Click OMV

Abstract

The present invention relates to a complex of an OMV, a vertebrate antimicrobial peptide (AMP), and an antigen, wherein the AMP is non-covalently complexed to the OMV and the antigen is conjugated to the AMP. Preferably, the antigen is covalently bound to the AMP. The present invention further relates to the induction of an immune response using the complex of the invention, and to methods for producing the complex of the invention.

Description

Technical Field

[0001] The present invention is in the field of vaccinology. The present invention relates to a platform technology using modified outer membrane vesicles (OMVs) for inducing or enhancing an immune response against an antigen, wherein the antigen is non-covalently attached to the OMV. The present invention further relates to a method for producing the OMVs of the present invention.

Background Art

[0002] Outer membrane vesicles (OMVs) are non-replicative structures released by Gram-negative bacteria that contain many important bacterial surface components in combination with pathogen-associated molecular patterns (PAMPs) that function as internal adjuvants by triggering an innate immune response. Such PAMPs preferably activate at least one of TLR2 and TLR4. OMVs are spherical nanostructures (50 - 250 nm) mainly composed of lipids, LPS, and outer membrane proteins, which efficiently present antigens to the immune system and generate a strong Ig and CD4+ T cell response. Outer membrane vesicles of Neisseria meningitidis have a long history of use as experimental vaccines against meningococcal disease. The use of OMVs has been proven to be safe. For example, the MenB vaccine containing OMVs is currently on the market.

[0003] A concept for next-generation OMVs based on engineered hypervesiculating strains with genetically detoxified LPS has been developed, eliminating the need for surfactant extraction to remove LPS. These native (n)OMVs readily produce and retain many loosely attached surface antigens, making these antigens further immunogenic. Heterogeneous non-meningococcal antigens can be further immunogenic by utilizing the presentation form of nOMVs. For this purpose, methods for heterogeneous surface presentation based on endogenous expression of antigens subsequently transported to the outer membrane of OMVs have been developed in the art. This method was used to express heterogeneous OspA on meningococcal OMVs. When mice were immunized with this OMV set, OMVs expressing OspA on their surface induced an OspA-specific antibody response (Salverda, Vaccine 34:1025-1033, 2016).

[0004] OMV carriers are proven and safe vaccine components. The OMV production process is scalable, yields high returns, uses chemically defined media, and is GMP compliant. A surfactant extraction step can be incorporated into the production process to remove LPS and increase vesicle release. Disadvantages of surfactant extraction include the removal of potential protective antigens from the outer membrane, such as loosely attached, surface-exposed lipoproteins, and reduced long-term stability of the OMV vaccine. Other techniques for obtaining OMV are known in the art, such as the use of chelating agents like EDTA, or genetically modified substances that loosen the outer membrane and increase OMV release, instead of surfactant extraction.

[0005] In addition, to manipulate the immune response and optimize safety, the endogenous adjuvant lipopolysaccharide (LPS) can be genetically detoxified. Such modifications may, for example, produce pentaacylated lipid A species exhibiting potent adjuvant activity and reduced endotoxin activity (Zariri, Sci Rep 6:36575, 2016).

[0006] OMVs possess excellent intrinsic immunostimulatory properties and can act as pathogen-mimicking adjuvants. Heterogeneous antigens can become even more immunogenic by taking the form of OMV presentation. In particular, OMVs are highly efficient in stimulating their uptake and processing by antigen-presenting cells due to their size and their diverse PAMP content. However, it is crucial that co-delivered antigens are taken up simultaneously; otherwise, antigen-presenting cells become activated and migratory before efficient antigen uptake occurs. For this reason, coupling of antigens to OMVs is desirable to produce an optimal immune response. This requires a method for heterologous surface presentation. However, the use of endogenous expression is limited by the need to obtain efficient expression and uptake of selected antigens into OMVs, and such expression and uptake can often be difficult to obtain. The need for compatibility with the biosynthetic mechanism of the bacterial outer membrane (OM) limits the efficient expression of many heterogeneous antigens. [Overview of the project]

[0007] Therefore, there remains a strong need for a versatile OMV platform for antigen presentation in this field. In particular, there is a need for an OMV platform that can be used to present a wide range of known and / or novel antigens without requiring antigen expression within OMV-producing cells.

[0008] overview In one embodiment, the present invention relates to a complex of an outer membrane vesicle (OMV), a vertebrate antimicrobial peptide (AMP), and an antigen, wherein the AMP is complexed with the OMV by non-covalent bonding, and the antigen is conjugated to the AMP.

[0009] Preferably, the antigen is covalently bound to AMP in a fusion protein containing the antigen and AMP in a single polypeptide chain.

[0010] Preferably, AMP is a cathelicidine, preferably a non-human cathelicidine, and more preferably mCRAMP.

[0011] Preferably, the antigen is an antigen associated with an infectious disease and / or tumor.

[0012] Preferably, the OMV is not OMV extracted with a surfactant, but rather naturally occurring OMV or native OMV, preferably native OMV.

[0013] Preferably, the OMV contains LPS that has been at least partially detoxified.

[0014] Preferably, OMV can be obtained from Gram-negative bacteria, and Gram-negative bacteria are a) Genetic modifications that cause bacteria to produce LPS with reduced toxicity, preferably reducing or eliminating the expression of at least one of the lpxL1, lpxL2, lpxA, lpxD, and lpxK genes or their homologs, and / or increasing the expression of at least one of the lpxP, lpxE, lpxF, and pagL genes, and b) Genetic modifications that increase vesicle formation, preferably reducing or eliminating the expression of the ompA gene or its homolog, more preferably the rmpM gene or its homolog. Preferably includes at least one of the following.

[0015] Preferably, OMV can be obtained from Gram-negative bacteria belonging to a genera selected from the group consisting of Neisseria, Bordetella, Escherichia, and Salmonella, and preferably the bacteria belong to a species selected from the group consisting of Neisseria meningitidis, Bordetella pertussis, Escherichia coli, and Salmonella enterica.

[0016] The present invention further relates to a pharmaceutical composition comprising a complex and pharmaceutically acceptable excipients as defined herein.

[0017] The present invention also relates to a complex or pharmaceutical composition as defined herein, for use as a pharmaceutical.

[0018] The present invention further relates to a complex or pharmaceutical composition as defined herein for use in a treatment comprising inducing or stimulating an immune response in a target to an antigen.

[0019] The present invention relates to an antigen conjugated to AMP as defined herein, preferably an antigen conjugated to AMP that is a fusion protein as defined herein.

[0020] The present invention relates to nucleic acids encoding fusion proteins as defined herein.

[0021] The present invention also relates to a host cell expressing a fusion protein as defined herein, preferably the host cell comprising a nucleic acid as defined herein.

[0022] The present invention relates to a method for producing a complex as defined herein, i) A step of culturing a population of Gram-negative bacteria as defined herein under conditions favorable for OMV production, ii) A step to recover the OMV produced in i), iii) The step of contacting the OMV recovered in ii) with AMP conjugated to an antigen as defined herein, under conditions favorable for the formation of a non-covalent complex between AMP and OMV, and vi) If necessary, take the step of recovering the complex. Regarding methods including [Modes for carrying out the invention]

[0023] definition Various terms relating to methods, compositions, uses, and other aspects of the present invention are used throughout the specification and claims. Such terms have their ordinary meanings in the art to which the present invention relates, unless otherwise indicated. Other specifically defined terms shall be construed in a manner consistent with the definitions provided herein. Any methods and materials similar to or equivalent to those described herein may be used in practice to test the present invention, but preferred materials and methods are described herein.

[0024] The methods of performing the prior art used in the method of the present invention will be apparent to those skilled in the art. Conventional techniques and conventions in molecular biology, biochemistry, computational chemistry, cell culture, recombinant DNA, bioinformatics, genomics, sequencing, and related fields are well known to those skilled in the art and are discussed, for example, in the following references: Sambrook et al. Molecular Cloning. A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, 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..

[0025] "A," "an," and "the": These singular terms include plural references unless the context clearly indicates otherwise. Thus, for example, reference to "a cell" includes combinations of two or more cells and the like.

[0026] As used herein, the term "about" is used to describe and account for small variations. For example, the term can refer to within ±10% or less, such as within ±5% or less, ±4% or less, ±3% or less, ±2% or less, ±1% or less, ±0.5% or less, ±0.1% or less, or ±0.05% or less. Further, amounts, ratios, and other numerical values are sometimes presented herein in a range format. Such range formats are used for convenience and brevity and are to be understood flexibly as including not only the numerical values explicitly specified as the limits of the range but also all individual numerical values or sub-ranges subsumed within that range as if each numerical value and sub-range were explicitly specified. For example, a ratio in the range of about 1 to about 200 is to be understood as including not only the explicitly mentioned limits of about 1 and about 200 but also individual ratios such as about 2, about 3, and about 4, as well as sub-ranges such as about 10 to about 50, about 20 to about 100, and others.

[0027] "And / or": The term "and / or" refers to situations where one or more of the cases mentioned may occur alone or in combination with at least one, and up to all, of the cases mentioned.

[0028] "Comprising": This term is to be interpreted as inclusive and open-ended and not exclusive. Specifically, this term and its variations mean that a specific feature, step, or component is included. These terms are not to be interpreted as excluding the presence of other features, steps, or components.

[0029] The terms “homology,” “sequence identity,” and similar terms are used herein without distinction. Sequence identity is defined herein as the relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by sequence comparison. In the art, “identity” also, as may be, the degree of sequence relevance between amino acid sequences or nucleic acid sequences, as determined by matching between a sequence of such sequences. The “similarity” between two amino acid sequences is determined by comparing the amino acid sequence of one polypeptide and its conserved substitution amino acids with the sequence of the second polypeptide. “Identity” and “similarity” can be readily calculated by known methods.

[0030] Sequence identity and sequence similarity can be determined by the alignment of two peptide sequences or two nucleotide sequences using a global or local alignment algorithm, depending on the lengths of the two sequences. Sequences of similar lengths are preferably aligned using a global alignment algorithm (e.g., Needleman-Bunsch) that optimally aligns the sequences over their entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g., Smith-Waterman). Sequences can thus be said to be "substantially identical" or "essentially similar" if they have at least a certain minimum percentage of sequence identity (as defined below) (when they are optimally aligned using default parameters, for example, by the GAP or BESTFIT program). GAP uses the Needleman-Bunsch global alignment algorithm to align two sequences over their full length, maximizing the number of matches and minimizing the number of gaps. Global alignment is appropriately used to determine sequence identity when two sequences have similar lengths. Generally, the default parameters for GAP are used, with a gap generation penalty of 50 (nucleotides) / 8 (proteins) and a gap elongation penalty of 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 alignment and sequence identity percentage scores can 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 the same parameters as above for GAP, or using default settings (for both "Needle" and "Water," and for both protein and DNA alignment, the default gap start penalty is 10.0 and the default gap extension penalty is 0.5; the default scoring matrix is ​​Blosum62 for protein and DNAFull for DNA), using open-source software such as EmbossWIN, version 2.10.0, either "Needle" (using the global Needleman-Bunsch algorithm) or "Water" (using the local Smith-Waterman algorithm). If sequences have substantially different total lengths, local alignment, such as that using the Smith-Waterman algorithm, is preferred.

[0031] Alternatively, the percentage of similarity or identity can be determined by searching public databases using algorithms such as FASTA or BLAST. Therefore, the nucleic acid and protein sequences of the present invention can be further used as "query sequences" to identify other family members or related sequences by searching public databases. Such searches can be performed using the BLASTn and BLASTx programs (version 2.0) from Altschul, et al. (1990) J. Mol. Biol. 215:403-10. A BLAST nucleotide search can be performed in the NBLAST program with a score of 100 and a word length of 12 to obtain nucleotide sequences homologous to the nucleic acid molecule of the present invention. A BLAST protein search can be performed in the BLASTx program with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the protein molecule of the present invention. For comparative purposes, Gapped BLAST can be used to obtain gap alignment, as described in Altschul et al., (1997) Nucleic Acids Res. 25(17): 3389-3402. When using the BLAST program and the Gapped BLAST program, the default parameters of each program (e.g., BLASTx and BLASTn) can be used. Please refer to the National Center for Biotechnology Information website at http: / / www.ncbi.nlm.nih.gov / .

[0032] Where necessary, in determining the degree of similarity of amino acids, a person skilled in the art may also take into account so-called "conservative" amino acid substitutions, as would be obvious to a person skilled in the art. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, the group of amino acids with aliphatic side chains are glycine, alanine, valine, leucine, and isoleucine; the group of amino acids with aliphatic hydroxyl side chains are serine and threonine; the group of amino acids with amide-containing side chains are asparagine and glutamine; the group of amino acids with aromatic side chains are phenylalanine, tyrosine, and tryptophan; the group of amino acids with basic side chains are lysine, arginine, and histidine; and the group of amino acids with sulfur-containing side chains are cysteine ​​and methionine. The preferred group of conservative amino acid substitutions are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, aspartic acid-glutamic acid, and asparagine-glutamine. The amino acid sequence substitution variants disclosed herein are those in which at least one residue in the disclosed sequence is removed and a different residue is inserted in its place. Preferably, the amino acid change is conservative. The preferred conservation 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.

[0033] As used herein, the terms “selectively hybridizing,” “selectively hybridizing,” and similar terms are intended to describe hybridization and washing conditions in which nucleotide sequences typically remain hybridized to one another, being at least 66%, at least 70%, at least 75%, at least 80%, more preferably at least 85%, and also more preferably at least 90%, preferably at least 95%, more preferably at least 98%, or even more preferably at least 99% homologous to one another. In other words, such hybridized sequences may have 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%, and also more preferably at least 90%, more preferably at least 95%, more preferably at least 98%, or even more preferably at least 99% sequence identity.

[0034] A preferred non-limiting example of such hybridization conditions is hybridization with 6×sodium chloride / sodium citrate (SSC) at about 45°C, followed by one or more washes with 1×SSC and 0.1% SDS at about 50°C, preferably about 55°C, preferably about 60°C, and more preferably about 65°C.

[0035] Highly stringent conditions include, for example, hybridization at approximately 68°C with 5×SSC / 5×Denhardt solution / 1.0%SDS, and washing at room temperature with 0.2×SSC / 0.1%SDS. Alternatively, washing may be performed at 42°C.

[0036] Those skilled in the art will know which conditions should be applied to stringent and highly stringent hybridization conditions. Further guidance on such conditions is readily available in the art, for example, in Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, NY, and Ausubel et al. (eds.), Sambrook and Russell (2001) "Molecular Cloning: A Laboratory Manual (3rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York 1995, Current Protocols in Molecular Biology, (John Wiley & Sons, NY)."

[0037] Naturally, polynucleotides that hybridize only to poly(A) sequences (such as the 3'-terminal poly(A) tract of mRNA) or to complementary T (or U) residue strands are not included in the polynucleotides of the present invention used to specifically hybridize to a portion of the nucleic acid, as they hybridize to any nucleic acid molecule having a poly(A) strand or their complements (e.g., virtually any double-stranded cDNA clone).

[0038] In this specification, “nucleic acid construct” or “nucleic acid vector” is understood to mean an artificial nucleic acid molecule obtained as a result of 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 include (a portion of) naturally occurring nucleic acid molecules. The term “expression vector” or “expression construct” refers to a nucleotide sequence that can result in gene expression in a host cell or host organism compatible with such a sequence. These expression vectors typically include at least a suitable transcriptional regulatory sequence and, optionally, a 3' transcription termination signal. Further factors necessary or useful for resulting in expression, such as expression enhancer elements, may also be present. An expression vector can be introduced into a suitable host cell and result in the expression of a coding sequence in an in vitro cell culture of the host cell. Expression vectors are suitable for replication in the host cells or host organisms of the present invention.

[0039] As used herein, the terms “promoter” or “transcriptional regulatory sequence” refer to a nucleic acid fragment that functions to control the transcription of one or more coding sequences, is located upstream of the transcription start site of the coding sequence with respect to the direction of transcription, and is structurally identified by the presence of a DNA-dependent RNA polymerase binding site, a transcription start site, any other DNA sequence including, but not limited to, transcription factor binding sites, repressor and activator protein binding sites, and any other nucleotide sequences known to those skilled 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 cells, preferably bacterial cells, under most physiological and developmental conditions. An “inducible” promoter is a promoter that is physiologically or developmentally regulated, for example, by the application of a chemical inducer.

[0040] The term “selection marker” is a term well known to those skilled in the art and is used herein to describe any genetic entity that, when expressed, can be used to select one or more cells having a selection marker. The term “reporter” may be used interchangeably with “marker,” but is primarily used to refer to visible markers such as green fluorescent protein (GFP). Selection markers may be dominant, recessive, or bidirectional.

[0041] As used herein, the term “operatably linked” refers to the linking of polynucleotide elements in a functional relationship. A nucleic acid is “operatably linked” if it is in a functional relationship with another nucleic acid sequence. For example, a transcriptional regulatory sequence is operatably linked to a coding sequence if it affects the transcription of that coding sequence. Operatably linked means that the linked DNA sequences are typically contiguous and, if necessary, contiguous and within a reading frame when it is required to link two protein-coding regions.

[0042] As used herein, the term "peptide" is defined as a chain of amino acid residues, typically having a defined sequence, but without reference to a specific mode of action, size, three-dimensional structure, or origin. 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 any peptide or protein comprising at least two amino acids linked by modified or unmodified peptide bonds. The term "peptide" can refer to short-chain molecules such as oligopeptides or oligomers, or long-chain molecules such as proteins.

[0043] Proteins / peptides can be linear, branched, or cyclic. Peptides may contain D amino acids, L amino acids, or combinations thereof. Peptides according to the present invention may contain modified amino acids. Therefore, peptides of the present invention may be modified by natural processes such as post-transcriptional modification or by chemical processes. Some examples of these modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent bonding with flavin, covalent bonding with heme, covalent bonding with nucleotides or nucleotide derivatives, covalent bonding to modified or unmodified carbohydrate moieties, bonding with lipids or lipid derivatives, covalent bonding with phosphatidylinositol, crosslinking, cyclization, disulfide bond formation, demethylation, cysteine ​​molecule formation, pyroglutamic acid formation, formylation, gamma-carboxylation, hydroxylation, iodization, methylation, oxidation, phosphorylation, and racemization. Therefore, any modification of a peptide that does not have the effect of eliminating the immunogenicity of the peptide is included in the scope of the present invention.

[0044] The term “gene” refers to a DNA fragment that contains a region (transcription region) that is transcribed into an RNA molecule (e.g., mRNA) within a cell, operably ligated to an appropriate regulatory region (e.g., a promoter). A gene typically contains several operably ligated fragments, e.g., a promoter, a 5' leader sequence, a coding region, and a 3' untranslated sequence (3' end) containing a polyadenylation site. “Genetic expression” refers to the process by which a DNA region operably ligated to an appropriate regulatory region, particularly a promoter, is transcribed into biologically active RNA, i.e., RNA that can be translated into a biologically active protein or peptide. When the term “homologous” is used to refer to the relationship between a given (recombinant) nucleic acid or polypeptide molecule and a given host organism or host cell, it is originally understood to mean that the nucleic acid or polypeptide molecule is produced by the same species, preferably the same variant or strain of host cell or host organism. If homologous to the host cell, nucleic acid sequences encoding polypeptides typically (but not always) operably ligate to (heterogeneous) promoter sequences outside their natural environment, and, where applicable, to (heterogeneous) secretory signal sequences and / or terminator sequences outside their natural environment. Regulatory sequences, signal sequences, terminator sequences, etc., are also understood to be homologous to the host cell.

[0045] The terms “heterogeneous” and “exogenous,” when used in relation to nucleic acids (DNA or RNA) or proteins, refer to nucleic acids or proteins that do not occur naturally as part of an organism, cell, genome, DNA sequence, or RNA sequence, or that are found in a different cell or at one or more locations within a genome, DNA sequence, or RNA sequence than in which they are found naturally. Heterogeneous and exogenous nucleic acids or proteins are not endogenous to the cell into which they are introduced, but are obtained, for example, from another cell or produced synthetically or by recombination. Generally, but not always, such nucleic acids encode proteins that are not normally produced by the cell in which the DNA is transcribed or expressed, i.e., exogenous proteins. Similarly, exogenous RNA may encode proteins that are not normally expressed in the cell in which the exogenous RNA is present. Heterogeneous / exogenous nucleic acids and proteins may also be called foreign nucleic acids or proteins. Any nucleic acid or protein that a person skilled in the art would recognize as foreign to the cell in which it is expressed is encompassed herein by the term heterogeneous or exogenous nucleic acid or protein. The terms heterogeneous and exogenous also apply to unnatural combinations of nucleic acid sequences or amino acid sequences, i.e., combinations in which at least two of the combined sequences are foreign to one another.

[0046] The term “immune response,” as used herein, refers to the production of antibodies and / or cells (such as T lymphocytes) that have and / or express or present an antigen and / or antigenic epitope on their surface, and that promote the degradation and / or inhibition of such entity. The expressions “effective immune defense response,” “immune defense,” and similar terms mean, for the purposes of the present invention, an immune response directed against one or more antigenic epitopes of a pathogen, a pathogen-infected cell, or a cancer cell, for the purpose of protecting a vaccinated subject from infection by a pathogen or from cancer. For the purposes of the present invention, protection from infection by a pathogen or from cancer includes not only complete prevention of infection or cancer, but also any detectable reduction in the degree or rate of infection or cancer in a vaccinated subject compared to, for example, an unvaccinated infected subject, or any detectable reduction in the severity of the disease or any symptom or condition resulting from infection or cancer. An effective immune defense response in the case of cancer includes eliminating cancer cells, thereby reducing the size of the tumor, or even eliminating the cancer altogether. Vaccination aimed at achieving this is also called therapeutic vaccination. Alternatively, an effective immune defense response may be induced in subjects who have never been infected with a pathogen and / or who are not infected with a pathogen or do not yet have cancer at the time of vaccination, and such vaccination can be called prophylactic vaccination.

[0047] In accordance with the present invention, the general use of the term “antigen” herein refers to any molecule that specifically binds to an antibody. The term also refers to any molecule or molecular fragment to which an MHC molecule can bind and which can be presented to a T cell receptor. An antigen may be a proteinaceous molecule, i.e., a polyamino acid sequence, which may optionally contain non-protein groups such as a carbohydrate and / or lipid portion, or an antigen may be a non-proteinaceous molecule, such as a carbohydrate. An antigen may be, for example, any portion of a naturally occurring or synthetically derived protein (peptide, partial protein, full-length protein), a cellular composition (whole cell, cell lysate, or disrupted cell), an organism (whole organism, lysate, or disrupted cell), or a carbohydrate or other molecule, or a portion thereof, which can elicit an antigen-specific immune response (humoral and / or cellular immune response) in a particular target, and the immune response may preferably be measurable by assay or method.

[0048] The term “antigen” is understood herein as a structural substance that functions as a target of a receptor in an adaptive immune response. Antigens thus function as targets of TCRs (T cell receptors) or BCRs (B cell receptors) or secreted forms of BCRs, i.e., antibodies. Antigens can therefore be proteins, peptides, carbohydrates, or other haptens, usually part of larger structures such as cells or virions. Antigens can originate from within the body ("self") or from the external environment ("non-self"). The immune system is normally thought to be non-reactive to “self” antigens under normal conditions, due to negative selection of T cells in the thymus, and to identify and attack only “non-self” invaders from the outside, or modified / harmful substances present in the body, for example, under disease conditions. Antigen structures that are targets of the cellular immune response are presented to T cells of the adaptive immune system by antigen-presenting cells (APCs) in the form of processed antigenic peptides via histocompatibility molecules. Depending on the type of antigen and histocompatibility molecule presented, several types of T cells may be activated. For T cell receptor (TCR) recognition, antigens are processed into small peptide fragments within the cell and presented to the T cell receptor by the major histocompatibility complex (MHC).

[0049] The term “immunogen” is used herein to describe an entity that contains or encodes at least one epitope of an antigen, and as a result, when administered to a subject, preferably together with an appropriate adjuvant, evokes a humoral and / or cellular immune response in the subject that is specific to the epitope and the antigen containing the epitope. An immunogen may be identical to an antigen or contain at least a portion of the antigen, e.g., a portion of the antigen containing an epitope. Thus, vaccinating a subject against a particular antigen means, in one embodiment, that an immune response is evoked against the antigen or its immunogenic portion as a result of administration of an immunogen containing at least one epitope of the antigen. Vaccination preferably results in a protective or therapeutic effect, where subsequent exposure to the antigen (or source of the antigen) evokes an immune response against the antigen (or source of the antigen), which reduces or prevents a disease or condition in the subject. The concept of vaccination is well known in the art. The immune response induced by the administration of the prophylactic or therapeutic composition of the present invention may be any detectable change in any aspect of the immune state (e.g., cellular response, humoral response, cytokine production) compared to the absence of vaccine administration.

[0050] In this specification, an “epitope” is defined as a single immunogenic site within a given antigen that is sufficient to provoke an immune response in a target. Those skilled in the art will recognize that the size and composition of T cell epitopes differ from those of B cell epitopes, and that T cell epitopes presented via the class I MHC pathway differ from those presented via the class II MHC pathway. Depending on the type of immune response, an epitope may be a linear arrangement or a three-dimensional epitope (a conserved binding region). An antigen may be as small as a single epitope or larger, and may contain multiple epitopes. Thus, the size of an antigen may be as small as about 5 to 12 amino acids (e.g., a peptide), or as large as a full-length protein, including a multimeric protein, protein complex, virion, particle, whole cell, whole microorganism, or a part thereof (e.g., a whole cell lysate or a microorganism extract).

[0051] In this specification, an adjuvant is understood to be an entity that, when administered to a human or animal subject in combination with an antigen to induce an immune response to the antigen within the subject, preferably stimulates the immune system, thereby causing, enhancing, or promoting an immune response to the antigen, without necessarily causing a specific immune response to the adjuvant itself. A preferred adjuvant enhances the immune response to a given antigen by at least 1.5, 2, 2.5, 5, 10, or 20 times compared to the immune response to the antigen under identical conditions but in the absence of the adjuvant. Tests for determining the statistical mean of the enhancement of the immune response to a given antigen induced by an adjuvant in a group of animal or human subjects compared to the corresponding control group are available in the art. Adjuvants can preferably enhance immune responses to at least two different antigens.

[0052] OMVs (also called "blebs") are typically spherical, bilayered membrane structures ranging in diameter from 20 to 250 nm (sometimes 10 to 500 nm) that are picked 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 at various positions, and largely reflects the structure of the bacterial outer membrane from which the OMV membrane was picked. The lumen of the OMV may contain various compounds derived from the periplasm or cytoplasm, such as proteins, RNA / DNA, and peptidoglycans (PGs), but unlike bacterial cells, OMVs do not have the ability to self-replicate. In the context of this invention, three types of OMVs are distinguishable according to their method of production. sOMVs are spontaneously occurring or native OMVs that are purified and concentrated from the culture supernatant by separating intact cells from already formed OMVs. dOMV, which is surfactant-derived OMV, is extracted from cells using surfactants such as deoxycholate, which also reduce the content of reactive LPS. After surfactant extraction, dOMV is separated from cells and cell debris and further purified and concentrated. Finally, the term native nOMV is used herein to distinguish these from wild-type, naturally occurring OMV and surfactant-extracted dOMV, for OMV produced from concentrated dead cells using non-surfactant cell disruption techniques, or extracted from cells using methods that do not use other (non-destructive) surfactants (e.g., using chelating agents such as EDTA).

[0053] Any reference in this specification to nucleotide or amino acid sequences accessible in public sequence databases refers to the version of the sequence registration available as of the filing date of this specification.

[0054] Detailed explanation The inventors have developed a method for directly presenting known antigens, and newly identified antigens, such as those from newly emerging pathogens, on an established and immunogenic OMV platform. OMVs can be pre-stockpiled, and novel target antigens, for example identified via bioinformatics, can be produced and attached using the method of the present invention, thus providing a rapid response platform.

[0055] This technology allows for the rapid deployment of an OMV platform, for example, in the case of an epidemic. The strength of the platform of the present invention lies in its strategy consisting of two components: OMV and antigen. The antigen and OMV are produced separately, and simply mixing the antigen and OMV allows the antigen to attach to the OMV by non-covalent bonding. For this purpose, the antigen has an antimicrobial peptide (AMP) as a "tag" or "anchor" to facilitate attachment to the OMV. A typical embodiment of the present invention is shown in Figure 1.

[0056] In a first aspect, the present invention thus relates to a complex of an outer membrane vesicle (OMV), an antimicrobial peptide (AMP), and an antigen. Preferably, the AMP is complexed with the OMV by non-covalent bonding, and the antigen is conjugated to the AMP. In the complex of the present invention, the AMP interacts with the membrane of the OMV. Preferably, the AMP is inserted into the lipid layer of the OMV. The antigen conjugated to the AMP remains at least partially exposed to the surface of the OMV. In this specification, it is understood that the AMP thus functions as an anchor portion, i.e., immobilizes the antigen on the surface of the OMV.

[0057] Antimicrobial peptides (AMPs) Preferably, the AMP is a vertebrate AMP. AMP is part of the innate immune system of vertebrates and is known to have a broad spectrum of antimicrobial activity against bacteria, enveloped viruses, and fungi (Kosciuczuk et al, Mol Biol Rep (2012) 39:10957-10970). AMP can penetrate or "punch holes" in the negatively charged membranes of pathogens. For this purpose, the AMP for use in the present invention preferably has several positively charged residues provided, for example, by arginine, lysine, or histidine in an acidic environment, and preferably the majority (e.g., >50%) are hydrophobic residues.

[0058] AMP may not be structured in free solution, but when partitioned into the OMV membrane, it folds into its final three-dimensional structure. This may have, for example, hydrophilic amino acid residues aligned along one side of a helical molecule and hydrophobic amino acid residues aligned along the opposite side of the molecule. The amphiphilic nature of the antimicrobial peptide allows it to be partitioned into the membrane lipid bilayer. The AMP used in the present invention is preferably a cationic peptide with amphiphilic properties that can penetrate the OMV membrane. Preferably, the AMP remains within the OMV membrane, i.e., does not partially or completely pass through the OMV membrane. Preferably, the AMP does not destroy or significantly destroy the formed OMV.

[0059] Preferably, the AMP for use in the present invention is mammalian AMP. Preferably, AMP is the active form of at least one of cathelicidin, alpha-defensin, beta-defensin, and regIII peptide. Preferably, AMP is the active form of cathelicidin. In this specification, it is understood that protein names such as the terms “cathelicidin,” “alpha-defensin,” “beta-defensin,” and “regIII peptide” are not limited to human peptides but also include ortholog peptides of other vertebrates, preferably other mammals. As a non-limiting example, the term “cathelicidin” includes human cathelicidin LL-37 and the mouse ortholog “cathelicidin-related” peptide mCRAMP.

[0060] AMP can be produced in an inactive form, which is activated by cleavage. For example, mammalian cathelicidins consist of three domains: a signal peptide, a cathelin domain, and an antimicrobial domain. The signal peptide is necessary for intracellular targeting to granules and is cleaved and removed by signal peptidases. The conserved cathelin domain, whose function is not well understood, remains attached to the antimicrobial domain during granule preservation. Cleavage between the cathelin domain and the antimicrobial domain releases a biologically active antimicrobial peptide. In this specification, the term AMP is understood to refer to the active form, i.e., the biologically active antimicrobial peptide.

[0061] The AMP for use in the present invention may be a naturally occurring peptide, a recombinant peptide, or a chemically synthesized peptide. The recombinant or synthetic AMP may be identical to the naturally occurring AMP. Alternatively, the recombinant (or synthetic) AMP for use in the present invention may involve one or more amino acid modifications compared to the amino acid sequence of its naturally occurring counterpart. The amino acid modification is preferably at least one of one or more amino acid deletions, additions, or substitutions.

[0062] The AMP of the present invention may contain deletions of one or more amino acid residues compared to its naturally occurring counterpart, preferably compared to its naturally occurring cathelicidine. The AMP may contain deletions of at least one, two, three, four, five, or ten amino acid residues. Preferably, the AMP containing the deletions of one or more amino acids maintains the ability to bind to (or complex with) OMV by non-covalent bonds. Preferably, the ability to bind to OMV by non-covalent bonds is equal to or preferably higher than the ability of its naturally occurring counterpart to bind to OMV.

[0063] Alternatively, or furthermore, AMP may include the addition of one or more amino acid residues compared to its naturally occurring counterpart, preferably compared to its naturally occurring cathelicidine. AMP may include the addition of at least one, two, three, four, five, or ten amino acid residues. Preferably, AMP including the addition of one or more amino acids maintains the ability to bind to (or complex with) OMV by non-covalent bonds. Preferably, the ability to bind to OMV by non-covalent bonds is equal to or preferably higher than the ability of its naturally occurring counterpart to bind to OMV.

[0064] Alternatively, or furthermore, AMP may contain one or more amino acid residue substitutions compared to its naturally occurring counterpart, preferably compared to its naturally occurring cathelicidine. AMP may contain at least one, two, three, four, five, ten, fifteen, or twenty amino acid residue substitutions. Preferably, AMP contains one or more conserved amino acid substitutions, preferably one or more conserved amino acid substitutions as defined herein. Preferably, recombinant AMP contains at least one, two, three, four, five, ten, fifteen, or twenty conserved amino acid substitutions. Preferably, AMP containing one or more amino acid substitutions retains the ability to bind to (or complex with) OMV by non-covalent bonds. Preferably, the ability to bind to OMV by non-covalent bonds is equal to or preferably higher than the ability of its naturally occurring counterpart to bind to OMV.

[0065] The ability of AMP to bind to (or form a complex with) OMV can be determined using any conventional method known in the art. Typical methods, such as quantitative dot blotting detailed in Example 1, are provided in the Examples section below, but are not limited to these. The ability of AMP to bind to OMV can be determined, for example, by coupling a PRN peptide or any other detectable moiety to AMP, and then, for example, by binding AMP to OMV on a dot blot. The PRN peptide or any other detectable moiety can then be detected quantitatively, for example, using a quantitative anti-PRN antibody detection method.

[0066] The sizes of AMP for use in the present invention are preferably about 10 to 100 amino acid residues, about 12 to 80 amino acid residues, about 12 to 50 amino acids, about 12 to 18 amino acid residues, about 39 to 80 amino acid residues, about 20 to 40 amino acid residues, or about 23 to 35 amino acid residues.

[0067] It is known in the art that AMPs do not have very conserved amino acid sequence identity. Instead, AMPs are mainly grouped based on the properties of their amino acids and / or the structures they form when they penetrate bacterial membranes. In addition, it is known in the art that AMPs have a wide range of structures. Therefore, it will be understood by those skilled in the art that the present invention is not limited to any specific AMP sequence or structure. Preferably, the AMPs for use in the present invention have a net charge of 0 to +7 and a hydrophobic percentage between 30 and 70%.

[0068] AMP can be a linear peptide that folds into an amphiphilic α-helix, or a small molecule having a beta-hairpin structure, and may be stabilized by one, two, or more disulfide bonds. Alternatively, AMP may further contain repeating proline motifs that form an extended polyproline-type structure. Preferred AMPs for use in the present invention have an amphiphilic helix structure. Preferably, AMPs for use in the present invention contain an amphiphilic cationic α-helix peptide.

[0069] Preferably, the AMP for use in the present invention may be or may be derived from a naturally occurring protein, where the inactive form of the protein contains a catheline domain. Preferably, the catheline domain is cleaved and removed to cause AMP activation.

[0070] The AMP used in the present invention may be a cathelicidine, preferably human cathelicidine, or an ortholog thereof. Human cathelicidine LL-37 is a peptide with broad antimicrobial activity, including both direct toxic effects and topical immunomodulatory effects against many different types of microorganisms (Xhindoli et al BBA 1858 (2016) 546-566). It performs its many different activities in a small amphiphilic helix structure, which is formed by epithelial cells and immune cells as a precursor prepro form. Its antimicrobial effect results from direct interaction with membranes, including the outer membrane of Gram-negative bacteria. The first step in this interaction is the binding of LPS, the major outer membrane lipid A, to the lipid A / internal central region. Since native OMV, for example, retains its LPS, the inventors have found that LL-37 can form suitable tags that can attach to a variety of antigens, thereby associating these antigens with OMV.

[0071] Since LL-37 is a human antigen, its combination with immunostimulatory OMV may produce an anti-autoimmune response. Therefore, preferably, AMP is a non-human cathelicidine. The non-human cathelicidine may be derived from a human cathelicidine, for example, a human cathelicidine containing one or more amino acid modifications. The non-human cathelicidine thus may include modified human cathelicidines. The non-human cathelicidine is preferably a cathelicidine derived from mouse, cattle, buffalo, horse, pig, sheep, goat, deer, chicken, fish, rhesus monkey, rat, guinea pig, or snake. Preferably, the cathelicidine is a mouse or rat cathelicidine, and preferably a mouse cathelicidine. If necessary, the mouse cathelicidine contains one or more amino acid modifications.

[0072] The cathelicidines for use in the present invention may be the proteins identified in Table 1 of Kosciuczuk et al. (above), incorporated herein by reference, or the cathelicidines identified in Table 1 of Kosciuczuk et al. above, having one or more amino acid modifications.

[0073] The AMP used in the present invention may be a protein selected from the group consisting of mCRAMP (CRAMP-1 / 2), LL-37, FALL-39, RL-37, rCRAMP, CAP-18, CAP-11, PR-39, Prophenin, PMAP-23, PMAP-36, PMAP-37, BMAP-27, BMAP-28, BMAP-34, Bac5, Bac7, Cathelicidin-AL, Fowlicidin-1, Fowlicidin-2, Fowlicidin-3, Cathelicidin-beta-1, Saha-CATH5, CATH1, and CATH2. Preferably, the AMP is mCRAMP, and may include modifications of one or more amino acids.

[0074] The sequences of naturally occurring AMPs are highly diverse. Therefore, the present invention is not limited to any AMP having a specific sequence. The AMP for use in the present invention may be a cathelicidine having the sequence disclosed in Figure 1 by Kosciuczuk et al., preferably the mature peptide sequence disclosed in Figure 1 by Kosciuczuk et al. In one embodiment, the sequence of AMP may have at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or about 100% sequence identity with SEQ ID NO: 1 (mCRAMP). mCRAMP is preferably, GLLRKGGEKIGEKLKKIGQKIKNFFQKLVPQPEQ (Sequence ID 1) It has an array of these.

[0075] In one embodiment, the sequence of AMP may have at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or about 100% sequence identity with respect to sequence number 13 (LL-37). LL-37 is preferably, LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES (Sequence ID 13) It has an array of these.

[0076] Preferably, AMP can penetrate the membrane of OMV and, by non-covalent bonding, can attach the antigen conjugated to AMP to OMV.

[0077] AMP-antigen conjugation In a preferred embodiment, the AMP is conjugated to an antigen, preferably an antigen described herein. The conjugation of the antigen to the AMP creates a conjugate that allows the AMP to enter the membrane of the OMV and thus enable the coupling or "immobilization" of the antigen to the OMV.

[0078] Antigens can be conjugated to AMP using any conventional means known in the art. Conjugations include covalent and non-covalent bonds. Preferably, the antigen is covalently bound to AMP.

[0079] The antigen can be conjugated to a portion of AMP that remains exposed on the surface of the OMV once AMP is inserted into the OMV membrane. Preferably, the antigen is conjugated to AMP in a manner that does not interfere with AMP's ability to bind to OMV. Preferably, and therefore, the antigen is conjugated to a hydrophilic residue of AMP, and more preferably, to a hydrophilic residue that is the solvent to which AMP is exposed when it enters the membrane (e.g., the OMV). The antigen may be conjugated to or adjacent to one or more cationic amino acid residues of AMP. Alternatively, or further, the antigen is conjugated to the C-terminus or N-terminus of AMP. Preferably, the antigen is conjugated to the N-terminus of the AMP peptide. In one embodiment, two or more antigen molecules are conjugated to a combination of the aforementioned sites on AMP. These two or more antigen molecules may be the same or different antigen molecules.

[0080] The antigen can be conjugated to AMP using any conventional chemical conjugation process, where the bond, preferably covalent, does not (significantly) reduce the AMP's ability to enter the OMV and immobilize the antigen to the OMV. For this purpose, AMP and antigen are first produced separately, and then AMP is coupled to the antigen, preferably covalently. Such covalent coupling can be carried out using any conventional means known to those skilled in the art. This method may be preferred in cases where AMP and antigen cannot be expressed as a single polypeptide, for example, when the antigen is an oligosaccharide or polysaccharide, and / or when the antigen requires chemical modification such as cyclization, but is not limited to these cases.

[0081] Preferred chemical conjugation methods include amide bond formation (e.g., using active esters and free amines), selective N-terminal ligation, native chemical ligation, and bioorthogonal ligation. Examples of bioorthogonal ligation methods include Michael addition (e.g., using maleimide and thiol, the thiol may be introduced via Trout's reagent), Diels-Alder cycloaddition, Huisgen cycloaddition, and cycloaddition using tetrazine or azide or trans-cyclooctene or strained cyclooctin or oxonolbornadiene. Such methods are widely known (see, for example, Bioconjugate Chem. 2015, 26, 2, 176-192, as well as doi.org / 10.1016 / j.cbpa.2013.07.031 and doi.org / 10.1016 / j.chembiol.2014.09.002), and the required reagents are often commercially available, sometimes even in the form of kits that include instructions for use.

[0082] The antigen may be directly conjugated to AMP or separated by a linker, preferably a linker as defined herein. If not expressed as a single fusion protein, the linker may be a specific amino acid sequence, a single amino acid, or another part such as ahx. The three-letter code ahx represents 6-aminohexanoic acid, also known as aminocaproic acid, abbreviated as Acp. Ahx is considered a linker portion that connects two further parts. In addition to ahx, other linkers may also be used, but are not limited to beta-alanine (beta-aminopropionic acid, also known as bAla), 4-aminobutyric acid (piperidine acid, also known as 4Abu), 3-aminoisobutyric acid (bAib), or other binding parts known in the art. Further examples of linkers are ethylene glycol-based, such as poly(ethylene glycol) (PEG) or oligo(ethylene glycol). PEG-based linkers are preferred due to their good solubility in water or other relevant solvents and their ease of handling. PEG linkers are often used to improve the renal clearance of peptides (Lang et al., Bioconjug. Chem. 2011 22(12): 2415-2422. doi: 10.1021 / bc200197h). Linkers are often defined by their function of connecting two further parts to each other to ensure their spatial proximity or to restrict the spatial position of each of them. Linkers provide a mechanical bond. Those skilled in the art will be able to select a suitable linker. For example, in N-terminal conjugation to peptides, alkyl chains or PEG with a free carboxylic acid moiety are suitable. In such cases, the other end of the PEG could be, for example, a protected amine or another orthogonal reactive moiety. Non-limiting examples of suitable PEG ends are amines, carboxylic acids, thiols, alcohols, aldehydes, azides, alkynes, or protected forms of any of these moieties.

[0083] Alternatively, AMP and antigen may be produced as a single polypeptide or a “fusion protein,” with a linker optionally present between AMP and antigen. Therefore, preferably, the antigen is covalently bound to AMP in a fusion protein containing the antigen and AMP in a single polypeptide chain. Preferably, the N-terminal fusion partner of the fusion protein is the antigen, and the C-terminal fusion partner of the fusion protein is AMP. Preferably, the fusion protein does not contain amino acid residues outside of AMP, antigen, and the linker as needed, as defined later herein.

[0084] The present invention also relates to an OMV comprising a fusion protein, wherein, firstly, preferably, the N-terminal fusion partner is an antigen, and secondly, preferably, the C-terminal fusion partner is an AMP capable of immobilizing the antigen on the OMV. Preferably, the OMV and the fusion protein are produced separately in different (micro)organisms, as may be. The first and second fusion partners may be separated by a linker.

[0085] The antigen can be bound to AMP by a linker (or spacer), preferably a mobile linker. Any conventional linker known in the art can be used to couple AMP to the antigen. Preferably, the linker does not interfere with, or substantially interferes with, AMP's ability to penetrate the OMV membrane. Furthermore, or otherwise, the linker does not interfere with, or substantially interferes with, the antigen's ability to elicit an immune response.

[0086] The linker can be a rigid linker or a mobile linker. The linker is preferably a mobile linker. The linker may first be conjugated, preferably covalently, to AMP, and then the antigen may be conjugated, preferably covalently, to the linker. Alternatively, the linker may first be conjugated, preferably covalently, to the antigen, and then AMP may be conjugated, preferably covalently, to the linker. Alternatively, a portion of the linker may be conjugated, preferably covalently, to AMP, and another portion of the linker may be conjugated, preferably covalently, to the antigen, and then both portions of the linker may be conjugated, preferably covalently, together. Alternatively, the linker may be produced as part of a fusion protein.

[0087] Preferably, the linker is a peptide linker. The linker may be a glycine-rich mobile linker. Preferably, the length of the linker is between 2 and 50 amino acid residues, between 3 and 40, between 4 and 30, or between 5 and 20. The linker may be one of the linkers specified in Table 1 of Chichili et al. (Protein Sci. 2013 Feb; 22(2): 153-167), which is incorporated herein by reference.

[0088] The linker may have a Gly-Ser sequence. Methods for selecting the linker according to the AMP and antigen are known to those skilled in the art. The linker may range from highly mobile linkers in the form of, for example, GGGSGGGSGGGS (SEQ ID NO: 12), (GGGGS)n (SEQ ID NO: 10), (GGS)n, (GS)n, and (G)n, to more rigid linkers in the form of (EAAAK)n (SEQ ID NO: 11), (SPKKKRKVEAS)n (SEQ ID NO: 2), or (SGSETPGTSESATPES)n (SEQ ID NO: 3), or (KSGSETPGTSESATPES)n (SEQ ID NO: 4), or any variant thereof, where n is preferably between 1 and 10, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0089] In some cases, one or more additional amino acid residues, such as tags and / or protease cleavage sites, are located between the antigen and AMP. If necessary, a his tag and / or twin-strep tag are present between the antigen and AMP. Alternatively, there may be protease cleavage sites, such as an HRV 3C recognition site, located between the antigen and AMP.

[0090] In another embodiment, the tag and / or protease cleavage site is not located between the antigen and AMP.

[0091] antigen The present invention is not limited to any specific antigen. Antigens for use in the present invention are preferably suitable for conjugation to AMP and subsequent presentation on the surface of OMV. The antigen may be at least one of sugars or peptides. Sugars may be, for example, oligosaccharides or polysaccharides. Similarly, peptides may be, for example, oligopeptides or long-chain molecules such as proteins. Preferably, the antigen is a peptide.

[0092] Antigenic peptides may be naturally occurring proteins or fragments thereof, preferably antigenic fragments thereof. Antigenic peptides may contain one or more modifications. As a non-limiting example, antigenic peptides may contain cysteine ​​at the N-terminus or C-terminus.

[0093] The antigen may consist of one or more epitopes obtained from the epitope database iedb.org (Vita et al, Nucleic Acids Res. 2015; 43 (Database issue): D405-D412 and periodic updates), or it may be any other antigen, such as a newly discovered antigen. Preferably, the antigen consists of one or more epitopes of antigens associated with infectious diseases or tumors. For example, an antigen fused to AMP may consist of one or more epitopes derived from antigens of pathogens and infectious agents such as viruses, bacteria, fungi, and protozoa.

[0094] Some examples of pathogenic viruses that cause infection or tumors and from which the antigen's epitope may originate include hepatitis (A, B, or C), herpesviruses (e.g., VZV, HSV-I, HAV-6, HSV-II, and CMV, Epstein-Barr virus), adenoviruses, SV40 virus (causing mesothelioma), influenza virus, flavivirus, Ebola virus, echovirus, rhinovirus, coxsackievirus, coronavirus, respiratory syncytial virus (RSV), mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, molluscum contagiosum virus, poliovirus, rabies virus, JC virus, arbovirus encephalitis virus, and human immunodeficiency virus (HIV virus, e.g., types I and II), human papillomavirus (HPV). Preferably, the antigen for use in the present invention comprises one or more epitopes of coronaviruses or enteroviruses. Preferably, the antigen for use in the present invention comprises one or more epitopes of a coronavirus. The coronavirus may be a virus of the genus Alphacoronavirus or Betacoronavirus, preferably a virus of the genus Betacoronavirus. The subgenus is preferably Salvecovirus or Merbecovirus. Preferably, the antigen for use in the present invention comprises one or more epitopes of a coronavirus selected from the group consisting of COVID-19 (SARS-CoV-2), SARS-CoV, MERS-CoV, HCoV-OC43 and HCoV-HKU1, HCoV-229E, and HCoV-NL63. The antigen for use in the present invention may comprise one or more epitopes of an enterovirus. The enterovirus is preferably at least one of poliovirus, coxsackievirus, echovirus, and rhinovirus. Preferably, the enterovirus is enterovirus 71 (EV71), and preferably the epitope is of enterovirus VP1 or VP2.

[0095] Some examples of pathogenic bacteria that cause infection and from which the antigenic epitope may originate include the genera Bordetella, Neisseria, Acinetobacter, Borrelia, Listeria, Escherichia, Chlamydia, Coxiella, and Rickettsia. This includes bacteria such as Mycobacteria, Staphylococcus, Streptococcus, Pneumonococcus, Meningococcus, Gonorrhea, Klebsiella, Proteus, Serratia, Pseudomonas, Legionella, Diphtheria, Salmonella, Bacilli, and bacteria that cause cholera, tetanus, botulism, anthrax, plague, leptospirosis, pertussis, and Lyme disease. A preferred Bordetella antigen may be a partactin protein or its antigenic fragment, the partactin protein preferably having at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or about 100% sequence identity with SEQ ID NO: 18.

[0096] Some examples of pathogenic fungi that cause infection and from which the antigenic epitope may originate include the genera Candida (e.g., Candida albicans, Candida krusei, Candida glabrata, Candida tropicalis), Cryptococcus neoformans, the genera Aspergillus (e.g., Aspergillus fumigatus, Aspergillus niger), fungi of the Mucorales order (Mucor, Absidia, Rhizopus), and Sporothrix schenkyi. This includes *Coccidiphyllum schenkii*, *Blastomyces dermatitidis*, *Paracoccidioides brasiliensis*, *Coccidioides immitis*, and *Histoplasma capsulatum*.

[0097] Some examples of pathogenic parasites that cause infection and from which the antigen epitope may originate include Entamoeba histolytica, Balantidium coli, Naegleria species, Fowleri species, Acanthamoeba sp., Giardia lambia, Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, and Toxoplasma gondii. This includes Plasmodium gondii and Plasmodium falciparis, the parasite responsible for tropical malaria.

[0098] Furthermore, or alternatively, the antigens for use in the present invention may include not only one or more epitopes of a broad range of tumor antigens, such as MAGE, BAGE, RAGE, GAGE, SSX-2, NY-ESO-1, CT antigen, CEA, PSA, p53, XAGE, and PRAME, but also virus-induced malignancies, including lymphomas induced by human papillomavirus (HPV), Kaposi's sarcoma herpesvirus (KSHV), and Epstein-Barr virus (EBV). Other examples of tumor antigens from which epitopes for use in the present invention may be derived may be a variety of ubiquitously expressed autoantigens known to be associated with cancer, such as p53, MDM-2, HDM2, and other proteins that play a role in the p53 pathway, molecules such as surviving, telomerase, cytochrome P450 isoform 1B1, Her-2 / neu, and CD19, as well as all so-called household proteins. Cancers that can be treated or prevented according to the present invention are selected from the following list: lung cancer, colon cancer, esophageal cancer, ovarian cancer, pancreatic cancer, skin cancer, stomach cancer, head and neck cancer, bladder cancer, sarcoma, prostate cancer, hepatocellular carcinoma, brain tumor, adrenal cancer, breast cancer, endometrial cancer, mesothelioma, kidney cancer, thyroid cancer, blood cancer, carcinoid, melanoma, parathyroid cancer, cervical cancer, neuroblastoma, Wilms' tumor, testicular cancer, pituitary cancer, and pheochromocytoma.

[0099] An antigen conjugated to an AMP may or may consist of one or more epitopes of infectious or tumor protein antigens exposed to a surface. For example, an antigen conjugated to an AMP may consist of or may consist of extracellular and / or surface-exposed domains of infectious or tumor protein antigens.

[0100] Preferably, the antigen coupled to AMP comprises or consists of one or more epitopes of a surface-exposed viral protein or lipoprotein surface-exposed domain. Preferably, one or more epitopes are of a coronavirus or enterovirus, preferably COVID-19 or EV71, and include, but are not limited to, the surface glycoprotein or "spike" of COVID-19 or the EV71 viral protein VP1 or VP2.

[0101] The antigen may comprise one or more epitopes of a viral spike protein. The antigen may be a viral spike protein or an antigenic fragment thereof. Preferred spike proteins or antigenic fragments thereof are obtained from coronaviruses, preferably selected from the group consisting of COVID-19 (SARS-CoV-2), SARS-CoV, MERS-CoV, HCoV-OC43 and HCoV-HKU1, HCoV-229E, and HCoV-NL63. Preferred spike proteins or antigenic fragments thereof are obtained from or derived from COVID-19.

[0102] The spike protein that can be used as an antigen in the complex of the present invention, preferably the spike protein of SARS-CoV-2, may be a pre-fusion or post-fusion structure. Preferably, the spike protein is the pre-fusion structure. The spike protein used as an antigen in the complex of the present invention may be a native protein or a modified protein, for example, a protein modified to increase its stability. The spike protein used as an antigen in the complex of the present invention may be a native or modified spike protein obtained from or derived from any of the SARS-CoV-2 strains, preferably a native or modified spike protein obtained from or derived from the SARS-CoV-2 strain Wuhan-Hu-1, GenBank MN908947.

[0103] Preferably, the spike protein is a modified protein having one or more amino acid substitutions. Preferably, the spike protein is a modified protein having one or more proline substitutions. Preferably, the spike protein is a protein with a modified conformation before fusion and contains two, three, four, five, or six proline substitutions. Preferably, the antigen in the complex of the present invention comprises one or more epitopes of the spike protein disclosed in Hsieh et al. (Science. 2020 Sep 18;369(6510):1501-1505). Preferably, the antigen in the complex of the present invention comprises or consists of the spike protein disclosed in Hsieh et al. (above).

[0104] Preferably, the antigen in the complex of the present invention is the SARS-CoV-2 spike protein having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 41. Preferably, the SARS-CoV-2 spike protein is encoded by a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 46. The spike protein may contain one or more substitutions. Preferably, the spike protein may contain substitutions at positions selected from the group consisting of F816, A891, A898, and A941. Preferably, the spike protein contains substitutions selected from the group consisting of F816P, A891P, A898P, and A941P. Preferably, the spike protein contains substitutions F816P, A891P, A898P, and A941P.

[0105] Preferably, the antigen in the complex of the present invention is the SARS-CoV-2 spike protein having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 44. Preferably, the antigen in the complex of the present invention is the SARS-CoV-2 spike protein encoded by a nucleotide sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 49.

[0106] The spike protein is preferably conjugated to AMP as defined herein, and preferably AMP is a cathelicidine as defined herein. The spike protein is preferably conjugated to mCRAMP as defined herein. Preferably, the spike protein is conjugated to mCRAMP using a linker, preferably linker GGGSGGGSGGGS (SEQ ID NO: 12).

[0107] Furthermore, a tag sequence and / or a protease cleavage site may be present before or after the amino acid sequence of the spike protein. The spike protein may be conjugated to at least one of the HRV 3C protease recognition site, the His tag, and the Twin strep tag. A spike protein conjugated to one or more tags and a protease recognition site may have at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 43. The spike protein conjugated to one or more tags and protease recognition sites is preferably encoded by a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 48.

[0108] The sequences of the proteins conjugated to AMP (and possibly the linker) preferably have at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 44.

[0109] A preferred conjugate of the present invention is a conjugation between the SARS-CoV-2 spike protein and mCRAMP. Preferably, the conjugate includes a linker between the spike protein and mCRAMP. Optionally, the conjugate further includes one or more tags, such as a His tag and a Twin strep tag. Furthermore, or or otherwise, the conjugate includes one or more protease cleavage sites, such as one or more HRV 3C recognition sites. A preferred conjugate of the present invention has at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 42. A preferred conjugate of the present invention is encoded by a nucleotide sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 47.

[0110] Preferably, the conjugate of the present invention does not contain a tag and / or protease recognition site. The conjugate may comprise or consist of an antigen, a linker, and an AMP as defined herein. Preferably, the linker is GGGSGGGSGGGS (SEQ ID NO: 12). Preferably, the spike protein has SEQ ID NO: 44. Preferably, the AMP is mCRAMP having SEQ ID NO: 1. Preferred conjugates of the present invention have at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 45. A preferred conjugate of the present invention is encoded by a nucleotide sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 50.

[0111] The EV71 VP1 protein may have at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or about 100% sequence identity with SEQ ID NOs. A preferred, preferably antigenic fragment of VP1 or VP2 may have at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or about 100% sequence identity with at least one of SEQ ID NOs.

[0112] The antigen coupled to AMP may or may consist of one or more epitopes of surface-exposed domains of surface-exposed bacterial proteins or lipoproteins. Preferably, surface-exposed domains of bacterial proteins or lipoproteins selected from any of the genera Bordetella, Neisseria, Acinetobacter, Borrelia, Coxiella, and any other pathogenic bacterial genera mentioned above.

[0113] AMP-antigen production The antigen and AMP can be produced separately and then conjugated after production. The antigen and / or AMP can be produced using a cell-free system. Such a cell-free system may be in vitro peptide synthesis, for example, liquid-phase peptide synthesis (LPPS) or solid-phase peptide synthesis (SPPS), but is not limited to these. Alternatively, the AMP and / or antigen can be purified from cells, tissues, or body fluids naturally containing the antigen or the AMP. Alternatively, the antigen and / or AMP can be produced in recombinant cells modified to express or overexpress the antigen and / or AMP. The AMP is preferably the AMP as defined above herein. Preferably, the AMP has a sequence having at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or about 100% sequence identity with respect to SEQ ID NO: 1.

[0114] Alternatively, AMP and antigen can be produced as a single polypeptide, i.e., as a fusion protein comprising a first fusion partner and a second fusion partner, wherein the first fusion partner is AMP and the second fusion partner is the antigen. If necessary, the fusion partners are separated by a linker. It is understood herein that the terms “first” and “second” do not specifically indicate that the respective fusion partners within the fusion protein are located at the N-terminus or C-terminus. The terms “first” and “second” are simply to indicate that the fusion protein contains at least two fusion partners, i.e., AMP and antigen. Preferably, the fusion protein comprises AMP, an optional linker, and the antigen in the N-terminus to C-terminus direction. Alternatively, the fusion protein comprises the antigen, an optional linker, and AMP in the N-terminus to C-terminus direction. The AMP of the fusion protein is preferably the AMP as defined herein. Preferably, the fusion protein contains an AMP having a sequence with at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or about 100% sequence identity with respect to SEQ ID NO: 1.

[0115] Fusion proteins can be produced using cell-free systems. Such cell-free systems may include in vitro peptide synthesis, for example, liquid-phase peptide synthesis (LPPS) or solid-phase peptide synthesis (SPPS), but are not limited to these. Alternatively, fusion proteins may be produced within recombinant cells. Recombinant cells or “host cells” expressing at least one of the AMPs, antigens, or fusion proteins described herein may be any suitable host cell. It is understood herein that cells expressing AMPs may be different from cells expressing antigens, i.e., they may originate from a different organism or a different cell type than the cells expressing antigens. Host cells may be transformed, transfected, transduced, etc., with nucleic acid constructs encoding the AMPs, antigens, or fusion proteins as defined herein. Thus, the term “host cell” means any cell type that is susceptible to transformation, transfection, transduction, etc., with such nucleic acid constructs.

[0116] Alternatively, the genome of the host cell may be modified to express or overexpress an endogenously encoded AMP or antigen. Alternatively, the genome of the host cell may be modified to express or overexpress a mutant of an endogenously encoded AMP or antigen, for example, by using site-directed editing techniques such as CRISR-Cas technology, but not limited to these. The term “host cell” further encompasses all offspring of a parent cell that are not identical to the parent cell due to mutations that occur during replication.

[0117] The choice of host cell may depend on the type of AMP, antigen, and / or fusion protein. The host cell can be any cell useful in the recombinant production of AMP, antigen, and / or fusion protein, such as a prokaryotic or eukaryotic cell.

[0118] The prokaryotic host cell can be any Gram-positive or Gram-negative bacterium. Gram-positive bacteria include, but are not limited to, the genera Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to, the genera Neisseria, Bordetella, Escherichia coli, Pseudomona, Campylobacter, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Salmonella, and Ureaplasma. Preferred Gram-negative bacteria are Neisseria, Bordetella, or Escherichia coli. The preferred Neisseria species is at least one of Neisseria meningitidis, Neisseria gonorrhoeae, or Neisseria lactamica. The preferred Bordetella species is at least one of Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica. The preferred prokaryotic host cell is Escherichia coli (E. coli).

[0119] Preferred eukaryotic host cells are animal cells, preferably vertebrate cells, preferably mammalian cells, and preferably human cells. Preferably, the cells are derived from a cell lineage, preferably an immortalized cell lineage.

[0120] The host cell may be transformed, transfected, or transduced with a nucleic acid construct encoding at least one of the antigen, AMP, and fusion protein. Preferably, the nucleic acid construct includes one or more regulatory elements that control the expression of at least one of the AMP, antigen, and fusion protein. Preferably, the regulatory elements include at least one promoter sequence. Those skilled in the art will understand that any promoter sequence suitable for expression in selected host cells may be used. Preferably, the promoter is at least one of a constitutively active promoter or an inductive promoter. In the case where the host cell is a bacterial host cell used for the expression of at least one of the AMP and fusion protein, the promoter is an inductive promoter, such as a chemoinductive promoter, but is not limited to that which.

[0121] The produced AMP, antigen, and / or fusion protein can be purified using any conventional means, including but not limited to one or more dialysis, filtration, or purification steps.

[0122] OMV Antigens conjugated to AMP can form complexes with any suitable OMV. OMVs are spherical buds of the outer membrane (OM) naturally produced by Gram-negative bacteria.

[0123] OMV (also known as “bleb”) for use in vaccines is traditionally prepared by surfactant extraction (dOMV purification process), in which surfactants such as deoxycholate are used to remove LPS and increase vesicular release. While LPS from most Gram-negative bacteria, such as Neisseria meningitidis, is highly toxic, a residual amount (approximately 1%) is required in OMV to maintain vesicular structure and for adjuvant activity. The first step in the interaction between AMP and OMV is presumed to be the binding of AMP to the lipid A / internal core region of LPS. Therefore, it is preferable for OMV to retain at least a portion of its LPS. Thus, OMV in the complex as defined herein is preferably not OMV extracted with a surfactant. However, it is understood that processes for preparing OMV that is not OMV extracted with a surfactant do not exclude the use of any surfactant. The use of low concentrations of surfactants and / or mild surfactants is not ruled out, as long as AMP is still able to immobilize the antigen on the extracted OMV, for example, as long as at least about 50, 60, 70, 80, 90, 95, or 99% of the AMP-conjugate antigen complexes with the extracted OMV compared to the amount of AMP-conjugate antigen that complexes with the naturally occurring OMV or supernatant OMV.

[0124] Preferably, the OMV that forms a complex with the antigen and AMP is a naturally occurring OMV or native OMV. The OMV is preferably a native OMV. The production of native OMVs is well known in the art and is described, for example, in Saunders et al. (1999, Infect Immun, 67, 113-119), van de Waterbeemd et al. (2012, Vaccine, 30: 3683-3690), and in brochure International Publication No. 2013006055. Methods for preparing sOMV are described, for example, in van de Waterbeemd et al. (2013, PLoS ONE, 8(1): e54314. doi:10.1371 / journal.pone.0054314) and Lee et al. (2007, Proteomics, 7: 3143-3153), all of which are incorporated herein by reference.

[0125] The LPS of OMV described herein may include LPS that has been at least partially detoxified. For example, LPS may have a modified oligosaccharide structure to eliminate possible epitopes that are thought to evoke an autoimmune response and / or increase dendritic cell binding and adjuvant activity. Furthermore or alternatively, LPS may have a modified lipid A moiety in which, for example, one or more acyl chains are shortened or absent compared to the wild-type lipid A moiety.

[0126] The OMV in the complex of the present invention can preferably be obtained from Gram-negative bacteria having a genetic modification selected from the group consisting of (i) a genetic modification that alters the lipopolysaccharide (LPS) biosynthesis pathway to obtain a variant that is less endotoxin and reactive, preferably; (ii) a genetic modification that increases OMV production by removing an outer membrane anchor protein; and (iii) a genetic modification that removes an immunomodulatory component that may induce an undesirable type of immune response. Furthermore or alternatively, the Gram-negative bacteria may include at least one of the following genetic modifications: (iv) a genetic modification that causes outer membrane retention of normally secreted antigens; and (v) a genetic modification that introduces the expression of a heterologous antigen derived from a pathogen other than the host OMV-producing strain.

[0127] Preferably, the OMV can be obtained from Gram-negative bacteria, which contain one or more genetic mutations that cause the bacteria to produce LPS with reduced (endogenous) toxicity. Preferably, the LPS retains at least a portion of its adjuvant activity. Preferably, the modification reduces or eliminates the expression of at least one of the lpxL1, lpxL2, lpxA, lpxD, and lpxK genes or their homologs. Preferably, the modification reduces or eliminates the expression of at least one of the endogenous lpxL1, lpxL2, lpxA, lpxD, and lpxK genes or their homologs.

[0128] Preferably, the Gram-negative bacteria have a genetic modification that reduces or eliminates the expression of the lpxL1 gene or its homolog, and the lpxL1 gene or its homolog preferably encodes a protein having at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 5.

[0129] Furthermore, or alternatively, OMV can be obtained from Gram-negative bacteria, which contain one or more genetic mutations that cause the bacteria to produce LPS with reduced (endo)toxicity, preferably the modification increases the expression of at least one of the lpxP, lpxA, lpxD, lpxE, lpxF, and pagL genes, or their homologs. Preferably the modification increases the expression of at least one of the heterologous lpxP, lpxA, lpxD, lpxE, lpxF, and PagL genes.

[0130] Preferably, the Gram-negative bacteria have a genetic modification that introduces or increases the expression of the lpxP gene or its homolog, the lpxP gene or its homolog preferably encodes a protein having at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 6.

[0131] Preferably, the Gram-negative bacteria have a genetic modification that introduces or increases the expression of the lpxA gene or its homolog, the lpxA gene or its homolog preferably encodes a protein having at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 7.

[0132] Preferably, the Gram-negative bacteria have a genetic modification that introduces or increases the expression of the lpxD gene or its homolog, the lpxD gene or its homolog preferably encodes a protein having at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 8.

[0133] Where necessary, the modified Gram-negative bacteria are modified to reduce or eliminate the expression of at least one of the endogenous lpxP, lpxA, lpxD, lpxE, lpxF, and pagL genes.

[0134] Gram-negative bacteria from which the OMV of the complex of the present invention can be obtained may include genetic modifications that reduce or eliminate the expression of genes encoding anchor proteins between the outer membrane and peptidoglycan in order to increase vesicle formation and thereby increase OMV yield.

[0135] Suitable gene modifications for this purpose include, for example, reducing or eliminating the expression of the OmpA homolog commonly found in Gram-negative bacteria, such as the RmpM protein in the genus Neisseria (Steeghs et al., 2002 Cell Microbiol, 4:599-611; van de Waterbeemd et al., 2010 Vaccine, 28:4810-4816). Therefore, preferably, the gene modification reduces or eliminates the expression of the ompA gene or its homolog, more preferably the rmpM gene or its homolog. Preferably, Gram-negative bacteria have a genetic modification that reduces or eliminates the expression of the rmpM gene or its homolog, the rmpM gene or its homolog preferably encoding a protein having at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 9. Eliminating RmpM expression preferably increases OMV release.

[0136] In one embodiment, a Gram-negative bacterium for the production of the complex OMV as defined herein includes a mutation that results in the retention of Prn93 (partactin of 93 kDa) in the outer membrane.

[0137] Gram-negative bacterial host cells for producing the OMV of the complex of the present invention may further have one or more genetic modifications that reduce or eliminate the expression of genes selected from the group consisting of cps, ctrA, ctrB, ctrC, ctrD, exbB, exbD, frpB, galE, htrB, msbB, lpbB, lpxK, lpxL1, nmb0033, opA, opC, rmpM, phoP, pilC, pmrE, pmrF, porA, porB, siaA, siaB, siaC, siaD, synA, synB, sync, tbpA, and tbpB, or their homologs, preferably cps and porB or their homologs. Many of these mutations are outlined in International Publication No. 02 / 09746.

[0138] The reduction in expression is preferably a reduction in expression compared to a bacterial host cell that is otherwise identical and does not involve genetic modification. Preferably, the genetic modification as defined herein reduces expression by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 100%. A 100% reduction is understood herein as loss of expression.

[0139] Gram-negative bacteria may include genetic modifications at the cps locus that preferably reduce or eliminate the expression of genes located at the cps locus, for example, the genes specified in Tables 2 and 3 of Harrison et al (Emerg Infect Dis. 2013 Apr; 19(4): 566-573), which are incorporated herein by reference. Preferably, the genetic modification at the cps locus results in a reduction or elimination of at least siaD expression.

[0140] Preferably, Gram-negative bacterial host cells for producing the OMV of the complex of the present invention may have one or more genetic modifications that reduce or eliminate the expression of a gene selected from the group consisting of lpxL1, porA, porB, rmpM, and siaD. Preferably, Gram-negative bacterial host cells for producing the OMV of the complex of the present invention may have one or more genetic modifications that reduce or eliminate the expression of a gene selected from the group consisting of lpxL1, porA, rmpM, and siaD.

[0141] Preferably, Gram-negative bacteria have genetic modifications that reduce or eliminate the expression of the siaD gene or its homolog, the siaD gene or its homolog preferably encoding a protein having at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 38. The reduction, preferably deletion, of siaD expression preferably results in the removal of capsular polysaccharides, thereby reducing the invasiveness of the bacteria.

[0142] Preferably, Gram-negative bacteria have a genetic modification that reduces or eliminates the expression of the porA gene or its homolog, the porA gene or its homolog preferably encodes a protein having at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 39.

[0143] Preferably, the Gram-negative bacteria have a genetic modification that reduces or eliminates the expression of the porB gene or its homolog, and the porB gene or its homolog preferably encodes a protein having at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 40.

[0144] Furthermore, or alternatively, Gram-negative bacteria for OMV production may include mutations that reduce or eliminate the toxicity of pertussis toxin (Ptx).

[0145] Gram-negative bacteria for producing OMVs that form part of the complex as defined herein preferably belong to a genera selected from the group consisting of Neisseria, Bordetella, Escherichia, and Salmonella. Preferred Neisseria species are at least one of Neisseria meningitidis, Neisseria gonorrhoeae, or Neisseria lactamica. Preferred Bordetella species are at least one of Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica. Preferably, the host cell is a bacterial host cell belonging to a species selected from the group consisting of Neisseria meningitidis, Bordetella pertussis, Escherichia coli, and Salmonella enterica. Preferred Neisseria meningitidis serotypes are serotypes A, B, C, W135, X, and Y, preferably serotype B. The preferred Neisseria meningitidis strain is H44 / 76.

[0146] Preferred Gram-negative bacterial cells for OMV production are Neisseria or Bordetella cells, as specified in the Examples section herein.

[0147] Preferably, the OMV-producing cells are Neisseria meningitidis having a mutation in at least one of the porB, rmpM, and lpxL1 genes. Preferably, the OMV-producing cells are Neisseria meningitidis having a mutation in at least one of the porA, rmpM, and lpxL1 genes. Preferably, the OMV-producing cells are Neisseria meningitidis having a mutation in at least one of the porB, rmpM, lpxL1, and cps genes. Preferably, the OMV-producing cells are Neisseria meningitidis having a mutation in at least one of the porA, rmpM, lpxL1, and cps genes. Preferably, the OMV-producing cells are Neisseria meningitidis having a mutation in at least one of the porA, rmpM, lpxL1, and siaD genes. Preferably, the OMV-producing cells are Neisseria meningitidis having a mutation in at least one of the porB, rmpM, LpxL1, and siaD genes.

[0148] In further embodiments, the present invention relates to a pharmaceutical composition comprising a complex as defined herein and a pharmaceutically acceptable excipient. The composition preferably comprises a pharmaceutically acceptable carrier, medium, or delivery vehicle conventionally known in the art (see, for example, "Handbook of Pharmaceutical Excipients", Rowe et al eds. 7th edition, 2012, www.pharmpress.com). Pharmaceutically acceptable stabilizers, osmotic agents, buffers, dispersants, etc., may also be incorporated into the pharmaceutical composition. The preferred form depends on the intended mode of administration and therapeutic use. The pharmaceutically acceptable carrier may be any suitable and non-toxic substance for delivering the active ingredient, i.e., the complex of the present invention, to the patient. Pharmaceutically acceptable carriers for parenteral delivery are exemplified by sterile buffered 0.9% NaCl or 5% glucose supplemented with 20% albumin as needed. Alternatively, the OMV containing the AMP-antigen conjugate may be suspended in phosphate-buffered saline (PBS). Preparations for parenteral administration must be sterile. The parenteral administration routes of the OMV complex of the present invention are consistent with known methods, for example, injection or infusion via intravenous, intraperitoneal, intramuscular, intranasal, intra-arterial, or intrafocal routes.

[0149] The OMV complex is preferably administered intranasally. In this embodiment, the pharmaceutical composition containing the OMV complex is preferably suited for intranasal administration. Intranasal or intranasal administration is understood herein as a route of administration in which the formulation is preferably blown through the nose. The composition containing the OMV complex of the present invention may be sprayed or dropped into at least one nostril, preferably both nostrils. The composition may be administered using a nasal dropper as defined herein below.

[0150] The complex or pharmaceutical composition may be administered sequentially by infusion or bolus injection. A typical pharmaceutical composition for intramuscular injection may be prepared, for example, to contain 1 to 10 ml of phosphate-buffered saline containing an effective dose of the OMV complex of the present invention. Methods for preparing parenterally administered compositions are well known in the art and are described in more detail in various sources, including, for example, "Remington: The Science and Practice of Pharmacy" (Ed. Allen, LV 22nd edition, 2012, www.pharmpress.com). The pharmaceutical composition is preferably a vaccine, preferably a cell-free vaccine.

[0151] The composition may include, for example, one or more additional adjuvants to further boost the immune response. The adjuvants may be organic or inorganic. Preferred inorganic adjuvants are aluminum salts, e.g., aluminum phosphate and aluminum hydroxide, but are not limited to these. Preferred organic adjuvants may be modified LPS, preferably modified Neisserial or Bordetella LPS, modified LOS, squalene, QS21, or monophosphoryl lipid A (MPL). Adjuvants may be selected from the group consisting of alum, aluminum hydroxide, aluminum phosphate, calcium hydroxide phosphate, paraffin oil, squalene, surfactants (e.g., Quil A), (plant) saponins, cytokines (e.g., IL-1, IL-2, or IL-12), complete Freund's adjuvants, and incomplete Freund's adjuvants. The use of specific adjuvants, the relative and absolute amounts of substances in the composition, and the dosage regimens for administration are known or determinable by those skilled in the art and can be adapted to the circumstances, such as the specific pathogen infection or persistent condition of the particular target being treated. Dosage regimens may include single doses, but may also include multiple doses, such as booster doses, and can be administered orally, intranasally, or parenterally, preferably intranasally or intramuscularly. Various dosage regimens for vaccination are known in the art and can be suitably adapted by those skilled in the art.

[0152] In some embodiments, the present invention relates to a complex or pharmaceutical composition as defined herein for use as a pharmaceutical. In other words, the present invention relates to the use as a pharmaceutical of at least one of the complexes and pharmaceutical compositions of the present invention. The present invention further relates to a method of treatment using at least one of the OMV complexes and pharmaceutical compositions as defined herein.

[0153] In another aspect, the present invention relates to the complex of the present invention or a pharmaceutical composition comprising the complex for the prevention or treatment of diseases, preferably infectious diseases, or tumors, related to the antigens defined herein above. Thus, in this aspect, the present invention relates to a method for vaccination against diseases, preferably infectious diseases, or tumors, or for the prevention or treatment thereof, by administering a therapeutic or prophylactic amount of the complex of the present invention (or a pharmaceutical composition comprising the complex of the present invention) to a subject in need of prevention or therapy. The present invention also relates to the use of the complex or a pharmaceutical composition as a pharmaceutical, preferably for vaccination against diseases, preferably infectious diseases, or tumors, or for the prevention or treatment thereof. In a further aspect, the present invention relates to the complex or a pharmaceutical composition defined herein for use in a treatment comprising inducing or stimulating an immune response in a subject to an antigen. Preferably, the treatment is for the prevention or treatment of infectious diseases or tumors related to the antigen present in the complex of the present invention, and the antigen is preferably the antigen defined herein above.

[0154] In some embodiments, the present invention relates to a complex as defined herein for use in immunotherapy. Preferably, the immunotherapy is for cancer or neurodegenerative diseases, including, for example, Alzheimer's disease or Parkinson's disease.

[0155] In some embodiments, the complex of the present invention is intended for use in preventing and / or suppressing the spread of infections such as bacterial or viral infections. The complex of the present invention can be used as a vaccine, for example, a vaccine against SARS-CoV-2, for example, but not limited to SARS-CoV-2.

[0156] In further embodiments, the present invention relates to an AMP as defined herein conjugated to an antigen as defined herein. Preferably, the AMP is covalently bound to the antigen using a linker, if necessary. The linker may be a linker as defined herein. The AMP conjugated to the antigen is preferably a single polypeptide. Thus, the present invention also relates to a fusion protein in which the first fusion partner is an AMP, preferably an AMP as defined herein, and the second fusion partner is preferably an antigen, preferably an antigen as defined herein.

[0157] The present invention further relates to recombinant host cells expressing AMP as defined herein. The same host cells may further express antigens, preferably antigens as defined herein. The host cells may be the host cells defined above herein. AMP and antigens may be part of a single fusion protein as defined herein.

[0158] The present invention further relates to a combination of a nucleic acid encoding AMP and a nucleic acid encoding an antigen. The combination of nucleic acids may be part of a single nucleic acid construct. The nucleic acid may further comprise one or more regulatory elements that control the expression of AMP and the antigen. AMP and the antigen may be part of a single fusion protein as defined herein. Means and methods for constructing expression constructs for protein expression in host cells as defined herein are generally well known in the art.

[0159] The present invention also relates to a method for producing a complex as defined herein. Preferably, the method comprises the steps of: i) culturing a population of Gram-negative bacteria as defined herein under conditions favorable for the production of OMV; ii) recovering the OMV produced in i); iii) contacting the OMV recovered in ii) with AMP conjugated to an antigen as defined herein under conditions favorable for the formation of a non-covalent complex between AMP and OMV; and vi) recovering the complex, if necessary. The production and purification / extraction of OMV can be carried out using any suitable method known in the art. Similarly, the production and purification / extraction of AMP / antigen / fusion protein can also be carried out using any suitable method known in the art. The method for producing the OMV of the complex of the present invention is more preferably a surfactant-free method as defined and described above herein.

[0160] In one embodiment, the present invention relates to a combination of OMV and AMP, where AMP is conjugated to an antigen.

[0161] In further embodiments, the present invention relates to a kit of parts comprising one vial containing an OMV, preferably an OMV as defined herein, and preferably an AMP conjugated to an antigen as defined herein. The kit may further comprise, for example, a second vial containing a pharmaceutical buffer. Alternatively or further, the kit of parts may comprise one vial containing an OMV, preferably an OMV as defined herein, and a second vial containing preferably an AMP conjugated to an antigen as defined herein. Alternatively or further, the kit of parts may comprise one vial containing an OMV, preferably an OMV as defined herein, one vial containing an AMP, preferably an AMP as defined herein, and one vial containing an antigen, preferably an antigen as defined herein.

[0162] Preferably, the volume of the vials in the kit does not exceed 100 mL, 50 mL, 20 mL, 10 mL, 5 mL, 4 mL, 3 mL, 2 mL, or 1 mL.

[0163] The reagents may be present in lyophilized form or in a suitable buffer. The kit may also contain any other components necessary to carry out the present invention, such as buffer, pipette, microtiter plate, injection needle and / or written instructions for use. Such other components of the kit of the present invention are known to those skilled in the art.

[0164] It is further understood that the use of the compositions in the treatment of medical conditions specified herein also includes the use of the compositions for the manufacture of pharmaceuticals for the corresponding medical treatments, and for methods of treating subjects suffering from such medical conditions by administering an effective amount of the compositions to the subject.

[0165] In a further embodiment, the present invention relates to a nasal spray bottle comprising a container for the composition of the present invention. The nasal spray bottle may be any nasal spray bottle described in the Art. Typically, the nasal spray bottle may comprise a pipette (with both ends open), a compression sphere, a container for holding the liquid, and a bottle cap. One end of the pipette is preferably placed in the container, and the compression sphere is preferably attached to the other end of the pipette. The free end of the pipette is held in the container containing the composition of the present invention, and when the compression sphere is compressed, the air inside the sphere is released into the container. Then, when the pressure on the compression sphere is released, the elasticity of the sphere causes it to return to its initial volume, creating a vacuum inside the sphere and filling the pipette with the composition of the present invention. By compressing the sphere again, preferably droplets of the composition can be released from the pipette. The pipette is usually attached to a bottle cap, and is usually attached in a sealing relationship to the center of the cap. A bottle cap with a pipette can be screwed onto a container, thereby creating an airtight liquid container capable of preventing liquid overflow.

[0166] In yet another embodiment, the present invention provides a nasal spray comprising a bottle or equivalent container containing the composition of the present invention. The nasal spray may be any nasal spray described in the prior art. Typically, the nasal spray comprises a bottle containing the composition of the present invention. The bottle more preferably comprises a portion for administering the composition into the nostrils. In this case, the solution may, in one embodiment, be ejected into the nostrils by any suitable means, for example, by a pump, by deformation of the bottle, or by using a suitable spraying agent.

[0167] In this document and its claims, the verb “comprise” and its conjugations are used in a non-restrictive sense, meaning that the items following the word are included, but items not specifically mentioned are not excluded.

[0168] All patents and references cited herein are thus incorporated herein by reference in their entirety.

[0169] The following examples are provided for illustrative purposes only and are not intended in any way to limit the scope of the invention. [Brief explanation of the drawing]

[0170] [Figure 1] An illustrative schematic diagram of one embodiment of the present invention. A) OMV and antigen are prepared separately and tagged together using AMP. B) OMV, mCRAMP and exemplary antigen (EV71 VP1 or partactin). [Figure 2-1] A) Dot blot: Association of partactin and OMV via linker mCRAMP or LL37. B) Dot blot: Binding of PRN and OMV from three different bacterial sources using mCRAMP linker. C) Quantitative analysis of PRN binding to OMV. [Figure 2-2] (As stated above.) [Figure 3]Total IgG antibody titer against EV71 VP1 protein. Mice were immunized with peptide and protein-based vaccine candidates on day 0 and day 28. Serum was collected on day 42 and tested for the presence of IgG antibodies against EV71 VP1 protein. The symbols shown represent the antibody titer in the serum of individual mice. [Figure 4] Antibody response in mice immunized with EV71 vaccine candidates. Mice were immunized with peptide and protein-based vaccine candidates on day 0 and day 28. Serum was collected on day 42 and tested for the presence of (A)IgG1 and (B)IgG2A antibodies against the EV71 VP1 protein. Serum was pooled from 5 mice out of a total of 10 mice per group. Data are expressed as mean ± SD. Results are from two consecutive sets of 2 pooled serum samples per group. [Figure 5] Total IgG antibody titers against the EV71 virus C4 genotype. Mice were immunized with peptide and protein-based vaccine candidates on day 0 and day 28. Serum was collected on day 42 and tested for the presence of IgG antibodies against the EV71 virus. The symbols shown represent the antibody titers in the serum of individual mice. [Figure 6] Serum anti-Prn antibody titers. Individual titers and their mean ± standard deviation are shown. * = Statistically significant difference compared to the placebo group. [Figure 7] Intranasal (A) and intramuscular (B) vaccination with OMV-spike potently induces the ability of mouse serum to neutralize SARS-CoV-2. VNT = viral neutralizing titer, in = intranasal, and im = intramuscular. [Figure 8] A) Intranasal vaccination using OMV-Spike strongly induces the ability of hamster serum to neutralize SARS-CoV-2, and B) vaccinated hamsters develop very few lung lesions after SARS-CoV-2 attacks. [Figure 9]A) Intramuscular (im) vaccination using OMV-Spike is not as efficient as intranasal (in) vaccination, but it does induce the ability of hamster serum to neutralize SARS-CoV-2, and B) vaccinated hamsters develop little to no lung lesions after SARS-CoV-2 attack. [Examples]

[0171] [Example 1] The virulence factor pertactin (PRN) from Bordetella pertussis was coupled to the human antimicrobial peptide LL-37 (SEQ ID NO: 22), or its mouse variant called mCRAMP (SEQ ID NO: 20). The coupled peptide was expected to bind PRN to OMV after simple mixing. As control proteins, PRN alone (SEQ ID NO: 19) and PRN coupled to a scrambled form of mCRAMP (SEQ ID NO: 21), which should not bind to OMV, were used. All proteins were provided with a His tag and produced as recombinant proteins. The OMV used was from Neisseria meningitidis (ΔPorB ΔRmpM ΔlpxL1 Δcps).

[0172] material and method Dot blot stock p69 0.35 mg / ml P69 mCRAMP 0.54 mg / ml p69 Scrambled mCRAMP 1.19 mg / ml OMV (MenB) 1.23 mg / ml

[0173] dot blot Two 1.5 μl dots were placed on nitrocellulose sections: one dot for PRN, PRN-LL37, PRN-mCRAMP, or PRN-Scrambled mCRAMP, and one dot for OMV. The nitrocellulose sections were then washed three times for 5 minutes each with 1 ml of Wst buffer (0.1 M Tris, 1.54 M NaCl, 5% Tween-80, pH=7.4). Next, the nitrocellulose sections were incubated with 5 μl of the same protein as the first dot. The staining procedure consisted of washing with Wst buffer, incubation with anti-his Ab in Wst buffer, washing with Wst buffer, incubation in wst-0.5% with anti-mouse IgG-AP, washing with Wst buffer, washing with MiliQ, incubation with AP mix, and re-washing with MiliQ. The amount of bound protein was determined using CLIQS software, in combination with a Bio-Rad dot blot instrument as needed. To determine the amount of OMV-binding protein, the intensity of stained dots was compared to the dilution series of control protein and OMV using a standard procedure.

[0174] result Dot blotting was used to demonstrate binding of other mCRAMP / LL-37 fusion proteins. Figures 2A and 2B show that PRN linked to LL-37 or mCRAMP binds to OMV, but PRN alone or PRN linked to scrambled mCRAMP does not. Figure 2C shows a strong correlation between the amount of OMV present in the dot blot and the amount of bound PRN-mCRAMP.

[0175] [Example 2] The inventors evaluated the induction of (neutralizing) antibodies in response to an antigen derived from enterovirus-71 bound to OMV. The antigen was produced together with C-terminally tagged LL-37 or a mouse ortholog of the human antimicrobial peptide LL-37 (mCRAMP). These proteins or peptides were individually combined with purified OMV to form OMV-antigen complexes.

[0176] material and method Outer membrane vesicles (Nonamen) Natural meningococcal OMV vaccines have been previously developed by the Dutch Vaccine Institute (NVI) / Institute for Translational Vaccineology (Intravacc) and consist of OMV derived from three meningococcal strains that have been engineered for advanced bleb formation (rmpM mutation), LPS toxicity removal (lpxL1 mutation), capsule loss (deletion of the entire locus), and PorB loss (gene deletion), as well as the expression of three different porA genes per strain. In this experiment, OMV from one strain (expressing PorA subtypes 14, 1, and 3) was used as the carrier.

[0177] antigen target The EV71 virus was evaluated as the first candidate. The linear epitopes of the EV71 viral proteins (VP1 and VP2) are well described in the literature. EV71 is the leading cause of hand, foot, and mouth disease (HFMD) and is a major problem in Asia. EV71 particles consist of a single RNA molecule protected by four viral capsid proteins VP1-VP4, of which VP1 contains many neutralizing epitopes and acts as the major immunogenic capsid protein; therefore, EV71-VP1 is an ideal target for vaccine development.

[0178] EV71 virus protein 1 This study investigated the feasibility of using OMV as a platform for viral vaccine development by coupling the complete EV71-C4 VP1 protein (NCBI accession number JN256062) with OMV. VP1 was ligated at the N-terminus to a 6×HIS tag for purification (SEQ ID NO: 23), and a human antimicrobial peptide (LL-37) or a mouse antimicrobial peptide (mCRAMP) was ligated at the C-terminus. The sequences of the recombinant proteins are shown in SEQ ID NO: 24 (VP1-mCRAMP) and SEQ ID NO: 25 (VP1-LL37), respectively. The complete protein is thought to associate with OMV via the LL-37 or mCRAMP ligated at the C-terminus. Protein expression was evaluated in 293 cells (mammalian expression) and Escherichia coli (E. coli).

[0179] The HIS-VP1-LL-37 protein was successfully produced by 293-6E cells. An estimated molecular weight of approximately 50 kDa was detected in cell culture supernatant and cell debris by Western blot analysis under reducing conditions (data not shown). The expression level of LL-37 was approximately 0.1–0.5 mg / L. Higher yields of the HIS-VP1-LL37 protein were achieved by expression in Escherichia coli (E. coli). The protein was obtained from inclusion bodies after denaturation and subsequent one-step purification using a Ni column. Approximately 70–85% pure protein at around 0.14–0.20 mg / ml was recovered from a 1-liter scale.

[0180] peptide Several papers have described linear peptide epitopes (1-3, 5) derived from EV71's VP1 and VP2 that induce antibodies after immunization in mice. Antibodies that recognize these selected peptides can neutralize the virus in vitro. Since several EV71 genotypes are publicly known, the inventors made a selection. For this purpose, the C4 and B4 genotypes have been the most prevalent in outbreaks that have occurred in the past decade. Table 1 shows the differences in peptide sequences between the C4 and B4 genotypes of the linear epitopes, along with the peptides used in this study.

[0181] [Table 1]

[0182] We developed various forms of these peptides combined with terminal cysteine, GS linker, His tag, mCRAMP, and / or LL37. All peptides (Table 1) were synthesized using in vitro synthesis (Pepscan, Lelystad, The Netherlands).

[0183] The peptide was associated with OMV via the LL37 (or mCRAMP) sequence. The ability of the peptide and VP1 protein to induce neutralizing antibodies after two immunizations (in combination with OMV or in the absence of OMV) was evaluated in mice.

[0184] Mouse model AC57BL / 6 mice were immunized with a panel of click-OMV vaccines. Ten mice in each group were vaccinated twice with each of the constructed vaccines, and the positive control group was immunized with inactivated EV71 virus. The vaccines (excluding the positive control) were mixed in PBS and kept at 37°C overnight. The following day (approximately 18 hours), all mice were immunized. This immunization was repeated after 4 weeks. Two weeks after the second immunization, the mice were sacrificed, and serum was collected from all mice. See Table 2 for the vaccination scheme and experimental setup.

[0185] [Table 2]

[0186] result Antibody levels against EV71 VP1 protein To determine whether immunized mice produced antibodies against the antigen, initial ELISA was performed on pooled serum against the EV71 VP1 protein and OMV present in the vaccine. High IgG titers against OMV were detected only in the group immunized with OMV (data not shown). Antibodies against the EV71 VP1 protein were also detected (data not shown). ELISA using EV71 VP1 protein coating was repeated using individual mouse serum (Figure 3). The total IgG response to the EV71 VP1 protein indicated that the negative group (PBS and OMV) did not produce IgG antibodies against EV71. The positive group (inactivated EV71) clearly induced IgG antibodies. Linking the protein to OMV in the presence of mCRAMP showed increased VP1-specific antibody production compared to an unlinked protein-OMV mixture. This increase was reduced when mice were immunized with a smaller amount of protein-linked OMV.

[0187] The levels of IgG1 and IgG2A, specific IgG subclasses against VP1, were determined by ELISA for further insight into the type of immune response induced. Typically, changes in the IgG2A to IgG1 ratio indicate a shift towards a more Th1-like response. In most groups, changes in subclass antibody titers were absent, except in mice immunized with VP1 protein linked to OMV by mCRAMP. In these groups, an increase in the IgG2A:IgG1 ratio was observed (Figure 4A+B).

[0188] Antibody levels against EV71 virus (C4 genotype) ELISA plates were coated with complete EV71 virus, and ELISA was performed to determine the amount of virus-specific antibodies in the serum. The results shown in Figure 5 confirm that antibodies against the virus were produced, and the same overall pattern of antibody response was observed. The highest titer was induced by the VP1 mCRAMP protein-OMV vaccine. Therefore, an increase in the antibody response against EV71 virus in mice can be induced by protein or peptide click-OMV vaccines.

[0189] conclusion - Peptides or proteins bound to OMVs enhance the antibody response in mice. - VP1(EV71) protein bound to OMV via mCRAMP induces a bias toward the Th1 response.

[0190] Therefore, this animal study demonstrates that coupling the EV71 antigen to the OMV platform increases the antibody response to the EV71 antigen and virus. VP1 protein linked via the antimicrobial peptide mCRAMP increased antibody production against VP1 protein and live virus compared to the unbound VP1-OMV vaccine.

[0191] [Example 3] The inventors investigated whether the immunogenicity of Prn can be enhanced by linking Prn to OMV via a linker peptide. Mice were immunized twice with the antigen alone, with the antigen mixed with OMV, or with the antigen coupled to OMV. Subsequently, antibody levels against the antigen in the serum were measured. Two different coupling peptides, namely mouse mCRAMP and human LL-37 peptide, were used for Prn coupled to Neisseria meningitidis (OMV).

[0192] material and method Administration of research substances The vaccine was administered to the groin area of ​​mice by sc injection (total volume 200 μL). Both vaccinations were administered on the right side using a needle and syringe.

[0193] Blood sample collection On day 42, blood was collected via the retinal artery during euthanasia into individually labeled tubes. Blood samples were left at room temperature for at least 30 minutes (but no more than 24 hours), and then centrifuged at 3500 rpm in an Eppendorf centrifuge at room temperature or at 3000 rpm for 15 minutes in an SL 40R centrifuge at room temperature, depending on the size of the tube. Serum was transferred to individually labeled tubes and stored below -20°C until analysis.

[0194] Analysis of anti-Prn antibody titers Serum levels of anti-Prn antibodies were measured using multiplex flow cytometry immunoassay.

[0195] Statistical analysis of the results Statistical significance testing between groups was performed for anti-Prn antibody titers. To detect potential differences between groups, the experimental group was compared to the placebo-treated group using the Kruskal-Wallis test and Dunn's test, and significant differences between means were determined. To detect potential differences between the group treated with OMV mixed with the antigen and the group treated with OMV coupled to the antigen, the Mann-Whitney U test was used, and the resulting p-values ​​were corrected for multiple testing using the Benjamini-Hochberg method. Statistical analysis was not performed on FACS data. All results were considered significant if p < 0.05.

[0196] result Serum anti-Prn titers Administration of Prn protein without OMV did not induce anti-Prn IgG (Figure 6). When Prn protein was administered mixed with OMV or coupled to OMV via mCRAMP, anti-Prn IgG levels were significantly elevated, indicating that the coupling method did not affect the immunogenicity of the antigen.

[0197] conclusion Mice were immunized with Bordetella pertussis antigen alone, mixed with OMV, or coupled to OMV. Administration of Prn protein with OMV already induced the production of anti-Prn IgG. Coupling of Prn with OMV via mCRAMP did not result in a further increase in anti-Prn antibody levels compared to Prn mixed with OMV. This indicates that the addition of Neisseria meningitidis OMV to Prn itself already increases the immunogenicity of Prn. This also indicates that the coupling method does not negatively interfere with the immunogenicity of the antigen.

[0198] [Example 4] As the antigen, we used a pre-fusion state SARS-CoV-2 spike protein with six proline substitutions, based on the HexaPro spike protein described in the paper by Hsieh et al (2020). An mCRAMP sequence was added to the C-terminus. The mCRAMP sequence enables spontaneous association between the spike protein and OMV after mixing. The immunogenicity of this SARS-CoV-2 vaccine concept was tested in mouse models after administration via the intranasal route and, for comparison, the intramuscular route. Mouse models provide good readout for the immunogenicity of these OMV vaccines. Syrian hamster models were used to measure protection. Various animal models, including Syrian hamsters, have been tested to date for studying SARS-CoV-2 infection. Using the results of SARS-CoV-2 model development studies, we defined the attack infection protocol in this study regarding the attack route, dose, and post-attack follow-up, and specified the selection of the Syrian hamster model to establish the efficacy of the novel SARS-CoV-2 vaccine candidate. In this case as well, both the intranasal and intramuscular pathways were compared.

[0199] Methods (mouse study) immunization BALB / c mice were immunized on days 0 and 21 via either the intranasal (in) or intramuscular (im) pathway. Blood was collected on days 0, 21, and 35 to evaluate the induction of SARS-CoV-2 specific neutralizing antibodies. Each group consisted of 10 mice.

[0200] The following groups were included: 1. Trissucrose in 2. OMV in 3. Spikes in 4. Spike mCRAMP in 5. OMV+ spikes in 6. OMV+ Spike mCRAMP in 7. Trissucrose im 8. Spikes IM 9. Spike mCRAMP im 10. OMV+ Spikes im 11. OMV+ Spike mCRAMP im

[0201] For intranasal immunization, 20 μl of the inoculum was divided into both nostrils using a pipette. For intramuscular immunization, 50 μl of the inoculum was injected into the lateral thigh.

[0202] The OMV dose used was 15 μg of protein per immunization. The spike and spike mCRAMP doses used were also 15 μg of protein per immunization.

[0203] OMV was isolated by EDTA extraction as described by van de Waterbeemd et al (2013). The spike protein was expressed in ExpiCHO-S cells and purified using a Twin-Strep column.

[0204] Serological analysis The virus neutralization (VN) assay was performed on samples collected during the study as follows. Briefly, the samples were heat-inactivated at 56°C for 30 minutes. Subsequently, serial 2-fold dilutions of the samples were prepared in triples in 96-well plates, starting with a 1:5 dilution ratio in infection medium. The sample dilutions were then incubated with a fixed amount of virus (200 TCID50 / well or 4000 TCID50 / ml) at 37°C for 1 hour to obtain the 1:10 starting dilution ratio of serum used in the assay. Next, the virus-antibody mixture was transferred to plates containing a Vero E6 cell culture monolayer, followed by an incubation period of 5-6 days at 37°C. The plates were then scored using the vitality marker WST8.

[0205] Results (mouse study) Viral neutralization titers were detected only in groups administered OMV in combination with either the spike protein or the spike mCRAMP protein, the latter group exhibiting the highest titers and the largest number of responders. Titers were not detected in groups administered with either the spike protein or spike mCRAMP alone. Overall results were similar after immunization via the in and im pathways (Figure 7).

[0206] Methods (Hamster Research) immunization Syrian hamsters were immunized on days 0 and 21 via intranasal (in) or intramuscular (im) pathways. During the study, animal body weight was measured and blood was collected to assess the induction of SARS-CoV-2 specific neutralizing antibodies. Three weeks after the second immunization (day 42), all animals were intranasally attacked with SARS-CoV-2 strain BetaCoV / Munich / BavPat1 / 2020 at a dose of 10^4.0 TCID50.

[0207] The following groups were included: 1. Trissucrose in 2. OMV in 3. Spikes in 4. Spike mCRAMP in 5. OMV+ spikes in 6. OMV+ Spike mCRAMP in 7. Trissucrose im 8. OMV im 9. Spikes im 10. Spike mCRAMP im 11. OMV+ Spikes im 12. OMV+ Spike mCRAMP im

[0208] Four days after the attack, half of the animals in each group were slaughtered by bleeding under isoflurane anesthesia and autopsies were performed, with the remaining half being slaughtered seven days after the attack.

[0209] pathology Macroscopic examination was performed at autopsy. All lung lobes were examined, the percentage of affected lung tissue was estimated dorsally, the macroscopic diagnosis was recorded, and the left lung lobe was inflated with 10% formalin and preserved. The trachea and turbinates were evaluated macroscopically and sampled for virology and histopathology. Relative lung weight was calculated. Histopathological analysis of selected tissues was performed on all animals. After fixation with 10% formalin, sections of the left lung and left turbinate, as well as sections of gastrointestinal tissue, were embedded in paraffin, and the tissue samples were stained for histological examination. Histopathological evaluation included aspects such as congestion, emphysema, presence of foreign bodies, hemorrhage, bronchioloalveolar hyperplasia and inflammation, and edema. Viral titers in the confluent layer of Vero E6 cells were determined using four 10-fold serial dilutions. For this purpose, serial dilutions of the samples (pharyngeal swabs and tissue homogenates) were prepared and incubated on Vero E6 monolayers at 37°C for 1 hour. The monolayers were washed and incubated at 37°C for 5 or 6 days, and scored for CPE using the survival marker WST8. Viral RNA was detected by PCR using pharyngeal swab and homogenized tissue samples. Viral neutralizing titers were determined as described above for mouse serum.

[0210] Results (Hamster Research) Viral neutralization titers were primarily detected in groups administered OMV in combination with either the spike protein or the spike mCRAMP protein. The OMV+spike mCRAMP group showed higher titers than the OMV+spike group. No titers were detected in groups administered OMV or spike alone, and only low titers were detected in some mice in the spike mCRAMP group without OMV. Post-SARS-CoV-2 attack, lung lesions were hardly detected in the OMV+spike and OMV+spike mCRAMP groups. Overall results were similar after immunization via the in and im pathways (Figures 8 and 9).

[0211] conclusion In both mouse and hamster models, virus-neutralizing antibodies are induced when the spike protein is combined with OMV. In the hamster model, when vaccination was performed with the spike protein combined with OMV, lung lesions were hardly observed after attack. Addition of a C-terminal mCRAMP tag increased the protective response in both models. Overall, these data indicate that (i) Neisseria OMV is an effective adjuvant / delivery system for the Covid-19 spike protein, and (ii) increased OMV association by the mCRAMP tag improves the protective response.

[0212] References

[0213] [Table 3-1]

[0214] [Table 3-2] The inventions described in the original claims of this application are listed below. [Invention 1] A complex comprising an outer membrane vesicle (OMV), a vertebrate antimicrobial peptide (AMP), and an antigen, wherein the AMP is complexed with the OMV by non-covalent bonding, and the antigen is conjugated to the AMP. [Invention 2] The complex according to Invention 1, wherein the antigen is covalently bound to the AMP in a fusion protein containing the antigen and the AMP in a single polypeptide chain. [Invention 3] The complex according to invention 1 or 2, wherein the AMP is a cathelicidine, preferably a non-human cathelicidine, and more preferably mCRAMP. [Invention 4] The complex according to any one of inventions 1 to 3, wherein the antigen is an antigen associated with infectious diseases and / or tumors. [Invention 5] The composite according to any one of Inventions 1 to 4, wherein the OMV is not OMV extracted with a surfactant, and preferably the OMV is naturally occurring OMV or native OMV, and preferably native OMV. [Invention 6] The composite according to any one of inventions 1 to 5, wherein the OMV comprises LPS that has been at least partially detoxified. [Invention 7] The aforementioned OMV can be obtained from Gram-negative bacteria, and the aforementioned Gram-negative bacteria, a) Genetic modifications that cause the bacteria to produce LPS with reduced toxicity, preferably reducing or eliminating the expression of at least one of the lpxL1, lpxL2, lpxA, lpxD, and lpxK genes or their homologs, and / or increasing the expression of at least one of the lpxP, lpxE, lpxF, and pagL genes, and b) Genetic modifications that increase vesicle formation, preferably reducing or eliminating the expression of the ompA gene or its homolog, more preferably the rmpM gene or its homolog. A composite according to any one of inventions 1 to 6, preferably comprising at least one of the above. [Invention 8] The composite according to Invention 7, wherein the Gram-negative bacteria belong to a genus selected from the group consisting of Neisseria, Bordetella, Escherichia, and Salmonella, and preferably the bacteria belong to a genus selected from the group consisting of Neisseria meningitidis, Bordetella pertussis, Escherichia coli, and Salmonella enterica. [Invention 9] A pharmaceutical composition comprising a complex described in any one of Inventions 1 to 8 and a pharmaceutically acceptable excipient. [Invention 10] A complex according to any one of Inventions 1 to 8 or a pharmaceutical composition according to Invention 9, for use as a pharmaceutical. [Invention 11] A complex according to any one of Inventions 1 to 8 or a pharmaceutical composition according to Invention 9, for use in a treatment that includes inducing or stimulating an immune response in a target to an antigen. [Invention 12] A complex for use according to invention 10 or 11, administered intranasally or intramuscularly. [Invention 13] An antigen conjugated to AMP according to any one of Inventions 1 to 4, wherein the antigen conjugated to AMP is a fusion protein according to any one of Inventions 2 to 4. [Invention 14] A nucleic acid encoding a fusion protein as described in any one of inventions 2 to 4. [Invention 15] A host cell expressing a fusion protein according to any one of Inventions 2 to 4, preferably comprising the nucleic acid described in Invention 14. [Invention 16] A method for producing a complex according to any one of inventions 1 to 8, i) The step of culturing a population of Gram-negative bacteria according to Invention 7 or 8 under conditions favorable for OMV production, ii) A step of recovering the OMV produced in i), iii) The step of contacting the OMV recovered in ii) with AMP conjugated to an antigen according to any one of Inventions 1 to 4, under conditions favorable for the formation of a non-covalent complex between the AMP and the OMV, and vi) If necessary, the step of recovering the composite. The method, including the method described above.

Claims

1. A complex comprising an outer membrane vesicle (OMV), a vertebrate antimicrobial peptide (AMP), and an antigen, wherein the AMP is complexed with the OMV by non-covalent bonding, the antigen is conjugated to the AMP, and the AMP is a cathelicidine.

2. The complex according to claim 1, wherein the antigen is covalently bound to the AMP in a fusion protein containing the antigen and the AMP in a single polypeptide chain.

3. The complex according to claim 1 or 2, wherein the cathelicidine is a non-human cathelicidine.

4. The complex according to claim 3, wherein the non-human catelicidine is mCRAMP.

5. The complex according to any one of claims 1 to 4, wherein the antigen is an antigen associated with infectious diseases and / or tumors.

6. The composite according to any one of claims 1 to 5, wherein the OMV is not OMV extracted with a surfactant.

7. The composite according to claim 6, wherein the OMV is a naturally occurring OMV or a native OMV.

8. The composite according to claim 6, wherein the OMV is a native OMV.

9. The composite according to any one of claims 1 to 8, wherein the OMV comprises LPS that has been at least partially detoxified.

10. The complex according to any one of claims 1 to 9, wherein the OMV can be obtained from Gram-negative bacteria.

11. The aforementioned Gram-negative bacteria, a) Genetic modification to cause the bacteria to produce LPS with reduced toxicity, and b) Genetic modification to increase vesicle formation Including at least one of the following: The composite according to claim 10.

12. The gene modification described in a) above is a gene modification that reduces or eliminates the expression of at least one of the lpxL1, lpxL2, lpxA, lpxD, and lpxK genes or their homologs, and / or increases the expression of at least one of the lpxP, lpxE, lpxF, and pagL genes. The gene modification described in b) above is a gene modification that reduces or eliminates the expression of the ompA gene or its homologs. The composite according to claim 11.

13. The complex according to claim 11, wherein the gene modification in b) is a gene modification that reduces or eliminates the expression of the rmpM gene or its homologs.

14. The complex according to any one of claims 11 to 13, wherein the Gram-negative bacteria belong to a genus selected from the group consisting of Neisseria, Bordetella, Escherichia, and Salmonella.

15. The complex according to claim 14, wherein the bacteria belong to a genus selected from the group consisting of Neisseria meningitidis, Bordetella pertussis, Escherichia coli, and Salmonella enterica.

16. A pharmaceutical composition comprising the complex according to any one of claims 1 to 15 and a pharmaceutically acceptable excipient.

17. A complex according to any one of claims 1 to 15 or a pharmaceutical composition according to claim 16, for use as a pharmaceutical.

18. A complex according to any one of claims 1 to 15 or a pharmaceutical composition according to claim 16, for use in a treatment comprising inducing or stimulating an immune response in a target to an antigen.

19. The complex for use according to claim 17 or 18, which is administered intranasally or intramuscularly.

20. An antigen conjugated to a cathelicidin according to any one of claims 1 to 5.

21. The antigen according to claim 20, wherein the antigen conjugated to the cathelicidin is the fusion protein according to any one of claims 2 to 5.

22. A nucleic acid encoding a fusion protein according to any one of claims 2 to 5.

23. A host cell expressing the fusion protein described in any one of claims 2 to 5.

24. The host cell according to claim 23, wherein the host cell contains the nucleic acid described in claim 22.

25. A method for producing the complex according to any one of claims 1 to 15, i) A step of culturing a population of Gram-negative bacteria in the complex according to any one of claims 10 to 15 under conditions favorable for OMV production, ii) the step of recovering the OMV produced in i), and iii) The step of contacting the OMV recovered in ii) with a cathelicidine conjugated with the antigen described in any one of claims 1 to 5, under conditions favorable for the formation of a non-covalent complex between the cathelicidine and the OMV. The method, including the method described above.

26. The above method further, iv) Step of recovering the composite The method according to claim 25, including the method described in claim 25.

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