FIELD OF THE INVENTION

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 LPS molecules that can be obtained from bioengineered meningococcal LPS mutants and that are useful as part of a whole cell vaccine, OMV vaccine or as purified LPS or lipid A molecules.

BACKGROUND OF THE INVENTION

Lipopolysaccharides (LPS), also known as bacterial endotoxin, are an abundant component of the outer membrane of gram-negative bacteria. During infection with gram-negative bacteria, LPS or more precisely the lipid A part of LPS activates the host's innate immune system (1-3). This activation occurs 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 (3,4). However, 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 (5, 6).

For complete activation of the TLR4/MD-2 complex, a lipid A structure with six acyl chains and two phosphate groups is critical (7). However, many bacterial species carry enzymes that can modify their lipid A structure either by changing the number of acyl chains or phosphate groups resulting in altered activation of the TLR4/MD-2 complex (8), even to the point of being an antagonist instead of an agonist as is observed for the tetra-acylatedE. colilipid IVa structure (7,9).

The TLR4/MD-2 complex is unique among the TLR family of receptors because it can signal through both the MyD88 as well as the TRIF pathway. Modified lipid A structures can induce select signalling by preferential recruitment of the MyD88 or TRIF adaptor molecules. Preferential signalling through the TRIF pathway, which triggers production of type I interferons, is thought to be important for vaccine adjuvants (10, 11). Monophosphoryl lipid A (MPLA) is an example of a modified lipid A that triggers a TRIF-biased signalling (11). MPLA is a heterogeneous lipid A mixture fromSalmonella minnesota, which has been chemically detoxified and is approved for the use as adjuvant in some vaccines (12). The main component of MPLA consists of a hexa-acylated 4′-monophosphoryl lipid A. Use of MPLA has the disadvantage that for its production a chemical treatment is needed in addition to LPS isolation from the bacteria and it only consists of the lipid A portion of LPS, making it water insoluble.

Neisseria meningitidistypically produces hexa-acylated LPS with phosphate and phosphoethanolamine groups appended to the 1 and 4′ position of the lipid A (13, 14). Heterologous expression of LPS modifying enzymes such as PagL or deletion of lipid A biosynthesis enzymes such as LpxL1 and LpxL2 has been used to detoxify the highly active meningococcal LPS (13, 15). Deletion of LpxL1 was shown to be an advantageous method for detoxifying meningococcal LPS when making meningococcal outer membrane vesicle vaccines, without the need to use a detergent to reduce excess LPS-related reactogenicity (16). However, the activity of this modified LPS has been reduced to the point that it barely induces any activation of the TLR4/MD-2 complex on human cells, making it less applicable as a stand-alone vaccine adjuvant (17). Heterologous expression of pagL inN. meningitidisresults in a different attenuated penta-acylated LPS structure, which is still capable of inducing TLR4 activation and induces a TRIF-biased cytokine production on a human monocytic cell line (13).

E.coliLpxP (Genbank accession number: U49787.1) is a cold shock-induced enzyme known to add a secondary 9-hexadecenoic acid (C16:1; palmitoleate) to the 2′ acyl chain of lipid A (27).

It is an object of the invention of providing modified LPS molecules useful as adjuvants, which have the optimal balance between retaining a sufficient amount of immune activation while limiting toxic side effects. The present invention addresses this problem by heterologous expression of LPS modifying enzymes in combination with targeted deletion of lipid A biosynthesis genes to provide a diverse set of meningococcal LPS structures with a broad range of TLR4/MD-2 activation capacities that are useful in a variety of prophylactic and therapeutic applications.

SUMMARY OF THE INVENTION

In a first aspect, the invention relates to a neisserial LPS having a hexa-acylated lipid A moiety, wherein the hexa-acylated lipid A moiety is modified as compared to the lipid A moiety of a wild-type neisserial LPS in that it has a palmitoleoyl (instead of a lauroyl) as secondary acyl chain bound to the primary acyl chain on the glucosamine at the non-reducing end of the lipid A moiety. Preferably, the LPS, except for the hexa-acylated lipid A moiety, has the structure of LPS ofNeisseria meningitidis, Neisseria gonorrhoeaeorNeisseria lactamica, whereby more preferably, theNeisseria meningitidis, Neisseria gonorrhoeaeorNeisseria lactamicais at least one of lgtB−and galE−and whereby most preferably, theNeisseria meningitidisis at least one of serogroup B and immunotype L3.

In a preferred neisserial LPS according to the invention, the hexa-acylated lipid A moiety has the structure of formula (I) (see below), wherein R1and R2, independently, are either —P(O)(OH)2, —[P(O)(OH)—O]2—H, —[P(O)(OH)—O]2—CH2CH2NH2, —[P(O)(OH)—O]3—CH2CH2NH2, —[P(O)(OH)—O]3—H or —P(O)(OH)—O—CH2CH2NH2, and wherein, preferably R1and R2, independently, are either —P(O)(OH)2, —[P(O)(OH)—O]2—H, —[P(O)(OH)—O]2—CH2CH2NH2or —[P(O)(OH)—O]3—CH2CH2NH2.

In a second aspect, the invention relates to a composition comprising a mixture of: i) a neisserial LPS with the above defined hexa-acylated lipid A moiety; and ii) a neisserial LPS with a penta-acylated lipid A moiety wherein the penta-acylated lipid A moiety is modified as compared to the lipid A moiety of a wild-type neisserial LPS in that it lacks the secondary acyl chain bound to the primary acyl chain on the glucosamine at the non-reducing end of the lipid A moiety. Preferably, the penta-acylated neisserial LPS, except for the penta-acylated lipid A moiety, has the structure of an LPS as defined above for the hexa-acylated LPS. More preferably, the penta-acylated lipid A moiety has the structure of formula (II) (see below), wherein R1and R2, independently, are either —P(O)(OH)2, —[P(O)(OH)—O]2—H, —[P(O)(OH)—O]2—CH2CH2NH2, —[P(O)(OH)—O]3-CH2CH2NH2, —[P(O)(OH)—O]3—H or —P(O)(OH)—O—CH2CH2NH2, and wherein, preferably R1 and R2, independently, are either —P(O)(OH)2, —[P(O)(OH)—O]2—H, —[P(O)(OH)—O]2—CH2CH2NH2or —[P(O)(OH)—O]3—CH2CH2NH2. Preferably, the composition comprises the hexa-acylated neisserial LPS and the penta-acylated neisserial LPS in a molar ratio in the range of 1:400 to 1:1.25.

In a second aspect, the invention relates to a genetically modified bacterium of the genusNeisseria, wherein the bacterium comprises: a) a genetic modification that reduces or eliminates the activity of a lipid A biosynthesis lauroyl acyltransferase encoded by an endogenous lpxL1 gene; and, b) a genetic modification that confers to the bacterium lipid A biosynthesis palmitoleoyltransferase activity. Preferably, the bacterium is a genetically modifiedNeisseria meningitidis, Neisseria gonorrhoeaeorNeisseria lactamica. Preferably, in the genetically modified bacterium, the endogenous lpxL1 gene is a gene encoding an LpxL1 protein having an amino acid sequence with at least 90% sequence identity with at least one of SEQ ID NO's: 1-3, and the genetic modification that confers to the bacterium lipid A biosynthesis palmitoleoyltransferase activity is a genetic modification that introduces the expression of a heterologous lpxP gene having a nucleotide sequence that encodes an LpxP lipid A palmitoleoyltransferase that has at least 89% amino acid sequence identity with at least one of SEQ ID NO's: 4-10. More preferably, the nucleotide sequence that encodes an LpxP lipid A palmitoleoyltransferase encodes a known cold shock-induced LpxP palmitoleoyl acyltransferase having an amino acid sequence selected from the group consisting of SEQ ID NO: 4-10.

The genetically modified bacterium of the invention, further preferably is genetically modified to express a heterologous antigen, whereby preferably, the heterologous antigen is expressed on the extracellular outer membrane surface of the bacterium. In a preferred embodiment, the genetically modified bacterium has a genetic modification that reduces or eliminates the expression of at least one of an endogenous lgtB gene and an endogenous galE gene.

Preferably a genetically modified bacterium according to the invention is aNeisseria meningitidisserogroup B, immunotype L3. More preferably, the bacterium isNeisseria meningitidisstrain H44/76 or a derivative thereof.

In a fourth aspect the invention pertains to a neisserial LPS according to the invention, or a composition comprising a mixture of hexa-acylated neisserial LPS and penta-acylated neisserial LPS according to the invention, wherein the LPS is obtainable or obtained from a genetically modified bacterium according to the invention.

In a fifth aspect, the invention relates to an OMV comprising a neisserial LPS of the invention, or a mixture of a hexa-acylated neisserial LPS and a penta-acylated neisserial LPS according to the invention. The OMV preferably is obtainable or obtained from a genetically modified bacterium according to the invention.

In a sixth aspect, the invention pertains to a composition comprising at least one of a neisserial LPS of the invention, a mixture of the invention, a genetically modified bacterium of the invention and an OMV of the invention. Preferably, the composition is a pharmaceutical composition further comprising a pharmaceutically accepted excipient. A preferred composition is an acellular vaccine comprising a neisserial LPS of the invention, a mixture of the invention, or an OMV of the invention. Another preferred composition is a whole cell vaccine comprising a bacterium of the invention. Preferably the compositions of the invention further comprises at least one non-neisserial antigen.

In a seventh aspect, the invention relates to a process for producing a neisserial LPS or a mixture of neisserial LPSs according to the invention. The process preferably comprises the steps of: a) cultivating a bacterium of the invention; and, b) optionally, at least one of extraction and purification of the LPS or of the mixture of LPSs.

In an eighth 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 bacterium of the invention, at a temperature in the range of 20-42° C.; 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. In a further preferred process, the bacterium is cultured at a temperature lower than 37° C.

In an ninth 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) a neisserial LPS a mixture of neisserial LPSs according to the invention, preferably in a process as defined hereinabove; and, ii) an OMV according to the invention, preferably in a process as defined hereinabove; and, b) formulating at least one of the neisserial LPS, the mixture of neisserial LPSs and the OMV, optionally with further vaccine components, into a vaccine formulation.

In a tenth 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 bacterium of the invention; and, ii) optionally, at least one of inactivation of the bacterium and formulation into a vaccine. In a preferred process, the bacterium is cultured at a temperature lower than 37° C.

In an eleventh aspect, the invention relates to a neisserial LPS of the invention, a composition comprising a mixture of neisserial LPSs of the invention, a bacterium of the invention, an OMV of the invention, or a composition of the invention, for use as a medicament.

In a twelfth aspect, the invention relates to a neisserial LPS of the invention or a composition comprising a mixture of neisserial LPSs of the invention, for use in a treatment comprising inducing or stimulating an immune response in a subject. Preferably, the neisserial LPS or the mixture is for a use as adjuvant.

In a preferred embodiment, the neisserial LPS or the mixture is used in a treatment further comprising the administration of an antigen together with the neisserial LPS and wherein the treatment is for preventing or treating an infectious disease or tumour associated with the antigen.

In another preferred embodiment, the neisserial LPS of the invention or a composition comprising a mixture of neisserial LPSs of the invention is for a use as a Toll-like receptor 4 (TLR4) agonist in an immunotherapy, wherein preferably, the immunotherapy is an immunotherapy of a cancer or of a neurodegenerative disease. In an alternative embodiment, the immunotherapy comprises a generalized immune stimulation for preventing and/or reducing the spread of diverse microbial infections and/or to suppress bacterial growth.

DESCRIPTION OF THE INVENTION

Definitions

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 nucleic acid (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.

“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 below). GAP uses the Needleman and Wunsch global alignment algorithm 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, Calif. 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). When sequences have a substantially different overall lengths, local alignments, such as those using the Smith Waterman algorithm, are preferred.

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 of the present invention 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 oxidoreductase nucleic acid molecules of the invention. BLAST protein searches can be performed with the BLASTx program, score=50, wordlength=3 to obtain amino acid sequences homologous to protein molecules of the invention. 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 www.ncbi.nlm.nih.gov/.

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, phenylalanine-tyrosine, 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 gln or his; Asp to glu; Cys to ser or ala; Gln to asn; Glu to asp; Gly to pro; His to asn or gln; Ile to leu or val; Leu to ile or val; Lys to arg; gln 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.

As used herein, the term “selectively hybridizing”, “hybridizes selectively” and similar terms are intended to describe conditions for hybridization and washing under which nucleotide sequences at least 66%, at least 70%, at least 75%, at least 80%, more preferably at least 85%, even more preferably at least 90%, preferably at least 95%, more preferably at least 98% or more preferably at least 99% homologous to each other typically remain hybridized to each other. That is to say, such hybridizing sequences may share at least 45%, at least 50%, at least 55%, at least 60%, at least 65, at least 70%, at least 75%, at least 80%, more preferably at least 85%, even more preferably at least 90%, more preferably at least 95%, more preferably at least 98% or more preferably at least 99% sequence identity.

A preferred, non-limiting example of such hybridization conditions is hybridization in 6×sodium chloride/sodium citrate (SSC) at about 45° C., followed by one or more washes in 1 X SSC, 0.1% SDS at about 50° C., preferably at about 55° C., preferably at about 60° C. and even more preferably at about 65° C.

Highly stringent conditions include, for example, hybridization at about 68° C. in 5×SSC/5×Denhardt's solution/1.0% SDS and washing in 0.2×SSC/0.1% SDS at room temperature. Alternatively, washing may be performed at 42° C.

The skilled artisan will know which conditions to apply for stringent and highly stringent hybridization conditions. Additional guidance regarding such conditions is readily available in the art, for example, in Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, N.Y.; and Ausubel et al. (eds.), Sambrook and Russell (2001) “Molecular Cloning: A Laboratory Manual (3rdedition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York 1995, Current Protocols in Molecular Biology, (John Wiley & Sons, N.Y.).

Of course, a polynucleotide which hybridizes only to a poly A sequence (such as the 3′ terminal poly(A) tract of mRNAs), or to a complementary stretch of T (or U) resides, would not be included in a polynucleotide of the invention used to specifically hybridize to a portion of a nucleic acid of the invention, since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA clone).

A “nucleic acid construct” or “nucleic acid vector” is herein understood to mean a man-made nucleic acid molecule resulting from the use of recombinant DNA technology. The term “nucleic acid construct” therefore does not include naturally occurring nucleic acid molecules although a nucleic acid construct may comprise (parts of) naturally occurring nucleic acid molecules. The terms “expression vector” or “expression construct” refer to nucleotide sequences that are capable of effecting expression of a gene in host cells or host organisms compatible with such sequences. These expression vectors typically include at least suitable transcription regulatory sequences and optionally, 3′ transcription termination signals. Additional factors necessary or helpful in effecting expression may also be present, such as expression enhancer elements. The expression vector will be introduced into a suitable host cell and be able to effect expression of the coding sequence in an in vitro cell culture of the host cell. The expression vector will be suitable for replication in the host cell or organism of the invention.

As used herein, the term “promoter” or “transcription regulatory sequence” refers to a nucleic acid fragment that functions to control the transcription of one or more coding sequences, and is located upstream with respect to the direction of transcription of the transcription initiation site of the coding sequence, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and 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. 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 or developmentally regulated, e.g. by the application of a chemical inducer.

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.

As used herein, 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 transcription regulatory sequence is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein encoding regions, contiguous and in reading frame.

The term “peptide” as used herein is defined as a chain of amino acid residues, usually having a defined sequence. As used herein the term peptide is interchangeable with the terms “polypeptide” and “protein”. In the context of the present invention, the term “peptide” is defined as being any peptide or protein comprising at least two amino acids linked by a modified or unmodified peptide bond. The term “peptide” refers to short-chain molecules such as oligopeptides or oligomers or to long-chain molecules such as proteins. A protein/peptide can be linear, branched or cyclic. The peptide can include D amino acids, L amino acids, or a combination thereof. A peptide according to the present invention can comprise modified amino acids. Thus, the peptide of the present invention can also be modified by natural processes such as post-transcriptional modifications or by a chemical process. Some examples of these modifications are: acetylation, acylation, ADP-ribosylation, amidation, covalent bonding with flavine, covalent bonding with a heme, covalent bonding with a nucleotide or a nucleotide derivative, covalent bonding to a modified or unmodified carbohydrate moiety, bonding with a lipid or a lipid derivative, covalent bonding with a phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, cysteine molecule formation, pyroglutamate formation, formylation, gamma-carboxylation, hydroxylation, iodination, methylation, oxidation, phosphorylation, racemization, hydroxylation, etc. Thus, any modification of the peptide which does not have the effect of eliminating the immunogenicity of the peptide, is covered within the scope of the present invention.

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′-nontranslated sequence (3′-end) comprising a polyadenylation site. “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, i.e. which is capable of being translated into a biologically active protein or peptide. The term “homologous” when used to indicate the relation between a given (recombinant) nucleic acid or polypeptide molecule and a given host organism or host cell, is understood to mean that in nature the nucleic acid or polypeptide molecule is produced by a host cell or organisms of the same species, preferably of the same variety or strain. If homologous to a host cell, a nucleic acid sequence encoding a polypeptide will typically (but not necessarily) be operably linked to another (heterologous) promoter sequence and, if applicable, another (heterologous) secretory signal sequence and/or terminator sequence than in its natural environment. It is understood that the regulatory sequences, signal sequences, terminator sequences, etc. may also be homologous to the host cell.

The terms “heterologous” and “exogenous” when used with respect to a nucleic acid (DNA or RNA) or protein refers to a nucleic acid or protein that does not occur naturally as part of the organism, cell, genome or DNA or RNA sequence in which it is present, or that is found in a cell or location or locations in the genome or DNA or RNA sequence that differ from that in which it is found in nature. Heterologous and exogenous nucleic acids or proteins are not endogenous to the cell into which it is introduced, but have been obtained from another cell or synthetically or recombinantly produced. Generally, though not necessarily, such nucleic acids encode proteins, i.e. exogenous proteins, that are not normally produced by the cell in which the DNA is transcribed or expressed. Similarly exogenous RNA encodes for proteins not normally expressed in the cell in which the exogenous RNA is present. Heterologous/exogenous nucleic acids and proteins may also be referred to as foreign nucleic acids or proteins. Any nucleic acid or protein that one of skill in the art would recognize as foreign to the cell in which it is expressed is herein encompassed by the term heterologous or exogenous nucleic acid or protein. The terms heterologous and exogenous also apply to non-natural combinations of nucleic acid or amino acid sequences, i.e. combinations where at least two of the combined sequences are foreign with respect to each other.

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, a pathogen-infected cell or a cancer cell so as to protect against infection by the pathogen or against cancer in a vaccinated subject. For purposes of the present invention, protection against infection by a pathogen or protection against cancer includes not only the absolute prevention of infection or cancer, but also any detectable reduction in the degree or rate of infection by a pathogen or of the cancer, or any detectable reduction in the severity of the disease or any symptom or condition resulting from infection by the pathogen or cancer in the vaccinated subject, for example as compared to an unvaccinated infected subject. An effective immunoprotective response in the case of cancer also includes clearing up the cancer cells, thereby reducing the size of cancer or even abolishing the cancer. Vaccination in order to achieve this is also called therapeutic vaccination. Alternatively, 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 or do not yet suffer from cancer at the time of vaccination, such vaccination can be referred to as prophylactic vaccination.

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. Antigens can be e.g. proteinaceous molecules, i.e. polyaminoacid sequences, optionally comprising non-protein groups such as carbohydrate moieties and/or lipid moieties or antigens can be e.g. molecules that are not proteinaceous such as carbohydrates. An antigen can be e.g. any 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 a carbohydrate or other molecule, 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.

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 or other hapten that is usually part of a larger structure, such as e.g. a cell or a virion. The antigen may originate from within the body (“self”) or from the external environment (“non-self”). The immune system is usually non-reactive against “self” antigens under normal conditions due to negative selection of T cells in the thymus and is supposed to identify and attack only “non-self” invaders from the outside world or modified/harmful substances present in the body under e.g. disease conditions. Antigens structures that are the target of a cellular immune response are presented by antigen presenting cells (APC) in the form of processed antigenic peptides to the T cells of the adaptive immune system via a histocompatibility molecule. Depending on the antigen presented and the type of the histocompatibility molecule, several types of T cells can become activated. For T-Cell Receptor (TCR) recognition, the antigen is processed into small peptide fragments inside the cell and presented to a T-cell receptor by major histocompatibility complex (MHC).

The term “immunogen” is used herein to describe an entity that comprises or encodes at least one epitope of an antigen such that when administered to a subject, preferably together with an appropriate adjuvant, elicits a specific humoral and/or cellular immune response in the subject against the epitope and antigen comprising the epitope. An immunogen can be identical to the antigen or at least comprises a part of the antigen, e.g. a part comprising an epitope of the antigen. Therefore, 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 an immunogen comprising at least one epitope of the antigen. Vaccination preferably results in a protective or therapeutic effect, wherein subsequent exposure to the antigen (or a source of the antigen) elicits an immune response against the antigen (or source) that reduces or prevents a disease or condition in the subject. 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.

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) and as large as: a full length protein, including multimeric proteins, protein complexes, virions, particles, whole cells, whole microorganisms, or portions thereof (e.g., lysates of whole cells or extracts of microorganisms).

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. The adjuvant preferably is capable of enhancing the immune response against at least two different antigens.

OMV (also referred to as “blebs”) are bi-layered membrane structures, usually spherical, with a diameter in the range of 20-250 nm (sometimes 10-500 nm), 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, OMV lack the ability to self-replicate. In the context of the present invention three type of OMV can be distinguished depending on the method of their production. sOMV are spontaneous or natural OMV that are purified and concentrated from culture supernatant, by separating intact cells from the already formed OMVs. Detergent OMV, dOMV, are extracted from cells with detergent, such as deoxycholate, which also reduces the content of reactogenic LPS. After detergent extraction dOMV are separated from cells and cellular debris and further purified and concentrated. Finally, the term native nOMV is used herein for OMV that are generated from concentrated dead cells with non-detergent cell disruption techniques, or that are extracted from cells with other (non-disruptive) detergent-free methods, to be able to clearly distinguish them from the wild-type spontaneous OMVs and from the detergent-extracted dOMV.

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.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a novel neisserial LPS having a hexa-acylated lipid A moiety and to mixtures thereof with the corresponding neisserial LPS having a penta-acylated lipid A moiety, which are useful in generating and/or stimulating immune responses. Penta-acylated LPS molecules obtained from lpxL1−mutant neisserial strains have reduced TLR4 activity as compared to the corresponding hexa-acylated LPS from the parent strain (13, 15). Interestingly, the present inventors have found that introduction of heterologous expression of an LpxP lipid A biosynthesis palmitoleoylacyltransferase into an lpxL1−mutant neisserial host stain conferred temperature-sensitive addition of a palmitoleoyl chain as sixth acyl chain to the lipid A. The addition this sixth acyl chain to the lipid A again increases the TLR4 activity of the LPS in a temperature sensitivity manner. The temperature sensitivity of the LpxP enzyme thus enables to prepare mixtures of penta- and hexa-acylated LPS of which the ratio between penta- and hexa-acylated molecules can be controlled. By selecting the time and/or temperature that the mutant strain is grown, it is feasible to increase or decrease the amount of hexa-acylated lipid A structure relative to the corresponding penta-acylated structure and thereby the TLR4 activity and cytokine profile. This provides a new approach of fine-tuning the immunological properties of neisserial LPS preparation, such as e.g. meningococcal OMV vaccines.

In a first aspect therefore, the invention pertains to a neisserial LPS having a hexa-acylated lipid A moiety, or to the hexa-acylated lipid A moiety itself. Preferably the hexa-acylated lipid A moiety is modified as compared to the lipid A moiety of a wild-type neisserial LPS in that it has a palmitoleoyl as secondary acyl chain bound to the primary acyl chain on the glucosamine at the non-reducing end of the lipid A moiety. Thus, compared to the hexa-acylated lipid A moiety of a wild-type neisserial LPS, the hexa-acylated lipid A moiety of the invention has palmitoleoyl instead of a lauroyl as secondary acyl chain bound to the primary acyl chain on the glucosamine at the non-reducing end of the lipid A moiety. Preferably, the neisserial LPS or the hexa-acylated lipid A moiety are isolated.

In a preferred neisserial LPS of the invention, the hexa-acylated lipid A moiety has the structure of formula (I):



wherein R1and R2, independently, are either —P(O)(OH)2, —[P(O)(OH)—O]2—H, —[P(O)(OH)—O]2—CH2CH2NH2, —[P(O)(OH)—O]2—CH2CH2NH2, —[P(O)(OH)—O]3—H or —P(O)(OH)—O-CH2CH2NH2, and wherein, preferably R1 and R2, independently, are either —P(O)(OH)2, —[P(O)(OH)—O]2—H, —[P(O)(OH)—O]2—CH2CH2NH2or —[P(O)(OH)—O]3—CH2CH2NH2.

In a second aspect, the invention relates to a composition comprising i) a neisserial LPS with a hexa-acylated lipid A moiety as defined herein above; and, ii) a neisserial LPS with a penta-acylated lipid A moiety wherein the penta-acylated lipid A moiety is modified as compared to the lipid A moiety of a wild-type neisserial LPS in that it lacks the secondary acyl chain bound to the primary acyl chain on the glucosamine at the non-reducing end of the lipid A moiety. Preferably, in the composition, the neisserial LPS with a penta-acylated lipid A moiety has a penta-acylated lipid A moiety with the structure of formula (II):



wherein R1and R2, independently, are either —P(O)(OH)2, —[P(O)(OH)—O]2—H, —[P(O)(OH)—O]2—CH2CH2NH2, —[P(O)(OH)—O]3—CH2CH2NH2, —[P(O)(OH)—O]3—H or —P(O)(OH)—O—CH2CH2NH2, and wherein, preferably R1 and R2, independently, are either —P(O)(OH)2, —[P(O)(OH)—O]2—H, —[P(O)(OH)—O]2—CH2CH2NH2or —[P(O)(OH)—O]3—CH2CH2NH2.

In a composition of the invention, the hexa-acylated neisserial LPS and the penta-acylated neisserial LPS are present in a molar ratio in the range of 1:400 to 1:1.25. The range of molar ratios of the hexa-acylated LPS to the penta-acylated LPS can have end points selected from 1:400, 1:200, 1:100, 1:50, 1:25, 1:10, 1:5, 1:2, 1:1.5 and 1:1.25. A molar ratio of hexa-acylated LPS to the penta-acylated LPS can suitably be determined by mass spectrometry. The composition can be any composition herein further described below and it can be produced in the desired ratio in methods further described herein below. It is further understood that when reference is made herein to a neisserial LPS of the invention, this neisserial LPS also includes any (composition comprising) mixture of the hexa-acylated neisserial LPS and the penta-acylated neisserial LPS described herein above. The higher the amount of the hexa-acylated LPS relative to the penta-acylated LPS, the higher the specific TLR4 agonist activity or the specific reactogenicity will be.

Except for their hexa-acylated or penta-acylated lipid A moieties described above, the neisserial LPS of the invention otherwise have the structure of a lipopolysaccharide that is obtained or obtainable from a bacterium of the genusNeisseria. Neisserial LPS are sometimes also referred to as lipooligosaccharides (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. The bacterium of the genusNeisseriafrom which the LPS of the invention are obtained or obtainable can be a wild typeNeisseria, or aNeisseriahaving one or more of the genetic modifications described herein below. The bacterium of the genusNeisseriapreferably is of a species selected fromNeisseria meningitidis, Neisseria gonorrhoeaeandNeisseria lactamica, whereby more preferably theNeisseria meningitidisis at least one of serogroup B and immunotype L3.

Preferably therefore, except for hexa-acylated or penta-acylated lipid A moieties, the remainder of the neisserial LPS of the invention have the structure of LPS ofNeisseria meningitidis, Neisseria gonorrhoeaeorNeisseria lactamicaor a strain of these species having a genetic modification as described herein below. More preferably, except for hexa-acylated or penta-acylated lipid A moieties, the remainder of the neisserial LPS of the invention have the structure of LPS ofNeisseria meningitidisthat is at least one of serogroup B and immunotype L3 or a strain of this serogroup and/or immunotype having a genetic modification as described herein below.

In a preferred embodiment the neisserial LPS of the invention have a modified oligosaccharide structure so as to remove possible epitopes that are suspected to provoke autoimmune responses, and/or to increase binding to dendritic cells and adjuvant activity. Preferably therefore, the neisserial LPS of the invention are obtained or obtainable from a bacterium of the genusNeisseriathat has a genetic modification that reduces or eliminates the expression of at least one of an endogenous lgtB gene and an endogenous galE gene. Neisserial lgtB genes encode enzymes having lacto-N-neotetraose biosynthesis glycosyl transferase activity and are described e.g. by Jennings et al. (Mol Microbiol, 1995, 18:729-740) and by Arkin et al. (J Bacteriol. 2001, 183: 934-941). LPS from neisserial strains with lgtB disruptions has been shown to target the DC-SIGN lectin receptor on dendritic cells (DC), thereby skewing T-cell responses driven by DC towards T helper type 1 activity (Steeghs et al. Cell Microbiol. 2006, 8:316-25). Neisserial galE genes encode enzymes having UDP-glucose 4-epimerase activity and are described by Jennings et al. (Mol Microbiol, 1993, 10:361-369) and by Lee et al. (Infect Immun. 1995, 63: 2508-2515).

In a third aspect, the invention relates to a genetically modified bacterium of the genusNeisseria. The bacterium preferably is a bacterium comprising a neisserial LPS having a hexa-acylated lipid A moiety according to the first aspect of the invention. The bacterium is preferably genetically modified in that it comprises: a) a genetic modification that reduces or eliminates the activity of a lipid A biosynthesis lauroyl acyltransferase encoded by an endogenous lpxL1 gene; and, b) a genetic modification that confers to the bacterium a lipid A biosynthesis palmitoleoyl acyltransferase. A genetic modification that reduces or eliminates the activity of the lauroyl acyltransferase can e.g. be a missense mutation such as described in Fransen et al. (2009, PLoS Pathogens 5(4): e1000396). Preferably, the genetic modification that reduces or eliminates the activity of the lipid A biosynthesis lauroyl acyltransferase is a modification that reduces or eliminates the expression of an endogenous lpxL1 gene. The genetically modified bacterium preferably is bacterium of a species selected fromNeisseria meningitidis, Neisseria gonorrhoeaeandNeisseria lactamica. More preferably, the genetically modifiedNeisseria meningitidisis at least one of serogroup B and immunotype L3, most preferably, the genetically modifiedNeisseria meningitidisis theNeisseria meningitidisH44/76 strain or a derivative thereof.

The endogenous lpxL1 gene to be modified preferably is a gene that encodes a lipid A biosynthesis lauroyl acyltransferase. lpxL1 genes have also been referred to as htrBI or msbB genes. The lpxL1 gene of which the expression is to be reduced or eliminated in the bacterium of the invention, preferably is a gene that encodes a LpxL1 (lipid A biosynthesis lauroyl acyltransferase) comprising an amino acid sequence with at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity with at least one of SEQ ID NO: 1 (LpxL1 ofNeisseria meningitidisH44/76), SEQ ID NO: 2 (LpxL1 ofNeisseria gonorrhoeae, Genbank WP_050158792) and SEQ ID NO: 3 (LpxL1 ofNeisseria lactamica, Genbank CBN86767). In a preferred embodiment, the expression of the lpxL1 gene is eliminated by inactivation of the gene, e.g. by disruption or deletion of the gene by methods known in the art per se.

The genetic modification that confers to the bacterium lipid A biosynthesis palmitoleoyl acyltransferase activity preferably is a genetic modification that introduces the expression of a heterologous 1pxP gene having a nucleotide sequence that encodes a lipid A biosynthesis palmitoleoyl acyltransferase. The LpxP lipid A biosynthesis palmitoleoyl acyltransferase is an enzyme that catalyses the transfer of a palmitoleoyl chain from palmitoleoyl-acyl carrier protein to the lipid A precursor Kdo2-lipid IVA(27).

Specifically, LpxP acylates the 2′ (R)-3-hydroxymyristoyl chain. InE.coli, LpxP activity is cold shock induced within 15 min following a shift of exponentially growing cells from 37 to 12° C. and peaks after 2 h and appearance of palmitoleoyltransferase activity is correlated with massive accumulation of lpxP mRNA at 12° C. (27). A preferred heterologous lpxP gene to be expressed in the bacterium of the invention is a gene that expresses a thermosensitive LpxP. A thermosensitive LpxP is herein understood as an LpxP of which the activity at 37° C. is less than 10, 5, 2, 1, 0.5, 0.2 or 0.1% of the activity at 12° C., as may e.g. be tested upon expression inE.coli.

The overall sequence conservation between the various LpxP homologs is rather high, with e.g. 90% sequence identity between the LpxP amino acid sequences ofE.coliandCitrobacter. A large number of LpxP amino acid sequences is available in public databases such as Genbank, including e.g. LpxP amino acid sequences from genera such asEscherichia, Citrobacter, Haemophilus, Klebsiella, Salmonella, SerratiaandShigella. A preferred nucleotide sequence for expression of LpxP lipid A biosynthesis palmitoleoyl acyltransferase activity in a genetically modified bacterium of the invention is a nucleotide sequence that encodes a LpxP palmitoleoyl acyltransferase comprising an amino acid sequence with at least 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity with at least one of SEQ ID NO: 4 (Escherichia coli, Genbank AAB66658), SEQ ID NO: 5 (Citrobacter youngae, Genbank EFE09970) SEQ ID NO: 6 (Haemophilus aegyptius, Genbank EGF15820), SEQ ID NO: 7 (Klebsiellasp., Genbank EFD82732), SEQ ID NO: 8 (Salmonella entericasubsp.entericaserovar Typhimurium, Genbank CBW18471), SEQ ID NO: 9 (Serratia plymuthica, Genbank EKF62147) and SEQ ID NO: 10 (Shigella flexneri, Genbank ADA74790). Preferably, the nucleotide sequence encodes a known thermosensitive or cold shock-induced LpxP palmitoleoyl acyltransferase e.g. having an amino acid sequence as set forth in any one of SEQ ID NO: 4-10.

In one embodiment the nucleotide sequence encodes a polypeptide with LpxP palmitoleoyl acyltransferase activity as it occurs in nature, e.g. as it can be isolated from a wild type source bacterium. Alternatively, the nucleotide sequence can encode engineered forms of any of the LpxP palmitoleoyl acyltransferases defined above and that comprise one or more amino acid substitutions, insertions and/or deletions as compared to the corresponding naturally occurring LpxP palmitoleoyl acyltransferase but that are within the ranges of identity or similarity as defined herein.

Preferably, a nucleotide sequence for expression of LpxP palmitoleoyl acyltransferase activity in a genetically modified bacterium of the invention is a nucleotide sequence that encodes a LpxP palmitoleoyl acyltransferase comprising in each of the invariable positions (that are indicated with a “*”, i.e. an asteriks in the sequence alignment of Table 4), the amino acid present in that invariable position. More preferably, the LpxP palmitoleoyl acyltransferase amino acid sequence also comprises in the strongly conserved positions (that are indicated with a “:”, i.e. a colon in the sequence alignment of Table 4) one of the amino acids that is present in the respective strongly conserved positions. Most preferably, the amino acid sequence further also comprises in the weakly conserved positions (that are indicated in the sequence alignment of Table 4 with a “.”, i.e. a dot) one of the amino acids that is present in the respective weakly conserved positions. Amino acid substitutions outside of these invariable and conserved positions are less likely to have a negative effect on the LpxP palmitoleoyl acyltransferase enzymatic activity.

The genetically modified bacterium of the invention further preferably has one or more genetic modifications selected from the group consisting of: (i) a genetic modification that alters the LPS biosynthesis pathway, preferably in order to further modify the endotoxicity and/or reactogenicity of the LPS; (ii) a genetic modification that causes outer membrane retention of normally secreted antigens; (iii) a genetic modification that increases OMV production by removing outer membrane anchor proteins; (iv) a genetic modification that removes immune-modulating components which may trigger an undesired type of immune response; and, (v) a genetic modification that introduces expression of heterologous antigens.

An LPS that is modified to have reduced endotoxicity is herein understood as an LPS that is modified to have less toxicity than the toxicity of a corresponding wild-type LPS. Preferably, the modified LPS has less than about 90, 80, 60, 40, 20, 10, 5, 2, 1, 0.5, or 0.2% of the toxicity of the corresponding wild-type LPS. The toxicities of wild-type and various modified LPS's with reduced toxicity may be determined in any suitable assay known in the art. A preferred assay for determining the toxicity, i.e. the biological activity of the LPS is IL-6 induction in the MM6 macrophage cell line (see par. 1.4 below).

A preferred genetic modification that alters the LPS biosynthesis pathway is a genetic modification that is selected from the group consisting of: a) a genetic modification that reduces or eliminates expression of at least one of an lptA gene, an lpxK gene and homologues of these genes; and b) a genetic modification that introduces or increases the expression of at least one of an lpxE gene, an lpxF gene and homologues of these genes.

A preferred genetic modification that increases OMV production is a genetic modification that reduces or eliminates expression of a gene encoding an anchor protein between outer membrane and peptidoglycan in order to increase vesicle formation and thereby increase OMV yield. A suitable genetic modification for this purpose e.g. reduces or eliminates expression of an OmpA homologue, which are commonly found in Gram-negative bacteria, e.g. the RmpM protein inNeisseria(Steeghs et al., 2002 Cell Microbiol, 4:599-611; van de Waterbeemd et al., 2010 Vaccine, 28:4810 - 4816). Thus, preferably, the genetically modified bacterium has a genetic modification reduces or eliminates expression of an rmpM gene or a homologue thereof.

Preferred genetic modifications that removes immune-modulating components which may trigger an undesired type of immune response are genetic modifications that reduces or eliminates the expression of at least one of an endogenous lgtB gene and an endogenous galE gene as described above.

Further preferred genetic modifications reduce or eliminate the expression of at least one gene selected from the group consisting of cps, ctrA, ctrB, ctrC, ctrD, exbB, exbD, frpB, lpbB, nmb0033, opA, opC, phoP, pilC, pmrE, pmrF, porA, porB, siaA, siaB, siaC, siaD, synA, synB, synC, tbpA, tbpB, and homologues of any of these genes. Many of these mutations are reviewed in WO02/09746.

In a further embodiment, the genetically modified bacterium of the invention, is further genetically modified to express a heterologous antigen. Preferably, the heterologous antigen is expressed on the extracellular outer membrane surface of the bacterium. The heterologous antigen can e.g. be an outer membrane protein from another bacterium, preferably from another Gram negative bacterium. Alternatively, the heterologous antigen can be fused to a protein that is expressed on the extracellular outer membrane surface of the bacterium, e.g. a neisserial outer membrane protein as are well known in the art per se.

Preferably, the heterologous antigen expressed by the genetically modified bacterium of the invention, comprises at least one epitope for inducing and/or enhancing an immune response against an antigen comprising the epitope. Preferably, a B-cell, humoral or antibody response is elicited by the epitope in the heterologous antigen. Preferably the epitope in the heterologous antigen elicits a protective and/or neutralizing antibody response. Alternatively and/or additionally, the heterologous antigen comprises epitopes that elicit a T cell response. A preferred T-cell response induced and/or enhanced by an immunogenic peptide comprises at least one of an HLA class I restricted CTL response and an HLA class II restricted Th response. More preferably the T-cell response consists of both an HLA class I restricted CTL response and simultaneously an HLA class II restricted Th response, and may be advantageously accompanied by a B-cell response.

The heterologous antigen can comprise one or more epitopes from a wide range of antigens of pathogens (infectious agents) and/or tumours. For example, the heterologous antigen may comprise one or more epitopes from antigens from pathogens and infectious agents such as viruses, bacteria, fungi and protozoa. Some examples of pathogenic viruses causing infections or tumours from which epitopes from antigens may be derived include: hepatitis (A, B, or C), herpes virus (e.g., VZV, HSV-I, HAV-6, HSV-II, and CMV, Epstein Barr virus), adenovirus, SV40 virus (causing mesothelioma), influenza virus, flaviviruses, ebola virus, echovirus, rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus (RSV), mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, molluscum virus, poliovirus, rabies virus, JC virus, arboviral encephalitis virus, and human immunodeficiency virus (HIV virus; e.g., type I and II), human papilloma virus (HPV). Some examples of pathogenic bacteria causing infections from which epitopes from antigens may be derived include:Borrelia, Listeria, Escherichia, Chlamydia, Coxiella, Rickettsial bacteria, Mycobacteria, Staphylococci, Streptocci, Pneumonococci, Meningococci, Gonococci, Klebsiella, Proteus, Serratia, Pseudomonas, Legionella, Diphtheria, Salmonella, Bacilli, Bordetella, bacteria causing Cholera, Tetanus, Botulism, Anthrax, Plague, Leptospirosis, Whooping cough and Lymes disease. Some examples of pathogenic fungi causing infections from which epitopes from antigens may be derived include:Candida(e.g.,albicans, krusei, glabrataandtropicalis),Cryptococcus neoformans, Aspergillus(e.g.,fumigatus, niger), fungi of the genusMucorales(Mucor, AbsidiaandRhizopus),Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitisandHistoplasma capsulatum. Some examples of pathogenic parasites causing infections from which epitopes from antigens may be derived include:Entamoeba histolytica, Balantidium coli, Naegleria, Fowleri, Acanthamoebasp.,Giardia Zambia, Cryptosporidiumsp.,Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondiiandPlasmodium falciparis.

In addition, the C-terminal fusion can comprise one or more epitopes from a wide range of tumour antigens, including e.g. MAGE, BAGE, RAGE, GAGE, SSX-2, NY-ESO-1, CT-antigen, CEA, PSA, p53, XAGE and PRAME but also virally induced malignancies, comprising Human papilloma virus (HPV), Kaposi sarcoma herpes virus (KSHV), Epstein Bar virus induced lymphoma's (EBV). Other examples of tumour antigens from which epitopes for use in the present invention may be derived are various ubiquitously expressed self-antigens that are known to be associated with cancer, which include e.g. p53, MDM-2, HDM2 and other proteins playing a role in p53 pathway, molecules such as surviving, telomerase, cytochrome P450 isoform 1B1, Her-2/neu, and CD19 and all so-called house hold proteins. Cancers that may be treated in accordance with the present invention are selected among the following list: lung, colon, esophagus, ovary, pancreas, skin, gastric, head and neck, bladder, sarcoma, prostate, hepatocellular, brain, adrenal, breast, endometrial, mesothelioma, renal, thyroid, hematological, carcinoid, melanoma, parathyroid, cervix, neuroblastoma, Wilms, testes, pituitary and pheochromocytoma cancers. In one embodiment, the heterologous antigen comprises or consists of one or more surface exposed epitopes from a proteinaceous antigen of an infectious agent or tumour. The heterologous antigen can e.g. comprises or consists of an extracellular and/or surface exposed domain of the proteinaceous antigen of an infectious agent or tumour.

In a fourth aspect, the invention relates to a hexa-acylated neisserial LPS as herein defined above or to (a composition comprising) a mixture of hexa- and penta-acylated neisserial LPS as herein defined above, wherein the LPS is obtained or obtainable from a genetically modified bacterium as herein defined above.

In a fifth aspect, the invention pertains to an OMV comprising the neisserial LPS as herein defined above. OMV (also known as “blebs”) 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 asN. meningitidisis highly toxic, yet residual amounts (approx. 1%) are needed in OMV to maintain vesicle structure and for adjuvant activity. However, the neisserial LPS of the invention combines reduced toxicity with useful adjuvant activity and therefore preferably remain present in the OMV to a much larger degree than the toxic wild type LPS. The detergent extraction process is therefore less suitable for producing OMV comprising the neisserial LPS of the present invention. An OMV comprising a neisserial LPS according to the invention 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 as long as most of the neisserial LPS according to the invention, i.e. at least 5, 10, 20, 50, 60, 70, 80, 90, 95 or 99% of the neisserial LPS, is maintained, e.g. as compared the amount of neisserial LPS present in spontaneous or supernatant OMV from an equal amount of the same culture.

A preferred OMV comprising the neisserial LPS of the invention is a supernatant or spontaneous OMV, i.e. sOMV as herein defined above, or a native OMV, i.e. nOMV as herein defined above. Methods for preparing nOMV are e.g. described in Saunders et al. (1999, Infect Immun, 67, 113-119), van de Waterbeemd et al. (2012, Vaccine, 30: 3683-3690) and in WO2013/006055 and 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. The OMV comprising a neisserial LPS of the invention are preferably obtained or obtainable from a genetically modified neisserial bacterium as herein defined above.

In a sixth aspect, the invention relates to a composition comprising at least one of the neisserial LPS, a genetically modified bacterium and an OMV as herein defined above. Preferably, the composition is a pharmaceutical composition further comprises a pharmaceutically acceptable excipient, carrier, medium or delivery vehicle as are conventionally known in the art (see e.g. “Handbook of Pharmaceutical Excipients”, Rowe et al eds. 7thedition, 2012, www pharmpress.com). Pharmaceutically acceptable stabilizing agents, osmotic agents, buffering agents, dispersing agents, and the like may also be incorporated into the pharmaceutical compositions. 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 to the patient. The “active ingredients of the invention” are herein understood to be one or more of a neisserial LPS, a genetically modified bacterium or an OMV as defined herein above.

Pharmaceutically acceptable carriers for parenteral delivery are exemplified by sterile buffered 0.9% NaCl or 5% glucose optionally supplemented with a 20% albumin. Alternatively, the active ingredients of the invention can be suspended in Phosphate buffer saline (PBS). Preparations for parental administration must be sterile. The parental route for administration of the active ingredients of the invention is in accord with known methods, e.g. injection or infusion by intravenous, intraperitoneal, intramuscular, and intraarterial or intralesional routes. 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 the active ingredients of the invention. 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).

In a preferred embodiment, the pharmaceutical composition of the invention is a vaccine. The vaccine can be an acellular vaccine preferably comprising at least one a neisserial LPS and an OMV as defined herein above. Alternatively, the vaccine can be a whole cell vaccine comprising at least a bacterium as herein defined above, wherein preferably the bacterium is inactivated or killed using means known in the art per se.

In a seventh aspect, the invention pertains to a process for producing a neisserial LPS of the invention. The process preferably comprises the steps of a) cultivating a genetically modified bacterium as herein defined above, preferably under condition conducive to the production of the LPS; and b) optionally, at least one of extraction and purification of the LPS. Methods for extraction of LPS are well known in the art (see e.g. 19). Methods for purification of LPS are described in the Examples herein and can e.g. include solid phase extraction (SPE) on reverse phase cartridges.

In the process, preferably, in step a), the temperature at which the genetically modified bacterium is cultured determines or is used to determine the molar ratio of the hexa-acylated neisserial LPS to the penta-acylated neisserial LPS of the invention. The optimal growth temperature forNeisseriais in the range of 35-37° C., at which temperature the temperature-sensitive LpxP enzyme has almost no activity. However, at temperatures below 25° C., when LpxP enzyme is most active,Neisseriawill stop growing. Depending on the desired TLR4 agonist activity, and the corresponding desired molar ratio of hexa- to penta-acylated neisserial LPS to be produced, the genetically modified bacterium is preferably cultured in step a) of the process at a temperature in a range limited at the lower end by 20, 21, 22, 23, 24 or 25° C. and limited at the higher end by 37, 38, 39, 40, 41 or 42° C. In step a) of the process, the genetically modified bacterium can thus be cultured at any temperature selected from 20, 21, 22, 23, 24, 25, 26, 27, 28, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 and 42° C. to produce a desired molar ratio of hexa- to penta-acylated neisserial LPS. For example, the inventors have found that when an lpxL1−mutantN. meningitidisexpressing theE. coliLpxP enzyme as used in the Examples, is grown at 25° C., a relative abundance of approximately 50% hexa-acyl lipid A is produced (i.e. a molar ratio of hexa- to penta-acylated neisserial LPS of about 1:2), whereas when grown at 37° C., extremely low amounts of hexa-acyl lipid

A were produced, estimated to be about 0.5% (i.e. a molar ratio of hexa- to penta-acylated neisserial LPS of about 1:200). Therefore, in one preferred embodiment of the process of the invention, in step a) the bacterium is cultured at a temperature lower than 37, 36.5, 36, 35.5, 35, 34.5, or 34° C. This ensures that at least some hexa-acylated neisserial LPS is produced, composition comprising only the penta-acylated LPS already being obtainable from single mutant lpxL1−Neisseria. It is further envisaged that other ratio's than those obtained in the Examples herein can be obtained at 25° C. and 37° C., when using other neisserial host strain and/or in combination with heterologous expression of other LpxP enzymes. In further embodiments of the process of the invention, step a) can comprises at least two phases wherein the bacterium is cultivated at different temperatures. Step a) in the process can e.g. comprise one or more phases at a temperature allowing more rapid accumulation of biomass (e.g. at least 32, 33, 34, 35, 36, or 37 ° C.), followed by one or more phases at a temperature allowing for palmitoylation of the penta-acylated neisserial LPS (e.g. not higher than 31, 30, 29, 28, 26, 25, 24, 23, 22, 21 or 20 ° C.). Alternatively, step a) in the process can start with a phase at a temperature allowing more rapid accumulation of biomass, followed by a phase during which the culture is cooled down to a temperature allowing for palmitoylation, whereby preferably the temperature is reduced along a predefined trajectory as a function of time or accumulated biomass.

In an eighth aspect, the invention relates to a process for producing an OMV of the invention. The process preferably comprises the steps of a) cultivating a genetically modified bacterium as herein defined above, preferably under condition conducive to the production of the OMV; b) optionally, extracting the OMV; and recovering the OMV, wherein the recovery at least comprises removal of the bacteria from the OMV. In the process, preferably, in step a), the temperature at which the genetically modified bacterium is cultured determines or is used to determine the molar ratio of the hexa-acylated neisserial LPS to the penta-acylated neisserial LPS of the invention as described above. Methods for preparing OMV, preferably detergent-free methods for preparing OMV, are described herein above. A preferred process for preparing OMV of the invention is thus a detergent-free process, e.g. a process for preparing nOMV or sOMV.

In a ninth aspect, the invention pertains to a process for producing an acellular vaccine as herein defined above. The process preferably comprises the steps of: a) producing at least one of: i) a neisserial LPS as herein defined above, preferably in a process for producing a neisserial LPS as herein defined above; and, ii) an OMV as herein defined above, preferably, in a process for producing an OMV as herein defined above; and, b) formulating at least one of the neisserial LPS and the OMV, optionally with further vaccine components, into a vaccine formulation.

In a tenth aspect, the invention relates to a process for producing a whole cell vaccine as herein defined above. The process preferably comprises the steps of: i) cultivating a genetically modified bacterium as herein defined; and, ii) optionally, at least one of inactivation of the bacterium and formulation into a vaccine.

In an eleventh aspect, the invention pertains to the use as medicament of at least one of a neisserial LPS of the invention, a genetically modified bacterium of the invention, an OMV of the invention, and a pharmaceutical composition of the invention.

In a twelfth aspect, the invention relates to a neisserial LPS of the invention for use in a treatment comprising inducing or stimulating an immune response in a subject.

Preferably, in the treatment, the neisserial LPS is used as adjuvant. A neisserial LPS of the invention can e.g. be included as adjuvant in a vaccine composition, preferably together with an antigen against which it is desirable to induce or stimulate an immune response. Preferably therefore, a neisserial LPS of the invention is used in a treatment comprising inducing or stimulating an immune response in a subject, wherein the treatment further comprises the administration of an antigen together with the neisserial LPS and wherein the treatment is for preventing or treating an infectious disease or tumour associated with the antigen, wherein the antigen preferably is an antigen as herein defined above.

In this aspect, the invention thus relates to a method for vaccination against, or for prophylaxis or therapy of an infectious disease or tumour, or for inducing or stimulating an immune response against an infectious disease or tumour. The method preferably at least comprises the step of administration of a therapeutically or prophylactically effective amount of an neisserial LPS of the invention or a pharmaceutical composition comprising said LPS, to a subject in need of said prophylaxis, therapy or immune response. The pharmaceutical composition comprising preferably also an antigen associated with the infectious disease or tumour, wherein the antigen preferably is an antigen as herein defined above.

In a thirteenth aspect, the invention relates to a neisserial LPS of the invention for use as a Toll-like receptor 4 (TLR4) agonist. Preferably, the neisserial LPS is used as TLR4 agonist in an immunotherapy. Preferably, the immunotherapy is an immunotherapy of a cancer (see e.g. 37) or of a neurodegenerative disease (see e.g. 35), including e.g. Alzheimer's disease or Parkinson's disease. Alternatively, the neisserial LPS is used as TLR4 agonist in a generalized immune stimulation for preventing and/or reducing the spread of (diverse) microbial infections and/or to suppress bacterial growth (see e.g. 36).

The advantage of the present invention is thus that the temperature sensitivity of the LpxP enzyme enables to prepare mixtures of penta- and hexa-acylated LPS of which the ratio between penta- and hexa-acylated molecules can be precisely controlled. By selecting the time and/or temperature that the mutant strain is grown, it is feasible to increase or decrease the amount of hexa-acylated lipid A structure relative to the corresponding penta-acylated structure and thereby modulate the TLR4 agonist activity of the neisserial LPS of the invention to the exact level of activity, and profile of subsequently released cytokines, required for a particular immunotherapeutic approach. Such controlled release of pro-inflammatory cytokines is important for using the neisserial LPS as an adjuvant, antigen and/or immunostimulatory molecule, since it is desirable to be able to induce or stimulate immunity without toxic effects. Often immune activation is correlated with more side effects due to unwanted inflammatory responses. In general, for vaccine application side effect levels are preferably kept as low as possible or more preferably be absent. However, for some specific immunotherapeutic applications such as those described above, the potential healing effects may outweigh considerable side effects. Therefore, even the high activity of ΔlpxL1-lpxP LPS grown at 25° C. could still be useful in such therapeutic approaches.

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. In addition, reference to an element by the indefinite article “a” or “an” does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article “a” or “an” thus usually means “at least one”.

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

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

