Modified meningococcal omvs

Abstract

The invention pertains to a genetically modified Neisseria bacterium comprising a lipopolysaccharide (LPS) having a lipid A moiety and a modified oligosaccharide core, wherein the modified oligosaccharide core comprises a first oligosaccharide chain coupled to heptose 1 and a second oligosaccharide chain coupled to heptose 2, wherein the first and second oligosaccharide chain comprise a lactose directly coupled to respectively heptose 1 and 2, thereby forming a Neisseria gonorrhoeae 2C7 epitope. The modified bacterium may express a further non-native N. gonorrhoeae antigen, such as MetQ, AniA or NspA. The modified Neisseria bacterium is not Neisseria gonorrhoeae. The bacterium is preferably Neisseria meningitidis, Neisseria lactamica or Neisseria cinerea. The invention further pertains to LPS and OMVs obtained from the genetically modified bacterium, as well as methods for producing said modified bacterium.

Description

[0001] P62015448wo

[0002] 1

[0003] Modified Meningococcal OMVs

[0004] Field of the invention

[0005] The present invention relates to the field of medicine, in particular to the fields of immunology, medical microbiology and vaccinology. The invention pertains to modified Neisseria meningitidis bacterium, as well as OMVs and LPS obtainable from the modified bacterium. The modified bacterium, OMV and / or LPS is preferably in particular suitable for the prevention and / or treatment of a Neisseria gonorrhoeae infection.

[0006] Background of the invention

[0007] Neisseria gonorrhoeae causes the sexually transmitted genitourinary infection gonorrhoeae. The World Health Organisation estimated that in 2020(1) 82.4 million new cases of gonorrhoeae had occurred worldwide. Untreated, or inadequately treated, gonorrhoeae can lead to pelvic inflammatory disease (PID), infertility, adverse pregnancy outcomes, elevated risk for HIV acquisition and transmission, and ongoing transmission of N. gonorrhoeae to sexual partners and neonates. Increased emergence of N. gonorrhoeae antimicrobial resistance (AMR) has heightened the possibility of future untreatable infections.

[0008] A vaccine against Neisseria gonorrhoeae has been proven difficult to develop. Protective immunity to infection is often not observed in humans, possibly due to high variability of key antigens, induction of blocking antibodies, or a large number of infections being relatively superficial and not inducing a strong immune response (Belcher et al, Front Immunol. 2023; 14:1-15).

[0009] A retrospective cohort study of individuals who had received an outer membrane vesicle (OMV) vaccine against N. meningitidis serogroup B (MeNZB) in New Zealand found vaccine effectiveness of 31 % in preventing a diagnosis of gonorrhoeae. Furthermore, MeNZB vaccination significantly protected against hospitalization from gonorrhoeae. Similarly, the incidence of gonorrhoeae in Cuba declined at the end of the 1990s following the introduction of an OMV vaccine against N. meningitidis. Cases of gonorrhoeae also decreased in Canada following mass vaccination with the 4CMenB, which contains MeNZB OMVs as one of its components (see Belcher et al, supra for review). While the mechanism of cross-protection of 4CMenB against gonococcus is not clear, sera from individuals vaccinated with 4CMenB were shown to react with gonococcal proteins in ELISA and Western blots (Semchenko et al, Clin Infect Dis. 2019 Sep 13;69(7):1101- 1111).

[0010] OMVs (also referred to as “blebs”) are bi-layered membrane structures, usually spherical, that are pinched off from the outer membrane of gram-negative bacteria. The OMV membrane contains phospholipids (PL) on the inside and lipopolysaccharides (LPS) and PL on the outside, mixed with membrane proteins in various positions, largely reflecting the structure of the bacterial outer membrane from which they pinched off. The lumen of the OMV may contain various compounds from the periplasm or cytoplasm, such as proteins, RNA / DNA, and peptidoglycan (PG), however, unlike bacterial cells, OMVs lack the ability to self-replicate. Three types of OMV can be P62015448wo

[0011] 2 distinguished depending on the method of their production. sOMV are spontaneous or natural OMVs that are purified and concentrated from culture supernatant, by separating intact cells from the already formed OMVs. Detergent OMVs, dOMVs, are extracted from cells with detergent, such as deoxycholate, which also reduces the content of reactogenic LPS. After detergent extraction dOMVs are separated from cells and cellular debris and further purified and concentrated. Finally, the term native nOMVs are OMVs generated from concentrated dead cells with non-detergent cell disruption techniques, or that are extracted from cells with other (non-disruptive) detergent-free methods (e.g. using chelating agents such as EDTA).

[0012] While Neisseria meningitidis OMVs could provide some level of cross-protection against Neisseria gonorrhoeae, there is still no effective vaccine available for the prevention and / or treatment of gonorrhoeae. There is therefore a need in the art for improved vaccine compositions and use of these compositions in vaccination against Neisseria gonorrhoeae.

[0013] Summary of the invention

[0014] The invention can be summarized in the following embodiments:

[0015] Embodiment 1 . A genetically modified Neisseria bacterium comprising a lipopolysaccharide (LPS) having a lipid A moiety and a modified oligosaccharide core, wherein the modified oligosaccharide core comprises a first oligosaccharide chain coupled to heptose 1 and a second oligosaccharide chain coupled to heptose 2, wherein the first and second oligosaccharide chain comprise a lactose directly coupled to respectively heptose 1 and 2, thereby forming a Neisseria gonorrhoeae 2C7 epitope, and wherein preferably at least one of: the first oligosaccharide chain is lacto-N-tetraose and the second oligosaccharide chain is lactose; and the first oligosaccharide chain is lactose and second oligosaccharide chain is lactose, wherein the modified Neisseria bacterium is not Neisseria gonorrhoeae, wherein preferably the bacterium is Neisseria meningitidis, Neisseria lactamica or Neisseria cinerea.

[0016] Embodiment 2. The genetically modified Neisseria bacterium according to embodiment 1 , wherein the bacterium is Neisseria meningitidis.

[0017] Embodiment 3. The genetically modified Neisseria bacterium according to embodiment 1 or 2, wherein the bacterium is modified by at least one of: i) increasing a1 ,3 glycosyltransferase (LgtG) activity; i) increasing N. gonorrhoeae p1 ,4 galactosyltransferase (LgtE) activity; and preferably iii) reducing p1 ,3 N-acetylglucosaminyltransferase(LgtA) activity activity and / or reducing a- galactosyl transferase (LgtC) activity. P62015448wo

[0018] 3

[0019] Embodiment 4. The modified Neisseria bacterium according to embodiment 3, wherein at least one of: i) LgtG glycosyltransferase activity is increased by introducing expression of an LgtG protein, preferably a Neisseria gonorrhoeae LgtG protein; ii) LgtE galactosyltransferase activity is increased by introducing expression of an LgtE protein having a substitution of at least one of position W151 and W183 of SEQ ID NO: 1 , preferably at least one of a W151 C substitution and a W183L substitution; and preferably iii) the LgtA N-acetylglucosaminyltransferase activity is reduced by decreasing expression of an LgtA protein and / or the LgtC a-galactosyl transferase activity is reduced by decreasing expression of an LgtC protein.

[0020] Embodiment 5. The modified Neisseria bacterium according to embodiment 3 or 4, wherein at least one of:

[0021] - the LgtE protein has at least 60% sequence identity with SEQ ID NO: 1 ;

[0022] - the LgtG protein has at least 60% sequence identity with SEQ ID NO: 2;

[0023] - the LgtA protein has at least 60% sequence identity with SEQ ID NO: 3 and

[0024] - the LgtC protein has at least 60% sequence identity with SEQ ID NO: 41 .

[0025] Embodiment 6. The modified Neisseria bacterium according to any one of embodiments 3 - 5, wherein LgtE glycosyltransferase activity is increased by at least one of: a) introducing into the bacterium a nucleic acid expressing a LgtE protein having at least 60% sequence identity with SEQ ID NO: 4, preferably having at least one of a cysteine at position 151 and a leucine at position 183; and b) modifying the genomic sequence encoding an endogenous LgtE protein having at least 60% sequence identity with SEQ ID NO: 1 , wherein preferably the modification results in a substitution of at least one of position W151 and W183, preferably a W151 C and / or a W183L substitution.

[0026] Embodiment 7. The modified Neisseria bacterium according to any one of embodiments 1 - 6, expressing a further N. gonorrhoeae antigen, preferably on the extracellular outer membrane surface of the bacterium, wherein preferably the further N gonorrhoeae antigen is at least one of:

[0027] - a N. gonorrhoeae MetQ protein, preferably having a least 60% sequence identity with SEQ ID NO: 5;

[0028] - a N. gonorrhoeae NspA protein, preferably having a least 60% sequence identity with SEQ ID NO: 6; and

[0029] - a N. gonorrhoeae AniA protein, preferably having a least 60% sequence identity with SEQ ID NO: 7.

[0030] Embodiment 8. The modified Neisseria bacterium according to any one of embodiments 1 - 7, further comprising at least one of: P62015448wo

[0031] 4

[0032] - a reduced expression of an LpxL1 protein, preferably having a least 60% sequence identity with SEQ ID NO: 8;

[0033] - a reduced expression of an RmpM protein, preferably having a least 60% sequence identity with SEQ ID NO: 9;

[0034] - a reduced expression of an PorA protein, preferably having a least 60% sequence identity with SEQ ID NO: 10; and

[0035] - a reduced expression of an SiaD protein, preferably having a least 60% sequence identity with SEQ ID NO: 11.

[0036] Embodiment 9. The modified Neisseria bacterium according to any one of embodiments 1 - 8, wherein the bacterium is Neisseria meningitidis strain H44 / 76, or a derivative thereof.

[0037] Embodiment 10. A Neisseria LPS molecule comprising a modified oligosaccharide core as defined in embodiment 1 , wherein the LPS is preferably obtainable from a bacterium according to any one of embodiments 1 - 9.

[0038] Embodiment 11. The Neisseria LPS molecule according to embodiment 10, wherein the lipid A moiety is a modified penta-acylated or tetra-acylated Lipid A moiety.

[0039] Embodiment 12. An OMV comprising the LPS of embodiment 10 or 11 , and preferably at least one of:

[0040] - a N gonorrhoeae MetQ protein, preferably having a least 60% sequence identity with SEQ ID NO: 5;

[0041] - a N gonorrhoeae NspA protein, preferably having a least 60% sequence identity with SEQ ID NO: 6; and

[0042] - a N gonorrhoeae AniA protein, preferably having a least 60% sequence identity with SEQ ID NO: 7.

[0043] Embodiment 13. An OMV obtainable from a bacterium as defined in any one of embodiments 1 - 8.

[0044] Embodiment 14. A composition comprising at least one of the bacterium as defined in any one of embodiments 1 - 9, an LPS as defined in embodiment 10 or 11 and / or an OMV as defined in embodiment 12 or 13 and a pharmaceutically accepted excipient, wherein preferably the bacterium is inactivated.

[0045] Embodiment 15. The composition of embodiment 14, wherein at least 90% of the LPS molecules in the compositions is a molecule as defined in embodiment 10 or 11 .

[0046] Embodiment 16. The composition of embodiment 14 or 15, further comprising an adjuvant. P62015448wo

[0047] 5

[0048] Embodiment 17. The composition of any one of embodiments 14 - 16, further comprising a non- neisserial antigen.

[0049] Embodiment 18. The composition according to any one of embodiments 14 - 17 for use in a medicament.

[0050] Embodiment 19. The composition according to any one of embodiments 14 - 17 for use in the treatment or prevention of a Neisseria gonorrhoeae infection.

[0051] Embodiment 20. A composition according to any one of embodiments 14 - 17, wherein the composition is an acellular vaccine comprising a neisserial LPS as defined in embodiments 10 or 11 and / or an OMV as defined in embodiment 12 or 13.

[0052] Embodiment 21. A composition according to any one of embodiments 14 - 17, wherein the composition is a whole cell vaccine comprising a bacterium as defined in any one of embodiments 1 - 9, and wherein the bacterium is inactivated.

[0053] Embodiment 22. A process for producing an OMV according to embodiment 12 or 13, wherein the process comprises the steps of: a) cultivating a bacterium as defined in any one of embodiments 1 - 8; b) optionally, extracting the OMV; and, c) recovering the OMV, wherein the recovery at least comprises removal of the bacteria from the OMV.

[0054] Embodiment 23. A process according to embodiment 22, wherein the process is a detergent-free process.

[0055] Detailed description of the invention

[0056] Definitions

[0057] Various terms relating to the methods, compositions, uses and other aspects of the present invention are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art to which the invention pertains, unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definition provided herein.

[0058] It is clear for the skilled person that any methods and materials similar or equivalent to those described herein can be used for practising the present invention.

[0059] Methods of carrying out the conventional techniques used in methods of the invention will be evident to the skilled worker. The practice of conventional techniques in molecular biology, P62015448wo

[0060] 6 biochemistry, computational chemistry, cell culture, recombinant DNA, bioinformatics, genomics, sequencing and related fields are well-known to those of skill in the art and are discussed, for example, in the following literature references: Green and Sambrook. Molecular Cloning. A Laboratory Manual, fourth Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2014; Ausubel et al.. Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1987 and periodic updates; and the series Methods in Enzymology, Academic Press, San Diego.

[0061] The singular terms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a combination of two or more cells, and the like. The indefinite article "a" or "an" thus usually means "at least one".

[0062] The term “and / or” refers to a situation wherein one or more of the stated cases may occur, alone or in combination with at least one of the stated cases, up to with all of the stated cases.

[0063] As used herein, the term “about” is used to describe and account for small variations. For example, the term can refer to less than or equal to ± (+ or -) 10%, such as less than or equal to ±5%, less than or equal to ±4%, less than or equal to ±3%, less than or equal to ±2%, less than or equal to ±1 %, less than or equal to ±0.5%, less than or equal to ±0.1 %, or less than or equal to ±0.05%. Additionally, amounts, ratios, and other numerical values are sometimes presented herein in a range format. It is to be understood that such range format is used for convenience and brevity and should be understood flexibly to include numerical values explicitly specified as limits of a range, but also to include all individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly specified. For example, a ratio in the range of about 1 to about 200 should be understood to include the explicitly recited limits of about 1 and about 200, but also to include individual ratios such as about 2, about 3, and about 4, and sub-ranges such as about 10 to about 50, about 20 to about 100, and so forth.

[0064] The term “comprising” is construed as being inclusive and open ended, and not exclusive. Specifically, the term and variations thereof mean the specified features, steps or components are included. These terms are not to be interpreted to exclude the presence of other features, steps or components.

[0065] The terms “homology”, “sequence identity” and the like are used interchangeably herein. Sequence identity is herein defined as a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleotide (polynucleotide) sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences. "Similarity" between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide. "Identity" and "similarity" can be readily calculated by known methods. The percentage sequence identity / similarity can be determined over the full length of the sequence.

[0066] As used herein “sequence identity” refers to the extent to which two optimally aligned polynucleotide or peptide sequences are invariant throughout a window of alignment of components, e.g., nucleotides or amino acids. An “identity fraction” for aligned segments of a test P62015448wo

[0067] 7 sequence and a reference sequence is the number of identical components which are shared by the two aligned sequences divided by the total number of components in reference sequence segment, i.e., the entire reference sequence or a smaller defined part of the reference sequence. “Percent identity” is the identity fraction times 100. “Sequence identity” and “sequence similarity” can be determined by alignment of two peptide or two nucleotide sequences using global or local alignment algorithms, depending on the length of the two sequences. Sequences of similar lengths are preferably aligned using a global alignment algorithms (e.g. Needleman Wunsch) which aligns the sequences optimally over the entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g. Smith Waterman). Sequences may then be referred to as "substantially identical” or “essentially similar” when they (when optimally aligned by for example the programs GAP or BESTFIT using default parameters) share at least a certain minimal percentage of sequence identity (as defined herein). The percent of sequence identity is preferably determined using the “BESTFIT” or “GAP” program of the Sequence Analysis Software Package™ (Version 10; Genetics Computer Group, Inc., Madison, Wis.). GAP uses the Needleman and Wunsch global alignment algorithm (Needleman and Wunsch, Journal of Molecular Biology 48:443-453, 1970) to align two sequences over their entire length (full length), maximizing the number of matches and minimizing the number of gaps. A global alignment is suitably used to determine sequence identity when the two sequences have similar lengths. Generally, the GAP default parameters are used, with a gap creation penalty = 50 (nucleotides) / 8 (proteins) and gap extension penalty = 3 (nucleotides) / 2 (proteins). For nucleotides the default scoring matrix used is nwsgapdna and for proteins the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). Sequence alignments and scores for percentage sequence identity may be determined using computer programs, such as the GCG Wisconsin Package, Version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121 -3752 USA, or using open source software, such as the program “needle” (using the global Needleman Wunsch algorithm) or “water” (using the local Smith Waterman algorithm) in EmbossWIN version 2.10.0, using the same parameters as for GAP above, or using the default settings (both for ‘needle’ and for ‘water’ and both for protein and for DNA alignments, the default Gap opening penalty is 10.0 and the default gap extension penalty is 0.5; default scoring matrices are Blossum62 for proteins and DNAFull for DNA). gap extension penalty is 0.5; default scoring matrices are Blossum62 for proteins and DNAFull for DNA). “BESTFIT” performs an optimal alignment of the best segment of similarity between two sequences and inserts gaps to maximize the number of matches using the local homology algorithm of Smith and Waterman (Smith and Waterman, Advances in Applied Mathematics, 2:482-489, 1981 , Smith et al., Nucleic Acids Research 11 :2205-2220, 1983). When sequences have a substantially different overall lengths, local alignments, such as those using the Smith Waterman algorithm, are preferred. Useful methods for determining sequence identity are also disclosed in Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo, H., and Lipton, D., Applied Math (1988) 48:1073. More particularly, preferred computer programs for determining sequence identity include the Basic Local Alignment Search Tool (BLAST) programs which are publicly available from National Center Biotechnology P62015448wo

[0068] 8

[0069] Information (NCBI) at the National Library of Medicine, National Institute of Health, Bethesda, Md. 20894; see BLAST Manual, Altschul et al., NCBI, NLM, NIH; Altschul et al., J. Mol. Biol. 215:403- 410 (1990); version 2.0 or higher of BLAST programs allows the introduction of gaps (deletions and insertions) into alignments; for peptide sequence BLASTX can be used to determine sequence identity; and, for polynucleotide sequence BLASTN can be used to determine sequence identity.

[0070] Alternatively percentage similarity or identity may be determined by searching against public databases, using algorithms such as FASTA, BLAST, etc. Thus, the nucleic acid and protein sequences described herein can further be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the BLASTn and BLASTx programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403 — 10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to nucleic acid molecules described herein. BLAST protein searches can be performed with the BLASTx program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to protein molecules described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17): 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., BLASTx and BLASTn) can be used. See the homepage of the National Center for Biotechnology Information at http: / / www.ncbi.nlm.nih.gov / .

[0071] Optionally, in determining the degree of amino acid similarity, the skilled person may also take into account so-called "conservative" amino acid substitutions, as will be clear to the skilled person. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagines and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulphur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalaninetyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Substitutional variants of the amino acid sequence disclosed herein are those in which at least one residue in the disclosed sequences has been removed and a different residue inserted in its place. Preferably, the amino acid change is conservative. Preferred conservative substitutions for each of the naturally occurring amino acids are as follows: Ala to ser; Arg to lys; Asn to gin or his; Asp to glu; Cys to ser or ala; Gin to asn; Glu to asp; Gly to pro; His to asn or gin; lie to leu or val; Leu to ile or val; Lys to arg; gin or glu; Met to leu or ile; Phe to met, leu or tyr; Ser to thr; Thr to ser; Trp to tyr; Tyr to trp or phe; and, Val to ile or leu.

[0072] The terms “protein", “peptide" or “polypeptide" are used interchangeably and refer to molecules consisting of a chain of amino acids, without reference to a specific mode of action, size, 3 dimensional structure or origin. A “fragment’ or “portion” of a protein may thus still be referred to P62015448wo

[0073] 9 as a “protein". An “isolated protein" is used to refer to a protein which is no longer in its natural environment, for example in vitro or in a recombinant bacterial host cell. The term "polypeptide" as used herein is defined as a chain of amino acid residues, usually having a defined sequence. The term "polypeptide" is defined as being any peptide or protein comprising at least two amino acids linked by a modified or unmodified peptide bond. The term "polypeptide" refers to short-chain molecules such as oligopeptides or oligomers or to long-chain molecules such as proteins. A polypeptide can be linear, branched or cyclic. The polypeptide can include D amino acids, L amino acids, or a combination thereof.

[0074] A “nucleic acid” or “polynucleotide” as used herein may include any polymer or oligomer of pyrimidine and purine bases, preferably cytosine, thymine, and uracil, and adenine and guanine, respectively (See Albert L. Lehninger, Principles of Biochemistry, at 793-800 (Worth Pub. 1982) which is herein incorporated by reference in its entirety for all purposes). Contemplated are any deoxyribonucleotide, ribonucleotide or nucleic acid component, and any chemical variants thereof, such as methylated, hydroxymethylated or glycosylated forms of these bases, and the like. The polymers or oligomers may be heterogeneous or homogenous in composition, and may be isolated from naturally occurring sources or may be artificially or synthetically produced. In addition, the nucleic acids may be DNA (optionally cDNA) or RNA, or a mixture thereof, and may exist permanently or transitionally in single-stranded or double-stranded form, including homoduplex, heteroduplex, and hybrid states.

[0075] An “isolated nucleic acid” is used to refer to a nucleic acid which is no longer in its natural environment, for example in vitro or in a recombinant bacterial or plant cell. A nucleic acid and / or protein may be at least one of a recombinant, synthetic or artificial nucleic acid and / or protein.

[0076] A “homologue” of a protein as used herein is a protein of the same species which performs the same or a similar function as the protein it is a homologue of. Homologous proteins may but need not be structurally related or are only partially structurally related. The homologue may differ from a sequence in the relevant polypeptide by less than 3, 5, 10, 15, 20 or more mutations (each of which may be a substitution, deletion or insertion of an amino acid residue). These mutations may be measured over a region of at least 30, for instance at least 40, 60 or 100 or more contiguous amino acids of the homologue.

[0077] An “orthologue” of a protein as used herein is a protein of a different species which performs the same or a similar function as the protein it is an orthologue of. Orthologous proteins may but need not be structurally related or are only partially structurally related. The orthologue may differ from a sequence in the relevant polypeptide by less than 3, 5, 10, 15, 20 or more mutations (each of which may be a substitution, deletion or insertion of an amino acid residue). These mutations may be measured over a region of at least 30, for instance at least 40, 60 or 100 or more contiguous amino acids of the orthologue.

[0078] The terms “nucleic acid construct”, “nucleic acid vector”, “vector” and “expression construct” are used interchangeably herein and is herein defined as a man-made nucleic acid molecule resulting from the use of recombinant DNA technology. The terms “nucleic acid construct” and “nucleic acid vector” therefore does not include naturally occurring nucleic acid molecules although P62015448wo

[0079] 10 a nucleic acid construct may comprise (parts of) naturally occurring nucleic acid molecules. The expression vector can be introduced into a suitable host cell, preferably bacterial host cell, preferably a Neisseria host cell, and is able to effect expression of the coding sequence in an in vitro cell culture of the host cell. The expression vector will preferably be suitable for replication in the host cell or organism of the invention.

[0080] The vector backbone may for example be a binary or superbinary vector (see e.g. U.S. Pat. No. 5,591 ,616, US 2002138879 and WO 95 / 06722), a co-integrate vector, a crossover construct, or a T-DNA vector, as known in the art and as described elsewhere herein, into which a (chimeric) gene is integrated or, if a suitable transcription regulatory sequence is already present, only a desired nucleic acid sequence (e.g. a coding sequence, an antisense or an inverted repeat sequence) is integrated downstream of the transcription regulatory sequence. Vectors can comprise further genetic elements to facilitate their use in molecular cloning, such as e.g. selectable markers, multiple cloning sites and the like. The term "selectable marker" is a term familiar to one of ordinary skill in the art and is used herein to describe any genetic entity which, when expressed, can be used to select for a cell or cells containing the selectable marker. The term "reporter" may be used interchangeably with marker, although it is mainly used to refer to visible markers, such as green fluorescent protein (GFP). Selectable markers may be dominant or recessive or bidirectional.

[0081] The term “gene” means a DNA fragment comprising a region (transcribed region), which is transcribed into an RNA molecule (e.g. an mRNA) in a cell, operably linked to suitable regulatory regions (e.g. a promoter). A gene will usually comprise several operably linked fragments, such as a promoter, a 5’ leader sequence, a coding region and a 3’ non-translated sequence (3’ end) comprising a polyadenylation site.

[0082] “Expression of a gene” refers to the process wherein a DNA region which is operably linked to appropriate regulatory regions, particularly a promoter, is transcribed into an RNA, which is biologically active, e.g. which is capable of being translated into a biologically active protein or peptide, or e.g. a regulatory non-coding RNA.

[0083] The term “operably linked” refers to a linkage of polynucleotide elements in a functional relationship. A nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter, or rather a transcription regulatory sequence, is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked may mean that the DNA sequences being linked are contiguous.

[0084] “Promoter” refers to a nucleic acid fragment that functions to control the transcription of one or more nucleic acids. A promoter fragment is preferably located upstream (5’) with respect to the direction of transcription of the transcription initiation site of the gene, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation site(s) and can further comprise any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter. P62015448wo

[0085] 11

[0086] A “constitutive” promoter is a promoter that is active in most tissues under most physiological and developmental conditions. An “inducible” promoter is a promoter that is physiologically (e.g. by external application of certain compounds) or developmentally regulated. A “tissue specific” promoter is only active in specific types of tissues or cells.

[0087] Optionally the term “promoter” may also include the 5’ UTR region (5’ Untranslated Region) (e.g. the promoter may herein include one or more parts upstream of the translation initiation codon of transcribed region, as this region may have a role in regulating transcription and / or translation).

[0088] A “3’ UTR” or “3’ non-translated sequence” (also often referred to as 3’ untranslated region, or 3’end) refers to the nucleic acid sequence found downstream of the coding sequence of a gene, which comprises for example a transcription termination site and optionally a polyadenylation signal (such as e.g. AAUAAA or variants thereof).

[0089] The term "immune response" as used herein refers to the production of antibodies and / or cells (such as T lymphocytes) that are directed against, and / or assist in the decomposition and / or inhibition of, a particular antigenic entity, carrying and / or expressing or presenting antigens and / or antigenic epitopes at its surface. The phrases "an effective immunoprotective response", "immunoprotection", and like terms, for purposes of the present invention, mean an immune response that is directed against one or more antigenic epitopes of a pathogen, or pathogen- infected cell so as to protect against infection by the pathogen in a vaccinated subject. A preferred pathogen in the context of the invention a Neisseria gonorrhoeae. For purposes of the present invention, protection against infection by a pathogen includes not only the absolute prevention of infection, but also any detectable reduction in the degree or rate of infection by a pathogen, or any detectable reduction in the severity of the disease or any symptom or condition resulting from infection by the pathogen in the vaccinated subject, for example as compared to an unvaccinated infected subject. An effective immunoprotective response can be induced in subjects that have not previously been infected with the pathogen and / or are not infected with the pathogen at the time of vaccination, such vaccination can be referred to as prophylactic vaccination.

[0090] According to the present invention, the general use herein of the term "antigen" refers to any molecule that binds specifically to an antibody. The term also refers to any molecule or molecular fragment that can be bound by an MHC molecule and presented to a T-cell receptor. An antigen can be e.g. proteinaceous molecule, i.e. a poly-amino acid sequence, optionally comprising nonprotein groups such as carbohydrate moieties and / or lipid moieties, or an antigen can be e.g. a molecule that is not proteinaceous such as a carbohydrate, preferably one or more oligosaccharides.

[0091] An antigen can be e.g. a portion of a protein (peptide, partial protein, full-length protein), wherein the protein is naturally occurring or synthetically derived, a cellular composition (whole cell, cell lysate or disrupted cells), an organism (whole organism, lysate or disrupted cells) or e.g. a carbohydrate, or a portion thereof, that is able to elicit an antigen-specific immune response (humoral and / or cellular immune response) in a particular subject, which immune response preferably is measurable via an assay or method. P62015448wo

[0092] 12

[0093] The term "antigen" is herein understood as a structural substance which serves as a target for the receptors of an adaptive immune response. An antigen thus serves as target for a TCR (T- cell receptor) or a BCR (B-cell receptor) or the secreted form of a BCR, i.e. an antibody. The antigen can thus be a protein, peptide, carbohydrate, oligosaccharide, that is usually part of a larger structure.

[0094] To vaccinate a subject against a particular antigen means, in one embodiment, that an immune response is elicited against the antigen or immunogenic portion thereof, as a result of administration of a composition comprising at least one epitope of the antigen. Vaccination preferably results in a protective or therapeutic effect, wherein subsequent exposure to the antigen elicits an immune response against the antigen that reduces or prevents a disease or condition in the subject, preferably prevents or reduces (the symptoms of) a Neisseria gonorrhoeae infection. The concept of vaccination is well-known in the art. The immune response that is elicited by administration of a prophylactic or therapeutic composition of the present invention can be any detectable change in any facet of the immune status (e.g., cellular response, humoral response, cytokine production), as compared to in the absence of the administration of the vaccine.

[0095] An “epitope” is defined herein as a (single) immunogenic site within a given antigen that is sufficient to elicit an immune response in a subject. Those of skill in the art will recognize that T cell epitopes are different in size and composition from B cell epitopes, and that T cell epitopes presented through the Class I MHC pathway differ from epitopes presented through the Class II MHC pathway. Epitopes can be linear sequences or conformational epitopes (conserved binding regions) depending on the type of immune response. An antigen can be as small as a single epitope, or larger, and can include multiple epitopes. As such, the size of an antigen can be as small as about 5-12 amino acids (e.g., a peptide), or a di-saccharide, and as large as: a full length protein, including multimeric proteins, a protein complex, an oligosaccharide, a LPS molecule, a bacterium, a particle, a whole cell, or a portion thereof (e.g., lysates of whole cells or extracts of bacteria).

[0096] An adjuvant is herein understood to be an entity, that, when administered in combination with an antigen to a human or an animal subject to raise an immune response against the antigen in the subject, stimulates the immune system, thereby provoking, enhancing or facilitating the immune response against the antigen, preferably without necessarily generating a specific immune response to the adjuvant itself. A preferred adjuvant enhances the immune response against a given antigen by at least a factor of 1.5, 2, 2.5, 5, 10 or 20, as compared to the immune response generated against the antigen under the same conditions but in the absence of the adjuvant. Tests for determining the statistical average enhancement of the immune response against a given antigen as produced by an adjuvant in a group of animal or human subjects over a corresponding control group are available in the art.

[0097] Any reference to nucleotide or amino acid sequences accessible in public sequence databases herein refers to the version of the sequence entry as available on the filing date of this document. P62015448wo

[0098] 13

[0099] Detailed description

[0100] The mechanism of protection of N. meningitidis OMVs against N. gonorrhoeae infection is still largely unknown. The inventors now constructed N. meningitidis OMVs capable of augmenting a N. meningitidis OMV-induced immune response against N. gonorrhoeae, by expressing a N. gonorrhoeae epitope on the outer membrane of the N. meningitidis OMVs. Amongst others, the invention therefore pertains to a N. gonorrhoeae epitope expressed on the outer membrane surface of a Neisseria species that is not Neisseria gonorrhoeae, such that the epitope is comprised in the membrane of OMVs pinched off from said bacterium.

[0101] Preferred modified Neisseria OMVs are OMVs obtainable from a Neisseria species that is not Neisseria gonorrhoeae. Preferably, the Neisseria OMV comprising an gonorrhoeae epitope expressed on the outer membrane is obtainable from at least one of Neisseria meningitidis, Neisseria lactamica and Neisseria cinerea, preferably an OMV obtainable from Neisseria meningitidis.

[0102] A preferred N. gonorrhoeae epitope is at least one of an epitope of a N. gonorrhoeae LPS, a N. gonorrhoeae MetQ protein, a N. gonorrhoeae AniA protein and an epitope of a N. gonorrhoeae NspA protein. Preferably, the epitope is an epitope from N. gonorrhoeae LPS, preferably a 2C7 epitope.

[0103] Genetically modified Neisseria bacterium

[0104] In an aspect, the invention pertains to a genetically modified Neisseria bacterium expressing a N. gonorrhoeae epitope on the extracellular outer membrane surface. The N. gonorrhoeae epitope is a heterologous (non-native) epitope. As the epitope is located in the Neisseria outer membrane, the N. gonorrhoeae epitope is also comprised in the Neisseria OMVs that are pinched off from the outer membrane. The genetically modified Neisseria bacterium is preferably not a modified Neisseria gonorrhoeae bacterium. Preferably, the genetically modified Neisseria bacterium is a modified Neisseria meningitidis, a modified Neisseria lactamica or a modified Neisseria cinerea bacterium. Preferably, the genetically modified bacterium is a genetically modified Neisseria meningitidis bacterium.

[0105] The invention is not limited to any particular Neisseria meningitidis strain. Preferably the Neisseria meningitidis strain does not express an LgtC a-galactosyl transferase. Preferably a genetically modified bacterium according to the invention is a Neisseria meningitidis serogroup B strain. The bacterium is preferably Neisseria meningitidis strain H44 / 76 or a derivative thereof.

[0106] The Neisseria bacterium of the invention is modified to express a Neisseria gonorrhoeae epitope on the extracellular outer membrane surface of the bacterium. The N. gonorrhoeae epitope is preferably an epitope of at least one of a N. gonorrhoeae LPS, a N. gonorrhoeae MetQ protein, a N. gonorrhoeae AniA protein and a N. gonorrhoeae NspA protein. The Neisseria bacterium is preferably modified to express an epitope that is specific to N. gonorrhoeae LPS. In addition or alternatively, the Neisseria bacterium may be genetically modified to express at least one of a N. gonorrhoeae MetQ protein, a N. gonorrhoeae AniA protein and a N. gonorrhoeae NspA protein. P62015448wo

[0107] 14

[0108] The modified Neisseria bacterium of the invention comprises one or more modifications that increase or decrease the expression of protein as defined herein.

[0109] Increasing protein activity and / or increasing protein expression in the Neisseria bacterium can be achieved using any conventional method known to the person skilled in the art. Non-limiting examples include introducing a nucleic acid comprising a sequence encoding the protein of interest (e.g. a protein as defined herein) into the Neisseria cell. The sequence can be maintained episomal or can be integrated into the genome of the cell. Preferably the nucleic acid is maintained, preferably maintained in the genome, after cell division. Preferably the sequence encoding the protein of interest is stably expressed in the modified Neisseria bacterium. The nucleotide sequence encoding the protein of interest may be integrated such that it becomes operably linked to a genomically encoded (endogenous) promoter. Alternatively, a nucleic acid comprising an expression cassette wherein the sequence encoding the protein of interest is operably linked to a regulatory sequence, is introduced into the Neisseria cell and the expression cassette is preferably subsequently integrated into the genome of the Neisseria cell or is maintained in an episome. The regulatory sequence is preferably a promoter sequence, preferably a constitutive promoter. A preferred promoter is the porA-nadA promoter, as described herein below.

[0110] The Neisseria genome, preferably the Neisseria meningitidis, Neisseria lactamica or Neisseria cinerea genome, can be modified to increase (introduce) protein expression, e.g. to increase (introduce) expression of a protein as defined herein. Optionally, to increase protein expression, the Neisseria genome can be modified at one or more specific genomic locations, e.g. by using a site-directed nuclease, optionally in combination with donor construct or template oligonucleotide. A preferred site-directed nuclease may be selected from the group consisting of a CRISPR-nuclease, a ZFNs and a TALEN.

[0111] Reducing protein expression or activity be achieved using any conventional method known to the person skilled in the art. Such methods include, but are not limited to, introducing a deletion in a regulatory sequence to reduce or knock out the expression of the operably linked protein. Alternatively or in addition, the sequence encoding the protein, preferably the genomic sequence, can be modified, e.g. by introducing one or more mutations. Such mutations preferably include at least one of a deletion, insertion and substitution. As a non-limiting example, a stop codon can be introduced, which results in the production of a truncated, and / or non-functional protein. Another non-limiting example is the deletion of part of, or the complete, sequence encoding to protein. Optionally, at least about 20%, 40%, 60%, 80% or 100% of the sequence encoding the protein can be deleted from the genome, preferably deleted from the Neisseria genome, preferably deleted from the Neisseria meningitidis, Neisseria lactamica or Neisseria cinerea genome. Optionally, the sequence encoding the protein can be replaced with the sequence of e.g. a marker, such as a selection marker. The methods preferably result in a reduction of protein expression or activity of about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or about 100%. Preferably the protein is inactive an / or not expressed, i.e. protein expression is knocked out. P62015448wo

[0112] 15

[0113] • 2C7 epitope

[0114] In an embodiment, the bacterium is genetically modified to produce an LPS molecule as defined herein. Preferably, at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or about 100% of the LPS molecules expressed in the modified bacterium are modified LPS molecules as defined herein. Optionally, at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or about 100% of the LPS molecules expressed in the genetically modified bacterium is a single type of the modified LPS molecule as defined herein. Hence optionally, the genetically modified bacterium produces a single form of the modified LPS molecule as described herein, i.e. produces a uniform LPS composition.

[0115] Preferably the modified LPS molecule comprises a Neisseria gonorrhoeae 2C7 epitope. The LPS molecule expressed in the genetically modified bacterium can be similar or identical to an unmodified Neisseria gonorrhoeae LPS molecule, e.g. as depicted in Figure 1 of Gulati et al (Front Immunol. 2019:10:321 , which is incorporated herein by reference in its entirety). Hence the invention pertains to a genetically modified Neisseria bacterium, wherein the bacterium is not Neisseria gonorrhoeae, and wherein the bacterium expresses a wild type Neisseria gonorrhoeae LPS molecule.

[0116] Preferably, the Neisseria bacterium is modified to express a modified LPS molecule as defined herein. The genetically modified bacterium thus may comprise an LPS molecule having a lipid A moiety and a modified oligosaccharide core, wherein the modified oligosaccharide core comprises the 2C7 epitope. The 2C7 epitope will thus be located on the exterior (i.e. extracellularly) of the modified bacterium. More in particular, the modified bacterium may comprise an LPS molecule, having a lipid A moiety and a modified oligosaccharide core, wherein the modified oligosaccharide core comprises a first oligosaccharide chain coupled to heptose 1 (hep1) and a second oligosaccharide chain coupled to heptose 2 (hep 2), and wherein the first and second oligosaccharide chain comprise a lactose directly coupled to respectively heptose 1 and 2, thereby forming the 2C7 epitope. A skilled person can select a suitable heptose, and a preferred heptose is g / ycero-D-manno-heptulose as is present in wildtype Neisseria LPS molecules.

[0117] The inventors discovered that modulating a specific combination of transferase activities in a Neisseria bacterium results in the expression of the LPS molecule comprising the N. gonorrhoeae epitope, preferably the 2C7 epitope. Preferably, the modified bacterium therefore is modified by at least one of increasing a1 ,3 glycosyltransferase (IgtG) activity, increasing p1 ,4 galactosyltransferase (IgtE) activity, and decreasing p1 ,3 N-acetylglucosaminyltransferase (IgtA) activity. Preferably, the modified bacterium of the invention is at least modified by increasing a1 ,3 glycosyltransferase (IgtG) activity and increasing p1 ,4 galactosyltransferase (IgtE) activity. Optionally the bacterium is further modified by decreasing p1 ,3 N-acetylglucosaminyltransferase (LgtA) activity. In addition or alternatively, the bacterium can be further modified by decreasing a- galactosyl transferase (LgtC) activity.

[0118] Preferably, activity is increased or reduced as compared to an unmodified control Neisseria bacterium. Preferably the control Neisseria bacterium is of the same species as the modified bacterium. Preferably the unmodified control Neisseria bacterium has the same genetic background P62015448wo

[0119] 16 as the modified bacterium, except that the control Neisseria bacterium does not have a modification to respectively increase or reduce activity. Equally, preferably expression is increased or reduced as compared to a unmodified control Neisseria bacterium. Preferably the control Neisseria bacterium is of the same species as the modified bacterium. Preferably the unmodified control Neisseria bacterium has the same genetic background as the modified bacterium, except that the control Neisseria bacterium does not have a modification to respectively increase or reduce expression.

[0120] Increasing a1 ,3 glycosyltransferase (IgtG) activity can be achieved using any conventional method known to the person skilled in the art. Preferably, the LgtG activity is increased by increasing or introducing expression of an LgtG protein. The LgtG protein (a1 ,3 lipooligosaccharide glycosyltransferase G) is a glucosyl transferase that forms a (preferably a 1-3) link between the Glc and Hep2. A preferred LgtG protein is an LgtG protein that is obtainable or derivable from a Neisseria species, preferably Neisseria gonorrhoeae. The LgtG protein may be a homologue or an orthologue of the Neisseria gonorrhoeae LgtG protein.

[0121] The amino acid sequence of the LgtG protein can be derived from any Neisseria gonorrhoeae strain, such as, but not limited to, the Neisseria gonorrhoeae strain FA19, FA1090, NCCP1 1945, MS11 and / or FA6140. Optionally, the amino acid sequence of the LgtG protein can be derived from the LgtG nucleotide sequence as encoded in the genome of FA (NZ_CP012026, complete genome FAW) and / or as encoded in the genome of FA1090 (NC_002946, complete genome FA1090).

[0122] The LgtG protein preferably comprises an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 2, preferably over the full length. SEQ ID NO: 2 is the LgtG protein of Neisseria gonorrhoeae strain FAW.

[0123] The skilled person can straightforwardly obtain the nucleotide sequence encoding a protein, e.g. by mapping the amino acids or stop signal to their respective codons. Optionally, those codons can be selected that achieve codon-optimized expression in the Neisseria bacterium.

[0124] Increasing IgtG activity can be achieved by introducing a nucleic acid comprising a nucleotide sequence encoding the LgtG glycosyltransferase. The nucleotide sequence encoding the LgtG glycosyltransferase can be the same or similar to the LgtG encoding sequence as comprised in, and / or expressed from, the Neisseria genome, preferably the Neisseria gonorrhoeae genome. The nucleotide sequence encoding the LgtG protein preferably has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 12, preferably over the full length. Optionally, the nucleotide sequence is comprised in an expression cassette and the expression cassette is introduced into the Neisseria cell,

[0125] The Neisseria bacterium is preferably modified to increase p1 ,4 LgtE galactosyltransferase activity. Preferably, increasing p1 ,4 LgtE galactosyltransferase activity results in the addition of a galactose p-1 ,4 to glucose, which glucose is attached to hep2 (the second oligosaccharide chain). The LgtE galactosyltransferase is preferably capable of extending at least one of the first and P62015448wo

[0126] 17 second oligosaccharide chain by the addition of galactose (p-1 ,4) to glucose. Hence preferably, the LgtE galactosyltransferase can extend the oligosaccharide chain attached to at least one of hep1 and hep2. Preferably, the LgtE galactosyltransferase introduced in the Neisseria bacterium can at least extend the p-chain that is attached to hep2. Preferably, the Neisseria bacterium is modified by introducing the expression of a p1 ,4 LgtE galactosyltransferase which can add a galactose (preferably p-1 ,4) to glucose attached to hep2.

[0127] The inventors discovered that, while Neisseria meningitidis does express an LgtE galactosyltransferase, said transferase appeared surprisingly incapable of extending the p-chain coupled to hep2, i.e. was incapable of extending the second oligosaccharide chain. Introducing a Neisseria gonorrhoeae LgtE galactosyltransferase into Neisseria meningitidis resulted in the extension of the hep2 p-chain. The inventors further deduced that a modification at two locations within the Neisseria meningitidis LgtE protein was sufficient to provide for a protein capable of adding a galactose p-1 ,4 to glucose at hep2.

[0128] A preferred LgtE protein is an LgtE protein that is obtainable or derivable from a Neisseria species, preferably Neisseria gonorrhoeae. The LgtE protein may be a homologue or orthologue of the Neisseria gonorrhoeae LgtE protein.

[0129] The amino acid sequence of the LgtE protein can be derived from any Neisseria gonorrhoeae strain, such as, but not limited to, the Neisseria gonorrhoeae strain FAW, FA1090, NCCP1 1945, MS11 and / or FA6140. Optionally, the amino acid sequence of the LgtE protein can be derived from the LgtE nucleotide sequence as encoded in the genome of FAW (NZ_CP012026, complete genome FAW) and / or as encoded in the genome of FA1090 (NC_002946, complete genome FA1090).

[0130] The LgtE protein preferably comprises an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 4, preferably over the full length. SEQ ID NO: 4 is the LgtE protein of Neisseria gonorrhoeae strain FAW. Preferably, the LgtE protein comprises at least one of a cysteine at position 151 and a leucine at position 183.

[0131] Alternatively or in addition, the LgtE protein can be obtainable or derivable from, Neisseria meningitidis, Neisseria lactamica or Neisseria cinerea, preferably Neisseria meningitidis, wherein the protein is modified such that it is capable of adding a galactose p-1 ,4 to glucose at hep2. Hence optionally, to increase LgtE galactosyltransferase expression wherein the LgtE is capable of adding galactose p-1 ,4 to glucose at hep2, the genome of the Neisseria bacterium can be modified such that the expressed LgtE protein comprises a modification at position 151 and / or position 183 of SEQ ID NO: 1 , or a position analogous thereto. Preferably the LgtE protein is derivable from Neisseria meningitidis. The LgtE protein preferably comprises an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 1 , and preferably comprises a modification of the tryptophan (W) amino acid residue at position 151 and / or at position 183. A preferred modification is a substitution of the tryptophan residue at position 151 and / or at position 183 of SEQ ID NO: 1. A preferred substitution is at least one of a W151 C and a W183L substitution. P62015448wo

[0132] 18

[0133] The LgtE protein can be, or can be obtainable or derivable from, a Neisseria lactamica or Neisseria cinerea LgtE protein, wherein the protein is modified such that it is capable of adding a galactose p-1 ,4 to glucose at hep2. The Neisseria lactamica or Neisseria cinerea LgtE protein can be a homologue or orthologue of the Neisseria meningitidis LgtE protein and modified at an position analogous to position W151 and / or W183 of SEQ ID NO: 1 .

[0134] Increasing IgtE activity can be achieved by introducing a nucleic acid comprising a nucleotide sequence encoding the LgtE galactosyltransferase, wherein the LgtE galactosyltransferase is capable of adding galactose p-1 ,4 to glucose at hep2. The nucleotide sequence encoding said LgtE galactosyltransferase can be the same or similar to the LgtE encoding sequence as comprised in, and / or expressed from, the Neisseria genome, preferably the Neisseria gonorrhoeae genome. The nucleotide sequence encoding the LgtE protein preferably has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 13, preferably over the full length. Alternatively, the introduced nucleic acid can comprise a nucleotide sequence encoding the LgtE galactosyltransferase that is capable of adding galactose p-1 ,4 to glucose at hep2, can be the similar to the LgtE encoding sequence as comprised in, and / or expressed from, the Neisseria meningitidis, Neisseria lactamica or Neisseria cinerea genome. The nucleotide sequence encoding the LgtE protein preferably has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 14, preferably over the full length, and wherein the nucleotide sequence comprises a mutation resulting in a modification of the expressed LgtE protein, preferably a modification of tryptophan amino acid residue at position 151 and / or at position 183 of SEQ ID NO: 1 , or a position analogous thereto. Preferably the mutation is at least one of a W151 C and W183L substitution. Optionally, the nucleotide sequence is comprised in an expression cassette and the expression cassette is introduced into the Neisseria cell,

[0135] Optionally, the Neisseria bacterium, preferably the Neisseria meningitidis, Neisseria lactamica or Neisseria cinerea bacterium is modified to reduce LgtA activity. The LgtA N- acetylglucosaminyltransferase is preferably a genomically encoded (endogenous) protein in Neisseria and is a N-acetylglucosaminyltransferase that provides for a GIcNAc p 1->3 Gal linkage, between GIcNac and Gal in the first oligosaccharide chain. Such linkage is absent in the Neisseria gonorrhoeae oligosaccharide core. Hence preferably to further mimic the Neisseria gonorrhoeae LPS structure, the modified bacterium of the invention comprises a reduced LgtA activity. Preferably, the LgtA activity is reduced at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100%. The reduction in activity is preferably in comparison to the non-modified (native) Neisseria bacterium. Preferably LgtA glycosyltransferase activity is absent. Reducing p1 ,3 N- acetylglucosaminyltransferase (LgtA) activity can be achieved using any conventional method known to the person skilled in the art. Preferably, the LgtA activity is reduced by decreasing or knock out the expression of the LgtA protein, preferably by deleting at least part of the sequence encoding the LgtA p1 ,3 N-acetylglucosaminyltransferase.

[0136] The modified bacterium preferably comprises a reduced expression of a protein having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or P62015448wo

[0137] 19

[0138] 100% sequence identity with SEQ ID NO: 3. SEQ ID NO: 3 is LgtA protein of Neisseria meningitidis, strain H44 / 76 (NC_017516). Reducing the expression of the LgtA protein can be achieved using any conventional method known to the person skilled in the art. Preferably the modified bacterium comprises a modification, preferably deletion, in the genomic sequence encoding a protein having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 3. Preferably the the modified bacterium comprises a modification, preferably deletion, in the genomic sequence encoding a p1 ,3 (LgtA) N- acetylglucosaminyltransferase, wherein the nucleotide sequence has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 15.

[0139] The Neisseria bacterium can be a bacterium that does not have a-galactosyl transferase (LgtC) activity, preferably does not express an a-galactosyl transferase (LgtC) protein. A nonlimiting example of such Neisseria bacterium is N. meningitidis H44 / 76 strain. Alternatively, the Neisseria bacterium can be modified to reduce LgtC activity, in addition or alternatively to reducing LgtA activity. Hence optionally, the Neisseria bacterium, preferably the Neisseria meningitidis, Neisseria lactamica or Neisseria cinerea bacterium is modified to reduce LgtC activity. The LgtC a- galactosyl transferase is preferably a genomically encoded (endogenous) protein in Neisseria and is an a-galactosyl transferase that provides for a Gal -> Gal linkage, thereby terminating hep1 with galactose (Gal), that can undergo sialy lation (Gulati et al, 2019, supra). The Gal - Gal linkage may interfere with mAb 2C7 binding and hence preferably the LgtC a-galactosyl transferase is not expressed. Preferably, the modified bacterium of the invention comprises a reduced LgtC activity. Preferably, the LgtC activity is reduced at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% in comparison to the non-modified (native) Neisseria bacterium. Preferably LgtC glycosyltransferase activity is absent. Reducing a-galactosyl transferase (LgtC) activity can be achieved using any conventional method known to the person skilled in the art. Preferably, the LgtC activity is reduced by decreasing or knock out the expression of the LgtC protein, preferably by deleting at least part of the sequence encoding the LgtC a-galactosyl transferase.

[0140] The modified bacterium preferably comprises a reduced expression of a protein having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 41. SEQ ID NO: 41 is LgtC protein of Neisseria meningitidis, strain M978. Reducing the expression of the LgtC protein can be achieved using any conventional method known to the person skilled in the art. Preferably the modified bacterium comprises a modification, preferably deletion, in the genomic sequence encoding a protein having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 41. Preferably the the modified bacterium comprises a modification, preferably deletion, in the genomic sequence encoding a a-galactosyl transferase (LgtC), wherein the nucleotide sequence has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 42. P62015448wo

[0141] 20

[0142] • MetQ epitope

[0143] In an embodiment, the Neisseria bacterium can be modified to express an epitope of the Neisseria gonorrhoeae MetQ protein. The MetQ protein of Neisseria gonorrhoeae is a known surface- expressed antigen and it elicits bactericidal and functional blocking antibodies (Semchenko EA et al, Infect Immun, 2017, 26;85(2): 1 -17).

[0144] The MetQ epitope could be part of a wild-type (of native) Neisseria gonorrhoeae MetQ protein. Alternatively, the epitope is comprised in a (MetQ) protein that comprises one or more mutations, which mutations preferably to not affect the conformation and / or amino acid sequence of the MetQ epitope. Preferably, the MetQ protein that is expressed in the modified Neisseria bacterium has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 5. Preferably, the MetQ protein expressed in the modified Neisseria bacterium is encoded by a nucleotide sequence that has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 16.

[0145] Optionally, expression of the native MetQ protein of the Neisseria bacterium is replaced for expression of a MetQ protein having an epitope of the Neisseria gonorrhoeae MetQ protein. Expression of the native MetQ protein can be replaced for expression of a Neisseria gonorrhoeae MetQ protein. Optionally, expression of the MetQ protein of respectively Neisseria meningitidis, Neisseria lactamica or Neisseria cinerea is replaced for a Neisseria gonorrhoeae MetQ protein. Optionally, the modified bacterium does not express a native MetQ protein.

[0146] Increasing expression of a MetQ protein comprising the Neisseria gonorrhoeae MetQ epitope can be achieved by introducing a specific mutation in a MetQ encoding sequence in Neisseria genome, preferably in the Neisseria meningitidis, Neisseria lactamica or Neisseria cinerea genome, such that the mutated MetQ protein comprises a MetQ Neisseria gonorrhoeae epitope.

[0147] Alternatively or in addition, increasing expression of a MetQ protein comprising the Neisseria gonorrhoeae MetQ epitope can be achieved by introducing a nucleic acid comprising a nucleotide sequence encoding said MetQ protein. The nucleotide sequence encoding said MetQ protein can be the same or similar to the MetQ protein encoding sequence as comprised in, and / or expressed from, the Neisseria genome, preferably the Neisseria gonorrhoeae genome. The nucleotide sequence encoding the MetQ protein preferably has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 16, preferably over the full length. Optionally, the Neisseria bacterium can be modified to comprise two or more copies of a nucleic acid comprising a nucleotide sequence encoding said MetQ protein. Optionally, the nucleotide sequence is comprised in an expression cassette and the expression cassette is introduced into the Neisseria cell,

[0148] Alternatively or in addition, (part of) a MetQ protein can be expressed as the N-terminal fusion partner of a fusion lipoprotein as described in WO 2016 / 193370. Alternatively or in addition, (part of) a MetQ protein can be coupled to a vertebrate antimicrobial peptide (AMP), preferably mCRAMP, for non-covalently coupling (the part of) the MetQ protein to the OMV. P62015448wo

[0149] 21

[0150] • AniA epitope

[0151] In an embodiment, the Neisseria bacterium can be modified to express an epitope of the Neisseria gonorrhoeae AniA protein. The outer membrane glycoprotein AniA (nitrate reductase) appears essential for the growth and survival of N. gonorrhoeae under oxygen-limiting conditions. It has also been demonstrated that an immune response is generated against AniA in response to gonococcal infection (Shewell LK et al, PLoS One (2017);12(8):1 -18).

[0152] The AniA epitope could be part of a wild-type (of native) Neisseria gonorrhoeae AniA protein. Alternatively, the epitope is comprised in a(n AniA) protein that comprises one or more mutations, which mutations preferably do not affect the conformation and / or amino acid sequence of the AniA epitope. Preferably, the AniA protein that is expressed in the modified Neisseria bacterium has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 7. Preferably, the AniA protein expressed in the modified Neisseria bacterium is encoded by a nucleotide sequence that has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 17.

[0153] Optionally, expression of the native AniA protein of the Neisseria bacterium is replaced for expression of an AniA protein having an epitope of the Neisseria gonorrhoeae AniA protein. Expression of the native AniA protein can be replaced for expression of a Neisseria gonorrhoeae Ani protein. Optionally, expression of the AniA protein of respectively Neisseria meningitidis, Neisseria lactamica or Neisseria cinerea is replaced for a Neisseria gonorrhoeae AniA protein. Optionally, the modified bacterium does not express a native AniA protein.

[0154] Increasing expression of an AniA protein comprising the Neisseria gonorrhoeae AniA epitope can be achieved by introducing a specific mutation in an AniA encoding sequence in Neisseria genome, preferably in the Neisseria meningitidis, Neisseria lactamica or Neisseria cinerea genome, such that the mutated AniA protein comprises a Neisseria gonorrhoeae AniA epitope.

[0155] Alternatively or in addition, increasing expression of an AniA protein comprising the Neisseria gonorrhoeae AniA epitope can be achieved by introducing a nucleic acid comprising a nucleotide sequence encoding said AniA protein. The nucleotide sequence encoding said AniA protein can be the same or similar to the AniA protein encoding sequence as comprised in, and / or expressed from, the Neisseria genome, preferably the Neisseria gonorrhoeae genome. The nucleotide sequence encoding the AniA protein preferably has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 17, preferably over the full length. Optionally, the Neisseria bacterium can be modified to comprise two or more copies of a nucleic acid comprising a nucleotide sequence encoding said AniA protein. Optionally, the nucleotide sequence is comprised in an expression cassette and the expression cassette is introduced into the Neisseria cell,

[0156] Alternatively or in addition, (part of) an AniA protein can be expressed as the N-terminal fusion partner of a fusion lipoprotein as described in WO 2016 / 193370. Alternatively or in addition, P62015448wo

[0157] 22

[0158] (part of) an AniA protein can be coupled to a vertebrate antimicrobial peptide (AMP), preferably mCRAMP, for non-covalently coupling the (part of the) AniA protein to the OMV.

[0159] • NspA epitope

[0160] In an embodiment, the Neisseria bacterium can be modified to express an epitope of the Neisseria gonorrhoeae NspA protein. The Neisserial surface protein A (NspA) protein of Neisseria gonorrhoeae is a highly conserved outer membrane protein and can elicit serum bactericidal activity.

[0161] The NspA epitope could be part of a wild-type (of native) Neisseria gonorrhoeae NspA protein. Alternatively, the epitope is comprised in a (NspA) protein that comprises one or more mutations, which mutations preferably to not affect the conformation and / or amino acid sequence of the NspA epitope. It is known in the art that NspA is a human-specific ligand of the complement inhibitor factor H (FH) (Lujan et al, Infect Immun. (2015);84(2):452-8. Therefore preferably, the NspA protein has a reduced factor H binding, such as the NspA from N. gonorrhoeae strain FA1090.

[0162] Preferably, the NspA protein that is expressed in the modified Neisseria bacterium has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 6. Preferably, the NspA protein expressed in the modified Neisseria bacterium is encoded by a nucleotide sequence that has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 18.

[0163] Optionally, expression of the native NspA protein of the Neisseria bacterium is replaced for expression of a NspA protein having an epitope of the Neisseria gonorrhoeae NspA protein. Expression of the native NspA protein can be replaced for expression of a Neisseria gonorrhoeae NspA protein, preferably a Neisseria gonorrhoeae NspA protein that does not, or not significantly, bind to human factor H. Optionally, expression of the NspA protein of respectively Neisseria meningitidis, Neisseria lactamica or Neisseria cinerea is replaced for a Neisseria gonorrhoeae NspA protein, wherein preferably the Neisseria gonorrhoeae NspA protein does not, or not significantly, bind to human factor H. Optionally, the modified bacterium does not express a native NspA protein.

[0164] Increasing expression of an NspA protein comprising the Neisseria gonorrhoeae NspA epitope can be achieved by introducing a specific mutation in an NspA encoding sequence in Neisseria genome, preferably in the Neisseria meningitidis, Neisseria lactamica or Neisseria cinerea genome, such that the mutated NspA protein comprises an NspA Neisseria gonorrhoeae epitope.

[0165] Alternatively or in addition, increasing expression of an NspA protein comprising the Neisseria gonorrhoeae NspA epitope can be achieved by introducing a nucleic acid comprising a nucleotide sequence encoding said NspA protein. The nucleotide sequence encoding said NspA protein can be the same or similar to the NspA protein encoding sequence as comprised in, and / or expressed from, the Neisseria genome, preferably the Neisseria gonorrhoeae genome, and optionally does not, or not significantly, bind to factor H. The nucleotide sequence encoding the NspA protein preferably has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, P62015448wo

[0166] 23

[0167] 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 18, preferably over the full length. Optionally, the Neisseria bacterium can be modified to comprise two or more copies of a nucleic acid comprising a nucleotide sequence encoding said NspA protein. Optionally, the nucleotide sequence is comprised in an expression cassette and the expression cassette is introduced into the Neisseria cell,

[0168] Alternatively or in addition, (part of) an NspA protein can be expressed as the N-terminal fusion partner of a fusion lipoprotein as described in WO 2016 / 193370. Alternatively or in addition, (part of) an NspA protein can be coupled to a vertebrate antimicrobial peptide (AMP), preferably mCRAMP, for non-covalently coupling (the part of) the NspA protein to the OMV.

[0169] The modified Neisseria bacterium may comprise one or more modifications to introduce a Neisseria gonorrhoeae epitope as described herein. The Neisseria bacterium may comprise a modification to express at least one of the 2C7 epitope, MetQ, AniA and NspA epitope. A schematic representation of this exemplary embodiment is shown in Figure 1. The Neisseria bacterium may comprise a modification to express at least one of a Neisseria gonorrhoeae MetQ, AniA and NspA protein. The Neisseria bacterium may comprise a modification to abolish expression of a native MetQ, AniA and NspA protein.

[0170] A preferred Neisseria bacterium is modified to express at least an LPS molecule as defined herein. The Neisseria bacterium comprising the LPS molecule may be modified to additionally comprise at least one of a MetQ, AniA and NspA epitope as described herein.

[0171] The modified Neisseria bacterium may comprise further modifications to e.g. reduce the toxicity of the LPS molecule and / or increase OMV yield. Optionally, the Neisseria Bacterium is modified to comprise additional antigens / epitopes.

[0172] The Neisseria bacterium could be modified to express additional epitopes of Neisseria gonorrhoeae, preferably similarly as described above for MetQ, NspA and AniA.

[0173] Optionally additional Neisseria gonorrhoeae epitopes / antigens are expressed on the extracellular outer membrane of the bacterium. Such additional epitope, or protein comprising such epitope, can be selected from the group consisting of MtrE, OpcA, ACP, PilQ, LptD, VacJ and BamA.

[0174] Preferably, the MtrE protein comprises an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99% or 100% sequence identity with SEQ ID NO: 19, preferably over the full length. The MtrE protein is preferably is encoded by an nucleotide sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99% or 100% sequence identity with SEQ ID NO: 20, preferably over the full length. Preferably, the OpcA protein comprises an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99% or 100% sequence identity with SEQ ID NO: 21 , preferably over the full length. The OpcA protein is preferably is encoded by an nucleotide sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99% or 100% sequence identity with SEQ ID NO: 22, preferably overthe full length. Preferably, the ACP protein comprises an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99% P62015448wo

[0175] 24 or 100% sequence identity with SEQ ID NO: 23, preferably over the full length. The ACP protein is preferably is encoded by an nucleotide sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99% or 100% sequence identity with SEQ ID NO: 24, preferably over the full length. Preferably, the PilQ protein comprises an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99% or 100% sequence identity with SEQ ID NO: 25, preferably over the full length. The PilQ protein is preferably is encoded by an nucleotide sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99% or 100% sequence identity with SEQ ID NO: 26, preferably over the full length. Preferably, the LptD protein comprises an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99% or 100% sequence identity with SEQ ID NO: 27, preferably over the full length. The LptD protein is preferably is encoded by an nucleotide sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99% or 100% sequence identity with SEQ ID NO: 28, preferably over the full length. Preferably, the VacJ protein comprises an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99% or 100% sequence identity with SEQ ID NO: 29, preferably over the full length. The VacJ protein is preferably is encoded by an nucleotide sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99% or 100% sequence identity with SEQ ID NO: 30, preferably over the full length. Preferably, the BamA protein comprises an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99% or 100% sequence identity with SEQ ID NO: 31 , preferably over the full length. The BamA protein is preferably is encoded by an nucleotide sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99% or 100% sequence identity with SEQ ID NO: 32, preferably over the full length.

[0176] The genetically modified Neisseria bacterium may comprise a further genetic modification selected from the group consisting of: (i) a genetic modification that alters the lipopolysaccharide (LPS) biosynthesis pathway, preferably in order to obtain less endotoxic and reactogenic variants; (ii) a genetic modification that increases OMV production by removing outer membrane anchor proteins; and (iii) a genetic modification that removes immune-modulating components which may trigger an undesired type of immune response. In addition or alternatively, the Gram-negative bacterium may comprise (iv) a genetic modification that causes outer membrane retention of normally secreted antigens and, (v) an additional genetic modification that introduces expression of heterologous antigens from other pathogens than the host OMV producing strain. Several of these exemplary modifications are described herein in more detail.

[0177] Optionally The Neisseria bacterium can be modified to express a non-Ne / sser / a antigen / epitope, preferably on the extracellular outer membrane of the bacterium.

[0178] Optionally, the Neisseria bacterium is modified to have a reduced expression of an endogenous Neisseria antigen. Optionally, the Neisseria meningitidis, Neisseria lactamica or the P62015448wo

[0179] 25

[0180] Neisseria cinerea bacterium is further modified to have a reduced expression of respectively a Neisseria meningitidis, Neisseria lactamica or a Neisseria cinerea antigen.

[0181] The Neisseria genetically modified bacterium can further have one or more genetic modifications that reduce or eliminate the expression of a gene selected from the group consisting of cps, ctrA, ctrB, ctrC, ctrD, exbB, exbD, frpB, galE, htrB, msbB, IpbB, IpxK, IpxLI, nmb0033, opA, opC, rmpM, phoP, piiC, pmrE, pmrF, porA, porB, siaA, siaB, siaC, siaD, synA, synB, sync, tbpA and tbpB, or homologues thereof, preferably SiaD and / or PorA, or homologues thereof. Many of these mutations are reviewed in W002 / 09746. Exemplary antigens that are preferably absent in the modified Neisseria bacterium are at least one of SiaD and PorA. PorA is an immunodominant antigen that is not present in gonococci and as such may divert the attention of the immune system.

[0182] Preferably, the SiaD and / or PorA expression is reduced by deleting at least part of the sequence encoding respectively the SiaD and PorA protein. Preferably, the SiaD and / or PorA expression is reduced at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100%. Preferably SiaD and / or PorA expression is absent. The modified bacterium preferably comprises a reduced expression of a protein having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with respectively SEQ ID NO: 11 or SEQ ID NO: 10. SEQ ID NO: 11 is SiaD protein of Neisseria meningitidis, strain H44 / 76 (NC_017516) and SEQ ID NO: 10 is PorA protein of Neisseria meningitidis, strain H44 / 76 (NC_017516).

[0183] Preferably the modified bacterium comprises a modification, preferably deletion, in the genomic sequence encoding a protein having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with respectively SEQ ID NO: 11 and / or SEQ ID NO: 10. Preferably the the modified bacterium comprises a modification, preferably deletion, in the genomic sequence encoding at least one of SiaD and PorA, wherein the nucleotide sequence has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with respectively SEQ ID NO: 33 and SEQ ID NO: 34.

[0184] • PagL, LpxL1, LpxL2,

[0185] In an embodiment, the Neisseria bacterium of the invention can be modified to express a heterologous enzyme or reduce expression of endogenous enzyme to reduce the toxicity of the produced LPS molecule. Non-limiting examples known in the art include reducing the expression of at least one of LpxL1 and LpxL2, and / or introducing the expression of PagL.

[0186] In an embodiment, the modified Neisseria bacterium, preferably the Neisseria meningitidis, Neisseria lactamica or Neisseria cinerea bacterium is further modified to reduce at least one of LpxL1 and LpxL2 activity. Preferably, the LpxL1 and / or LpxL2 activity is reduced at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100%. Preferably LxpL1 and / or LpxL2 activity is absent. Reducing at least one of LpxL1 and LpxL2 activity can be achieved using any conventional method known to the person skilled in the art. Preferably, the LpxL1 and / or LpxL2 activity is reduced by decreasing or knock out the expression of respectively the LpxL1 or LpxL2 protein, preferably P62015448wo

[0187] 26 by deleting at least part of the sequence encoding respectively the LpxL1 or LpxL2 protein. Preferably at least the expression of LpxL1 is knocked out.

[0188] The modified bacterium preferably comprises a reduced expression of a protein having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 8 and / or SEQ ID NO: 35. SEQ ID NO: 8 is LpxL1 protein of Neisseria meningitidis, strain H44 / 76 (NC_017516) and SEQ ID NO: 35 is LpxL2 protein of Neisseria meningitidis, strain H44 / 76 (NC_017516).

[0189] Reducing the expression of the LpxL1 and / or LpxL2 protein can be achieved using any conventional method known to the person skilled in the art. Preferably the modified bacterium comprises a modification, preferably deletion, in the genomic sequence encoding a protein having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with respectively SEQ ID NO: 8 and / or SEQ ID NO: 35. Preferably the the modified bacterium comprises a modification, preferably deletion, in the genomic sequence encoding a LxpL1 or a LpxL2, wherein the nucleotide sequence has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with respectively SEQ ID NO: 36 or SEQ ID NO: 37.

[0190] Alternatively or in addition, the modified Neisseria bacterium comprises a modification to increase PagL expression. Preferably, the PagL protein that is expressed in the modified Neisseria bacterium has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 38. Preferably, the PagL protein expressed in the modified Neisseria bacterium is encoded by a nucleotide sequence that has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 39. Preferably the PagL protein is obtained from Bordetella, preferably Bordetella bronchiseptica or Bordetella pertussis or Bordetella parapertussis.

[0191] Increasing expression of a PagL protein can be achieved by introducing a nucleic acid comprising a nucleotide sequence encoding said PagL protein. The nucleotide sequence encoding said PagL protein can be the same or similar to the PagL protein encoding sequence as comprised in, and / or expressed from, the Bordetella genome, preferably at least one of the Bordetella bronchiseptica or Bordetella pertussis or Bordetella parapertussis genome. Optionally, the nucleotide sequence is comprised in an expression cassette and the expression cassette is introduced into the Neisseria cell,

[0192] The nucleotide sequence encoding the PagL protein preferably has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 39, preferably over the full length.

[0193] • rmpM

[0194] The modified Neisseria bacterium described herein may be modified to increase OMV yield. A nonlimiting example known in the art to increase OMV production is to reduce RmpM expression. RmpM links the outer membrane to the peptidoglycan layer, and which absence loses the outer P62015448wo

[0195] 27 membrane leading to increased OMV release. Reducing RmpM expression therefore allows for the isolation of native OMVs at a higher yield without the need for detergent extraction.

[0196] In an embodiment, the modified Neisseria bacterium, preferably the Neisseria meningitidis, Neisseria lactamica or Neisseria cinerea bacterium is further modified to reduce RmpM activity. Preferably, the RmpM activity is reduced at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100%. Preferably RmpM activity is absent. Reducing RmpM activity can be achieved using any conventional method known to the person skilled in the art. Preferably, the RmpM activity is reduced by decreasing or knock out the expression of the RmpM protein, preferably by deleting at least part of the sequence encoding the RmpM protein.

[0197] The modified bacterium preferably comprises a reduced expression of a protein having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 9. SEQ ID NO: 9 is RmpM protein of Neisseria meningitidis, strain H44 / 76 (NC_017516).

[0198] Preferably the modified bacterium comprises a modification, preferably deletion, in the genomic sequence encoding a protein having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 9. Preferably the the modified bacterium comprises a modification, preferably deletion, in the genomic sequence encoding a RmpM, wherein the nucleotide sequence has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 40.

[0199] Preferably, the modified bacterium comprises a deletion of LgtA. In addition or alternatively, the native AniA, MetQ and NspA are replaced for Neisseria gonorrhoeae AniA, MetQ and NspA. In addition or alternatively, the bacterium is modified to express Neisseria gonorrhoeae LgtG and LgtE. Preferably, the modified bacterium comprises a deletion of PorA, SiaD, LpxL1 , LgtA and RmpM. In addition or alternatively, the native AniA, MetQ and NspA are replaced for Neisseria gonorrhoeae AniA, MetQ and NspA. In addition or alternatively, the bacterium is modified to express Neisseria gonorrhoeae LgtG and LgtE.

[0200] Preferably, the bacterium is modified to reduce or delete the expression of the native proteins LgtA, AniA, MetQ and NspA. In addition, the bacterium is modified to introduce the expression of Neisseria gonorrhoeae AniA, MetQ, NspA, LgtG and LgtE. Preferably, the bacterium is modified to reduce or delete the expression of the native proteins PorA, SiaD, LpxL1 , LgtA, RmpM, AniA, MetQ and NspA. In addition, the bacterium is modified to introduce the expression of Neisseria gonorrhoeae AniA, MetQ, NspA, LgtG and LgtE.

[0201] Modified neisserial LPS molecule

[0202] In an aspect, the invention pertains to a Neisseria lipopolysaccharide (LPS) molecule that is modified to comprise a Neisseria gonorrhoeae epitope. Preferably, the LPS molecule of the invention is a Neisseria meningitidis, Neisseria lactamica, or Neisseria cinerea LPS that is modified to comprise a Neisseria gonorrhoeae epitope. The invention preferably pertains to a Neisseria meningitidis LPS that is modified to comprise a Neisseria gonorrhoeae epitope. Hence the invention P62015448wo

[0203] 28 pertains to a Neisseria meningitidis LPS having a lipid A moiety and a modified oligosaccharide core, wherein the modified oligosaccharide core comprises a Neisseria gonorrhoeae LPS epitope. The neisserial LPS is sometimes also referred to as lipooligosaccharide (LOS) due to the fact that they differ from the LPS of the Enterobacteriaceae by lacking the O side chains. In the context of the invention the terms “LPS” and “LOS” are however interchangeable. For reasons of consistency we shall further refer to LPS.

[0204] The oligosaccharide core is preferably modified to comprise the Neisseria gonorrhoeae 2C7 LPS epitope. Hence the modified Neisseria LPS, preferably the modified Neisseria meningitidis LPS comprises a Neisseria gonorrhoeae 2C7 epitope. The 2C7 epitope has been well- characterized in the art (see e.g. Gulati et al, J Infect Dis. 1996;174(6):1223-37, and Yamasaki et al, J Biol Chem, 1999;274(51):36550-8). The epitope is an oligosaccharide epitope on gonococcal LPS which is expressed on >95% of fresh clinical isolates (Gulati et al, 1996, supra) and thus well- conserved. The 2C7 epitope is recognized by a monoclonal antibody named mAb 2C7.

[0205] Preferably, the oligosaccharide core of the Neisseria LPS is modified to comprise a first oligosaccharide chain coupled to heptose 1 (hep1) and a second oligosaccharide chain coupled to heptose 2 (hep2), thereby forming the 2C7 epitope. The first oligosaccharide chain preferably comprises a lactose (galactose (Gal) - glucose (Glc) ) coupled to hep1 , preferably a Gal 1 -> 4 Glc 1 -> 4 hep1 structure, preferably a Gal p1 -> 4 Glc p1 -> 4 hep1 structure. The lactose on hep1 may be elongated. Preferably, the lactose on hep1 is not elongated with a galactose. Hence preferably, the first oligosaccharide chain coupled to heptose 1 does not comprise a di-galactose, preferably does not comprise Gal-Gal-GIc. Optionally, the first oligosaccharide chain structure coupled hep1 is lacto-N-tetraose (Gal-GIcNac-Gal-GIc). Preferably, the lactose on hep1 is not further elongated, i.e. the first oligosaccharide chain consists of lactose.

[0206] The second oligosaccharide chain preferably comprises a lactose coupled to heptose 2 (hep2). Preferably, the second oligosaccharide chain comprises a Gal 1 -> 4 Glc 1 -> 3 Hep2 structure, preferably a Gal p1 -> 4 Glc p1 -> 3 Hep2 structure. Preferably, the lactose on hep2 is not further elongated, i e. the second oligosaccharide chain consists of lactose.

[0207] The modified oligosaccharide core of the Neisseria LPS of invention preferably comprises a di-lactose structure. Preferably the modified oligosaccharide core comprises a lactose on hep2 and a lactose on hep1 , wherein the lactose on hep2 and hep1 are not further elongated. Put differently, the first oligosaccharide chain consists of lactose and the second oligosaccharide chain consists of lactose. Alternatively, the first oligosaccharide chain is elongated after the lactose and the second oligonucleotide chain is not elongated after the lactose. The elongated first oligosaccharide chain is preferably lacto-N-tetraose. P62015448wo

[0208] 29

[0209] In some embodiments the LPS molecule of the invention is represented by general formula (1): wherein

[0210] LipA is lipid A;

[0211] X1is H or -C(=0)CH3; preferably X1is H;

[0212] X2is H or -PO3CH2CH2NH2 (PEA); preferably X2is H;

[0213] X3is H or -C(=O)CH2NH2; preferably X3is H;

[0214] X4is H or PEA or Glcal ; preferably X4is H;

[0215] R1comprises a lactose moiety; and

[0216] R2comprises a lactose moiety.

[0217] In preferred embodiments each of X1, X2, X3, and X4is H. Lipid A as represented by LipA can be comprised in general formula (1) as it is in naturally occurring Neisseria lipopolysaccharides. Alternatively, lipid A as represented by LipA can be comprised in general formula (1) as being a modified lipid A moiety having a reduced number of acyl chains, preferably a penta-acylated lipid A moiety. In some embodiments the compound of general formula (1) is represented by general formula (2):

[0218] P62015448wo

[0219] 30 wherein

[0220] X5is H, galactosyl such as Galal , or Neu5Ac2-6Gala1 ; preferably X5is H;

[0221] X6is H or Galp1-4GlcNAcp1 ; preferably X6is H;

[0222] X7is H or Galp1-4GlcNAcp1 ; preferably X7is H; X8is H, galactosyl such as Galal , or Neu5Ac2-6Gala1 ; preferably X8is H.

[0223] A compound of general formula (2) is preferably of general formula (3):

[0224] Preferred compounds of general formula (1), (2), or (3) have a stereochemistry as represented in general formula (1s), (2s), or (3s), respectively. P62015448wo

[0225] 31

[0226] In highly preferred embodiments the LPS molecule of the invention is represented by general formula (2s) or (3s). P62015448wo

[0227] 32

[0228] The modified LPS may further comprise the structure of Neisseria meningitidis, Neisseria lactamica or Neisseria cinerea LPS, preferably the structure of Neisseria meningitidis LPS. The two heptose residues (hep1 and hep2) are via two 2-keto-3-deoxy-mannooctulosonic acid (KDO) moieties attached to the lipid A moiety. Preferably, the modified LPS molecule may comprise a N. gonorrhoeae epitope and the N. meningitidis, N. lactamica or N. cinerea lipid A moiety. Preferably, the modified LPS molecule comprises a N. meningitidis lipid A moiety. The Neisseria, preferably the Neisseria meningitidis, lipid A moiety can be an unmodified (wild type) lipid A moiety or a modified lipid A moiety. An exemplary embodiment is depicted in Figure 2A.

[0229] The unmodified N. meningitidis lipid A moiety is hexa-acylated. The unmodified Neisseria meningitidis lipid A moiety is known in the art and is depicted e.g. in Figure 2B (taken from Zariri et al, Sci Rep (2016):6:36575, which is incorporated herein by reference in its entirety).

[0230] Modification of the N. meningitidis lipid A moiety has been described in the art to reduce LPS toxicity. The lipid A moiety of LPS may activates the host’s innate immune system through binding of LPS to the pattern recognition receptor Toll-like receptor 4 / myeloid differentiation factor 2 (TLR4 / MD-2) complex, which starts a signalling cascade leading to cytokine production necessary to clear the infection. Overstimulation of this signalling cascade and overproduction of the inflammatory cytokines is detrimental to the host and can lead to life-threatening conditions such as septic shock. For complete activation of the TLR4 / MD-2 complex, a lipid A moiety with six acyl chains and two phosphate groups appears to be critical. Neisseria meningitidis typically produces hexa-acylated LPS with phosphate and phosphoethanolamine groups appended to the 1 and 4’ position of the lipid A moiety. Mutation of LPS modifying enzymes such as deletion of lipid A biosynthesis enzymes such as LpxL1 and LpxL2 has been used to detoxify N. meningitidis LPS (see e.g. Zariri et al, supra).

[0231] Preferably, the modified LPS of the invention comprises a modification of the lipid A moiety to reduce toxicity. The modified lipid A moiety preferably comprises a penta- instead of hexa- acylated lipid A moiety. The skilled person knows how to produce a penta- acylated Neisseria meningitidis lipid A moiety, e.g. by deletion of LpxL1 or LpxL2 expression. Preferably, the lipid A moiety is modified such that the secondary lauroyl chain is missing from the non-reducing end of the GlcN disaccharide, e.g. by deleting LpxL1 expression. It is known that deletion of LpxL1 expression prevents the addition of C12 to the primary linked acyl chain at the 2’-position. Alternatively or in addition, the lipid A moiety is modified such that the secondary lauroyl chain is missing from the reducing end of the GlcN disaccharide, e.g. by deleting LpxL2 expression. The person skilled in the art knows that deletion of LpxL2 expression prevents the addition of C12 to the primary linked acyl chain at the 2 position. Alternatively or in addition, the lipid A moiety is modified such that the C12 is missing at the 3 position. It is known that expression of the heterologous Bordetella PagL removes the acyl chain from the 3 position.

[0232] Hence preferably the lipid A moiety of the LPS of the invention comprises a modified oligosaccharide core as described herein and a modified Ne / sser / a meningitidis lipid A moiety. The modified lipid A moiety is preferably a penta-acylated lipid A moiety. The lipid A moiety is preferably P62015448wo

[0233] 33 modified, as compared to the wild-type Neisseria meningitidis hexa-acylated lipid A moiety, in that the lipid A moiety lacks at least one of:

[0234] - the acyl chain linked to the primary linked acyl chain at the 2’ position;

[0235] - the acyl chain linked to the primary linked acyl chain at the 2 position; and

[0236] - the acyl chain at the 3-position.

[0237] The deletion of one acyl chain from the wild type Neisseria meningitidis lipid A moiety thus results in a penta-acylated Lipid A moiety. Optionally, the modified lipid A moiety is a tetra-acylated lipid A moiety, i.e. by deletion of two acyl chains from the wild type lipid A moiety. The deletion of two acyl chains may be achieved by at least one of: deletion of LpxL1 and LpxL2 expression; deletion of LpxL1 expression and introducing PagL expression; and deletion of LpxL2 expression and introducing PagL expression

[0238] The lipid A moiety can be modified, as compared to the wild-type Neisseria meningitidis hexa-acylated lipid A moiety, in that the lipid A moiety lacks at least one of: the acyl chain linked to the primary linked acyl chain at the 2’ position and the acyl chain linked to the primary linked acyl chain at the 2 position; the acyl chain linked to the primary linked acyl chain at the 2’ position and the acyl chain at the 3-position; and the acyl chain linked to the primary linked acyl chain at the 2 position and the acyl chain at the 3-position.

[0239] The skilled person understands that the modified LPS of the invention is not limited to the modifications described herein to reduce LPS toxicity. Additional or alternative modification include e.g. the deletion of IptA expression to remove phosphethanolamine in lipid A. The modified Neisseria meningitidis LPS of the invention may have additional or alternative modifications, while maintaining the modified oligosaccharide core forming the Neisseria gonorrhoeae 2C7 epitope. Optionally, the Neisseria LPS is not a wild type Neisseria gonorrhoeae LPS.

[0240] The LPS molecule of the invention may comprise a Neisseria meningitidis hexa-acylated lipid A moiety and a modified oligosaccharide core, wherein the first and second oligosaccharide chain is lactose. The LPS molecule may comprise a modified Neisseria meningitidis penta- or tetraacylated lipid A moiety and a modified oligosaccharide core, wherein the first and second oligosaccharide chain is lactose, or wherein the first oligosaccharide chain is lacto-N-tetraose and the second oligosaccharide chain is lactose.

[0241] In a preferred embodiment, the LPS is obtainable from a genetically modified bacterium as described herein.

[0242] OMV

[0243] In an aspect, the invention pertains to a Neisseria OMV comprising a Neisseria gonorrhoeae epitope on the extracellular outer membrane surface. The Neisseria OMV is preferably not Neisseria gonorrhoeae OMV, preferably the Neisseria OMV is at least one of a Neisseria meningitidis, Neisseria lactamica and Neisseria cinerea OMV. P62015448wo

[0244] 34

[0245] The OMV of the invention preferably comprises a Neisseria gonorrhoeae epitope as defined herein. Preferably, the OMV comprises at least one of an LPS molecule, MetQ protein, NspA protein and an AniA protein as defined herein. The OMV may comprise at least two or more Neisseria gonorrhoeae epitopes as defined herein. Preferably, the OMV comprises at least an LPS molecule as defined herein. Preferably, the OMV comprising the LPS molecule, further comprises at least one of a MetQ protein, NspA protein and AniA protein as defined herein.

[0246] The OMV may further have a reduced amount of an endogenous SiaD protein and / or OmpA protein as described herein, wherein preferably the amount is reduced as compared to a control Neisseria bacterium. Optionally, the OMV may further lack the at least one of a SiaD protein and an OmpA protein as described herein.

[0247] Preferably the OMV is obtainable or obtained by a genetically modified bacterium as described herein.

[0248] OMVs for use in vaccines have traditionally been prepared by detergent extraction (a dOMV purification process), wherein detergents such deoxycholate are used to remove LPS and increase vesicle release. The LPS of most Gram-negative bacteria, such as N. meningitidis is highly toxic, yet residual amounts (approx. 1 %) are needed in OMV to maintain vesicle structure and for adjuvant activity. It is preferred that the OMV maintains at least part of its LPS. An OMV as defined herein is therefore preferably is not a detergent-extracted OMV. It is understood however, that a process for preparing an OMV that is not a detergent-extracted OMV does not exclude the use of any detergents. The use of low concentration of detergent and / or the use of mild detergents are not excluded

[0249] Preferably, the OMV as defined herein is a spontaneous OMV or a native OMV. The OMV is preferably a native OMV. The production of native OMV is well-known in the art, and has e.g. been described in Saunders et al. (1999, Infect Immun, 67, 113-1 19), van de Waterbeemd et al. (2012, Vaccine, 30: 3683-3690) and in WO2013006055. Methods for preparing sOMV are e.g. described in van de Waterbeemd et al. (2013, PLoS ONE, 8(1): e54314. doi:10.1371 / journal. pone.0054314) and in Lee et al. (2007, Proteomics, 7: 3143-3153), all of which are incorporated herein by reference.

[0250] Composition

[0251] In an aspect, the invention pertains to a composition comprising at least one of a genetically modified Neisseria bacterium as defined herein, an OMV as defined herein, and an LPS molecule as defined herein. Preferably, the genetically modified Neisseria bacterium is inactivated.

[0252] The composition comprises an LPS molecule as defined herein. Preferably, the composition comprises LPS molecules, wherein at least about 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% of the LPS molecules in the composition are LPS molecules as defined herein. The LPS molecules can be comprised in the OMV or in the genetically modified bacterium. Optionally, the LPS molecules are purified, i.e. the molecules are at least separated from the genetically modified bacterium producing the LPS molecule. Neisseria gonorrhoeae produces a variety of LPS molecules, e.g. as taught in Gulati S et al (Front Immunol. 2019:10:321). The LPS in the P62015448wo

[0253] 35 composition, optionally comprised in the OMV and / or genetically modified bacterium, preferably does not comprise a first oligosaccharide chain having, or consisting of, Galactose-Galactose- Glucose, wherein Glucose is coupled to hep1 .

[0254] Preferably, the composition is a pharmaceutical composition comprising at least one of the OMV, LPS and genetically modified Neisseria bacterium as defined herein and a pharmaceutically accepted excipient. The composition preferably comprises a pharmaceutically acceptable carrier, medium or delivery vehicle as are conventionally known in the art (see e.g. “Handbook of Pharmaceutical Excipients”, Rowe et al eds. 7th edition, 2012, www.pharmpress.com). Pharmaceutically acceptable stabilizing agents, osmotic agents, buffering agents, dispersing agents, and the like may also be incorporated into the pharmaceutical composition. The preferred form depends on the intended mode of administration and therapeutic application. The pharmaceutical carrier can be any compatible, non-toxic substance suitable to deliver the active ingredients, i.e. at least one of the OMV, LPS and genetically modified Neisseria bacterium to the patient. Pharmaceutically acceptable carriers for parenteral delivery are exemplified by sterile buffered 0.9% NaCI or 5% glucose optionally supplemented with a 20% albumin. Alternatively, at least one of the OMV, LPS and genetically modified Neisseria bacterium can be suspended in Phosphate buffer saline (PBS). Preparations for parental administration must be sterile. The parental route for administration of the composition is in accord with known methods, e.g. injection or infusion by intravenous, intraperitoneal, intramuscular, intranasal, intraarterial or intralesional routes.

[0255] The pharmaceutical composition may be administrated continuously by infusion or by bolus injection, preferably a bolus injection. A typical pharmaceutical composition for intramuscular injection would be made up to contain, for example, 1 - 10 ml of phosphate buffered saline comprising the effective dosages of at least one of the OMV, LPS and genetically modified Neisseria bacterium as defined herein. Methods for preparing parenterally administrable compositions are well known in the art and described in more detail in various sources, including, for example, “Remington: The Science and Practice of Pharmacy” (Ed. Allen, L. V. 22nd edition, 2012, www.pharmpress.com).

[0256] The pharmaceutical composition as defined herein is preferably a vaccine, preferably an a- cellular vaccine. The vaccine preferably comprises an OMV as defined herein. Preferably, the pharmaceutical composition is for use in the protection against a Neisseria gonorrhoeae infection. The pharmaceutical composition is preferably for use in the protection against a Neisseria gonorrhea infection, wherein the Neisseria gonorrhea expresses at least one of the 2C7 epitope, the N. gonorrhoeae MetQ protein, the N. gonorrhoeae NspA protein and the N. gonorrhoeae AniA protein. Preferably, the pharmaceutical composition is for use in the protection against a Neisseria gonorrhea infection, wherein the Neisseria gonorrhea expresses the 2C7 epitope.

[0257] The composition may comprise one or more adjuvants e.g. to further boost an immune response. The adjuvant may be an organic or inorganic adjuvant. A preferred inorganic adjuvant is an aluminium salt, such as, but not limited to aluminium phosphate and aluminium hydroxide. A preferred organic adjuvant may be a modified LPS, preferably modified neisserial or bordetella LPS, P62015448wo

[0258] 36 modified LOS, squalene, QS21 , or monophosphoryl lipid A (MPL). The adjuvant may be selected from the group consisting of alum, aluminum hydroxide, aluminum phosphate, calcium phosphate hydroxide, paraffin oil, squalene, detergents (e.g. Quil A), (plant) saponins, cytokine (e.g. IL-1 , IL- 2, or IL-12), Freund's complete adjuvant and Freund's incomplete adjuvant. The use of specific adjuvants, the relative and absolute amounts of substances in the compositions and the doses regimen for the administration are known or may be determined by the skilled person and may be adapted for the circumstances such as the disease progression of the Neisseria gonorrhoeae infection or the status of the particular subject to be treated. The doses regimen may comprise a single dose but may also comprise multiple doses, for instance booster doses, and may preferably be administered orally, intranasally or parenterally, preferably intranasally or intramuscularly. Various doses regimens for vaccination purposes are known in the art and may be suitably adapted by the skilled person.

[0259] The pharmaceutical composition may comprise traces of an inorganic adjuvant. Alternatively, the pharmaceutical composition does not comprise an inorganic adjuvant, preferably does not comprise an aluminium salt, such as, but not limited to aluminium phosphate and aluminium hydroxide.

[0260] The LPS of the invention preferably functions as an antigen in a pharmaceutical composition. Alternatively or in addition, the LPS of the invention may function as an adjuvant in a pharmaceutical composition. Optionally the LPS is combined with a Neisseria or non-Neisseria antigen, wherein the LPS in the composition functions as an adjuvant, optionally in addition to providing a specific immune response against Neisseria gonorrhea. Hence optionally, the LPS as defined herein can function both as an adjuvant and a vaccine antigen.

[0261] In an aspect the invention pertains to a pharmaceutical composition as defined herein for use as a medicament. The invention thus pertains to the use as medicament of at least one of an OMV as defined herein, an LPS molecule as defined herein and a genetically modified Neisseria bacterium as defined herein. The genetically modified Neisseria bacterium is preferably an inactivated bacterium. The bacterium can be activated using any means known to the person skilled in the art, preferably the bacterium is heat-inactivated.

[0262] The invention further concerns a method of treatment using at least one of the (pharmaceutical composition comprising) an OMV as defined herein, an LPS molecule as defined herein and a genetically modified Neisseria bacterium as defined herein. Preferably, the invention concerns a method of treatment using a (pharmaceutical composition comprising) an OMV as defined herein.

[0263] In an aspect, the invention pertains to a (pharmaceutical composition comprising) at least one of an OMV as defined herein, the LPS molecule as defined herein and the genetically modified Neisseria bacterium as defined herein for the prevention or treatment of a Neisseria gonorrhea infection. Preferably, the invention pertains to a (pharmaceutical composition comprising) an OMV as defined herein for the prevention or treatment of a Neisseria gonorrhea infection. In this aspect, the invention thus relates to a method for vaccination against, or for prophylaxis or therapy of a Neisseria gonorrhea infection by administration of a therapeutically or prophylactically effective P62015448wo

[0264] 37 amount of at least one of an OMV as defined herein, the LPS molecule as defined herein and the genetically modified Neisseria bacterium as defined herein, to a subject in need of prophylaxis or therapy.

[0265] The invention also relates to a (pharmaceutical composition comprising) at least one of an OMV as defined herein, an LPS molecule as defined herein and a genetically modified Neisseria bacterium as defined herein for use as a medicament, preferably a medicament for vaccination against, or for prophylaxis or therapy of a Neisseria gonorrhea infection. Preferably, the invention relates to a (pharmaceutical composition comprising) at least an OMV as defined herein for use as a medicament, preferably a medicament for vaccination against, or for prophylaxis or therapy of a Neisseria gonorrhea infection.

[0266] In a further aspect, the invention concerns a (pharmaceutical composition comprising) at least one of an OMV as defined herein, an LPS molecule as defined herein and a genetically modified Neisseria bacterium as defined herein for use in a treatment comprising inducing or stimulating an immune response in a subject against a Neisseria gonorrhoeae antigen, preferably a Neisseria gonorrhoeae antigen as defined herein. Preferably the treatment is for preventing or treating a Neisseria gonorrhoeae infection.

[0267] In an aspect, the (pharmaceutical composition comprising) at least one of an OMV as defined herein, an LPS molecule as defined herein and a genetically modified Neisseria bacterium as defined herein is for use in preventing and / or reducing the spread of a Neisseria gonorrhoeae infection. The pharmaceutical composition thus may be vaccine.

[0268] The Neisseria gonorrhea infection as defined herein is preferably an infection with a Neisseria gonorrhea strain expressing at least one of a N. gonorrhea MetQ, AniA and NspA protein and / or expressing the 2C7 epitope. Hence preferably, the N. gonorrhea strain expresses at least N. gonorrhea MetQ. In addition or alternatively, the N. gonorrhea strain expresses at least N. gonorrhea AniA. In addition or alternatively, the N. gonorrhea strain expresses at least N. gonorrhea NspA. In addition or alternatively, the N. gonorrhea strain expresses at least the 2C7 epitope. The Neisseria gonorrhea strain can be a strain that is resistant to ceftriaxone treatment.

[0269] In an aspect, the invention relates to a process for producing a LPS molecule according to the invention. The process preferably comprises the steps of: a) cultivating a genetically modified Neisseria bacterium of the invention; and, b) optionally, at least one of extraction and purification of the LPS.

[0270] In an aspect, the invention relates to a process for producing an OMV according to the invention. The process preferably comprises the steps of: a) cultivating a genetically modified Neisseria bacterium of the invention; b) optionally, extracting the OMV; and, c) recovering the OMV, wherein the recovery at least comprises removal of the bacteria from the OMV. A preferred process for producing OMV is a detergent-free process.

[0271] In an aspect, the invention relates to a process for producing an acellular vaccine of the invention. The process preferably comprises the steps of: a) producing at least one of: i) an LPS molecule according to the invention, preferably in a process as defined herein; and, ii) an OMV P62015448wo

[0272] 38 according to the invention, preferably in a process as defined herein; and, b) formulating at least one of the LPS and the OMV, optionally with further vaccine components, into a vaccine formulation.

[0273] In a ninth aspect, the invention relates to a process for producing a whole cell vaccine of the invention, wherein the process comprises the steps of: i) cultivating a genetically modified Neisseria bacterium of the invention; and, ii) optionally, at least one of inactivation of the bacterium and formulation into a vaccine.

[0274] It is understood that the use of a (pharmaceutical composition comprising) at least one of an OMV as defined herein, an LPS molecule as defined herein and a genetically modified Neisseria bacterium as defined herein in prevention or treatment of an infection as specified herein also includes the use for the manufacture of a medicament for the corresponding medical treatment, as well as, methods for treating a subject suffering from, or at risk of, a Neisseria gonorrhoeae infection as specified herein by administering an effective amount of the composition to the subject.

[0275] Genetically modified bacterium

[0276] In an aspect, the invention pertains to a gram-negative bacterium that is modified to express the Neisseria gonorrhoeae 2C7 epitope. Preferably, the bacterium is a genetically modified Neisseria bacterium as defined herein above. The invention is however not limited to a genetically modified Neisseria bacterium. The inventors discovered that expression of N. gonorrhoeae p1 ,4 galactosyltransferase (LgtE) results in the addition of lactose to heptose 2, therefore elucidating an important step in the production of the 2C7 epitope. Heterologous expression of the LgtE galactosyltransferase as defined herein in a non-Neisseria bacterium thus can result in, or provides for an important step of, the production of the 2C7 epitope. The invention therefore further pertains to a genetically modified gram-negative bacterium expressing a heterologous N. gonorrhoeae LgtE. Such preferred modified gram-negative bacterium is at least one of E. coli and Bordetella. A preferred Bordetella is at least one of Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica. Remodeling E.coli LPS would preferably further require truncation of the oligosaccharide to the inner core 2 KDO - 2 Hep region, and additional expression of the meningococcal icsA-icsB glycosyltransferases, to generate the same oligosaccharide acceptor structure for the 2C7 digalactose as found in Neisseria gonorrhoeae. It is further understood herein that the invention as described above for a genetically modified Neisseria bacterium, equally applies to a non-Neisseria genetically modified bacterium expressing LgtE.

[0277] In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded.

[0278] All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.

[0279] The present invention has been described above with reference to a number of exemplary embodiments as shown in the drawings. Modifications and alternative implementations of some P62015448wo

[0280] 39 parts or elements are possible, and are included in the scope of protection as defined in the appended claims.

[0281] The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.

[0282] Table 1. SEQ ID NOs P62015448wo

[0283] 40

[0284] Figure legend

[0285] Figure 1 Schematic representation of an exemplary embodiment of the invention. Neisseria meningitidis bacteria are genetically modified to express the Neisseria gonorrhoeae AniA, NspA, MetQ and 2C7 LPS antigens on the extracellular surface. The OMVs pinching off from these bacteria equally comprise the N. gonorrhoeae antigens.

[0286] Figure 2 A) Schematic representation of an embodiment of the invention. The Neisseria meningitidis LPS structure is modified to comprise the Neisseria gonorrhoeae epitope. B) Wild-type Neisseria meningitidis Lipid A moiety, depicting the enzymes that can modify the lipid A structure. Deletion of LpxL1 or LpxL2 omit the addition of the C12 acyl chain to the primary linked acyl chain at respectively the 2’ or 2 position. Heterologous Bordetella bronchiseptica PagL expression removes the acyl chain from the 3-position. Deletion of IptA expression removes the lipid A phosphethanolamine.

[0287] Figure 3 Deconvoluted ESI-FT mass spectra of intact LOS isolated from OMV samples. “NmBB” stands for “N. meningitidis Backbone mutant", NmBB+A for “NmBB with AniA”, NmBB+M for “NmBB with MetQ”, NmBB+N for “NmBB with NspA”, NmBB+L for “NmBB with 2C7 epitope”, NmBB+4G for “NmBB with AniA, MetQ, NspA, 2CT and MenB NZ stands for “N. meningitidis New Zealand (wild-type)”

[0288] Figure 4 BLAST alignment Neisseria meningitidis LgtE (bottom sequence) and Neisseria gonorrhoeae (top sequence) LgtE. The single point mutations are highlighted grey and the boxed regions were further analysed.

[0289] Figure 5 The three IgtE hybrids were expressed in the NmBB backbone strain through replacement of the meningococcal IgtE gene, and expression of the 2C7 LPS epitope was tested by ELISA. LgtE Hybrid 1 (H1) has 8 amino acid changes in helix P62015448wo

[0290] 41 a2 and a2-3 loop, plus 2 amino acid changes in helix a3 and E232G, LgtE Hybrid 2 (H2) comprises the mutations W151 C and W183L and LgtE Hybrid 3 (H3) has 8 amino acid changes in helix a2 and a2-3 loop, 2 amino acid changes in helix a3, W151 C, W183L and E232G. The results are shown for two clones of each hybrid, i.e. H1 C1 and H1 C2 (hybrid 1 clones 1 and 2), H2C1 and H2C2 (hybrid 2 clone 1 and 2) and H3C1 and H3C2 (hybrid 3 clones 1 and 2). NmBB is N.m. strain H44 / 76 AporA, AsiaD, AlpxLI , ArmpM (negative control), the “predecessor” is Nm strain H44 / 76 AporA, AsiaD, AlpxLI , aniA:: aniA N.g. FAW, metQ:: metQ N.g. FAW, IgtA :: IgtG N.g. FAW, nspA:: nspA N.g. FA1090, Alpt3, ArmpM (negative control), and NmBB+4G is N.m. strain H44 / 76 AporA, StrepR, AsiaD, AlpxLI , aniA::aniA N.g. FAW, metQ::metQ N.g. FAW, ArmpM, nspA::nspA N.g. FA1090, lgtA::lgtG N.g. FAW, lpt3::lgtE N.g. FAW (positive control).

[0291] Figure 6 A) Serum antibody response against Neisseria gonorrhoeae strain FA19, B) Serum antibody response against 2C7_LOS of NM-OMV_NG-4G, C) 2C7 antibody binding against Neisseria gonorrhoeae strains FAW, F62 and FA1090 and D) Antigenspecific response after vaccination with NM-OMV_NG-4G OMVs, left, middle and right corresponds to the response against AniA, MetQ and NspA, respectively.

[0292] Figure 7 Colonization after vaccination with PBS, NM-OMV, NM-OMV_NG-2C7, NM- OMV_NG-MetQ or NM-OMV_NG-4G and subsequent challenge with Neisseria gonorrhoeae strain FAW.

[0293] Figure 8 Antibody responses to whole-cell and purified antigens of Neisseria gonorrhoeae following immunization with OMVs expressing gonococcal antigens. (A-B) IgG antibodies against whole cell Ng. Whole-cell ELISA showing high antibody titers against N. gonorrhoeae strains A) FAW and B) FA1090 in the high dose OMV groups (Bexsero is a control). (C-D) ELISA against purified 2C7 LPS, C) 1 :1000 serum, D) 1 :10000 serum. (E-F) Combination of low dose OMVs with alum adjuvant E) FAW and F) FA1090, compared to low dose OMVs alone.

[0294] Figure 9 Serum bactericidal antibody (SBA) titers against Neisseria gonorrhoeae strains following immunization with OMVs expressing gonococcal antigens. P62015448wo

[0295] 42

[0296] Examples

[0297] Example 1.1

[0298] The aim of the construction process was to obtain a meningococcal strain capable of producing high amounts of safe and immunogenic OMVs expressing gonococcal antigens. To this extent, two types of mutations were introduced; (1) deletions that improve safety and OMV production, and (2) introducing gonococcal antigens, by replacement of meningococcal antigens.

[0299] Selection of surface-exposed, functional, immunogenic and conserved gonococcal antigens for expression in N. meningitidis:

[0300] MetQ: surface-exposed neisserial lipoprotein, methionine-binding component in ABC transport system, previously shown to induce bactericidal and function-blocking antibodies

[0301] AniA: OMP required for denitrification pathway, expressed during anaerobic growth, previously shown to induce bactericidal and function-blocking antibodies

[0302] NspA: OMP that binds the complement inhibitor factor H, previously shown to induce bactericidal antibodies when expressed in meningococci. A version of NspA was used that doesn’t bind factor H.

[0303] 2C7 LPS epitope: di-lactose inner core structure expressed by >95% of gonococcal clinical isolates, known to induce bactericidal antibodies and shown to be protective in a mouse model.

[0304] Strain construction was started using a porA deletion mutant of Neisseria meningitidis (N.m.) H44 / 76. To improve safety of laboratory handling, the meningococcal capsule was removed by deletion of siaD. LPS endotoxicity was reduced by deleting IpxLI , which result in a penta-acylated rather than hexa-acylated lipid A. Increased blebbing was obtained by the deletion of rmpM, which connects the outer membrane to the peptidoglycan layer.

[0305] For heterologous expression, gonococcal antigens aniA, metQ, nspA, and the 2C7 epitope of gonococcal LPS were selected. For the first three, the meningococcal gene was replaced by its gonococcal homologue. In order to achieve high and stable expression, a previously described strong promoter was added upstream of the start codon for all three homologous genes inserted. This hybrid promoter contains the porA promoter with the 18 bp sequence between the -35 and - 10 box replaced by the 17 bp sequence between the -35 and -10 box from the nadA promoter of N.m. MC58. This promoter is herein referred to as the ‘porA-nadA promoter’. P62015448wo

[0306] 43

[0307] In order to obtain the 2C7 epitope on meningococcal LPS, two adjustments were made to the meningococcal LPS biosynthesis pathway (replacement of IgtA by gonococcal IgtG and replacement of Ipt3 by gonococcal IgtE). We expected that inactivation of IgtA and insertion of an active copy of IgtG would result in the 2C7 epitope, which contains two lactose (Gal-GIc) moieties, each attached to a heptose. However, this strategy did not work. We initially reasoned that this might be caused by the presence of phosphoethanolamine (PEA) at the same heptose position, which could block the addition the new lactose chain. We therefore inactivated Ipt3, thereby removing the PEA. This however also did not result in lactose addition. Surprisingly, the introduction of the gonococcal IgtE gene (in the position of Ipt3, as this gene was not required anyway) enabled the addition of Gal to Glc at both lactose groups, thereby forming the 2C7 LPS epitope (see Results below).

[0308] Summarizing, five meningococcal genes were deleted or inactivated (PorA, RmpM, LpxL1 , SiaD, LgtA), three meningococcal genes were replaced by gonococcal genes (AniANg, NspANg, MetQNg), and two gonococcal gene were inserted (LgtGNg, LgtENg).

[0309] N. meningitidis H44 / 76-derived strain for heterologous gonococcal OMV vaccine

[0310] • Knockouts: PorA, RmpM, LpxL1 , SiaD, LgtA

[0311] • Replacements: AniANg, NspANg, MetQNg

[0312] • Insertions: LgtGNg, LgtENg

[0313] • All mutations introduced marker-free

[0314] The strain construction was done using a two-step marker-free mutagenesis technique. This requires a strain resistant to streptomycin for the counterselection step. Since N.m. H44 / 76 is not resistant to streptomycin, a spontaneous StrepR mutant was selected for by plating N.m. H44 / 76 AporA cells on GC medium containing 500 pg / ml Strep. Resistant colonies were isolated and their rpsL gene (that codes for ribosomal protein S12) was sequenced, showing that they contained the amino acid change K88R in rpsL. This mutation is known to confer streptomycin resistance.

[0315] The marker-free mutagenesis method is based on two-step selection / counterselection of a cassette that confers erythromycin resistance (EryR) and streptomycin sensitivity (StrepS). For the first step, a so-called 1stcrossover construct is designed that consists of the EryR-StrepS cassette, flanked by DNA bordering the region to be replaced / deleted in the N.m. genome (from here on referred to as ‘upstream flank’ and ‘downstream flank’). A neisserial DUS sequence is added in order to ensure uptake of the DNA. When a plasmid containing this 1st crossover construct is linearized and presented to N.m. cells in the presence of MgCI2, selection for erythromycin resistance results in isolation of cells with a genomic insertion of the EryR-StrepS cassette These cells are resistant the erythromycin, but sensitive to streptomycin (because streptomycin sensitivity is dominant over the genomic streptomycin resistance). For the counterselection step, a 2nd crossover construct is P62015448wo

[0316] 44 designed that is identical to the 1st crossover construct, except that the EryR-StrepS cassette is either deleted or replaced by a DNA sequence to be inserted. Positive transformants that carry the EryR-StrepS cassette in their genome are subjected to transformation with a linearized plasmid carrying the 2nd crossover construct. Selection for streptomycin resistance then results in the isolation of cells that have lost their genomic EryR-StrepS cassette as a result of recombination between linearized plasmid and the N.m. genome. Depending on the plasmid used, this results in deletion of the region between the upstream and downstream flank or insertion of a DNA sequence present in between these flanks in the 2ndcrossover construct.

[0317] Results

[0318] Expression of the NG (Neisseria Gonorrhea) antigens in NM (Neisseria Meningitidis) OMVs was confirmed by proteomics. The relative abundance of the inserted NG antigens was determined by mass spectrometry. As shown in Table 2 below, the prepared NM OMVs expressed these NG antigens.

[0319] Table 2. NG antigen detection in NM OMVs

[0320] “eOMV” stands for native OMV, “sOMV” stands for spontaneous OMV

[0321] To verify expression of the required dilactose structure forming the 2C7 epitope, mass spectrometry on LPS was performed. The deconvoluted ESI- FT mass spectra of intact LOS isolated from OMV samples are shown in Figure 3. Compositions proposed for the ion peaks observed are listed in Table 3 below. These composition proposals were further supported by MS / MS analysis of main LPS ion peaks.

[0322] It was found that the LPS molecular mass shifts observed were in line with those expected from the introduced genetic mutations. In particular, MS data were consistent with the presence of pentaacylated LPS in all OMV samples originating from bacteria with an LpxL1 mutation, and two P62015448wo

[0323] 45 additional hexoses in the oligosaccharide core of LPS from strains with replacement of meningococcal LgtA with gonococcal LgtG and expression of gonococcal LgtE.

[0324] Table 3. Compositions proposed for the ion peaks P62015448wo

[0325] 46 P62015448wo

[0326] 47

[0327] Example 1.2

[0328] Structure-based study of N. meningitidis and N. gonorrhoeae LgtE enzymes to identify residues dictating the distinct functions of the two enzymes.

[0329] Sequence of mutations introduced in the genome of NM H44 / 76 (AporA, StrepR)

[0330] 1 Deletion of siaD

[0331] 2 Deletion of IpxLI

[0332] 3 Replacement of aniA by aniA from N.g. FA

[0333] 4 Replacement of metQ by metQ from N.g. FA19

[0334] 5 Replacement of IgtA by IgtG from N.g. FAW

[0335] 6 Replacement of nspA by nspA from N.g. FA19

[0336] 7 Deletion of Ipt3

[0337] 8 Deletion of rmpM

[0338] 9 Replacement of nspA N.g. FAW by nspA from N.g. FA1090

[0339] 10 Replacement of IgtG N.g. FAW (inactive) by active version

[0340] 11 Insertion of IgtE N.g. FAW at former location of Ipt3

[0341] N. gonorrhoeae (Ng) LOS contains 3 OS chains. The OS chains branch from two heptose residues attached to lipid A via two KDO molecules. The genes responsible for the addition of most sugars have been defined. Among these IgtE encodes a glycosyl transferase responsible for the addition of galactose p-1 ,4 to glucose, which is one of the two Ng epitopes (2C7 epitope) that do not react with human glycosphingolipids (GSL) antigens (Gulati et al, 2009, supra). While LgtE enzymes from Ng and Nm are highly homologous, we discovered that they have slightly different specificities, as only the Ng LgtE could extend the p-chain attached to Hep II, which is required for 2C7 expression.

[0342] The aim was to engineer Nm to make it express the Ng 2C7 epitope. Thus, a structure-based study has been performed to i) identify key residues differing between Ng and Nm LgtE enzymes, ii) evaluate their possible impact on the enzyme function, iii) generate a short list of residues to be mutated in Nm LgtE to test 2C7 expression. As such, we could pinpoint the crucial residues determining the difference between Nm and Ng LgtE functionality.

[0343] LgtE is a glycosyl transferase family 25 (GT25) protein belonging to the GT-A superfamily fold. Its structure is characterized by a central p-sheet core flanked by a smaller one and several alpha helix motifs. The loop connecting p3 and p4 motifs accommodates the DxD motif (EDD in Nm and Ng LgtE) and defines the LgtE active site.

[0344] In the present study Nm H44 / 76 and Ng FAW LgtE protein sequences were considered. Given the absence of experimentally solved structures for Nm H44 / 76 and Ng FAW LgtE, AlphaFold2 was chosen as predictive algorithm to model Nm and Ng LgtE structures. The LgtE active site was identified in both Nm and Ng LgtE proteins by aligning the predicted structures with the experimental P62015448wo

[0345] 48 fold of a member of a glycosyl transferase family 7 (GT7) protein in complex with the catalytic metal ion and donor and acceptor sugars (PDB ID 4M4K). GT7 and GT25 protein families are both classified as inverting enzymes with GT-A fold (Breton C. et al, Glycobiology, 2006;16(2):29R- 37R.). Thus, we could reasonable predict conservation of the relative position of cofactors in GT7 and GT25 active sites.

[0346] The BLAST sequence-based alignment of Nm H44 / 76 and Ng FA LgtE proteins showed that most of the single amino-acid differences are located on helix a2 and the a2-a3 loop, whereas single point mutations are spread along the entire sequence.

[0347] A list of potentially relevant residues was established by pinpointing the locations of single-point mutations on their 3D structures. Varied residues found on helix a2 and the a2-a3 loop appeared to be in close proximity to the sugar donor binding site, suggesting their involvement in its recruitment. The positioning of K74E and E60G on helix a3, along with E232G (from Nm to Ng), implied a role in stabilizing the folding of helix a2 and the a2-a3 loop region. Notably, W151 C and W183L (from Nm to Ng) delineated a portion of the sugar acceptor (i.e., LPS, Persson et al, Nat Struct Biol, 2001 ;8(2):166-75) binding site suggesting their contribution to determining the reaction product due to their different steric hindrance.

[0348] To test the 2C7 expression in Nm H44 / 76, three sets of mutations were performed involving the following residues combinations:

[0349] • Hybrid clone 1 . helix a2 and a2-a3 loop (8 substitutions in total, see also alignment Fig. 4, depicting the substitutions in the helix a2 and a2-a3 loop) + E60G and K74E in helix a3 + E232G facing the a2-a3 loop;

[0350] • Hybrid clone 2. W151 C and W183L, i.e., residues part of the sugar acceptor binding site;

[0351] • Hybrid clone 3. Both selections (13 substitutions in total) at the same time

[0352] Results

[0353] The results are shown in Figure 5. Introduction of only W151 C and W183L is sufficient to achieve 2C7 expression in Nm. The modelling described above had predicted that W151 and W183 in Nm LgtE are part of the LPS binding site, and their substitution in Ng LgtE could reduce steric hindrance, which might explain the extended specificity to the Hep2-Glc chain.

[0354] The sequential experimental cloning steps included the knockout of N.m. Lpt3 in the final strains. Initially, we had inactivated the Ipt3 gene because we hypothesized that the presence of PEA at the same heptose position as the lactose moiety could interfere with 2C7 expression. To check whether this was indeed the case, we also made the following strains: P62015448wo

[0355] 49

[0356] Hybrid 2 without Ipt3: N.m. H44 / 76 AporA, AsiaD, AlpxLI , aniA:: aniAN s FA19, metQ:: metQN s FA19, IgtA -> lgtGNs ^ (active),ns pA : : nSpAN9 FA1090,A|pt3 ArmpM, lgtE::hybrid2

[0357] Hybrid 2 with Ipt3: N.m. H44 / 76 AporA, AsiaD, AlpxLI , ArmpM, IgtA:: |gtGNs FAi9(acti e) |gtE: :hybrid2 Expression of the 2C7 LPS epitope was tested by ELISA. The results clearly confirmed that 2C7 expression did not require an inactivation of Ipt3 (data not shown).

[0358] Example 1.3

[0359] To determine whether MetQ expression could be further increased, an additional gene copy was introduced in the N. meningitidis strains.

[0360] In more detail, the additional strain construction started from the three previously constructed N.m. H44 / 76-derived strains as described in example 1.1 ;

[0361] 1 . N.m. H44 / 76 AporA, StrepR, AsiaD, AlpxLI , metQ:: metQNg <FAI9), ArmpM

[0362] 2. N.m. H44 / 76 AporA, StrepR, AsiaD, AlpxLI , ArmpM, IgtA:: IgtG Ng (FA19, active version), Ipt3:: IgtENg (FA19)

[0363] 3. N.m. H44 / 76 AporA, StrepR, AsiaD, AlpxLI , aniA::aniANg (FAi9>, metQ::metQNg (FAi9>, ArmpM, nspA::nspANg (FA1090), lgtA::lgtGNg (FA19, active version) , Ipt3: : IgtENg (FA19)

[0364] These three strains are annotated as;

[0365] 1 . NmBB+M; Neisseria meningitidis backbone with metQ (M) replaced by gonococcal metQ

[0366] 2. NmBB+2C7; Neisseria meningitidis backbone expressing the gonococcal 2C7 LPS-epitope

[0367] 3. NmBB+4G; Neisseria meningitidis backbone expressing four gonococcal antigens (aniA, metQ and nspA replaced by gonococcal homologues and 2C7 expressed)

[0368] These strains were then amended as follows: For all three strains, the meningococcal opaB coding sequence was replaced by gonococcal metQ and with a stabilized, high expression promoter. In addition, meningococcal metQ in NmBB+2C7 was replaced by gonococcal metQ. For NmBB+M and NmBB+4G, the high expression stabilized promoter that was initially introduced together with gonococcal metQ (P1) was replaced by a new version (P2). The resulting strains are

[0369] 1 . N.m. H44 / 76 AporA, StrepR, AsiaD, AlpxLI , ArmpM, opaB:: metQN g <FAI9), metQ:: metQN g (FA19)

[0370] 2. N.m. H44 / 76 AporA, StrepR, AsiaD, AlpxLI , ArmpM, IgtA:: IgtG N g (FA19, active version), Ipt3:: IgtEN g (FA19), opaB:: metQN.g. (FA19), metQ:: metQN g <FAI9)

[0371] 3. N.m. H44 / 76 AporA, StrepR, AsiaD, AlpxLI , aniA::aniAN g <FAI9), ArmpM, nspA::nspAN g (FA1090), lgtA::lgtG N g (FA19, active version), lpt3::lgtEN g <FAI9), opaB:: metQN g <FAI9), metQ::metQN g <FAI9)

[0372] These three final strains are annotated as follows; P62015448wo

[0373] 50

[0374] 1 . NmBB+M+M; backbone strain with gonococcal metQ expressed from two loci

[0375] 2. NmBB+2C7+M+M; backbone strain with gonococcal 2C7 LPS-epitope and with gonococcal metQ expressed from two loci

[0376] 3. NmBB+4G+M; backbone strain expressing four gonococcal antigens (aniA, metQ and nspA replaced by gonococcal homologues, with expression of gonococcal metQ from two loci, and 2C7 expressed). Note that the abbreviation of this strain contains only one ‘M’ for metQ, because the other ‘M’ is included in the ‘4G’ part of the abbreviation.

[0377] Results

[0378] Inactivated cell suspensions of the starting strains, intermediates (with opaB replaced by metQ), and final strains were first analyzed for total protein content using Peterson’s modification of the Lowry method (Peterson, G.L., Analyt. Biochem., 83, 346 (1977) ). Subsequently, proteome analysis was carried out by LC-MS. In order to make an accurate estimate of the amount of MetQ in each sample, a MetQ signature peptide of known quantity was mixed with each sample before analysis by LC-MS. Results of MetQ quantification are provided in Table 4 below. The results show that two copies of gonococcal metQ gene indeed result in a higher expression level.

[0379] Table 4. Estimate of MetQ quantity based on LC-MS measurement. Mass% calculated using the total protein content as measured by the Lowry-Peterson method. P1 = porA-nadA promoter, P2 = stabilized porA promoter. CV = coefficient of variation. Samples were measured in triplicate. P62015448wo

[0380] 51

[0381] Example 2: Immunization and challenge studies with meningococcal OMV vaccines carrying gonococcal antigens

[0382] Mouse immunogenicity study

[0383] Example 2. 1

[0384] Four Neisseria gonorrhoeae (NG) antigens were expressed by introducing their genes into the Nm chromosome, thereby replacing their meningococcal homologues: AniA, NspA, MetQ and introducing the 2C7 LPS epitope. Gene replacements were introduced using a marker-free allelic replacement method, and placed behind a hybrid porA / nadA promotor for stable, high expression, as described in example 1 . As these antigens are no targets for currently used antibiotics, they have the potential also to be effective against antibiotic-resistant strains. Several strains were constructed that each contain one NG antigen and a strain was constructed containing all 4 antigens (annotated as 4G). Hence the following OMVs were produced:

[0385] • NM-OMV_NG-MetQ: N. meningitidis OMV expressing N. gonorrhoeae MetQ

[0386] • NM-OMV_NG-AniA: N. meningitidis OMV expressing N. gonorrhoeae AniA

[0387] • NM-OMV_NG-NspA: N. meningitidis OMV expressing N. gonorrhoeae NspA

[0388] • NM-OMV_NG-2C7: N. meningitidis OMV expressing N. gonorrhoeae 2C7 epitope

[0389] • NM-OMV_NG-4G: N. meningitidis OMV expressing N. gonorrhoeae MetQ, AniA, NspA and the 2C7 epitope

[0390] After production of OMV and confirming expression ofthe antigens in the OMV, the different vaccine candidates were tested in immunogenicity experiments in mice. Mice were immunized on day 0, day 21 and day 42 with 25 pg OMV without any additional adjuvant via subcutaneous injection. On day 63, blood was collected for assessment of induction of antibodies against gonococci.

[0391] Results

[0392] Antibody titers were determined against heat inactivated gonococcal strain FA (Figure 6A). Administration of all OMV vaccines induced a significant increase in antibody titers against gonococci when compared with the PBS group. Compared to the group immunized with NM-OMV, P62015448wo

[0393] 52 vaccination with NM-OMV_NG-MetQ and NM-OMV_NG-4G induced a significant increase of specific antibody responses. The NM-OMV_NG-MetQ group also had a significantly higher response than the Bexsero control group. The antibody response induced by NM-OMV_NG-AniA and NM-OMV_NG-NspA was similar to NM-OMV . The NM-OMV_NG-2C7 induced antibody response was also similar to the NM-OMV group against FAW.

[0394] Since expression of the gonococcal 2C7 epitope by NM_NG-2C7 and NM_NG-4G vaccine strains was confirmed by mass spectrometry (see example 1 , above), LPS was isolated from the NM_NG- 4G strain. Serum antibody responses to LPS of this strain were measured in mice vaccinated with NM-OMV_NG-MetQ, NM-OMV_NG-2C7 and NM-OMV_NG-4G. Compared to PBS, Bexsero and NM-OMV, vaccination with NM-OMV_NG-2C7 and NM-OMV_NG-4G induced a significant increase in antibody response (figure 6B) to 2C7-LPS. As expected, NM-OMV_NG-MetQ did not induce a significantly higher response to 2C7-LPS than NM-OMV from the backbone strain or Bexsero.

[0395] Later analysis showed that the strain FAW does not contain the 2C7 epitope (Figure 6C), which explains why no increased antibody response was induced by the OMVs containing 2C7 LPS. While the far majority (>95%) of clinical Ng isolates express the 2C7 epitope, it has been previously shown that this epitope is lost upon passage in the lab. A similar observation was seen for F62. Figure 6C further shows that the Neisseria meningitidis strain NM-OMV_NG-4G and Neisseria gonorrhea strain FA1090 bind equally strong to the 2C7 antibody, confirming that NM-OMV_NG-4G properly expresses the 2C7 epitope. Finally, vaccination with NM-OMV_NG-4G also induced antigenspecific antibody responses against purified AniA, metQ and NspA proteins (Figure 6D).

[0396] Example 2.2

[0397] To test the concept of the invention, NM-OMV_NG-MetQ and NM-OMV_NG-4G was further evaluated in a challenge experiment. As control groups PBS and NM-OMV were included. In the challenge experiment female mice were vaccinated with 25 pg OMV without additional adjuvant on day 0, day 21 and day 42. Twenty-one days after the last vaccination, animals were infected with NG strain F62, and clearance of infection was monitored in time by taking vaginal swab cultures on day 1 , 3, 5 and 7 after infection.

[0398] Results

[0399] In Figure 7, it can be seen that mice vaccinated with NM-OMV clear the infection much more efficiently compared to PBS vaccinated animals, demonstrating the cross-protective capacity of NM against NG.

[0400] Animals vaccinated with NM-OMV_NG-MetQ or NM-OMV_NG-4G have a higher percentage of cleared infection 7 days after challenge compared to animals vaccinated with NM-OMV, showing the additive effect of including NG antigens in the NM-OMV. At the end of the experiment (day 7) the differences between the groups became more pronounced. P62015448wo

[0401] 53

[0402] In addition, no adverse effects were observed following administration of any of the OMV preparations or negative control tested in this study. In contrast, immunization of mice with the control 4Bexsero by SC injection typically resulted in a subcutaneous mass at the immunization site with skin excoriation resulting from scratching. However, no excoriations or irritation was associated with any of the OMVs tested in this current study.

[0403] Altogether the available data clearly demonstrate the capacity of NM based OMVs to induce crossprotection against NG, and that replacing NM antigens like MetQ and LPS with their NG counterparts (e.g. the 2C7 epitope), improves the NG specific response.

[0404] Example 2.3

[0405] In order to investigate the ability of the engineered meningococcal OMVs to provide a cross-reactive antibody response against gonococci, a mouse immunogenicity study was performed. Mice received three intramuscular injections with three-week intervals. Two dose levels were used, 15 pg (low dose) and 45 pg (high dose), with OMVs carrying different combinations of the four selected gonococcal antigens. Sera were collected two weeks after the final immunization. The OMVs carrying the different antigen combinations are indicated in table 5 below.

[0406] Table 5. The four hybrid OMVs tested. Meningococcal antigens replaced by gonococcal versions.

[0407] NmBB-Ng_2C7: AporA, AsiaD, AlpxLI , ArmpM, IgtA:: lgtGN9FA19, Ipt3:: lgtENaFA19

[0408] NmBB-Ng_2C7+M+M_(2xMetQ): AporA, AsiaD, AlpxLI , ArmpM, IgtA:: lgtGN-aFA19, Ipt3:: lgtENa

[0409] FA19, opaB:: metQNsFA19, metQ:: metQNsFA19 P62015448wo

[0410] 54

[0411] NmBB-Ng+M+M (2xMetQ): AporA, AsiaD, AlpxLI , ArmpM, opaB:: metQN 9 FA19, metQ:: metQN 9 FA19NmBB-Ng_4G+M_(MetQ): AporA, AsiaD, AlpxLI , aniA:: aniAN 9 FA19, ArmpM, nspA:: nspAN 9 FA1090, IgtA:: lgtGN 9 FA19, Ipt3:: lgtEN 9 FA19, opaB:: metQN 9 FA19, metQ:: metQN 9 FA19

[0412] Results

[0413] High antibody titers could be measured against whole cells of N.gonorrhoeae strains FA and FA1090 for both the low and high dose OMV groups, which was much higher than the positive control Bexsero (Figure 8A-B). The different OMVs generally induced similar antibody levels, with the exception of 2C7-expressing OMVs. Interestingly in the 2C7-positive strain FA1090, OMVs containing the 2C7 antigen induced higher IgG titers as compared to the OMVs that did not contain the 2C7 antigen (Fig. 8B), indicating the superior potency of the 2C7 antigen. Against the 207- negative strain FAW this difference was absent (Fig. 8A), further demonstrating specificity of the LPS response. The specific anti-2C7 response with OMVs expressing this epitope was also clearly seen when purified 2C7 LPS was used as test antigen (Figure 8C-D).

[0414] While all OMVs induced high antibody levels against purified gonococcal MetQ protein, the levels were higher with OMVs expressing the gonococcal version. Against the other two introduced gonococcal antigens AniA and NspA lower antibody levels were found, which were similar for OMVs with or without the gonococcal version (data not shown). Using a panel of 1 1 WHO gonococcal reference strains in the whole cell ELISA, a broadly cross-reactive antibody response was seen The 2C7-specific OMV response was at least comparable, but generally higher in the 2C7-positive strains (data not shown).

[0415] Surprisingly, combinations of the low dose OMVs with alum adjuvant resulted in lower antibody levels (Figure 8E-F).

[0416] Taken together, high IgG antibody titers against Ng whole cells were induced in all Nm-Ng-OMV groups, comparable IgG antibody levels for both low and high dose OMV-groups and lower antibody levels after inclusion of aluminum hydroxide adjuvant. Specific antibodies were induced against isolated antigens MetQ, AniA, NspA and 2C7 LPS. The specific antibody levels induced against MetQ and 2C7 LPS were higher with OMVs expressing the Ng-version. Notably, expression of 2C7 LPS in OMVs leads to higher antibody levels against Ng whole cells expressing the 2C7 LPS. There is a broadly cross-reactive antibody response against panel of 11 WHO reference strains.

[0417] Example 2.4

[0418] Evaluation of serum bactericidal antibody (SBA) activity P62015448wo

[0419] 55

[0420] Method

[0421] A glycerol stock of the gonococcal strain to be tested was thawed, inoculated in 6 ml GC broth with 1 % IsoVitaleX (Becton Dickinson) and incubated at 37°C, 200 RPM, and 5% CO2 until OD590 reached 0.2 - 0.4. Cells were then diluted to ~8 x 10exp4 CFU / ml in Hank’s Balanced Salt Solution with Ca2+ and Mg2+ (HBSS, ThermoFisher Scientific), and 1 % BSA (Sigma). The exact dilution varied per strain and was assessed prior to execution of the assay. 20 pl heat-inactivated, pooled mouse serum was serially diluted in HBSS + 1 % BSA in a 96-well U bottom plate (Greiner) and then mixed with 20 pl diluted bacteria. After incubation for 15’ at 37°C and 5% CO2, the 40 pl mix of serum and bacteria was mixed with 40 pl IgG / IgM depleted human complement (Pel-Freeze) diluted to 5% - 20% in HBSS + 1 % BSA. Like bacterial dilution, complement dilution varied per strain and was determined prior to the SBA (>80% survival compared to control without complement). The mix of serum, bacteria, and complement was then incubated for 45’ as before. As controls, each row of the plate contained a well without complement (serum control), without serum (complement control), and without serum and complement (bacteria only). After incubation, cells were vigorously mixed by adding 100 pl GCII broth. A 10 pl drop was then plated on large, square plates with GC (Becton Dickinson) supplemented with IsoVitaleX (ThermoFisher). The plates were tilted 90° immediately afterwards, so that the drops rolled down, ensuring an even spread of bacteria in the resulting lane. Plates were incubated for 18-24 hours (37°C, 5% CO2) after which bacterial colonies were photographed and manually counted. Serum dilutions with >50% killing compared to the complement control were scored as positive and the highest serum dilution with this amount of killing was scored as the SBA titer.

[0422] Initial SBA attempts unexpectedly showed high SBA titers for placebo sera, indicating the presence of something else than antibodies involved in bacterial killing. Pre-treatment of sera with anti-mouse IgM bound to agarose (Sigma) eliminated this effect (data not shown) and all sera were therefore IgM depleted prior to the SBA. Briefly, four volumes of anti-mouse IgM agarose was centrifuged, washed in PBS, and then mixed with one volume of serum. After overnight incubation at 4°C and 1200 RPM in a benchtop shaker, agarose was removed by centrifugation using a Spin- X 0.22 pm filter tube (Cornig).

[0423] Results

[0424] It was tested whether the engineered OMVs could induce serum bactericidal activity (SBA) against N.gonorrhoeae. Using the test strains FAW, FA1090 and MS11 , SBA titers were seen already with the backbone OMVs, indicating that native Nm OMVs can elicit a bactericidal antibody response against N. gonorrhoeae.

[0425] As compared to the backbone Nm-OMV, the titers against FA1090 significantly increased for Nm- Ng-2C7. Such drastic increase in titer was not observed against FAW, showing that a specific bactericidal response against this 2C7 LPS epitope was induced. This difference was also apparent for F62 strains with and without 2C7, although the overall titers were lower in this strain background. No such difference was seen for the OMVs with only MetQ as additional antigen. P62015448wo 56

[0426] Against a panel of six different WHO reference strains, a broad SBA response was observed, especially for the group receiving OMVs with all four gonococcal antigens (data not shown) All in all, high bactericidal response was observed against strain FA1090 for OMV groups expressing 2C7 LPS, lower bactericidal response against strain FAW, and broad bactericidal response against panel of WHO reference strains for Nm-Ng_4G OMV groups.

Claims

1. P62015448wo57Claims1 . A genetically modified Neisseria bacterium comprising a lipopolysaccharide (LPS) having a lipid A moiety and a modified oligosaccharide core, wherein the modified oligosaccharide core comprises a first oligosaccharide chain coupled to heptose 1 and a second oligosaccharide chain coupled to heptose 2, wherein the first and second oligosaccharide chain comprise a lactose directly coupled to respectively heptose 1 and 2, thereby forming a Neisseria gonorrhoeae 2C7 epitope, and wherein preferably at least one of: the first oligosaccharide chain is lacto-N-tetraose and the second oligosaccharide chain is lactose; and the first oligosaccharide chain is lactose and second oligosaccharide chain is lactose, wherein the modified Neisseria bacterium is not Neisseria gonorrhoeae, wherein preferably the bacterium is Neisseria meningitidis, Neisseria lactamica or Neisseria cinerea.

2. The genetically modified Neisseria bacterium according to claim 1 , wherein the bacterium is Neisseria meningitidis.

3. The genetically modified Neisseria bacterium according to claim 1 or 2, wherein the bacterium is modified by at least one of: i) increasing a1 ,3 glycosyltransferase (LgtG) activity; i) increasing N. gonorrhoeae p1 ,4 galactosyltransferase (LgtE) activity; and preferably iii) reducing p1 ,3 N-acetylglucosaminyltransferase(LgtA) activity and / or reducing a- galactosyl transferase (LgtC) activity.

4. The modified Neisseria bacterium according to claim 3, wherein at least one of: i) LgtG glycosyltransferase activity is increased by introducing expression of an LgtG protein, preferably a Neisseria gonorrhoeae LgtG protein; ii) LgtE galactosyltransferase activity is increased by introducing expression of an LgtE protein having a substitution of at least one of position W151 and W183 of SEQ ID NO: 1 , preferably at least one of a W151 C substitution and a W183L substitution; and preferably iii) the LgtA N-acetylglucosaminyltransferase activity is reduced by decreasing expression of an LgtA protein and / or the LgtC a-galactosyl transferase activity is reduced by decreasing expression of an LgtC protein and / or wherein preferably at least one of:- the LgtE protein has at least 60% sequence identity with SEQ ID NO: 1 ;- the LgtG protein has at least 60% sequence identity with SEQ ID NO: 2;- the LgtA protein has at least 60% sequence identity with SEQ ID NO: 3 and- the LgtC protein has at least 60% sequence identity with SEQ ID NO: 41 .P62015448wo585. The modified Neisseria bacterium according to claim 3 or 4, wherein LgtE glycosyltransferase activity is increased by at least one of: a) introducing into the bacterium a nucleic acid expressing a LgtE protein having at least 60% sequence identity with SEQ ID NO: 4, preferably having at least one of a cysteine at position 151 and a leucine at position 183; and b) modifying the genomic sequence encoding an endogenous LgtE protein having at least 60% sequence identity with SEQ ID NO: 1 , wherein preferably the modification results in a substitution of at least one of position W151 and W183, preferably a W151 C and / or a W183L substitution.

6. The modified Neisseria bacterium according to any one of claims 1 - 5, expressing a further N. gonorrhoeae antigen, preferably on the extracellular outer membrane surface of the bacterium, wherein preferably the further N gonorrhoeae antigen is at least one of:- a N. gonorrhoeae MetQ protein, preferably having a least 60% sequence identity with SEQ ID NO: 5;- a N. gonorrhoeae NspA protein, preferably having a least 60% sequence identity with SEQ ID NO: 6; and- a N. gonorrhoeae AniA protein, preferably having a least 60% sequence identity with SEQ ID NO: 7.

7. The modified Neisseria bacterium according to any one of claims 1 - 6, further comprising at least one of:- a reduced expression of an LpxL1 protein, preferably having a least 60% sequence identity with SEQ ID NO: 8;- a reduced expression of an RmpM protein, preferably having a least 60% sequence identity with SEQ ID NO: 9;- a reduced expression of an PorA protein, preferably having a least 60% sequence identity with SEQ ID NO: 10; and- a reduced expression of an SiaD protein, preferably having a least 60% sequence identity with SEQ ID NO: 1 1.

8. A Neisseria LPS molecule comprising a modified oligosaccharide core as defined in claim 1 , wherein the LPS is preferably obtainable from a bacterium according to any one of claims 1 - 7, and wherein preferably the lipid A moiety is a modified penta-acylated or tetra-acylated Lipid A moiety.

9. An OMV comprising the LPS of claim 8, and preferably at least one of:- a N gonorrhoeae MetQ protein, preferably having a least 60% sequence identity with SEQ ID NO: 5;P62015448wo59- a N gonorrhoeae NspA protein, preferably having a least 60% sequence identity with SEQ ID NO: 6; and- a N gonorrhoeae AniA protein, preferably having a least 60% sequence identity with SEQ ID NO: 7, wherein preferably the OMV is obtainable from a bacterium as defined in any one of claims 1 - 7.

10. A composition comprising at least one of the bacterium as defined in any one of claims 1 - 79, an LPS as defined in claim 8 and / or an OMV as defined in claim 9 and a pharmaceutically accepted excipient, wherein preferably the bacterium is inactivated and wherein preferably at least 90% of the LPS molecules in the composition is a molecule as defined in claim 9.

11. The composition of claim 10, further comprising an adjuvant and / or optionally further comprising a non-neisserial antigen.

12. The composition according to claim 10 or 11 for use in a medicament.

13. The composition according to claims 11 or 12 for use in the treatment or prevention of aNeisseria gonorrhoeae infection.

14. A composition according to any one of claim 11 or 12, wherein the composition is at least one of i) an acellular vaccine comprising a neisserial LPS as defined in claim 8 and / or an OMV as defined in claim 9; and ii) a whole cell vaccine comprising a bacterium as defined in any one of claims 1 - 7, and wherein the bacterium is inactivated.

15. A process for producing an OMV according to claim 9, wherein the process comprises the steps of: a) cultivating a bacterium as defined in any one of claims 1 - 7; b) optionally, extracting the OMV; and, c) recovering the OMV, wherein the recovery at least comprises removal of the bacteria from the OMV, wherein preferably the process is a detergent-free process.

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