African swine fever (Asf) virus vaccine

Abstract

The present invention describes immunogenic compositions containing immunogenic polypeptides of African swine fever (ASF) virus, including immunogenic compositions containing antigens other than ASF viral antigens, including antigens that can be used to immunize against pathogens that cause diarrheal disease. Methods of eliciting an immune response using the disclosed immunogenic compositions and methods of treating ASF infection are also described.

Description

[Technical Field]

[0001] This invention generally relates to compositions that induce an immune response to African swine fever (ASF) virus. In particular, this invention relates to immunogenic compositions (e.g., vaccines) comprising an immunogenic polypeptide of the ASF virus. The immunogenic compositions may further contain antigens other than the ASF virus antigen. Methods for inducing an immune response using the immunogenic compositions disclosed herein, and methods for treating ASF infection, are also described herein. [Background technology]

[0002] African swine fever (ASF) is a viral disease of pigs that causes high mortality in domesticated pigs, while being asymptomatic in natural suid reservoir hosts. The disease causes significant economic losses that are unavoidable in the absence of an effective vaccine, and the available methods of controlling the disease are isolation of affected areas and slaughter of infected animals. ASF is caused by the ASF virus (ASFV), a double-stranded DNA virus with a complex molecular structure. It is the only virus belonging to the Asfarviridae family and the only DNA virus transmitted by soft ticks of the arthropod genus Ornithodoros. In Africa, the soft tick Ornithodoros moubata is involved in the silvatic transmission cycle of the virus, while in Europe, O. erraticus is involved. In Europe, wild boars that suffer from acute illness similar to that of domesticated pigs appear to be involved in the transmission cycle.

[0003] The disease caused by this virus was first identified in Kenya in the 1920s. Initially confined to Africa, it spread to Europe in the mid-20th century, and subsequently to South America and the Caribbean. The disease was eradicated from Europe (with the exception of Sardinia) in the 1990s through thorough control and eradication programs. However, in 2007, it reappeared in Africa and the Caucasus region, particularly Georgia, and by 2014 reached the eastern part of the EU. Recent reports indicate increasing infections in EU member states, Poland, the Baltic states, and most recently, Moldova. Because there is no effective vaccine, African Swine Fever (ASF) poses a serious threat to all European countries. The epidemiological complexity of ASF is clearly demonstrated in East and Southern Africa, and genetic characterization of ASFV based on sequence variations in the C-terminal region of the B646L gene encoding the major capsid protein p72 has revealed the existence of 22 genotypes. Recently, a new genotype XXIII has been reported, sharing a common ancestor with genotypes IX and X, including isolates circulating in East African countries and the Republic of Congo. This review summarizes the current state of knowledge regarding ASFV.

[0004] ASFV is a large enveloped virus with an icosahedral morphology and an average diameter of 200 nm. Its viral genome consists of a single molecule of double-stranded DNA closed by linear covalent bonds. The genome length varies between 170 and 190 kbp depending on the isolate, encoding 151 to 167 open reading frames. The ASFV replication cycle primarily takes place in the cytoplasm, but the nucleus also serves as a site for viral DNA synthesis in its early stages. The degradation of the lamina network near the site where the viral genome begins replication, and the redistribution of several nuclear proteins, suggest the existence of a sophisticated mechanism controlling nuclear mechanisms during viral infection.

[0005] Viral gene transcription is highly regulated. Four classes of mRNA, including immediate, early, intermediate, and late-type transcripts, have been identified based on their characteristic accumulation dynamics. Immediate and early-type genes are expressed before the initiation of DNA replication, while intermediate and late-type genes are expressed afterward. The presence of intermediate genes suggests a cascade model of ASFV gene expression regulation. Enzymes necessary for DNA replication are expressed immediately after the virus enters the cytoplasm from a partially uncoated core particle, using enzymes and other factors packaged within the viral particle. Viral morphogenesis takes place in the viral factory, where the major late stages of DNA replication also occur.

[0006] ASFV particles have an icosahedral morphology consisting of several concentric domains: the inner core, formed by the central genome, contains a nucleoid, which is covered by a thick protein layer called the core shell; an inner lipid envelope surrounding the core; and finally, the capsid, which is the outermost layer of the intracellular virion. The extracellular virion has an even outerter envelope, which is acquired when the virus buds from the cell membrane. However, the importance of this envelope is unclear.

[0007] Current approaches to ASF vaccines are broadly divided into two camps. The current approach by the USDA and DHS focuses on improved live vaccines, believing that viral replication within cells is absolutely essential for a defense response. However, cutting-edge prototype vaccines in this category have recently been announced to take "at least eight years from approval to use," raising many safety concerns. Recent studies have demonstrated that highly effective gene-deletion ASF variant vaccines replicate weakly in pigs, but provide protection from lethal exposure in vaccinated animals.

[0008] While promising, there are problems with using attenuated live strains in US vaccines, and currently, primary macrophages are required to culture the vaccine virus. There is a popular belief that China attempted to replicate this vaccine but with negative results, but this has not yet been confirmed.

[0009] The second vaccine, developed at the Pirbright Institute in the UK, was also promising, offering protection from the fatal consequences of ASF infection without restricting viral replication. In short, this two-dose vaccine incorporated eight recombinant adenoviruses expressing eight unique ASF proteins into a single vaccine. Administration of this vaccine appears to yield similar results to previous studies using fewer proteins.

[0010] Subunit vaccines are euthanized products, in contrast to live vaccines. Due to the difficulties associated with clearly defining the targets of protective proteins and producing vaccines that provide broad protection against multiple strains, these vaccines have been largely ignored.

[0011] Therefore, improved treatments for individuals exhibiting clinical symptoms associated with ASF virus infection and methods to prevent the spread of infection remain necessary. [Overview of the Initiative]

[0012] The present invention provides an immunogenic composition containing African swine fever (ASF) virus antigen, particularly as part of a subunit vaccine.

[0013] In embodiments, a method for producing immunogenic polypeptides and / or peptides derived from ASF virus by mixing or co-expressing them with an adjuvant is disclosed. The immunogenic composition may comprise one or more polypeptides and / or adjuvants described herein. For example, the immunogenic composition may comprise other antigens that can be used in immunization against pathogens causing other diseases, such as antigens derived from non-ASF virus pathogens.

[0014] In embodiments, a process for producing polypeptides is disclosed, comprising the step of culturing host cells transformed with nucleic acids described herein under conditions that induce polypeptide expression. In relevant embodiments, ASF virus proteins may be expressed by recombinant technology and used to develop immunogenic compositions comprising recombinant antigenic subunits, where such expressed polypeptides are produced using baculovirus / insect cell methodologies.

[0015] In one embodiment, a process for producing nucleic acids, wherein the nucleic acids encoding proteins or polypeptides derived from the ASF virus are prepared (at least partially) by chemical synthesis. In a related embodiment, the process includes amplifying the nucleic acids using a primer-based amplification method (e.g., PCR).

[0016] In another embodiment, a process for producing a protein complex is disclosed, comprising administering an ASF virus-derived polypeptide or a fragment thereof to a target. In a related embodiment, this process comprises mixing the ASF virus-derived polypeptide with a pharmaceutically acceptable carrier or diluent. In a further related embodiment, the composition may comprise the polypeptides described in SEQ ID NOs: 6 (p30 / p54 fusion protein), 8 (p72 protein), 10 (p30 protein), 12 (p54 protein), and 17 (hemagglutinin protein). In a further related embodiment, the polypeptide composition comprises SEQ ID NOs: 6 and 17.

[0017] In an embodiment, a method of inducing an immunological response in a subject is disclosed, which includes administering the composition of the present disclosure. In a related aspect, the method further includes administering an adjuvant. In a further related aspect, the method includes administering an immunogenic composition to the subject via a local, parenteral or mucosal route.

[0018] In one aspect, the administration may be multiple times, where the first immunogenic composition and the second immunogenic composition are the same. In another aspect, the first immunogenic composition and the second immunogenic composition are different.

[0019] In one aspect, the administration is performed two or more times.

[0020] In an embodiment, a method for treating an infection by ASF virus is disclosed, which includes administering a therapeutically effective amount of the immunogenic composition described herein to a subject that needs it.

[0021] In one aspect, a plurality of therapeutically effective doses of the immunogenic composition are administered to the subject.

[0022] In a related aspect, the method includes mucosal administration of a therapeutically effective amount of a first immunogenic composition containing one or more ASF virus antigens, and local or parenteral administration of a therapeutically effective amount of a second immunogenic composition containing one or more ASF virus antigens.

[0023] In one aspect, a plurality of therapeutically effective doses of the immunogenic composition are administered to the subject. In another aspect, the immunogenic composition contains distinct non-ASF virus antigens.

[0024] In one aspect, the composition contains the ASF virus p30 / p54 fusion protein.

[0025] In a related embodiment, the composition comprises ASF virus hemagglutinin protein. In a further related embodiment, the composition comprises administering a composition comprising ASF virus p30 / p54 fusion protein and ASF virus hemagglutinin protein.

[0026] In one embodiment, the subject is a pig. In a related embodiment, the proteins are administered substantially simultaneously or sequentially.

[0027] These and other embodiments of the disclosed subject matter will be readily apparent to those skilled in the art in the context of this disclosure. Detailed description of the invention [Modes for carrying out the invention]

[0028] Before describing the compositions, methods, and methodologies of the present invention, it should be understood that the present invention is not limited to the specific compositions, methods, and experimental conditions described herein. Furthermore, it should be understood that the terms used herein are solely for the purpose of describing specific embodiments and are not intended to limit the scope of the present invention, as it is limited only to the appended claims.

[0029] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include multiple references unless the context clearly indicates otherwise. For example, a reference to “a nucleic acid” includes one or more nucleic acids and / or compositions of the type described herein, which would be apparent to those skilled in the art by reading this disclosure, etc.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention pertains. Methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, and modifications and alterations will be understood to be included within the spirit and scope of immediate disclosure.

[0031] Where used herein, “about,” “approximately,” “substantially,” and “significantly” are understood by those skilled in the art and vary to some extent depending on the context in which they are used. Where there is a use of a term that is not clear to those skilled in the art when considering the context in which the term is used, “about” and “approximately” mean less than plus or minus 10% of the particular term, and “substantially” and “significantly” mean more than plus or minus 10% of the particular term. In embodiments, a composition “contains,” “comprises,” or “consist essentially of” a particular component or group of components, but a person skilled in the art would understand the latter to mean that the claim is limited to a specified material or process that does not “significantly affect the basic and novel properties” of the claimed invention.

[0032] As used herein, the term "ASF" refers to members of the genus Asfivirus in the family Asfarviridae, Africa; these are African swine fever viruses. The term ASF encompasses strains of all gene groups of the virus. Currently, ASF strains are classified into 24 gene groups (Gx-Gxn) based on the nucleotide sequence of the p72 / B646L gene. The term ASF also includes isolates that have not been characterized at the time of filing.

[0033] The terms “polypeptide” and “protein” refer to polymers of amino acid residues and are not limited to the minimum length of the product. Therefore, peptides, oligopeptides, dimers, polymers, etc., are included in the definition. Both full-length proteins and their fragments are also included in the definition. The terms also include post-expression modifications of the polypeptide, such as glycosylation, acetylation, and phosphorylation. Furthermore, for the purposes of this disclosure, “polypeptide” refers to a protein that includes modifications (generally inherently conserved) to its native sequence, such as deletions, additions, and substitutions, as long as the protein maintains the desired activity. These modifications may be intentional, such as those caused by site-directed mutagenesis, or accidental, such as mutations in the host producing the protein or errors during PCR amplification.

[0034] "Substantially purified" generally refers to isolating a substance (compound, polynucleotide, protein, polypeptide, polypeptide composition) to constitute the majority of the sample in which it is present. Typically, in a sample, substantially purified components make up about 50%, 80-85%, or 90-95% of the sample. Techniques for purifying target polynucleotides and polypeptides are well known in the art and include, for example, ion exchange chromatography, affinity chromatography, and density precipitation.

[0035] "Isolated," when referring to polypeptides, means that the molecule in question is distinct from the whole organism or cell in which it is found in nature, or exists in the substantial absence of other biological macromolecules of the same type. With respect to polynucleotides, the term "isolated" refers to a nucleic acid molecule that lacks all or part of the sequences normally associated in nature, or a sequence that exists in nature but has associated heterologous sequences, or a molecule that has been cleaved from a chromosome.

[0036] As used herein, the terms “label” and “detectable label” refer to detectable molecules, including but not limited to radioisotopes, phosphors, chemiluminescent substances, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, chromophores, dyes, metal ions, metal sols, and ligands (e.g., biotin or hapten). The term “phosphor” refers to a substance or part thereof that can exhibit fluorescence within a detectable range. Specific examples of labels that can be used include fluorescein, rhodamine, dansyl, umbelliferone, Texas Red, luminol, acradium esters, NADPH, and α-β-galactosidase.

[0037] "Homologousity" refers to the percentage of identity between two polynucleotides or two polypeptide segments. Two nucleic acid sequences or two polypeptide sequences are "substantially homologous" to each other if they exhibit at least about 50% sequence identity, at least about 75% sequence identity, at least about 80%–85% sequence identity, at least about 90% sequence identity, and at least about 95%–98% sequence identity over a given length of the molecule. In this specification, substantially homologous also includes sequences that exhibit complete identity with a particular sequence.

[0038] Generally, "identity" refers to the exact correspondence between two polynucleotide or polypeptide sequences, either nucleotide-to-nucleotide or amino acid-to-amino acid. The identity percentage can be determined by directly comparing the sequence information of two molecules. This is done by aligning the sequences, counting the exact number of matches between the two aligned sequences, dividing by the length of the shorter sequence, and multiplying the result by 100. The analysis can be assisted using readily available computer programs. For example, ALIGN, Dayhoff, MO in Atlas of Protein Sequence and Structure, Dayhoff, ed., 5 Suppl. 3:353-358, National Biomedical Research Foundation, Washington, DC, which adapts Smith and Waterman Advances in Appl. Math. 2:482-489, 1981, for peptide analysis. Programs for determining nucleotide sequence identity are available in the Wisconsin Sequence Analysis Package, Version 8 (available from Genetics Computer Group, Madison, Wis.), and include programs such as BESTFIT, FASTA, and GAP, which also rely on the Smith and Waterman algorithm. These programs are readily available with the default parameters recommended by the manufacturer and listed in the Wisconsin Sequence Analysis Package mentioned above. For example, the percentage of identity of a particular nucleotide sequence to a reference sequence can be determined using the Smith and Waterman homology algorithm, with a default scoring table and a 6-nucleotide gap penalty.

[0039] Another method for establishing percentage identity in the context of this disclosure is to use the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, CA). The Smith-Waterman algorithm can be employed from this package family, with default parameters used for the scoring table (e.g., gap-open penalty 12, gap-extension penalty 1, gap 6). The "Match" value from the generated data reflects the "sequence identity." Other suitable programs for calculating percentage identity or similarity between sequences are generally known in the art; for example, another alignment program, BLAST, is used with default parameters. For example, BLASTN and BLASTP can be used with the following default parameters: genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swiss protein+Spupdate+PIR. Details of these programs are readily available.

[0040] Alternatively, homology can be determined by hybridization of polynucleotides under conditions that form stable double helixes between homologous regions, followed by digestion with single-strand specific nucleases, and sizing of the digested fragments. Substantially homologous DNA sequences can be identified, for example, in Southern hybridization experiments under stringent conditions defined for their particular system. Defining appropriate hybridization conditions is within the scope of the art of the art.

[0041] As used herein to describe nucleic acid molecules, “recombinant” means a polynucleotide of genomic, cDNA, viral, semi-synthetic, or synthetic origin that, by its origin or manipulation, is not associated with all or part of the polynucleotides with which it naturally associates. As used with respect to proteins or polypeptides, “recombinant” means a polypeptide produced by the expression of a recombinant polynucleotide. Generally, the gene of interest is cloned and expressed in a transformed organism, as further described below. The host organism expresses the foreign gene and produces a protein under expression conditions.

[0042] The term "transformation" refers to the insertion of exogenous polynucleotides into a host cell, regardless of the method used for such insertion. Examples include direct incorporation, transduction, or f-mating. The exogenous polynucleotides may be maintained as non-integrated vectors, such as plasmids, or they may be integrated into the host genome.

[0043] "Recombinant host cell," "host cell," "cell," "cell line," "cell culture," and other such terms referring to microorganisms or higher eukaryotic cell lines cultured as single cells refer to cells that can or have been used as recipients of recombinant vectors or other transfected DNA, and include offspring of the original transfected cells.

[0044] A “coding sequence” or a sequence that “codes” a selected polypeptide is a nucleic acid molecule that, when placed under the control of an appropriate regulatory sequence (or “regulatory element”), is transcribed (in the case of DNA) and translated in vivo into a polypeptide (in the case of mRNA). The boundaries of a coding sequence may be determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. Coding sequences include, but are not limited to, cDNA from viruses, prokaryotic or eukaryotic mRNA, genomic DNA sequences from viral or prokaryotic DNA, and even synthetic DNA sequences. A transcription termination sequence may be located at 3' of the coding sequence.

[0045] Typical "regulatory elements" include, but are not limited to, transcription promoters, transcription enhancer elements, transcription termination signals, polyadenylation sequences (located at 3' of the translation stop codon), sequences for optimizing translation initiation (located at 5' of the coding sequence), and translation termination sequences.

[0046] The term "nucleic acid" includes DNA and RNA, as well as their analogues, such as those containing modified backbones (e.g., phosphorothioates), and peptide nucleic acids (PNAs). This disclosure provides nucleic acids containing sequences complementary to those described above (e.g., for antisense or probing purposes).

[0047] "Operatively linked" refers to the arrangement of elements configured so that the components described in this way perform their normal function. Therefore, a given promoter operably linked to a coding sequence can result in the expression of the coding sequence if the appropriate enzyme is present. The promoter does not need to be contiguous with the coding sequence as long as it functions to direct its expression. Therefore, for example, even if there is an intervening sequence that has not yet been transcribed between the promoter sequence and the coding sequence, the promoter sequence can be considered "operably linked" to the coding sequence.

[0048] "Encoded" refers to a nucleic acid sequence that encodes a polypeptide sequence, and the polypeptide sequence or a portion thereof includes an amino acid sequence of at least 3-5 amino acids, at least 8-10 amino acids, and at least 15-20 amino acids derived from the polypeptide encoded by the nucleic acid sequence.

[0049] An “expression cassette” or “expression construct” refers to an assembly that can direct the expression of a sequence(s) or gene(s) of interest. An expression cassette generally includes regulatory elements such as promoters that are operablely linked to the sequence(s) or gene(s) of interest (to direct transcription), and often also includes polyadenylated sequences. In embodiments, the expression cassettes described herein may be contained within a plasmid construct. In addition to the components of an expression cassette, a plasmid construct may also include one or more selectable markers, signals that enable the plasmid construct to exist as single-stranded DNA (e.g., an M13 origin), at least one multiplexing site, and a “mammalian” origin (e.g., SV40 or an adenovirus origin).

[0050] "Purified polynucleotides" refer to the polynucleotide or fragment of interest that is essentially free, for example, containing less than 50%, less than 70%, and at least less than 90% of the protein in which the polynucleotide is naturally associated. Techniques for purifying the polynucleotide of interest are well known in the art and include, for example, disruption of polynucleotide-containing cells with chaotropic agents, ion exchange chromatography, affinity chromatography, and separation of polynucleotides from proteins by density precipitation.

[0051] The term "transfection" refers to the uptake of foreign DNA by a cell. When foreign DNA is introduced inside the cell membrane, the cell is considered "transfected." Many transfection techniques are commonly known in this field. Such techniques are used to introduce one or more foreign DNAs into a suitable host cell. The term refers to both the stable and transient uptake of genetic material, including the uptake of peptides or antibody-bound DNA.

[0052] A "vector" is a device that can introduce a nucleic acid sequence into a target cell (e.g., viral vectors, non-viral vectors, microparticle carriers, and liposomes). Typically, "vector constructs," "expression vectors," and "gene transfer vectors" refer to any nucleic acid construct that can induce the expression of a desired nucleic acid and introduce a nucleic acid sequence into a target cell. Therefore, this term includes cloning and expression vehicles, as well as viral vectors.

[0053] A "fragment" refers to a molecule consisting of only a portion of the intact full-length sequence and structure. Polypeptide fragments may include C-terminal deletions, N-terminal deletions, and / or internal deletions of the native polypeptide. Polypeptide fragments generally contain at least about 5 to 10 consecutive amino acid residues of the full-length molecule, at least about 15 to 25 consecutive amino acid residues of the full-length molecule, and at least about 20 to 50 or more consecutive amino acid residues of the full-length molecule, or any integer between 5 amino acids and the number of amino acids in the full-length sequence, provided that the fragment retains the ability to elicit a desired biological response. Nucleic acid fragments may include 5'-deletions, 3'-deletions, and / or internal deletions of nucleic acids. Nucleic acid fragments generally contain at least approximately 5 to 1000 consecutive nucleotide bases of the full-length molecule, and may contain at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250, or at least 500 consecutive nucleotides of the full-length molecule, or any integer between 5 nucleotides and the number of nucleotides in the full-length sequence. Such fragments are useful for hybridization, amplification, production of immunogenic fragments, or nucleic acid immunization.

[0054] An "immunogenic fragment" means a fragment of an immunogen containing one or more epitopes that can modulate an immune response or act as an adjuvant to a co-administered antigen. Such fragments can be identified using epitope mapping techniques well known in the art. For example, linear epitopes can be determined by simultaneously synthesizing a number of peptides on a solid support (the peptides corresponding to parts of protein molecules) and reacting them with an antibody while the peptides are attached to the support. Such techniques are known in the art and are described, for example, in U.S. Patent No. 4,708,886, which is described in its entirety in Patent No. 4,708,871 and is incorporated herein by reference. Similarly, conformational epitopes can be readily identified by determining the spatial conformation of amino acids, for example, by X-ray crystallography or two-dimensional nuclear magnetic resonance.

[0055] For the purposes of this disclosure, immunogenic fragments will typically be at least about 2 amino acids long, about 5 amino acids long, and at least about 10 to about 15 amino acids long. There is no definitive upper limit to the length of the fragments, and they may even consist of nearly the entire length of the protein sequence or fusion proteins containing two or more epitopes.

[0056] In this specification, the term “epitope” generally refers to a site on an antigen recognized by a T cell receptor and / or antibody. In embodiments, it is a short peptide derived from or part of a protein antigen. However, the term is intended to also include peptides having glycopeptides or carbohydrate epitopes. Multiple different epitopes may be supported by a single antigen molecule. The term “epitope” also includes modified sequences of amino acids or glycans that stimulate a reaction that recognizes an entire organism. It is advantageous that the selected epitope is an epitope of an infectious agent that causes an infectious disease.

[0057] Epitopes can be generated without excessive experimentation from knowledge of the amino acid sequence of a peptide or polypeptide and its corresponding DNA sequence, as well as from the properties of specific amino acids (e.g., size, charge, etc.) and a codon dictionary. Guidelines for determining whether a protein will stimulate a reaction include: peptide length—peptides are approximately 8 or 9 amino acids long to fit into MHC class I complexes, and approximately 13 to 25 amino acids long to fit into class II MHC complexes. This length is the minimum for a peptide to bind to an MHC complex. In some embodiments, peptides may be longer than these lengths, as cells may cleave them. The peptide contains a suitable anchor motif and can bind to various class I or class II molecules with sufficient specificity to trigger an immune response. This can be done without excessive experimentation by comparing the sequence of the protein in question with the published structures of peptides associated with MHC molecules. Thus, those skilled in the art can identify the epitope of interest by comparing the sequence of a protein with sequences listed in protein databases.

[0058] As used herein, the term “T cell epitope” generally refers to a peptide structural feature capable of inducing a T cell response, and the term “B cell epitope” generally refers to a peptide structural feature capable of inducing a B cell response.

[0059] An “immunological response” to an antigen or composition refers to the expression of a humoral and / or cellular immune response in a subject to an antigen present in the composition of interest. In this disclosure, “humoral immune response” refers to an immune response mediated by antibody molecules, while “cellular immune response” refers to an immune response mediated by T lymphocytes and / or other leukocytes. One important aspect of cellular immunity is the antigen-specific response by cytolytic T cells (CTLs). CTLs have specificity for peptide antigens that are encoded by major histocompatibility complexes (MHC) and presented by binding to proteins expressed on the cell surface. CTLs help induce and promote the destruction of intracellular microorganisms and the lysis of cells infected with such microorganisms. Another aspect of cellular immunity is the antigen-specific response by helper T cells. Helper T cells stimulate and concentrate the activity of nonspecific effector cells against cells that have peptide antigens bound to MHC molecules on their surface. “Cellular immune response” also refers to the production of the following: "Cellular immune response" also refers to the production of cytokines, chemokines, and other such molecules produced by activated T cells and / or other leukocytes, including those derived from CD4+ and CD8+ T cells.

[0060] Compositions or vaccines that induce a cellular immune response sensitize vertebrate targets by binding to MHC molecules on the cell surface and presenting antigens. The cell-mediated immune response is directed towards the cell presenting the antigen on its surface, or its vicinity. Furthermore, antigen-specific T lymphocytes may be generated to provide future protection to the immunized host.

[0061] The ability of specific antigens to stimulate cell-mediated immunological responses can be determined by numerous assays, including lymphocyte proliferation (lymphocyte activation) assays and CTL cytotoxicity assays, as well as by evaluating antigen-specific T lymphocytes in sensitized subjects. Such assays are well known in the art. Recent methods for measuring cell-mediated immune responses include measuring intracellular cytokines or cytokine secretion by T cell populations, or measuring epitope-specific T cells.

[0062] Thus, the immunological response as used herein may stimulate the production of antibodies (for example, neutralizing antibodies that bind to and block pathogens such as bacterial toxins or viruses that invade and replicate in cells, typically protecting cells from infection and destruction). The antigen of interest may also induce the production of CTLs. Therefore, an immunological response may include one or more actions of: antibody production by B cells; and / or activation of suppressor T cells and / or memory / effector T cells that specifically target the antigen or antigen(s) present in the composition or vaccine of interest. These responses neutralize infectivity and / or mediate antibody-complement or antibody-dependent cytotoxicity (ADCC), providing protection to the immunized host. Such responses can be determined using standard immunoassays and neutralization assays well known in the art. The mammalian innate immune system also recognizes and responds to the molecular characteristics of pathogenic organisms through the activation of Toll-like receptors and similar receptor molecules on immune cells. When the innate immune system is activated, various maladaptive immune response cells are activated, producing, for example, various cytokines, lymphakines, and chemokines. Cells activated by the innate immune response include immature and mature dendritic cells of the monocyte and squamous epithelial lineages (MDC, PDC), as well as γ, δ, α, and β T cells, B cells, etc. Therefore, this disclosure also intends to describe immune responses that include both innate and adaptive responses.

[0063] An "immunogenic composition" is a composition containing an antigenic molecule, which, when administered to a subject, elicits a humoral and / or cellular immune response to the target antigenic molecule in the subject.

[0064] The term “immunogenic” protein or polypeptide refers to an amino acid sequence that elicits an immunological response as described above. Where used herein, “immunogenic” protein or polypeptide includes the full-length sequence of the protein in question, including precursors and mature forms, their analogues, or their immunogenic fragments.

[0065] Gene transfer or gene delivery refers to a method or system for reliably inserting target DNA or RNA into a host cell. Such methods may result in transient expression of introduced non-integrated DNA, extrachromosomal replication and expression of introduced replicons (e.g., episomes), or integration of introduced genetic material into the host cell's genomic DNA. Gene transfer vectors include, but are not limited to, bacterial plasmid vectors, viral vectors, non-viral vectors, alphaviruses, poxviruses, and vacciniavirus-derived vectors. When used in immunization, such gene transfer vectors are sometimes called vaccines or vaccine vectors.

[0066] In this specification, the term “derived from” is used to identify the original source of the molecule, but does not mean to limit the method of producing the molecule, for example, by chemical synthesis or recombinant means.

[0067] Generally, a viral polypeptide is either (i) encoded by the open reading frame of the virus's polynucleotide (viral polynucleotide), or (ii) "derived" from a specific polypeptide of the virus (viral polypeptide) if it exhibits sequence identity with the viral polypeptide as described above.

[0068] A polynucleotide "derived" from a specified sequence refers to a polynucleotide sequence containing a sequence of approximately at least about 6 nucleotides, at least about 8 nucleotides, at least about 10-12 nucleotides, and at least about 15-20 nucleotides that corresponds to, i.e., is identical to or complementary to, a region of the specified nucleotide sequence. A derived polynucleotide is not necessarily physically derived from the nucleotide sequence of interest and can be produced by any method, including but not limited to chemosynthesis, replication, reverse transcription, or transcription, based on the information provided by the base sequence of the region(s) from which the polynucleotide originates. Thus, the polynucleotide may exhibit either sense or antisense orientation of the original polynucleotide.

[0069] The ASF polynucleotides, oligonucleotides, nucleic acids, proteins, polypeptides, or peptides defined above are molecules derived from the ASF virus, including, but not limited to, any of the various isolates of the ASF virus. These molecules do not need to be physically derived from the specific isolate in question; they may be synthetically or recombinantly produced.

[0070] Genomic DNA consists of 168 open reading frames (ORFs). Some of these proteins are derived from larger precursors resulting from post-translational modifications of precursor proteins. In particular, the p30, p54, p72, and hemagglutinin polypeptides encoded by ASF virus ORFs, as well as their variants, their immunogenic fragments, and nucleic acids encoding such polypeptides, variants, or immunogenic fragments, may be used in the implementation of the disclosed subject.

[0071] The target nucleic acid and protein sequences are also publicly known for many ASF virus isolates. Representative nucleic acid sequences for p30, p54, p72, and hemagglutinin are shown in SEQ ID NOs: 1 (p30 / p54 fusion), 7 (p72), 9 (p30), 11 (p54), 13 (hemagglutinin), and 14 (hemagglutinin). Representative amino acid sequences for p30, p54, p72, and hemagglutinin are shown in SEQ ID NOs: 6 (p30 / p54 fusion), 8 (p72), 10 (p30), 12 (p54), and 17 (hemagglutinin). Additional representative sequences, including ASF virus sequences and their encoded polypeptides from ASF virus isolates, are listed in the National Center for Biotechnology Information (NCBI) database. For example, see the GenBank entries, though not limited to these: CBw46759.1; ACJ61575.1; MH735140.1; MH601419.1; MH727102.1; KF834194.1; LC322015.1; MH735142; MH681419.1; KM609342.1; FR682468.1; KJ380910.1; FR682468.1;

[0071] MH722357; MH68419.1; MH713612.1; LC322016.1; KF834193.1 (All of these sequences (entered up to the filing date of this application) are incorporated herein by reference.)

[0072] As used herein, the terms “p30,” “p54,” “p72,” or “hemagglutinin” with respect to AFS virus polypeptides refer to polypeptides containing sequences homologous or identical to the “p30,” “p54,” “p72,” or “hemagglutinin” polypeptides of the ASF virus, containing sequences exhibiting at least about 80–100% sequence identity, and any percentage of identity within these ranges, for example, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity. Capsid polypeptides may be encoded by either the same strain of ASF virus or different strains of ASF virus.

[0073] As used herein, the term “p30 / p54 fusion protein” means a protein containing sequences homologous or identical to the ASF virus-encoding p30 protein and p54 protein derived from the ASF virus, and containing sequences that exhibit at least approximately 80–100% sequence identity with them, and any percentage identity within this range, for example, sequences that exhibit 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity with them.

[0074] An "antigen" refers to a molecule containing one or more epitopes (linear, conformational, or both) that stimulate the host's immune system to elicit a humoral and / or cellular antigen-specific response. This term is used interchangeably with the term "immunogen." Typically, B cell epitopes contain at least about five amino acids, but may also contain three to four. T cell epitopes, such as CTL epitopes, contain at least about seven to nine amino acids, and helper T cell epitopes contain at least about twelve to twenty amino acids. Typically, epitopes contain about seven to fifteen amino acids, e.g., nine, ten, twelve, or fifteen. The term "antigen" can refer to both subunit antigens (i.e., antigens isolated and independent from the whole organism to which the antigen naturally associates) and dead, attenuated, or inactivated bacteria, viruses, fungi, parasites, or other microorganisms. Antibodies such as anti-idiotype antibodies or their fragments, or synthetic peptide mimotopes that mimic antigens or antigenic determinants, are also included in the definition of antigen as used herein. Similarly, oligonucleotides or polynucleotides that express antigens or antigenic determinants in vivo, such as in the application of gene therapy or DNA immunization, are also included in the definition of antigen as used herein.

[0075] The term "antibody" encompasses polyclonal antibody preparations and monoclonal antibody preparations, as well as preparations including hybrid antibodies, modified antibodies, chimeric antibodies and humanized antibodies, and hybrid (chimeric) antibody molecules and any functional fragments obtained from such molecules, the fragments of which retain the specific binding properties of the parent antibody molecule.

[0076] The terms "hybridize" and "hybridization" refer to the formation of a complex between nucleotide sequences that have sufficient complementarity to form a complex via Watson-Crick base pairing. When a primer "hybridizes" with a target (template), such a complex (or hybrid) is stable enough to perform the priming function required by DNA polymerase, for example, to initiate DNA synthesis.

[0077] As used herein, “biological sample” means a tissue or liquid sample isolated from a subject, including, but not limited to, blood, plasma, serum, feces, urine, bone marrow, bile, cerebrospinal fluid, lymph, skin samples, external secretions from skin, respiratory tract, intestinal tract, and urogenital tract, tears, saliva, milk, blood cells, organs, biopsies, and conditioning media resulting from the proliferation of cells and tissues in culture media, such as recombinant cells and cellular components, as well as samples of in vitro cell culture components. In particular, ASF virus may be obtained from biological samples including, but not limited to, blood, serum, spleen, liver, lungs, lymph nodes, tonsils, and kidneys.

[0078] The term "subject" refers to all members of the family Suidae, including the pig (sus domesticus), though this is not an exhaustive definition. This term does not indicate a specific age; therefore, it is intended to cover both adult and newborn individuals.

[0079] The terms “mutant,” “analog,” and “mutein” refer to biologically active derivatives of a reference molecule that retain desired activity, such as antigenic activity that induces an immune response to ASF. Generally, the terms “mutant” and “analog” refer to compounds that have a native polypeptide sequence and structure with one or more amino acid additions, substitutions (generally of conserved nature) and / or deletions from the native molecule, provided that the modifications do not destroy the biological activity, and are “substantially homologous” to the reference molecule as defined below. Generally, the amino acid sequence of such an analog has a high degree of sequence homology with respect to the reference sequence, for example, if the two sequences are aligned, they have amino acid sequence homology of 50% or more, generally 60% to 70% or more, and even more particularly 80% to 85% or more, for example, at least 90% to 95% or more. Often, the analog contains the same number of amino acids but includes substitutions as described herein. The term “mutein” further includes compounds containing only amino and / or imino molecules, polypeptides containing one or more analogs of amino acids (e.g., including unnatural amino acids), polypeptides having substituted links, and polypeptides having one or more amino acid-like molecules, including but not limited to other modifications known in the art, such as naturally occurring and unnatural (e.g., synthetic), cyclized, branched molecules. The term also includes molecules containing one or more N-substituted glycine residues ("peptoids") and other synthetic amino acids or peptides. (See, for example, U.S. Patents 5,831,005; 5,877,278; and 5,977,301). In embodiments, the analog or mutein has at least the same antigenic activity as the native molecule. Methods for producing polypeptide analogs and muteins are known in the art and are further described below.

[0080] As explained above, analogs generally involve essentially conserved substitutions, i.e., substitutions that occur within the family of amino acids whose side chains are related. Specifically, amino acids are generally divided into four families: (1) acidic - aspartic acid and glutamic acid; (2) basic - lysine, arginine, histidine; (3) nonpolar - alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) non-charged - glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. For example, it can be reasonably predicted that similar conservative substitutions, such as replacing leucine with isoleucine or valine, aspartic acid with glutamic acid, threonine with serine, or amino acids with structurally related amino acids, will not have a significant impact on biological activity. For example, the target polypeptide may contain approximately 5 to 10 conserved or non-conserved amino acid substitutions, or approximately 15 to 25 conserved or non-conserved amino acid substitutions, or any integer between 5 and 25, as long as the desired function of the molecule remains intact. Those skilled in the art can easily determine the range of the target molecule that allows for variation by referring to the Hopp / Woods plot or Kyte-Doolittle plot, which are well known in the art.

[0081] As used herein, the terms “multiepitope fusion antigen” or “multiepitope fusion protein” refer to a polypeptide in which multiple ASF virus antigens are part of a single, continuous chain of amino acids, which does not exist in nature. The ASF virus antigens may be directly linked to one another by peptide bonds or separated by intervening amino acid sequences. The fusion antigen may contain the p30 / p54 ASF virus-coding polypeptide or a fragment thereof. The fusion antigen may also contain sequences exogenous to the ASF virus. Furthermore, the sequences present may be derived from multiple genotypes and / or isolates of the ASF virus.

[0082] In the context of immunogenic compositions, "therapeutably effective amount" means the amount of immunogen (e.g., immunogenic polypeptide, fusion protein, polyprotein, or nucleic acid encoding an antigen) that induces an immunological response for antibody production or for the treatment or prevention of ASF infection. Such a response generally results in the expression of an antibody-mediated immune response and / or a secretory or cellular immune response to the composition in the subject. Typically, such a response includes, but is not limited to, one or more actions of: production of antibodies from any of the immunological classes such as immunoglobulin A, D, E, G, or M; proliferation of B and T lymphocytes; supply of activation, proliferation, and differentiation signals to immunological cells; and proliferation of helper T cells, suppressor T cells, and / or cytotoxic T cells and / or γ, δ-T cell populations.

[0083] For the purposes of this disclosure, the “effective dose” of the adjuvant is the amount that enhances the immunological response to the co-administered antigen or nucleic acid encoding the antigen.

[0084] In this specification, “treatment” means any of the following: (i) prevention of infection or reinfection, as in conventional vaccines; (ii) reduction or elimination of symptoms; and (iii) substantial or complete elimination of the pathogen in question. Treatment may be preventive (before infection) or therapeutic (after infection).

[0085] Before describing this disclosure in detail, it should be understood that the implementation of this disclosure will employ conventional methods of virology, microbiology, molecular biology, recombinant DNA technology, and immunology, unless otherwise specified. Such techniques are well described in the literature. Many methods and materials similar or equivalent to those described herein may be used in the claimed implementation of the invention, but the materials and methods described herein are not.

[0086] This disclosure includes compositions and methods for immunizing a subject against ASF infection. This disclosure provides immunogenic compositions comprising nucleic acids encoding capsid proteins and / or other immunogenic polypeptides derived from one or more strains of ASF virus, and compositions comprising immunogenic polypeptides derived from one or more strains of ASF virus. The immunogenic polypeptides used in the practice of this subject may include ASF virus-derived polypeptides comprising multiple epitope fusion antigens. Furthermore, the immunogenic composition may include one or more adjuvants or nucleic acids encoding adjuvants, wherein the immunogenic polypeptide is mixed or co-expressed with the adjuvants. The immunogenic composition may also include additional antigens other than ASF virus antigens, such as antigens that may be used in immunization against pathogens causing diarrheal diseases.

[0087] To further the understanding of the disclosed subject matter, a more detailed discussion is provided below regarding the production of nucleic acids and polypeptides for use in immunogenic compositions, and methods for using such compositions in the treatment or prevention of ASF infection. Structural polypeptides, non-structural polypeptides, polyproteins

[0088] The immunogenic compositions described herein may comprise one or more polypeptides derived from one or more genotypes and / or isolates of the ASF virus. Polypeptides that may be used in the practice of the subject disclosed herein include structural proteins, non-structural proteins, and polyproteins. Such polypeptides may be full-length proteins or variants thereof or immunogenic fragments thereof that can induce an immune response against the ASF virus.

[0089] Polypeptides in an immunogenic composition may be encoded by any region of the ASF virus genome. Multiple polypeptides may be included in the immunogenic composition. Such a composition may include polypeptides from the same ASF virus isolate, or polypeptides from different strains and isolates, including isolates having any of the various ASF virus genotypes, in order to provide higher protection against a wide range of ASF virus genotypes. Multiple virus strains of ASF virus are known, and multiple polypeptides containing epitopes derived from any of these strains can be used in an immunogenic composition.

[0090] The antigens used in the immunogenic compositions of this disclosure may be present in the composition as individual, distinct polypeptides. Generally, the recombinant proteins of this disclosure are expressed as GST fusion proteins and / or His-tagged fusion proteins. Multiepitope fusion protein

[0091] The immunogenic compositions described herein may also comprise multiple epitope fusion proteins. Such fusion proteins comprise multiple epitopes derived from two or more viral polypeptides of one or more genotypes and / or isolates of the ASF virus. Multiple epitope fusion proteins offer two main advantages: firstly, polypeptides that may be unstable or poorly expressed on their own may be aided by the addition of a suitable hybrid partner to overcome the problem; secondly, commercial production is simplified because only one expression and purification is required to produce two antigenically useful polypeptides.

[0092] The polypeptides in the fusion protein may be derived from the same ASF virus isolate, or from different strains and isolates, including isolates with any of the various ASF virus genotypes, in order to provide higher protection against a wide range of viral genotypes. Multiple viral strains of ASF virus are known, and epitopes derived from any of these strains may be used in the fusion protein.

[0093] It is well known that any given species can vary from organism to organism, and that a given organism, such as a virus, can have numerous different strains. For example, as described above, the ASF virus has at least 24 gene groups. Generally, antigenic determinants can have high homology in terms of amino acid sequence, and the degree of homology is generally 30% or more, or 40% or more, when aligned. Fusion proteins may also contain multiple copies of the epitope, and one or more polypeptides of the fusion protein contain sequences containing exact copies of the same epitope. Furthermore, polypeptides may be selected based on specific viral clades prevalent in the particular geographical area in which the vaccine composition containing the fusion is used. It is readily apparent that the subject fusions provide an effective means of treating infection in a wide variety of situations.

[0094] Multiple epitope fusion antigens are of the formula NH2-A-{-XL-} n -B--COOH [where X is the amino acid sequence of the ASF virus antigen or a fragment thereof; L is any linker amino acid sequence; A is any N-terminal amino acid sequence; B is any C-terminal amino acid sequence; and n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15] can be represented as -B--COOH [where X is the amino acid sequence of the ASF virus antigen or a fragment thereof; L is any linker amino acid sequence; A is any N-terminal amino acid sequence; B is any C-terminal amino acid sequence; and n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15].

[0095] If the --X-- portion has a leader peptide sequence in its wild type, it may or may not be included in the multiple epitope fusion antigen. In some embodiments, the other leader peptides of the --X-- portion located at the N-terminus of the hybrid protein are deleted, i.e., the leader peptide of X1 is retained, but X2...X n This is omitted. This is equivalent to removing all leader peptides and using the leader peptide of X1 as site-A-.

[0096] For each example of n in (--XL-), the linker amino acid sequence -L- may or may not be present. For example, when n=2, the hybrids may be NH2--X1-L1-X2-L2-COOH, NH2--X1--X2--COOH, NH2--X1-L1-X2--COOH, NH2--X1--X2-L2-COOH, etc. The linker amino acid sequence(singular or plural)-L- is typically short, for example, 20 or fewer amino acids (i.e., 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples include short peptide sequences to facilitate cloning, polyglycine linkers (Gly, where n=2, 3, 4, 5, 6, 7, 8, 9, 10 or more), and histidine tags (His n Examples include [where n=3, 4, 5, 6, 7, 8, 9, 10 or more]). Other suitable linker amino acid sequences will be apparent to those skilled in the art. Useful linkers include GSGGGG, the Gly-Ser dipeptide formed from the BamHI restriction site to aid in cloning and manipulation, and the (Gly)4 tetrapeptide, a typical polyglycine linker. Furthermore, protease substrate sequences may be added (e.g., TEV protease: ENLYFQG).

[0097] -A- is an arbitrary N-terminal amino acid sequence. This is typically a short sequence, for example, 40 amino acids or less (i.e., 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). For example, it could be a leader sequence to direct protein transport or a short peptide sequence to facilitate cloning and purification (e.g., histidine-tagged His n Examples include [where n = 3, 4, 5, 6, 7, 8, 9, 10 or more]). Other suitable N-terminal amino acid sequences will be obvious to those skilled in the art. If X1 lacks its own N-terminal methionine, then -A- is an oligopeptide that provides the N-terminal methionine (e.g., having 1, 2, 3, 4, 5, 6, 7 or 8 amino acids).

[0098] --B-- is an arbitrary C-terminal amino acid sequence. This is typically short, for example, 40 or fewer amino acids (i.e., 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). For example, a sequence that directs protein transport, a short peptide sequence that facilitates cloning or purification (e.g., His n Examples include sequences that enhance protein stability (where n = 3, 4, 5, 6, 7, 8, 9, 10 or more), or sequences that enhance protein stability. Other suitable C-terminal amino acid sequences include His, if preceded by a TEV protease substrate sequence. n It will be obvious to those skilled in the art that sequences can be removed (for example, ENLYFQGHis n ).

[0099] The individual antigens (individual --X-- portions) of the immunogenic composition within a multiple epitope fusion antigen may come from one or more strains or one or more M types. If n=2, for example, X2 may come from the same strain or type as X1, or from a different strain or type. If n=3, the strains may be (i) X1=X2=X3, (ii) X1=X2≠X3, (iii) X1≠X2=X3, (iv) X1≠X2≠X3, or (v) X1=X3≠X2, etc.

[0100] When multiple epitope fusion antigens are used, individual antigens within the fusion protein (i.e., individual --X-- portions) may originate from one or more strains. For n=2, for example, X2 may be from the same strain as X1 or from a different strain. For n=3, the strains may be (i) X1=X2=X3, (ii) X1=X2≠X3, (iii) X1≠X2=X3, (iv) X1≠X2≠X3, or (v) X1=X3≠X2, etc.

[0101] Accordingly, in embodiments, antigenic determinants from different ASF virus strains may be present. Representative multiepitope fusion proteins for use in this disclosure, comprising polypeptides derived from ASF virus isolates, are discussed below. However, it should be understood that multiepitope fusion proteins containing other epitopes from the ASF virus genome, or multiepitope fusion proteins containing epitopes in different configurations, may also be found for use in immunogenic compositions such as those disclosed.

[0102] In certain embodiments, the fusion protein comprises one or more capsid and / or minor structural polypeptides derived from one or more isolates of the ASF virus.

[0103] In another embodiment, the fusion protein comprises ASF virus polypeptides derived from two or more virus strains.

[0104] In all fusions described herein, the viral regions do not need to be in the order in which they occur naturally. Furthermore, each region may be derived from the same or different ASF virus isolates. The various ASF virus polypeptides present in the various fusions described above may be full-length polypeptides or parts thereof.

[0105] If necessary, the fusion protein, or the individual components of these proteins, may also contain other amino acid sequences such as amino acid linkers or signal sequences, as well as ligands useful for protein purification, such as glutathione-S-transferase or staphylococcal protein A. nucleic acid

[0106] Nucleic acids for use as disclosed herein may be derived from any of the various regions of the ASF virus genome, for example, in polypeptide production.

[0107] Representative sequences of the ASF virus are known, including sequence numbers 1, 7, 9, 11, 13, and 14.

[0108] Any of these sequences, as well as their fragments and variants that can be used in nucleic acid immunization to induce an immune response against ASF virus, will find use in the present method. Accordingly, the present disclosure provides variants of the above sequences exhibiting at least about 80–100% sequence identity, including any percentage identity within these ranges, such as 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, and 100% sequence identity. The present disclosure also provides polynucleotides encoding immunogenic fragments of ASF virus polypeptides derived from any of the above sequences or variants thereof. The polynucleotides may also include coding sequences of naturally occurring polypeptides or synthetic sequences that do not exist in nature.

[0109] A polynucleotide may contain fewer base sequences than the entire ASF virus genome, or it may contain the entire base sequence of the viral genomic DNA.

[0110] In the embodiment, the polynucleotide comprises one or more ASF virus sequences encoding p30, p54, p72 and / or hemagglutinin proteins of one or more isolated strains of ASF virus.

[0111] In embodiments, this disclosure provides polynucleotides encoding the multiepitope fusion proteins described herein. The multiepitope fusion proteins may include sequences derived from one or more genotypes and / or isolates of ASF virus.

[0112] The nucleic acids described herein may be prepared by many methods (e.g., chemical synthesis from a genome or cDNA library, from the organism itself, etc.) and may take various forms (e.g., single-stranded, double-stranded, vectors, probes, etc.). In embodiments, the nucleic acids are prepared in a substantially pure form (i.e., substantially free from nucleic acids of other host cells or non-host cells).

[0113] For example, nucleic acids may be obtained by screening cDNA and / or genomic libraries from virus-infected cells, or by inducing genes from vectors known to contain them. For example, the polynucleotide of interest may be isolated from a genomic library derived from viral DNA present, for example, in hair or blood samples from an infected individual. Alternatively, ASF viral nucleic acids may be isolated from biological samples taken from an infected mammal or infected individual. Amplification methods such as PCR may be used to amplify the polynucleotide from any of the ASF viral genomic DNA encoding it. Alternatively, the polynucleotide may be synthesized in the laboratory, for example, using an automated synthesizer. The nucleotide sequence may be designed with appropriate codons for a specific amino acid sequence of interest. Generally, codons suitable for the host in which the sequence will be expressed are selected. The complete sequence of the polynucleotide of interest may be assembled from duplicate oligonucleotides prepared by standard methods and assembled into a complete coding sequence. The polynucleotide may be RNA or single-stranded or double-stranded DNA. In embodiments, the polynucleotide is isolated without other components such as proteins and lipids.

[0114] Therefore, a particular nucleotide sequence may be obtained from a vector containing the desired sequence, or, where appropriate, fully or partially synthesized using various oligonucleotide synthesis techniques known in the art, such as site-directed mutagenesis and polymerase chain reaction (PCR) techniques. In particular, one method for obtaining a nucleotide sequence encoding a desired sequence is to anneal a complementary set of overlapping synthetic oligonucleotides produced by a conventional automated polynucleotide synthesizer, then ligate them with a suitable DNA ligase, and amplify the ligated nucleotide sequence by PCR. Primer sequences may include, but are not limited to, SEQ ID NOs: 2, 3, 4, 5, 15, and 16. Production of immunogenic polypeptides

[0115] Polypeptides described herein may be prepared by any suitable method (e.g., recombinant expression, purification from cell culture, chemosynthesis, etc.) and in various forms (e.g., native, fusion, non-glycosylated, lipid-containing, etc.). Such polypeptides include naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art. Polypeptides are prepared in substantially pure forms (i.e., substantially free from other host cell proteins or non-host cell proteins).

[0116] Polypeptides may be synthesized chemically by conventional methods using one of several techniques known to those skilled in the peptide technology. Generally, these methods employ the sequential addition of one or more amino acids to a growing peptide chain. Typically, the amino or carboxyl group of the first amino acid is protected with a suitable protecting group. The protected or derivatized amino acid can then be made available in solution by attaching it to an inert solid support or by adding the next amino acid in the sequence having a well-protected complementary (amino or carboxyl) group under conditions that allow for the formation of an amide bond. The protecting group is then removed from the newly added amino acid residue, and the next amino acid (well-protected) is added, and so on. After the desired amino acids have been linked in the appropriate order, the remaining protecting groups (and the solid support, if using solid-phase synthesis techniques) are removed sequentially or simultaneously to obtain the final polypeptide. By easily modifying this general procedure, for example, multiple amino acids can be added to a growing chain at once by coupling a protected tripeptide with a well-protected dipeptide (under conditions that do not racemize the chiral center) to form a pentapeptide after deprotection.

[0117] Typical protecting groups include t-butyloxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl (Fmoc), benzyloxycarbonyl (Cbz), p-toluenesulfonyl (Tx), 2,4-dinitrophenyl; benzyl (Bzl), biphenylisopropyloxycarbonyl, t-amyloxycarbonyl, isobomyloxycarbonyl, o-bromobenzyloxycarbonyl, cyclohexyl, isopropyl, acetyl, and o-nitrophenylsulfonyl. Typical solid supports are crosslinked polymer supports. These may include divinylbenzene crosslinked-styrene polymers, such as divinylbenzene-hydroxymethylstyrene copolymer, divinylbenzene-chloromethylstyrene copolymer, and divinylbenzene-benzhydrylaminopolystyrene copolymer.

[0118] The polypeptides of this disclosure can also be chemically prepared by other methods, such as simultaneous multiplex peptide synthesis.

[0119] Alternatively, the immunogenic polypeptides, polyproteins, and multiepitope fusion proteins described above may be produced recombinantly. Once the coding sequences of the desired proteins are isolated or synthesized, they can be cloned into any suitable vector or replicon for expression. Numerous cloning vectors are known to those skilled in the art, and the selection of a suitable cloning vector is a matter of choice. Various bacterial, yeast, plant, mammalian, and insect expression systems are available in the art, and any such expression system may be used. Optionally, the polynucleotides encoding these proteins may be translated in a cell-free translation system. Such methods are well known in the art.

[0120] Examples of recombinant DNA vectors for cloning and host cells that may be transformed include bacteriophage λ (Escherichia coli), pBR322 (Escherichia coli), pACYC177 (Escherichia coli), pKT230 (Gram-negative bacteria), pGV1106 (Gram-negative bacteria), pLAFRI (Gram-negative bacteria), pME290 (non-Escherichia coli Gram-negative bacteria), pHV14 (Escherichia coli and Bacillus subtilis), pBD9 (Bacillus subtilis), pIJ61 (Yeast (Streptomyces)), pUC6 (Yeast (Streptomyces)), YIp5 (Yeast (Streptomyces)), YCpl9 (Yeast (Streptomyces)), and bovine papillomavirus (mammalian cells).

[0121] Insect cell expression systems, such as baculovirus systems, may also be used and are known to those skilled in the art, for example, as described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987). Materials and methods for baculovirus / insect cell expression systems are commercially available, in particular, in the form of kits ("MaxBac" kits) from Invitrogen, San Diego, CA.

[0122] Plant expression systems may also be used to produce immunogenic proteins. Generally, such systems use virus-based vectors to transfect heterologous genes into plant cells.

[0123] Viral systems, such as vaccinia-based infection / transfection systems, are also used in conjunction with the subject matter disclosed herein. In this system, cells are first transfected in vitro with a vaccinia virus recombinant encoding bacteriophage T7 RNA polymerase. This polymerase exhibits exquisite specificity, transcribing only templates containing the T7 promoter. After infection, cells are transfected with the target DNA driven by the T7 promoter. The polymerase expressed in the cytoplasm from the vaccinia virus recombinant transcribes the transfected DNA into RNA, which is then translated into protein by the host's translation mechanism. This method provides a large amount of high-level, transient cytoplasmic production of RNA and its translation products.

[0124] The gene may be under the control of a promoter, a ribosome binding site (in the case of bacterial expression), and optionally an operator (collectively referred to herein as “control” element) so that the DNA sequence encoding the desired immunogenic polypeptide is transcribed into RNA in host cells transformed with a vector containing this expression construct. The coding sequence may or may not include a signal peptide or a leader sequence. In the subject matter of the invention disclosed herein, both naturally occurring signal peptides and heterologous sequences may be used. The leader sequence may be removed by the host in post-translational processing. See, for example, U.S. Patents 4,431,739; 4,425,437; and 4,338,397 (each of which is incorporated herein in its entirety by reference). Such sequences include, but are not limited to, tpa leaders and honeybee mellitin signal sequences.

[0125] Other regulatory sequences that enable the regulation of protein sequence expression in relation to host cell proliferation may also be desirable. Such regulatory sequences are known to those skilled in the art, and examples include those that turn gene expression on or off in response to chemical or physical stimuli, including the presence of regulatory compounds. Other types of regulatory elements may also be present in the vector, such as enhancer sequences.

[0126] The control sequence and other regulatory sequences may be ligated to the coding sequence before insertion into the vector. Alternatively, the coding sequence may be directly cloned into an expression vector that already contains the control sequence and appropriate restriction sites.

[0127] In embodiments, it may be necessary to modify the coding sequence so that it can be bound to the control sequence in an appropriate orientation; that is, to maintain an appropriate reading frame. It may also be desirable to prepare mutants or analogs of immunogenic polypeptides. Mutants or analogs may be prepared by deleting a portion of the protein-coding sequence, inserting a sequence, and / or substituting one or more nucleotides within the sequence. Techniques for modifying nucleotide sequences, such as site-directed mutagenesis, are well known to those skilled in the art.

[0128] Next, appropriate host cells are transformed using the expression vector. Numerous mammalian cell lines are known in the art, including, but are not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and other cells, as well as immortalized cell lines available from the American Type Culture Collection (ATCC). Similarly, bacterial hosts such as Escherichia coli, Bacillus subtilis, and Streptococcus would also be used with the expression constructs of the present invention. Yeast hosts useful in the subject matter as disclosed include, in particular, Saccharomyces cerevisiae, Candida albicans, Candida maltosa, Hansenula polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Pichia guillerimondii, Pichia pastoris, Schizosaccharomyces pombe, and Yarrowia lipolytica. Insect cells that can be used in conjunction with baculovirus expression vectors include, in particular, Aedes aegypti, Autographa californica, Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda, and Trichoplusia ni.

[0129] Depending on the selected expression system and host, the proteins disclosed herein are produced by growing host cells transformed with the expression vector under conditions in which the protein of interest is expressed. The selection of appropriate growth conditions is within the scope of the art of the art. The cells are then destroyed using chemical, physical, or mechanical means that lyse the cells while maintaining the ASF virus immunogenic polypeptide substantially intact. The intracellular protein may also be obtained by removing components from the cell wall or membrane, for example, using a surfactant or organic solvent, so that leakage of the immunogenic polypeptide occurs.

[0130] For example, methods for disrupting cells for use with subjects such as those disclosed herein include, but are not limited to, sonication or ultrasonic irradiation; agitation; liquid or solid extrusion; heat treatment; freeze-thaw; drying; explosive decompression; osmotic shock; treatment with lytic enzymes including proteases such as trypsin, neuraminidase and lysozyme; alkaline treatment; and the use of surfactants and solvents such as bile salts, sodium dodecyl sulfate, Triton, NP40, and CHAPS. The specific technique used to disrupt cells is a matter of choice and will depend on the type of cells used expressing polypeptides, culture conditions, and pretreatment.

[0131] After disrupting the cells, the cellular debris is generally removed by centrifugation, and the ASF virus immunogenic polypeptides produced within the cells are further purified using standard purification techniques such as column chromatography, ion exchange chromatography, size exclusion chromatography, electrophoresis, HPLC, immunoadsorption, affinity chromatography, and immunoprecipitation.

[0132] For example, one method for obtaining intracellular ASF virus immunogenic polypeptides disclosed herein includes affinity purification, such as by immunoaffinity chromatography using a specific antibody. The selection of a suitable affinity resin is within the scope of the art for those skilled in the art. After affinity purification, the immunogenic polypeptide may be further purified by any of the conventional techniques well known in the art, such as the techniques described above.

[0133] It may be desirable to simultaneously produce multiple polypeptides (e.g., structural and / or non-structural proteins from one or more viral strains or viral polypeptides combined with polypeptide adjuvants). The production of two or more different polypeptides may be readily achieved, for example, by transfecting host cells together with constructs encoding different polypeptides. Co-transfection may be trans or cis, i.e., achieved using separate vectors or a single vector encoding the polypeptides. When a single vector is used, polypeptide expression may be driven by a single set of regulatory elements, or the polypeptide-encoding sequence may be present on the vector in individual expression cassettes controlled by individual regulatory elements.

[0134] Polypeptides described herein may be bound to a solid support. Examples of solid supports that may be used in practice with the subject matter disclosed herein include nitrocellulose (e.g., in the form of a membrane or microtiter well); polyvinyl chloride (e.g., a sheet or microtiter well); polystyrene latex (e.g., beads or microtiter plates); polyvinylidene fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and other substrates.

[0135] Typically, first, a solid-phase component (e.g., one or more ASF virus antigens) is reacted with the solid-phase component under appropriate binding conditions such that its components are sufficiently immobilized on the support. Sometimes, the immobilization of the antigen on the support may be facilitated by first binding the antigen to a protein with better binding properties. Suitable coupling proteins include serum albumins including bovine serum albumin (BSA), keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, and macromolecules such as other proteins well-known to those skilled in the art, but are not limited thereto. Other molecules that can be used to bind the antigen to the support include polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, etc. Such molecules and methods of binding these molecules to the antigen are well-known to those skilled in the art.

[0136] If desired, the polypeptide can be labeled using conventional techniques. Suitable labels include fluorophores, chromophores, radioactive atoms (especially, 32 P and 125 I), reagents with high electron density, enzymes, and ligands having specific binding partners. Enzymes are usually detected by their activity. For example, horseradish peroxidase is usually detected by its ability to convert 3,3’,5,5’-tetramethylbenzidine (TMB) into a blue dye and can be quantified by a spectrophotometer. A "specific binding partner" refers to a protein that can bind a ligand molecule with high specificity, such as an antigen and its specific monoclonal antibody. Other specific binding partners include biotin and avidin or streptavidin, IgG and protein A, and numerous receptor-ligand couples known in the art. As disclosed herein, a single label or a combination of labels can be used.

[0137] Once formulated, the compositions as disclosed herein can be administered directly to the subject (e.g., as described above) or, alternatively, using the methods as described above, delivered ex vivo to cells derived from the subject. immunogenic composition

[0138] This disclosure also provides compositions comprising one or more immunogenic polypeptides and / or polyprotein multiepitope fusion proteins described herein. Different polypeptides, polyproteins, and multiepitope fusion proteins may be mixed together in a single formulation. In such combinations, the antigen of the immunogenic composition may be present in multiple polypeptides, or multiepitope polypeptides, or polyproteins.

[0139] The immunogenic composition may consist of a mixture of polypeptides, which may be delivered sequentially using the same or different vehicles. Antigens may be administered individually or in combination in immunogenic compositions, for example, prophylactic (i.e., to prevent infection) or therapeutic (to treat infection). The immunogenic composition may be administered multiple times to achieve the desired effect (e.g., one or more "boost" doses following a "prime" dose). The same composition may be administered in one or more priming and one or more boosting stages. Alternatively, different compositions may be used for priming and boosting.

[0140] Immunogenic compositions generally contain one or more "pharmaceutically acceptable excipients or vehicles," such as water, physiological saline, glycerol, or ethanol. Furthermore, auxiliary substances such as humectants or emulsifiers and pH buffers may be present in such vehicles.

[0141] Immunogenic compositions typically include, in addition to the components described above, one or more "pharmaceutically acceptable carriers." These include any carriers that do not themselves induce the production of antibodies harmful to the organism receiving the composition. Suitable carriers are typically large, slowly metabolized polymers such as proteins, polysaccharides, polylactic acid, polyglycolic acid, high molecular weight amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes). Such carriers are well known to those skilled in the art. The composition may also contain diluents such as water, saline, or glycerol. Furthermore, auxiliary substances such as wetting agents or emulsifiers and pH buffers may be present. A thorough discussion of pharmaceutically acceptable components is available in Gennaro (2000) Remington: The Science and Practice of Pharmacy. 20th ed., ISBN: 0683306472.

[0142] Medicinally acceptable salts may also be used in compositions such as those disclosed herein, including, for example, inorganic salts such as hydrochlorides, hydrobroms, phosphates, or sulfates, and organic salts such as acetates, propions, malons, or benzoates. Particularly useful protein substrates are serum albumin, keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus toxoid, and other proteins well known to those skilled in the art. Compositions such as those disclosed may also contain, alone or in combination with, liquids or excipients such as water, saline, glycerol, dextrose, or ethanol, as well as substances such as wetting agents, emulsifiers, or pH buffers. Antigens may also be adsorbed onto or encapsulated within liposomes or particulate carriers such as PLGs, or otherwise conjugated.

[0143] To enhance immunogenicity, the antigen may be bound to a carrier protein. This is particularly useful in compositions in which sugar or carbohydrate antigens are used.

[0144] Examples of carrier proteins include, but are not limited to, bacterial toxins or toxoids, such as diphtheria toxoid or tetanus toxoid. CRM197 diphtheria toxoid may also be used. Other carrier polypeptides include N. meningitidis outer membrane protein (European Patent Application Publication EP-A-0372501), synthetic peptides (European Patent Application Publications EP-A-0378881 and EP-A-0427347), heat shock proteins (International Publications WO93 / 17712 and WO94 / 03208), pertussis protein (International Publication WO 98 / 58668 and European Patent Application Publication EP-A-0471177), H. influenzae-derived protein D (International Publication WO 00 / 56360), cytokines (International Publication WO 91 / 01146), lymphokines, hormones, growth factors, toxins A or B derived from C. difficile (International Publication WO 00 / 61761), and iron uptake proteins such as transferrin (International Publication WO 01 / 72337). If the mixture contains capsular saccharides from both serigraph A and C, the MenA saccharide:MenC saccharide ratio (w / w) may be greater than 1 (e.g., 2:1, 3:1, 4:1, 5:1, 10:1 or greater). Different saccharides may be conjugated to the same or different types of carrier proteins. Any suitable conjugation reaction may be used, and any suitable linker may be used as needed.

[0145] The immunogenic composition containing the disclosed vaccine may be administered in combination with other immunomodulators. For example, the vaccine disclosed herein may contain an adjuvant. The adjuvant includes, but is not limited to, one or more of the adjuvants described below. Mineral-containing composition

[0146] Mineral-containing compositions suitable for use as adjuvants disclosed herein include mineral salts such as aluminum salts and calcium salts. Salts disclosed herein include mineral salts such as hydroxides (e.g., oxyhydroxides), phosphates (e.g., hydroxyphosphates, orthophosphates), sulfates, or mixtures of different mineral compounds (e.g., mixtures of phosphates and hydroxide adjuvants (optionally including excess phosphates)), and the compounds take any suitable form (e.g., gels, crystals, amorphous materials). Mineral-containing compositions may also be formulated as particles of metal salts (International Publication No. WO00 / 23105).

[0147] Aluminum salts are Al 3+ The dosage may be included in the vaccine in such a way that it is 0.2 to 1.0 mg per dose.

[0148] In embodiments, the aluminum-based adjuvant for use as disclosed is alum (potassium aluminum sulfate (AlK(SO4)2)) or an alum derivative formed in situ by mixing an antigen in phosphate buffer with alum and then titrating and precipitating it with a base such as ammonium hydroxide or sodium hydroxide.

[0149] Another aluminum-based adjuvant for use in the vaccine formulation of the present invention is aluminum hydroxide adjuvant (Al(OH)3) or crystalline aluminum oxyhydroxide (AlOOH), which is an excellent adsorbent and is approximately 500 m 2 It has a surface area of ​​ / g. Alternatively, aluminum phosphate adjuvants (AlPO4) or aluminum hydroxyphosphate are provided, which contain phosphate groups instead of some or all of the hydroxyl groups of the aluminum hydroxide adjuvant. In embodiments, the aluminum phosphate adjuvants provided herein are amorphous and soluble in acidic, basic, and neutral media.

[0150] In embodiments, the adjuvants disclosed herein include both aluminum phosphate and aluminum hydroxide. In one embodiment, the adjuvant has a greater amount of aluminum phosphate than aluminum hydroxide, for example, the weight ratio of aluminum phosphate to aluminum hydroxide is 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or higher. More specifically, the aluminum salt in the vaccine is present in amounts of 0.4 to 1.0 mg per vaccine dose, or 0.4 to 0.8 mg per vaccine dose, or 0.5 to 0.7 mg per vaccine dose, or about 0.6 mg per vaccine dose.

[0151] Generally, the choice of aluminum-based adjuvants (one or more), or the ratio of multiple aluminum-based adjuvants such as aluminum phosphate to aluminum hydroxide, is selected by optimizing the intermolecular electrostatic attraction so that the antigen has opposite charges to the adjuvant at the desired pH. For example, aluminum phosphate adjuvant (iep=4) adsorbs lysozyme at pH 7.4 but not albumin. If albumin is the target, aluminum hydroxide adjuvant would be selected (i.e., 11.4). Alternatively, pre-treating aluminum hydroxide with a phosphate lowers its isoelectric point, making it a preferred adjuvant for more basic antigens. Oil-emulsion

[0152] Suitable oil-emulsion compositions for use as adjuvants include squalene-water emulsions such as MF59 (formulated as submicron particles using a microfluidizer, containing 5% squalene, 0.5% TWEEN® 80, and 0.5% SPAN® 85). See International Publication No. WO90 / 14837. MF59 is used as an adjuvant in the FLUAD™ influenza virus trivalent subunit vaccine.

[0153] The adjuvants used in the composition are submicron oil-in-water emulsions. The submicron oil-in-water emulsions for use herein may be squalene / water emulsions optionally containing various amounts of MTP-PE, for example, the oil-in-water emulsion may contain 4-5% w / v squalene, 0.25-1.0% w / v TWEEN® 80 (polyoxyethylene sorbitan monooleate), and / or 0.25-1.0% SPAN® 85 (sorbitan trioleate), and optionally N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(β-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine The emulsion may also contain yl-sn-glycero-3-huydroxyphosphosphoryloxy)-ethylamine (MTP-PE), for example, a submicron oil-in-water emulsion known as "MF59" (International Publication No. WO90 / 14837; U.S. Patent Nos. 6,299,884 and 6,451,325). MF59 contains 4-5% w / v squalene (e.g., 4.3%), 0.25-0.5% w / v TWEEN® 80, and 0.5% w / v SPAN® 85, and optionally, various amounts of MTP-PE formulated into submicron particles using a microfluidizer such as the Model 110Y microfluidizer (Microfluidics, Newton, Mass.). For example, MTP-PE may be present in amounts of approximately 0-500 μg / dose, 0-250 μg / dose, and 0-100 μg / dose. As used herein, the term "MF59-0" refers to the above-mentioned submicron oil-in-water emulsion lacking MTP-PE, while the term "MF59-MTP" refers to a formulation containing MTP-PE. For example, "MF59-100" contains 100 mu g of MTP-PE per dose.Another submicron oil-in-water emulsion for use herein, MF69, contains 4.3% w / v squalene, 0.25% w / v TWEEN® 80, and 0.75% w / v SPAN® 85, and optionally MTP-PE. Yet another submicron oil-in-water emulsion is MF75, also known as SAF, which contains 10% squalene, 0.4% TWEEN® 80, 5% Pluronic® block polymer L121, and thr-MDP, and is also microfluidized into a submicron emulsion. MF75-MTP refers to an MF75 formulation containing MTP, for example, 100-400 μg of MTP-PE per dose.

[0154] Submicron oil-in-water emulsions for use in this composition, methods for producing the same, and immunostimulants such as muramil peptides are described in detail in International Publication No. WO90 / 14837 and U.S. Patents No. 6,299,884 and 6,451,325.

[0155] Complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA) may also be used as adjuvants. Saponin preparations

[0156] Saponin preparations may also be used as adjuvants. Saponins are a heterogeneous group of sterol glycosides and triterpenoid glycosides found in the bark, leaves, stems, roots, and even flowers of a wide range of plant species. Saponins isolated from the bark of Quillaia saponaria Molina have been widely studied as adjuvants. Saponins are also commercially available from Smilax ornata (sarsaparilla), Gypsophila paniculata (brides veil), and Saponaria officinalis (Saponaria officianalis) (soap root). Saponin adjuvant preparations include purified preparations such as QS21, as well as lipid preparations such as ISCOM.

[0157] Saponin compositions are purified using high-performance thin-layer chromatography (HP-TLC) and reverse-phase high-performance liquid chromatography (RP-HPLC). Specific purified fractions obtained using these techniques have been identified as containing QS7, QS17, QS18, QS21, QH-A, QH-B, and QH-C. In embodiments, the saponin is QS21. A method for producing QS21 is disclosed in U.S. Patent No. 5,057,540. Saponin preparations may also contain sterols such as cholesterol (see International Publication No. WO96 / 33739).

[0158] Combinations of saponins and cholesterol may be used to form unique particles called immunostimulatory complexes (ISCOMs). ISCOMs typically also contain phospholipids such as phosphatidylethanolamine or phosphatidylcholine. Any known saponin may be used in ISCOMs. In embodiments, ISCOMs comprise one or more of Quil A, QHA, and QHC. ISCOMs are further described in European Patent Application Publication EP0109942, International Publication WO96 / 11711, and International Publication WO96 / 33739. Optionally, ISCOMs may not contain (additional) detergents (one or more). See International Publication WO00 / 07621. Bacteria or microbial derivatives

[0159] Adjuvants suitable for use as disclosed herein include bacterial or microbial derivatives such as:

[0160] (1) Non-toxic derivatives of intestinal bacterial lipopolysaccharide (LP)

[0161] Such derivatives include monophosphoryl lipid A (MPL) and 3-O-deacylated MPL (3dMPL). 3dMPL is a mixture of 3-de-O-acylated monophosphoryl lipid A having 4, 5, or 6 acylated chains. One “small particle” form of 3-De-O-acylated monophosphoryl lipid A is disclosed in European Patent Application EP 0 689 454. Such “small particles” of 3dMPL are small enough to be aseptically filtered through a 0.22 micron membrane (see European Patent Application Publication EP 0 689 454). Other non-toxic LPS derivatives include monophosphoryl lipid A mimics, such as aminoalkylglucosaminide phosphate derivatives, such as RC-529.

[0162] (2) Lipid A derivatives

[0163] Lipid A derivatives include E. coli-derived lipid A derivatives such as OM-174.

[0164] (3) Immunostimulatory oligonucleotides

[0165] Immunostimulant oligonucleotides suitable for use as adjuvants may include nucleotide sequences containing a CpG motif (a sequence consisting of unmethylated cytosine followed by guanosine, linked by a phosphate bond). Bacterial double-stranded RNA or oligonucleotides containing palindromic or poly(dG) sequences have also been shown to be immunostimulant.

[0166] CpG may contain nucleotide modifications / analogs such as phosphorothioate modifications, and may be double-stranded or single-stranded. Optionally, guanosine may be substituted with an analog such as 2'-deoxy-7-deazaguanosine.

[0167] The CpG sequence may be directed to a TLR9 such as the motif GTCGTT or TTCGTT. The CpG sequence may be specific to induce a Thl immune response, such as CpG-A ODN, or specific to induce a B cell response, such as CpG-B ODN. In this embodiment, the CpG is CpG-A ODN.

[0168] In some embodiments, the CpG oligonucleotide may be constructed such that its 5' end is accessible for receptor recognition. Optionally, two CpG oligonucleotide sequences may be joined at their 3' ends to form an "immunomer".

[0169] (4) ADP-ribosylated toxin and its detoxified derivatives

[0170] Bacterial ADP-ribosylated toxins and their detoxified derivatives may be used as adjuvants. In embodiments, the proteins may be derived from Escherichia coli (i.e., E. coli heat-unstable enterotoxin "LT"), cholera ("CT"), or pertussis ("PT"). The use of detoxified ADP-ribosylated toxins as mucosal adjuvants is described in International Publication WO95 / 17211, and as parenteral adjuvants in International Publication WO98 / 42375. In embodiments, the adjuvants are detoxified LT variants such as LT-K63, LT-R72, and LTR192G. Bioadhesives and Mucoadhesives

[0171] Bioadhesives and mucosal adhesives may also be used as adjuvants. Suitable bioadhesives include esterified hyaluronic acid microspheres and adhesives such as polyacrylic acid, polyvinyl alcohol, polyvinylpyrrolidone, polysaccharides, and cross-linked derivatives of carboxymethylcellulose. Chitosan and its derivatives can also be used as adjuvants. For example, see International Publication No. WO99 / 27960. Muramyl peptide

[0172] Examples of muramyl peptides suitable for use as adjuvants include N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetylnormuramyl-L-alanyl-D-isoglutamine (nor-MDP), and N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(l'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine MTP-PE). Imidazoquinoline compounds

[0173] Examples of imidazoquinoline compounds suitable for use as adjuvants include imiquimod and its analogues (see, for example, U.S. Patents Nos. 4,689,338, 5,389,640, 5,268,376, 4,929,624, 5,266,575, 5,352,784, 5,494,916, 5,482,936, 5,346,905, 5,395,937, 5,238,944, and 5,525,612). Thiosemiccarbazone compounds

[0174] Examples of thiosemicarbazone compounds suitable for use as adjuvants, as well as methods for formulation, manufacturing, and screening of these compounds, are all included in International Publication No. W004 / 60308. Thiosemicarbazone is particularly effective in stimulating human peripheral blood mononuclear cells for cytokine production, such as TNF-α. Tryptanthrin compounds

[0175] Examples of tryptanthrin compounds suitable for use as adjuvants disclosed herein, as well as methods for formulation, manufacturing, and screening of these compounds, all include those described in International Publication No. WO04 / 64759. Tryptanthrin compounds are particularly effective in stimulating human peripheral blood mononuclear cells for cytokine production, such as TNF-α.

[0176] A combination of one or more aspects of the adjuvants identified above may be applied to compositions such as those disclosed herein. For example, the following adjuvant compositions may be used:

[0177] (1) Saponin and oil-in-water emulsion (WO99 / 11241); (2) Saponin (e.g., QS21) + non-toxic LPS derivative (e.g., 3dMPL) (see WO94 / 00153); (3) Saponin (e.g., QS21) + non-toxic LPS derivative (e.g., 3dMPL) + cholesterol; (4) Saponin (e.g., QS21) + 3dMPL + IL-12 (optionally + sterol) (WO98 / 57659); (5) Combination of 3dMPL with e.g. QS21 and / or oil-in-water emulsion (see European Patent Applications 0835318, 0735898 and 0761231); (6) 10% squalane, 0.4% SAF containing TWEEN® 80, 5% Pluronic® Block Polymer L121, and thr-MDP, which can be formed into a submicron emulsion with microfluidics or into a large particle size emulsion with vortexing. (7) RIBI® Adjuvant System (RAS), (Ribi Immunochem), containing 2% squalene, 0.2% TWEEN® 80, and one or more bacterial cell wall components from the group consisting of monophospholipid A (MPL), trehalose dimicholate (TDM), and cell wall skeleton (CWS), MPL+CWS(DETOX®); and (8) one or more mineral salts (such as aluminum salts) + non-toxic derivatives of LPS (such as 3dPML). (9) One or more mineral salts (e.g., aluminum salts) + one or more immunostimulant oligonucleotides (e.g., nucleotide sequences containing a CpG motif) + one or more detoxified ADP-ribosylated toxins (e.g., LT-K63 and LT-R72), (10) Inulin and inulin acetate preparations (see, for example, International Publication WO 2013 / 110050, which is incorporated herein in its entirety). Other antigens

[0178] The compositions disclosed herein may optionally contain one or more additional polypeptide antigens not derived from ASF viral proteins. Such antigens may include bacterial, viral, or parasitic antigens.

[0179] In some embodiments, the ASF virus antigens include orthomyxovirus (influenza), pneumovirus (RSV), paramyxovirus (PIV and mumps), morbillivirus (measles), togavirus (rubella), enterovirus (polio), HBV, coronavirus (SARS), and varicella-zoster virus (VZV), Epstein-Barr virus (EBV), pneumococcus (Streptococcus pneumoniae), meningococcus (Neisseria meningitides), Streptococcus pyogenes (Group A Streptococcus), Moraxella catarrhalis, Bordetella pertussis, Staphylococcus aureus, and Clostridium tetani. It is combined with one or more antigens, including (but not limited to) antigens derived from bacteria or viruses such as tetanus (tetani), Cornynebacterium diphtheriae (diphtheria), Haemophilus influenzae B (Hib), Pseudomonas aeruginosa, Streptococcus agalactiae (Group B Streptococcus), and Escherichia coli.

[0180] In other embodiments, the ASF virus antigen may include, but is not limited to, Neisseria meningitides, Streptococcus pyogenes (Group A Streptococcus), Moraxella catarrhalis, Bordetella pertussis, Staphylococcus aureus, Staphylococcus epidermis, Clostridium tetani (tetanus), Cornynebacterium diphtheriae (diphtheria), Haemophilus influenzae B (Hib), Pseudomonas aeruginosa, and Klebsiella pneumoniae. It is combined with one or more antigens, including *Streptococcus pneumophila*, *Streptococcus agalactiae* (Group B Streptococcus), *Enterococcus faecalis*, *Helicobacter pylori*, *Clamydia pneumoniae*, *Orthomyxovirus (Influenza)*, *Pneumovirus (RSV)*, *Paramyxovirus (PIV and Mumps)*, *Morbillivirus (Measles)*, *Togavirus (Rubella)*, *Enterovirus (Polio)*, *HBV*, *Coronavirus (SARS)*, *Varicella varicella-zoster virus (VZV)*, *Epstein-Barr virus (EBV)*, and *Cytomegalovirus (CMV)*.

[0181] In other embodiments, the ASF virus antigen is combined with one or more antigens useful in a vaccine designed to protect an individual from pathogens that cause diarrheal diseases. Such antigens include, but are not limited to, rotavirus, Shigella spp., enterotoxigenic Escherichia coli (ETEC), Vibrio cholerae, and Campylobacter jejuni antigens. In embodiments, one or more norovirus antigens may be derived from Norwalk virus, Snow Mountain virus, and / or Hawaii virus and are combined with rotavirus antigen in an immunogenic composition.

[0182] Antigens that can be found to be used in conjunction with this composition include, but are not limited to, one or more antigens defined below, or antigens derived from one or more pathogens defined below: bacterial antigen

[0183] Preferred bacterial antigens disclosed herein include proteins, polysaccharides, lipopolysaccharides, and outer membrane vesicles, which may be isolated, purified, or derived from bacteria. Furthermore, bacterial antigens may include bacterial lysates and inactivated bacterial preparations. Bacterial antigens may be produced by recombinant expression. Bacterial antigens include epitopes that can be exposed on the surface of a bacterium at at least one stage of its life cycle. Bacterial antigens may be conserved across multiple serotypes. Bacterial antigens include antigens derived from one or more of the bacteria specified below, as well as examples of specific antigens specified below.

[0184] Neisseria meningitides: Meningitis antigens may include proteins purified from or derived from N. meningitides serogroups such as A, C, W135, Y, and / or B (as identified in References 1–7), saccharides (including polysaccharides, oligosaccharides, or liposaccharides), or outer membrane vesicles. Meningitis protein antigens may be selected from adhesion, autotransporters, toxins, iron-gathering proteins, and membrane-related proteins (e.g., integral outer membrane proteins).

[0185] Streptococcus pneumoniae: Pneumococcal antigens may include sugars (including polysaccharides or oligosaccharides) and / or proteins derived from Streptococcus pneumoniae. Sugar antigens may be selected from serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F. Protein antigens may be selected from proteins identified in International Publication Nos. WO98 / 18931, WO98 / 18930, U.S. Patent Nos. 6,699,703, 6,800,744, International Publication Nos. WO97 / 43303, and WO97 / 37026. The pneumococcus protein may be selected from the polyhistidine triad family (PhtX), the choline-binding protein family (CbpX), CbpX cleavage proteins, the LytX family, LytX cleavage proteins, CbpX cleavage-LytX cleavage chimeric proteins, pneumolysin (Ply), PspA, PsaA, Spl28, SplOl, Spl30, Spl25, or Spl33.

[0186] Streptococcus pyogenes (Group A streptococcus): Group A streptococcal antigens may include proteins identified in International Publication WO 02 / 34771 or International Publication WO 2005 / 032582 (including GAS 40), fusions of GAS M protein fragments (including those described in International Publication WO 02 / 094851), fibronectin-binding protein (Sfbl), streptococcal heme-associated protein (Shp), and streptoricin S (SagA).

[0187] Moraxella catarrhalis: Moraxella antigens include the antigens identified in International Publications WO 02 / 18595 and WO 99 / 58562, the outer membrane protein antigen (HMW-OMP), the C-antigen, and / or LPS.

[0188] Bordetella pertussis: Pertussis antigens include petussis holotoxin (PT) and filamentous hemagglutinin (FHA) derived from Bordetella pertussis, and optionally combinations with pertactin and / or agglutinogen 2 and 3 antigens.

[0189] Staphylococcus aureus: Staphylococcus aureus antigens include Staphylococcus aureus type 5 and type 8 capsular polysaccharides and are optionally bound to antigens derived from non-toxic recombinant Pseudomonas aeruginosa exotoxin A (e.g., STAPHVAX®), or surface proteins, invasin (leucocidin, kinase, hyaluronidase), surface factors that inhibit phagocytic cell entrapment (capsule, protein A), carotenoids, catalase production, protein A, coagulation factors, and / or membrane-damaging toxins (optionally detoxified) that lyse eukaryotic cell membranes (hemolysin, leukotoxin, leucocidin).

[0190] Staphylococcus epidermis: Staphylococcus epidermis antigens include slime-associated antigens (SAA).

[0191] Clostridium tetani (tetanus): Tetanus antigen, comprising tetanus toxoid (TT), may be used as a carrier protein in combination with / conjugated to the compositions of this disclosure.

[0192] Cornynebacterium diphtheriae (diphtheria): The diphtheria antigen is CRM 197 This includes diphtheria toxins, including detoxified forms such as [specific examples of diphtheria toxins]. Furthermore, antigens that can modulate, inhibit, or associate ADP-ribosylation are intended for use in combination / co-administration / conjugation with the compositions of this disclosure. Diphtheria toxoids may be used as carrier proteins.

[0193] Haemophilus influenzae B (Hib): Hib antigens include Hib saccharide antigens.

[0194] Pseudomonas aeruginosa: Pseudomonas antigens include endotoxin A, Wzz protein, Pseudomonas LPS, more specifically LPS isolated from PAO1 (O5 serotype), and / or outer membrane proteins (including outer membrane protein F (OprF)).

[0195] Legionella pneumophila. The bacterial antigen may be derived from Legionella pneumophila.

[0196] Streptococcus agalactiae (Group B Streptococcus): Group B Streptococcus antigens include proteins or glycan antigens identified in International Publications WO 02 / 34771, WO 03 / 093306, WO 04 / 041157, or WO 2005 / 002619 (including proteins GBS 80, GBS 104, GBS 276, and GBS 322, and glycan antigens derived from serotypes la, lb, la / c, II, III, IV, V, VI, VII, and VIII).

[0197] Neiserria gonorrhoeae: Gonorrhea antigens include Por (or porin) proteins such as PorB, transfer-binding proteins such as TbpA and TbpB, opacity proteins (such as Opa), reductivable proteins (Rmp), and outer membrane vesicle (OMV) preparations (see, for example, International Publications WO99 / 24578, WO99 / 36544, WO99 / 57280, and WO02 / 079243).

[0198] Chlamydia trachomatis: Chlamydia trachomatis antigens include antigens derived from serotypes A, B, Ba and C (pathogens of trachoma, which causes blindness), serotypes L1, L2 and L3 (associated with lymphogranuloma of the inguinal region), and serotype DK. Chlamydia trachoma antigens may also include antigens identified in International Publication WO 00 / 37494, International Publication WO 03 / 049762, International Publication WO 03 / 068811, or International Publication WO 05 / 002619, including PepA (CT045), LcrE (CT089), ArtJ (CT381), DnaK (CT396), CT398, OmpH-like (CT242), L7 / L12 (CT316), OmcA (CT444), AtosS (CT467), CT547, Eno (CT587), HrtA (CT823), and MurG (CT761).

[0199] Treponema pallidum (syphilis): Syphilis antigens include the TmpA antigen.

[0200] Haemophilus ducreyi (cause of chancre): The Ducrey antigen contains an outer membrane protein (DsrA).

[0201] Enterococcus faecalis or Enterococcus faecium: The antigens include trisaccharide repeats provided in U.S. Patent No. 6,756,361 or other Enterococcus-derived antigens.

[0202] Helicobacter pylori: H. pylori antigens include Cag, Vac, Nap, HopX, HopY, and / or urease antigens.

[0203] Staphylococcus saprophyticus: The antigen contains 160kDa hemagglutinin of the S. saprophyticus antigen.

[0204] Yersinia enterocolitica antigen contains LPS.

[0205] Escherichia coli (E. coli): E. coli antigens may be derived from enterotoxigenic Escherichia coli (ETEC), enteroaggregative Escherichia coli (EAggEC), diffusely adhering E. coli (DAEC), enteropathogenic Escherichia coli (EPEC), and / or enterohemorrhagic Escherichia coli (EHEC).

[0206] Bacillus anthracis (Anthrax Bacillus): The B. anthracis antigen may be optionally detoxified and selected from component A (lethal factor (LF) and edema factor (EF)), both of which may share a common component B known as the protective antigen (PA).

[0207] Yersinia pestis (plague): The plague antigen includes the Fl capsule antigen.

[0208] Mycobacterium tuberculosis: Mycobacterium tuberculosis antigens include lipoproteins, LPS, BCG antigen, optionally a fusion protein of antigen 85B (Ag85B) and / or ESAT-6 formulated into cationic lipid vesicles, Mycobacterium tuberculosis (Mtb) isocitrate dehydrogenase-associated antigen, and / or MPT51 antigen.

[0209] Rickettsia: Antigens include outer membrane proteins, including outer membrane protein A and / or B (OmpB).

[0210] Listeria monocytogenes. The bacterial antigen may be derived from Listeria monocytogenes.

[0211] Chlamydia pneumoniae: Antigens include those identified in International Publication No. WO 02 / 02606.

[0212] Vibrio cholerae: Antigens include proteinase antigens, LPS, particularly the lipopolysaccharide of Vibrio cholerae II, the O1 Inaba O-specific polysaccharide, the antigens of V. cholera 0139 and the IEM108 vaccine, and / or Zonula occludens toxin (Zot).

[0213] Salmonella typhi (typhoid fever): The antigen contains a capsular polysaccharide that includes a conjugate (Vi, i.e., vax-TyVi).

[0214] Borrelia burgdorferi (Lyme disease): Antigens include lipoproteins (such as OspA, OspB, Osp C, and Osp D), other surface proteins such as OspE-related proteins (Erps), decorin-binding proteins (such as DbpA), and antigen variant VI proteins such as antigens associated with P39 and P13 VlsE antigen variant proteins.

[0215] Porphyromonas gingivalis: The antigen includes P. gingivalis outer membrane protein (OMP).

[0216] Klebsiella: Antigens include OMP containing OMP A, or polysaccharides optionally bound to tetanus toxoid.

[0217] Further bacterial antigens of this disclosure may be any of the capsular antigens, polysaccharide antigens, or protein antigens described above. Further bacterial antigens may also include outer membrane vesicle (OMV) preparations. Furthermore, the antigens may include live, attenuated, and / or purified versions of any of the aforementioned bacteria. The antigens of this disclosure may be derived from Gram-negative or Gram-positive bacteria. The antigens of this disclosure may be derived from aerobic or anaerobic bacteria.

[0218] Furthermore, one of the above bacterial-derived saccharides (polysaccharide, LPS, LOS, or oligosaccharide) is used as a carrier protein (e.g., CRM). 197 ) may be conjugated to other drugs or antigens, such as ). Such conjugations may be directly conjugated by reductive amination of the carbonyl moiety on the saccharide with the amino group on the protein, as described in U.S. Patent No. 5,360,897. Alternatively, the saccharide may be conjugated via a linker having succinamide or other linkages, for example. Viral antigens

[0219] Viral antigens suitable for use in compositions such as those disclosed include subunit preparations, viral proteins that may be purified, isolated from, or derived from viruses, and virus-like particles (VLPs). Viral antigens may be derived from viruses grown in cell culture or on other substrates. Alternatively, viral antigens may be recombinantly expressed. Viral antigens include epitopes exposed on the viral surface at at least one stage of their life cycle. Viral antigens may be conserved across multiple serotypes or isolates. Viral antigens include antigens derived from one or more of the viruses defined below, as well as specific antigen examples specified below.

[0220] Orthomyxovirus: The orthomyxovirus antigen may be derived from orthomyxoviruses such as influenza A, B, and C. The orthomyxovirus antigen may be selected from one or more viral proteins containing one or more of the following: hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix protein (M1), membrane protein (M2), and transcriptase components (PB1, PB2, and PA). In embodiments, the antigen includes HA and NA.

[0221] The influenza antigen may be derived from an influenza strain during an epidemic period (one year). Alternatively, the influenza antigen may be derived from a strain that has the potential to cause a pandemic (i.e., an influenza strain with new hemagglutinin compared to the hemagglutinin of currently circulating strains, or an influenza strain that is pathogenic in birds and can be transmitted horizontally in human populations, or an influenza strain that is pathogenic to humans).

[0222] Paramyxoviridae viruses: Viral antigens may be derived from paramyxoviridae viruses such as pneumovirus (RSV), paramyxovirus (PIV), and morbillivirus (measles).

[0223] Pneumovirus: The viral antigen may be derived from pneumoviruses such as respiratory syncytial virus (RSV), bovine respiratory syncytial virus, mouse pneumonia virus, and turkic rhinotracheitis virus. In embodiments, the pneumovirus is RSV. The pneumovirus antigen may be selected from one or more of the following proteins: surface protein fusion (F), glycoprotein (G) and small hydrophobic protein (SH), matrix proteins M and M2, nucleocapsid proteins N, P and L, and non-structural proteins NS1 and NS2. The pneumovirus antigen may include F, G and M. The pneumovirus antigen may also be incorporated into or derived from a chimeric virus. For example, a chimeric RSV / PIV virus may contain components of both RSV and PIV.

[0224] Paramyxovirus: The viral antigen may be derived from paramyxoviruses such as parainfluenza viruses types 1-4 (PIV), mumps, Sendai virus, Simian virus 5, bovine parainfluenza virus, and Newcastle disease virus. In embodiments, the paramyxovirus is PIV or mumps. The paramyxovirus antigen may be selected from one or more of the following proteins: hemagglutinin-neuraminidase (HN), fusion proteins Fl and F2, nucleoprotein (NP), phosphoprotein (P), large protein (L), and matrix protein (M). The paramyxovirus protein may include HN, Fl, and F2. The paramyxovirus antigen may also be compounded with or derived from a chimeric virus. For example, a chimeric RSV / PIV virus may contain components of both RSV and PIV. Commercial mumps vaccines contain attenuated live mumps virus and are used monovalent or in combination with a measles-rubella vaccine (MMR).

[0225] Morbillivirus: Viral antigens may be derived from morbilliviruses such as measles. Morbillivirus antigens may be selected from one or more of the following proteins: hemagglutinin (H), glycoprotein (G), fusion factor (F), large protein (L), nucleoprotein (NP), polymerase phosphoprotein (P), and matrix (M). Commercially available measles vaccines contain attenuated live measles virus and are usually used in combination with mumps and rubella (MMR).

[0226] Picornavirus: Viral antigens may be derived from picornaviruses, such as enteroviruses, rhinoviruses, heparnaviruses, cardioviruses, and aphthoviruses. Antigens derived from enteroviruses such as poliovirus may also be used.

[0227] Enterovirus: The viral antigen may be derived from enteroviruses such as poliovirus types 1, 2, or 3, coxsackie A viruses types 1-22 and 24, coxsackie B viruses types 1-6, echovirus (ECHO) viruses types 1-9, 11-27 and 29-34, and enterovirus types 68-71. In some embodiments, the enterovirus may be poliovirus. The enterovirus antigen may contain one or more of the following capsid proteins: VP1, VP2, VP3, and VP4. Commercially available polio vaccines include inactivated polio vaccine (IPV) and oral poliovirus vaccine (OPV).

[0228] Heparnavirus: The viral antigen may be derived from heparnaviruses such as hepatitis A virus (HAV). Commercially available HAV vaccines include inactivated HAV vaccines.

[0229] Togavirus: The viral antigen may be derived from a togavirus such as rubivirus, alphavirus, or arterivirus. A rubivirus-derived antigen, such as rubella virus, may also be used. Togavirus antigens may be selected from El, E2, E3, C, NSP-1, NSPO-2, NSP-3, or NSP-4. Togavirus antigens include El, E2, or E3. Commercially available rubella vaccines contain live cold-adapted virus and are typically used in combination with the mumps and measles vaccine (MMR).

[0230] Flavivirus: The viral antigen may be derived from flaviviruses, such as tick-borne encephalitis (TBE), dengue fever (type 1, 2, 3, or 4), yellow fever, Japanese encephalitis, West Nile encephalitis, St. Louis encephalitis, Russian spring-summer encephalitis, or Poissant encephalitis. The flavivirus antigen may be selected from PrM, M, C, E, NS-1, NS-2a, NS2b, NS3, NS4a, NS4b, and NS5. The flavivirus antigen may include PrM, M, and E. Commercially available TBE vaccines contain an inactivated virus vaccine.

[0231] Pestivirus: The viral antigen may be derived from a pestivirus, such as bovine viral diarrhea (BVDV), classical swine fever (CSFV), or border disease (BDV).

[0232] Hepadnavirus: The viral antigen may be derived from hepadnaviruses such as hepatitis B virus. The hepadnavirus antigen may be selected from surface antigens (L, M, and S) and core antigens (HBc, HBe). Commercial HBV vaccines contain a subunit vaccine that includes the surface antigen S protein.

[0233] Hepatitis C virus: The viral antigen may be derived from hepatitis C virus (HCV). The HCV antigen may be selected from one or more peptides from El, E2, E1 / E2, NS345 polyprotein, NS345-core polyprotein, core, and / or non-structural regions.

[0234] Rhabdovirus: Viral antigens may be derived from rhabdoviruses such as lyssavirus (rabies virus) and becyclovirus (VSV). Rhabdovirus antigens can be selected from glycoproteins (G), nucleoproteins (N), large proteins (L), and non-structural proteins (NS). Commercially available rabies virus vaccines contain viricidal agents grown in human diploid cells or rhesus monkey fetal lung cells.

[0235] Caliciviridae: Viral antigens may originate from Caliciviridae viruses, such as Norwalk virus, Hawaii virus, and Snow Mountain virus (Norwalk-like viruses).

[0236] Coronavirus: The viral antigen may be derived from coronavirus, SARS, human respiratory coronavirus, avian infectious bronchitis (IBV), mouse hepatitis virus (MHV), and porcine infectious gastroenteritis virus (TGEV). The coronavirus antigen may be selected from the spike (S), envelope (E), matrix (M), nucleocapsid (N), and hemagglutinin-esterase glycoprotein (HE). In embodiments, the coronavirus antigen is derived from the SARS virus. The SARS virus antigen is described in International Publication No. WO 04 / 92360;.

[0237] Retroviruses: Viral antigens may be derived from retroviruses such as oncovirus, lentivirus, or spumavirus. Oncovirus antigens may be derived from HTLV-1, HTLV-2, or HTLV-5. Lentiviral antigens may be derived from HIV-1 or HIV-2. Retroviral antigens may be selected from gag, pol, env, tax, tat, rex, rev, nef, vif, vpu, and vpr. HIV antigens may be selected from gag (p24gag and p55gag), env (gpl60 and gp41), pol, tat, nef, rev, vpu, miniproteins (e.g., p55gag and gpl40v deletion). HIV antigens may be derived from one or more of the following strains: HIV IIIb HIV SF2 HIV LAV HIV LAI HIV MN HIV-1 CM235 HIV-1 US4 .

[0238] Reovirus: The viral antigen may be derived from a reovirus such as orthoreovirus, rotavirus, orbivirus, or cortivirus. The reovirus antigen may be selected from structural proteins λ1, λ2, λ3, μ1, μ2, σ1, σ2, or σ3, or non-structural proteins σNS, μNS, or σ1. The reovirus antigen may be derived from rotavirus. The rotavirus antigen may be selected from VP1, VP2, VP3, VP4 (or cleavage products VP5 and VP8), NSP1, VP6, NSP3, NSP2, VP7, NSP4, or NSP5. The rotavirus antigen may include VP4 (or cleavage products VP5 and VP8) and VP7. For example, see International Publication Nos. WO 2005 / 021033, WO 2003 / 072716, WO 2002 / 11540, WO 2001 / 12797, WO 01 / 08495, WO 00 / 26380, and WO 02 / 036172 (their entirety is incorporated into this book by reference).

[0239] Parvovirus: The viral antigen may be derived from a parvovirus such as parvovirus Bl9. The parvovirus antigen may be selected from VP-1, VP-2, VP-3, NS-1, and NS-2. In one embodiment, the parvovirus antigen is the capsid protein VP-2.

[0240] Hepatitis delta virus (HDV): The viral antigen may be derived from HDV, particularly from HDV-derived delta antigen (see, for example, U.S. Patent No. 5,378,814).

[0241] Hepatitis E virus (HEV): The viral antigen may be derived from HEV.

[0242] Hepatitis G virus (HGV): The viral antigen may be derived from HGV.

[0243] Human herpesviruses: Viral antigens may be derived from human herpesviruses such as herpes simplex virus (HSV), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus 6 (HHV6), human herpesvirus 7 (HHV7), and human herpesvirus 8 (HHV8). Human herpesvirus antigens may be selected from immediate-type early protein (α), early protein (β), and late protein (γ). HSV antigens may be derived from HSV-1 or HSV-2 strains. HSV antigens may be selected from glycoproteins gB, gC, gD, and gH, fusion protein (gB), or immune evasion proteins (gC, gE, or gl). VZV antigens may be selected from core, nucleocapsid, tegument, or envelope proteins. Attenuated VZV vaccines are commercially available. The EBV antigen may be selected from the initial antigen (EA) protein, the viral capsid antigen (VCA), and the membrane antigen (MA) glycoprotein. The CMV antigen may be selected from the capsid protein, the envelope glycoprotein (e.g., gB and gH), and the tegument protein.

[0244] Papovavirus: The antigen may be derived from papovaviruses such as papillomavirus and poliomavirus. Papillomaviruses include HPV serotypes 1, 2, 4, 5, 6, 8, 11, 13, 16, 18, 31, 33, 35, 39, 41, 42, 47, 51, 57, 58, 63, and 65. In embodiments, the HPV antigen is derived from serotype 6, 11, 16, or 18. The HPV antigen may include capsid proteins (LI) and (L2), or E1-E7, or fusions thereof. Polyomaviruses include BK virus and JK virus. The polyomavirus antigen may be selected from VP1, VP2, or VP3.

[0245] Circovirus: The antigen may be derived from circoviruses such as porcine circovirus (PCV) 1, PCV 2, PCV 3, and PCV 4. fungal antigen

[0246] Suitable fungal antigens may be derived from one or more of the following fungi:

[0247] The fungal antigen may be derived from dermatophytes, including the following: Epidermophyton floccusum, Microsporum audouini, Microsporum canis, Microsporum distortum, Microsporum equinum, Microsporum gypsum, Microsporum nanum, Trichophyton concentricum, Trichophyton equinum, Trichophyton gallinae, and Trichophyton gypseum. Trichophyton gypseum), Trichophyton megnini, Trichophyton mentagrophytes, Trichophyton quinckeanum, Trichophyton rubrum, Trichophyton schoenleini, Trichophyton tonsurans, Trichophyton verrucosum, T. verrucosum var. album, var. discoides, var. ochraceum, Trichophyton violaceum (Trichophyton This includes *Violaceum* and / or *Trichophyton faviforme*. .

[0248] The fungal pathogens include Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, Aspergillus terreus, Aspergillus sydowi, Aspergillus flavatus, Aspergillus glaucus, Blastoschizomyces capitatus, Candida albicans, Candida enolase, and Candida tropicalis. Candida tropicalis, Candida glabrata, Candida krusei, Candida parapsilosis, Candida stellatoidea, Candida kusei, Candida parakwsei, Candida lusitaniae, Candida pseudotropicalis, Candida guilliermondi, Cladosporium carrionii, Coccidioides immitis, Blastomyces dermatidis (dermatidis), Cryptococcus neoformans, Geotrichum clavatum, Histoplasma capsulatum, Klebsiella pneumoniaePneumoniae, Paracoccidioides brasiliensis, Pneumocystis carinii, Pythium insidiosum, Pityrosporum ovale, Saccharomyces cerevisae, Saccharomyces boulardii, Saccharomyces pombe, Scedosporium apiosperum, Sporothrix schenckii, Trichosporon beigelii, Toxoplasma gondii gondii), Penicillium marneffei, Malassezia spp., Fonsecaea spp., Wangiella spp., Sporothrix spp., Basidiobolus spp., Conidiobolus spp., Rhizopus spp., Mucor spp., Absidia spp., Mortierella spp., Cunninghamella spp., Saxenaea spp., Alternaria spp.) Curvularia spp., Helminthosporium spp., Fusarium spp., Aspergillus spp., Penicillium spp., Monolinia spp., Rhizoctonia spp., PaecilomycesIt may also be derived from the genera Pithomyces spp. and Cladosporium spp.

[0249] Processes for producing fungal antigens are well known in the art (see U.S. Patent No. 6,333,164). One method, comprising the steps of: obtaining living fungal cells; obtaining fungal cells with substantially or partially removed cell walls; rupturing the fungal cells with substantially or partially removed cell walls; obtaining an insoluble fraction; and extracting and separating a solubilized fraction from the insoluble fraction. Respiratory Antigens

[0250] Compositions such as those disclosed herein may contain one or more antigens derived from pathogens that cause respiratory diseases. For example, respiratory antigens may be derived from respiratory viruses such as orthomyxovirus (influenza), pneumovirus (RSV), paramyxovirus (PIV), morbillivirus (measles), togavirus (rubella), VZV, and coronavirus (SARS). Respiratory antigens may also be derived from bacteria that cause respiratory diseases such as Streptococcus pneumoniae, Pseudomonas aeruginosa, Bordetella pertussis, Mycobacterium tuberculosis, Mycoplasma pneumoniae, Chlamydia pneumoniae, Bacillus anthracis, and Moraxella catarrhalis. Examples of specific antigens derived from these pathogens are as described above.

[0251] The immunogenic compositions disclosed herein may be prepared in various forms. For example, a composition may be prepared as a liquid solution or suspension for injection. A solid form suitable for solution or suspension in a liquid vehicle before injection may also be prepared (e.g., lyophilized composition or spray lyophilized composition). A composition may be prepared for topical administration, for example, as an ointment, cream or powder. A composition may be prepared for oral administration, for example, as a tablet or capsule or as a spray. A composition may be prepared for pulmonary administration, for example, as a fine powder or spray in an inhaler. A composition may be prepared as a suppository or pessary. A composition may be prepared for administration to the nasal cavity, ear or eye, for example, as a drop. The preparation of such pharmaceutical compositions is within the realm of general art for those skilled in the art. See, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 18th edition, 1990.

[0252] The compositions may be in the form of kits, designed so that the combined compositions are reconstituted immediately before administration to a patient. Such kits may, in liquid form, comprise one or more antigens or nucleic acids encoding such antigens, and any additional antigens and adjuvants as described herein.

[0253] An immunogenic composition containing a disclosed polypeptide antigen is a vaccine composition. The pH of such a composition is 6–8, about 7, and the pH may be maintained by the use of a buffer. The composition may be sterile and / or pyrogen-free. The composition may be isotonic with respect to the subject. The vaccines according to this disclosure may be used prophylactically or therapeutically, but are typically prophylactic and may be used to treat animals (including farming, game, companion, and experimental mammals).

[0254] The immunogenic composition used as a vaccine comprises an immunologically effective amount of an antigen(s) and / or nucleic acid encoding an antigen(s), and, as may be, other components. “Immunologically effective amount” means that, in either a single dose or a series of doses, that amount administered to an individual is effective in treating or preventing disease. This amount varies depending on the health and physical condition of the individual being treated, age, taxonomy of the individual being treated (e.g., pig, cattle), the individual’s immune system’s ability to synthesize antibodies, the desired level of protection, the vaccine formulation, the veterinarian’s assessment of the medical condition, and other relevant factors. The amount is expected to fall within a relatively broad range determined by routine testing. Administration

[0255] The compositions disclosed herein are generally administered directly to the subject. Direct delivery is by parenteral injection (e.g., subcutaneous, intraperitoneal, intravenous, intramuscular, or intrastitial space of tissue), or by mucosal administration, such as rectal, oral (e.g., tablets, sprays), vaginal, topical, transdermal (e.g., see International Publication WO99 / 27961), or transdermal (e.g., see International Publication WO02 / 074244 and International Publication W002 / 064162), nasal (e.g., see International Publication W003 / 028760), oral, ear, lung, or other mucosal administration. Immunogenic compositions may also be administered topically by direct transfer to the skin surface. Topical administration may be achieved without the use of instruments or by bringing the immunogenic composition into contact with bare skin using a bandage or bandage-like instrument (e.g., see U.S. Patent No. 6,348,450).

[0256] In embodiments, the mode of administration may be parenteral, mucosal, or a combination of mucosal and parenteral immunity. In one embodiment, the mode of administration is parenteral, mucosal, or a combination of mucosal and parenteral immunity in a total of one to two vaccinations at intervals of one to three weeks. In one embodiment, the route of administration includes, but is not limited to, oral administration, intramuscular administration, and a combination of oral and intramuscular administration.

[0257] It has already been demonstrated that mucosal and systemic immune responses to antigens such as Helicobacter pylori antigens can be enhanced by systemic boosting immunity following mucosal priming. In one embodiment, a method for treating an ASF virus infection comprises mucosal administration of a first immunogenic composition containing one or more ASF virus antigens to a subject in need, followed by parenteral administration of a therapeutically effective amount of a second immunogenic composition containing one or more ASF virus antigens.

[0258] The immunogenic composition may be used to induce systemic immunity and / or mucosal immunity, or to induce enhanced systemic immunity and / or mucosal immunity.

[0259] In this embodiment, the immune response is characterized by the induction of a serum IgG and / or intestinal IgA immune response.

[0260] As described above, the prime-boost method may be employed in which one or more gene vectors and / or polypeptide antigens are delivered in a “priming” step, followed by one or more second gene vectors and / or polypeptide antigens being delivered in a “boosting” step. In certain embodiments, priming and boosting with one or more gene vectors or polypeptide antigens as described herein are followed by additional boosting with one or more polypeptide-containing compositions (e.g., polypeptides containing ASF virus antigens).

[0261] In any method including co-administration, the various compositions may be delivered in any order. Therefore, in embodiments involving the delivery of multiple different compositions or molecules, the nucleic acids do not all need to be delivered before the polypeptides. For example, the priming step may include the delivery of one or more polypeptides, and the boosting step may include the delivery of one or more nucleic acids and / or one or more polypeptides. Multiple polypeptide administrations may be followed by multiple nucleic acid administrations, and polypeptide and nucleic acid administrations may be carried out in any order. Therefore, one or more gene transfer vectors and one or more polypeptides described herein can be co-administered in any order and via any route of administration. Accordingly, any combination of polynucleotides and polypeptides described herein may be used to induce an immune response. Dosage Regime

[0262] Dosage treatment can follow a single-dose schedule or a multi-dose schedule. Multi-dose schedules may be used in primary and / or booster immunization schedules. In multi-dose schedules, various doses may be administered via the same or different routes, for example, parenteral prime and mucosal boost, or mucosal prime and parenteral boost.

[0263] In embodiments, the dosage regime enhances the activity of the antibody response, resulting in an antibody with neutralizing properties. An in vitro neutralization assay may be used to test for the neutralizing antibody.

[0264] There is a strong correlation between serum antibody levels and protection against disease caused by the ASF virus. Tests to determine the effectiveness of the immune response

[0265] One method for evaluating the effectiveness of a therapeutic treatment includes monitoring infection after administration of the composition as disclosed. Another method for evaluating the effectiveness of a prophylactic treatment includes monitoring the immune response to the antigen in the composition as disclosed after administration of the composition.

[0266] Another method for evaluating the immunogenicity of the constituent proteins of the immunogenic compositions of this disclosure is to recombinantly express the proteins and screen patient serum or mucosal secretions by immunoblotting. A positive reaction between the protein and patient serum indicates that the patient has previously had an immune response to the protein in question, i.e., the protein is immunogenic. This method may also be used to identify immunodominant proteins and / or epitopes.

[0267] Another method for confirming the effectiveness of a therapeutic treatment includes monitoring infection after administration of the compositions of this disclosure. Another method for confirming the effectiveness of a prophylactic treatment includes monitoring both systemic (e.g., monitoring IgG1 and IgG2a production levels) and mucosal (e.g., monitoring IgA production levels) immune responses to antigens in the compositions of this disclosure after administration of the compositions. Typically, serum-specific antibody responses are determined post-immunization but pre-exposure, while mucosal-specific antibody responses are determined post-immunization and post-exposure.

[0268] The immunogenic compositions of this disclosure may be evaluated in in vitro and in vivo animal models prior to host development. Particularly useful mouse models include those in which intraperitoneal immunization is followed by intraperitoneal challenge or intratranasal challenge.

[0269] The efficacy of the immunogenic compositions of this disclosure may also be determined in vivo by exposing infected model animals, such as guinea pigs, mice, or rhesus monkeys, to the immunogenic compositions. The immunogenic compositions may or may not be derived from the same strain as the exposed strain. In embodiments, the immunogenic compositions may be derived from the same strain as the exposed strain.

[0270] In vivo efficacy models include, but are not limited to, (i) mouse infection models using human strains, (ii) mouse disease models using strains adapted to mice, such as strains that are particularly pathogenic in mice, and (iii) primate models using human isolates. Human challenge models supported by the NIH and the Centers for Disease Control and Prevention (CDC) are also available.

[0271] The immune response may be either a TH1 immune response or a TH2 immune response, or both. The immune response may be an improved, enhanced, or altered immune response. The immune response may be either a systemic immune response or a mucosal immune response, or both. In some embodiments, the immune response is an enhanced systemic and / or mucosal response.

[0272] Enhanced systemic immunity and / or mucosal immunity are reflected in enhanced TH1 and / or TH2 immune responses. In embodiments, the enhanced immune response includes increased production of IgG1 and / or IgG2a and / or IgA. In embodiments, the mucosal immune response is a TH2 immune response. In one embodiment, the mucosal immune response includes increased production of IgA.

[0273] Activated TH2 cells enhance antibody production, making them valuable in responding to extracellular infections. Activated TH2 cells may secrete one or more of IL-4, IL-5, IL-6, and IL-10. The TH2 immune response may lead to the production of IgG1, IgE, IgA, and memory B cells for future protection.

[0274] The TH2 immune response can include one or more increases in cytokines associated with the TH2 immune response (such as IL-4, IL-5, IL-6, and IL-10), or one or more increases in the production of IgGl, IgE, IgA, and memory B cells. In embodiments, the enhanced TH2 immune response includes an increase in IgGl production.

[0275] The TH1 immune response can include one or more of an increase in CTLs, one or more increases in cytokines associated with the TH1 immune response (such as IL-2, IFNγ, and TNFβ), an increase in activated macrophages, an increase in NK activity, or an increase in the production of IgG2a. In embodiments, the enhanced TH1 immune response includes an increase in IgG2a production.

[0276] The immunogenic compositions of the present disclosure, particularly immunogenic compositions comprising one or more antigens of the present disclosure, may be used alone or in combination with other antigens, optionally together with an immunomodulatory agent that can induce a Thl response and / or a Th2 response.

[0277] The immunogenic compositions of the present disclosure may also include one or more immunomodulatory agents such as mineral salts such as aluminum salts and oligonucleotides containing CpG motifs. In embodiments, the immunogenic composition includes both an aluminum salt and an oligonucleotide containing a CpG motif. Alternatively, the immunogenic composition includes an ADP-ribosylating toxin such as a detoxified ADP-ribosylating toxin and an oligonucleotide containing a CpG motif. In one aspect, the one or more immunomodulatory agents include an adjuvant. The adjuvant may further be selected from one or more of the groups consisting of the TH1 adjuvants and TH2 adjuvants described above.

[0278] The immunogenic composition of this composition can induce both humoral and cell-mediated immune responses to effectively address infection. This immune response can induce long-lasting (e.g., neutralizing) antibodies and cell-mediated immunity that can respond rapidly upon exposure to one or more infectious antigens. As an example, evidence of neutralizing antibodies in a blood sample of interest is considered a surrogate parameter of protection because the formation of neutralizing antibodies is critically important for viral elimination in Mycobacterium tuberculosis infection. Use of immunogenic compositions as pharmaceuticals

[0279] This disclosure also provides compositions for use as pharmaceuticals. The pharmaceuticals may be able to elevate the immune response in mammals (i.e., they are immunogenic compositions) and may be vaccines. This disclosure also provides the use of the compositions in the manufacture of pharmaceuticals for elevating the immune response in mammals. The pharmaceuticals may be vaccines. In embodiments, the vaccines are used to prevent and / or treat intestinal infections such as gastroenteritis, including acute gastroenteritis. Gastroenteritis may result from an imbalance in the movement of ions and / or water, which leads to both watery diarrhea and / or intestinal peristalsis and / or motility (vomiting).

[0280] This disclosure provides a method for inducing or enhancing an immune response using the above-described compositions. The immune response may be defensive and may induce antibody and / or cell-mediated immunity (including systemic immunity and mucosal immunity). The immune response may include a booster response.

[0281] This disclosure also provides a method for enhancing an immune response in a mammal, comprising the step of administering an effective amount of a composition of this disclosure. The immune response may be defensive and may include antibody and / or cell-mediated immunity. In embodiments, the immune response may include one or both of a TH1 immune response and a TH2 immune response. The method may also enhance a booster response. kit

[0282] This disclosure also provides a kit comprising one or more containers of the compositions described herein. The compositions, as well as the individual antigens, may be in liquid form or lyophilized. Suitable containers for the compositions include, for example, bottles, vials, syringes, and test tubes. The containers may be formed from a variety of materials, including glass or plastic. The containers may have a sterile access port (for example, the container may be an intravenous solution bag or vial with a stopper that can be punctured with a subcutaneous needle).

[0283] The kit may further include a second container containing a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or glucose solution. It may also include other materials useful to the end user, such as other pharmaceutically acceptable formulation solutions, diluents, filters, needles and syringes or other delivery devices. The kit may further include a third component containing an adjuvant.

[0284] The kit may also include a package insert containing instructions on how to induce immunity or treat an infection. The package insert may be an unapproved draft or an approved package insert by the Food and Drug Administration (FDA) or other regulatory agency.

[0285] In one embodiment, the delivery device is pre-filled with an immunogenic composition as disclosed herein. Method for producing ASF virus-specific antibodies

[0286] The ASF virus polypeptides described herein can be used to produce ASF virus-specific polyclonal and monoclonal antibodies that specifically bind to or are selective to the ASF virus antigen. Polyclonal antibodies can be produced by administering ASF virus polypeptides to mammals such as mice, rabbits, goats, and horses. Serum from immunized animals is collected and precipitated, for example, with ammonium sulfate, and then the antibodies are purified from the plasma by chromatography, including affinity chromatography. Techniques for producing and processing polyclonal antiserum are known in the art.

[0287] Monoclonal antibodies against ASF virus-specific epitopes present in polypeptides can also be readily produced. Normal B cells derived from mammals, such as mice immunized with ASF virus polypeptide, can be fused with, for example, HAT-sensitive mouse myeloma cells to produce hybridomas. Hybridomas producing ASF virus-specific antibodies may be identified using RIA or ELISA and isolated by cloning or limiting dilution in semi-solid agar. Clones producing ASF virus-specific antibodies can be isolated by further screening.

[0288] Antibodies against ASF virus epitopes, i.e., monoclonal and polyclonal antibodies (polyclonal) from serum, are particularly useful for detecting the presence of ASF virus antigens in samples, such as serum samples from subjects infected with ASF virus. Immunoassays for ASF virus antigens can utilize one antibody or multiple antibodies. For example, an immunoassay for ASF virus antigens may use a monoclonal antibody directed towards an ASF virus epitope, a combination of monoclonal antibodies directed towards one or more ASF virus polypeptide epitopes, multiple monoclonal antibodies directed towards different ASF virus polypeptide epitopes, a polyclonal antibody directed towards the same ASF virus antigen, a polyclonal antibody directed towards different ASF virus antigens, or a combination of monoclonal and polyclonal antibodies. The immunoassay protocol may be based on, for example, a competitive method, a direct reaction method, or a sandwich method using labeled antibodies. Labeling can be, for example, fluorescent, chemiluminescent, or radioactive.

[0289] Polyclonal or monoclonal antibodies may further be used to isolate ASF virus particles or antigens by an immunoaffinity column. The antibodies may be immobilized on a solid support, for example by adsorption or covalent bonding, so that the antibodies retain their immunoselective activity. Optionally, spacer groups may be included so that the antigen-binding sites of the antibodies remain accessible. The immobilized antibodies may be used to bind ASF virus particles or antigens from biological samples such as blood or plasma. The bound ASF virus particles or antigens are recovered from the column matrix, for example, by a change in pH.

[0290] All patent documents cited herein are incorporated herein by reference in their entirety. [Examples]

[0291] Example 1. Production of baculovirus protein subunits The recombinant baculovirus protein expression system was based on the nucleic acid sequence of the target ASF virus protein. The final sequence was optimized for expression in in-house Spodoptera fulgiperda (Sf9) insect cells to ensure that the appropriate restriction endonuclease site was present at the end of the sequence.

[0292] To prepare a plasmid vector containing the target sequence, the pBacPAK8 cloning vector (Clontech Laboratories, Inc., Mountain View, CA) was used. This plasmid vector contains a flanking sequence homologous to the linear BestBac 2.0 baculovirus vector (Expression Systems, Davis, CA). When this plasmid, containing ASF virus p30, p30 / p54, p72, and / or hemagglutinin inserts, was co-introduced into a linear BestBac 2.0 baculovirus backbone and Sf9 cells, homologous recombination resulted in the exchange of the H3 insert with the baculovirus polyhedrin gene. As a result, a baculovirus containing the ASF virus sequence expressed under the control of the polyhedrin promoter was obtained. Cells and viruses were cultured in Expression Systems medium (Media ES 99-300) prepared to be free of animal-derived components. Gentamicin solution was added from the purchased stock solution (Gibco Cat #15710) to a final concentration of 10 μg / ml. At the final harvest, the infected cultures were centrifuged to remove cells, and the supernatant was collected. The supernatant was treated with a 0.2 micron sterile disposable filter. The premaster culture medium was titrated to determine the final concentration.

[0293] Sf9 cells are scaled up to production levels using glass or sterilized disposable plastic containers. When the final cell culture volume required for production is reached, virus infection occurs in the same container in which the final passage of the cells was prepared. Mixing of the culture is achieved by shaking or agitating the container or by using a low-shear type impeller. The stirring speed and intensity are adjusted to maintain the cells in suspension without causing excessive shear or foaming that could lead to cell breakage.

[0294] The virus solution is inactivated with β-propiolactone (BPL) at a final concentration of 0.2 - 0.3%. Prior to inactivation, the pH of the disrupted fluid is adjusted to 7.5 - 8.0 using 2 - 10 N NaOH as the base or 10 - 38% HCl or 10% nitric acid as the acid. The disrupted fluid is warmed to room temperature for 1 - 18 hours before adding BPL. BPL is added at the above concentrations with stirring. After adding BPL, the virus solution is transferred to an inactivation container in a "bottom to bottom" transfer process to ensure that all of the liquid has come into contact with BPL. The disrupted liquid is cultured with stirring at 17 - 27°C for 18 - 48 hours. After the inactivation process is complete, the pH is adjusted to 7.0 - 7.5 with an acid or base as described above. The inactivated virus solution is stored at 2 - 8°C until further processed. The antigen is prepared with a water / oil / water (WOW) adjuvant.

[0295] Example 2. Field evaluation of an ASF antigen-based vaccine The antigen was produced as described above (i.e., hemagglutinin, SEQ ID NO: 17 and p30 / p54 fusion protein, SEQ ID NO: 6).

[0296] Eight commercial farms were selected for enrollment in this study. The livestock used on these farms came from other farms that were not infected with ASF. Experimental design

[0297] Subjects, exclusion and discontinuation criteria

[0298] All animals used in this study were clearly healthy at the start of the study. Pigs suffering from illness, ill thrift, serious trauma, or lameness were excluded according to standard management procedures.

[0299] Randomization

[0300] Pigs that met all inclusion criteria and none of the exclusion criteria were randomly assigned to either one of two treatment groups or a placebo group. An equal number of pigs and 25% placebo were assigned to each pen from each treatment group.

[0301] Treatment of illness

[0302] Sick animals were treated according to the farm's standard operating procedures or treatment protocols. All causes of pathological conditions were documented.

[0303] Blood samples were collected from animals suspected of having ASF (e.g., severe lethargy, discoloration of the limbs) and from deceased animals. An initial screening for the presence of ASF antigens was performed using a rapid test kit, and then the same blood samples were subjected to confirmatory testing. Treatment group: [Table 1]

[0304] Individual animals were used as experimental units. Each pig was double-tagged (one on each ear) and mixed with an equal number of pigs from different vaccination groups and control groups within each enclosure. The enclosures used were located within a single building. Vaccination

[0305] The vaccinated group received one of two vaccines, followed by two doses of 1 mL administered three weeks apart. Blood collection for serological testing: [Table 2] Collected data and results

[0306] Animals were monitored daily for adverse events, starting on day 0 and continuing until day 21 after the second vaccination. All adverse events were recorded. Particular attention was paid to swelling / inflammation, pain, redness, abscesses, lumps, lesions, and warmth at the injection site. Other adverse events recorded included, but were not limited to, lameness, lack of appetite, and general abnormal behavior.

[0307] All serum samples were tested for the presence of antibodies against the ASF p54 antigen using the Biochek African Swine Fever Antibody Test Kit. This test was intended to determine seroconversion to the experimental vaccine by examining the immune response to the p54 portion of the p30 / p54 fusion protein in the vaccine formulation.

[0308] All samples were tested for the presence of antibodies against the ASF p72 antigen using Ingenza PPA CROM. This served as a screening test to detect whether the experimental animals had been exposed to the ASF virus.

[0309] All samples were tested for the presence of antibodies against ASF p30(32), p62, and p72 antigens using ID Screen ASF Indirect ELISA. This test was intended to determine seroconversion to the experimental vaccine by examining the immune response to the p30 portion of the p30 / p54 fusion protein in the vaccine formulation.

[0310] Of the three assays performed on samples collected during the study period, two, Biocheck and ID Screen, were used to evaluate the animals' immunological response to vaccination. Each of these tests measured different percentages of p30 / p54 fusion protein in the vaccine formulation. The ID Screen assay, which measures the p30(32) antigen, showed an immune response during vaccination and the observation period. conclusion

[0311] Immune response

[0312] Evaluating the immunological response of animals administered the test vaccine containing the p30 / p54 fusion protein, we can conclude that in both treatment groups 1 and 2, vaccination induced an immune response to the p30(32) antigen as tested with the ID Screen Indirect kit. Increased antibody concentrations were detected in naive farm around day 21 of the study in treatment group 1 and were very similar between the two treatment groups in blood samples taken on day 42 of the study.

[0313] These results indicate that this drug induces an immune response to ASF antigen.

[0314] Although the present invention has been described with reference to the above embodiments, it will be understood that modifications and variations are entailed within the spirit and scope of the invention. Accordingly, the present invention is limited only by the following claims. The present invention includes the following embodiments: <Aspect 1> A method for producing immunogenic polypeptides and / or peptides derived from African swine fever (ASF) virus, comprising optionally mixing or co-expressing the immunogenic polypeptides and / or peptides with one or more adjuvants. <Aspect 2> The method according to embodiment 1, comprising culturing host cells transformed with nucleic acids under conditions that induce the expression of the polypeptide and / or peptide. <Aspect 3> The method according to embodiment 2, wherein the immunogenic polypeptide and / or peptide comprises a recombinant subunit vaccine, and such expressed polypeptide and / or peptide is produced using a baculovirus / insect cell methodology. [Claim 4] <Aspect 4> The method according to embodiment 1, wherein the polypeptide and / or peptide comprises the amino acid sequence described in SEQ ID NOs: 6, 8, 10, 12, 17 or a combination thereof. <Aspect 5> The method according to embodiment 2, wherein the nucleic acid encoding the immunogenic polypeptide and / or peptide derived from the ASF virus is prepared by chemical synthesis. <Pattern 6> The method according to embodiment 5, wherein the nucleic acid encoding the immunogenic polypeptide and / or peptide derived from ASF is produced using a primer-based amplification method. <Aspect 7> The method according to embodiment 6, wherein the primer-based amplification method is PCR. <Aspect 8> An immunogenic composition comprising the polypeptides described in SEQ ID NOs: 6, 8, 10, 12, 17 or any combination thereof. <Aspect 9> A method for inducing an immunological response in a subject, comprising administering the composition described in Embodiment 8. <Aspect 10> The method according to embodiment 9, further comprising administering an adjuvant. <Aspect 11> The method according to embodiment 10, wherein the immunogenic composition is administered to the subject via local administration, parenteral administration, or mucosal administration. <Aspect 12> The method according to embodiment 9, wherein the administration is performed by multiple doses. <Aspect 13> The method according to embodiment 12, wherein the first immunogenic composition and the second immunogenic composition are the same. <Aspect 14> The method according to embodiment 12, wherein the first immunogenic composition and the second immunogenic composition are different. <Aspect 15> A method for treating infection by African swine fever virus, comprising administering a therapeutically effective amount of the immunogenic composition described in embodiment 8 to a subject in need thereof. <Aspect 16> The method according to embodiment 15, wherein the composition comprises an ASF virus p30 / p54 fusion protein. <Aspect 17> The method according to embodiment 15, wherein the composition comprises ASF virus hemagglutinin protein. <Aspect 18> The method according to embodiment 15, wherein the composition comprises administering a composition comprising an ASF virus p30 / p54 fusion protein and an ASF virus hemagglutinin protein. <Aspect 19> The method according to embodiment 15, wherein the subject is a pig. <Aspect 20> The method according to embodiment 18, wherein the proteins are administered substantially simultaneously or sequentially.

Claims

1. A method for producing an immunogenic polypeptide and / or immunogenic peptide derived from African swine fever (ASF) virus, wherein the polypeptide and / or peptide comprises the amino acid sequence described in SEQ ID NO: 6, and comprises culturing nucleic acid-transformed host cells under conditions that induce the expression of the polypeptide and / or peptide, wherein the polypeptide produces a neutralizing antibody. method.

2. The method according to claim 1, wherein the immunogenic polypeptide and / or peptide is mixed with or co-expressed with one or more adjuvants.

3. The method according to claim 2, wherein the immunogenic polypeptide and / or peptide comprises a recombinant subunit vaccine, and such expression polypeptide and / or peptide is produced using a baculovirus / insect cell methodology.

4. The method according to claim 2, wherein the nucleic acid encoding the immunogenic polypeptide and / or immunogenic peptide derived from African swine fever (ASF) virus is prepared by chemical synthesis.

5. The method according to claim 4, wherein the nucleic acid encoding the immunogenic polypeptide and / or immunogenic peptide derived from African swine fever (ASF) virus is produced using a primer-based amplification method.

6. The method according to claim 5, wherein the primer-based amplification method is PCR.

7. An immunogenic composition comprising a polypeptide containing the amino acid sequence shown in Sequence ID No.

6.

8. A method for inducing an immunological response in a subject, comprising administering the composition according to claim 7, The aforementioned subjects are excluding humans, method.

9. The method according to claim 8, further comprising administering an adjuvant.

10. The method according to claim 9, wherein the immunogenic composition is administered to the subject via local administration, parenteral administration, or mucosal administration.

11. The method according to claim 8, wherein the administration is performed by multiple doses.

12. The multiple administrations are two administrations, and the immunogenic composition is a first immunogenic composition for the first administration and a second immunogenic composition for the second administration, The method according to claim 11, wherein the first immunogenic composition and the second immunogenic composition are the same.

13. The multiple administrations are two administrations, and the immunogenic composition is a first immunogenic composition for the first administration and a second immunogenic composition for the second administration, The method according to claim 11, wherein the first immunogenic composition and the second immunogenic composition are different.

14. The procedure involves administering a therapeutically effective amount of the immunogenic composition described in claim 7 to a subject in need thereof. The aforementioned subjects are excluding humans, Methods for treating infections caused by African swine fever virus.

15. The method according to claim 14, wherein the composition comprises an ASF virus p30 / p54 fusion protein.

16. The method according to claim 14, wherein the composition comprises ASF virus hemagglutinin protein.

17. The method according to claim 14, wherein the composition comprises administering a composition comprising an ASF virus p30 / p54 fusion protein and an ASF virus hemagglutinin protein.

18. The method according to claim 14, wherein the subject is a pig.

19. The method according to claim 17, wherein the proteins are administered simultaneously or sequentially.

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