Recombinant circovirus capsid-virus-like particle (VLP): compositions, methods, and uses

Abstract

To provide a mammalian expression system for producing recombinant porcine circovirus type 2 (PCV2) virus-like particles (VLPs).SOLUTION: A mammalian expression system includes a mammalian cell and a plasmid that comprises a PCV2 gene encoding a capsid protein. The PCV2 gene is codon optimized, and the mammalian cell is transfected with the plasmid. The expression system produces recombinant PCV2 VLPs, such as PCV2d VLPs. Also, provided herein are a method for producing porcine circovirus type 2 (PCV2) virus-like particles (VLPs), as well as a PCV2 VLP generated by the method.SELECTED DRAWING: Figure 1A

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to and the benefit of U.S. Patent Application No. 62 / 837,758, entitled "Recombinant Circovirus Capsid - Virus - Like Particle (VLP): Compositions, Methods and Uses", which is hereby incorporated by reference in its entirety.

[0002] 1. Field This application relates to compositions comprising porcine circovirus - 2 (PCV2), particularly PCV2 virus - like particles (VLPs).

Background Art

[0003] 2. Background Circoviruses are small, non - enveloped icosahedral viruses that contain a small, circular, covalently closed single - stranded DNA (ssDNA) genome. Circoviruses have been identified in various species and are known to cause infections in birds, aquatic animals, and terrestrial animals (1, 2). Due to changes in capsid morphology and genomic constitution, the Circoviridae family has recently been classified into two distinct genera, Circovirus and Cyclovirus (3). The Circovirus genus includes porcine circovirus type 1 (PCV1), type 2 (PCV2), and type 3 (PCV3) (2). Genomic sequences related to the Cyclovirus genus are associated with several vertebrate and invertebrate species, but the definitive host recognition for this group remains unclear (2). PCV2 infection is a cause of significantly high mortality among pigs as a causative agent of porcine circovirus - associated diseases (PCVAD), and is also associated with porcine dermatitis nephropathy syndrome (PDNS) and porcine reproductive disorders (1, 4).

[0004] The diameter of virion particles is approximately 19 nm, and the size of the PCV genome ranges from 1.7 kb to 2.3 kb. The circular nature of the genome gives rise to the name of the viral family Circoviridae. The evolutionary history has been investigated and a detailed phylogenetic tree and changes in capsid surface structure can be explained (5 - 8). Initial cryo-EM image reconstructions of several natural circoviruses have shown that the capsid has T = 1 icosahedral symmetry (9). The PCV genome encodes one structural capsid protein (CP). Expression and purification of the PCV2b CP protein from E. coli have shown that it self-assembles to mimic the overall morphology of 70 infectious virions. The crystal structure of this virus-like particle (VLP) has visualized that the CP fold is a standard viral jelly roll crystal structure consisting of two four-stranded β-sheets (10, 11). The loops connecting the β-strands form features on the virus surface and may contain antigenic epitopes. PCV2b CP has also been expressed and purified as VLPs from Trichoplasia ni insect cells (11). Cryo-EM image reconstruction of this VLP has shown that the N-terminus is located inside the capsid, and the authors concluded that the antigenic properties associated with the N-terminus are likely the result of the N-terminus transiently externalizing from the capsid via a process called viral "breathing" (11 - 13). The externalization of the N-terminus may play an important role in the viral life cycle.

[0005] The amino acid sequences of GenBank's PCV2 deposits are classified into four different genotypes (PCVa - d) (14). PCV2a was the dominant global genotype until 2003 when a genotype shift to PCV2b was observed (15, 16). The PCV2c genotype may be extinct as there are only three deposits in GenBank. In 2013, Wei et al. reported a large number of PCV2d sequences and deposited them in GenBank (5). Further reports and deposits of PCV2d sequences have led to approximately 84 out of 320 deposits in GenBank (7). The increase in PCV2d deposits may be the result of PCV2d emerging and becoming the dominant genotype in Asia, Europe, North and South America. This increase may be the result of an escape mutant from vaccination (17). As a result, PCV2d may represent a second global genotype shift that may not respond to the current vaccination programs against PCV2 (7). Despite a significant number of phylogenetic studies of PCV2 genotypes and the newly emerging importance of PCV2d in the global swine industry, there are no reports explaining the structure of the PCV2d capsid.

[0006] Considering the emergence of PCV2d, a deeper understanding of PCV2 genotypes and their differences is necessary for the development of treatments for the diseases caused by these types of viruses. These and other concerns are addressed by the present application.

Summary of the Invention

[0007] 3. Summary In a first aspect, a mammalian expression system for producing recombinant porcine circovirus type 2 (PCV2) virus - like particles (VLPs) is provided. The expression system includes a mammalian cell and a plasmid containing a PCV2 gene encoding a capsid protein. The PCV2 gene is codon - optimized and the mammalian cell is transfected with the plasmid. The expression system produces recombinant PCV2 VLPs.

[0008] In another aspect, the mammalian cell is a human embryonic kidney-293 (HEK-293) mammalian cell.

[0009] In another aspect, the PCV2 gene contains a recognition site for NheI, a Kozak sequence, and a recognition site for NotI. The recognition site for NheI and the Kozak sequence are upstream from the start codon of the PCV2 gene, and the recognition site for NotI is incorporated after the stop codon of the PCV2 gene.

[0010] In another aspect, most of the recombinant PCV2 VLPs produced are present in the nucleus of the mammalian cell.

[0011] In another aspect, the capsid protein contains the amino acid sequence of SEQ ID NO: 2. In another aspect, the capsid protein is encoded by the nucleotide sequence of SEQ ID NO: 1.

[0012] In another aspect, the capsid protein is modified with a secretion signal sequence introduced at the NH2 terminus of the capsid protein. In a further aspect, the capsid protein contains the amino acid sequence of SEQ ID NO: 4. In a further aspect, the capsid protein is encoded by the nucleotide sequence of SEQ ID NO: 3.

[0013] In another aspect, the recombinant PCV2 VLPs produced are selected from the group consisting of PCV2a VLPs, PCV2b VLPs, PCV2c VLPs, PCV2d VLPs, and PCV2e VLPs. In a further aspect, the recombinant PCV2 VLPs produced are PVC2d VLPs.

[0014] In another aspect, the plasmid is pcDNA3.4-PCV2. In another aspect, the PCV2 gene is codon-optimized using the codon-optimized amino acid sequence of FIG. 1A.

[0015] In a second aspect, a method for producing porcine circovirus type 2 (PCV2) virus-like particles (VLPs) is provided. In this method, a suspension of cultured mammalian cells is provided. The mammalian cells are transfected with a plasmid containing a PCV2 gene encoding a capsid protein. Sodium valproate (VPA) is added to the transfected mammalian cells, and the addition of VPA inhibits cell proliferation. The transfected mammalian cells are centrifuged and washed. Next, the centrifuged mammalian cells are suspended in a phosphate-buffered saline (PBS) solution. A plurality of freeze and thaw cycles are performed on the mammalian cells, and then the mammalian cells are sonicated in a plurality of cycles. Next, two consecutive centrifugation cycles of the mammalian cells are performed to produce PCV2 VLPs, and most of the produced PCV2 VLPs are present in the nuclei of the mammalian cells.

[0016] In another aspect of this method, the step of centrifuging and washing the transfected mammalian cells includes centrifuging the mammalian cells at 2,000×g for 15 minutes, washing the mammalian cells with a PBS solution, and centrifuging the mammalian cells again at 2,000×g for 15 minutes.

[0017] In another aspect of this method, the centrifuged mammalian cells are frozen at about -80 °C and thawed at about 37 °C during the freeze and thaw cycles.

[0018] In another aspect of this method, the first of the two consecutive centrifugation cycles is performed at 2,000×g for 15 minutes, and the second of the two consecutive centrifugation cycles is performed at 8,000×g for 15 minutes.

[0019] In another aspect of this method, the PCV2 VLPs are purified by ultracentrifugation.

[0020] In another aspect of this method, the mammalian cells are human embryonic kidney-293 (HEK-293) mammalian cells.

[0021] In another aspect of this method, the PCV2 gene contains a recognition site for NheI, a Kozak sequence, and a recognition site for NotI. The recognition site for NheI and the Kozak sequence are upstream from the start codon of the PCV2 gene, and the recognition site for NotI is incorporated after the stop codon of the PCV2 gene.

[0022] In another aspect of this method, the plasmid is pcDNA3.4-PCV2.

[0023] In another aspect of this method, the produced PCV2 VLPs are selected from the group consisting of PCV2a VLPs, PCV2b VLPs, PCV2c VLPs, PCV2d VLPs, and PCV2e VLPs. In a further aspect, the produced PCV2 VLPs are PVC2d VLPs.

[0024] In a third aspect, PCV2 VLPs produced by the above method are provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] 4. BRIEF DESCRIPTION OF THE DRAWINGS

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BEST MODE FOR CARRYING OUT THE INVENTION

[0026] 5. DETAILED DESCRIPTION The practice of the various embodiments of this application, unless otherwise indicated, can utilize conventional methods in chemistry, biochemistry, molecular biology, immunology, and pharmacology within the skill of the art. Such techniques are explained in detail in the literature. See, for example, Remington’s Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990), Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.), and Handbook of Experimental Immunology, Vols. I-IV (D.M. Weir and C.C. Blackwell, eds., 1986, Blackwell Scientific Publications), Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989), Short Protocols in Molecular Biology, 4th ed. (Ausubel et al. eds., 1999, John Wiley & Sons), Molecular Biology Techniques: An Intensive Laboratory Course, (Ream et al., eds., 1998, Academic Press), PCR (Introduction to Biotechniques Series), 2nd ed. (Newton & Graham eds., 1997, Springer Verlag), Fundamental Virology, Second Edition (Fields & Knipe eds., 1991, Raven Press, New York).

[0027] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entireties, respectively.

[0028] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to "VLP" can include a mixture of two or more such VLPs.

[0029] Definitions As used herein, "virus-like particle" or "VLP" refers to a non-replicating viral capsid. VLPs are generally composed of one or more viral proteins, such as, but not limited to, capsid, coat, shell, surface and / or envelope proteins, or proteins called particle-forming polypeptides derived from these proteins. VLPs can be formed spontaneously upon recombinant expression of the protein in a suitable expression system. Methods for producing specific VLPs will be considered in more detail below. The presence of VLPs after recombinant expression of viral proteins can be detected using conventional techniques known in the art, such as electron microscopy, biophysical characterization, and homologs. See, for example, Baker et al., Biophys. J. (1991) 60:1445-1456, Hagensee et al., J. Virol. (1994) 68:4503-4505. For example, VLPs can be isolated by density gradient centrifugation and / or identified by characteristic density banding (e.g., in the examples). Alternatively, cryo-electron microscopy can be performed on vitrified aqueous samples of the VLP preparation in question, and images can be recorded under appropriate exposure conditions. Further methods of VLP purification include, but are not limited to, chromatography techniques such as affinity, ion exchange, size exclusion, and reverse phase procedures.

[0030] As used herein, the terms "hybrid" or "chimeric" refer to a molecule (e.g., a protein or VLP) containing portions from at least two different proteins. It will be apparent that the hybrid or chimeric molecules described herein may include full-length proteins fused to additional heterologous polypeptides (full-length or a portion thereof), and portions of proteins fused to additional heterologous polypeptides (full-length or a portion thereof). It will also be apparent that the hybrid or chimeric molecules may include wild-type or mutant sequences in any one, several, or all of the heterologous domains.

[0031] "Antigen" refers to a molecule containing one or more epitopes (i.e., "antigenic epitopes") in linear, conformational, or both forms that stimulate the host immune system to generate a humoral and / or cellular antigen-specific response. In one or more embodiments, the epitope comprises about 7-15 amino acids, such as 9, 10, 12, or 15 amino acids. This term includes polypeptides containing modifications such as deletions, additions, and substitutions (generally conservative in nature) compared to the native sequence, as long as the protein maintains its ability to induce an immunological response as defined herein. These modifications may be artificial, such as by site-directed mutagenesis, or may be accidental, such as by mutations in the host producing the antigen.

[0032] An "immunogenic composition" is a composition containing an antigen molecule such that administration of the composition to a subject results in the generation of a humoral and / or cellular immune response in the subject against the antigen molecule of interest.

[0033] "Purified" or "purification" generally refers to isolating a substance (compound, polynucleotide, protein, polypeptide, polypeptide composition) such that it constitutes a majority percentage of the sample in which it is present. Typically, in a sample, the "purified" component constitutes 50% of the sample, preferably 80% - 85%, or more preferably 90 - 95%. Techniques for purifying the polynucleotides and polypeptides of interest are well known in the art and include, for example, ion exchange chromatography, affinity chromatography, and sedimentation according to density.

[0034] A "coding sequence" or a sequence that "encodes" a selected polypeptide is a nucleic acid molecule that, when placed under the control of appropriate regulatory sequences (or "control elements"), is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo. The boundaries of the coding sequence are determined by the start codon at the 5' (amino) terminus and the translation stop codon at the 3' (carboxy) terminus. Coding sequences can include, but are not limited to, cDNA derived from viral, prokaryotic, or eukaryotic mRNA, genomic DNA sequences derived from viral or prokaryotic DNA, and also synthetic DNA sequences. Transcription termination sequences may be located on the 3' side of the coding sequence. Typical "control elements" include, but are not limited to, transcription promoters, transcription enhancer elements, transcription termination signals, polyadenylation sequences (located 3' to the translation stop codon), sequences for optimizing translation initiation (located 5' to the coding sequence), and translation termination sequences, and / or sequence elements that control open chromatin structure. See, for example, McCaughan et al. (1995) PNAS USA 92:5431 - 5435, Kochetov et al (1998) FEBS Letts. 440:351 - 355.

[0035] The term "nucleic acid" molecule can include, but is not limited to, prokaryotic sequences, eukaryotic mRNA, cDNA derived from eukaryotic mRNA, genomic DNA sequences derived from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. This term also captures sequences containing any of the known base analogs of DNA and RNA.

[0036] As used herein to describe a nucleic acid molecule, "recombinant" means a polynucleotide of genomic, cDNA, semi-synthetic, or synthetic origin that, by virtue of its origin or manipulation, (1) is not associated with all or a portion of the polynucleotide with which the polynucleotide would be associated in nature and / or (2) is linked to a polynucleotide other than the polynucleotide to which the polynucleotide would be linked in nature. The term "recombinant" as used with respect to a protein or polypeptide means a polypeptide produced by the expression of a recombinant polynucleotide. The terms "recombinant host cell," "host cell," "cell," "cell line," "cell culture," and other such terms denoting a prokaryotic microorganism or eukaryotic cell line cultured as a unicellular entity are used interchangeably and refer to a cell that has been or can be used as a recipient for a recombinant vector or other transfer DNA and includes the progeny of the original transfected cell. It is understood that the progeny of a single parental cell need not be precisely identical in morphology or in genomic or total DNA complement to the original parent, due to accidental or deliberate mutation. Progeny of a parental cell that are sufficiently similar to the parental cell to be characterized by related properties such as the presence of a nucleotide sequence encoding a desired peptide are included within the scope of this definition and are encompassed by the above terms.

[0037] "Codon optimization" or "codon-optimized" generally refers to a genetic engineering approach that utilizes synonymous codon changes to increase protein production.

[0038] Techniques for determining the "similarity" of amino acid sequences are well known in the art. Generally, "similarity" means that when the amino acids of two or more polypeptides are accurately compared at appropriate positions, the amino acids are identical or possess similar chemical and / or physical properties such as charge or hydrophobicity. Next, the so-called "similarity ratio" can be determined between the compared polypeptide sequences. Techniques for determining the identity of nucleic acid and amino acid sequences are also well known in the art and include determining the nucleotide sequence of the mRNA of the gene (usually via a cDNA intermediate), and determining the amino acid sequence it encodes, and comparing this with a second amino acid sequence. Generally, "identity" refers to the exact match of nucleotides to nucleotides or amino acids to amino acids of the respective two polynucleotide or polypeptide sequences.

[0039] Two or more polynucleotide sequences can be compared by determining their "identity rate." Similarly, two or more amino acid sequences can be compared by determining their "identity rate." Regardless of whether it is a nucleic acid or a peptide sequence, the identity rate of two sequences is generally described as the number of exact matches between the two aligned sequences divided by the length of the shorter sequence and multiplied by 100. An approximate alignment of nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981). This algorithm was developed by Dayhoff, Atlas of Protein Sequences and Structure, M.O. Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research Foundation, Washington, D.C., USA, and can be extended for use in peptide sequences using a scoring matrix normalized by Gribskov, Nucl. Acids Res. 14(6):6745-6763 (1986). Suitable programs for calculating the identity or similarity rate between sequences are generally known in the art.

[0040] A "vector" can introduce a gene sequence into a target cell (e.g., bacterial plasmid vectors, viral vectors, non-viral vectors, particulate carriers, and liposomes). Typically, "vector constructs," "expression vectors," and "gene transfer vectors" mean any nucleic acid construct that can induce the expression of one or more target sequences in a host cell. Thus, the term includes cloning and expression vehicles, as well as viral vectors. This term is used interchangeably with the terms "nucleic acid expression vector" and "expression cassette."

[0041] As used herein, "subject" generally refers to any member of the subphylum Chordata, including, but not limited to, humans and other primates, such as chimpanzees and other apes and monkey species; agricultural animals such as cows, sheep, pigs, goats and horses; domesticated mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs; birds including domesticated birds such as chickens, turkeys and other poultry, ducks, geese, and the like, wild birds and game birds. The term does not denote a particular age. Thus, it is intended to encompass both adult and neonatal individuals. This system described herein is intended for use in any of the above vertebrate species since all immune systems of these vertebrates function similarly.

[0042] "Pharmaceutically acceptable" or "pharmacologically acceptable" means that the material is biologically or otherwise desirable, i.e., the material can be administered to an individual in a formulation or composition without causing any unacceptable biological effects or interacting harmfully with any of the components of the composition in which it is contained.

[0043] As used herein, "treatment" refers to any of (i) prevention of infection or reinfection similar to traditional vaccines, (ii) reduction or elimination of symptoms, and (iii) substantial or complete elimination of the causative pathogen of the problem. Treatment can be carried out prophylactically (before infection) or therapeutically (after infection).

[0044] As used herein, the term "adjuvant" refers to a compound that, when used in combination with a particular immunogen (e.g., VLP) in a formulation, enhances, or otherwise alters or modifies, the resulting immune response. Modification of the immune response includes enhancement or broadening of the specificity of either or both of the antibody and cellular immune responses. Modification of the immune response may also mean reducing or suppressing a particular antigen-specific immune response.

[0045] As used herein, an "effective amount" generally refers to an amount of the VLPs of one or more embodiments of the present application that is sufficient to induce immunity, prevent and / or ameliorate an infectious disease, or reduce at least one symptom of an infectious disease, and / or enhance the effectiveness of another dose of VLPs. An effective amount may refer to an amount of VLPs sufficient to delay or minimize the onset of an infectious disease. An effective amount may also refer to an amount of VLPs that provides a therapeutic effect in the treatment or management of an infectious disease. Further, an effective amount is an amount that provides a therapeutic effect in the treatment or management of an infectious disease, alone or in combination with other therapeutic agents, with respect to the VLPs of one or more embodiments of the present application. An effective amount may also be an amount sufficient to enhance the subject's (e.g., human) own immune response to subsequent exposure to an infectious agent. The level of immunity can be monitored, for example, by plaque neutralization, complement fixation, enzyme-linked immunosorbent, or micro-neutralization assays, for example, by measuring the amount of neutralizing secretion and / or serum antibodies. In the case of a vaccine, an "effective amount" is one that prevents a disease and / or reduces the severity of symptoms.

[0046] As used herein, the term "effective amount" refers to an amount of VLPs necessary or sufficient to achieve a desired biological effect. The effective amount of a composition is an amount that achieves the selected result, and such amount can be determined as a matter of routine experimentation by one of ordinary skill in the art. For example, an effective amount for preventing, treating and / or ameliorating an infectious disease may be an amount that, when exposed to the VLPs of the present application, causes activation of the immune system and results in the development of an antigen-specific immune response. This term is also synonymous with "sufficient amount".

[0047] As used herein, the term "multivalent" refers to VLPs having multiple antigenic proteins against multiple types or strains of infectious agents.

[0048] As used herein, the terms "defensive immune response" or "defensive response" refer to an immune response mediated by antibodies against an infectious agent that is found in vertebrates (e.g., humans) and that prevents or ameliorates an infection, or reduces at least one symptom thereof. The VLPs of the present application can, for example, neutralize an infectious agent, prevent the infectious agent from entering cells, prevent the replication of the infectious agent, and / or stimulate the production of antibodies that protect host cells from infection and destruction. This term can also refer to an immune response mediated by T lymphocytes and / or other leukocytes against an infectious agent that is found in vertebrates (e.g., humans) and that prevents or ameliorates influenza infection, or reduces at least one symptom thereof.

[0049] As used herein, the term "vaccine" refers to a formulation containing the VLPs of the present application that is in a form that can be administered to a vertebrate and that induces immunity to prevent and / or ameliorate an infection, and / or reduce at least one symptom of an infection, and / or induce a defensive immune response sufficient to enhance the effectiveness of another dose of VLPs. Typically, a vaccine includes a conventional saline or buffered aqueous media in which the composition of the present application is suspended or dissolved. In this form, the composition of the present application can be conveniently used to prevent, ameliorate, or otherwise treat an infection. When introduced into a host, the vaccine can elicit an immune response including, but not limited to, the production of antibodies and / or cytokines, and / or the activation of cytotoxic T cells, antigen-presenting cells, helper T cells, dendritic cells, and / or other cellular responses.

[0050] In one or more embodiments, the present application relates to methods of making and using virus-like particles (VLPs) of viruses of the family Circoviridae [e.g., Porcine circovirus-2 (PCV2), its different genotypes and serotypes or other members of the family], compositions comprising the virus-like particles (VLPs), and vaccines based on the virus-like particles (VLPs) (e.g., monovalent, multivalent, single particle universal or multivalent, single particle mosaic or modified chimeric compositions), as well as their use for therapeutic delivery [(e.g., small molecules, nucleic acids, antibodies, enzymes (nanocarriers, nanobodies)], diagnosis, immunomodulatory functions and therapeutic markers. In particular, the present disclosure includes strategies and methods used for the development of novel monovalent, multivalent or universal porcine circovirus vaccines that can protect pigs from infection by one or more serotypes, clades or antigenic variants of the genus Porcine circovirus. Also described herein are VLP production methods (e.g., secretory systems) that produce VLPs presenting specific antigenic constructs or modifications. These VLPs are characterized by native or chimeric epitopes of a three-dimensional structure associated with the generation of an enhanced neutralizing immune response against Porcine circovirus or other viral agents. Monovalent, bivalent, multivalent, universal or chimeric (e.g., different serotypes and genotypes such as PCV2a, PCV2b, PCV2c, PCV2d and PCV2e) single particle VLPs are constructed and used to formulate vaccine compositions that enable immunization and subsequent protection against one or more serotypes or antigenically distinct viruses (e.g., serotypes from Asia, Europe or North America, etc.) VLPs, and native, modified or redesigned capsid monomers allow for the conjugation / conjugation of different molecular entities to the outer surface of the particle (small or large molecular entity), or the encapsidation of such molecular entities within the structure of the particle via the disassembly and reconstruction or alternative packaging methods of the VLPs.

[0051] Furthermore, VLPs are also used for the diagnosis or treatment indicators of infectious diseases. VLP vaccines can be produced in suspension cultures of eukaryotic cells and can be retained intracellularly or released into the culture medium. After purification, concentration, and formulation, the vaccine can be administered via any suitable route, such as either the mucosal or parenteral route, and induces an immune response that can protect against any or all of the serotypes of porcine circovirus, antigen variants, etc. VLPs including therapeutic, immunomodulatory functions and diagnostic applications are also provided.

[0052] These and other aspects of the compositions and methods of the present invention are described in further detail below with reference to the accompanying drawings and examples, in which one or more exemplary embodiments and / or arrangements of PCV2 VLPs are shown. The compositions and methods of this application are not limited to any of the exemplary embodiments and / or arrangements shown. It should be understood that the compositions and methods as shown in the accompanying drawings are merely examples of the compositions and methods of this application, and they can be embodied in various forms as recognized by those skilled in the art.

[0053] According to one or more embodiments of the present application, a mammalian expression system for producing recombinant porcine circovirus type 2 (PCV2) virus-like particles (VLPs) is provided. The expression system includes mammalian cells and a plasmid containing a PCV2 gene encoding a capsid protein. The PCV2 gene is codon-optimized and the mammalian cells are transfected with the plasmid. The expression system produces recombinant PCV2 VLPs. In at least one embodiment, the mammalian cells are human embryonic kidney-293 (HEK-293) mammalian cells.

[0054] In one or more embodiments, the PCV2 gene of the expression system can include a recognition site for NheI, a Kozak sequence, and a recognition site for NotI. The recognition site for NheI and the Kozak sequence are upstream of the start codon of the PCV2 gene, and the recognition site for NotI is incorporated after the stop codon of the PCV2 gene.

[0055] In at least one embodiment, most of the recombinant PCV2 VLPs produced are present in the nucleus of mammalian cells.

[0056] In one or more embodiments, the capsid protein comprises the amino acid sequence of SEQ ID NO: 2. In at least one embodiment, the capsid protein is encoded by the nucleotide sequence of SEQ ID NO: 1.

[0057] In at least one embodiment, the capsid protein is modified with a secretion signal sequence introduced at the NH2 terminus of the capsid protein. In one or more further embodiments, the capsid protein comprises the amino acid sequence of SEQ ID NO: 4. In one or more further embodiments, the capsid protein is encoded by the nucleotide sequence of SEQ ID NO: 3.

[0058] In one or more embodiments, the recombinant PCV2 VLPs produced may be at least one of the following: PCV2a VLPs, PCV2b VLPs, PCV2c VLPs, PCV2d VLPs, and PCV2e VLPs. In a preferred embodiment, the recombinant PCV2 VLPs produced are PVC2d VLPs.

[0059] In at least one embodiment, the plasmid is pcDNA3.4-PCV2. In one or more embodiments, the PCV2 gene is codon-optimized using the codon-optimized amino acid sequence of FIG. 1A.

[0060] In one or more embodiments of the present application, a method for producing porcine circovirus type 2 (PCV2) virus-like particles (VLPs) is provided. In this method, a suspension of cultured mammalian cells is provided. The mammalian cells are transfected with a plasmid containing a PCV2 gene encoding a capsid protein. Sodium valproate (VPA) salt is added to the transfected mammalian cells, and the addition of VPA salt inhibits cell growth. The transfected mammalian cells are centrifuged and washed. Next, the centrifuged mammalian cells are suspended in a phosphate buffered saline (PBS) solution. At least one freeze-thaw cycle, preferably multiple freeze-thaw cycles, is performed on the mammalian cells, and then the mammalian cells are sonicated in multiple cycles. In one or more embodiments, two consecutive centrifugation cycles of the mammalian cells are then performed to produce PCV2 VLPs. In at least one embodiment, most of the produced PCV2 VLPs are present in the nucleus of the mammalian cells.

[0061] In at least one embodiment, the step of centrifuging and washing the transfected mammalian cells can include centrifuging the mammalian cells at 2,000×g for 15 minutes, washing the mammalian cells with a PBS solution, and centrifuging the mammalian cells again at 2,000×g for 15 minutes.

[0062] In one or more embodiments, the centrifuged mammalian cells can be frozen at about -80°C and thawed at about 37°C during the freeze-thaw cycle.

[0063] In at least one embodiment, the first of two consecutive centrifugation cycles is performed at 2000×g for 15 minutes, and the second of two consecutive centrifugation cycles is performed at 8,000×g for 15 minutes.

[0064] In at least one embodiment, the PCV2 VLPs are purified by ultracentrifugation.

[0065] In at least one embodiment, the plasmid is pcDNA3.4-PCV2. In one or more embodiments of this method, the produced PCV2 VLPs are selected from the group consisting of PCV2a VLPs, PCV2b VLPs, PCV2c VLPs, PCV2d VLPs, and PCV2e VLPs. In at least one preferred embodiment, the produced PCV2 VLPs are PVC2d VLPs. In at least one embodiment, PCV2 VLPs produced by the above method are provided.

[0066] In summary, according to one or more embodiments, the present application discloses a mammalian construct system for PCV2 VLPs (e.g., PCV2d VLPs). The present application further discloses a structural analysis based on cryo-electron microscopy reconstruction that reveals the external and internal features of the VLPs, which has important significance for the development of novel PCV2 vaccines and the utilization of the particles as nanovehicles for delivering various molecular entities for prophylactic, therapeutic, immunotherapeutic, immunization (heterologous, homologous, multivalent), and diagnostic applications. These and other examples related to PCV2 VLPs are described in more detail below.

Examples

[0067] 6. Examples 6.1. Example 1 Materials and Methods Cells, capsid gene, plasmid, and antibody. A suspension culture of Expi293F human cells (Life Technologies, CA) was grown in serum-free Expi293 expression medium (Life Technologies, CA) at 37°C in a 5% CO2 environment and stirred at 150 rpm in a Erlenmeyer flask. The porcine circovirus type 2 (PCV2) gene encoding the capsid protein (RQ3 strain) was chemically synthesized using a codon-optimized sequence (amino acid sequence shown in Fig. 1A) by Blue Heron Technologies (Bothell, WA). Recognition sites for NheI and Kozak sequences were added immediately upstream of the start codon, and a recognition site for NotI was incorporated after the stop codon. The synthesized Cap gene was recovered from the transfer plasmid by double digestion with NheI and NotI restriction enzymes, gel purified, and subcloned into the mammalian expression plasmid pcDNA3.4 digested with the same enzymes. The ligated plasmid was transformed into MAX Efficiency Stbl2 Cells (Life Technologies), and the correct clone was identified by restriction enzyme analysis and verified by sequencing.

[0068] Production and purification of virus-like particles (VLPs). PCV2 VLPs were produced in a suspension culture of Expi293F mammalian cells after transient transfection with plasmid pcDNA3.4-PCV2 (Figs. 1A - 1D). Expi293F cells were seeded at 2×10 6Seeded at a concentration of cells / ml and cultured for 16 hours before transfection. Plasmid DNA (1 μg / ml) was diluted with Opti-MEM in a volume corresponding to 5% of the total volume of the culture. Separately, polyethyleneimine (PEI) was prepared with an equal volume of Opti-MEM (4 μg / ml). After incubating at room temperature for 5 minutes, the PEI solution was added dropwise to the tube containing DNA in 300 drops, incubated at RT for 30 minutes, and then the mixture was dropped into the cell suspension. Twenty-four hours after transfection, sodium valproate (VPA) was added to the cell culture to a final concentration of 3.75 mM to inhibit cell growth. Seventy-two hours after transfection, the cells were pelleted by centrifugation at 2,000 × g for 15 minutes, then washed once with phosphate-buffered saline (PBS), and rotated again at 2,000 × g for 15 minutes. The cell pellet was resuspended in PBS and then subjected to 3 cycles of freezing (-80 °C) and thawing (37 °C). Subsequently, the cells were further fragmented by 3 cycles of sonication and clarified by 2 consecutive centrifugations, first at 2,000 × g for 15 minutes and then at 8,000 × g for 15 minutes. By this procedure, a part of the VLPs was released into the cytoplasm, but most of the particles seemed to be trapped inside the cell nucleus (data not shown). The PCV2 VLPs contained in the clarified supernatant were further purified by ultracentrifugation on a 2-layer CsCl density gradient. The lower layer, 5 ml of 1.4 g / ml CsCl, and the upper layer, 10 ml of 1.25 g / ml CsCl, both prepared with 10 mM Tris-HCl (pH 7.9). The sample was loaded onto the gradient and rotated at 140,000 × g for 4 hours at 15 °C using an SW28 rotor (Beckman Coulter, CA). The VLPs appeared as an opaque band at the interface of the 1.25 and 1.4 g / ml CsCl layers and were collected by puncturing the tube with an 18G needle and syringe. The collected solution was mixed with 37% CsCl in 10 mM Tris-HCl (pH 7.9) to a final volume of 12 ml and then rotated at 155,000 × g for 16 hours at 15 °C using an SW 41Ti rotor (Beckman Coulter, CA). The VLPs were detected at the bottom of the tube and collected as described above.The collected VLP materials were dialyzed overnight at 10°C against 10 mM Tris-HCl pH 7.9 and 150 mM NaCl using a Slide-A-Lyzer Cassette. The purified PCV2 VLPs were concentrated and buffer-exchanged into phosphate-buffered saline (PBS) using an Amicon Utra-4 centrifugal filter device (Merck Millopore, MA). The PCV2 VLP samples were stored at -80°C in 50 - 100 μl aliquots.

[0069] Western blot and Coomassie blue staining. The purified VLPs were mixed with loading buffer, heated at 100°C for 5 minutes, and electrophoresed on a 4 - 12% Bis-Tris SDS-polyacrylamide gel (Life Technologies, CA). The protein loading amounts were 1 μg for future Coomassie staining and 0.5 μg for Western blot. After electrophoretic separation, the gel was stained with Coomassie blue or with the protein electrophoretically transferred to a 0.45 μm nitrocellulose membrane (Life Technologies LC2001). Next, the membrane was blocked with 5% non-fat milk in TBST (10 mM Tris-HCl, 130 mM NaCl, and 0.05% Tween-20, pH 7.4) for 1 hour at 20°C, and then incubated overnight at 20°C in primary rabbit anti-porcine circovirus antibody (Cab 183908, Abcam, UK) diluted in blocking buffer. The membrane was washed three times with TBST and then incubated for 2 hours with secondary antibody (goat anti-rabbit IgG HRP 332 conjugated, 1:1,000) diluted in blocking buffer. Finally, the membrane was washed three times with TBST and developed using an ECL Western blot system (Life Technologies, CA) according to the manufacturer's instructions. The stained gel and immunoblot images were acquired using a FluorChem Imager device (Protein Simple, CA).

[0070] 337 Negative staining and TEM examination. 5 μL of the pCV2-2 VLP sample was applied to a CF200-CU carbon film 200 mesh copper grid (Electron Microscopy Science) for 1 minute, then the grid was washed with 200 μl of 50 mM sodium cacodylate buffer, and then immediately pulled with 50 μl of 0.5% uranyl acetate for 1 minute. This grid was examined by a JOEL2100 transmission electron microscope operating at 200 kV equipped with an Orius 2048×2048 pixel CCD (Gatan Inc., Pleasanton, CA).

[0071] Cryo-EM data collection. A frozen hydrated sample of PCV2d VLP was prepared on a Quantifoil R 2 / 2, 200 mesh copper grid (Electron Microscopy Science). 4 μl of a pre-screened sample of VLP was applied to the blotted grid for 3 seconds and rapidly frozen in liquid ethane using an FEI Vitrobot instrument. This grid was stored in liquid nitrogen until data collection. Data were collected cryogenically on an FEI Titan Krios operating at 300 kV equipped with a Gatan K2 camera after a GIF quantum energy filter with a width of 15 eV. Data collection was performed using the Leginon package software (36).

[0072] Image reconstruction. The motion of the particles was corrected using the MotionCor2 package (37). Default parameters and dose weighting were used for correction except for the patch 5 option, and the first frame of each movie was discarded during alignment. The particles were automatically selected using Gautomatch v0.53, and the estimation of the contrast transfer function (CTF) was performed on the aligned micrographs using Gctf v0.50 (38). Relion 3.0 was used to extract 23,358 300x300 pixel particles from the dose-weighted micrographs using the coordinates identified by Gautomatch. Reference-free 2D classification was performed using Relion 3.0. Options other than the default for this step included a diameter of 250 Å and required 128 classes (39). An initial model was generated at a resolution of 60 Å using the molmap function of UCSF Chimera together with the PCV2 crystal structure (PDB entry 3R0R) (40). 3D classification was performed on 7196 particles using Relion 3.0 with a diameter of 250 Å, 3 classes, and C1 symmetry. A single class containing 4,442 particles showed the highest resolution. These particles were used for high-resolution image reconstruction with Relion 3.0. Again, a diameter of 250 Å and I1 symmetry were used together with the remaining default parameters of Relion 3.0. A binary mask was created using the relion_mask_create program of Relion 3.0. The binary mask for post-processing was generated as follows. 1) High-resolution image reconstruction was low-pass filtered to 15 Å resolution using relion_image_handler (Relion 3.0), 2) The lowest threshold, which is noise not related to the PCV2 capsid, was identified for this volume using UCSF Chimera, 3) This volume was converted to a binary mask using relion_mask_create (Relion 3.0) with the identified threshold, mask dilation by 7 pixels, and 2 soft-edge pixels. Next, the resulting mask was inspected in UCSF Chimera to confirm the absence of internal cavities.

[0073] The local resolution was calculated with the program MonoRes. The same binary mask used during post - processing in Relion was used for the calculation. A resolution range of 2.8 Å to 6.0 Å was used (41).

[0074] Improvement of the structure 376. The atomic coordinates for the crystal structure of the PCV2b crystal structure (PDB entry 3R0R) were changed using Coot (42). The biological matrix required to generate virus - like particles was present in the PDB and was used by Coot to generate VLPs of PCV2d. UCSF Chimera was used to manually dock the VLPs into the symmetry - averaged image reconstruction. The resulting coordinates were repeatedly improved using phenix.real_space_refine from the Phenix software package with non - crystallographic symmetry (NCS) constraints applied, and manual fitting in Coot (43).

[0075] Sequence alignment and evolutionary coupling Using the PCV2d sequences and a protein Blast search with the general name of the organism, Porcine circovirus 2 (taxid:85708) filter, a total of 1,966 PCV2 sequences were generated (44). Sequences with partial sequences, names containing "putative", "P3", "unknown", and "P27.9" were manually removed. Sequence alignment was performed on the remaining sequences using MUSCLE with default parameters (45). Sequences that generated gaps, had more than 10 amino acids with different sequences, or did not have sequences similar to them were manually removed. This was done to remove possible errors / artifacts that could have occurred during the sequencing process (i.e., artificial recombination during PCR with Taq polymerase (46)). To remove identical sequences, several rounds of alignment and deletion were performed. As a result, 1,377 sequences remained. The final round of alignment was performed using Clustal Omega with default parameters (19). Due to the limited range of Expect (E) values present in the sequences (i.e., the sequences were too similar), the evolutionary coupling calculation could not be successfully performed using the EV coupling server 394 (evfold.org). As a result, plmc was used to generate coevolution and covariation within the sequences. The fields and L2 lambda of the coupling used were 0.01 and 16.0, respectively, and a maximum of 100 iterations were performed. This result was converted to be visualized in EVzoom using the script provided by MATLAB 2019 and the plmc program (22). The obtained matrix was visualized in EVzoom (22), and the structural covariance was visually confirmed in UCSF Chimera (40).

[0076] Results Recombinant PCV2 VLPs constructed in mammalian cells. The PCV2d capsid protein (CP) gene (GenBank: ABX71783.1) was subcloned into the mammalian expression plasmid pcDNA 3.4 to produce recombinant PCV2d VLPs in mammalian cells (Figure 1A). When a suspension culture of HEK-293 mammalian cells was transfected with the plasmid, CP was produced and VLPs were constructed. To date, PCV2 VLPs have been produced using insect cells, yeast, or E. coli (11), so the expression of VLPs using mammalian cells provides a substrate similar to that utilized by natural PCV2 infection. During purification, most of the VLPs or CP were determined to be in the cell nucleus, but some were detected in the cytoplasm of infected cells. This suggests that the VLPs can exit the nucleus via a potential nuclear export signal that could not be clearly identified, presumably using bioinformatics tools (data not shown). Analysis of the purified PCV2 VLPs and their protein composition was confirmed by SDS-PAGE and Western blot analysis (Figures 1B and C). Further examination by negative staining electron microscopy revealed homogeneous spherical particles with smooth edges and a slightly rough surface with a diameter of approximately 19 nm (Figure 1D). The CP described in this study possesses the amino acid sequences identified from a number of recently isolated and reported PCV2d virus genome entries in GenBank, such as C / 2013 / 3 (AWD32058.1) isolated in Taiwan, W233-12 (BBE28610.1) isolated in Japan, CN-FJC011 (AVZ66019.1) isolated in China, and England / 15-P0222-09-14 (AATN97185.1) isolated in the UK.

[0077] Cryo-electron microscopy and image reconstruction of PCV2d VLPs. The icosahedral cryo-EM image reconstruction of purified PCV2d VLPs was determined at a resolution of 3.3 Å (Figure 2A). A detailed inspection of the image reconstruction shows that the molecular envelope of the side chains is of sufficient quality to model atomic coordinates. Coordinates from the PCV2b crystal structure were manually fitted to the image reconstruction and appropriate changes were made to reflect the PCV2d amino acid sequence. The fitted model was improved by repeating several times the automatic refinement by Phenix and manual adjustment using the program Coot (Figure 2B). The final refinement statistics are shown in Table 1 below.

[0078]

Table 1

[0079] Subunits from the PCV2a, b, and d genotype structures were superimposed to generate an average root mean square deviation plot of equivalent Cα atoms (9, 18). Regions showing the greatest diversity correspond to two surface-exposed loops consisting of amino acids 85 - 91 (loop CD) and 188 - 194 (loop GH) (Figure 2C).

[0080] Sequence conservation among the four genotypes can be useful for the generation of a universal vaccine against PCV2. There are a number of publications that have compared the amino acid sequences of PCV2 CP to gain insights into the evolution of the PCV2 capsid (6, 7). However, the inventors are not aware of studies in which sequence alignment information has been mapped to the atomic coordinates of the capsid to understand how the observed mutations correlate with structure and / or immune evasion. A total of 1,377 non-redundant PCV2 CP sequences were subjected to sequence alignment by the Clustal Omega server (19). A WebLogo diagram was generated using the sequence alignment to observe sequence conservation (Figure 3A). The horizontal axis of the alignment shows the amino acids, and the vertical axis shows the observed frequency of the amino acids. The sequences with the greatest diversity (GenBank accession: AVZ65995.1 and ALK0432.1) share 74.4% sequence identity. The WebLogo diagram successfully shows the frequency of occurrence of the most well-known amino acids at each position, but suppresses / excludes the frequency of occurrence of less well-known amino acids. In fact, mutations can be observed in the hydrophobic core of the protein, at the subunit interface, and in the residues on the outer and inner surfaces of the capsid. Furthermore, a detailed examination of the aligned sequences shows that only Met1 and Arg147 are completely conserved. The observed sequence diversity suggests that the capsid can tolerate mutations at almost all positions while leaving the infectious virus intact. To visualize the sequence conservation / variability at each amino acid position, the ConSurf server was used to plot the sequence alignment information onto the PCV2d atomic coordinates (Figure 3B) (20). This figure shows a significant degree of non-conserved mutations that the capsid has undergone. The regions colored red have the greatest degree of change, while the regions colored blue have the least degree of change. The highly conserved residues are scattered throughout the structure. However, two sets of residues form patches on the surface of the capsid. These include Tyr55, Thr56, Met71 and Arg73, and Pro82, Thr170, Gln188, Thr189 and Val193 (Figure 3C).The presence of these patches suggests that it may be possible to generate a vaccine that can neutralize all available genotypes of PCV2 if antibody production can be induced by these amino acids (Figure 3C).

[0081] Evolutionary coupling mutations distinguish PCV2 genotypes. Through the deposition of numerous CP sequences in GenBank, we were able to query whether any of the amino acid positions were evolutionarily constrained (21). For evolutionarily constrained amino acids, a mutation in one amino acid requires a mutation in another amino acid. In the simplest case, this may be because the amino acids are packing against each other within the protein structure. Therefore, mutating to a large amino acid at one position may require mutating to a small amino acid at a second position to generate proper packing. Such information can be used to predict protein folding or to identify functionally important sites (21 - 23). Evolutionary coupling (EC) measurements determined from 1,377 unique sequences indicate that two independent positions within the structure show coupling. The first position is composed of amino acids 53 and 215, and the second position is composed of amino acids 77, 80, 89, 90, 190, and 191 (Figure 3C). Surprisingly, differences in the sequences at the second position are involved in the structural diversity observed between PCV2a and PCV2b / d for loops CD and GH (Figure 2C) (9, 18). The EC results may indicate that the coupled residues are functionally related. Calculations of solvent-exposed surface area at the GetArea server (curie.utmb.edu) show that residues 77, 80, 89, and 90 are more than 75% buried, whereas residues 190 and 191 are solvent-exposed (24). Residues 190 and 191 serve to define neutralizing epitopes on the surface of the capsid.

[0082] Array changes on the surface of the capsid are a response to neutralizing antibodies. Using the atomic coordinates of PCV2d, the amino acids on the surface of the VLP with side chains exposed to the solvent were identified (53, 55 - 56, 58 - 64, 70 - 71, 73, 75, 77 - 78, 82 - 83, 85, 88 - 89, 102, 113, 123, 127, 131 - 137, 148, 155, 156, 158, 161, 166, 168 - 170, 188 - 191, 194, 204, 206 - 208, 210, 229 - 234). Since the side chains provide the surface for antibody interaction, these amino acids could be antigenic determinants. The continuous sequences that could represent linear epitopes are shown in Figures 4A - 4H. Next, the amino acids that are most likely to vary in these regions were identified (53, 57, 59, 60, 63, 75, 77, 88, 89, 131, 133, 134, 136, 169, 190, 191, 211, 215, and 232). The variations could be the result of escape mutants from antibody neutralization. The presence of mutations as an escape response to antibody neutralization is supported by antigen subtyping experiments by Lefebvre et al. and Saha et al., where the ability of a panel of neutralizing monoclonal antibodies to bind to different strains of PCV2 was tested. This study showed that amino acid positions 59, 63, 88, 89, 130, 133, 206, and 210 are involved in antibody binding discrimination (25, 26). A study by Franz o et al. that described the evolution of PCV2 before and after the introduction of vaccination identified changes in amino acids 59, 191, 206, 210, 228, and 232 (27). Except for amino acid 130, which is buried at the subunit - subunit interface, these amino acids are on the surface of the capsid and are subject to mutations (Figures 4A - 4H).

[0083] The inner region of the PCV2 VLP can be used to package materials for nanotechnology. A nearly central reconstructed slice was extracted from the cryo - EM image reconstruction, and the density traces of pixel values were calculated in the horizontal and vertical directions. Based on the central slice of the reconstruction, the outer diameter of the PCV2 VLP is approximately 18.5 nm, assuming it is approximately spherical for calculation. Thus, the outer volume is 3.3×10 3 nm3 is estimated to be. The inner diameter using the same spherical estimate has a diameter of about 13 nm. Thus, the volume of the inner region is about 1.2×10 3 nm 3 . Scanning of the central slice of the 3D molecular volume showed that the inner region was occupied. The density is lower than that of the capsid shell, which is likely to represent the cumulative disordered matrix N-terminus of the 60 CP units 200 that make up the entire VLP.

[0084] Discussion The porcine circovirus type 2 (PCV2) genome encodes four known proteins: a replicase (ORF1) involved in genome replication, a capsid protein (ORF2) involved in the production of the capsid shell, and ORF3 and ORF4, which are thought to play roles in the regulation of cell apoptosis (28, 29). Phylogenetic analysis of the PCV2 CP sequences shows here that four genotypes are globally distributed (PCV2a - d) (14). PCV2b is considered the currently dominant genotype. However, the recent increase in the number of PCV2d CP deposits in GenBank suggests that there may be a genotype shift from PCV2b to PCV2d (5). The increase in the number of PCV2d registrations may be the result of escape mutants in response to vaccination (17). To address the potential shift to the PCV2d genotype and the possibility that this new genotype may be resistant to vaccines available on the market, the present application establishes a mammalian expression system for producing large amounts of PCV2d virus - like particles (VLPs). The present application provides the first system in which mammalian cells have been utilized to produce large amounts of VLPs. Since the mammalian expression system is similar to cells naturally infected with PCV2, it is particularly advantageous for the expression of PCV2 in E. coli or baculovirus, and it becomes possible to study the details of the PCV2 capsid from the perspective of the viral replication cycle. For example, the N - terminus of the CP has multiple nuclear localization signals, and it is predicted that virus assembly occurs in the nucleus of infected cells where the ssDNA genome is replicated (30). However, it was previously unknown whether the PCV2 capsid could exit the intact nucleus and be released from infected cells.

[0085] As a result, in one or more embodiments, the present expression system can help address whether the constructed capsid can exit the nucleus. In one or more embodiments, the dominant fraction of VLPs in the present expression system is located within the cell nucleus. This is unexpected because no nuclear export signal (NES) within the CP sequence was identified in the bioinformatics search (data not shown). Thus, the PCV2 capsid may potentially possess an NES that could play an important role in the viral life cycle. However, nucleocytoplasmic transport can be reduced by redirecting the translation of the capsid protein to the secretory pathway by introducing a secretory signal sequence at the NH2 terminus of the capsid protein. This genetic modification results in increased capsid protein synthesis, VLP construction, and release of particles into the culture medium.

[0086] Cryo-EM image reconstruction and its structural analysis show that VLPs expressed in mammals, baculoviruses, and Escherichia coli are hardly distinguishable. Comparing the atomic coordinates of PCV2a, b, and d, differences in the three-dimensional structures of the surface-exposed loops consisting of amino acids 86 - 91 (loop CD), 131 - 136 (loop EF), and 188 - 194 (loop GH) are identified (Figs. 2A - 2C). The structures of PCV2b and PCV2d are more similar to each other than either is to PCV2a. These differences are due to evolutionary coupling mutations in these loops (Fig. 3B) and provide a structural explanation for the antigenic discontinuous mutations observed in PCV2a - PCV2b in 2003 (15, 16). In the expansion of symmetry followed by focused classification of capsid subunits, no class that can show post-translational modification of the capsid is identified (data not shown). However, mass spectrometry may be needed to further address the possibility of post-translational modification of PCV2 VLPs generated from mammalian expression systems. Alignment of 1,377 unique PCV2 CP sequences shows that only two amino acids (Met1 and Arg147) are completely conserved and the remaining positions in the sequence are subject to mutations. This indicates a remarkable flexibility of the capsid structure that undergoes mutations while maintaining the infectious virus, highlighting the ability of PCV2 to evade antibody neutralization. The atomic coordinates obtained from cryo-EM image reconstruction identify sites on the capsid surface that may be targets for antibody neutralization. In the present application, continuous regions on the capsid surface that can serve as epitopes potentially inducing neutralizing antibodies against several genotypes are identified. Peptides showing sequence changes may represent escape mutants from antibody neutralization. Therefore, using the described expression protocol, capsids can be generated that are constructed as multivalent mosaics presenting neutralizing epitopes of several genotypes simultaneously. Positions showing no variability may represent regions on the viral capsid that could be ideal for universal vaccine design.

[0087] The response of mammals to VLPs constructed can more closely mimic the immune response to actual infection. Mammalian VLP expression systems can provide highly effective recombinant vaccines. For example, it has been found that the antibody responses of pigs inoculated with recombinant Cap protein derived from baculovirus are different from those of pigs that have experienced natural infection (31, 32). Pigs inoculated with the recombinant vaccine preferentially recognized only the largest polypeptide fragment, CP (43 - 233), while experimentally infected pigs and pigs with PDNS showed strong reactivity against the CP oligopeptide 169 - 180. The smaller peptide is common to both PCV2a and PCV2b subtypes and can serve as a decoy to divert the defense response from the larger 43 - 233 peptide.

[0088] Currently used vaccines are produced using capsids derived from the PCV2a genome, and several studies have reported failures in immunization as a result of PCV2d infection (7, 33, 34). Furthermore, there is always a possibility that vaccine efficacy is low due to genomic shift, and this factor must be considered in future vaccine development. G. Franzo et al. (27) studied the vaccine-derived selective pressure induced by vaccination. They reported that high mutation rates at amino acid positions 59, 191, 206 for PCV2a and 131, 228 for PCV2b had previously reduced the binding of antibodies that had been bound to the capsid, which could potentially cause immune escape from vaccine protection (Figure 4) (35). Therefore, a platform for expressing PCV2d VLPs is guaranteed. Thus, according to at least one embodiment, the present application discloses an expression for producing PVC2d VLPs in mammalian cells.

[0089] In one or more embodiments, the VLP expression system of the present application is further applicable to translation. The 40 amino acids at the N-terminus of CP are rich in arginine and are very positively charged, and are presumed to interact with the ssDNA genome during virus morphogenesis. This motif is located in the internal space of the VLP and seems to be available for binding specific tags (or simply absorbing negatively charged small molecules or oligonucleotides), and the particles can be utilized as nanoparticles for carrying therapeutically or immunomodulatory moieties directed to specific tissues. Alternatively, the N-terminus of PCV2 can be changed to hydrophobic amino acids to create an internal hydrophobic environment for hydrophobic molecules. Such nanostructures can be utilized as diagnostic antigens or in vaccine formulations. Further, by replacing surface-exposed amino acids with thiol-containing residues or unnatural amino acids, conjugation of small, medium or large molecules useful for heterologous vaccination, drug delivery, therapeutic treatment, diagnosis, etc. may be made possible.

[0090] According to one or more embodiments, the expression system of the present application can produce a large amount of PVC2d VLP in mammalian cells (human fetal kidney: HEK293). This system enables rapid study of the PCV2 life cycle in a background that closely mimics the natural host of PCV2. As will be described in more detail below, cryo-EM image reconstruction of the VLP was determined to be at a resolution of 3.3 Å and was used to identify potential antigenic epitopes that can be used in vaccine design or small therapeutic molecule delivery formulations. Comparison of 1,377 unique PCV2 CP entries in GenBank showed that all amino acid positions except two had mutations. However, two groups of amino acids that form different patches on the surface of the capsid show limited sequence variation. Vaccines that can direct antibodies to these patches may serve as universal vaccines against PCV2.

[0091] 6.2. Example 2 A vaccination-challenge study to evaluate the efficacy of the porcine circovirus 2 vaccine. Experimental design. Blood samples were obtained from 120 male and female piglets at approximately 1 week of age at the breeding farm of the source. These blood samples were analyzed for the presence of PCV-2 by quantitative real-time polymerase chain reaction (qRT-PCR) assay to discard all positive piglets. These qRT-PCR PCV-2 negative samples were tested for antibodies against PCV-2 using a commercially available enzyme-linked immunosorbent assay (ELISA). Next, 72 two-week-old male and female piglets with the lowest levels of PCV-2 antibodies (all the tested piglets were from the same litter and were negative) were selected and transported to the experimental breeding farm at the test site. Upon arrival at the facility, the animals were weighed and the next day, randomly assigned to six treatment groups of 12 pigs each based on PCV-2 S / P titer (at the time of screening), body weight, sex, and the parity of female pigs. Thereafter, the pigs were housed in four rooms to acclimatize for 10 days.

[0092] At 21+ / -3 days of age (day 0 of the study), the pigs were immunized by the intramuscular (IM) route on the right side of the neck. Piglets from treatment group T01 were immunized with 1 mL of vaccine PIGONE (porcine circovirus type 2 (PCV2), strain VQ2610, 1×10 9 ~5×10 9 copies of DNA equivalent to virus pre-activation). Piglets from treatment group T02 were immunized with 2 mL of vaccine PIGONE (porcine circovirus type 2 (PCV2), strain VQ2610, 1×10 9 ~5×10 9Animals were immunized with DNA of the copy (hereinafter referred to as "PIGONE" in this specification). Animals from T03 were immunized with 2 mL of vaccine Huve-PCV2 (6 animals with Huve-PCV2_A and 6 animals with Huve-PCV2_B). Huve-PCV2_A contains the PCV2d ORf2 protein expressed by eukaryotic cells, and Huve-PCV2_B contains the PCV2d VLP expressed by mammalian cells. Animals from treatment group T04 were immunized with 1 mL of vaccine HVP-DNA (HVP-DNA contains the PCV2b ORF2 DNA vaccine), and animals from treatment T05 were immunized with 1 mL of the commercial vaccine Ingelvac CIRCOFLEX (inactivated baculovirus-expressed PCV2 ORf2) (Boehringer Ingelheim). Phosphate-buffered saline (PBS) was administered to piglets from treatment group T06 and used as a negative control group. On the 21st day of the study (SD21), at about 6 weeks of age, the pigs were immunized by the intramuscular (IM) route in the left-sided muscle of the neck. Pigs from treatment group T01 were immunized with 1 mL of vaccine PIGONE, pigs from treatment group T02 were immunized with 2 mL of vaccine PIGONE, animals from treatment group T03 were immunized with 2 mL of vaccine Huve-PCV2 (6 animals with Huve-PCV2_A and 6 animals with Huve-PCV2_B), and pigs from treatment group T04 were immunized with 1 mL of vaccine HVP-DNA. Groups T05 and T06 were immunized by intramuscular injection with 1 mL of PBS. The pigs were clinically examined before each vaccination.

[0093] On the 35th day of the study at approximately 8 weeks of age (WOA), all animals from the six treatment groups were challenged intranasally (IN) with 3 mL of an inoculum of the PCV-2b strain Sp-10-7-54-13 at 104.95 TCID50 / mL.

[0094] Blood samples were collected weekly throughout the study period for serology (ELISA) and / or detection of PCV-2 in serum (qRT-PCR). Additionally, nasal and rectal swabs were obtained weekly after challenge for determination of the PCV-2 excretion profile (if necessary). After challenge, the clinical signs of the animals were scored weekly, and the body weights were recorded at the time of the first vaccination, challenge, and necropsy.

[0095] In 12 WOA (SD63 and SD64), all pigs from the five treatment groups were euthanized. At necropsy, the tonsils, tracheobronchial lymph nodes, mesenteric lymph nodes, and superficial inguinal lymph nodes were collected for histopathological examination and detection of PCV-2 in the tissues (if necessary). The experimental plan is shown in Table 2.

[0096] Allocation of treatments. Animals were allocated into six groups (T01, T02, T03, T04, T05, or T06) of 12 animals according to the PCV-2 antibody titer at screening, body weight on arrival date, sex, and parity of female pigs. The variables were ranked and then classified and used to assign pigs to the six treatment groups in six blocks. Animals from treatments T01, T02, T03, T04, and T05 were randomly mixed in three rooms (Rooms 5, 6, and 7), and four animals from each group were placed in each room. Treatment T06 was in a separate room (Room 8).

[0097] Animals from group T03 (Huve-PCV2) were divided into two subgroups, T03_A (Huve-PCV2_A) and T03_B (Huve-PCV2_B). This was done randomly at SD0, and two animals from T03 in each room of each subgroup were selected.

[0098] The order of entry into the rooms was established as follows. Immunization phase: Room 8 - Room 7 - Room 6 - Room 5, Challenge phase: Room 5 - Room 6 - Room 7 - Room 8.

[0099] The room accommodating the PBS group (T06) was the first room to be entered during the immunization phase, and thus the first room where sham vaccination and challenge were performed. After challenge, access was gained to the room where the vaccinated animals were located before entering the room where the control PBS group (T06) was located.

[0100]

Table 2

[0101] Results of the preliminary stage. Health event. Animal #472 (PIGONE 2 mL) from group T01 was found dead at SD32. An autopsy was performed and fibrinous pericarditis was reported. The cause of death was probably due to bacterial sepsis and was not related to the test item.

[0102] Clinical observation of individual animals. All pigs were evaluated for depression, body condition, and dyspnea on the vaccination days before vaccination (SD0 and SD21). Additionally, the animals were also scored for clinical signs weekly after challenge, on days 35, 41, 49, 56, and 63 before challenge. During the study period, the clinical signs of the study animals were not recorded.

[0103] Body weight. Body weight was recorded from all pigs when they arrived at the experimental facility for the purpose of treatment allocation, at the first vaccination (SD0), at the time of challenge (SD35), and at the time of autopsy (SD63). The average daily weight gain (ADWG) was calculated for the periods from the first vaccination - challenge, challenge - autopsy, and the first vaccination - autopsy. The change in body weight was observed in Figure 5 and the ADWG was observed in Figure 6.

[0104] The PBS group (T06) had a slightly higher average body weight at SD35 and SD63, and a higher ADWG was observed in the three periods compared to the other groups, but this is probably not statistically different.

[0105] Serology. The presence of antibodies against PCV - 2 in the blood was tested at study days SD0, SD7±1, SD13, SD20, SD29 before vaccination, SD35, SD41, SD49, SD56, and SD63 before challenge using a commercially available ELISA IngezimCirco IgG kit. The results were expressed as S / P titers.

[0106] The kinetics of the antibodies are shown in Figure 7. Seven treatment groups are represented, and treatment T03 (Huve - PCV2) is divided into two subgroups, Huve - PCV2_A (T03_A) and Huve - PCV2_B (T03_B).

[0107] In the SD0 (initial vaccination), the S / P values of all the research animals were in the range of 0.2 - 1, and the overall average value was 0.71.

[0108] The boosting effect of the second vaccination was observed at SD29 in three groups: PIGONE 1 mL and 2 mL (T01 and T02), and Huve - PCV2_B (T03).

[0109] On the challenge day, the groups of CIRCOFLEX (inactivated baculovirus - expressed PCV2 Orf2) (T05), PBS (T06), Huve - PCV2_A (T03_A), and HVP - DNA (T04) showed low S / P values of 0.38 - 0.4. After the challenge, the S / P values generally decreased, and a mild response was observed in some vaccinated groups at SD56 or SD63.

[0110] The only group with a high antibody response after the second - advanced vaccination was Huve - PCV2_B (T03_B).

[0111] Viremia. The PCV - 2 DNA in serum samples obtained on the pre - vaccination research day SD0, and on SD35, SD41, SD49, SD56, and SD63 before the challenge was detected and quantified using the commercially available qRT - PCR kit LSI VetMAX Porcine circovirus type 2 - Quantification (Life Technologies, reference code QPCV). The results were expressed as PCV - 2 copy number log 10 / mL, and classified as negative (<3 log 10 copies / mL), non - quantifiable positive (3 log 10 copies / mL - 4 log10 copies / mL) or positive (≥4 log 10 copies / mL).

[0112] Viremia results considering a viral load >3 log 10 copies / mL.

[0113] The results are shown in Figures 8 and 9 as the percentage of quantitatively positive and non-quantitatively positive animals, and the mean value of quantitatively positive animals (>4 log 10 copies / mL).

[0114] Overall, a low percentage of positive animals (>3 log 10 copies / mL) was observed during the challenge phase. Treatments T01 and T02 vaccinated with PIGONE (1 mL or 2 mL) showed numerically higher percentages of positive animals at SD49 and SD56, but in neither case did they reach 50% positive (quantitative + non-quantitative). CIRCOFLEX (inactivated baculovirus-expressed PCV2 ORf2) (T04) had no positive (quantitative or non-quantitative) animals during the challenge phase.

[0115] HUVE-PCV2_B (T03_B), CIRCOFLEX (inactivated baculovirus-expressed PCV2 ORF2) (T05), and PBS (T06) had no quantitatively positive animals (>4 log 10 copies / mL) during the challenge phase. The remaining groups had 1 - 3 positive animals on at least one sampling day during the challenge phase, and the day with positive animals in many groups was SD56. The mean value of quantitatively positive animals ranged from 4.12 log 10 to 5.34 log 10 PCV-2 copies / mL.

[0116] Results of viremia considering a viral load >2 log 10 copies / mL.

[0117] The results are shown in Figures 10 and 11 as the percentage of viremic animals and the mean value of all animals (>2 log 10 copies / mL).

[0118] Considering all animals including samples below the detection limit, a higher proportion of positives is observed in all study groups. The PIGONE groups (T01 or T02) continued to be numerically high-proportion positive groups with SD49 (PIGONE 1 mL, T01), SD56 and SD63 (PIGONE 2 mL, T02), and subsequently HVPDNA (T04).

[0119] The average viral load considering all animals with qRT-PCR values was in the range of 2.19 - 4.83 log 10 copies / mL.

[0120] Groups T01 (PIGONE 1 mL), T02 (PIGONE 2 mL), T03_B (Huve-PCV2_B), and T04 (HVP-DNA) showed an average value > 2 log 10 copies / mL during the 4 sampling days after challenge. The Huve-PCV2_A group (T03_A) and the PBS control group (T06) showed positive values for 3 days from SD49 to SD63, while CIRCOFLEX (inactivated baculovirus-expressed PCV2 ORf2) (T05) showed viremic animals for 2 days at 1 week (SD41) and at necropsy (SD63) after challenge.

[0121] At SD35, one animal from group T02 (PIGONE 2 mL) had a viral load of 2.76 log 10 copies / mL. This animal was negative for qRT-PCR at animal selection, SD0, SD7, SD14, and SD21. Also, it was negative at SD41 and SD49 after challenge. During challenge, its viral load was > 3 log 10 copies / mL at SD56 and SD63.

[0122] Serology. The S / P value at SD0 was higher than in previous studies due to maternally-derived antibodies. Thus, a clear seroconversion after vaccination could not be confirmed. PIGONE 1 mL and 2 mL (T01, T02) and Huve-PCV2_B (T03_B) clearly showed seroconversion after the second vaccination compared to PBS (T06). CIRCOFLEX (inactivated baculovirus-expressed PCV2 Orf2) (T05) and the HVP-DNA vaccine (T04) did not show an antibody response. They had an antibody profile similar to that of the PBS group.

[0123] The lack of seroconversion in the CIRCOFLEX (inactivated baculovirus-expressed PCV2 Orf2) group (T05) was expected considering the serological values at the time of vaccination. The CIRCOFLEX (inactivated baculovirus-expressed PCV2 Orf2) protection is based on cellular immunity. Usually, in the field situation, when animals with maternally-derived antibodies are vaccinated with CIRCOFLEX (inactivated baculovirus-expressed PCV2 Orf2), no serological response is observed.

[0124] The Huve-PCV2 subgroup could not be compared to the other groups as n was half that of the other groups (6 animals / subgroup). However, when compared among themselves, the serological response of T03_B was higher, so the results were different.

[0125] Viremia. At SD35 before challenge, one animal from the PIGONE 2 mL (T02) group had viremia, below the detection limit (viral load: 2.76 log 10(copies / mL). This animal was negative for qRT-PCR at animal selection, SD0, SD7, SD14, and SD21. Also, it was negative at SD41 and SD49 after challenge. An attempt was made to sequence this DNA at SD35, but it was not possible due to low virus load. Two possible explanations for this are as follows. 1) Subclinical infection by field PCV-2 strains during the immune stage with very low virus load, and 2) Detection of PCV-2 DNA contamination rather than the whole virus. The cause is unknown.

[0126] Generally, PCV-2 inoculation induced mild subclinical infection because low virus load was observed during challenge. Thus, it was determined that all qRT-PCR results, including those "below the limit of detection" (2 - 3 log 10 copies / mL), should be considered. The PIGONE 1 mL (T01), PIGONE 2 mL (T02), and HVP-DNA (T04) groups showed a higher proportion of positive animals. The Huve-PCV2_B (T03_B), CIRCOFLEX (inactivated baculovirus-expressed PCV2 ORf2) (T05), and PBS (T06) groups had no / very low proportions of viremic animals throughout the challenge stage. The mean value was <3 log 10 copies / mL. When comparing the Huve-PCV2 (T03) subgroups among them, subgroup T03_B numerically had a lower proportion of viremic animals, and the virus load was average from SD49 to SD63.

[0127] Information on the challenge day. The inoculum was prepared as follows. The inoculum was thawed, mixed, and then divided into five vials of approximately 50 mL / vial. The titration on the challenge day was 104.95 TCID50. The vials were shared among rooms. That is, one vial was started and no new vials were used until it was finished, so the same one vial could be used in two rooms. The inoculum vials were frozen until use.

[0128] Order of challenge: The PBS group (T06) was challenged first (room 8), followed by the vaccinated groups (rooms 7, 6, and 5). Potential for room effects: The room for PBS (T06) had a lower animal density than the other three rooms for the vaccinated groups. There were 12 or 20 animals / room respectively. The four rooms had equivalent space and accommodation conditions (temperature, humidity, etc.).

[0129] Further embodiments According to one or more embodiments, the present application discloses the construction of recombinant porcine circovirus capsids or virus-like particles (VLPs) with specific structural and antigenic properties, wherein the capsid monomers, which are one or more of the components, incorporate sequences that confer the ability to encapsulate or mount monovalent or multivalent vaccine attributes and / or molecular entities with different biological activities. In at least one embodiment, the recombinant capsid or VLP comprises a secretion signal sequence genetically linked to the capsid coding sequence that enables the release and / or secretion of the particles from the production cells into the culture medium.

[0130] In one or more embodiments, the recombinant capsid or VLP has sequences encoding surface motifs and loops that are mutated, altered, or modified, and thus the antigenic properties of the constructed VLPs are novel, different, or chimeric.

[0131] In one or more embodiments of the recombinant capsid of the VLP, the construction and / or production is carried out using two or more distinct capsid protein monomers, resulting in VLPs of a mixed composition (mosaic or chimera) with specific antigenic and biological properties. In one or more embodiments, the recombinant capsid or VLP comprises a modified surface that enables the conjugation of different molecular entities to the surface of the VLP.

[0132] In one or more embodiments, the recombinant capsid or VLP contains NH2-terminal or internal amino acids of mutated, deleted, and / or modified capsid monomers that allow for the incorporation of small molecules, nucleic acids, or other molecular moieties in vitro during construction within the cell producing the VLP or by hiding and reconstructing the VLP.

[0133] In one or more embodiments, the resulting VLP targets specific tissues or cells to deliver molecular entities and exert biological activity.

[0134] In at least one embodiment, the present application also discloses a DNA construct comprising a sequence encoding a capsid protein in all its sequences and forms used to construct the VLP. A DNA construct comprising sequences encoding various forms of capsid monomers. In one or more embodiments, the present application further discloses a method for producing a VLP, which includes introducing one or more of the DNA constructs into a host cell under conditions such that the cell produces the VLP. In at least one embodiment, the host cell is a eukaryotic cell selected from the group consisting of mammalian, yeast, insect, plant, amphibian, and avian cells. In one or more embodiments, the DNA construct is introduced into the cell and integrated into the cell genome or evenly divides and separates into daughter cells, resulting in continuous production of the VLP.

[0135] In at least one embodiment, the present application discloses a VLP produced by the above method.

[0136] In one or more embodiments, an immunogenic composition comprising at least one VLP according to any of the above embodiments is provided. In at least one embodiment, the immunogenic composition further comprises an adjuvant. In one or more embodiments, a method of generating an immune response against one or more porcine circoviruses in an animal is provided. The method comprises administering to the animal an effective amount of the immunogenic composition. In at least one embodiment of the method, the composition is administered mucosally, intradermally, subcutaneously, intramuscularly, or orally. In at least one embodiment, the immune response vaccinates the animal against multiple serotypes or antigenic variants of one or more porcine circoviruses. In at least one embodiment of the method, the animal is a pig.

[0137] In one or more embodiments, the recombinant capsids or VLPs of the present application comprise a molecule having pharmaceutical or biological activity. In at least one embodiment, the recombinant capsid or VLP comprises a pharmaceutical composition for VLP drug delivery, heterologous immunization, and / or immunomodulation.

[0138] In one or more embodiments of a method for generating an immune response against one or more porcine circoviruses in an animal, the immunogenic composition is administered to a subject as a therapeutic or prophylactic treatment. The methods of the present invention provide for the effective use of the compositions of the present application in a subject. In at least one embodiment, the subject is a human or an animal.

[0139] Sequence Listing SEQ ID NO: 1. Nucleotide sequence of wild-type PCV2 capsid. SEQ ID NO: 2. Amino acid sequence of wild-type PCV2 capsid. SEQ ID NO: 3. Nucleotide sequence of a modified PCV2 capsid having a secretion signal sequence. SEQ ID NO: 4. Amino acid sequence of a modified PCV2 capsid having a secretion signal sequence. SEQ ID NO: 5. Nucleotide sequence of Pcdna3.4-PCV2. (Figure 1A).

[0140] Accession No. 1. Porcine circovirus type 2 strain PCV2 capsid Length: 705 Type: DNA ATGACGTATC CAAGGAGGCG TTTCCGCAGA CGAAGACACC GCCCCCGCAG CCATCTTGGC CAGATCCTCC GCCGCCGCCC CTGGCTCGTC CACCCCCGCC ACCGTTACCG CTGGAGAAGG AAAAATGGCA TCTTCAACAC CCGCCTCTCC CGCACCATCG GTTATACTGT CAAGAAAACC ACAGTCAGAA CGCCCTCCTG GAATGTGGAC ATGATGAGAT TTAATATTAA TGATTTTCTT CCCCCAGGAG GGGGCTCAAA CCCCCTCACT GTGCCCTTTG AATACTACAG AATAAGGAAG GTTAAGGTTG AATTCTGGCC CTGCTCCCCA ATCACCCAGG GTGACAGGGG AGTGGGCTCC ACTGCTGTTA TTCTAGATGA TAACTTTGTA ACAAAGGCCA ATGCCCTAAC CTATGACCCC TATGTAAACT ACTCCTCCCG CCATACCATA ACCCAGCCCT TCTCCTACCA CTCCCGGTAC TTTACCCCGA AACCTGTCCT TGATAGGACA ATCGATTACT TCCAACCCAA TAACAAAAGA AATCAACTCT GGCTGAGACT ACAAACTACT GGAAATGTAG ACCATGTAGG CCTCGGCACT GCGTTCGAAA ACAGTATCTA CGACCAGGAC TACAATATCC GTATAACCAT GTATGTACAA TTCAGAGAAT TTAATCTTAA AGACCCCCCA CTTAACCCTA AGTGA

[0141] Accession No. 2. Porcine circovirus type 2 strain PCV2 capsid Length: 234 Type: Amino acid M T Y P R R R F R R R R H R P R S H L G Q I L R R R P W L V H P R H R Y R W R R K N G I F N T R L S R T I G Y T V K K T T V R T P S W N V D M M R F N I N D F L P P G G G S N P L T V P F E Y Y R I R K V K V E F W P C S P I T Q G D R G V G S T A V I L D D N F V T K A N A L T Y D P Y V N Y S S R H T I T Q P F S Y H S R Y F T P K P V L D R T I D Y F Q P N N K R N Q L W L R L Q T T G N V D H V G L G T A F E N S I Y D Q D Y N I R I T M Y V Q F R E F N L K D P P L N P K

[0142] Sequence number 3. Modified porcine circovirus type 2 (PCV2) capsid Length: 750 Type: DNA ATGGAGAAAA TAGTGCTTCT TTTTGCAATA GTCAGTCTTG TTAAAAGTAC GTATCCAAGG AGGCGTTTCC GCAGACGAAG ACACCGCCCC CGCAGCCATC TTGGCCAGAT CCTCCGCCGC CGCCCCTGGC TCGTCCACCC CCGCCACCGT TACCGCTGGA GAAGGAAAAA TGGCATCTTC AACACCCGCC TCTCCCGCAC CATCGGTTAT ACTGTCAAGA AAACCACAGT CAGAACGCCC TCCTGGAATG TGGACATGAT GAGATTTAAT ATTAATGATT TTCTTCCCCC AGGAGGGGGC TCAAACCCCC TCACTGTGCC CTTTGAATAC TACAGAATAA GGAAGGTTAA GGTTGAATTC TGGCCCTGCT CCCCAATCAC CCAGGGTGAC AGGGGAGTGG GCTCCACTGC TGTTATTCTA GATGATAACT TTGTAACAAA GGCCAATGCC CTAACCTATG ACCCCTATGT AAACTACTCC TCCCGCCATA CCATAACCCA GCCCTTCTCC TACCACTCCC GGTACTTTAC CCCGAAACCT GTCCTTGATA GGACAATCGA TTACTTCCAA CCCAATAACA AAAGAAATCA ACTCTGGCTG AGACTACAAA CTACTGGAAA TGTAGACCAT GTAGGCCTCG GCACTGCGTT CGAAAACAGT ATCTACGACC AGGACTACAA TATCCGTATA ACCATGTATG TACAATTCAG AGAATTTAAT CTTAAAGACC CCCCACTTAA CCCTAAGTGA

[0143] Accession No. 4. Modified Porcine circovirus type 2 strain PCV2 capsid Length: 249 Type: Amino acid M E K I V L L F A I V S L V K S T Y P R R R F R R R R H R P R S H L G Q I L R R R P W L V H P R H R Y R W R R K N G I F N T R L S R T I G Y T V K K T T V R T P S W N V D M M R F N I N D F L P P G G G S N P L T V P F E Y Y R I R K V K V E F W P C S P I T Q G D R G V G S T A V I L D D N F V T K A N A L T Y D P Y V N Y S S R H T I T Q P F S Y H S R Y F T P K P V L D R T I D Y F Q P N N K R N Q L W L R L Q T T G N V D H V G L G T A F E N S I Y D Q D Y N I R I T M Y V Q F R E F N L K D P P L N P K

[0144] Accession number 5. Pcdna3.4-PCV2. (Figure 1A). Length: 6778 Type: DNA [Sequence Listing 1-1] TIFF2025090677000004.tif197169[Sequence Listing 1-2] TIFF2025090677000005.tif245167[Sequence Listing 1-3] TIFF2025090677000006.tif139169

[0145] The present invention should not be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying drawings. Such modifications are intended to be included within the scope of the appended claims.

[0146] Patents, patent applications, and publications are cited throughout this application, and in particular, their disclosures, including all chemical structures disclosed, are incorporated herein by reference. The citation of such publications or documents is not intended as an admission that any of the foregoing is prior art relevant hereto, nor does it constitute any admission as to the content or date of these publications or documents. All references cited herein are incorporated by reference to the same extent as if each individual publication, patent application, or patent were specifically and individually indicated to be incorporated by reference.

[0147] The foregoing specification is considered to be sufficient to enable one of ordinary skill in the art to practice the invention. Various modifications of the invention in addition to those shown and described herein will become apparent to those of ordinary skill in the art from the foregoing description and fall within the scope of the appended claims.

[0148] Exemplary systems and methods are described in the following items. Item 1: A mammalian expression system for producing recombinant porcine circovirus type 2 (PCV2) virus-like particles (VLPs), the expression system comprising a mammalian cell, and a plasmid comprising a PCV2 gene encoding a capsid protein, wherein the PCV2 gene is codon-optimized, the mammalian cell is transfected with the plasmid, and the expression system produces recombinant PCV2 VLPs. Item 2: The expression system according to Item 1, wherein the mammalian cell is a human embryonic kidney-293 (HEK-293) mammalian cell. Item 3: The PCV2 gene comprises a recognition site for NheI, a Kozak sequence, and a recognition site for NotI, wherein the recognition site for NheI and the Kozak sequence are upstream of the start codon of the PCV2 gene, and the recognition site for NotI is incorporated after the stop codon of the PCV2 gene, the expression system according to Item 1 or 2. Item 4: The expression system according to any one of Items 1 to 3, wherein most of the produced recombinant PCV2 VLPs are present in the nucleus of mammalian cells. Item 5: The expression system according to any one of Items 1 to 4, wherein the capsid protein contains the amino acid sequence of SEQ ID NO: 2. Item 6: The expression system according to any one of Items 1 to 5, wherein the capsid protein is encoded by the nucleotide sequence of SEQ ID NO: 1. Item 7: The expression system according to any one of Items 1 to 4, wherein the capsid protein is modified with a secretion signal sequence introduced at the NH2 terminus of the capsid protein. Item 8: The expression system according to Item 7, wherein the capsid protein contains the amino acid sequence of SEQ ID NO: 4. Item 9: The expression system according to Item 7 or 8, wherein the capsid protein is encoded by the nucleotide sequence of SEQ ID NO: 3. Item 10: The expression system according to any one of Items 1 to 9, wherein the produced recombinant PCV2 VLPs are selected from the group consisting of PCV2a VLPs, PCV2b VLPs, PCV2c VLPs, PCV2d VLPs, and PCV2e VLPs. Item 11: The expression system according to any one of Items 1 to 10, wherein the produced recombinant PCV2 VLPs are PVC2d VLPs. Item 12: The expression system according to any one of Items 1 to 11, wherein the plasmid is pcDNA3.4-PCV2. Item 13: The expression system according to any one of Items 1 to 12, wherein the PCV2 gene is codon-optimized using the codon-optimized amino acid sequence of FIG. 1A. Item 14: A method for producing porcine circovirus type 2 (PCV2) virus-like particles (VLPs), comprising: providing a suspension of cultured mammalian cells; transfecting the mammalian cells with a plasmid containing a PCV2 gene encoding a capsid protein; Adding sodium valproate (VPA) to the transfected mammalian cells, wherein the addition of the sodium VPA inhibits cell growth, and adding; Centrifuging and washing the transfected mammalian cells; Suspending the centrifuged mammalian cells in a phosphate buffered saline (PBS) solution; Performing a plurality of freeze and thaw cycles on the mammalian cells; Sonication of the mammalian cells in a plurality of cycles; Performing two consecutive centrifugation cycles of the mammalian cells to produce the PCV2 VLP, and; A method, wherein most of the produced PCV2 VLP is present in the nucleus of the mammalian cells. Item 15: The step of centrifuging and washing the transfected mammalian cells is Centrifuging the mammalian cells at 2,000 × g for 15 minutes; Washing the mammalian cells with a PBS solution; Centrifuging the mammalian cells again at 2,000 × g for 15 minutes, the method according to item 14. Item 16: The method according to item 14 or 15, wherein the centrifuged mammalian cells are frozen at about -80°C and thawed at about 37°C during the freeze and thaw cycles. Item 17: The method according to any one of items 14 to 16, wherein the first of the two consecutive centrifugation cycles is performed at 2,000 × g for 15 minutes, and the second of the two consecutive centrifugation cycles is performed at 8,000 × g for 15 minutes. Item 18: The method according to any one of items 14 to 17, further comprising purifying the PCV2 VLP by ultracentrifugation. Item 19: The method according to any one of items 14 to 18, wherein the mammalian cells are human embryonic kidney - 293 (HEK - 293) mammalian cells. Item 20: The PCV2 gene contains a recognition site for NheI, a Kozak sequence, and a recognition site for NotI, The recognition site for NheI and its Kozak sequence are upstream from the start codon of the PCV2 gene, and the recognition site for NotI is incorporated after the stop codon of the PCV2 gene, by the method according to any one of items 14 to 19. Item 21: The method according to any one of items 14 to 20, wherein the plasmid is pcDNA3.4-PCV2. Item 22: The method according to any one of items 14 to 21, wherein the produced PCV2 VLP is selected from the group consisting of PCV2a VLP, PCV2b VLP, PCV2c VLP, PCV2d VLP, and PCV2e VLP. Item 23: The method according to any one of items 14 to 22, wherein the produced PCV2 VLP is PVC2d VLP. Item 24: PCV2 VLP produced by the method according to any one of items 14 to 23.

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Claims

1. 1. A mammalian expression system for producing recombinant porcine circovirus type 2 (PCV2) virus-like particles (VLPs), comprising: A mammalian cell; a plasmid comprising a PCV2 gene encoding a capsid protein, wherein the PCV2 gene is codon-optimized; said mammalian cell being transfected with said plasmid; A mammalian expression system, wherein said expression system produces recombinant PCV2 VLPs.

2. 2. The expression system of claim 1, wherein the mammalian cell is a human embryonic kidney-293 (HEK-293) mammalian cell.

3. the PCV2 gene contains a recognition site for NheI, a Kozak sequence, and a recognition site for NotI; 3. The expression system of claim 1, wherein the recognition site for NheI and the Kozak sequence are upstream from the start codon of the PCV2 gene, and the recognition site for NotI is incorporated after the stop codon of the PCV2 gene.

4. The expression system according to any one of claims 1 to 3, wherein the majority of the produced recombinant PCV2 VLPs are present in the nucleus of the mammalian cell.

5. The expression system according to any one of claims 1 to 4, wherein the capsid protein comprises the amino acid sequence of SEQ ID NO:

2.

6. The expression system according to any one of claims 1 to 5, wherein the capsid protein is encoded by the nucleotide sequence of SEQ ID NO:

1.

7. 5. The expression system according to claim 1, wherein the capsid protein is modified with a secretory signal sequence introduced at the NH2-terminus of the capsid protein.

8. The expression system of claim 7 , wherein the capsid protein comprises the amino acid sequence of SEQ ID NO:

4.

9. 9. An expression system according to claim 7 or 8, wherein the capsid protein is encoded by the nucleotide sequence of SEQ ID NO:

3.

10. 10. The expression system according to any one of claims 1 to 9, wherein the produced recombinant PCV2 VLP is selected from the group consisting of PCV2a VLP, PCV2b VLP, PCV2c VLP, PCV2d VLP, and PCV2e VLP.

11. The expression system according to any one of claims 1 to 10, wherein the recombinant PCV2 VLP produced is a PVC2d VLP.

12. The expression system according to any one of claims 1 to 11, wherein the plasmid is pcDNA3.4-PCV2.

13. 13. The expression system according to any one of claims 1 to 12, wherein the PCV2 gene is codon-optimized using the codon-optimized amino acid sequence of Figure 1A.

14. 1. A method for producing porcine circovirus type 2 (PCV2) virus-like particles (VLPs), comprising: Providing a suspension of cultured mammalian cells; transfecting said mammalian cells with a plasmid comprising a PCV2 gene encoding a capsid protein; adding valproic acid (VPA) sodium salt to the transfected mammalian cells, wherein the addition of the VPA sodium salt inhibits cell proliferation; Centrifugation and washing of the transfected mammalian cells; suspending the centrifuged mammalian cells in a phosphate buffered saline (PBS) solution; subjecting the mammalian cells to multiple freezing and thawing cycles; sonicating the mammalian cells for multiple cycles; performing two successive centrifugation cycles of the mammalian cells to produce the PCV2 VLPs; The method, wherein the majority of the PCV2 VLPs produced are present in the nucleus of the mammalian cell.

15. centrifugation and washing the transfected mammalian cells, Centrifuging the mammalian cells at 2,000×g for 15 minutes; washing the mammalian cells with a PBS solution; and centrifuging the mammalian cells again at 2,000 x g for 15 minutes.

16. 16. The method of claim 14 or 15, wherein the centrifuged mammalian cells are frozen at about -80°C and thawed at about 37°C during the freeze and thaw cycle.

17. 17. The method according to any one of claims 14 to 16, wherein a first of the two successive centrifugation cycles is carried out at 2,000 x g for 15 minutes and a second of the two successive centrifugation cycles is carried out at 8,000 x g for 15 minutes.

18. The method of any one of claims 14 to 17, further comprising purifying the PCV2 VLPs by ultracentrifugation.

19. The method of any one of claims 14 to 18, wherein the mammalian cells are human embryonic kidney-293 (HEK-293) mammalian cells.

20. the PCV2 gene contains a recognition site for NheI, a Kozak sequence, and a recognition site for NotI; The method according to any one of claims 14 to 19, wherein the recognition site for NheI and the Kozak sequence are upstream from the start codon of the PCV2 gene, and the recognition site for NotI is incorporated after the stop codon of the PCV2 gene.

21. The method of any one of claims 14 to 20, wherein the plasmid is pcDNA3.4-PCV2.

22. 22. The method of any one of claims 14 to 21, wherein the produced PCV2 VLPs are selected from the group consisting of PCV2a VLPs, PCV2b VLPs, PCV2c VLPs, PCV2d VLPs, and PCV2e VLPs.

23. The method of any one of claims 14 to 22, wherein the produced PCV2 VLPs are PCV2d VLPs.

24. A PCV2 VLP produced by the method of any one of claims 14 to 23.

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