Recombinant circovirus capsid-virus-like particles (VLPs): compositions, methods and uses

Abstract

Provided herein is a mammalian expression system for producing recombinant porcine circovirus type 2 (PCV2) virus-like particles (VLPs). The expression system includes mammalian cells and a plasmid containing a PCV2 gene encoding a capsid protein. The PCV2 gene is codon-optimized, and mammalian cells are transfected with the plasmid. The expression system produces recombinant PCV2 VLPs, such as PCV2d VLPs. Also provided herein are methods for producing porcine circovirus type 2 (PCV2) virus-like particles (VLPs), as well as PCV2 VLPs produced by the methods.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. patent application Ser. No. 62 / 837,758, entitled "Recombinant Circovirus Capsid-Virus-Like Particle (VLP): Compositions, Methods and Uses," which is incorporated herein by reference in its entirety.

[0002] 1. Field The present application relates to compositions comprising porcine circovirus-2 (PCV2), in particular PCV2 virus-like particles (VLPs). [Background technology]

[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, and terrestrial animals (1, 2). Variations in capsid morphology and genome organization have recently led to the classification of the Circoviridae family into two separate genera, Circovirus and Cyclovirus (3). The genus Circovirus includes porcine circovirus type 1 (PCV1), type 2 (PCV2), and type 3 (PCV3) (2). Genome sequences related to the Cyclovirus genus have been associated with several vertebrate and invertebrate species, but definitive host recognition for this group is still unknown (2). PCV2 infection is responsible for a significant high mortality rate among pigs as the causative agent of porcine circovirus-associated disease (PCVAD), and has also been linked to porcine dermatitis and nephropathy syndrome (PDNS) and porcine reproductive disorders (1, 4).

[0004] The diameter of the virion particle is about 19 nm, and the size of the genome of PCV ranges from 1.7 kb to 2.3 kb. The circular nature of the genome has led to the name of the virus family Circoviruses. The evolutionary history has been investigated, allowing detailed phylogenetic trees and changes in the capsid surface structure to be described (5-8). The first cryo-EM image reconstructions of several native circoviruses showed that the capsid has an icosahedral symmetry with T=1 (9). The PCV genome encodes one structural capsid protein (CP). Expression and purification of the PCV2b CP protein from E. coli showed that it self-assembles to mimic the overall morphology of 70 infectious viruses. The crystal structure of this virus-like particle (VLP) visualized the CP fold as that of a canonical viral jelly roll consisting of two four-stranded β-sheets (10, 11). The loops connecting the β-strands form a feature on the viral surface and may contain antigenic epitopes. PCV2b CP was also expressed as a VLP from Trichoplasia ni insect cells and purified (11). Cryo-EM image reconstruction of this VLP showed that the N-terminus was located inside the capsid, and the authors concluded that the antigenic properties associated with the N-terminus were likely the result of the N-terminus being transiently externalized from the capsid through a process called viral "respiration" (11-13). Externalization of the N-terminus may play an important role in the viral life cycle.

[0005] The amino acid sequences of PCV2 accessions in GenBank have been classified into four different genotypes (PCVa-d) (14). PCV2a was the predominant global genotype until 2003, when a genotypic shift to PCV2b was observed (15, 16). The PCV2c genotype may have become extinct, as only three deposits exist in GenBank. In 2013, Wei et al. reported and deposited a large number of PCV2d sequences in GenBank (5). Further reporting and deposits of PCV2d sequences have resulted in approximately 320 deposits of 84 in GenBank (7). The increase in deposits of PCV2d sequences may be the result of PCV2d emerging and becoming the predominant genotype in Asia, Europe, and North and South America. This increase may be the result of vaccination escape variants (17). As a result, PCV2d may represent a second global genotypic shift that may not respond to currently implemented vaccination regimes against PCV2 (7). Despite a significant number of phylogenetic studies of PCV2 genotypes, and the emerging importance of PCV2d in the global swine industry, no reports exist describing the structure of the PCV2d capsid.

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

[0007] 3. Overview 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 embodiment, the mammalian cell is a human embryonic kidney-293 (HEK-293) mammalian cell.

[0009] In another embodiment, the PCV2 gene comprises 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 embodiment, the majority of the produced recombinant PCV2 VLPs are present in the nucleus of the mammalian cell.

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

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

[0013] In another embodiment, the recombinant PCV2 VLP produced is selected from the group consisting of PCV2a VLP, PCV2b VLP, PCV2c VLP, PCV2d VLP, and PCV2e VLP. In a further embodiment, the recombinant PCV2 VLP produced is a PCV2d VLP.

[0014] In another embodiment, the plasmid is pcDNA3.4-PCV2. In another embodiment, 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. Valproic acid (VPA) sodium salt is added to the transfected mammalian cells, and the addition of VPA sodium salt inhibits cell growth. The transfected mammalian cells are centrifuged and washed. The centrifuged mammalian cells are then suspended in a phosphate buffered saline (PBS) solution. Multiple freeze and thaw cycles are performed on the mammalian cells, and the mammalian cells are then sonicated for multiple cycles. Two successive centrifugation cycles of the mammalian cells are then performed to produce PCV2 VLPs, and the majority of the produced PCV2 VLPs are present in the nucleus of the mammalian cells.

[0016] In another aspect of this method, the step of centrifuging and washing the transfected mammalian cells comprises 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 embodiment of this method, the centrifuged mammalian cells are frozen at about -80°C and thawed at about 37°C during a freeze and thaw cycle.

[0018] In another embodiment of this method, a first of two successive centrifugation cycles is carried out at 2,000 x g for 15 minutes and a second of two successive centrifugation cycles is carried out at 8,000 x g for 15 minutes.

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

[0020] In another embodiment of this method, the mammalian cell is a human embryonic kidney-293 (HEK-293) mammalian cell.

[0021] In another embodiment of this method, the PCV2 gene comprises 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 embodiment of this method, the plasmid is pcDNA3.4-PCV2.

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

[0024] In a third aspect, there is provided a PCV2 VLP produced by the above method. [Brief description of the drawings]

[0025] 4. Brief description of the drawings [Figure 1A]Expression of PCV2 virus-like particles in mammalian cells. Figure 1A) Plasmid generated for expression of PCV2d capsid protein. The codon-optimized PCV2d capsid gene was synthesized (Blue Heron Technologies, Bothell, WA) and cloned into the expression vector pcDNA3.4 (Fisher Scientific). Figure 1B) Protein expression was performed in transiently transfected suspension cultures of Expi293 cells (Life Technologies). SDS-PAGE analysis of purified PCV2d VLPs (1 μg protein) and stained with Coomassie Blue. Figure 1C) SDS-PAGE analysis of purified PCV2d VLPs (0.5 μg protein) transferred to nitrocellulose membrane and Western blot probed with primary rabbit anti-PCV2 capsid polyclonal antibody (Cab 183908, Abcam, UK). Figure 1D) Negative stain electron micrograph of purified VLPs stained with uranyl acetate. Particle size is approximately 19 nm in diameter. [Figure 1B] Same as above. [Figure 1C] Same as above. [Figure 1D] Same as above. [Figure 2A] Structural study of PCV2d VLP. Figure 2A) Icosahedral cryo-EM image reconstruction of purified PCV2d VLP colored according to local resolution. The gradient color map on the left indicates the color resolution. Figure 2B) Ribbon diagram of atomic coordinates with one asymmetric unit colored in cyan. Figure 2C) Structural overlay of PCV2a (dark blue), PCV2b (cyan), and PCV2d (yellow). Loops are labeled according to the β-strands they connect. Diagram generated using UCSF ChimeraX (47). [Figure 2B] Same as above. [Figure 2C] Same as above. [Figure 3A]Sequence comparison of 1,377 PCV2 capsid protein accessions plotted on PCV2d atomic coordinates. Figure 3A) Sequence alignment by the Clustal Omega server was used to generate a WebLogo diagram to show sequence variation. The horizontal axis of the alignment indicates the amino acid, and the vertical axis indicates its observed frequency. The bars connecting amino acids 77, 80, 99, 91, 190, 19 (black), and 53, 215 (gray) represent the evolutionary coupling clusters shown in panel D. Figure 3B) Space-filling model of PCV2d atomic coordinates with modified color-coding scheme of ConSurf. The color bar at the bottom indicates the degree of conservation as determined by the ConSurf server. Yellow boxes indicate insufficient data as determined by the server (1 indicates poor conservation, 9 indicates a highly conserved mutation). The top right quadrant of the VLP surface was removed to show sequence conservation inside the capsid. Images produced with UCSF ChimeraX and colored using flat illumination. Figure 3C) Highly conserved amino acid patches on the capsid surface (amino acids 82, 170, 188, 189, and 193 in green; amino acids 55, 56, 51, and 73 in blue). Antibodies against these residues may possess broadly neutralizing capabilities. Figure 3D) Ribbon diagram of the PCV2d subunit. Aberrant residues are evolutionarily joined together as determined using the plmc. MATLAB 2019, and EVzoom programs. Diagram generated using UCSF ChimeraX (47). [Figure 3B] Same as above. [Figure 3C] Same as above. [Figure 3D] Same as above. [Figure 4A] Antibody neutralization site. Top) Space-filling model of PCV2d atomic coordinates with surface-exposed amino acids colored in cyan, sequence-variable amino acids in purple, and antibody-binding amino acids in yellow. Center) WebLogo diagram of 11 amino acids including the solvent-exposed amino acids. Bottom) Amino acids on the surface of the capsid. [Figure 4B] Same as above. [Figure 4C] Same as above. [Figure 4D] Same as above. [Figure 4E] Same as above. [Figure 4F] Same as above. [Figure 4G] Same as above. [Figure 4H] Same as above. [Diagram 5] Body weight change during the study phase for each treatment group: T01 (PIGONE 1 mL), T02 (PIGONE 2 mL), T03_A (Huve-PCV2_A), T03_B (Huve-PCV2_B), T04 (HVP-DNA), T05 (CIRCOFLEX [inactivated baculo-expressed PCV2 ORf2]) and T06 (PBS). The Y-axis represents body weight in Kg and the X-axis represents study days from SD0 to SD63. [Figure 6] Average daily weight gain during the study period for each treatment group: T01 (PIGONE 1 mL), T02 (PIGONE 2 mL), T03_A (Huve-PCV2_A), T03_B (Huve-PCV2_B), T04 (HVP-DNA), T05 (CIRCOFLEX (inactivated baculo-expressed PCV2 ORf2)) and T06 (PBS). The Y-axis represents weight gain in gr, and the X-axis represents the study periods SD0–SD35, SD35–SD63, and SD0–SD63. [Figure 7] Serological changes during the study phase for each treatment group: T01 (PIGONE 1 mL), T02 (PIGONE 2 mL), T03_A (Huve-PCV2_A), T03_B (Huve-PCV2_B), T04 (HVP-DNA), T05 (CIRCOFLEX [inactivated baculo-expressed PCV2 ORf2]) and T06 (PBS). The Y-axis represents S / P values ​​and the X-axis represents study days from SD0 to SD63. [Figure 8] Percentage of quantified positive and non-quantified positive animals during the challenge phase for each treatment group: T01 (PIGONE 1 mL), T02 (PIGONE 2 mL), T03_A (Huve-PCV2_A), T03_B (Huve-PCV2_B), T04 (HVP-DNA), T05 (CIRCOFLEX (inactivated baculo-expressed PCV2 ORf2)) and T06 (PBS). Y-axis represents percentage of animals and X-axis represents study days from SD35 to SD63. [Figure 9] Mean viral load (>4 log10 copies / mL) of quantified positive animals during the challenge phase for each treatment group: T01 (PIGONE 1 mL), T02 (PIGONE 2 mL), T03_A (Huve-PCV2_A), T03_B (Huve-PCV2_B), T04 (HVP-DNA), T05 (CIRCOFLEX (inactivated baculo-expressed PCV2 ORf2)) and T06 (PBS). Y-axis represents viral load and X-axis represents study days from SD41 to SD63. [Figure 10] Percentage of quantified positive, non-quantified positive, and below detection limit animals during the challenge phase for each treatment group: T01 (PIGONE 1 mL), T02 (PIGONE 2 mL), T03_A (Huve-PCV2_A), T03_B (Huve-PCV2_B), T04 (HVP-DNA), T05 (CIRCOFLEX (inactivated baculo-expressed PCV2 ORf2)) and T06 (PBS). Y-axis represents percentage of animals and X-axis represents study days from SD35 to SD63. [Figure 11] Mean viral load (>2log10 copies / mL) of all animals during the challenge phase for each treatment group: T01 (PIGONE 1 mL), T02 (PIGONE 2 mL), T03_A (Huve-PCV2_A), T03_B (Huve-PCV2_B), T04 (HVP-DNA), T05 (CIRCOFLEX (inactivated baculo-expressed PCV2 ORf2)) and T06 (PBS). Y-axis represents viral load and X-axis represents study days from SD35 to SD63. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] 5. Detailed Description The practice of the various embodiments of the present application will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, immunology and pharmacology, within the skill of the art, such techniques being fully explained in the literature. 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 (DMWeir and CC 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; and 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 entirety.

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

[0029] definition As used herein, "virus-like particle" or "VLP" refers to a non-replicating viral shell. 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 derived from these proteins called particle-forming polypeptides. VLPs can form naturally upon recombinant expression of proteins in an appropriate expression system. Methods for producing specific VLPs are discussed in more detail below. The presence of VLPs following recombinant expression of viral proteins can be detected using conventional techniques known in the art, such as by electron microscopy, biophysical characterization, and the like. See, e.g., 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., Examples). Alternatively, cryo-electron microscopy can be performed on vitrified aqueous samples of the VLP preparation in question and images recorded under appropriate exposure conditions. Additional methods of VLP purification include, but are not limited to, chromatographic techniques such as affinity, ion exchange, size exclusion, and reverse phase procedures.

[0030] As used herein, the term "hybrid" or "chimeric" refers to a molecule (e.g., a protein or VLP) that contains 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 portions thereof) and portions of proteins fused to additional heterologous polypeptides (full-length or portions thereof). It will also be apparent that the hybrid or chimeric molecules may include wild-type or mutant sequences in any one, some or all of the heterologous domains.

[0031] "Antigen" refers to a molecule that contains one or more epitopes (i.e., "antigenic epitopes"), linear, conformational, or both, that stimulate the host's 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. The term includes polypeptides that contain modifications, such as deletions, additions, and substitutions (generally conservative in nature), compared to the native sequence, so long as the protein retains the ability to elicit an immunological response as defined herein. These modifications may be artificial, such as by site-directed mutagenesis, or may be accidental, such as by mutation of the host that produces the antigen.

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

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

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

[0035] "Nucleic acid" molecules can include, but are 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. The term also captures sequences that contain any of the known base analogs of DNA and RNA.

[0036] "Recombinant" as used herein to describe a nucleic acid molecule means a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin that, by its origin or manipulation, (1) is not associated with all or a portion of a polynucleotide with which it is naturally associated, and / or (2) is linked to a polynucleotide other than the polynucleotide with which it is naturally linked. The term "recombinant" as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide. "Recombinant host cell," "host cell," "cell," "cell line," "cell culture," and other such terms referring to prokaryotic microorganisms or eukaryotic cell lines cultured as unicellular organisms are used interchangeably and refer to cells that can be or have been used as recipients of recombinant vectors or other transfer DNA, including the progeny of the original transfected cell. It is understood that the progeny of a single parent cell may not necessarily be completely identical in morphology or in genomic or total DNA complement to the original parent due to accidental or artificial mutation. Progeny of the parental cell that are sufficiently similar to the parent to be characterized by the relevant property, such as the presence of a nucleotide sequence encoding a desired peptide, are included in the progeny intended by this definition and are encompassed by the above term.

[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. In general, "similarity" means that when comparing amino acids exactly at the appropriate positions of two or more polypeptides, the amino acids are identical or possess similar chemical and / or physical properties, such as charge or hydrophobicity. A so-called "percent similarity" can then 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 the amino acid sequence it encodes, and comparing this to a second amino acid sequence. In general, "identity" refers to the exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotide or polypeptide sequences, respectively.

[0039] Two or more polynucleotide sequences can be compared by determining their "percent identity". Similarly, two or more amino acid sequences can be compared by determining their "percent identity". The percent identity of two sequences, whether nucleic acid or peptide sequences, is generally described as the number of exact matches between the two aligned sequences divided by the length of the shorter sequence multiplied by 100. 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 can be extended for use with peptide sequences using a scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, MO Dayhoff ed., 5 suppl.3:353-358, National Biomedical Research Foundation, Washington, DC, USA, and normalized by Gribskov, Nucl.Acids Res.14(6):6745-6763 (1986). Suitable programs for calculating the percent identity or similarity between sequences are generally known in the art.

[0040] A "vector" is capable of introducing 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" refer to any nucleic acid construct capable of inducing the expression of one or more sequences of interest in a host cell. Thus, the term includes cloning and expression vehicles, as well as viral vectors. The 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, including non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cows, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs; domestic birds such as chickens, turkeys and other poultry, ducks, geese and the like, wild birds and game birds. The term does not indicate a particular age. Thus, it is intended to encompass both adult and newborn individuals. The system described herein is intended for use with any of the above vertebrate species, as the immune systems of all of these vertebrates function similarly.

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

[0043] As used herein, "treatment" refers to either (i) prevention of infection or reinfection, similar to traditional vaccines, (ii) reduction or elimination of symptoms, and (iii) substantial or complete elimination of the pathogen in question. Treatment can be prophylactic (before infection) or therapeutic (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 immune response includes enhancing or broadening the specificity of either or both antibody and cellular immune responses. Modification of immune response may also mean reducing or suppressing a particular antigen-specific immune response.

[0045] As used herein, an "effective dose" generally refers to an amount of the VLP of one or more embodiments of the present application sufficient to induce immunity, prevent and / or ameliorate an infection, or reduce at least one symptom of an infection, and / or enhance the effectiveness of another dose of the VLP. An effective dose may refer to an amount of the VLP sufficient to delay or minimize the onset of an infection. An effective dose may also refer to an amount of the VLP that provides a therapeutic effect in the treatment or management of an infection. Additionally, an effective dose is an amount that, with respect to the VLP of one or more embodiments of the present application, alone or in combination with other therapeutic agents, provides a therapeutic effect in the treatment or management of an infection. An effective dose may also be an amount sufficient to enhance a subject's (e.g., a human's) own immune response to subsequent exposure to an infectious agent. The level of immunity can be monitored, for example, by measuring the amount of neutralizing secretory and / or serum antibodies, for example, by plaque neutralization, complement fixation, enzyme-linked immunosorbent, or microneutralization assays. In the case of a vaccine, an "effective dose" is one that prevents 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. An effective amount of a composition is an amount that achieves a 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 necessary to cause activation of the immune system upon exposure to the VLPs of the present application, resulting in the generation of an antigen-specific immune response. This term is also synonymous with "sufficient amount."

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

[0048] As used herein, the term "protective immune response" or "protective response" refers to an antibody-mediated immune response to an infectious agent that is found in a vertebrate (e.g., human) and prevents or ameliorates infection or at least one symptom thereof. The VLPs of the present application can, for example, stimulate the production of antibodies that neutralize the infectious agent, block the infectious agent from entering a cell, block the replication of the infectious agent, and / or protect host cells from infection and destruction. The term may also refer to an immune response mediated by T lymphocytes and / or other white blood cells to an infectious agent that is found in a vertebrate (e.g., human) and prevents or ameliorates influenza infection or at least one symptom thereof.

[0049] As used herein, the term "vaccine" refers to a formulation containing the VLPs of the present application in a form that can be administered to a vertebrate and induces immunity to prevent and / or ameliorate an infectious disease, and / or induces a protective immune response sufficient to alleviate at least one symptom of an infectious disease, and / or enhance the effectiveness of another dose of the VLPs. Typically, the vaccine comprises a conventional saline or buffered aqueous medium 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 infectious disease. 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 viruses of the Circoviridae family (e.g., Porcine Circovirus-2 (PCV2), its different genotypes and serotypes or other members of the family), compositions comprising virus-like particles (VLPs), and methods of making and using those VLPs, including the creation and production of vaccines based on virus-like particles (VLPs) (e.g., monovalent, multivalent, single particle universal or multivalent, single particle mosaic or modified chimeric compositions), and their use for therapeutic delivery (e.g., small molecules, nucleic acids, antibodies, enzymes (nanocarriers, nanobodies)], diagnostic, immunomodulatory functions and therapeutic indications. In particular, the present disclosure includes strategies and methods used for the development of novel monovalent, multivalent or universal porcine circovirus vaccines capable of protecting pigs from infection with one or more serotypes, clades or antigenic variants of the Porcine Circovirus genus. Also provided herein are VLP production methods that produce VLPs displaying specific antigenic configurations or modifications. (e.g., secretion systems) have also been described. These VLPs feature natural or chimeric epitopes in conformations associated with the generation of enhanced neutralizing immune responses against porcine circovirus or other viral agents. Single particle monovalent, bivalent, multivalent, universal or chimeric (e.g., different serotypes and genotypes such as PCV2a, PCV2b, PCV2c, PCV2d and PCV2e) VLPs are constructed and used to formulate vaccine compositions that allow immunization and subsequent protection against one or more serotypes or antigenically distinct viruses (e.g., Asian, European or North American serotypes, etc.) VLPs, natural, modified or redesigned capsid monomers allow the linkage / conjugation of different molecular entities to the outer surface of the particle (small or large molecular entities) or the encapsidation of such molecular entities within the structure of the particle via disassembly and reassembly of the VLP or alternative packaging methods.

[0051] In addition, VLPs are also used for diagnostic or therapeutic indications 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, for example, either mucosal or parenteral, to induce an immune response that can protect against any or all of the porcine circovirus serotypes, antigenic variants, etc. Also provided are VLPs with therapeutic, immunomodulatory functions, and diagnostic applications.

[0052] These and other aspects of the compositions and methods of the present invention are described in more detail below with reference to the accompanying drawings and examples, in which one or more illustrated embodiments and / or configurations of PCV2 VLPs are shown. The compositions and methods of the present application are not limited to the illustrated embodiments and / or configurations. It should be understood that the compositions and methods as shown in the accompanying drawings are merely exemplary of the compositions and methods of the present application, which may 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 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. In at least one embodiment, the mammalian cell is a human embryonic kidney-293 (HEK-293) mammalian cell.

[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 from 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, the majority of the produced recombinant PCV2 VLPs are present in the nucleus of the mammalian cell.

[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 secretory 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 VLP produced may be at least one of the following: PCV2a VLP, PCV2b VLP, PCV2c VLP, PCV2d VLP, and PCV2e VLP. In a preferred embodiment, the recombinant PCV2 VLP produced is a PCV2d VLP.

[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. Valproic acid (VPA) sodium salt is added to the transfected mammalian cells, and the addition of VPA sodium salt inhibits cell growth. The transfected mammalian cells are centrifuged and washed. The centrifuged mammalian cells are then suspended in a phosphate buffered saline (PBS) solution. At least one freeze and thaw cycle, preferably multiple freeze and thaw cycles, are performed on the mammalian cells, and then the mammalian cells are sonicated for 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, the majority 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 x 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.

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

[0063] In at least one embodiment, the first of the two successive centrifugation cycles is performed at 2000×g for 15 minutes, and the second of the two successive 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 PCV2 VLP produced is selected from the group consisting of PCV2a VLP, PCV2b VLP, PCV2c VLP, PCV2d VLP, and PCV2e VLP. In at least one preferred embodiment, the PCV2 VLP produced is a PVC2d VLP. In at least one embodiment, a PCV2 VLP produced by the above method is provided.

[0066] In summary, according to one or more embodiments, the present application discloses mammalian construct systems of PCV2 VLPs (e.g., PCV2d VLPs). The present application further discloses structural analyses based on cryo-electron microscopy reconstructions that reveal external and internal characteristics of the VLPs, which have important implications for the development of novel PCV2 vaccines and the use of the particles as nano-vehicles to deliver a variety of molecular entities for prophylactic, therapeutic, immunotherapeutic, immunization (heterogeneous, homogeneous, multivalent), and diagnostic applications. These analyses and other examples related to PCV2 VLPs are described in more detail below. EXAMPLES

[0067] 6. Working Example Example 1 material and method Cells, capsid genes, plasmids and antibodies. Suspension cultures of Expi293F human cells (Life Technologies, CA) were grown in serum-free Expi293 expression medium (Life Technologies, CA) at 37°C in a 5% CO2 environment and agitated at 150 rpm in Erlenmeyer flasks. The porcine circovirus type 2 (PCV2) gene encoding the capsid protein (strain RQ3) was chemically synthesized using a codon-optimized sequence (amino acid sequence shown in Figure 1A) by Blue Heron Technologies (Bothell, WA). A recognition site for NheI and a Kozak sequence were added immediately upstream from the start codon, and a recognition site for NotI was incorporated after the stop codon. The synthesized Cap gene was recovered from the transport plasmid by double digestion with NheI and 289 NotI restriction enzymes and subcloned into the mammalian expression plasmid pcDNA3.4 cut with the same enzymes after gel purification. Ligated plasmids were transformed into MAX Efficiency Stbl2 Cells (Life Technologies), and correct clones were identified by restriction enzyme analysis and verified by sequencing.

[0068] Production and purification of virus-like particles (VLPs). PCV2 VLPs were produced in suspension cultures of Expi293F mammalian cells after transient transfection with the plasmid pcDNA3.4-PCV2 (Figures 1A-1D). Expi293F cells were cultured at 2 x 10 6Cells were seeded at a concentration of 1000 x g / ml and cultured for 16 h before transfection. Plasmid DNA (1 μg / ml) was diluted in a volume of Opti-MEM equivalent to 5% of the total volume of the culture. Separately, polyethyleneimine (PEI) was prepared in an equal volume of Opti-MEM (4 μg / ml). After 5 min of incubation at room temperature, the PEI solution was added 300 drops to the tube containing the DNA, and after 30 min of incubation at RT, the mixture was added dropwise to the cell suspension. 24 h after transfection, valproic acid sodium salt (VPA) was added to the cell culture to a final concentration of 3.75 mM to inhibit cell growth. 72 h after transfection, cells were pelleted by centrifugation at 2,000 x g for 15 min, then washed once with phosphate-buffered saline (PBS) and spun again at 2,000 x g for 15 min. The cell pellet was resuspended in PBS and then subjected to three freeze (-80 °C) and thaw (37 °C) cycles. The cells were then further fragmented by three cycles of sonication and clarified by two successive centrifugations, first at 2,000×g for 15 min and then at 8,000×g for 15 min. This procedure released a portion of the VLPs into the cytoplasm, but the majority of the particles appeared to be trapped within the cell nuclei (data not shown). The PCV2 VLPs contained in the clarified supernatant were further purified by ultracentrifugation on a two-layer CsCl density gradient: bottom layer, 5 ml of 1.4 g / ml CsCl, and top layer, 10 ml of 1.25 g / ml CsCl, both prepared in 10 mM Tris-HCl (pH 7.9). The sample was loaded onto the gradient and spun at 140,000×g for 4 h at 15° C. using an SW28 rotor (Beckman Coulter, CA). 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 spun at 155,000×g for 16 h at 15° C. using a SW 41Ti rotor (Beckman Coulter, CA). VLPs were detected at the bottom of the tube and collected as described above.The collected VLP material was dialyzed overnight at 10 °C against 10 mM Tris-HCl pH 7.9 and 150 mM NaCl using a Slide-A-Lyzer Cassette. Purified PCV2 VLPs were concentrated and buffer exchanged into phosphate-buffered saline (PBS) using Amicon Utra-4 centrifugal filter devices (Merck Millopore, MA). PCV2 VLP samples were stored in 50-100 μl aliquots at -80 °C.

[0069] Western blot and Coomassie blue staining. Purified VLPs were mixed with loading buffer, heated at 100 °C for 5 min, and run on 4–12% Bis-Tris SDS-polyacrylamide gels (Life Technologies, CA). Protein loading was 1 μg for prospective Coomassie staining and 0.5 μg for Western blots. After electrophoretic separation, gels were stained with Coomassie blue or proteins electrophoretically transferred to 0.45 μm nitrocellulose membranes (Life Technologies LC2001). The membranes were then blocked with 5% nonfat milk in TBST (10 mM Tris-HCl, 130 mM NaCl, and 0.05% Tween-20, pH 7.4) for 1 h 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 with secondary antibody (goat anti-rabbit IgG HRP 332 conjugated, 1:1,000) diluted in blocking buffer for 2 h. Finally, the membrane was washed three times with TBST and developed with an ECL Western Blot System (Life Technologies, CA) according to the manufacturer's instructions. Stained gel and immunoblot images were acquired using a FluorChem Imager instrument (Protein Simple, CA).

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

[0071] Cryo-EM data collection. Frozen hydrated samples of PCV2d VLPs were prepared on Quantifoil R 2 / 2, 200 mesh copper grids (Electron Microscopy Science). A 4 μl prescreened sample of VLPs was applied to the blotted grid for 3 s and flash frozen in liquid ethane using an FEI Vitrobot instrument. The 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 followed by a GIF quantum energy filter with a width of 15 eV. Data collection was performed using the Leginon software suite (36).

[0072] Image reconstruction. Particle motion was corrected using the MotionCor2 package (37). Default parameters and dose weighting were used for correction, except for the patch5 option, and the first frame of each movie was discarded during alignment. Particles were automatically selected using Gautomatch v0.53, and contrast transfer function (CTF) estimation was performed on 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 coordinates identified by Gautomatch. Reference-free 2D classification was performed with Relion 3.0. Nondefault options for this step included a diameter of 250 Å and 128 classes were requested (39). An initial model was generated at 60 Å resolution using the PCV2 crystal structure (PDB entry 3R0R) with the molmap function in UCSF Chimera (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 with 4,442 particles showed the best resolution. These particles were used for high-resolution image reconstruction in Relion 3.0. Again, a diameter of 250 Å and I1 symmetry were used with the remaining default parameters in Relion 3.0. Binary masks were created using the relion_mask_create program in Relion 3.0. Binary masks for post-processing were generated as follows: 1) the high-resolution image reconstruction was low-pass filtered to 15 Å resolution using relion_image_handler (Relion 3.0); 2) a minimum threshold that was noise unrelated 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. The resulting mask was then inspected on a UCSF Chimera to ensure that no internal cavities were present.

[0073] Local resolution was calculated with the program MonoRes. The same binary mask used during postprocessing in Relion was used in the calculations. A resolution range of 2.8 Å to 6.0 Å was used (41).

[0074] Structural improvements 376. The atomic coordinates for the crystal structure of PCV2b crystal structure (PDB entry 3R0R) were refined using Coot (42). The biological matrices required to generate virus-like particles are present in the PDB and were used by Coot to generate VLPs of PCV2d. The VLPs were manually docked into the symmetrized image reconstruction using UCSF Chimera. The resulting coordinates were iteratively refined 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 A protein Blast search using the sequence of PCV2d and the common name of the organism, porcine circovirus 2 (taxid:85708) filter, generated a total of 1,966 PCV2 sequences (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 had no sequence similar to them were manually removed. This was done to remove spurious errors / artifacts that may have occurred during the sequencing process (i.e., artificial recombination during PCR with Taq polymerase (46)). Several rounds of alignment and deletion were performed to remove identical sequences. 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 are too similar), we could not successfully perform evolutionary coupling calculations using the EV coupling server394 (evfold.org). As a result, we used plmc to generate coevolution and covariation within the sequences. The L2 lambdas for fields and couplings used were 0.01 and 16.0, respectively, and we ran up to 100 iterations. The results were converted for visualization in EVzoom using MATLAB 2019 and scripts provided by the plmc program (22). The resulting matrices were visualized in EVzoom (22), and structural covariances were visually confirmed with UCSF Chimera (40).

[0076] result 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). Transfection of suspension cultures of HEK-293 mammalian cells with the plasmid results in the production of CP and assembly of VLPs. To date, PCV2 VLPs have been produced using insect cells, yeast, or E. coli (11), so expression of VLPs using mammalian cells provides a substrate similar to that utilized by native PCV2 infection. During purification, the majority of the VLPs or CPs were determined to be in the nucleus of the cells, although some were detected in the cytoplasm of infected cells. This suggests that VLPs can exit the nucleus, possibly via a cryptic nuclear export signal that could not be clearly identified 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 (Figure 1B and C). Further examination by negative staining electron microscopy showed homogeneous spherical particles with smooth edges and slightly rough surfaces with a diameter of approximately 19 nm (Figure 1D). The CP described in this study harbors amino acid sequences identified from a number of recently isolated and reported PCV2d virus genome accessions 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 United Kingdom.

[0077] Cryo-electron microscopy and image reconstruction of PCV2d VLP. An icosahedral cryo-EM image reconstruction of purified PCV2d VLP was determined at 3.3 Å resolution (Figure 2A). Close inspection of the image reconstruction indicates that the molecular envelope of the side chains is of sufficient quality to model the atomic coordinates. Coordinates from the PCV2b crystal structure were manually fitted to the image reconstruction and appropriate modifications were performed to reflect the PCV2d amino acid sequence. The fitted model was improved through several iterations of automated refinement with Phenix and manual adjustments 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 overlaid to generate mean-square standard deviation plots of equivalent Cα atoms ( 9 , 18 ).The regions showing the greatest diversity correspond to two surface-exposed loops consisting of amino acids 85 to 91 (loop CD) and 188 to 194 (loop GH) (Fig. 2C ).

[0080] Sequence conservation among the four genotypes may aid in the generation of a universal vaccine for PCV2. There have been numerous publications comparing the amino acid sequences of PCV2 CP to gain insight into the evolution of the PCV2 capsid (6, 7). However, we are not aware of any 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). The sequence alignment was used to generate a WebLogo diagram to observe sequence conservation (Figure 3A). The horizontal axis of the alignment indicates the amino acid, and the vertical axis indicates the observed frequency of the amino acid. The sequences with the greatest diversity (GenBank accessions: AVZ65995.1 and ALK0432.1) share 74.4% sequence identity. The WebLogo diagram successfully shows the frequency of occurrence of the most known amino acids at each position, while suppressing / eliminating the frequency of less known amino acids. Indeed, mutations can be observed in residues within the hydrophobic core of the protein, at the intersubunit interface, as well as on the external and internal surfaces of the capsid. Furthermore, a closer inspection of the aligned sequences shows that only Met1 and Arg147 are fully conserved. The observed sequence diversity suggests that the capsid can tolerate mutations at almost all positions while remaining an infectious virus. To visualize the sequence conservation / variability at each amino acid position, the sequence alignment information was plotted onto the PCV2d atomic coordinates using the ConSurf server (Figure 3B) (20). This diagram shows the considerable degree of non-conserved mutations that the capsid has undergone. The extent of change is greatest in the regions colored in red, while the extent of change is least in the regions colored in blue. 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, as well as Pro82, Thr170, Gln188, Thr189 and Val193 (Figure 3C).The presence of these patches suggests that if antibody production could be induced with these amino acids, it may be possible to generate a vaccine capable of neutralizing all available genotypes of PCV2 (Figure 3C).

[0081] Evolutionary coupling mutations distinguish PCV2 genotypes. The large number of CP sequence deposits in GenBank allowed us to query whether any of the amino acid positions are evolutionarily suppressed (21). For evolutionarily suppressed amino acids, mutation of one amino acid requires mutation of the other amino acid. In the simplest case, this may be because the amino acids pack against each other in the protein's structure. Thus, a mutation to a larger amino acid at one position requires a mutation to a smaller amino acid at the second position for proper packing to occur. 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 in the structure exhibit 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, sequence differences at the second position are responsible for the structural diversity observed between PCV2a and PCV2b / d for loops CD and GH (Figure 2C) (9, 18). EC results may indicate that coupled residues are functionally related. Solvent-accessible surface area calculations on the GetArea server (curie.utmb.edu) indicate 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 help define neutralizing epitopes on the surface of the capsid.

[0082] Sequence changes on the surface of the capsid are a response to neutralizing antibodies. Using the atomic coordinates of PCV2d, we identified amino acids on the surface of the VLP that have solvent-exposed side chains (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). These amino acids may be antigenic determinants because the side chains provide a surface for antibody interaction. Contiguous sequences that may represent linear epitopes are shown in Figures 4A-4H. We then identified the most variable amino acids in these regions (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., in which a panel of neutralizing monoclonal antibodies was tested for their ability to bind different strains of PCV2. This study indicates that amino acid positions 59, 63, 88, 89, 130, 133, 206, and 210 are involved in the discrimination of antibody binding (25, 26). A study by Franzo et al. describing the evolution of PCV2 before and after the introduction of vaccination identified changes at amino acids 59, 191, 206, 210, 228, and 232 (27). With the exception of amino acid 130, which is embedded in the subunit-subunit interface, these amino acids are located on the surface of the capsid and undergo mutations (Figures 4A–4H).

[0083] The inner region of the PCV2 VLP can be used to package materials for nanotechnology. A roughly central reconstructed slice was extracted from the cryo-EM image reconstruction and 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 an approximately spherical shape for the calculations. Thus, the outer volume is 3.3 × 10 3 nm3 The inner diameter, using the same spherical assumption, has a diameter of about 13 nm. The volume of the inner region is therefore estimated to be about 1.2 × 10 3 nm 3 Scanning of a central slice of the 3D molecular volume showed that the inner region is occupied at a lower density than the capsid shell, which likely represents the cumulative disordered matrix N-terminus of the 60 CP units that make up the entire VLP.

[0084] Consideration The porcine circovirus 2 genome encodes four known proteins: a replicase (ORF1) involved in genome replication, a capsid protein (ORF2) involved in generating the capsid shell, and ORF3 and ORF4 that are thought to play a role in regulating cell apoptosis (28, 29). Phylogenetic analysis of PCV2 CP sequences indicates that four genotypes are globally distributed here (PCV2a-d) (14). PCV2b is currently thought to be the predominant genotype. However, a recent increase in the number of PCV2d CP deposits in GenBank suggests that there may be a genotypic shift from PCV2b to PCV2d (5). The increase in the number of PCV2d accessions 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 present on the market, this application establishes a mammalian expression system to produce large quantities of PCV2d virus-like particles (VLPs). This application provides the first system in which mammalian cells have been utilized to produce large quantities of VLPs. Mammalian expression systems are particularly advantageous for E. coli or baculovirus expression, as they are more similar to cells naturally infected with PCV2, allowing the study of the details of the PCV2 capsid in the context of the viral replication cycle. For example, the N-terminus of the CP harbors multiple nuclear localization signals, predicting that viral assembly occurs in the nucleus of infected cells where the ssDNA genome is replicated (30). However, it was previously unknown whether the PCV2 capsid can leave the intact nucleus and be released from infected cells.

[0085] As a result, in one or more embodiments, the expression system can help address whether assembled capsids can exit the nucleus. In one or more embodiments, the predominant fraction of VLPs in the expression system is located in the nucleus of cells. This is unexpected, since bioinformatics searches do not identify nuclear export signals (NES) in the CP sequence (data not shown). Thus, PCV2 capsids may carry a cryptic NES that may play an important role in the viral life cycle. However, nucleocytoplasmic transport can be reduced by redirecting the translation of capsid protein to the secretory pathway by introducing a secretion signal sequence at the NH2-terminus of the capsid protein. This genetic modification results in increased capsid protein synthesis, VLP assembly, and release of particles into the culture medium.

[0086] Cryo-EM image reconstructions and their structural analysis show that mammalian, baculovirus and E. coli expressed VLPs are nearly indistinguishable. Comparison of the atomic coordinates of PCV2a, b and d identifies conformational differences in the surface-exposed loops consisting of amino acids 86–91 (loop CD), 131–136 (loop EF) and 188–194 (loop GH) (Figures 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 (Figure 3B), providing a structural explanation for the antigenic discontinuity observed in PCV2a–PCV2b in 2003 (15, 16). Symmetry extension followed by focused classification of capsid subunits does not identify classes that may indicate post-translational modifications of the capsid (data not shown). However, mass spectrometry may need to be used to further address possible post-translational modifications on 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 highlights the ability of PCV2 to evade antibody neutralization, as it shows the remarkable flexibility of the capsid structure to undergo mutations while maintaining infectious virus. Atomic coordinates obtained from cryo-EM image reconstructions identify sites on the capsid surface that may be potential targets for antibody neutralization. In this application, continuous regions on the surface of the capsid are identified that may serve as epitopes that could potentially elicit neutralizing antibodies against several genotypes. Peptides that show sequence changes may represent escape mutants from antibody neutralization. Thus, the described expression protocol can be used to generate capsids constructed as multivalent mosaics that simultaneously present neutralizing epitopes of several genotypes. Positions that show no variability may represent regions on the viral capsid that may be ideal for universal vaccine design.

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

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

[0089] In one or more embodiments, the VLP expression system of the present application is further adapted for translation. The N-terminal 40 amino acids of the CP are rich in arginine, highly positively charged, and are predicted to interact with the ssDNA genome during viral morphogenesis. This motif is located in the interior space of the VLP and appears to be available for binding specific tags (or simply absorbing negatively charged small molecules or oligonucleotides), allowing the particle to be utilized as a nanoparticle for carrying therapeutic or immunomodulatory moieties targeted to specific tissues. Alternatively, the N-terminus of PCV2 can be modified 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. Additionally, replacement of surface-exposed amino acids with thiol-containing residues or unnatural amino acids may allow for the conjugation of small, medium, or large molecules useful for heterologous vaccination, drug delivery, therapeutic treatment, diagnosis, etc.

[0090] According to one or more embodiments, the expression system of the present application can produce large quantities of PVC2d VLPs in mammalian cells (human embryonic kidney: HEK293). This system allows for rapid study of the PCV2 life cycle in a background that closely mimics its natural host. As described in more detail below, cryo-EM image reconstruction of the VLPs 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 shows that all amino acid positions have undergone mutations, except for two amino acids. However, two groups of amino acids that form distinct patches on the surface of the capsid show limited sequence changes. A vaccine that can target antibodies to these patches can serve as a universal vaccine for PCV2.

[0091] 6.2. Example 2 Vaccination-challenge study to evaluate the efficacy of porcine circovirus 2 vaccines. Experimental Design. At approximately 1 week of age, blood samples from 120 male and female piglets were obtained at the source farm. 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 to PCV-2 using a commercially available enzyme-linked immunosorbent assay (ELISA). 72 2-week-old male and female piglets that tested negative for PCV-2 by qRT-PCR (all of the piglets tested were from negative litters) and had the lowest levels of PCV-2 antibodies were then selected and transported to the experimental farm at the study site. Upon arrival at the facility, the animals were weighed and the next day randomly distributed into 6 treatment groups of 12 pigs each based on PCV-2 S / P titers (at screening), weight, sex, and parity of the sows. The pigs were then housed in 4 rooms for 10 days of acclimation.

[0092] At 21 + / - 3 days of age (study day 0), pigs were immunized by the intramuscular (IM) route on the right side of the neck. Piglets from treatment group T01 received 1 mL of the vaccine PIGONE (Porcine Circovirus Type 2 (PCV2), VQ2610 strain, equivalent to 1 × 10 virus preactivation). 9 ~5×10 9 Piglets from treatment group T02 were immunized with 2 mL of the vaccine PIGONE (Porcine circovirus type 2 (PCV2), strain VQ2610, equivalent to 1 × 10 copies of virus preactivation). 9 ~5×10 9Animals from treatment group T03 were immunized with 2 mL of vaccine Huve-PCV2 (6 with Huve-PCV2_A and 6 with Huve-PCV2_B). Huve-PCV2_A contains PCV2d ORf2 protein expressed by eukaryotic cells, and Huve-PCV2_B contains PCV2d VLP expressed by mammalian cells. Animals from treatment group T04 were immunized with 1 mL of vaccine HVP-DNA (HVP-DNA contains PCV2b ORF2 DNA vaccine), and animals from treatment T05 were immunized with 1 mL of commercial vaccine Ingelvac CIRCOFLEX (inactivated baculo-expressed PCV2 ORf2) (Boehringer Ingelheim). Piglets from treatment group T06 were administered phosphate-buffered saline (PBS) and served as a negative control group. On study day 21 (SD21), at approximately 6 weeks of age, pigs were immunized by the intramuscular (IM) route on the left side 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 with Huve-PCV2_A and 6 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. Pigs were clinically examined before each vaccination.

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

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

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

[0096] Distribution of treatments. Animals were distributed into six groups of 12 animals (T01, T02, T03, T04, T05 or T06) according to PCV-2 antibody titers at screening, weight on the day of arrival, sex and parity of the sows. The variables were ranked and then classified and used to allocate pigs to the six treatment groups in blocks of six. Animals from treatments T01, T02, T03, T04 and T05 were randomly mixed in three rooms (rooms 5, 6 and 7) with four animals from each group placed per 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), randomly at SD0, selecting two animals from T03 in each room for each subgroup.

[0098] The room entry order 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 housing the PBS group (T06) was the first room entered during the immunization phase and therefore the first room in which mock vaccination and challenge took place. After challenge, access was given to the room containing the vaccinated animals before entering the room containing the control PBS group (T06).

[0100] [Table 2]

[0101] Preliminary results. Health Event: Animal #472 (PIGONE 2mL) from group T01 was found dead at SD32. A necropsy was performed and fibrous pericarditis was reported. The cause of death was likely bacterial sepsis and was unrelated to the study item.

[0102] Clinical Observations of Individual Animals. All pigs were evaluated for depression, body condition and respiratory distress on the vaccination days (SD0 and SD21) prior to vaccination. In addition, animals were also scored for clinical signs weekly post-challenge, pre-challenge on study days 35, 41, 49, 56 and 63. No clinical signs were recorded for study animals during the study phase.

[0103] Body weight. Body weight was recorded from all pigs upon arrival at the experimental facility for treatment assignment, at first vaccination (SD0), at challenge (SD35), and at necropsy (SD63). Average daily weight gain (ADWG) was calculated for the first vaccination-challenge, challenge-necropsy, and first vaccination-necropsy periods. Body weight changes are observed in Figure 5 and ADWG in Figure 6.

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

[0105] Serology. Presence of antibodies against PCV-2 in blood was tested with a commercial ELISA IngezimCirco IgG kit on study days SD0, SD7±1, SD13, SD20, SD29 before vaccination, and SD35, SD41, SD49, SD56 and SD63 before challenge. Results were expressed as S / P titers.

[0106] Antibody kinetics are shown in Figure 7. Seven treatment groups are represented, with treatment T03 (Huve-PCV2) divided into two subgroups: Huve-PCV2_A (T03_A) and Huve-PCV2_B (T03_B).

[0107] At SD0 (first vaccination), S / P values ​​for all study animals ranged from 0.2 to 1, with an overall mean value of 0.71.

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

[0109] On the day of challenge, CIRCOFLEX (inactivated baculovirus-expressed PCV2 ORf2) (T05), PBS (T06), Huve-PCV2_A (T03_A), and HVP-DNA (T04) groups showed low S / P values ​​ranging from 0.38 to 0.4. After challenge, the S / P values ​​decreased overall, and mild reactions were observed in some vaccinated groups at SD56 or SD63.

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

[0111] Viremia. The commercially available qRT-PCR kit LSI VetMAX Porcine Circovirus Type 2-Quantification (Life Technologies, ref. code QPCV) was used to detect and quantitate PCV-2 DNA in serum samples obtained on study days SD0 before vaccination, SD35, SD41, SD49, SD56 and SD63 before challenge. Results were expressed as PCV-2 copy numbers log 10 / mL, negative (<3 log 10 copies / mL), non-quantitative positive (3log 10 copies / mL ~ 4log10 copies / mL) or positive (≥ 4log 10 The results were classified as (copies / mL).

[0112] Viral load >3log 10 Results of viremia considering copies / mL.

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

[0114] Overall, a low percentage of positive animals (>3 log 10 copies / mL) are observed during the challenge phase. Treatments T01 and T02 vaccinated with PIGONE (1 mL or 2 mL) show a numerically higher percentage of positive animals at SD49 and SD56, but in both cases reach 50% positivity (quantified + non-quantified). CIRCOFLEX (inactivated baculo-expressed PCV2 ORf2) (T04) had no positive (quantified or non-quantified) animals during the challenge phase.

[0115] HUVE-PCV2_B (T03_B), CIRCOFLEX (inactivated baculo-expressed PCV2 ORF2) (T05) and PBS (T06) produced quantifiable positive animals (>4 log 10 The remaining groups had 1–3 positive animals on at least one sampling day during the challenge phase, with most groups having positive animals on days with an SD of 56. The mean number of quantified positive animals was 4.12 log 10 ~5.34log 10 The PCV-2 copies / mL ranged from 10 to 15.

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

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

[0118] Considering all animals with samples below the detection limit, a higher percentage of positives is observed in all study groups. The PIGONE group (T01 or T02) continues to be the group with a numerically higher percentage of positives in SD49 (PIGONE 1 mL, T01), SD56 and SD63 (PIGONE 2 mL, T02), followed by HVPDNA (T04).

[0119] Considering all animals with qRT-PCR values, the mean viral load ranged from 2.19 to 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 a mean >2 log 10 Copies / mL are shown. The Huve-PCV2_A group (T03_A) and the PBS control group (T06) showed positive values ​​for 3 days from SD49 to SD63, whereas CIRCOFLEX (inactivated baculo-expressed PCV2 ORf2) (T05) showed viraemic animals for 2 days, 1 week after challenge (SD41) and at necropsy (SD63).

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

[0122] Serology. S / P values ​​at SD0 were higher than in previous studies due to maternal antibodies. This does not allow to confirm clear seroconversion after vaccination. PIGONE 1mL and 2mL (T01, T02) as well as Huve-PCV2_B (T03_B) show clear seroconversion after the second vaccination compared to PBS (T06). CIRCOFLEX (inactivated baculo-expressed PCV2 ORf2) (T05) and HVP-DNA vaccine (T04) do not show antibody response. They have similar antibody kinetics to those from the PBS group.

[0123] The lack of seroconversion in the CIRCOFLEX (inactivated baculo-expressed PCV2 ORf2) group (T05) was expected considering the serological value at the time of vaccination. CIRCOFLEX (inactivated baculo-expressed PCV2 ORf2) protection is based on cell-mediated immunity. Usually, in field conditions, no serological response is observed in animals with maternally derived antibodies when vaccinated with CIRCOFLEX (inactivated baculo-expressed PCV2 ORf2).

[0124] The Huve-PCV2 subgroup cannot be compared with the other groups because n is half that of the other groups (6 animals / subgroup). However, when compared between them, the results are different due to the higher serological response of T03_B.

[0125] Viremia. On SD35 before challenge, one animal from the PIGONE 2mL (T02) group had viremia below the limit of detection (viral load: 2.76 log 10The highest viral load was 100 copies / mL. This animal was negative for qRT-PCR at animal selection, SD0, SD7, SD14, and SD21, and after challenge at SD41 and SD49. An attempt to sequence this DNA at SD35 was not possible due to the low viral load. Two possible explanations for this are: 1) subclinical infection with a field PCV-2 strain during the immunization phase when viral loads are very low, and 2) PCV-2 DNA contamination is being detected, but not whole virus. Cause unknown.

[0126] In general, PCV-2 vaccination induced mild subclinical infections due to the low viral loads observed during challenge, which were “below the limit of detection” (2–3 log 10 It was decided that all qRTPCR results should be considered, including the number of copies / mL. PIGONE 1mL (T01), PIGONE 2mL (T02) and HVP-DNA (T04) groups show a higher percentage of positive animals. Huve-PCV2_B (T03_B), CIRCOFLEX (inactivated baculo-expressed PCV2 ORf2) (T05) and PBS (T06) groups show an absent / very low percentage of viremic animals throughout the challenge phase. Mean values ​​are <3log 10 Comparing the Huve-PCV2 (T03) subgroups among them, subgroup T03_B had a numerically lower percentage of viraemic animals with an average viral load ranging from SD49 to SD63.

[0127] Challenge day information. The inoculum was prepared as follows: the inoculum was thawed, mixed, and then split into 5 vials of approximately 50 mL / vial. The titer on the day of challenge was 104.95 TCID50. Vials were shared between rooms, i.e., one vial was started and a new vial was not used until it was finished, allowing two rooms to use one identical vial. 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). Possible room effect: The PBS (T06) room had a lower animal density than the other three rooms in the vaccinated groups; 12 or 20 animals / room, respectively. The four rooms were comparable in spatial and housing 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) that contain unique structural and antigenic properties, where one or more of the constituent capsid monomers incorporate sequences that confer monovalent or polyvalent vaccine attributes and or the ability to encapsulate or mount molecular entities with distinct biological activities. In at least one embodiment, the recombinant capsid or VLP comprises a secretory signal sequence genetically linked to the capsid coding sequence that allows release and / or secretion of the particle from the producing cell into the culture medium.

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

[0131] In one or more embodiments of the recombinant capsids of the VLPs, construction and / or production is performed using two or more distinct capsid protein monomers, resulting in VLPs of mixed composition (mosaic or chimeric) with unique antigenic and biological properties. In one or more embodiments, the recombinant capsids or VLPs comprise modified surfaces that allow for conjugation of different molecular entities to the surface of the VLP.

[0132] In one or more embodiments, the recombinant capsid or VLP contains mutated, deleted, and / or modified NH2-terminal or internal amino acids of the capsid monomer that allow for the incorporation of small molecules, nucleic acids or other molecular moieties during assembly in cells that produce the VLP, or in vitro by shedding and reassembly of the VLP.

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

[0134] In at least one embodiment, the present application also discloses a DNA construct comprising a sequence encoding the capsid protein in all its sequences and forms used to construct the VLP. A DNA construct comprising a sequence encoding various forms of capsid monomers. In one or more embodiments, the present application further discloses a method of producing a VLP, the method comprising 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 either integrated into the cell genome or equally divided and separated into daughter cells, resulting in the continuous production of VLPs.

[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 is provided comprising at least one VLP according to any of the above embodiments. 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 an effective amount of the immunogenic composition to the animal. 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 molecules having pharmaceutical or biological activity. In at least one embodiment, the recombinant capsids or VLPs comprise pharmaceutical compositions for VLP drug delivery, heterologous immunization, and / or immunomodulation.

[0138] In one or more embodiments of the method for generating an immune response against one or more porcine circoviruses in an animal, the immunogenic composition is administered to the subject as a therapeutic or prophylactic treatment. The method of the present invention provides for the effective use of the composition of the present application in the 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 wild-type PCV2 capsid. SEQ ID NO:3. Nucleotide sequence of modified PCV2 capsid with secretory signal sequence. SEQ ID NO: 4. Amino acid sequence of modified PCV2 capsid with secretory signal sequence. SEQ ID NO:5. Nucleotide sequence of Pcdna3.4-PCV2. (Figure 1A).

[0140] SEQ ID NO: 1. Porcine circovirus-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] SEQ ID NO:2. Porcine circovirus-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-2 Strain PCV2 Capsid Length: 249 Type: Amino Acid MEKIVLLFAIVSLVKSTYPRR RFRRRRHRPRSHLGQILRRRP WLVHPRHRYRWRRKNGIFNTR LSRTIGYTVKKTTVRTPSWNV DMMRFNINDFLPPGGGSNPLT VPFEYYRIRKVKVEFWPCSPI TQGDRGVGSTAVILDDNFVTK ANALTYDPYVNYSSRHTITQP FSYHSRYFTPKPVLDRTIDYF QPNNKRNQLWLRLQTTGNVDH VGLGTAFENSIYDQDYNIRIT MYVQFREFNLKDPPLNPK

[0144] SEQ ID NO:5. Pcdna3.4-PCV2. (Figure 1A). Length:6778 Type:DNA [Sequence Table 1-1] TIFF0007649250000003.tif197169[Sequence Table 1-2] TIFF0007649250000004.tif245167[Sequence Table 1-3] TIFF0007649250000005.tif139169

[0145] The present invention should not be limited in scope by the specific embodiments described herein. Indeed, various modifications of the present invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and 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 their disclosures, including all disclosed chemical structures, are incorporated herein by reference. Citation of the above publications or documents is not intended as an admission that any of the foregoing is relevant prior art, nor does it constitute any admission as to the contents 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 was specifically and individually indicated to be incorporated by reference.

[0147] The foregoing written specification is considered to be sufficient to enable one skilled 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 skilled 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 sections: Item 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; The mammalian cell is transfected with the plasmid, A mammalian expression system, wherein the expression system produces recombinant PCV2 VLP. 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 contains a recognition site for NheI, a Kozak sequence, and a recognition site for NotI; 3. The expression system according to item 1 or 2, 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 integrated after the stop codon of the PCV2 gene. Item 4: The expression system according to any one of items 1 to 3, wherein the majority of the produced recombinant PCV2 VLPs are present in the nucleus of a mammalian cell. Item 5: The expression system according to any one of Items 1 to 4, wherein the capsid protein comprises 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 secretory signal sequence introduced into the NH2-terminus of the capsid protein. Item 8: The expression system described in Item 7, wherein the capsid protein comprises 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 VLP is selected from the group consisting of PCV2a VLP, PCV2b VLP, PCV2c VLP, PCV2d VLP, and PCV2e VLP. Item 11: The expression system according to any one of items 1 to 10, wherein the produced recombinant PCV2 VLP is a PVC2d VLP. 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 cell with a plasmid containing a PCV2 gene encoding a capsid protein; adding valproic acid (VPA) sodium salt to the transfected mammalian cells, wherein the addition of 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; A method wherein the majority of the PCV2 VLPs produced are present in the nucleus of the mammalian cell. Item 15: The step of centrifuging and washing the transfected mammalian cells comprises: Centrifugation of the mammalian cells at 2,000×g for 15 minutes; Washing the mammalian cells with PBS solution; and centrifuging the mammalian cells again at 2,000 x g for 15 minutes. 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 a freeze and thaw cycle. Item 17: The method according to any one of items 14 to 16, wherein a first of two successive centrifugation cycles is carried out at 2,000 × g for 15 minutes and a second of the two successive centrifugation cycles is carried out 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 VLPs 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; 20. The method according to any one of items 14 to 19, wherein the recognition site for NheI and the Kozak sequence are located upstream from the start codon of the PCV2 gene, and the recognition site for NotI is integrated after the stop codon of the PCV2 gene. 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 VLPs are selected from the group consisting of PCV2a VLPs, PCV2b VLPs, PCV2c VLPs, PCV2d VLPs, and PCV2e VLPs. Item 23: The method according to any one of items 14 to 22, wherein the produced PCV2 VLPs are PVC2d VLPs. Item 24: PCV2 VLPs 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; the capsid protein comprises the amino acid sequence of SEQ ID NO:4 and / or the capsid protein is encoded by the nucleotide sequence of SEQ ID NO:3, said mammalian cell being transfected with said plasmid; A mammalian expression system, wherein the expression system produces recombinant PCV2 VLPs, and the capsid protein is modified with a secretory signal sequence introduced at the NH2-terminus of the capsid protein.

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 of claim 1 , wherein the recombinant PCV2 VLP produced is a PCV2d VLP.

5. 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, said capsid protein comprising the amino acid sequence of SEQ ID NO:4 and / or encoded by the nucleotide sequence of SEQ ID NO:3, said capsid protein being modified with a secretion signal sequence introduced at the NH2-terminus of said 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 said mammalian cells to produce said PCV2 VLPs, in the order listed.

6. Centrifuging 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.

7. 7. The method of claim 5 or 6, wherein the centrifuged mammalian cells are frozen at -80°C and thawed at 37°C during the freeze and thaw cycle.

8. 8. The method according to any one of claims 5 to 7, 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.

9. The method of claim 5, further comprising purifying the PCV2 VLPs by centrifugation at 140,000 xg for 4 hours after the final centrifugation.

10. The method of any one of claims 5 to 9, wherein the mammalian cells are human embryonic kidney-293 (HEK-293) mammalian cells.

11. 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 5 to 10, 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.

12. The method of any one of claims 5 to 11, wherein the produced PCV2 VLPs are PCV2d VLPs.

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