African Swine Fever (ASF) Virus Vaccine

Abstract

The present invention describes an immunogenic composition containing an immunogenic polypeptide of the African Swine Fever (ASF) virus, comprising an immunogenic composition containing an antigen other than the ASF virus antigen, which includes an antigen that can be used for immunization against pathogens causing diarrhea. Additionally, a method for inducing an immune response with the disclosed immunogenic composition and a method for treating an ASF infection are described.

Description

Technology Field

[0001] The present invention generally relates to a composition that induces an immune response against the African Swine Fever (ASF) virus. In particular, the present invention relates to an immunogenic composition (e.g., a vaccine) comprising an immunogenic polypeptide of the ASF virus. Additionally, the immunogenic composition may contain antigens other than the ASF virus antigen. Furthermore, a method for inducing an immune response and a method for treating an ASF infection are described using the immunogenic composition disclosed herein. Background Technology

[0002] African Swine Fever (ASF) is a viral disease of pigs that causes high mortality rates in domestic pigs while remaining asymptomatic in natural reservoir hosts, such as wild boars. Without an effective vaccine, it causes unavoidable and significant economic losses, and available disease control methods include the isolation of affected areas and the culling 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 member of the Asfarviridae family and the only DNA virus transmitted by arthropods, specifically soft ticks of the Ornithodoros genus. The soft tick (Ornithodoros moubata) is involved in the sylvatic transmission cycle of the African virus and O. erraticus in Europe. Wild boars suffering from acute diseases similar to those in domestic pigs appear to be associated with the transmission cycle in Europe.

[0003] The disease caused by this virus was first identified in Kenya in the 1920s. It was then confined to Africa until it spread to Europe in the mid-20th century, and later to South America and the Caribbean. The disease was eradicated from Europe (excluding Sardinia) in the 1990s through rigorous control and eradication programs. However, in 2007, the disease spread again from Africa to the Caucasus, particularly Georgia, and reached the eastern territories of the European Union in 2014. Recent reports on the disease include a list of increasing cases in EU countries, Poland, the three Baltic republics, and, more recently, Moldova. Because there is no vaccine with protective efficacy, ASF poses a serious threat to all European countries. The epidemiological complexity of ASF was clearly demonstrated in East and Southern Africa, where genetic characteristics of ASFV based on sequence variations in the C-terminal region of the B646L gene encoding the major capsid protein p72 revealed the existence of 22 genotypes. Recently, a new genotype, genotype XXIII, was described, which shares a common ancestor with genotypes IX and X, including isolates circulating in East African countries and the Republic of Congo. This review paper 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. The viral genome consists of a single molecule of linear, covalently close-ended, double-stranded DNA. The genomes of different isolates vary in length from 170 to 190 kbp and encode 151 to 167 open read frames. The ASFV replication cycle occurs primarily in the cytoplasm, although the nucleus is also the site of viral DNA synthesis in the early stages. The disassembly of the lamina network near the replication initiation site of the viral genome and the redistribution of various nuclear proteins suggest the existence of sophisticated mechanisms regulating the nuclear machinery during viral infection.

[0005] The transcription of viral genes is strongly regulated. Four types of mRNAs, including immediate-early, early, intermediate, and late transcripts, were identified by distinct accumulation kinetics. Immediate-early and early genes are expressed prior to the start of DNA replication, whereas intermediate and late genes are expressed later. The presence of intermediate genes suggests a cascade model for the regulation of ASFV gene expression. Enzymes required for DNA replication are expressed immediately after viral entry from the uncoated core particle into the cytoplasm, utilizing enzymes and other factors packed within the viral particle. Viral morphogenesis occurs in the viral factory where the major late stages of DNA replication take place.

[0006] ASFV particles have an icosahedral shape composed of several concentric domains: an inner core formed by the central genome, which is a nucleoid coated with a thick protein layer called the core shell; an inner lipid envelope surrounding the core; and finally, a capsid, which is the outermost layer of the intracellular virion. The extracellular virion has an additional outer envelope formed when the virus buds through the plasma membrane. However, the importance of this envelope is unclear as it is not required for infectivity.

[0007] Current approaches to ASF vaccines are broadly divided into two "camps." The current approach taken by the USDA and DHS focuses on modified live vaccines, based on the belief that viral replication inside cells is absolutely necessary to generate a protective response. However, the most advanced prototype vaccines in this category are recently stated to take "at least eight years from approval to use" and entail many safety issues. Recent research has demonstrated that a highly effective gene-deleted ASF mutant vaccine replicates weakly in pigs but protects vaccinated animals from lethal attacks.

[0008] While it is promising, the use of live attenuated strains for the US vaccine is problematic, and the culture of the vaccine virus currently requires the use of primary macrophages. There are colloquial reports of China's attempts to replicate the vaccine that caused negative results, but this needs to be verified.

[0009] A second vaccine developed by the Pirbright Laboratory in the UK has also shown promise, providing protection against the fatal consequences of ASF infection without limiting viral replication. Simply put, this two-dose vaccine was developed by combining eight individual recombinant adenoviruses expressing eight distinct ASF proteins into a single vaccine. When administered, the results appear similar to those observed in previous studies using fewer proteins.

[0010] In contrast to the use of live methods, subunit vaccines are dead products due to the difficulties associated with describing protective protein targets and generating vaccines that provide broad protection against multiple strains, and these vaccines have been largely ignored.

[0011] Therefore, there remains a need for improved therapies to treat subjects exhibiting clinical symptoms associated with ASF virus infection and methods to prevent the spread of infection. The problem to be solved

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

[0013] In the examples, a method for preparing an ASF virus-derived immunogenic polypeptide and / or peptide that may be mixed with or co-expressed with adjuvants 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 for immunization against other disease-causing pathogens, such as antigens derived from non-ASF virus pathogens.

[0014] In an example, a method for producing a polypeptide is disclosed, comprising the step of culturing host cells transformed with the nucleic acid described herein under conditions that induce polypeptide expression. In a related aspect, an ASF virus protein may be expressed by recombinant technology and may be used to develop an immunogenic composition comprising a recombinant antigen subunit, wherein such expressed polypeptide is produced using a baculovirus / insect cell methodology.

[0015] In one aspect, a method for preparing a nucleic acid is disclosed in which the nucleic acid encoding an ASF virus-derived protein or polypeptide is prepared (at least partially) by chemical synthesis. In a related aspect, the method comprises the step of amplifying the nucleic acid using a primer-based amplification method (e.g., PCR).

[0016] In another aspect, a method for preparing a protein complex is disclosed, comprising the step of administering an ASF virus-derived polypeptide or a fragment thereof to a subject. In a related aspect, the method comprises the step of mixing the ASF virus-derived polypeptide with a pharmaceutically acceptable carrier or diluent. In a further related aspect, the composition may comprise the polypeptides presented in SEQ ID NO: 6 (p30 / p54 fusion protein), 8 (p72 protein), 10 (p30 protein), 12 (p54 protein), and 17 (hemagglutinin protein). In another related aspect, the polypeptide composition comprises SEQ ID NO: 6 and 17.

[0017] In an example, a method for inducing an immunological response in a subject is disclosed, comprising the step of administering a composition of the present invention. In a related aspect, the method further comprises the step of administering an immunoadjuvant. In a further related aspect, the method comprises the step of administering an immunogenic composition to a subject via a local, parenteral, or mucosal route.

[0018] In one aspect, the administration may be multiple administrations, wherein the first immunogenic composition and the second immunogenic composition are identical. In another aspect, the first immunogenic composition and the second immunogenic composition are different.

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

[0020] In an example, a method for treating an infection caused by an ASF virus is disclosed, comprising the step of administering a therapeutically effective amount of the immunogenic composition described herein to a subject who requires treatment for an infection caused by an ASF virus.

[0021] In one aspect, an immunogenic composition of multiple therapeutically effective doses is administered to a subject.

[0022] In a related aspect, the method comprises the steps of administering a therapeutically effective amount of a first immunogenic composition containing one or more ASF virus antigens to the mucosa and administering a therapeutically effective amount of a second immunogenic composition containing one or more ASF virus antigens topically or parenterally.

[0023] In one aspect, multiple therapeutically effective doses of an immunogenic composition are administered to a subject. In another aspect, the immunogenic composition contains a separate non-ASF viral antigen.

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

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

[0026] In one aspect, the subject is a pig. In a related aspect, the protein is administered substantially simultaneously or sequentially.

[0027] These and other embodiments of the disclosed invention will readily come to mind to a person skilled in the art in the context of the invention. means of solving the problem

[0028] Before describing the composition, method, and methodology, it should be understood that the present invention is not limited to the specific composition, method, and experimental conditions described, and that such composition, method, and conditions may vary. Furthermore, it should be understood that the technical terms used herein are merely for describing specific embodiments and are not intended to be limiting, as the scope of the invention is limited only to the appended claims.

[0029] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include the plural forms unless the context clearly indicates otherwise. Accordingly, for example, a reference to “nucleic acid” includes one or more nucleic acids, and / or a composition of the type described in this specification that would be apparent to a person skilled in the art upon reading this specification, etc.

[0030] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as generally understood by a person skilled in the art to which the present invention pertains. Any method and material similar or equivalent to that described herein may be used in the practice or testing of the present invention, and it will be understood that modifications and variations are included within the spirit and scope of the present invention.

[0031] As used herein, “about,” “approximately,” “substantially,” and “significantly” will be understood by a person skilled in the art and will 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 a person skilled in the art in the context in which such term is used, “about” and “approximately” will mean plus or minus <10% of a specific term, and “substantially” and “significantly” will mean plus or minus >10% of a specific term. In the embodiments, a composition may “contain,” “include,” or “essentially consist” of a specific component or group of components, wherein a person skilled in the art will understand the latter to mean that it is limited to the material or step specified in the claim and “materially not affecting the basic and novel feature(s) of the claimed invention.”

[0032] As used herein, the term "ASF" refers to a member of the genus Asfivirus of the family Asfaviridae of African swine fever virus. The term ASF includes strains within all genogroups of the virus. Currently, ASF strains are divided into 24 genogroups (Gx-Gxn) based on the sequencing of the p72 / B646L gene. Additionally, the term ASF includes isolates not characterized at the time of filing.

[0033] The terms “polypeptide” and “protein” refer to macromolecules of amino acid residues and are not limited to the minimum length of the product. Thus, peptides, oligopeptides, dimers, multimers, etc. are included within the definition. Both full-length proteins and fragments thereof are included within the definition. Additionally, the term includes post-expression modifications of polypeptides, e.g., glycosylation, acetylation, phosphorylation, etc. Furthermore, for the purposes of the present invention, “polypeptide” refers to a protein that includes modifications such as deletions, additions, and substitutions (generally virtually conserved) to the natural sequence, provided that the protein maintains the desired activity. Such modifications may be intentional, such as through site-directed mutagenesis, or accidental, such as through mutations in the host producing the protein or errors resulting from PCR amplification.

[0034] "Substantially purified" generally refers to the separation of a substance (compound, polynucleotide, protein, polypeptide, polypeptide composition) such that the substance comprises the majority of the sample in which it is present. Generally, in a sample, the substantially purified component comprises about 50%, about 80%–85%, or about 90–95% of the sample. Techniques for purifying polynucleotides and polypeptides of interest are well known in the art and include, for example, ion-exchange chromatography, affinity chromatography, and density-based sedimentation.

[0035] When referring to polypeptides, "isolated" means that the indicated molecule is separated and discrete from the whole organism or cell where it is not actually found or where other biological macromolecules of the same type are substantially present. With respect to polynucleotides, the term "isolated" refers to a nucleic acid molecule that is, in fact, generally lacking the associated sequence in whole or in part; a sequence that is, in fact, present but has an associated heterogeneous sequence; or a molecule isolated from a chromosome.

[0036] As used herein, the terms “label” and “detectable label” refer to detectable molecules including, but not limited to, radioactive isotopes, fluorophores, chemiluminescent agents, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, chromophores, dyes, metal ions, metal sols, ligands (e.g., biotin or haptens). The term “fluorophor” refers to a substance or part thereof capable of exhibiting fluorescence within a detectable range. Specific examples of labels that may be used include fluorescein, rhodamine, dansyl, umbelliferone, Texas red, luminol, acradimum esters, NADPH, and α-β-galactosidase.

[0037] "Homology" indicates percent identity between two polynucleotides or two polypeptide portions. Two nucleic acid or two polypeptide sequences are "substantially homologous" to each other when, for a molecule of a defined length, the sequences 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. As used herein, substantially homology also refers to a sequence that exhibits complete identity with the specified sequence.

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

[0039] Another method for establishing percent identity in the context of the present invention is to use the MPSRCH package of the program, copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok and distributed by IntelliGenetics, Inc. (Mountain View, CA). In this package family, the Smith-Waterman algorithm may be used when the basic parameters are applied to a score table (e.g., a gap-opening penalty of 12, a gap-extending penalty of 1, and a gap of 6). From the generated data, the "Match" value indicates "sequence identity." Other suitable programs for calculating percent identity or similarity between sequences are generally known in the art; for example, another alignment program is BLAST used with basic parameters. For example, BLASTN and BLASTP may be used with the following basic parameters: Gene Code = Standard; Filter = None; Strand = Both; Cutoff = 60; Expectation = 10; Matrix=BLOSUM62; Description=50 sequences; Alignment Criteria=High score; Database=Non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS Translation+Swiss Protein+Spupdate+PIR. Details regarding these programs are readily available.

[0040] Alternatively, homology can be determined by hybridization of polynucleotides under conditions forming a stable duplex between homologous regions, followed by degradation by single-strand-specific nuclease(s), and determination of the size of the degraded fragment. Substantially homologous DNA sequences can be identified, for example, in Southern hybridization experiments under harsh conditions defined for a specific system. Defining appropriate hybridization conditions is within the scope 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 is not related in whole or in part to the polynucleotides substantially associated by its origin or manipulation. As used with respect to proteins or polypeptides, the term “recombinant” means a polypeptide produced by the expression of a recombinant polynucleotide. Generally, the gene of interest is cloned and then expressed in a transformed organism, as further described below. The host organism expresses the foreign gene to produce a protein under expression conditions.

[0042] The term "transformation" refers to the insertion of an exogenous polynucleotide into a host cell, regardless of the method used for insertion. Examples include direct uptake, transduction, or mating. The exogenous polynucleotide may be maintained as a non-integrated vector, e.g., a plasmid, or alternatively, 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 unicellular entities refer to cells that may or have been used as recipients for a recombinant vector or other transferred DNA, and include the original progeny of the transfected original cell.

[0044] A "coding sequence" or a sequence "encoding" a selected polypeptide is a nucleic acid molecule that is transcribed into a polypeptide (in the case of DNA) and translated (in the case of mRNA) in vivo under the control of an appropriate regulatory sequence (or "control element"). The boundaries of the coding sequence may be determined by a start codon at the 5' (amino) end and a translation stop codon at the 3' (carboxy) end. The coding sequence may include, but is not limited to, cDNA from viral, 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 the 3' end relative to the coding sequence.

[0045] General “control elements” include, but are not limited to, transcription promoters, transcription enhancer elements, transcription termination signals, polyadenylation sequences (located at 3’ for translation stop codons), sequences for optimizing translation initiation (located at 5’ for coding sequences), and translation termination sequences.

[0046] The term "nucleic acid" includes DNA and RNA, and analogs thereof such as those containing modified backbones (e.g., phosphorothioates, etc.), and also peptide nucleic acids (PNAs), etc. The present invention provides a nucleic acid comprising a sequence complementary to those described above (e.g., for antisense or probing purposes).

[0047] "Operatorily linked" indicates an arrangement of elements configured so that the component described in this way performs its general function. Therefore, a given promoter operably linked to a coding sequence can influence the expression of the coding sequence in the presence of the appropriate enzyme. As long as the promoter functions to direct its expression, it does not need to be adjacent to the coding sequence. Thus, for example, an intermediate sequence that has not been translated but has not yet been transcribed may exist between the promoter sequence and the coding sequence, and the promoter sequence can still be considered "operatorily linked" to the coding sequence.

[0048] “Encoded by” indicates a nucleic acid sequence encoding a polypeptide sequence, wherein the polypeptide sequence or part thereof contains an amino acid sequence of at least 3 to 5 amino acids, at least 8 to 10 amino acids, and at least 15 to 20 amino acids from the polypeptide encoded by the nucleic acid sequence.

[0049] "Expression cassette" or "expression construct" refers to an assembly capable of directing the expression of sequence(s) or gene(s) of interest. An expression cassette generally includes control elements such as a promoter (to direct its transcription) operably linked to the sequence(s) or gene(s) of interest, as described above, and often includes a polyadenylation sequence. In the examples, the expression cassette described herein may be contained within a plasmid construct. In addition to the components of the expression cassette, the plasmid construct may also include one or more selectable markers, a signal allowing the plasmid construct to exist as single-stranded DNA (e.g., an M13 replication origin), at least one multiple cloning site, and a "mammalian" replication origin (e.g., an SV40 or adenovirus replication origin).

[0050] "Purified polynucleotide" refers to a polynucleotide of interest or a fragment thereof that is essentially non-existent, for example, contains less than about 50%, less than about 70%, and less than about 90% of the naturally bound protein. Techniques for purifying the polynucleotide of interest are well known in the art and include, for example, disruption of cells containing the polynucleotide with a chaotropic agent and separation of the polynucleotide(s) and protein by ion-exchange chromatography, affinity chromatography, and density-based sedimentation.

[0051] The term "transfection" is used to denote the uptake of foreign DNA by a cell. When exogenous DNA is introduced into the cell membrane, the cell is "transfected." Numerous transfection techniques are generally known in the art. These techniques can be used to introduce one or more exogenous DNA segments into suitable host cells. The term refers to both stable and transient uptake of genetic material and includes the uptake of peptide- or antibody-linked DNAs.

[0052] A "vector" can deliver a nucleic acid sequence to a target cell (e.g., viral vectors, non-viral vectors, particulate carriers, and liposomes). Generally, "vector construct," "expression vector," and "gene delivery vector" refer to any nucleic acid construct capable of directing the expression of a nucleic acid of interest and delivering a nucleic acid sequence to a target cell. Thus, the terms include cloning and expression vehicles, and viral vectors.

[0053] "Fragment" means a molecule consisting of only a part of the complete full-length sequence and structure. A fragment of a polypeptide may include C-terminal deletions, N-terminal deletions, and / or internal deletions of the natural polypeptide. A fragment of a polypeptide will generally include at least about 5-10 consecutive amino acid residues of the full-length molecule, at least about 15-25 consecutive amino acid residues of the full-length molecule, and at least about 20-50 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 (in question) must retain the ability to induce a desired biological response. A fragment of a nucleic acid may include 5'-deletions, 3'-deletions, and / or internal deletions of the nucleic acid. The nucleic acid fragment will generally contain at least about 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 of the full-length sequence. Such fragments may be useful for hybridization, amplification, generation of immunogenic fragments, or nucleic acid immunization.

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

[0055] For the purposes of the present invention, the immunogenic fragment will generally be at least about 2 amino acid lengths, at least about 5 amino acid lengths, and at least about 10 to about 15 amino acid lengths. There is no critical upper limit for the length of the fragment, which may include nearly the entire length of the protein sequence, or even a fusion protein containing two or more epitopes.

[0056] As used herein, the term “epitope” generally refers to a site on an antigen recognized by T-cell receptors and / or antibodies. In the examples, this is a short peptide derived from or as part of a protein antigen. However, the term is also intended to include peptides having glycopeptide and carbohydrate epitopes. Several different epitopes may be carried by a single antigen molecule. Additionally, the term “epitope” includes a modified sequence of amino acids or carbohydrates that stimulates a response recognizing the entire organism. It is advantageous if the selected epitope is an epitope of an infectious agent that causes an infectious disease.

[0057] Epitopes can be generated without excessive experimentation from the characteristics of specific amino acids (e.g., size, charge, etc.) and codon dictionaries, as well as from knowledge of the amino acids of a peptide or polypeptide and its corresponding DNA sequence. Several guidelines for determining whether a protein will stimulate a response include peptide length: the peptide is approximately 8 or 9 amino acids long to fit into an MHC Class I complex and approximately 13–25 amino acids long to fit into a Class II MHC complex. This length is the minimum length required for the peptide to bind to the MHC complex. On the other hand, peptides can be longer than these lengths because cells can cleave the peptide. The peptide may contain a suitable anchor motif that enables it to bind to various Class I or Class II molecules with sufficiently high specificity to trigger an immune response. This can be done without excessive experimentation by comparing the sequence of the protein of interest with the publicly available structure of the peptide associated with the MHC molecule. Thus, a skilled technician can identify the epitope of interest by comparing the protein sequence with sequences listed in protein databases.

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

[0059] An “immunological response” to an antigen or composition is the occurrence of a humoral and / or cellular immune response to an antigen present in the composition of interest in a subject. For the purposes of the present invention, a “humoral immune response” refers to an immune response mediated by antibody molecules, whereas a “cellular immune response” is a response mediated by T-lymphocytes and / or other leukocytes. One important aspect of cellular immunity involves antigen-specific responses by cytolytic T-cells (“CTLs”). CTLs are encoded by the major histocompatibility complex (MHC) and possess specificity for peptide antigens presented in relation to proteins expressed on the cell surface. CTLs help induce and promote the destruction of intracellular microorganisms or the lysis of cells infected by these microorganisms. Another aspect of cellular immunity involves antigen-specific responses by helper T-cells. Helper T-cells play a role in stimulating the function of non-specific effector cells toward cells that display peptide antigens associated with MHC molecules on their surfaces, and helping to focus their activity. Additionally, the "cellular immune response" refers to the production of cytokines, chemokines, and other such molecules by activated T-cells and / or other leukocytes, including those originating from CD4+ and CD8+ T-cells.

[0060] Compositions or vaccines that induce a cellular immune response can serve to sensitize vertebrate subjects by presenting antigens associated with MHC molecules on the cell surface. The cell-mediated immune response is directed toward or near the cell presenting the antigen on its surface. Additionally, it generates antigen-specific T-lymphocytes, allowing for the future protection of the immunized host.

[0061] The ability of a specific antigen to stimulate a cell-mediated immunological response can be determined by a number of analyses, such as lymphoproliferative (lymphocyte activation) analysis, CTL cytotoxic cell analysis, or analysis of antigen-specific T-lymphocytes in sensitized subjects. These analyses are well known in the art. Recent methods for measuring cell-mediated immune responses include the measurement of intracellular cytokines or cytokine secretion by T-cell populations or by epitope-specific T-cells.

[0062] Accordingly, the immunological response used herein may be one that stimulates the production of antibodies (e.g., neutralizing antibodies that block bacterial toxins and pathogens, such as viruses entering cells, binding to toxins and pathogens, and replicating, generally protecting cells from infection and destruction). Additionally, the antigen of interest may induce the production of CTLs. Accordingly, the immunological response may include one or more of the following effects: the production of antibodies by B cells; and / or the activation of suppressor T-cells and / or memory / authoritative T-cells specifically directed toward the antigen or antigens present in the composition of interest or vaccine. Such a response may serve to protect the immunized host by neutralizing infectivity and / or mediating antibody-complement or antibody-dependent cell cytotoxicity (ADCC). Such a response may be determined using standard immunoassays and neutralization assays well known in the art. Additionally, the mammalian innate immune system recognizes and responds to the molecular characteristics of pathogenic organisms through the activation of Toll-like receptors and similar receptor molecules on immune cells. Upon activation of the innate immune system, various non-adaptive immune response cells are activated to produce, for example, various cytokines, lymphokines, and chemokines. Cells activated by the innate immune response include immature and mature dendritic cells of monocytes and plasmacytoid lineages (MDC, PDC), as well as gamma, delta, alpha, and beta T cells and B cells, etc. Accordingly, the present invention also considers an immune response involving both innate and adaptive responses.

[0063] "Immunogenic composition" is a composition comprising an antigen molecule such that administration of the composition to a subject causes the occurrence of a humoral and / or cellular immune response to the antigen molecule of interest in the subject.

[0064] The term “immunogenic” protein or polypeptide refers to an amino acid sequence that induces the immunological response described above. As used herein, the “immunogenic” protein or polypeptide comprises the full length sequence of said protein, including precursor and mature forms, analogs thereof, or immunogenic fragments thereof.

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

[0066] The term “derived from” is used herein to identify the original source of a molecule, but does not imply limiting the method by which the molecule is produced, which may be, for example, by chemical synthesis or recombination methods.

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

[0068] The polynucleotide derived from the “specified sequence” represents a polynucleotide sequence comprising a continuous sequence of at least about 6 nucleotides, at least about 8 nucleotides, at least about 10-12 nucleotides, and at least about 15-20 nucleotides corresponding to the region of the specified nucleotide sequence, i.e., identical or complementary. The derived polynucleotide does not necessarily have to be physically derived from the nucleotide sequence of interest, but may be produced by any method including, but not limited to, chemical synthesis, replication, reverse transcription, or transcription based on information provided by the sequence of bases of the region(s) from which the polynucleotide is derived. Thus, it may exhibit the sense or antisense orientation of the original polynucleotide.

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

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

[0071] Nucleic acid and protein sequences of interest for multiple ASF virus isolates are also known. Representative p30, p54, p72, and hemagglutinin nucleic acid sequences are presented at SEQ ID NOs: 1 (p30 / p54 fusion), 7 (p72), 9 (p30), 11 (p54), 13 (hemagglutinin), and 14 (hemagglutinin). Representative p30, p54, p72, and hemagglutinin amino acid sequences are presented at 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 NCBI (National Center for Biotechnology Information) database. For example, GenBank entry: CBw46759.1; Refer to, but not limited to, ACJ61575.1; MH735140.1; MH601419.1; MH727102.1; KF834194.1; LC322015.1; MH735142; MH681419.1; KM609342.1; FR682468.1; KJ380910.1; FR682468.1; MH722357; MH68419.1; MH713612.1; LC322016.1; KF834193.1, and all sequences (entered as of the filing date of this application) are incorporated herein by reference.

[0072] As used herein, the terms “p30,” “p54,” “p72,” or “hemagglutinin” relating to AFS virus polypeptides refer to polypeptides comprising sequences homologous or identical to the “p30,” “p54,” “p72,” or “hemagglutinin” polypeptides of the ASF virus, including any percent 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, 100% sequence identity with respect to the same, and including sequences having at least about 80-100% sequence identity with respect to the same, or different, strains of the ASF virus. The capsid polypeptide may be encoded by the same strain of the ASF virus or different strains of the ASF virus.

[0073] As used herein, the term “p30 / p54 fusion protein” refers to a protein comprising a sequence homologous or identical to a p30 ASF virus-encoded p30 and p54 protein derived from an ASF virus, and includes a sequence having at least about 80-100% sequence identity with it, including any percent 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, 100% sequence identity.

[0074] "Antigen" refers to a molecule containing one or more epitopes (linear, morphological, or both) that stimulate the host immune system to elicit a humoral and / or cellular antigen-specific response. This term is interchangeable with the term "immunogen." Generally, B-cell epitopes contain at least about 5 amino acids, but can be as small as 3–4 amino acids. T-cell epitopes, such as CTL epitopes, will contain at least about 7–9 amino acids, and helper T-cell epitopes will contain at least about 12–20 amino acids. Generally, epitopes will contain about 7 to 15 amino acids, e.g., 9, 10, 12, or 15 amino acids. The term "antigen" refers to both subunit antigens (i.e., antigens separated and discretized from the whole organism to which the antigen is effectively bound), and dead, attenuated, or inactivated bacteria, viruses, fungi, parasites, or other microorganisms. Antibodies, such as anti-idiotype antibodies, or fragments thereof, and synthetic peptide mimotopes capable of mimicking antigens or epitopes are also captured under the definition of antigen used herein. Similarly, oligonucleotides or polynucleotides that express antigens or epitopes in vivo, as in gene therapy and DNA immunization applications, are also included in the definition of antigen herein.

[0075] The term "antibody" includes polyclonal and monoclonal antibody preparations, and preparations including hybrid antibodies, modified antibodies, chimeric antibodies and humanized antibodies, and hybrid (chimeric) antibody molecules and any functional fragments obtained from these molecules, wherein these fragments 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 are sufficiently complementary to form a complex through Watson-Crick base pairing. When a primer "hybridizes" with a target (template), this complex (or hybrid) is stable enough, for example, to provide the priming function required by DNA polymerase to initiate DNA synthesis.

[0077] As used herein, “biological sample” refers to, for example, samples of tissue or body fluid isolated from a subject, including but not limited to blood, plasma, serum, fecal matter, urine, bone marrow, bile, spinal fluid, lymph fluid, skin samples, skin, external secretions of the respiratory tract, intestines and genitourinary tract, tears, saliva, milk, blood cells, organs, and biopsies, and also samples of in vitro cell culture components, for example, recombinant cells and cell components, including but not limited to culture media resulting from the growth of cells and tissues in culture media. In particular, the ASF virus may be obtained from biological samples including but not limited to blood, serum, spleen, liver, lungs, lymph nodes, tonsils, and kidneys.

[0078] "Subject" means any member of the family Suidae, including but not limited to pigs of the genus Sus domesticus. The term does not indicate a specific age. Therefore, both adult and newborn individuals are included.

[0079] The terms “variant,” “analogous,” and “mutein” refer to biologically active derivatives of a reference molecule that retain a desired activity, such as antigenic activity, to induce an immune response against ASF. Generally, the terms “variant” and “analogous” refer to compounds having a natural polypeptide sequence and structure having one or more amino acid additions, substitutions (generally substantially conserved), and / or deletions relative to the natural molecule, provided that the modification does not impair biological activity and is “substantially homologous” to the reference molecule defined below. Generally, the amino acid sequences of such analogs will have a high degree of sequence homology to the reference sequence when the two sequences are aligned, e.g., 50% or more, generally 60%–70% or more, even more particularly 80%–85% or more, e.g., at least 90%–95% or more. Often, analogs will contain the same number of amino acids, but will include substitutions as described herein. The term “mutane” further comprises compounds comprising only amino and / or imino molecules, polypeptides containing one or more amino acid-like molecules including but not limited to naturally occurring and non-naturally occurring (e.g., synthetic), cyclic, branched molecules, etc., polypeptides having substituted bonds, and other variations known in the art. Additionally, the term comprises molecules comprising one or more N-substituted glycine residues (“peptoids”) and other synthetic amino acids or peptides. (See, e.g., US Patent Nos. 5,831,005; 5,877,278; and 5,977,301). In the examples, the analogs or mutains have at least the same antigenic activity as the natural molecules. Methods for preparing polypeptide analogs and mutains are known in the art and are further described below.

[0080] As described above, analogs generally involve virtually conservative substitutions, that is, substitutions occurring within the series of related amino acids in the side chain. Specifically, amino acids are generally divided into four series: (1) acidic—aspartate and glutamate; (2) basic—lysine, arginine, histidine; (3) non-polar—alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar—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 separate substitutions of leucine to isoleucine or valine, aspartate to glutamate, or threonine to serine, or similar conservative substitutions of amino acids to structurally related amino acids, will not significantly affect biological activity. For example, the polypeptide of interest may contain up to about 5 to 10 conservative or non-conservative amino acid substitutions, or even up to about 15 to 25 conservative or non-conservative amino acid substitutions, or any integer between 5 and 25, as long as the desired function of the molecule is maintained. A person skilled in the art can easily determine the region of the molecule of interest to which changes may be allowed by referring to Hopp / Woods and Kyte-Doolittle plots, which are well known in the art.

[0081] As used herein, the terms “multiple epitope fusion antigen” or “multiple epitope fusion protein” refer to a polypeptide in which multiple ASF virus antigens are part of a single continuous amino acid chain, which does not occur in nature. ASF virus antigens may be directly linked to each other by peptide bonds or separated by an amino acid sequence. The fusion antigen may contain a p30 / p54 ASF virus-encoded polypeptide or a fragment thereof. Additionally, the fusion antigen may contain a sequence exogenous to the ASF virus. Additionally, the sequences present may be derived from multiple genotypes and / or isolates of the ASF virus.

[0082] In the context of immunogenic compositions, "therapeutic effective dose" refers to an amount of immunogen (e.g., immunogenic polypeptides, fusion proteins, polyproteins, or nucleic acids encoding antigens) that will induce an immunological response for antibody production or the treatment or prevention of ASF infection. Such a response will generally result in the occurrence of an antibody-mediated and / or secretory or cellular immune response to the composition in the subject. Generally, such a response includes, but is not limited to, one or more of the following effects: production of antibodies from any one of the immunological classes such as immunoglobulins A, D, E, G, or M; proliferation of B and T lymphocytes; provision of activation, growth, and differentiation signals to immunological cells; expansion of helper T cell, suppressor T cell, and / or cytotoxic T cell and / or γ,δ-T cell populations.

[0083] For the purposes of the present invention, the "effective amount" of the adjuvant will be an amount that enhances the immunological response to the co-administered antigen or the nucleic acid encoding the antigen.

[0084] As used herein, “treatment” refers to any one of (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. Treatment may be performed prophylactically (before infection) or therapeutically (after infection).

[0085] Before describing the invention in detail, it should be understood that the practice of the invention will utilize conventional methods of virology, microbiology, molecular biology, recombinant DNA techniques, and immunology unless otherwise indicated, all of which are within the ordinary art of the art. Such techniques are sufficiently described in the literature. Although many methods and materials similar or equivalent to those described herein may be used in the practice of the claimed invention, the materials and methods are described herein.

[0086] The present invention includes a composition and a method for immunizing a subject against ASF infection. The present invention provides an immunogenic composition comprising a nucleic acid encoding a capsid protein and / or other immunogenic polypeptide from one or more ASF virus strains, and a composition comprising an immunogenic polypeptide derived from one or more ASF virus strains. The immunogenic polypeptide used in the practice of the present invention may comprise an ASF virus-derived polypeptide comprising a multiple epitope fusion antigen. Additionally, the immunogenic composition may comprise one or more adjuvants or a nucleic acid encoding an adjuvant, wherein the immunogenic polypeptide is mixed with or co-expressed with the adjuvant. Additionally, the immunogenic composition may comprise additional antigens other than ASF virus antigens, such as antigens that can be used for immunization against pathogens causing diarrhea.

[0087] To aid in understanding the disclosed invention, 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 for the treatment or prevention of ASF infection.

[0088] Structural polypeptides, non-structural polypeptides, and polyproteins

[0089] 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 invention disclosed herein include structural proteins, non-structural proteins, and polyproteins. These polypeptides may be full-length proteins, variants thereof, or immunogenic fragments capable of inducing an immune response against the ASF virus.

[0090] Polypeptides within the immunogenic composition may be encoded by any region of the ASF virus genome. Multiple polypeptides may be included in the immunogenic composition. To provide increased protection against a wide range of ASF virus genotypes, such compositions may include polypeptides from the same ASF virus isolate or different strains and isolates, including isolates having any one of the various ASF virus genotypes. Multiple virus strains of ASF virus are known, and multiple polypeptides containing epitopes derived from any one of these strains may be used in the immunogenic composition.

[0091] The antigens used in the immunogenic composition of the present invention may be present in the composition as individual separate polypeptides. Generally, the recombinant proteins of the present invention are represented as GST-fusion proteins and / or His-tagged fusion proteins.

[0092] Multiepitope fusion protein

[0093] Additionally, the immunogenic compositions described herein may comprise a multiple epitope fusion protein. 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 provide two main advantages: first, polypeptides that are unstable or may be poorly expressed on their own can be helped by adding a suitable hybrid partner to overcome the problem; second, commercial manufacturing is simplified because only a single expression and purification step is required to produce two polypeptides that are both antigenically useful.

[0094] The polypeptide of the fusion protein may be derived from the same ASF virus isolate or different strains and isolates, including isolates having any one of various ASF virus genotypes, to provide increased protection against a wide range of viral genotypes. Multiple viral strains of ASF virus are known, and an epitope derived from any one of these strains may be used for the fusion protein.

[0095] It is well known that the species of any given organism differ from individual organism to individual, and that a given organism, such as a virus, can have multiple different strains. For example, as described above, the ASF virus contains at least 24 gene families. Generally, epitopes can have a high degree of homology in terms of amino acid sequences, and this degree of homology is typically greater than 30% or greater than 40% when aligned. Additionally, fusion proteins may contain multiple copies of epitopes, wherein one or more polypeptides of the fusion protein contain sequences containing exact copies of the same epitope. Furthermore, polypeptides may be selected based on the endemicity of specific viral clades in the specific geographical region where the vaccine composition containing the fusion agent will be used. It is readily apparent that the target fusion agent provides an effective method for treating infections in a wide variety of situations.

[0096] The multiple epitope fusion antigen is formula NH2-A-{-XL-} n It 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.

[0097] If the --X-- portion has a leader peptide sequence of its wild-type form, it may be included in the multiple epitope fusion antigen or omitted. In one embodiment, the leader peptides will be deleted except for that of the --X-- portion located at the N-terminus of the hybrid protein; that is, the leader peptide of X1 will be retained, but X2... X nThe leader peptide of will be omitted. This is equivalent to deleting all leader peptides and using the leader peptide of X1 as a partial -A-.

[0098] For each n of (--XL-), the linker amino acid sequence -L- may be present or absent. For example, when n=2, the hybrid 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(s) -L- will generally be 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 that facilitate cloning, poly-glycine linkers (Gly, where n=2, 3, 4, 5, 6, 7, 8, 9, 10 or more), and histidine tags (His n , where n=3, 4, 5, 6, 7, 8, 9, 10 or more). Other suitable linker amino acid sequences will be obvious to a person skilled in the art. Useful linkers are GSGGGG, which contains a Gly-Ser dipeptide formed from the BamHI restriction site to aid in cloning and manipulation, and a (Gly)4 tetrapeptide, a common poly-glycine linker. Additionally, protease substrate sequences may be added (e.g., TEV protease: ENLYFQG).

[0099] -A- is any N-terminal amino acid sequence. This is generally a short sequence, 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). Examples include leader sequences directing protein trafficking or short peptide sequences that facilitate cloning or purification (e.g., histidine tags His with n=3, 4, 5, 6, 7, 8, 9, 10 or more). n It includes ). Other suitable N-terminal amino acid sequences will be obvious to a person skilled in the art. If X1 does not have its own N-terminal methionine, -A- is an oligopeptide that provides N-terminal methionine (e.g., having 1, 2, 3, 4, 5, 6, 7, or 8 amino acids).

[0100] --B-- is any C-terminal amino acid sequence. This is generally a short sequence, 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). Examples include sequences directing protein trafficking, or short peptide sequences that facilitate cloning or purification (e.g., His with n=3, 4, 5, 6, 7, 8, 9, 10 or more). n ), or includes a sequence that enhances protein stability. Other suitable C-terminal amino acid sequences are included when preceded by a TEV protease substrate sequence (e.g., ENLYFQGHis n ), such His nIt will be obvious to a person skilled in the art that the sequence may be removed.

[0101] Individual antigens (individual --X-- parts) of the immunogenic composition within the multiple epitope fusion antigen may be derived from one or more strains or one or more M types. For example, when n=2, X2 may be from the same strain or type as X1 or from a different strain or type. When n=3, the strains may be (i) X1=X2=X3, (ii) X1=X2 is not equal to X3, (iii) X1 is not equal to X2=X3, (iv) X1 is not equal to X2 and not equal to X3, or (v) X1=X3 is not equal to X2, etc.

[0102] When multiple epitope fusion antigens are used, individual antigens (i.e., individual --X-- parts) within the fusion protein may originate from one or more strains. For example, when n=2, X2 may be from the same strain as X1 or from a different strain. When n=3, the strains may be (i) X1=X2=X3, (ii) X1=X2 is not equal to X3, (iii) X1 is not equal to X2=X3, (iv) X1 is not equal to X2 and not equal to X3, or (v) X1=X3 is not equal to X2, etc.

[0103] Accordingly, in the examples, epitopes from different ASF virus strains may be present. Representative multiple epitope fusion proteins for use in the present invention, comprising polypeptides derived from ASF virus isolates, are discussed below. However, it should be understood that multiple epitope fusion proteins comprising other epitopes derived from the ASF virus genome or multiple epitope fusion proteins comprising epitopes of different arrangements may also be used in the disclosed immunogenic compositions.

[0104] In certain embodiments, the fusion protein comprises one or more capsid and / or small structural polypeptides from one or more isolates of the ASF virus.

[0105] In another embodiment, the fusion protein comprises an ASF virus polypeptide from one or more virus strains.

[0106] In all fusions described herein, the viral regions do not need to be in a naturally occurring order. Additionally, 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.

[0107] If desired, 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, and ligands useful for protein purification, such as glutathione-S-transferase and staphylococcal protein A.

[0108] nucleic acids

[0109] For example, the nucleic acid to be used in polypeptide generation as disclosed herein may be derived from any one of the various regions of the ASF virus genome.

[0110] Representative sequences from the ASF virus are known, including sequence numbers 1, 7, 9, 11, 13, and 14.

[0111] Any one of these sequences, and fragments and variants thereof, which can be used for nucleic acid immunization to induce an immune response against the ASF virus, will be used in the present method. Accordingly, the present invention comprises variants of said sequences exhibiting at least about 80-100% sequence identity with the same, including any percent 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. Additionally, the present invention provides a polynucleotide or a variant thereof encoding an immunogenic fragment of an ASF virus polypeptide derived from any one of said sequences. Furthermore, the polynucleotide may include a coding sequence for a naturally occurring polypeptide or may be an artificial sequence that does not occur in nature.

[0112] The polynucleotide may contain less than the entire ASF virus genome, or alternatively, may contain the sequence of the entire viral genome DNA.

[0113] In the examples, the polynucleotide comprises one or more ASF virus sequences encoding p30, p54, p72 and / or hemagglutinin proteins of one or more isolates of ASF virus.

[0114] In the examples, the present invention provides a polynucleotide encoding a multiple epitope fusion protein as described herein. The multiple epitope fusion protein may comprise a sequence from one or more genotypes and / or isolates of the ASF virus.

[0115] The nucleic acid according to the present invention can be prepared in various ways (e.g., by chemical synthesis, from a genome or cDNA library, from the organism itself, etc.) and can take various forms (e.g., single strand, double strand, vector, probe, etc.). In the examples, the nucleic acid is prepared in a substantially pure form (i.e., substantially free of other host cell or non-host cell nucleic acids).

[0116] For example, nucleic acids can be obtained by screening cDNA and / or genomic libraries from virus-infected cells or by deriving genes from vectors known to contain them. For example, the polynucleotide of interest can be isolated from a genomic library derived from viral DNA present in, for example, hair or blood samples from an infected individual. Alternatively, ASF viral nucleic acids can be isolated from infected mammals or from biological samples collected from infected individuals. Polynucleotides can be amplified from the ASF viral genomic DNA encoding them using amplification methods such as PCR. Alternatively, polynucleotides can be synthesized in a laboratory, for example, using an automated synthesizer. The nucleotide sequence can be designed with appropriate codons for the desired specific amino acid sequence. Generally, codons preferred for the host in which the sequence is intended to be expressed will be selected. The complete sequence of the polynucleotide of interest is assembled from nested oligonucleotides prepared by standard methods and can be assembled into a complete coding sequence. The polynucleotide can be RNA or single- or double-stranded DNA. In the examples, the polynucleotide is isolated without other components such as proteins and lipids.

[0117] Accordingly, a specific nucleotide sequence may be obtained from a vector containing the desired sequence, or, where appropriate, synthesized wholly or partially using various oligonucleotide synthesis techniques known in the art, such as site-directed mutagenesis and polymerase chain reaction (PCR) techniques. In particular, one method of obtaining a nucleotide sequence encoding the desired sequence is to anneale a complementary set of overlapping synthetic oligonucleotides produced on a conventional automated polynucleotide synthesizer, ligate them with a suitable DNA ligase, and amplify the ligated nucleotide sequence via PCR. Primer sequences may include, but are not limited to, SEQ ID NOs: 2, 3, 4, 5, 15, and 16.

[0118] production of immunogenic polypeptides

[0119] The polypeptides described herein may be prepared by any suitable method (e.g., recombinant expression, purification from cell cultures, chemical synthesis, etc.) and in various forms (e.g., natural, fused, non-glycosylated, lipidated, etc.). These polypeptides include naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Methods for preparing these polypeptides are well known in the art. The polypeptides are prepared in a substantially pure form (i.e., substantially free of other host cell or non-host cell proteins).

[0120] Polypeptides can be chemically and conveniently synthesized by any one of several techniques known to those skilled in the art of peptides. Generally, these methods involve 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 by a suitable protecting group. Then, the protected or derivatized amino acid may be attached to an inert solid support under conditions allowing for the formation of an amide bond, or may be utilized in solution by adding the next amino acid to a sequence having a suitably protected complementary (amino or carboxyl) group. Then, operations such as removing the protecting group from the newly added amino acid residue and adding the next amino acid (suitably protected) are performed. After linking the desired amino acids into a suitable sequence, any remaining protecting groups (and, if a solid-phase synthesis technique is used, all solid supports) are removed sequentially or simultaneously to obtain the final polypeptide. By a simple variation of this general method, for example, by combining a protected tripeptide with a suitably protected dipeptide (under conditions that do not racemize the chiral center) to form a deprotected pentapeptide, it is possible to add one or more amino acids to a growing chain at a time.

[0121] Common protecting groups include t-butyloxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl (Fmoc), benzyloxycarbonyl (Cbz); p-toluenesulfonyl (Tx); 2,4-dinitrophenyl; benzyl (Bzl); biphenylisopropyloxycarboxy-carbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl, o-bromobenzyloxycarbonyl, cyclohexyl, isopropyl, acetyl, o-nitrophenylsulfonyl, etc. Common solid supports are cross-linked polymer supports. These may include divinylbenzene cross-linked styrene-based polymers, for example, divinylbenzene-hydroxymethylstyrene copolymer, divinylbenzene-chloromethylstyrene copolymer, and divinylbenzene-benzhydrylaminopolystyrene copolymer.

[0122] In addition, the polypeptide of the present invention can be chemically prepared by other methods, such as a simultaneous multiple peptide synthesis method.

[0123] Alternatively, the immunogenic polypeptides, polyproteins, and multiple epitope fusion proteins described above may be recombinantly generated. Once the coding sequence for the desired protein is isolated or synthesized, it may be cloned into any suitable vector or replicon for expression. Many 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 of these expression systems 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.

[0124] Examples of recombinant DNA vectors for cloning and host cells that can be transformed by them include bacteriophage λ (E. coli), pBR322 (E. coli), pACYC177 (E. coli), pKT230 (Gram-negative bacteria), pGV1106 (Gram-negative bacteria), pLAFR1 (Gram-negative bacteria), pME290 (non-E. coli Gram-negative bacteria), pHV14 (E. coli and Bacillus subtilis), pBD9 (Bacillus), pIJ61 (Streptomyces), pUC6 (Streptomyces), YIp5 (Saccharomyces), YCp19 (Saccharomyces), and bovine papilloma virus (mammalian cells).

[0125] In addition, insect cell expression systems, such as baculovirus systems, may be used and are known to those skilled in the art, as described, for example, 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 kit form, particularly from Invitrogen, San Diego, CA ("MaxBac" kit).

[0126] In addition, immunogenic proteins can be produced using plant expression systems. Generally, these systems use virus-based vectors to transfect plant cells with heterologous genes.

[0127] Viral systems, such as vaccinia-based infection / transfection systems, will also be used in conjunction with the invention disclosed herein. In this system, cells are first transfected in vitro with a vaccinia virus recombinant encoding bacteriophage T7 RNA polymerase. This polymerase exhibits sophisticated specificity in that it transcribes only the template containing the T7 promoter. After infection, cells are transfected with DNA of interest 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 translation machinery. This method provides high levels of transient cytoplasmic production of large amounts of RNA and its translation product(s).

[0128] To ensure that a DNA sequence encoding a desired immunogenic polypeptide is transcribed into RNA in a host cell transformed by a vector containing this expression construct, the gene may be placed under the control of a promoter, a ribosome binding site (for bacterial expression), and optionally, an operator (referred to collectively herein as the “control” element). The coding sequence may or may not contain a signal peptide or a leader sequence. In conjunction with the invention disclosed herein, both naturally occurring signal peptides or heterogeneous sequences may be used. The leader sequence may be removed by the host during post-translational processing. References, e.g., US Patent Nos. 4,431,739; 4,425,437; and 4,338,397, are incorporated herein by reference in their entirety. Such sequences include, but are not limited to, tpa leaders and honeybee melittin signal sequences.

[0129] Other regulatory sequences that allow for the regulation of protein sequence expression for host cell growth may also be desirable. Such regulatory sequences are known to those skilled in the art and include, for example, turning gene expression on or off in response to chemical or physical stimuli, including the presence of a regulatory compound. Other types of regulatory elements, such as enhancer sequences, may also be present in the vector.

[0130] Control sequences and other regulatory sequences can be ligated to the coding sequence before being inserted into the vector. Alternatively, the coding sequence can be directly cloned into an expression vector that already contains the control sequence and a suitable restriction site.

[0131] In the examples, it may be necessary to modify the coding sequence so that it can be attached to the control sequence in a suitable orientation, that is, to maintain a suitable read frame. Additionally, it may be desirable to generate a mutant or analog of the immunogenic polypeptide. The mutant or analog may be produced by deletion of part of the sequence encoding the protein, insertion of the sequence, and / or substitution of 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.

[0132] Next, suitable host cells are transformed using an expression vector. Many mammalian cell lines are known in the art and include immortalized cell lines available from the ATCC (American Type Culture Collection), such as but not limited to Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney (COS) cells, and human hepatocellular carcinoma cells (e.g., Hep G2). Similarly, bacterial hosts such as E. coli, Bacillus subtilis, and Streptococcus spp. will be used with the expression construct of the present invention. Yeast hosts useful for the disclosed invention include, in particular, Saccharomyces cerevisiae, Candida albicans, Candida maltosa, Hansenula polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Pichia guilliermondii, Pichia pastoris, Schizosaccharomyces pombe, and Yarrowia lipolytica. Insect cells for use with baculovirus expression vectors include, in particular, Aedes aegypti, Autographa californica, Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda, and Trichoplusia ni.

[0133] Depending on the selected expression system and host, the protein disclosed herein is produced by growing host cells transformed by the expression vector described above under conditions in which the protein of interest is expressed. The selection of suitable growth conditions is within the scope of the art. Then, the cells are destroyed using chemical, physical, or mechanical methods that lyse the cells but retain the ASF virus immunogenic polypeptide substantially intact. Additionally, intracellular proteins may be obtained by removing components from the cell wall or membrane, for example, using a detergent or organic solvent, so as to cause leakage of the immunogenic polypeptide.

[0134] For example, methods for destroying cells for use with the invention disclosed herein include, but are not limited to: sonication or ultrasonication; agitation; liquid or solid extrusion; heat treatment; freeze-thaw; desiccation; explosive decompression; osmotic shock; treatment with lytic enzymes including proteases such as trypsin, neuraminidase, and lysozyme; alkali treatment; and the use of detergents and solvents such as bile salts, sodium dodecyl sulfate, Triton, NP40, and CHAPS. The specific technique used to destroy the cells is largely a matter of choice and will depend on the cell type in which the polypeptide is expressed, culture conditions, and any pretreatments used.

[0135] After cell destruction, cellular debris is generally removed by centrifugation, and the ASF virus immunogenic polypeptide generated within the cell is further purified using standard purification techniques such as, but not limited to, column chromatography, ion-exchange chromatography, size-exclusion chromatography, electrophoresis, HPLC, immunoabsorption techniques, affinity chromatography, and immunoprecipitation.

[0136] For example, one method for obtaining the intracellular ASF virus immunogenic polypeptide disclosed herein includes affinity purification, such as immunoaffinity chromatography using a specific antibody. The selection of a suitable affinity resin is within the scope of the art. After affinity purification, the immunogenic polypeptide may be further purified using conventional techniques well known in the art, for example, by any one of the techniques described above.

[0137] It may be desirable to generate multiple polypeptides simultaneously (e.g., viral polypeptides combined with structural and / or non-structural proteins from one or more viral strains or polypeptide adjuvants). The generation of two or more different polypeptides can be easily achieved, for example, by co-transfecting a host cell with a construct encoding different polypeptides. Co-transfection can be achieved trans or cis-transfected, that is, using separate vectors or using a single vector encoding the polypeptide. When a single vector is used, the expression of the polypeptide can be driven by a single set of control elements, or alternatively, the sequence encoding the polypeptide may exist on the vector of individual expression cassettes regulated by individual control elements.

[0138] The polypeptides described in this specification may be attached to a solid support. Solid supports that may be used in practice with the invention disclosed in this specification include nitrocellulose (e.g., in the form of a membrane or microtiter well); polyvinyl chloride (e.g., in the form of a sheet or microtiter well); polystyrene latex (e.g., beads or microtiter plates); polyvinylidene fluoride; diazotized paper; nylon membranes; and substrates such as activated beads, self-reactive beads, etc.

[0139] Generally, a solid support is first reacted with a solid phase component (e.g., one or more ASF virus antigens) under suitable binding conditions so that the component is sufficiently immobilized on the support. Sometimes, the immobilization of the antigen on the support can be enhanced by first binding the antigen to a protein having better binding properties. Suitable binding proteins include, but are not limited to, macromolecules such as serum albumin including bovine serum albumin (BSA), keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, and other proteins well known to those skilled in the art. Other molecules that may be used to bind the antigen to the support include polysaccharides, polylactic acid, polyglycolic acid, high molecular weight amino acids, amino acid copolymers, etc. Methods for binding these molecules to the antigen are well known to those skilled in the art.

[0140] If desired, polypeptides can be labeled using conventional techniques. Suitable labels include fluorophores, chromophores, and radioactive atoms (especially, 32 P and 125I includes electron-dense reagents, enzymes, and ligands having specific binding partners. Enzymes are generally detected by their activity. For example, horseradish peroxidase is generally detected by its ability to convert 3,3',5,5'-tetramethylbenzidine (TMB) into a blue pigment that can be quantified by a spectrophotometer. "Specific binding partners" refer to proteins capable of binding to ligand molecules with high specificity, such as in the case of antigens and monoclonal antibodies specific thereto. Other specific binding partners include biotin and avidin or streptavidin, IgG and protein A, and numerous receptor-ligand couples known in the art. Single labels or combinations of labels may be used as disclosed herein.

[0141] Once formulated, the composition disclosed herein may be administered directly to a subject (e.g., as described above) or, alternatively, delivered ex vivo to cells derived from a subject using a method such as that described above.

[0142] Immunogenic composition

[0143] In addition, the present invention provides a composition comprising one or more of the immunogenic polypeptides and / or polyproteins 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 one or more polypeptides, or multiepitope polypeptides, or polyproteins.

[0144] The immunogenic composition may comprise a mixture of polypeptides, which may be delivered sequentially using the same or different vehicles. The antigen may be administered, for example, as a prophylactic (i.e., to prevent infection) or therapeutic (to treat infection) immunogenic composition, either individually or in combination. The immunogenic composition may be provided one or more times to achieve the desired effect (e.g., one or more "boosts" following a "prime" administration). The same composition may be administered in one or more priming steps and one or more boosting steps. Alternatively, different compositions may be used for priming and boosting.

[0145] The immunogenic composition will generally contain one or more "pharmaceutically acceptable excipients or vehicles," such as water, saline solution, glycerol, ethanol, etc. Additionally, auxiliary substances such as wetting agents or emulsifiers, pH buffering agents, etc. may be present in these vehicles.

[0146] The immunogenic composition will generally comprise one or more "pharmaceutically acceptable carriers" in addition to the components mentioned above. These include any carriers that do not induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are generally large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, high molecular weight amino acids, amino acid copolymers, and lipid aggregates (e.g., oil droplets or liposomes). These carriers are well known to those skilled in the art. Additionally, the composition may contain diluents such as water, saline solution, glycerol, etc. Additionally, auxiliary substances such as wetting agents or emulsifiers, pH buffers, etc. may be present. A detailed discussion of pharmaceutically acceptable components is available in Gennaro (2000) Remington: The Science and Practice of Pharmacy. 20th ed., ISBN: 0683306472.

[0147] Pharmaceutically acceptable salts, for example, inorganic salts such as hydrochloride, hydrobromide, phosphate, or sulfate, and salts of organic acids such as acetate, propionate, malonate, or benzoate may also be used in the compositions disclosed herein. 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. Additionally, the disclosed compositions may contain, alone or in combination, liquids or excipients such as water, saline solution, glycerol, dextrose, ethanol, etc., and substances such as wetting agents, emulsifiers, or pH buffers. Additionally, antigens may be adsorbed onto, captured within, or bound to particulate carriers such as PLGs and liposomes.

[0148] Antigens can be conjugated to carrier proteins to enhance immunogenicity. This is particularly useful in compositions where sugar or carbohydrate antigens are used.

[0149] The carrier protein may include, but is not limited to, bacterial toxins or toxoids such as diphtheria or tetanus toxoid. CRM 197 Diphtheria toxoid may be used. Other carrier polypeptides include N. meningitidis outer membrane protein (EP-A-0372501), synthetic peptides (EP-A-0378881 and EP-A-0427347), heat shock protein (WO 93 / 17712 and WO 94 / 03208), pertussis protein (WO 98 / 58668 and EP-A-0471177), protein D from H. influenzae (WO 00 / 56360), cytokine (WO 91 / 01146), lymphokine, hormone, growth factor, toxin A or B from C. difficile (WO 00 / 61761), iron-absorbing protein such as transferring (WO 01 / 72337), etc. If the mixture contains capsular saccharides from both serigraphs A and C, the ratio of MenA saccharides to MenC saccharides (w / w) may be 1 or greater (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. If necessary, any suitable conjugation reaction may be used with any suitable linker.

[0150] An immunogenic composition comprising the disclosed vaccine may be administered together with other immunomodulators. For example, the vaccine disclosed herein may comprise an adjuvant. The adjuvant comprises one or more of the following types of adjuvants described below, but is not limited thereto.

[0151] mineral-containing composition

[0152] The mineral-containing composition suitable for use as an adjuvant disclosed herein comprises inorganic salts such as aluminum salts and calcium salts. The salts disclosed herein comprise inorganic salts such as hydroxides (e.g., oxyhydroxide), phosphates (e.g., hydroxyphosphate, orthophosphate), sulfates, etc., or mixtures of different inorganic compounds (e.g., a mixture of phosphate and hydroxide adjuvants, optionally with an excess amount of phosphate) that take any suitable form (e.g., gel, crystalline, amorphous, etc.). Additionally, the mineral-containing composition may be formulated as particles of metal salts (WO00 / 23105).

[0153] Aluminum salts are Al 3+ It may be included in the vaccine in a dose of 0.2 to 1.0 mg per dose.

[0154] In the examples, the aluminum-based adjuvant for use as disclosed is alum (aluminum potassium sulfate (AlK(SO4)2)), or an alum derivative formed in situ by mixing an antigen in a phosphate buffer with alum and then titrating and precipitating with a base such as ammonium hydroxide or sodium hydroxide.

[0155] Another aluminum-based adjuvant for use in the vaccine formulation of the present invention is an aluminum hydroxide adjuvant (Al(OH)3) or approximately 500 m 2It is crystalline aluminum oxyhydroxide (AlOOH), which is an excellent adsorbent having a surface area of ​​1 / g. Alternatively, aluminum hydroxyphosphate containing phosphate groups instead of some or all of the hydroxyl groups of aluminum hydroxide adjuvants (AlPO4) or aluminum hydroxyphosphate is provided. In the examples, the aluminum phosphate adjuvants provided herein are amorphous and soluble in acidic, basic, and neutral media.

[0156] In the examples, the adjuvant disclosed herein comprises both aluminum phosphate and aluminum hydroxide. In one aspect, the adjuvant has an amount of aluminum phosphate greater than aluminum hydroxide, such as an aluminum phosphate to aluminum hydroxide weight ratio of 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 9:1 or greater. More particularly, the aluminum salt in the vaccine is present at 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.

[0157] Generally, the aluminum-based adjuvant(s), or the ratio of multiple aluminum-based adjuvants such as aluminum phosphate to aluminum hydroxide, is selected by optimizing the electrostatic attraction between molecules so that the antigen has an opposite charge to the adjuvant at the desired pH. For example, the aluminum phosphate adjuvant (iep=4) adsorbs lysozyme but does not adsorb albumin at pH 7.4. If albumin is the target, the aluminum hydroxide adjuvant will be selected (i.e., 11.4). Alternatively, pretreatment of aluminum hydroxide with phosphate lowers its isoelectric point, making it a preferred adjuvant for more basic antigens.

[0158] oil-emulsion

[0159] Oil-emulsion compositions suitable for use as adjuvants may include squalene-water emulsions, for example, MF59 (5% squalene, 0.5% TWEEN 80™, and 0.5% SPAN 85™, formulated into submicron particles using a microfluidizer). See WO90 / 14837. MF59 is used as an adjuvant in the FLUAD™ influenza virus trivalent subunit vaccine.

[0160] In particular, the adjuvant for use in the composition is a submicron oil-in-water emulsion. The submicron oil-in-water emulsion for use herein is a squalene / water emulsion containing optionally varying amounts of MTP-PE, such as a submicron oil-in-water emulsion containing 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-(beta-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE), e.g., a submicron oil-in-water emulsion known as "MF59" (International Publication No. WO90 / 14837; US Patent No. It may be (Section 6,299,884 and Section 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 contains varying amounts of MTP-PE formulated into submicron particles using a microfluidizer such as a Model 110Y microfluidizer (Microfluidics, Newton, Mass.). For example, MTP-PE may be present in amounts of about 0-500 μg / dose, 0-250 μg / dose, and 0-100 μg / dose. As used herein, the term "MF59-0" refers to the submicron oil-in-water emulsion lacking MTP-PE, whereas the term MF59-MTP refers to a formulation containing MTP-PE. For example, "MF59-100" contains 100 μg MTP-PE per volume, etc. 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. Another submicron oil-in-water emulsion contains 10% squalene, 0.MF75, also known as SAF, contains 4% TWEEN 80™, 5% Pluronic-blocking polymer L121, and thr-MDP, and is also microfluidized into a submicron emulsion. MF75-MTP represents an MF75 formulation containing MTPs such as 100-400 μg MTP-PE per dose.

[0161] Immunostimulants such as muramyl peptides for use in submicron oil-in-water emulsions, methods for preparing the same, and compositions are described in detail in International Publication No. WO90 / 14837 and US Patents No. 6,299,884 and No. 6,451,325.

[0162] Complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA) can also be used as adjuvants.

[0163] Saponin formulation

[0164] Saponin formulations can also be used as adjuvants. Saponins are a heterologous 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 the Quillaia saponaria Molina tree have been widely studied as adjuvants. Additionally, saponins can be commercially obtained from Smilax ornata (sarsaparilla), Gypsophila paniculata (brides veil), and Saponaria officinalis (soap root). Saponin adjuvant formulations include purified formulations such as QS21 and lipid formulations such as ISCOMs.

[0165] The saponin composition was purified using High Performance Thin Layer Chromatography (HP-TLC) and Reversed Phase High Performance Liquid Chromatography (RP-HPLC). Specific purified fractions were identified using these techniques, including QS7, QS17, QS18, QS21, QH-A, QH-B, and QH-C. In the example, the saponin is QS21. A method for preparing QS21 is disclosed in US Patent No. 5,057,540. Additionally, the saponin formulation may contain sterols such as cholesterol (Ref. WO96 / 33739).

[0166] A combination of saponins and cholesterol can be used to form unique particles called Immunostimulating Complexes (ISCOMs). ISCOMs generally also contain phospholipids such as phosphatidylethanolamine or phosphatidylcholine. All known saponins can be used for ISCOMs. In the examples, ISCOMs contain one or more of Quil A, QHA, and QHC. ISCOMs are further described in EP0109942, WO96 / 11711, and WO96 / 33739. Optionally, ISCOMs may be free of additional detergent(s). See WO00 / 07621.

[0167] Bacteria or microbial derivatives

[0168] Immunostimulants suitable for use as disclosed in this specification include the following bacteria or microbial derivatives:

[0169] (1) Non-toxic derivatives of Enterobacter lipopolysaccharides (LPs)

[0170] These derivatives include monophosphoryl lipid A (MPL) and 3-O-deacylated MPL (3dMPL). 3dMPL is a mixture of 3-de-O-acylated monophosphoryl lipid A and 4, 5, or 6-acylated chains. One "small particle" form of 3-de-O-acylated monophosphoryl lipid A is disclosed in EP 0 689 454. These "small particles" of 3dMPL are small enough to be sterile filtered through a 0.22-micron membrane (Ref. EP 0 689 454). Other non-toxic LPS derivatives include aminoalkyl glucosaminid phosphate derivatives, such as monophosphoryl lipid A mimics like RC-529.

[0171] (2) Lipid A derivative

[0172] Lipid A derivatives include derivatives of lipid A from E. coli such as OM-174.

[0173] (3) Immunostimulating oligonucleotide

[0174] Immunostimulating oligonucleotides suitable for use as adjuvants may include nucleotide sequences containing a CpG motif (a sequence containing unmethylated cytosines followed by guanosine and connected by phosphate bonds). Additionally, bacterial double-stranded RNA or oligonucleotides containing palindromic or poly(dG) sequences have been shown to be immunostimulating.

[0175] CpGs may include nucleotide modifications / analogs such as phosphorothioate modifications and may be double-stranded or single-stranded. Optionally, guanosine may be replaced with an analog such as 2'-deoxy-7-deaguaguanosine.

[0176] The CpG sequence may relate to TLR9, such as the motif GTCGTT or TTCGTT. The CpG sequence may be specific for inducing a Th1 immune response, such as CpG-A ODN, or may be more specific for inducing a B cell response, such as CpG-B ODN. In the examples, the CpG is CpG-A ODN.

[0177] In the examples, the CpG oligonucleotides may be configured so that their 5' ends are accessible for receptor recognition. Optionally, two CpG oligonucleotide sequences may be attached to their 3' ends to form "immunomers".

[0178] (4) ADP-ribosylated toxin and its detoxified derivatives.

[0179] Bacterial ADP-ribosylated toxin and its detoxified derivatives may be used as adjuvants. In the examples, the protein may be derived from E. coli (i.e., E. coli heat-unstable enterotoxin "LT"), cholera ("CT"), or pertussis ("PT"). The use of detoxified ADP-ribosylated toxin as a mucosal adjuvant is described in WO95 / 17211, and as a parenteral adjuvant is described in WO98 / 42375. In the examples, the adjuvant is a detoxified LT mutant such as LT-K63, LT-R72, and LTR192G.

[0180] Bioadhesives and Mucoadhesives

[0181] Bioadhesives and mucoadhesives may also be used as adjuvants. Suitable bioadhesives include esterified hyaluronic acid microspheres or mucoadhesives such as cross-linked derivatives of polyacrylic acid, polyvinyl alcohol, polyvinylpyrrolidone, polysaccharides, and carboxymethylcellulose. Chitosan and its derivatives may also be used as adjuvants. For example, WO99 / 27960.

[0182] Muramyl Peptides

[0183] Examples of muramyl peptides suitable for use as adjuvants include N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-1-alanyl-d-isoglutamine (nor-MDP), and N-acetylmuramyl-1-alanyl-d-isoglutaminyl-1-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE).

[0184] imidazoquinoline compound.

[0185] Examples of imidazoquinoline compounds suitable for use as adjuvants include imiquimod and analogs thereof (see, e.g., US Patent 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).

[0186] Thiosemicabazone compound.

[0187] Examples of thiosemicabazone compounds, and methods for formulation, preparation, and screening of compounds suitable for use as immunoadjuvants, include those described in WO04 / 60308. Thiosemicabazone is particularly effective for stimulating human peripheral blood mononuclear cells for the production of cytokines such as TNF-α.

[0188] Tryptanthrin compound.

[0189] Examples of tripantrin compounds, and methods for formulation, preparation, and screening of compounds suitable for use as immunoadjuvants disclosed herein, include those described in WO04 / 64759. Tripantrin compounds are particularly effective for stimulating human peripheral blood mononuclear cells for the production of cytokines such as TNF-α.

[0190] A combination of one or more aspects of the above-identified adjuvants may be applied to the compositions disclosed herein. For example, the following adjuvant compositions may be used:

[0191] (1) Saponin and oil-in-water emulsion (WO99 / 11241); (2) Saponin (e.g., QS21) + non-toxic LPS derivative (e.g., 3dMPL) (Ref. WO94 / 00153); (3) Saponin (e.g., QS21) + non-toxic LPS derivative (e.g., 3dMPL) + cholesterol; (4) Saponin (e.g., QS21) + 3dMPL + IL-12 (optional + sterol) (WO98 / 57659); (5) E.g., combination of QS21 and / or oil-in-water emulsion with 3dMPL (Ref. European Patent Applications 0835318, 0735898 and 0761231); (6) SAF containing 10% squalene, 0.4% TWEEN 80™, 5% Pluronic-blocking polymer L121, and thr-MDP, which is microfluidized into a submicron emulsion or vortexed to produce a larger particle size emulsion; (7) Ribi™ adjuvant system (RAS), (Ribi Immunochem), containing 2% squalene, 0.2% TWEEN 80™, and MPL+CWS (Detox™), which is one or more bacterial cell wall components from the group consisting of monophosphoryl lipid A (MPL), trehalose dimicolate (TDM), and cell wall skeleton (CWS); and (8) one or more inorganic salts (e.g., aluminum salts) + non-toxic derivatives of LPS (e.g., 3dPML), (9) one or more inorganic salts (e.g., aluminum salts) and one or more immunostimulatory oligonucleotides (e.g., nucleotide sequences containing CpG motifs) and one or more detoxified ADP-ribosylated toxins (e.g., LT-K63 and LT-R72), (10) inulin and inulin acetate formulations (see, e.g., WO 2013 / 110050, in its entirety incorporated herein).

[0192] Additional antigens

[0193] The compositions disclosed herein may optionally comprise one or more additional polypeptide antigens not derived from ASF virus proteins. Such antigens include bacterial, viral, or parasitic antigens.

[0194] In one embodiment, the ASF virus antigen is 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), Streptococcus pneumoniae, Neisseria meningitides, Streptococcus pyogenes (Group A Streptococcus), Moraxella It is combined with one or more antigens derived from bacteria or viruses such as Moraxella catarrhalis, Bordetella pertussis, Staphylococcus aureus, Clostridium tetani (tetanus), Corynebacterium diphtheriae (diphtheria), Haemophilus influenzae B (Hib), Pseudomonas aeruginosa, Streptococcus agalactiae (Group B streptococcus), and E. coli, but is not limited thereto.

[0195] In another embodiment, the ASF virus antigen is Neisseria meningitides, Streptococcus pneumoniae, Streptococcus pyogenes (Group A Streptococcus), Moraxella cataralis, Bordetella pertussis, Staphylococcus aureus, Staphylococcus epidermis, Clostridium tetani (tetanus), Corynebacterium diphtheria (diphtheria), Haemophilus influenzae B (Hib), Pseudomonas aeruginosa, Legionella pneumophila, Streptococcus agalactiae (Group B Streptococcus), Enterococcus faecalis, Helicobacter pylori, Chlamydia pneumoniae It is combined with one or more antigens, including but not limited to pneumoniae), orthomyxovirus (influenza), pneumovirus (RSV), paramyxovirus (PIV and mumps), morbilivirus (measles), togavirus (rubella), enterovirus (polio), HBV, coronavirus (SARS), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), and cytomegalovirus (CMV).

[0196] In another embodiment, an ASF virus antigen is combined with one or more antigens useful in a vaccine designed to protect an individual from pathogens causing diarrheal disease. These antigens include, but are not limited to, rotavirus, Shigella spp., enterotoxigenic Escherichia coli (ETEC), Vibrio cholerae, and Campylobacter jejuni antigens. In an embodiment, one or more norovirus antigens may be derived from Norwalk virus, Snow Mountain virus, and / or Hawaiian virus and are combined with a rotavirus antigen in an immunogenic composition.

[0197] Antigens that can be used with the present composition include, but are not limited to, one or more of the antigens listed below, or antigens derived from one or more of the pathogens listed below:

[0198] Bacterial antigen

[0199] Suitable bacterial antigens disclosed herein include proteins, polysaccharides, lipopolysaccharides, and outer membrane vesicles that may be isolated, purified, or derived from bacteria. Additionally, bacterial antigens may include bacterial lysates and inactivated bacterial formulations. Bacterial antigens may be generated by recombinant expression. Bacterial antigens include epitopes that may be exposed on the surface of bacteria during at least one stage of their life cycle. Bacterial antigens may be conserved across multiple serotypes. Bacterial antigens include antigens derived from one or more of the bacteria presented below and specific antigen examples identified below.

[0200] Neisseria meningitides: Meningitides antigens may comprise proteins (e.g., as identified in References 1-7), sugars (including polysaccharides, oligosaccharides, or lipopolysaccharides), or outer membrane vesicles purified or derived from N. meningitides serogroups such as A, C, W135, Y, and / or B. Meningitides protein antigens may be selected from adhesions, autotransporters, toxins, Fe-acquiring proteins, and membrane-associated proteins (e.g., integral outer membrane proteins).

[0201] Streptococcus pneumoniae: Streptococcus pneumoniae antigens may include sugars (including polysaccharides or oligosaccharides) and / or proteins 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. The protein antigen may be selected from proteins identified in WO 98 / 18931, WO 98 / 18930, US Patent No. 6,699,703, US Patent No. 6,800,744, WO 97 / 43303, and WO 97 / 37026. The Streptococcus pneumoniae protein may be selected from the Poly Histidine Triad family (PhtX), the Choline-binding protein family (CbpX), CbpX truncates, the LytX family, LytX truncates, CbpX truncate-LytX truncate chimeric protein, pneumolysin (Ply), PspA, PsaA, Sp128, Sp101, Sp130, Sp125, or Sp133.

[0202] Streptococcus pyogenes (Group A Streptococcus): Group A Streptococcus antigens may include proteins identified in WO 02 / 34771 or WO 2005 / 032582 (including GAS 40), fusions of fragments of GAS M protein (including those described in WO 02 / 094851), fibronectin binding protein (Sfb1), streptococcal heme-associated protein (Shp), and streptolysin S (SagA).

[0203] Moraxella catarrhalis: Moraxella antigens include the antigens identified in WO 02 / 18595 and WO 99 / 58562, outer membrane protein antigen (HMW-OMP), C-antigen, and / or LPS.

[0204] Bordetella pertussis: Pertussis antigens include pertussis holotoxin (PT) and filamentous hemagglutinin (FHA) from B. pertussis, and optionally include combinations with pertactin and / or agglutinogens 2 and 3 antigens.

[0205] Staphylococcus aureus: Staph aureus antigens include non-toxic recombinant Pseudomonas aeruginosa exotoxin A, e.g. S. aureus type 5 and 8 capsule polysaccharides optionally conjugated to STAPHVAX™, or antigens derived from surface proteins, invasins (leukocidin, kinase, hyaluronidase), surface factors inhibiting phagocytic engulfment (capsule, protein A), carotenoids, catalase production, protein A, coagulase, clotting factor, and / or membrane-damaging toxins (hemolysins, leukotoxins, leukocidin) that lyse eukaryotic cell membranes (optional detoxified).

[0206] Staphylococcus epidermis: S. epidermis antigens include slime-associated antigens (SAA).

[0207] Clostridium tetani (tetanus): Tetanus antigens include tetanus toxoid (TT) and can be used as carrier proteins combined with / conjugated with the composition of the present invention.

[0208] Corynebacterium diphtheria (Diphtheria): Diphtheria antigen is detoxified CRM 197 It includes diphtheria toxins containing [specific component]. Additionally, antigens capable of regulating, inhibiting, or inducing ADP ribosylation are considered for combination / co-administration / conjugation with the compositions of the present invention. Diphtheria toxoids can be used as carrier proteins.

[0209] Haemophilus influenzae B (Hib): Hib antigens include Hib saccharide antigens.

[0210] Pseudomonas aeruginosa: Pseudomonas antigens include endotoxin A, Wzz protein, P. aeruginosa LPS, more particularly LPS isolated from PAO1 (O5 serotype), and / or outer membrane proteins including outer membrane protein F (OprF).

[0211] Legionella pneumophila: Bacterial antigens can originate from Legionella pneumophila.

[0212] Streptococcus agalactiae (Group B Streptococcus): Group B Streptococcus antigens include protein or glycan antigens identified in 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 Ia, Ib, Ia / c, II, III, IV, V, VI, VII, and VIII).

[0213] Neiserria gonorrhoeae: Gonorrhoeae antigens include Por (or porin) proteins (e.g., PorB), transfer-binding proteins (e.g., TbpA and TbpB), opaque proteins (e.g., Opa), reduction-modifiable proteins (Rmp), and outer membrane endoplasmic reticulum (OMV) preparations (see, e.g., WO99 / 24578, WO99 / 36544, WO99 / 57280, WO02 / 079243).

[0214] Chlamydia trachomatis: Chlamydia trachomatis antigens include antigens derived from serotypes A, B, Ba, and C (preparations of trachoma, which causes blindness), serotypes L1, L2 & L3 (associated with Lymphogranuloma venereum), and serotype DK. Additionally, Chlamydia trachomatis antigens may include antigens identified in WO 00 / 37494, WO 03 / 049762, WO 03 / 068811, or 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).

[0215] Treponema pallidum (Syphilis): The syphilis antigen contains the TmpA antigen.

[0216] Haemophilus ducreyi (causes chancroid): The ducreyi antigen contains the outer membrane protein (DsrA).

[0217] Enterococcus faecium or Enterococcus faecium: The antigen comprises a trisaccharide repeat or another Enterococcus-derived antigen provided in US Patent No. 6,756,361.

[0218] Helicobacter pylori: H. pylori antigens include Cag, Vac, Nap, HopX, HopY, and / or urease antigens.

[0219] Staphylococcus saprophyticus: The antigen contains the 160 kDa hemagglutinin of the S. saprophyticus antigen.

[0220] Yersinia enterocolitica antigens contain LPS.

[0221] E. coli: E. coli antigens may originate from enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAggEC), diffusely adhering E. coli (DAEC), enteropathogenic E. coli (EPEC), and / or enterohemorrhagic E. coli (EHEC).

[0222] Bacillus anthracis (anthrax): B. anthracis antigens are randomly detoxified and can be selected from A-components (lethal factor (LF) and edema factor (EF)), both of which may share a common B-component known as the protective antigen (PA).

[0223] Yersinia pestis (plague): The plague antigen contains the F1 capsule antigen.

[0224] Mycobacterium tuberculosis: Tuberculosis antigens include lipoproteins, LPS, BCG antigens, fusion proteins of antigen 85B (Ag85B) and / or ESAT-6 optionally formulated from cationic lipid vesicles, Mycobacterium tuberculosis (Mtb) isocitrate dehydrogenase-associated antigens, and / or MPT51 antigens.

[0225] Rickettsia: The antigen contains outer membrane proteins including outer membrane proteins A and / or B (OmpB).

[0226] Listeria monocytogenes: Bacterial antigens can originate from Listeria monocytogenes.

[0227] Chlamydia pneumoniae: Antigens include those identified in WO 02 / 02606.

[0228] Vibrio cholerae: Antigens include proteinase antigens, LPS, particularly lipopolysaccharides of Vibrio cholerae II, O1 Inaba O-specific polysaccharides, V. cholerae O139, antigens of the IEM108 vaccine, and / or Zonula occludens toxin (Zot).

[0229] Salmonella typhi (typhoid fever): The antigen contains a capsule polysaccharide containing a conjugate (Vi, i.e., vax-TyVi).

[0230] Borrelia burgdorferi (Lyme disease): Antigens include lipoproteins (e.g., OspA, OspB, OspC, and OspD), other surface proteins such as OspE-associated proteins (Erps), decorin-binding proteins (e.g., DbpA), and antigenically variable VI proteins such as antigens associated with P39 and P13 VlsE antigen variant proteins.

[0231] Porphyromonas gingivalis: The antigen contains the P. gingivalis outer membrane protein (OMP).

[0232] Klebsiella: The antigen comprises an OMP containing OMP A, or a polysaccharide optionally conjugated to tetanus toxoid.

[0233] Additional bacterial antigens of the present invention may be capsule antigens, polysaccharide antigens, or any of the protein antigens mentioned above. Additionally, additional bacterial antigens may include outer membrane vesicle (OMV) preparations. Additionally, antigens include live, attenuated, and / or purified versions of any of the bacteria mentioned above. Antigens of the present invention may be derived from Gram-negative or Gram-positive bacteria. Antigens of the present invention may be derived from aerobic or anaerobic bacteria.

[0234] In addition, any one of the above bacteria-derived sugars (polysaccharides, LPS, LOS, or oligosaccharides) is a carrier protein (e.g., CRM). 197 It may be conjugated to other preparations or antigens such as ). Such conjugation may be direct conjugation affected by reductive amination of the carbonyl moiety on the sugar to the amino group on the protein, as provided in US Patent No. 5,360,897. Alternatively, the sugar may be conjugated through a linker such as succinamide or other bonds.

[0235] viral antigen

[0236] Viral antigens suitable for use in the disclosed compositions include purified subunit formulations, viral proteins that may be isolated, purified, or derived from viruses, and virus-like particles (VLPs). Viral antigens may be derived from viruses replicated in cell cultures or other substrates. Alternatively, viral antigens may be recombinantly expressed. Viral antigens comprise epitopes that are exposed on the surface of the virus during 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 presented below and specific antigen examples identified below.

[0237] Orthomyxovirus: Viral antigens may be derived from orthomyxoviruses such as influenza A, B, and C. Orthomyxovirus antigens may be selected from one or more viral proteins comprising hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix protein (M1), membrane protein (M2), and one or more transcriptionase components (PB1, PB2, and PA). In the examples, the antigens comprise HA and NA.

[0238] Influenza antigens can originate from interpandemic (annual) influenza strains. Alternatively, influenza antigens can originate from strains likely to cause an epidemic (i.e., influenza strains with novel hemagglutinins compared to the hemagglutinins of currently circulating strains, influenza strains pathogenic in avian subjects and likely to spread horizontally in human populations, or influenza strains pathogenic to humans).

[0239] Paramyxoviridae viruses: Viral antigens can originate from paramyxoviridae viruses such as pneumovirus (RSV), paramyxovirus (PIV), and morbilivirus (measles).

[0240] Pneumovirus: The viral antigen may be derived from pneumoviruses such as respiratory syncytial virus (RSV), bovine respiratory syncytial virus, mouse pneumovirus, and Turkey rhinotracheitis virus. In the examples, the pneumovirus is RSV. The pneumovirus antigen may be selected from one or more of the following proteins, including a surface protein fusion (F), a glycoprotein (G) and a 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. Additionally, the pneumovirus antigen may be formulated from or derived from a chimeric virus. For example, a chimeric RSV / PIV virus may contain components of both RSV and PIV.

[0241] Paramyxovirus: The viral antigen may be derived from paramyxoviruses such as parainfluenza virus types 1-4 (PIV), mumps, Sendai virus, Simian virus 5, bovine parainfluenza virus, and Newcastle disease virus. In the examples, 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 F1 and F2, nucleoprotein (NP), phosphoprotein (P), large protein (L), and matrix protein (M). The paramyxovirus proteins may include HN, F1, and F2. Additionally, the paramyxovirus antigen may be formulated from or derived from a chimeric virus. For example, a chimeric RSV / PIV virus may contain components of both RSV and PIV. Commercially available mumps vaccines contain live attenuated mumps virus in a monovalent form or in combination with the measles and rubella vaccine (MMR).

[0242] Morbilivirus: Viral antigens may be derived from morbiliviruses such as measles. Morbilivirus 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 generally contain live attenuated measles virus combined with mumps and rubella (MMR).

[0243] Picornavirus: Viral antigens can be derived from picornaviruses such as enteroviruses, rhinoviruses, heparnaviruses, cardioviruses, and aphthoviruses. Antigens derived from enteroviruses, such as poliovirus, may be used.

[0244] Enterovirus: The viral antigen may be derived from enteroviruses such as poliovirus type 1, 2, or 3, Coxsackie A virus types 1 to 22 and 24, Coxsackie B virus types 1 to 6, Echovirus (ECHO virus) types 1 to 9, 11 to 27, and 29 to 34, and enteroviruses 68 to 71. In the examples, the enterovirus may be poliovirus. The enterovirus antigen may include one or more of the following capsid proteins VP1, VP2, VP3, and VP4. Commercially available polio vaccines include inactivated polio vaccines (IPV) and oral poliovirus vaccines (OPV).

[0245] Heparnavirus: Viral antigens can be derived from heparnaviruses such as hepatitis A virus (HAV). Commercially available HAV vaccines contain inactivated HAV vaccines.

[0246] Togavirus: Viral antigens may be derived from togaviruses such as rubivirus, alphavirus, or arterivirus. Antigens derived from rubiviruses, such as rubella virus, may be used. Togavirus antigens may be selected from E1, E2, E3, C, NSP-1, NSPO-2, NSP-3, or NSP-4. Togavirus antigens include E1, E2, or E3. Commercially available rubella vaccines generally contain a live cold-adapted virus combined with the mumps and measles vaccine (MMR).

[0247] Flavivirus: Viral antigens may be derived from flaviviruses such as tick-borne encephalitis (TBE), dengue fever (types 1, 2, 3, or 4), yellow fever, Japanese encephalitis, West Nile encephalitis, St. Louis encephalitis, Russian spring-summer encephalitis, and Powassan encephalitis. Flavivirus antigens may be selected from PrM, M, C, E, NS-1, NS-2a, NS2b, NS3, NS4a, NS4b, and NS5. Flavivirus antigens may include PrM, M, and E. Commercially available TBE vaccines contain inactivated viral vaccines.

[0248] Pestivirus: Viral antigens can originate from pestiviruses such as bovine viral diarrhea (BVDV), classic swine fever (CSFV), or Border disease (BDV).

[0249] Hepadnavirus: Viral antigens can be derived from hepadnaviruses, such as the hepatitis B virus. Hepadnavirus antigens can be selected from surface antigens (L, M, and S) and core antigens (HBc, HBe). Commercially available HBV vaccines contain subunit vaccines that include the surface antigen S protein.

[0250] Hepatitis C virus: Viral antigens may be derived from hepatitis C virus (HCV). HCV antigens may be selected from one or more peptides from E1, E2, E1 / E2, NS345 polyprotein, NS 345-core polyprotein, core, and / or non-structural regions.

[0251] Rhabdovirus: Viral antigens may be derived from rhabdoviruses such as Lyssavirus (rabies virus) and Vesiculovirus (VSV). Rhabdovirus antigens may be selected from glycoproteins (G), nucleoproteins (N), large proteins (L), and non-structural proteins (NS). Commercially available rabies virus vaccines contain dead viruses grown in human diploid cells or fetal rhesus lung cells.

[0252] Caliciviridae; viral antigens can be derived from Caliciviridae such as Norwalk virus and Norwalk-like viruses such as Hawaii virus and Snow Mountain virus.

[0253] Coronavirus: Viral antigens may be derived from coronavirus, SARS, human respiratory coronavirus, avian infectious bronchitis (IBV), mouse hepatitis virus (MHV), and porcine transmissible gastroenteritis virus (TGEV). Coronavirus antigens may be selected from spike (S), envelope (E), matrix (M), nucleocapsid (N), and hemagglutinin-esterase glycoprotein (HE). In the examples, coronavirus antigens are derived from SARS virus. SARS virus antigens are described in WO 04 / 92360.

[0254] Retrovirus: Viral antigens may be derived from retroviruses such as oncoviruses, lentiviruses, or spumaviruses. Oncovirus antigens may be derived from HTLV-1, HTLV-2, or HTLV-5. Lentivirus antigens may be derived from HIV-1 or HIV-2. Retrovirus 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 (gp160 and gp41), pol, tat, nef, rev, vpu, and miniproteins (e.g., p55 gag and gp140v deletions). 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 .

[0255] Reovirus: Viral antigens may be derived from reoviruses such as orthoreovirus, rotavirus, orbivirus, or coltivirus. Reovirus antigens may be selected from structural proteins λ1, λ2, λ3, μ1, μ2, σ1, σ2, or σ3, or non-structural proteins σNS, μNS, or σ1s. Reovirus antigens may be derived from rotavirus. Rotavirus antigens may be selected from VP1, VP2, VP3, VP4 (or cleaved products VP5 and VP8), NSP1, VP6, NSP3, NSP2, VP7, NSP4, or NSP5. Rotavirus antigens may include VP4 (or cleaved products VP5 and VP8), and VP7. See, for example, WO 2005 / 021033, WO 2003 / 072716, WO 2002 / 11540, WO 2001 / 12797, WO 01 / 08495, WO 00 / 26380, WO 02 / 036172; incorporated herein by reference in its entirety.

[0256] Parvovirus: The viral antigen may be derived from a parvovirus such as parvovirus B19. The parvovirus antigen may be selected from VP-1, VP-2, VP-3, NS-1, and NS-2. In the example, the parvovirus antigen is the capsid protein VP-2.

[0257] Delta hepatitis virus (HDV): Viral antigens may be derived from HDV, and in particular, may be delta-antigens from HDV (see, exe, US Patent No. 5,378,814).

[0258] Hepatitis E virus (HEV): Viral antigens can originate from HEV.

[0259] Hepatitis G virus (HGV): Viral antigens can be derived from HGV.

[0260] Human Herpesvirus: Viral antigens may be derived from human herpesviruses such as Herpes Simplex Viruses (HSV), Varicella-Zerosinus 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 the immediate-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 escape protein (gC, gE, or gI). VZV antigens can be selected from core, nucleocapsid, tetragument, or envelope proteins. Live attenuated VZV vaccines are commercially available. EBV antigens can be selected from glycoproteins of the initial antigen (EA) protein, viral capsid antigen (VCA), and membrane antigen (MA). CMV antigens can be selected from capsid proteins, envelope glycoproteins (e.g., gB and gH), and tetragument proteins.

[0261] Papovaviruses: The antigen may be derived from papovaviruses, such as papillomaviruses and polyomaviruses. Papovaviruses 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 the examples, the HPV antigen is derived from serotype 6, 11, 16, or 18. The HPV antigen may include capsid proteins (L1) and (L2), or E1-E7, or a fusion thereof. Polyomaviruses include BK viruses and JK viruses. The polyomavirus antigen may be selected from VP1, VP2, or VP3.

[0262] Circovirus: Antigens can be derived from circoviruses such as porcine circovirus (PCV) 1, PCV 2, PCV 3, and PCV 4.

[0263] Fungal antigens

[0264] Suitable fungal antigens may be derived from one or more of the fungi presented below.

[0265] The fungal antigens are Epidermophyton floccosum, Microsporum audouini, Microsporum canis, Microsporum distortum, Microsporum equinum, Microsporum gypseum, Microsporum nanum, Trichophyton concentricum, Trichophyton equinum, Trichophyton gallinae, Trichophyton gypseum, Trichophyton megnini, Trichophyton mentagrophytes, and Trichophyton It may be derived from dermatophytes including Trichophyton quinckeanum, Trichophyton rubrum, Trichophyton schoenleini, Trichophyton tonsurans, Trichophyton verrucosum, T. verrucosum var. album, var. discoides, var. ochraceum, Trichophyton violaceum, and / or Trichophyton faviforme.

[0266] The fungal pathogens are Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, Aspergillus terreus, Aspergillus sydowi, Aspergillus flavatus, Aspergillus glaucus, Blastoschizomyces capitatus, Candida albicans, Candida enolase, Candida tropicalis, Candida glabrata, and Candida krusei. Candida parapsilosis, Candida stellatoidea, Candida kusei, Candida parakwsei, Candida lusitaniae, Candida pseudotropicalis, Candida guilliermondi, Cladosporium carrionii, Coccidioides immitis, Blastomyces dermatidis, Cryptococcus neoformans, Geotrichum clavatum, Histoplasma capsulatum, Klebsiella Klebsiella pneumoniae,Paracoccidioides brasiliensis, Pneumocystis carinii, Pythium insidiosum, Pityrosporum ovale, Saccharomyces cerevisae, Saccharomyces boulardii, Saccharomyces pombe, Scedosporium apiosperum, Sporothrix schenckii, Trichosporon beigelii, Toxoplasma gondii, Penicillium marneffei, Malassezia spp., Fonsecaea spp., Wangiella spp., Sporothrix spp., Basidiobulus 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., Paecilomyces spp., Pithomyces spp.,and can be derived from Cladosporium spp.

[0267] Methods for producing fungal antigens are well known in the art (see US Patent No. 6,333,164). In one method, a solubilizing fraction extracted and separated from an insoluble fraction obtainable from a fungal cell in which the cell wall has been substantially or at least partially removed is characterized by comprising the steps of: obtaining a live fungal cell; obtaining a fungal cell in which the cell wall has been substantially or at least partially removed; rupturing the fungal cell in which the cell wall has been substantially or at least partially removed; obtaining an insoluble fraction; and extracting and separating a solubilizing fraction from the insoluble fraction.

[0268] Respiratory antigens

[0269] The compositions disclosed herein may comprise 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), morbilivirus (measles), togavirus (rubella), VZV, and coronavirus (SARS). Respiratory antigens may be derived from bacteria that cause respiratory diseases, such as Streptococcus pneumoniae, Pseudomonas aeruginosa, Bordetella pertussis, Mycobacterium tuberculousis, Mycoplasma pneumoniae, Chlamydia pneumoniae, Bacillus anthracis, and Moraxella catarrhalis. Examples of specific antigens derived from these pathogens are described above.

[0270] The immunogenic compositions disclosed herein may be prepared in various forms. For example, the composition may be prepared as an injectable, such as a liquid solution or suspension. A solid form suitable for a solution or suspension in a liquid vehicle may also be prepared prior to injection (e.g., a lyophilized composition or a spray-lyophilized composition). The composition may be prepared for topical administration, such as an ointment, cream, or powder. The composition may be prepared for oral administration, such as a tablet, capsule, or spray. The composition may be prepared for pulmonary administration, such as by an inhaler, using a fine powder or spray. The composition may be prepared as a suppository or pessary. The composition may be prepared for nasal, aural, or ocular administration, such as drops. Formulations of such pharmaceutical compositions are within the general scope of the art. See, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 18th edition, 1990.

[0271] The composition may be in the form of a kit designed to be reconstituted immediately before the combined composition is administered to a patient. Such a kit may comprise one or more antigens in liquid form or nucleic acids encoding such antigens, and any one of the additional antigens and adjuvants described herein.

[0272] An immunogenic composition comprising the disclosed polypeptide antigen is a vaccine composition. The pH of such composition is 6 to 8, and is about 7. 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 to a subject. The vaccine according to the present invention may be used prophylactically or therapeutically, but will generally be prophylactic and may be used to treat animals (including farm, game, companion, and laboratory mammals).

[0273] The immunogenic composition used as a vaccine comprises an immunologically effective amount of antigen(s) and / or nucleic acid encoding the antigen(s), and any other components as needed. "Immunologically effective amount" means that the administration of that amount to an individual, either as a single dose or as part of a series, is effective for treatment or prevention. This amount depends on the health and physical condition of the individual to be treated, age, the taxonomic group of the individual to be treated (e.g., pig, cattle, etc.), the ability of the individual's immune system to synthesize antibodies, the desired degree of protection, the formulation of the vaccine, the assessment of the medical condition by the treating veterinarian, and other relevant factors. This amount is expected to fall within a relatively broad range that can be determined through routine testing.

[0274] administration

[0275] The compositions disclosed herein will generally be administered directly to a subject. Direct delivery may be achieved by parenteral injection (e.g., subcutaneous, intraperitoneal, intravenous, intramuscular, or interstitial space of tissues), or by administration to mucous membranes, e.g., rectum, orally (e.g., tablet, spray), vaginally, topically, transdermally (see, e.g., WO99 / 27961), or transcutaneously (see, e.g., WO02 / 074244 and WO02 / 064162), intranasally (see, e.g., WO03 / 028760), ocularly, eardrum, lung, or other mucous membranes. Additionally, the immunogenic composition may be administered topically by direct delivery to the surface of the skin. Topical administration may be achieved by bringing naked skin into contact with the immunogenic composition without using any device, or by using a bandage or bandage-like device (see, e.g., US Patent No. 6,348,450).

[0276] In the examples, the method of administration may be parenteral, mucosal, or a combination of mucosal and parenteral immunization. In one aspect, the method of administration is parenteral, mucosal, or a combination of mucosal and parenteral immunization with a total of 1-2 vaccinations at intervals of 1-3 weeks. In one aspect, the route of administration includes, but is not limited to, oral delivery, intramuscular delivery, and a combination of oral and intramuscular delivery.

[0277] It has already been demonstrated that mucosal and systemic immune responses to antigens such as Helicobacter pylori antigens can be enhanced through systemic boosting immunization following mucosal priming. In the example, a method for treating an infection caused by the ASF virus comprises the step of mucosally administering a first immunogenic composition containing one or more ASF virus antigens to a subject in need, and then parenterally administering a second immunogenic composition containing one or more ASF virus antigens in a therapeutically effective amount.

[0278] Immunogenic compositions can be used to induce systemic and / or mucosal immunity to induce enhanced systemic and / or mucosal immunity.

[0279] In the examples, the immune response is characterized by the induction of serum IgG and / or intestinal IgA immune responses.

[0280] As mentioned above, the prime-boost method may be used where one or more gene transfer vectors and / or polypeptide antigens are delivered in the "priming" step, and subsequently, one or more second gene transfer vectors and / or polypeptide antigens are delivered in the "boosting" step. In certain embodiments, priming and boosting using one or more gene transfer vectors or polypeptide antigens described herein are followed by further boosting using one or more polypeptide-containing compositions (e.g., polypeptides containing ASF virus antigens).

[0281] In any method involving co-administration, various compositions may be delivered in any order. Accordingly, in embodiments involving the delivery of multiple different compositions or molecules, the nucleic acids do not all need to be delivered prior to the polypeptides. For example, a priming step may include the delivery of one or more polypeptides, and a boosting step may include the delivery of one or more nucleic acids and / or one or more polypeptides. Multiple nucleic acid administrations or polypeptide and nucleic acid administrations may be performed in any order after multiple polypeptide administrations. Accordingly, one or more of the gene delivery vectors described herein and one or more of the polypeptides described herein may be co-administered in any order through any administration route. Accordingly, any combination of polynucleotides and polypeptides described herein may be used to induce an immune response.

[0282] Dosage and Usage

[0283] Dose therapy may follow a single-dose schedule or a multiple-dose schedule. Multiple doses may be used in a primary immunization schedule and / or a booster immunization schedule. In a multiple-dose schedule, various doses may be delivered by the same or different routes, e.g., parenteral prime and mucosal boost, mucosal prime and parenteral boost, etc.

[0284] In the example, the dosage method enhances the binding affinity of the antibody reaction leading to an antibody with neutralizing properties. An in-vitro neutralization assay can be used to test for neutralizing antibodies.

[0285] There is a strong case for a correlation between serum antibody levels and protection against disease caused by the ASF virus.

[0286] Tests to determine the efficacy of the immune response

[0287] One method for evaluating the efficacy of therapeutic treatment includes monitoring infection after administration of the disclosed composition. One method for evaluating the efficacy of prophylactic treatment includes monitoring the immune response to an antigen in the disclosed composition after administration of the composition.

[0288] Another method for evaluating the immunogenicity of a component protein of the immunogenic composition of the present invention is to recombinantly express the protein and screen patient serum or mucosal secretions by immunoblotting. A positive reaction between the protein and the patient serum indicates that the patient has previously elicited an immune response to the protein. That is, the protein is an immunogen. Additionally, this method can be used to identify immunodominant proteins and / or epitopes.

[0289] Another method for confirming the efficacy of therapeutic treatment involves monitoring infection after administration of the composition of the present invention. One method for confirming the efficacy of prophylactic treatment involves monitoring both the systemic immune response to antigens within the composition of the present invention (e.g., monitoring IgG1 and IgG2a production levels) and the mucosal immune response (e.g., monitoring IgA production levels) after administration of the composition. Generally, serum-specific antibody responses are determined post-immunization but pre-challenge, whereas mucosal-specific antibody responses are determined post-immunization and post-challenge.

[0290] The immunogenic compositions of the present invention can be evaluated in in vitro and in vivo animal models prior to host. Particularly useful mouse models include mouse models that perform intraperitoneal or intranasal attacks following intraperitoneal immunization.

[0291] In addition, the efficacy of the immunogenic composition of the present invention can be determined in vivo by attacking an infected animal model, e.g., guinea pigs, mice, or rhesus macaques, with the immunogenic composition. The immunogenic composition may or may not be derived from the same strain as the attacking strain. In the examples, the immunogenic composition may be derived from the same strain as the attacking strain.

[0292] In vivo efficacy models include, but are not limited to: (i) models of murine infection using human strains; (ii) models of murine disease using mouse-adapted strains, such as particularly virulent strains in mice; and (iii) primate models using human isolates. Human attack models supported by the NIH and the Center for Disease Control (CDC) may also be used.

[0293] The immune response may be one or both of a TH1 immune response and a TH2 immune response. The immune response may be an improved, enhanced, or altered immune response. The immune response may be one or both of a systemic and mucosal immune response. In the examples, the immune response is an enhanced systemic and / or mucosal immune response.

[0294] Enhanced systemic and / or mucosal immunity manifests in an enhanced TH1 and / or TH2 immune response. In the examples, the enhanced immune response includes an increase in the production of IgG1 and / or IgG2a and / or IgA. In the examples, the mucosal immune response is a TH2 immune response. In one aspect, the mucosal immune response includes an increase in the production of IgA.

[0295] Activated TH2 cells enhance antibody production, making them valuable for responding to extracellular infections. Activated TH2 cells can secrete one or more of IL-4, IL-5, IL-6, and IL-10. The TH2 immune response can trigger the production of IgG1, IgE, IgA, and memory B cells for future protection.

[0296] The TH2 immune response may include an increase in one or more of cytokines associated with the TH2 immune response (e.g., IL-4, IL-5, IL-6, and IL-10), or an increase in the production of one or more of IgG1, IgE, IgA, and memory B cells. In the examples, the enhanced TH2 immune response will include an increase in IgG1 production.

[0297] The TH1 immune response may include an increase in CTLs, an increase in one or more of cytokines associated with the TH1 immune response (e.g., IL-2, IFNγ, and TNFβ), an increase in activated macrophages, an increase in NK activity, or an increase in the production of IgG2a. In the examples, the enhanced TH1 immune response will include an increase in IgG2a production.

[0298] The immunogenic composition of the present invention, in particular, an immunogenic composition comprising one or more antigens of the present invention, may be used alone or optionally in combination with other antigens with an immunomodulator capable of inducing Th1 and / or Th2 responses.

[0299] Additionally, the immunogenic composition of the present invention may comprise one or more immunomodulators, such as an inorganic salt like an aluminum salt and an oligonucleotide containing a CpG motif. In the examples, the immunogenic composition comprises both an aluminum salt and an oligonucleotide containing a CpG motif. Alternatively, the immunogenic composition comprises an ADP ribosylated toxin, such as detoxified ADP ribosylated toxin, and an oligonucleotide containing a CpG motif. In one aspect, one or more immunomodulators comprise an adjuvant. The adjuvant may be selected from one or more of the group consisting of TH1 adjuvants and TH2 adjuvants further discussed above.

[0300] The immunogenic composition of the present invention can induce both humoral and cell-mediated immune responses to effectively treat an infection. This immune response can induce long-lasting (e.g., neutralizing) antibodies and cell-mediated immunity capable of responding rapidly upon exposure to one or more infectious antigens. For example, evidence of neutralizing antibodies in a subject's blood sample is considered a surrogate parameter for protection because their formation is critically important for viral clearance in TBE infection.

[0301] Use of immunogenic compositions as pharmaceuticals

[0302] Furthermore, the present invention provides a composition for use as a pharmaceutical agent. The pharmaceutical agent may induce an immune response in mammals (i.e., is an immunogenic composition) and may be a vaccine. Additionally, the present invention provides a use of the composition of the present invention in the manufacture of a pharmaceutical agent for inducing an immune response in mammals. The pharmaceutical agent may be a vaccine. In the examples, a vaccine is used to prevent and / or treat intestinal infections such as gastroenteritis, including acute gastroenteritis. Gastroenteritis may cause watery diarrhea and / or intestinal peristalisis and / or motility (vomiting) due to an imbalance in ion and / or water transport.

[0303] The present invention provides a method for inducing or enhancing an immune response using the composition described above. The immune response may be protective and may induce antibodies and / or cell-mediated immunity (including systemic and mucosal immunity). The immune response includes a booster response.

[0304] Additionally, the present invention provides a method for inducing an immune response in a mammal, comprising the step of administering an effective amount of a composition of the present invention. The immune response may be protective and may include antibodies and / or cell-mediated immunity. In the examples, the immune response includes one or both of a TH1 immune response and a TH2 immune response. The method may induce a booster response.

[0305] Kit

[0306] Additionally, the present invention provides a kit comprising one or more containers of the compositions described herein. The compositions may be in liquid form, such as individual antigens, or may be lyophilized. Suitable containers for the compositions include, for example, bottles, vials, syringes, and test tubes. The containers may be formed from various materials, including glass or plastic. The containers may have a sterile access port (for example, the containers may be intravenous solution bags or vials having a stopper that can be punctured with a hypodermic injection needle).

[0307] The kit may further comprise a second container containing a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or dextrose solution. Additionally, it may contain other materials useful to the end-user, including other pharmaceutically acceptable formulation solutions such as buffers, diluents, filters, needles, and syringes or other delivery devices. The kit may further comprise a third component containing an adjuvant.

[0308] Additionally, the kit may include a package insert containing written instructions regarding an immune induction method or an infection treatment method. The package insert may be an unapproved draft package insert or a package insert approved by the FDA (Food and Drug Administration) or other regulatory agencies.

[0309] In the examples, the delivery device is pre-charged with the immunogenic composition disclosed herein.

[0310] Method for producing ASF virus-specific antibodies

[0311] Using the ASF virus polypeptide described herein, ASF virus-specific polyclonal and monoclonal antibodies that specifically bind to / selectively bind to the ASF virus antigen, respectively, can be produced. Polyclonal antibodies may be produced by administering the ASF virus polypeptide to mammals such as mice, rabbits, goats, or horses. Serum is collected from immunized animals, and antibodies are precipitated, for example, with ammonium sulfate and then purified from plasma by chromatography including affinity chromatography. Techniques for producing and processing polyclonal antisera are known in the art.

[0312] Monoclonal antibodies against ASF virus-specific epitopes present in the polypeptide can also be readily generated. Normal B cells of mammals, such as mice immunized with ASF virus polypeptide, can fuse with, for example, HAT-sensitive mouse myeloma cells to produce hybridomas. Hybridomas producing ASF virus-specific antibodies can be identified using RIA or ELISA and isolated by cloning on semi-solid agar or by limiting dilution. Clones producing ASF virus-specific antibodies are isolated by further screening.

[0313] Antibodies against ASF virus epitopes, namely monoclonal and polyclonal antibodies from serum (polyclonal), are particularly useful for detecting the presence of ASF virus antigens in samples such as serum samples from ASF virus-infected subjects. Immunoassays for ASF virus antigens may utilize a single antibody or multiple antibodies. Immunoassays for ASF virus antigens may use, for example, a monoclonal antibody against an ASF virus epitope, a combination of monoclonal antibodies against the epitope of one ASF virus polypeptide, a monoclonal antibody against the epitope of different ASF virus polypeptides, a polyclonal antibody against the same ASF virus antigen, a polyclonal antibody against different ASF virus antigens, or a combination of monoclonal and polyclonal antibodies. Immunoassay protocols may be based, for example, on competition, direct reaction, or, for example, on a sandwich-type assay using labeled antibodies. Labels may be, for example, fluorescent, chemiluminescent, or radioactive.

[0314] Polyclonal or monoclonal antibodies may be further used to isolate ASF virus particles or antigens by an immunoaffinity column. The antibodies may be attached to a solid support, for example, by adsorption or covalent bonding, so that the antibodies maintain their immunoselective activity. Optionally, spacer groups may be included to keep the antigen-binding sites of the antibodies accessible. Then, ASF virus particles or antigens from biological samples, such as blood or plasma, can be bound using the immobilized antibodies. The bound ASF virus particles or antigens are recovered from the column matrix, for example, by a change in pH.

[0315] All patent literature cited in the present invention is incorporated herein by reference in its entirety. Specific details for implementing the invention

[0316] Examples

[0317] Example 1. Production of Baculovirus Protein Subunits

[0318] The recombinant baculovirus protein expression system was based on the nucleic acid sequence of the targeted ASF virus protein. The final sequence was optimized for expression in in-house Spodoptera prugiferda insect cells (Sf9) such that a suitable restriction endonuclease site is present at the end of the sequence.

[0319] A plasmid vector containing the target sequence was prepared using the pBacPAK8 cloning vector (Clontech Laboratories, Inc., Mountain View, CA). The plasmid vector contains a side sequence homologous to the linear BestBac 2.0 baculovirus vector (Expression Systems, Davis, CA) so that when a plasmid containing ASF virus p30, p30 / p54, p72, and / or hemagglutinin inserts was co-transfected into Sf9 cells with the linear BestBac 2.0 baculovirus backbone (Expression Systems, Davis, CA), homologous recombination would exchange the H3 insert for the baculovirus polyhedrin gene. Subsequently, the resulting baculoviruses containing the ASF virus sequences expressed under the control of the polyhedrin promoter were harvested. Cells and viruses were grown in culture media obtained from an expression system (Media ES 99-300) formulated without animal-derived components. Gentamicin solution was added from the purchased stock solution (Gibco Cat #15710) to a final concentration of 10 μg / ml. At final harvest, the infected culture was centrifuged to remove cells, and the supernatant was collected. The supernatant was processed through a 0.2-micron sterile disposable filter. The final concentration was determined by titrating the pre-master culture.

[0320] Sf9 cells are scaled up to production volume using glass or sterile disposable plastic containers. Once the final cell culture volume required for production is reached, viral infection occurs in the same container prepared for the final passage of cells. Culture mixing is achieved by shaking / rocking the container or using a low-shear impeller design. The mixing speed and intensity are adjusted to maintain the cells in a suspension without generating foaming that causes excessive shear or cell destruction.

[0321] Viral fluids are inactivated with beta-propiolactone (BPL) at a final concentration of 0.2–0.3%. Before inactivation, the pH of the disrupted fluid is adjusted to 7.5–8.0 using 2–10N NaOH as a base or 10–38% HCl or 10% nitric acid as an acid. The disrupted fluid may be warmed to room temperature for 1–18 hours prior to the addition of BPL. BPL is added at the concentrations specified above while mixing. After the addition of BPL, the viral fluid is transferred to an inactivation container using a "bottom-to-bottom" transfer process to ensure that all fluid has come into contact with BPL. The disrupted fluid is incubated at 17–27°C for 18–48 hours while stirring. After the inactivation process is complete, the pH is adjusted to 7.0–7.5 using the aforementioned acid or base. The inactivated viral fluid is stored at 2–8°C until further processing. Prepare the antigen using WOW (Water / Oil / Water) adjuvant.

[0322] Example 2. Field Evaluation of ASF Antigen-Based Vaccine

[0323] The antigens were prepared as described above (i.e., hemagglutinin, SEQ ID NO: 17 and p30 / p54 fusion protein, SEQ ID NO: 6).

[0324] Eight commercial farms were selected for this study. The animals used on the farms came from other farms that were not infected with ASF.

[0325] Experimental Design

[0326] Inclusion, Exclusion, and Withdrawal Criteria

[0327] All animals used in this study were clearly healthy at the start of the study. Pigs suffering from disease, poor health, significant trauma, or lameness were excluded in accordance with normal management practices.

[0328] Randomization

[0329] Pigs that met all inclusion criteria and had no exclusion criteria were randomly assigned to one of two treatment groups or a placebo group. An equal number of pigs from each treatment group and 25% of the placebo were assigned to each pen.

[0330] Handling of Sick

[0331] Sick animals were treated according to farm standard operating procedures or treatment protocols. Morbidities of all causes were noted and recorded.

[0332] Blood was collected from animals suspected of having ASF (severe lethargy, discoloration of the extremities, etc.) and / or dead animals using a rapid test kit for initial screening for the presence of ASF antigens, and the same blood samples were subsequently submitted for confirmatory testing.

[0333] Treatment group:

[0334]

[0335] Each animal was an experimental unit. Each pig was double-tagged (one tag on each ear) and mixed with an equal number of pigs from different vaccination and control groups in each pen. The pens used were located in a single building.

[0336] Vaccination

[0337] The animals in the vaccinated group received one of two vaccines and were administered 1 mL twice at 3-week intervals.

[0338] Blood collection for serological testing:

[0339]

[0340] Collected Data and Results

[0341] 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. Special attention was paid to swelling / inflammation at the injection site, soreness, redness, abscesses, lumps, lesions, and warmth. Other observations to be recorded included, but were not limited to, lameness, lack of thriftiness, anorexia, and lack of normal general behavior.

[0342] 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 conducted 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.

[0343] 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 experimental animals had been exposed to the ASF virus.

[0344] 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 testing the immune response to the p30 portion of the p30 / p54 fusion protein in the vaccine formulation.

[0345] Of the three analyses performed on the samples collected during the study period, two were intended to evaluate the animals' immunological responses to vaccination, BioCheck, and ID Screen. Each of these tests measured different portions of the p30 / p54 fusion protein in the vaccine formulation. The ID Screen analysis, which measures the p30 (32) antigen, was found to demonstrate an immune response during the vaccination process and the study observation period.

[0346] conclusion

[0347] immune response

[0348] When evaluating the immunological response of animals administered a test vaccine containing p30 / p54 fusion proteins in both treatment groups 1 and 2, it can be concluded that the vaccination induced an immune response to the p30 (32) antigen tested by the ID Screen Indirect kit. An increase in the presence of antibodies began around day 21 of the study for treatment group 1 and was detected in the rustic farm, and antibody concentrations were very similar between the two treatment groups in blood collections on day 42 of the study.

[0349] Based on these results, the formulation induces an immune response against ASF antigens.

[0350] Although the present invention has been described with reference to the above embodiments, it will be understood that variations and changes are included within the spirit and scope of the present invention. Accordingly, the present invention is limited only by the following claims.

Claims

Claim 1 A method for preparing an African swine fever (ASF) virus-derived immunogenic polypeptide and / or peptide, the method comprising: (a) culturing a host cell transformed with nucleic acid under conditions that induce expression of said polypeptide and / or peptide, wherein the polypeptide and / or peptide comprises the amino acid sequence presented in SEQ ID NO: 6; and (b) mixing or co-expressing said immunogenic polypeptide and / or peptide with one or more adjuvants. Claim 2 A method according to claim 1, wherein the immunogenic polypeptide and / or peptide and one or more immunoadjuvants are further mixed with at least one pharmaceutically acceptable excipient or vehicle selected from the group consisting of water, saline solution, glycerol, ethanol, wetting agents, emulsifiers, pH buffering substances, and combinations thereof. Claim 3 A method according to claim 2, wherein the immunogenic polypeptide and / or peptide comprises a recombinant subunit vaccine, and such expressed polypeptide and / or peptide is generated using a baculovirus / insect cell methodology. Claim 4 A method according to claim 1, wherein one or more immunoadjuvants are selected from the group consisting of mineral-containing compositions, oil-emulsions, saponin formulations, non-toxic derivatives of enterobacter lipopolysaccharides (LPs), lipid A derivatives, immunostimulating oligonucleotides, ADP-ribosylated toxins and detoxified derivatives thereof, bioadhesives and mucosal adhesives, muramyl peptides, imidazoquinoline compounds, thiosemicabazone compounds, tripantrin compounds, non-ASF antigens, and combinations thereof. Claim 5 A method according to claim 1, characterized in that the ASF virus-derived immunogenic polypeptide and / or nucleic acid encoding the peptide is prepared by chemical synthesis. Claim 6 A method according to claim 5, characterized in that the nucleic acid encoding the ASF virus-derived immunogenic polypeptide and / or peptide is generated using a primer-based amplification method. Claim 7 A method according to claim 6, characterized in that the primer-based amplification method is PCR. Claim 8 An immunogenic composition comprising the polypeptide presented in SEQ ID NO: 6 and at least one pharmaceutically acceptable excipient or vehicle. Claim 9 A pharmaceutical composition for immunizing a subject against African swine fever (ASF) virus, comprising the immunogenic composition of claim 8 as an active ingredient. Claim 10 A pharmaceutical composition according to claim 9, further comprising an adjuvant. Claim 11 A pharmaceutical composition according to claim 9, characterized in that the pharmaceutical composition is administered to a subject via topical, parenteral, or mucosal administration. Claim 12 A pharmaceutical composition according to claim 10, further comprising at least one pharmaceutically acceptable excipient or vehicle. Claim 13 A pharmaceutical composition characterized in that, in claim 10, the adjuvant is a WOW (Water / Oil / Water) adjuvant. Claim 14 delete Claim 15 delete Claim 16 delete Claim 17 A pharmaceutical composition according to claim 9, characterized in that the pharmaceutical composition further comprises an ASF virus hemagglutinin protein. Claim 18 delete Claim 19 A pharmaceutical composition characterized in that, in claim 9, the subject is a pig. Claim 20 delete

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