour, day, week, month, or year. In some embodiments, the composition may be administered continuously or systemically.

[0309] The pharmaceutical compositions described herein may be provided in a kit that includes the pharmaceutical composition (e.g., in a container) and instructions for use thereof. The kit may contain one or more containers, in which each container contains a different composition (e.g., one container with a first OMV and one container with a second OMV). The instructions enclosed with the kit may be used to instruct a user to perform a method as described herein.

[0310] Also contemplated herein is immunogenic composition (e.g., a vaccine) that includes a composition containing OMVs as described herein and a pharmaceutically acceptable carrier. The immunogenic composition may further include an adjuvant. The compositions may further include an adjuvant such that, when administered to a subject in conjunction with outer membrane vesicles, an increased or enhanced immune response to the antigen or antigens present on the surface of the OMVs is observed. The compositions may further include an adjuvant such that reduces reactogenicity to the OMV.

[0311] The composition may include an aluminum salt adjuvant. Suitable aluminum salt adjuvant includes hydroxides, phosphates or mixtures thereof. The salts can take any suitable form (e.g. gel, crystalline, amorphous etc.) with adsorption of the antigen to the salt being preferred. In an embodiment the adjuvant is an aluminum salt adjuvant, for example aluminum hydroxide. In an embodiment the adjuvant is aluminum hydroxide. In an embodiment, the OMVs are adsorbed onto aluminum hydroxide. Compositions may further include excipients, e.g., sodium salts (e.g. sodium chloride) to provide tonicity. Other salts that may be present include potassium chloride, potassium dihydrogen phosphate, disodium phosphate dehydrate, magnesium chloride, calcium chloride etc. Compositions may further include detergent e.g., TWEEN™ (polysorbate).

[0312] The composition can include a Toll-like receptor agonist adjuvant. For example, the composition can include a Toll-like receptor 4 (TLR4) agonist adjuvant or a TLR9 agonist adjuvant. The composition can include a saponin adjuvant.

[0313] Compositions may include one or more buffers. Typical buffers include: a phosphate buffer; a Tris buffer; a borate buffer; a succinate buffer; a histidine buffer (particularly with an aluminum hydroxide adjuvant); or a citrate buffer.

[0314] Immunogenic compositions according to the invention may either be prophylactic (e.g..prevent infection) or therapeutic (e.g., to treat infection). The immunogenic compositions include an immunologically effective amount of antigens, wherein the antigens are present on the surface of the OMVs.

[0315] Formulations for immunogenic composition compositions as described herein can be prepared using standard pharmaceutical formulation chemistries and methodologies that are readily available to PATENT

[0316] ATTORNEY DOCKET NO: 51838-002WO3 the reasonably skilled artisan. For example, OMVs as described herein can be combined with one or more pharmaceutically acceptable excipients or vehicles. Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances and the like, may be present in the excipient or vehicle. These excipients, vehicles and auxiliary substances are generally pharmaceutical agents that do not induce an immune response in the individual receiving the composition, and which may be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, polyethyleneglycol, hyaluronic acid, glycerol and ethanol. Pharmaceutically acceptable salts can also be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. A thorough discussion of pharmaceutically acceptable excipients, vehicles and auxiliary substances is available in Remington’s Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).

[0317] Such compositions may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable compositions may be prepared, packaged, or sold in unit dosage form, such as in ampoules or in multi-dose containers containing a preservative. Compositions may include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such compositions may further include one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a composition for parenteral administration, the active ingredient is provided in dry (e.g., a powder or granules) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition. The compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may include, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1 ,3-butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer’s solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or diglycerides.

[0318] Other parentally-administrable compositions that are useful include those which include the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation may include pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.

[0319] Alternatively, the OMVs described herein may be encapsulated, adsorbed to, or associated with particulate carriers. Suitable particulate carriers include those derived from polymethyl methacrylate polymers, as well as PLG microparticles derived from poly(lactides) and poly(lactide-co-glycolides) (see, e.g., Jeffery et al. Pharm. Res. 10:362-368, 1993). Other particulate systems and polymers can also be used, for example, polymers such as polylysine, polyarginine, polyornithine, spermine, spermidine, as well as conjugates of these molecules. PATENT

[0320] ATTORNEY DOCKET NO: 51838-002WO3

[0321] The formulated compositions will include an amount of two or more OMVs described herein that are sufficient to elicit an immunological response. An immunogenic amount can be readily determined by one of skill in the art. Such an amount will fall in a relatively broad range that can be determined through routine trials.

[0322] Compositions can include a mixture of distinct outer membrane vesicles as described herein. For example, immunogenic compositions may include outer membrane vesicles from, e.g., 2, 3, 4, 5, 6, 7, 8, or more distinct strains as described herein.

[0323] Methods of Treatment

[0324] The compositions described herein may be used to treat or prevent gonorrhea. Efficacy of prophylactic and therapeutic treatment can be tested by monitoring the N. gonorrhoeae infection after administration of an immunogenic composition. The protective effect of immunization can be tested by monitoring immune responses against immunogenic proteins in the outer membrane vesicles or other antigens after administration of the composition or immunogenic composition. Immunogenicity of compositions can be determined by administering them to test subjects and then determining standard serological parameters (e.g. levels / concentration of anti-OMV IgG and presence of functional antibodies). These immune responses will generally be determined after administration of the composition and compared to values determined before administration of the composition. Where more than one dose of the composition is administered, more than one post-administration determination may be made. The immunogenic composition may be considered efficacious if gonococcal infection at specific anatomical sites is reduced / lower in subjects that received the immunogenic composition, compared to subjects administered a control / placebo composition. In an embodiment the immunogenic composition reduces the risk of infection by at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or more, e.g., in a population of subjects.

[0325] In a further aspect is provided a method for the treatment or prevention of disease caused by Neisseria (for example N. gonorrhoeae) in a subject in need thereof by administering to the subject a therapeutically effective amount of an immunogenic composition as described herein.

[0326] In a further aspect is provided a method for immunizing a subject in need thereof against Neisseria by administering an immunologically effective amount of the immunogenic composition to the subject.

[0327] In a further aspect is provided a method for generating an immune response in a subject by administering an immunogenic composition as described herein to a subject. In a further aspect is provided a method for generating an immune response against Neisseria infection (for example N. gonorrhoeae infection) in a subject by administering an immunogenic composition to a subject.

[0328] Dosage treatment can be a single dose schedule or a multiple dose schedule. In a further aspect is provided a use of the immunogenic composition, wherein at least two doses of the composition are administered to a subject. Multiple doses may be used in a primary immunization schedule and / or in a booster immunization schedule. A primary dose schedule may be followed by a booster dose schedule. Therefore, in a further embodiment is provided the use of the immunogenic composition wherein at least two doses of the composition are administered to a subject, and at least one dose is a booster dose. PATENT

[0329] ATTORNEY DOCKET NO: 51838-002WO3

[0330] In a further aspect is provided the immunogenic composition for use, the method or the use wherein the subjects are adolescents and / or adults (e.g. young adults).

[0331] In some embodiments, the immunogenic composition is administered via an intramuscular or intraperitoneal route. In an embodiment the immunogenic composition is administered intramuscularly. In an embodiment the route of administration remains unchanged between the first and any subsequent immunization.

[0332] The methods may also include optional administration of a primary immunization regimen and subsequent booster doses of OMVs as described herein. The primary immunization regimen is defined by the number of doses administered and time intervals between the doses. The primary immunization regimen is optimized to achieve an optimal initial protection in newly immunized subjects. The number of doses in a primary immunization regimen is typically 1 -4. The time period for the completion of a primary immunization regimen is 1 -12 months. These primary immunization doses can be administered at, for example, 1 -11 months after the initial immunization, as determined to be appropriate by one skilled in the art. Thus, in various examples, one or more primary immunization doses can be administered at 2-10, 3-9, 4-8, 5-7, or 6 months after the initial immunization. Booster doses can be administered following the primary immunization regimen to increase the longer-term duration of protection. Booster doses can be administered at 1 -10 years following the first dose of the primary series and are typically a single dose. The amount of OMVs present in all primary immunization doses and booster doses is typically the same, but can vary and be, for example, an amount as described elsewhere herein, or optionally can be 5-20%, e.g., 10%, of the amount of the initial dose.

[0333] Compositions as described herein can be delivered to a mammalian subject (e.g., a human or other mammal described herein) using a variety of known routes and techniques. For example, a composition can be provided as an injectable solution, suspension, or emulsion, and administered via intramuscular, subcutaneous, intradermal, intracavity, parenteral, epidermal, intraarterial, intraperitoneal, or intravenous injection using a conventional needle and syringe, or using a liquid jet injection system. Compositions can also be administered topically to skin or mucosal tissue, such as nasally, intratracheally, intestinal, rectally, or vaginally, or provided as a finely divided spray suitable for respiratory or pulmonary administration. Other modes of administration include oral administration, suppositories, and active or passive transdermal delivery techniques.

[0334] The compositions described herein can be administered to a subject (e.g., a human subject that has or is at risk of developing a N. gonnorhoeae infection) in an amount that is compatible with the dosage formulation and that will be prophylactically and / or therapeutically effective. An appropriate effective amount will fall in a relatively broad range but can be readily determined by one of skill in the art by routine trials.

[0335] Compositions may be prepared into unit-dose or multiple-dose preparations by those skilled in the art using a pharmaceutically acceptable carrier and / or excipient according to a method known in the art. PATENT

[0336] ATTORNEY DOCKET NO: 51838-002WO3

[0337] Kits

[0338] The invention also includes kits that can be used to carry out the methods. Thus, kits can include a composition as described herein, e.g., one or more, e.g., two or more OMVs, as described herein, optionally in the form of an immunogenic composition including an adjuvant, such as, for example, aluminum hydroxide. In some examples, the OMVs are present in a container (e.g., a glass vial) in liquid form (e.g., in water or a buffered salt solution, such as, 10 mM sodium phosphate, pH 6.5 or 7.0, and 154 mM sodium chloride. In other examples, the OMVs are present in a container (e.g., a glass vial) in lyophilized form. In such examples, the kit may optionally also include a diluent (e.g., water or a buffered salt solution) for reconstitution of the lyophilized OMVs into liquid form prior to administration. The OMVs may also be present in another formulation, as described herein, or as is known to be acceptable in the art. The amount of OMVs and, optionally, adjuvant present in the compositions of the present kits can be, for example, as described above. Thus, for example, the kits can include OMVs in an amount to facilitate the administration of a dose as described herein.

[0339] The kit components can be provided in dosage form to facilitate administration and can optionally include materials required for administration and / or instructions for subject treatment consistent with, for example, the methods described herein.

[0340] For example, the kit can include instructions for use, which guides the user (e.g., the physician) with respect to the administration of the OMV immunogenic composition (e.g., at the point of care location).

[0341] The kit may be packaged in materials suitable for storage in a refrigerator at a temperature of between 35°F and 46°F (2°C and 8°C). The desired average refrigerator immunogenic composition storage temperature is ~40°F (~5°C). Exposure to temperatures outside these ranges may result in reduced immunogenic composition potency and increased risk of immunogenic composition-preventable diseases.

[0342] EXAMPLES

[0343] The following examples are intended to illustrate the invention. These are not meant to limit the invention in any way.

[0344] Example 1. Phylogenetic analysis of outer membrane proteins of Neisseria gonorrhoeae

[0345] N. gonorrhoeae is a pathogenic bacterium that can cause infections in humans. N. gonorrhoeae has developed resistance to many antibiotic treatments, posing a challenge to treatment of its infections. Several immunogenic compositions have been developed that can reduce the risk of a N. gonorrhoeae infection. Some of these immunogenic compositions include OMVs isolated from N. gonorrhoeae, or related species. However, the immunogenic compositions have limited efficacy and do not protect individuals from a wide variety of N. gonorrhoeae strains.

[0346] An immunogenic composition that combined evolutionarily distant antigens from different N. gonorrhoeae strains could increase the number of strains against which an immunogenic composition provides protection. From the N. gonorrhoeae genome, 45 proteins of interest were identified that potentially localized to outer membranes. Of these 45 proteins, twenty were either detected at OMVs or PATENT

[0347] ATTORNEY DOCKET NO: 51838-002WO3 had a reported serum bactericidal activity. Five of these proteins were selected for phylogenetic analyses: PorB, FetA, NspA, TbpB, and NHBA.

[0348] 153 full-length N. gonorrhoeae genomes were compiled. Sequences for each full-length protein of interest were then compared using Geneious Prime 2024.0 software. Diversity scores of each protein were calculated as described in Marjuki et al. (mBio, 10(5) :e01668-19, 2019) and are shown in Table 1 below. Phylogenetic trees were generated using Geneious Prime 2024.0 software and analyzed. At least two distinct clusters of strains emerged from the phylogenetic trees for each protein, suggesting that each pair of proteins could cover or be representative of the greatest number of circulating strains when choosing a protein from each cluster. When comparing PorB across the different strains, similar results were obtained whether comparing full-length protein or only comparing the surface-exposed major variable regions. Thus, only full-length protein sequences were used for the remaining analyses.

[0349] Table 1. Diversity scores of selected N. gonorrhoeae proteins

[0350] Example 2. Evaluation of immunogenicity of native OMVs for protection against sialylated bacteria

[0351] N. gonorrhoeae can be sialylated or non-sialylated in vivo. In particular lipooligosaccharides on the outer membrane of the N. gonorrhoeae can be sialylated. Sialylation is an important driver of N. gonorrhoeae infection, transmission, and colonization in its host. For example, sialylated bacteria promote asymptomatic infections as well as transmission from males to females, whereas non-sialylated bacteria promote symptomatic infection in males and transmission from females to males. Thus, it was hypothesized that an effective N. gonorrhoeae vaccine should induce antibodies that kill both sialylated and non-sialylated N. gonorrhoeae. It was also hypothesized that sialylated N. gonorrhoeae may not be targeted by antibodies against non-sialylated LOS.

[0352] OMVs of six strains of N. gonorrhoeae (FA1090, F62, WHO-O, WHO-F, FA19, and SK92-679) were isolated from cultured bacteria. Mice were inoculated with the isolated OMVs at a dose of 20 pg / dose at weeks 0 and 3. At week 5, serum was collected from the mice and pooled.

[0353] A panel of 15 test strains of N. gonorrhoeae (WHO-Z, MS11 , WHO-W, F-18, FA19, WHO-L, WHO-M, WHO-O, WHO-N, WHO-F, FA1090, WHO-P, WHO-V, WHO-Y, and SK92-679) were separately cultured with or without 2 pg / mL cytidine Monophospho-N-Acetylneuraminic acid (CMP-NANA) to produce sialylated and non-sialylated bacteria, respectively.

[0354] Serum from the inoculated mice was then tested in a serum bactericidal assay (SBA) with human complement and each of the sialylated and non-sialylated strains of N. gonorrhoeae bacteria. Sialylated bacteria were more difficult to kill than non-sialylated bacteria by sera in SBA (FIGS. 1 A-1 D). This trend was observed both for strains expressing PorB. IA and for strains expressing PorB. IB. Serum from mice PATENT

[0355] ATTORNEY DOCKET NO: 51838-002WO3 that were not inoculated with OMVs was not effective at killing bacteria by SBA, suggesting that the killing was due to inoculation of mice with OMVs (FIG. 2).

[0356] A similar protocol was performed where individual mice were inoculated with OMVs from multiple strains of N. gonorrhoeae. Sera from mice that were inoculated with OMVs from WHO-F and FA1090 N. gonorrhoeae or from WHO-F, SK92-679, and FA1090 N. gonorrhoeae exhibited a greater coverage of strains that were killed by SBA than sera from mice that were inoculated with OMVs from a single strain (FIG. 3).

[0357] Bexsero was a previous vaccine candidate for N. gonorrhoeae. Although Bexsero was effective in inducing an immune response that could result in killing of 9 of 12 non-sialylated test strains of N. gonorrhoeae by SBA, the sera were unable to induce killing of any test strains that were grown in the presence of CMP-NANA (i.e., sialylated bacteria) (FIGS. 4A and 4B). Natively-extracted OMVs from N. gonorrhoeae induced comparable killing of non-sialylated bacteria to the Bexsero vaccine (FIGS. 5A and 5B); however, unlike the Bexsero vaccine, these natively extracted OMVs were effective in killing some test strains of sialylated N. gonorrhoeae by SBA (FIG. 6).

[0358] Example 3. Evaluation of lipidoligosaccharides

[0359] LOS are the immunodominant antigen present in native OMVs. To assist in the rationale for selection of strains of N. gonorrhoeae to be used in a vaccine, LOS structures for different N. gonorrhoeae (FA1090, F62, WHO-O, WHO-F, WHO-K, FA19, and SK92-679) were analyzed.

[0360] The strains were evaluated for the presence of LOS structures recognized by commercially available 2C7 and 4C8 antibodies (FIG. 7). Additionally, silver staining was used to estimate length of LOS alpha chains (FIG. 8A and 8B).

[0361] It was then hypothesized that western blots could be used to probe for specific LOS antibodies in immune serum. Mice were vaccinated with Bexsero and sera were collected from the mice. WHO-O and WHO-F N. gonorrhoeae were grown in the presence or absence of CMP-NANA to generate sialylated and non-sialylated N. gonorrhoeae, respectively. 0.1 pg of lysates of sialylated and non-sialylated WHO-O and WHO-F N. gonorrhoeae were loaded on a gel. The gel was stained with serum from the mice vaccinated with Bexsero or with anti-2C7 antibodies. The serum was able to identify 4HexG- LOS, which is present on both Bexsero immunogens and WHO-O N. gonorrhoeae; the anti-2C7 antibodies detected WHO-F bacteria (FIGS. 9A and 9B).

[0362] Finally, pellets of N. gonorrhoeae strain FA1090 and N. gonorrhoeae strain FA19 were analyzed by mass spectrometry (FIGS. 10A and 10B)

[0363] Summary results from these analyses are shown in FIGS. 11 A and 11 B.

[0364] Example 4. Comparison of native OMVs and detergent-extracted OMVs

[0365] Antibodies can recognize both outer membrane proteins (OMPs) and LOS structures as key antigens to trigger bactericidal activities.

[0366] It was hypothesized that detergent-extracted OMVs (dOMVs) have about a 10-fold reduction of LOS and could steer the immune system to generate more anti-OMP antibodies, whose bactericidal activities are less susceptible to N. gonorrhoeae sialylation. In addition, dOMVs, which are extracted from PATENT

[0367] ATTORNEY DOCKET NO: 51838-002WO3 bacterial pellets, were hypothesized to potentially have a different antigen composition as compared to spontaneously secreted nOMV.

[0368] It was confirmed that WHO-F dOMVs were indeed significantly different from native OMVs (nOMVs) in both LOS and OMP contents. dOMVs exhibited lower levels of LOS and PorB protein but increased levels of NHBA and MetQ, compared to nOMVs.

[0369] WHO-F N. gonorrhoeae bacteria were cultured in a 1 L flask and then centrifuged for 10 minutes at 1000 x g. The supernatant was then centrifuged at 5000 x g for 10 minutes. This second supernatant was passed through a 0.2 pm filter to collect nOMVs. dOMVs were isolated from the pellet generated from the 1000 x g centrifugation. This pellet was resuspended in 0.1 M Tris-HCI, 10 mM ethylenediaminetetraacetic Acid (EDTA), 0.5% sodium deoxycholate (DOC). The resuspended pellet was incubated at 37°C at 200 rotations per minute for 30 minutes. This solution was centrifuged at 5000 x g for 10 minutes, and the resulting supernatant was passed through a 0.2 pm filter to collect dOMVs. OMVs were further purified using tangential flow filtration (SARTOCON® 300K), Polish (CAPTO™ Core 400), and concentrated (Amicon 100K). Coomassie staining and western blots revealed that nOMVs and dOMVs had different levels of OMPs such as PorB, NspA, NHBA, and MetQ (FIGS. 12A and 12B). For example, nOMVs exhibited higher levels of PorB and NspA than dOMVs, whereas dOMVs exhibited higher levels of MetQ and NHBA than nOMVs. Likewise, nOMVs and dOMVs exhibited differing levels of LOS as analyzed by silver staining (FIG. 13).

[0370] Next, the culturing protocol was modified to include a test culture of WHO-F N. gonorrhoeae bacteria that were cultured with 20 pg / mL CMP-NANA to generate sialylated nOMVs (Sia-nOMVs) and sialylated dOMVs (Sia-dOMVs). Additionally, the OMV purification was modified to include a 1000 U / L benzonase nuclease treatment step for 24-72 hours at 4°C before tangential flow filtration for nOMVs and Sia-nOMVs. Purification of dOMVs and Sia-dOMVs was modified to include a detergent removal step (using a Detergent Removal Spin Column (Thermo Scientific)), a benzonase treatment step as for purification of Sia-nOMVs and nOMVs, and an additional tangential flow filtration step in that order, after the first tangential flow filtration step. Coomassie staining revealed that sialylation had a minimal impact on OMP composition in WHO-F nOMVs and dOMVs (FIG. 14). Silver staining and western blots with 2C7 antibodies revealed that dOMVs consistently had fewer LOS species than nOMV did and revealed that sialylation did not affect LOS quantity in either nOMVs or dOMVs (FIG. 15).

[0371] Enzyme-linked immunosorbent assays (ELISAs) were also used to assess LOS and OMP presence on N. gonorrhoeae or N. gonorrhoeae OMVs with and without sialic acid. N. gonorrhoeae were grown in the presence or absence of 2 pg / mL sialic acid and then centrifuged at 500 x g for 5 minutes. The pellets were resuspended in phosphate buffered saline (PBS). ELISAs were performed by coating wells at 2°C - 8°C overnight or at 37°C for 1.5 - 2 hours. The wells were then blocked for 10 minutes at room temperature. Epitopes were then detected with primary antibodies (at room temperature for 1.5 - 2 hours), horseradish peroxidase-conjugated secondary antibodies (at room temperature for 1 hour), and developed with 3,3',5,5'-tetramethylbenzidine (TMB) substrate (at room temperature for 30 minutes). ELISAs confirmed that dOMVs consistently had fewer LOS than nOMV and confirmed that sialylation did not affect LOS quantity in nOMV or dOMV (FIG. 16). PATENT

[0372] ATTORNEY DOCKET NO: 51838-002WO3

[0373] ELISAs also were used to assess accumulation of outer membrane proteins on sialylated and non-sialylated nOMVs and dOMVs. These assays revealed that dOMV had fewer PorB proteins but a greater number of NHBA and MetQ proteins compared to nOMVs; western blots confirmed these findings (FIG. 17A and 17B). LOS and protein levels were then compared across sialylated and non-sialylated bacteria, nOMVs, and dOMVs. Sialylation decreased detection of LOS and PorB. IA protein on N. gonorrhoeae bacteria (FIG. 18). Bacteria showed greater accumulation than nOMVs of exposed FetA protein and less accumulation of BamA and TamA proteins; nOMVs showed the greatest accumulation of exposed NspA proteins (FIG. 19). Data are summarized in FIG. 20.

[0374] Titers generated from dOMVs and nOMVs were compared by SBAs. Surprisingly, even though dOMVs had more OMPs present than nOMVs did, nOMVs induced greater SBA titers in both sialylated and non-sialylated contexts (FIGS. 21 A and 21 B).

[0375] Example 5. Biochemical Characterization of nOMVs from Genetically Engineered FA1090 Mutants

[0376] N. gonorrhoeae of strain FA1090 were mutated to knock-out reduction modifiable protein (rmpM) gene or to knock-out both rmpM and lipid A biosynthesis lauroyl acyltransferase (IpxLI ) genes. These mutations caused a global change in protein accumulation at nOMVs as assessed by SDS-PAGE (FIG. 22). nOMVs from genetically engineered FA1090 exhibited different levels of NHBA and MetQ compared to WT nOMVs; specifically, nOMVs from ArmpM bacteria had more NHBA protein than nOMVs from WT bacteria, and nOMVs from ArmpM Alpxll bacteria had more MetQ protein than nOMVs from WT bacteria (FIG. 23 A and 23B). Mutations also affected protein levels of BamA, TamA, FetA, NspA, and TbpB proteins (FIG. 24).

[0377] Example 6. Evaluation of Recombinant Proteins as Vaccine Components

[0378] Recombinant proteins were assessed for their ability to induce a bactericidal immune response. Rabbits were inoculated with one of nine different recombinant proteins (NHBA, MetQ, BamA, TamA, PorBIA, PorBIB, TbpB, FetA, or NspA). Serum was collected and the bactericidal activity was assessed by human SBA. The recombinant proteins were able to elicit bactericidal elicit bactericidal antibodies toward N. gonorrhoeae (FIGS. 25A and 25B). However, recombinant protein-induced SBA titers were low in non-sialylated SBA assays, and the titers reduced to close to background in sialylated SBA assays. This suggests that recombinant proteins may be most effective when combined with antigens that can produce an immune response effective against sialylated bacteria.

[0379] Example 7. Process Development

[0380] A process was developed to generate research-grade OMVs. A stock of N. gonorrhoeae bacteria were pre-cultured and then moved to a larger volume for production. OMVs were than harvested, as described above. After filtration, the OMVs were treated with nuclease (e.g., benzonase) as described in Example 4. The OMVs were then purified with tangential flow filtration (ultrafiltration and diafiltration), polished with CAPTO™ Core 400 multipmodal chromatography resin, purified with another round of tangential flow filtration, and then filtered with a sterile filter. This led to OMVs with an acceptable concentration, size distribution, purity, and yield (FIGS. 26A-26D). PATENT

[0381] ATTORNEY DOCKET NO: 51838-002WO3

[0382] Several filters for tangential flow filtration were analyzed; SARTOCON ® Slice 50, HYDROSART ®, 50 cm2, (Sartorius) emerged as the top-choice due to its pore size and processing time (FIG. 27).

[0383] N. gonorrhoeae of strain FA1090 were grown in the following conditions: •Inoculation: optical density at 600 nm (OD600) = 0.352 •Medium: modified American Type Culture Collection (ATCC) medium •pH: 7.0 + / - 0.05 (0.5M NaOH + 0.5M NaHCO3) •Temperature: 37°C •Atmosphere: 95% Air, 5% CO2 •Agitation: 300rpm - 900rpm •Dissolved oxygen (DO): 30% (Csc - agitation, air, 02) •Glucose: 88 g / L at the start point •Harvest time: stationary growth phase (monitor OD600)

[0384] It was noted that DO sharply decreased immediately following inoculation. Increasing agitation could have helped increase DO but only lasted for a couple of hours. The heavy foam generated during fermentation was hypothesized to significantly reduce DO. Although antifoam was not preferred for nOMV production, heavy foam significantly impacted oxygen transfer rate during fermentation. It was determined that antifoam was desirable for upstream processes. The effect of including Antifoam 204 (Sigma) in bacterial cultures was assessed. Antifoam 204 did not impact growth of N. gonorrhoeae of strain FA1090, nOMV characteristics, or nOMV size (FIGS. 28A-28D).

[0385] Other Embodiments

[0386] All publications, patents, and patent applications mentioned in the above specification are hereby incorporated by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

[0387] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the invention that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims.

[0388] Other embodiments are within the claims.

Claims

PATENTATTORNEY DOCKET NO: 51838-002WO3CLAIMS1 . A composition comprising outer membrane vesicles (OMVs) from a plurality of strains of Neisseria gonorrhoeae (N. gonorrhoeae), wherein the composition comprises a first OMV isolated from a first strain of N. gonorrhoeae and a second OMV from a second strain of N. gonorrhoeae that is different from the first strain.

2. The composition of claim 1 , wherein the first strain expresses a first major outer membrane protein (PorB) protein and the second strain expresses a second PorB protein having at most 95% sequence identity to the first PorB protein.

3. The composition of claim 2, wherein the second PorB protein has at most 75% sequence identity to the first PorB protein.

4. The composition of claim 2 or 3, wherein the first strain expresses a IB allele of PorB protein (PorB. IB) and the second strain expresses a IA allele of PorB protein (PorB.IA).

5. The composition of any one of claims 1 -4, wherein the first strain expresses a first neisserial heparin binding antigen (NHBA) protein, and the second strain expresses a second NHBA protein having at most 99% sequence identity to the first NHBA protein.

6. The composition of claim 5, wherein the second NHBA protein has at most 95% sequence identity to the first NHBA protein.

7. The composition of any one of claims 1 -6, wherein the first strain expresses a first neisserial surface protein A (NspA) protein, and the second strain expresses a second NspA protein having at most 99.5% sequence identity to the first NspA protein.

8. The composition of claim 7, wherein the second NspA protein has at most 95% sequence identity to the first NspA protein.

9. The composition of any one of claims 1 -8, wherein the first strain expresses a first transferrin binding protein B (TbpB) protein, and the second strain expresses a second TbpB protein having at most 95% sequence identity to the first TbpB protein.

10. The composition of claim 9, wherein the second TbpB protein has at most 75% sequence identity to the first TbpB protein.11 . The composition of any one of claims 1 -10, wherein the first strain expresses a first ferric enterobactin transporter (FetA) protein, and the second strain expresses a second FetA protein having at most 98% sequence identity to the first FetA protein.PATENTATTORNEY DOCKET NO: 51838-002WO312. The composition of claim 11 , wherein the second FetA protein has at most 95% sequence identity to the first FetA protein.

13. The composition of any one of claims 1 -12, wherein the first strain and / or the second strain comprises a genetic modification that reduces or abolishes expression and / or function of a lipid A biosynthesis lauroyl acyltransferase (IpxLI ) gene, mRNA, and / or polypeptide.

14. The composition of any one of claims 1 -13, wherein each of the strains comprises a genetic modification that reduces or abolishes expression and / or function of IpxLI .

15. The composition of claim 13 or 14, wherein the genetic modification that reduces or abolishes expression and / or function of IpxLI is a mutation of IpxLI .

16. The composition of claim 13 or 14, wherein the genetic modification that reduces or abolishes expression and / or function of IpxLI suppresses IpxLI in a strain that expresses wild-type IpxLI .

17. The composition of any one of claims 13-16, wherein the genetic modification that reduces or abolishes expression and / or function of IpxLI comprises a point mutation, a knock-out, or a mutation to a promoter.

18. The composition of any one of claims 1 -17, wherein the first strain and / or the second strain comprises a genetic modification that reduces or abolishes expression and / or function of a reduction modifiable protein (rmpM) gene, mRNA, and / or polypeptide.

19. The composition of any one of claims 1 -18, wherein each of the strains comprises a genetic modification that reduces or abolishes expression and / or function of rmpM.

20. The composition of claim 18 or 19, wherein the genetic modification that reduces or abolishes expression and / or function of rmpM is a mutation of rmpM.21 . The composition of claim 18 or 19, wherein the genetic modification that reduces or abolishes expression and / or function of rmpM suppresses rmpM in a strain that expresses wild-type rmpM.

22. The composition of any one of claims 18-21 , wherein the genetic modification that reduces or abolishes expression and / or function of rmpM comprises a point mutation, a knock-out, or a mutation to a promoter.

23. The composition of any one of claims 1 -22, further comprising a third OMV from a third strain of N. gonorrhoeae that is different from both the first strain and the second strain.PATENTATTORNEY DOCKET NO: 51838-002WO324. The composition of claim 23, wherein the first strain expresses a first PorB protein, the second strain expresses a second PorB protein, and the third strain expresses a third PorB, wherein the third PorB protein has at most 95% sequence identity to the first and / or the second PorB protein.

25. The composition of claim 24, wherein the third PorB protein has at most 75% sequence identity to the first and / or the second PorB protein.

26. The composition of claim 24 or 25, wherein the third strain expresses a PorB. IB, and the first strain and / or the second strain expresses a PorB.IA.

27. The composition of claim 24 or 25, wherein the third strain expresses a PorB.IA, and the first strain and / or the second strain expresses a PorB. IB.

28. The composition of any one of claims 23-27, wherein the first strain expresses a first NHBA protein, the second strain expresses a second NHBA protein, and the third strain expresses a third NHBA protein, wherein the third NHBA protein has at most 99% sequence identity to the first and / or the second NHBA protein.

29. The composition of claim 28, wherein the third NHBA protein has at most 95% sequence identity to the first and / or the second NHBA protein.

30. The composition of any one of claims 23-29, wherein the first strain expresses a first NspA protein, the second strain expresses a second NspA protein, and the third strain expresses a third NspA protein, wherein the third NspA protein has at most 99.5% sequence identity to the first and / or the second NspA protein.31 . The composition of claim 30, wherein the third NspA protein has at most 95% sequence identity to the first and / or the second NspA protein.

32. The composition of any one of claims 23-31 , wherein the first strain expresses a first TbpB protein, the second strain expresses a second TbpB protein, and the third strain expresses a third TbpB, wherein the third TbpB protein has at most 95% sequence identity to the first and / or the second TbpB protein.

33. The composition of claim 32, wherein the third TbpB protein has at most 75% sequence identity to the first and / or the second TbpB protein.

34. The composition of any one of claims 23-33, wherein the first strain expresses a first FetA protein, the second strain expresses a second FetA protein, and the third strain expresses a third FetA, wherein the third FetA protein has at most 98% sequence identity to the first and / or the second FetA protein.PATENTATTORNEY DOCKET NO: 51838-002WO335. The composition of claim 34, wherein the third FetA protein has at most 95% sequence identity to the first and / or the second FetA protein.

36. The composition of any one of claims 23-35, wherein the third strain comprises a genetic modification that reduces or abolishes expression and / or function of rmpM.

37. The composition of claim 36, wherein the genetic modification that reduces or abolishes expression and / or function of rmpM in the third strain is a mutation of rmpM.

38. The composition of claim 36 or 37, wherein the genetic modification that reduces or abolishes expression and / or function of rmpM in the third strain suppresses rmpM in a strain that expresses wild-type rmpM.

39. The composition of any one of claims 36-38, wherein the genetic modification that reduces or abolishes expression and / or function of rmpM in the third strain comprises a point mutation, a knock-out, or a mutation to a promoter.

40. The composition of any one of claims 1 -39, wherein at least one OMV was extracted using detergent.41 . The composition of any one of claims 1 -39, wherein at least one OMV was naturally secreted.

42. The composition of any one of claims 1 -41 , comprising OMVs from a plurality of strains of N. gonorrhoeae.

43. The composition of any one of claims 1 -42, wherein at least one of the OMVs comprises a sialic acid moiety.

44. A composition comprising a first OMV isolated from a first strain of N. gonorrhoeae that was incubated with sialic acid or a precursor of sialic acid.

45. The composition of claim 44, wherein the first OMV is a native OMV.

46. The composition of claim 44 or 45, further comprising a second OMV isolated from a second strain of N. gonorrhoeae that was incubated with sialic acid or a precursor of sialic acid.

47. The composition of claim 46, wherein the second OMV is a native OMV.PATENTATTORNEY DOCKET NO: 51838-002WO348. The composition of claim 46 or 47, wherein the second strain of N. gonorrhoeae comprises a genetic modification that reduces or abolishes expression and / or function of a rmpM gene, mRNA, and / or polypeptide.

49. The composition of any one of claims 44-48, wherein the first strain of N. gonorrhoeae comprises a genetic modification that reduces or abolishes expression and / or function of a rmpM gene, mRNA, and / or polypeptide.

50. The composition of any one of claims 44-49, further comprising a third OMV from a third strain of N. gonorrhoeae.51 . The composition of claim 50, wherein the third OMV is a detergent-extracted OMV.

52. The composition of claim 50 or 51 , wherein the third OMV comprises PorB.IA, PorB.IB, translocator assembly module A (TamA), lipopolysaccharide transport protein D (LptD), p-barrel assembly machinery A (BamA), NspA, FetA, or opacity (Opa) proteins.

53. The composition of claim 52, wherein the third OMV comprises PorB.IA, PorB.IB, LptD, BamA, or NspA.

54. The composition of any one of claims 50-53, wherein the third strain of N. gonorrhoeae is different from the first strain of N. gonorrhoeae.

55. The composition of any one of claims 48-54, wherein the third strain of N. gonorrhoeae is different from the second strain of N. gonorrhoeae.

56. The composition of any one of claims 48-55, wherein the second strain of N. gonorrhoeae is different from the first strain of N. gonorrhoeae.

57. The composition of any one of claims 50-53, wherein the third strain of N. gonorrhoeae is the same strain as the first strain of N. gonorrhoeae.

58. The composition of any one of claims 50-53, wherein the third strain of N. gonorrhoeae is the same strain as the second strain of N. gonorrhoeae.

59. The composition of any one of claims 44-58, wherein the composition further comprises OMVs from a plurality of strains of N. gonorrhoeae.PATENTATTORNEY DOCKET NO: 51838-002WO360. A composition comprising OMVs from N. gonorrhoeae, wherein the composition comprises a first OMV isolated from a first strain of N. gonorrhoeae and a second OMV from a second strain of N. gonorrhoeae, wherein the first OMV is a native OMV and the second OMV is a detergent-extracted OMV.61 . The composition of claim 60, wherein the first strain of N. gonorrhoeae was incubated with sialic acid or a precursor of sialic acid.

62. The composition of claim 60 or 61 , wherein the first OMV comprises 2HexG+, 3HexG+, 4HexG+, 4HexG-, 5HexG+, and / or 5HexG- lipooligosaccharides (LOS) on a surface of the OMV.

63. The composition of any one of claims 60-62, wherein expression of NHBA, MetQ, TbpB, and / or Pilin proteins on a surface of the first OMV is reduced following extraction of the first OMV.

64. The composition of any one of claims 60-63, wherein the second OMV comprises PorB.IA, PorB.IB, TamA, LptD, BamA, NspA, FetA, and / or Opa proteins on a surface of the OMV.

65. The composition of any one of claims 60-64, wherein expression of PorB.IA, PorB.IB, TamA, LptD, BamA, NspA, FetA, and / or Opa proteins on a surface of the second OMV is increased following the detergent extraction of the second OMV.

66. The composition of any one of claims 60-65, wherein expression of NHBA, MetQ, TbpB, and / or Pilin proteins on a surface of the second OMV is reduced following detergent extraction of the second OMV.

67. The composition of any one of claims 60-66, wherein accumulation of LOS on a surface of the second OMV is reduced following detergent extraction of the second OMV.

68. The composition of any one of claims 60-67, wherein the second OMV comprises PorB.IA, PorB.IB, LptD, BamA, or NspA.

69. The composition of any one of claims 60-68, further comprising a third OMV isolated from a third strain of N. gonorrhoeae that was incubated with sialic acid or a precursor of sialic acid.

70. The composition of claim 69, wherein the third OMV is a native OMV.71 . The composition of claim 69 or 70, wherein the third strain of N. gonorrhoeae comprises a genetic modification that reduces or abolishes expression and / or function of a rmpM gene, mRNA, and / or polypeptide.

72. The composition of any one of claims 69-71 , wherein the third strain of N. gonorrhoeae is different from the first strain of N. gonorrhoeae.PATENTATTORNEY DOCKET NO: 51838-002WO373. The composition of any one of claims 69-72, wherein the third strain of N. gonorrhoeae is different from the second strain of N. gonorrhoeae.

74. The composition of any one of claims 69-73, wherein the second strain of N. gonorrhoeae is different from the first strain of N. gonorrhoeae.

75. The composition of any one of claims 69-71 , wherein the second strain of N. gonorrhoeae is the same strain as the first strain of N. gonorrhoeae.

76. The composition of any one of claims 69-71 , wherein the third strain of N. gonorrhoeae is the same strain as the second strain of N. gonorrhoeae.

77. The composition of any one of claims 60-76, wherein the first strain of N. gonorrhoeae comprises a genetic modification that reduces or abolishes expression and / or function of a rmpM gene, mRNA, and / or polypeptide.

78. The composition of any one of claims 1 -77, further comprising a recombinant protein.

79. The composition of claim 78, wherein the recombinant protein is NHBA, L-methionine binding lipoprotein (MetQ), BamA, TamA, LptD, TbpB, FetA, NspA, Opa, or Pilin.

80. The composition of claim 79, wherein the recombinant protein is NHBA, MetQ, BamA, TamA, LptD, or TbpB.81 . The composition of claim 80, wherein the recombinant protein is NHBA, MetQ, or TbpB.

82. The composition of claim 81 , wherein the recombinant protein is TbpB.

83. The composition of claim 81 , wherein the recombinant protein is NHBA.

84. The composition of any one of claims 1 -83, further comprising an adjuvant.

85. The composition of claim 84, wherein the adjuvant is alum, a Toll-like receptor 4 (TLR4) agonist, a TLR9 agonist, or saponin.

86. The composition of claim 85, wherein the adjuvant is alum.

87. A pharmaceutical composition comprising the composition of any one of claims 1 -86.PATENTATTORNEY DOCKET NO: 51838-002WO388. An immunogenic composition comprising the composition of any one of claims 1 -86 or the pharmaceutical composition of claim 87.

89. A method of treating or preventing a N. gonorrhoeae infection in a subject, the method comprising administering to the subject the composition of any one of claims 1 -86, the pharmaceutical composition of claim 87, or the immunogenic composition of claim 88.

90. A method of producing an immune response in a subject, the method comprising administering to the subject the composition of any one of claims 1 -86, the pharmaceutical composition of claim 87, or the immunogenic composition of claim 88.91 . The method of claim 89 or 90, wherein the method comprises administering a plurality of doses of the composition.

92. The method of any one of claims 89-91 , wherein the method provides protection against N. gonorrhoeae for at least one year.

93. The method of any one of claims 89-92, wherein the composition provides resistance to at least 80% of genetically characterized N. gonorrhoeae strains.

94. The method of any one of claims 89-93, wherein the composition is administered to a population of subjects and reduces the risk of infection of N. gonorrhoeae in the population of subjects by at least 50%.

95. A composition comprising a cell from a first strain of N. gonnorhoeae and a cell from a second strain of N. gonnorhoeae, wherein the second strain is different from the first strain.

96. The composition of claim 95, further comprising a cell from a third strain of N. gonnorhoeae, wherein the third strain is different from both the first strain and the second strain.

97. A method of making an isolated OMV from a first strain of N. gonorrhoeae, the method comprising:(a) incubating a N. gonorrhoeae with sialic acid or a precursor of sialic acid; and(b) isolating an OMV from the N. gonorrhoeae.

98. The method of claim 97, wherein isolating the OMV comprises native extraction of the OMV.

99. The method of claim 98, wherein the native extraction comprises centrifuging a culture of N. gonorrhoeae and collecting a first supernatant.10O.The method of claim 99, wherein the culture is centrifuged at about 1000 x g for about 5 minutes.PATENTATTORNEY DOCKET NO: 51838-002WO3101 . The method of claim 99 or 100, wherein the native extraction further comprises centrifuging the first supernatant and collecting a second supernatant.102.The method of claim 101 , wherein the culture is centrifuged at about 5000 x g for about 10 minutes.

103. The method of any one of claims 99-102, wherein the native extraction further comprises filtering the second supernatant.

104. The method of claim 103, wherein the second supernatant is filtered using an about 0.2 pm filter.

105. The method of any one of claims 99-104, wherein the native extraction further comprises tangential flow filtration, multimodal chromatography, and / or concentration.

106. The method of any one of claims 97-105, wherein the N. gonorrhoeae comprises a genetic modification that reduces or abolishes expression and / or function of a rmpM gene, mRNA, and / or polypeptide.

107. An OMV from N. gonorrhoeae produced by the method of any one of claims 97-106.

108. An engineered OMV from N. gonorrhoeae comprising a sialic acid moiety.

109. The engineered OMV of claim 108, wherein at least 50% of LOS on a surface of the OMV are sialylated.

110. The engineered OMV of claim 108 or 109, wherein the sialic acid moiety is on a 2HexG+, 3HexG+, 4HexG+, 4HexG-, 5HexG+, and / or 5HexG- LOS.

111. The engineered OMV of any one of claims 108-110, wherein the N. gonorrhoeae comprises a genetic modification that reduces or abolishes expression and / or function of a rmpM gene, mRNA, and / or polypeptide.

112. The engineered OMV of any one of claims 108-111 , wherein the OMV is isolated.

113. The engineered OMV of any one of claims 108-112, wherein the OMV is a native OMV.

114. A method of identifying a first candidate strain of N. gonorrhoeae as suitable for producing an immunogenic OMV, the method comprising:(a) culturing the first candidate strain of N. gonorrhoeae in the presence of sialic acid or a sialic acid precursor;(b) collecting a first OMV from the N. gonorrhoeae;PATENTATTORNEY DOCKET NO: 51838-002WO3(c) inoculating a mammal with the OMV;(d) collecting serum from the mammal;(e) performing an assay to determine an activity of the first OMV on a test strain of N. gonorrhoeae; and(f) identifying the candidate strain as suitable for producing an immunogenic OMV if the activity of the isolated first OMV on the test strain exceeds a threshold.

115. The method of claim 114, further comprising:(i) culturing a second candidate strain of N. gonorrhoeae in the absence of sialic acid or a sialic acid precursor; and(ii) collecting a second OMV from the second candidate strain of N. gonorrhoeae, wherein the mammal is also inoculated with the second OMV.

116. The method of claim 115, wherein collecting the second OMV comprises isolating the second OMV.

117. The method of claim 115, wherein isolating the second OMV comprises isolating an OMV by a detergent extraction.

118. The method of any one of claims 115-117, further comprising repeating steps (e) and (f) with a plurality of test strains of N. gonorrhoeae.

119. The method of claim 118, wherein at least 5, 10, 15, or 17 test strains of N. gonorrhoeae are tested.

120. The method of claim 118 or 119, wherein the candidate strains are identified as suitable for producing an immunogenic OMV if the activity of the isolated OMVs on the test strain exceeds an activity threshold for a threshold percent of strains.121 . The method of any one of claims 118-120, wherein the first candidate strain and the second candidate strain are identified as suitable for producing an immunogenic OMV if the activity of the first OMV and the second OMV on the test strain exceeds an activity threshold for a threshold percent of strains.

122. The method of claim 121 , wherein the threshold percent of strains is at least 50%.

123. The method of any one of claims 115-122, wherein the second candidate strain of N. gonorrhoeae comprises a genetic modification that reduces or abolishes expression and / or function of a rmpM gene, mRNA, and / or polypeptide.

124. The method of any one of claims 114-123, wherein collecting the first OMV comprises isolating the first OMV.PATENTATTORNEY DOCKET NO: 51838-002WO3125. The method of claim 124, wherein isolating the first OMV comprises isolating a native OMV.

126. The method of any one of claims 114-125, wherein the test strain of N. gonorrhoeae was incubated with sialic acid or a sialic acid precursor prior to step (e).

127. The method of any one of claims 114-126, further comprising repeating steps (e) and (f) with a plurality of test strains of N. gonorrhoeae.

128. The method of claim 127, wherein at least 5, 10, 15, or 17 test strains of N. gonorrhoeae are tested.

129. The method of claim 127 or 128, wherein the first candidate strain is identified as suitable for producing an immunogenic OMV if the activity of the isolated first OMV on the test strain exceeds an activity threshold for a threshold percent of strains.

130. The method of any one of claims 127-129, wherein the first candidate strain is identified as suitable for producing an immunogenic OMV if the activity of the isolated first OMV on the test strain exceeds an activity threshold for a threshold percent of strains.131 . The method of claim 130, wherein the threshold percent of strains is at least 50%.

132. The method of any one of claims 114-131 , wherein the assay comprises mixing the serum with N. gonorrhoeae of the test strain and complement protein and determining a titer of the serum required to kill a threshold percent of bacteria of the test strain.

133. The method of claim 132, wherein the candidate strain is identified as suitable for producing an immunogenic OMV if the titer is below a threshold titer.

134. The method of any one of claims 114-133, wherein the first candidate strain of N. gonorrhoeae comprises a genetic modification that reduces or abolishes expression and / or function of a rmpM gene, mRNA, and / or polypeptide.

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