Porcine circovirus type 2 (PCV2) immunogenic compositions and methods of stimulating an immune response to PCV2

Abstract

Bacterial vaccine vectors, cell cultures, immunogenic compositions, and methods for stimulating an immune response against Porcine Circovirus Type 2 (PCV2) infection in a host animal susceptible to infection by PCV2. Vaccine vectors comprising a recombinant bacterium comprising a nucleic acid encoding a conserved PCV2 capsid protein, which is configured to self-assemble, after expression, into a non -infectious PCV2 virus-like particle to stimulate an immune response against PCV2 infection in a host animal susceptible to infection by PCV.

Description

[0001] PORCINE CIRCOVIRUS TYPE 2 (PCV2) IMMUNOGENIC COMPOSITIONS AND METHODS OF STIMULATING AN IMMUNE RESPONSE TO PCV2

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] The present application claims the priority benefit of U.S. Provisional Patent Application Serial No. 63 / 609,549, filed December 13, 2023, entitled PORCINE CIRCOVIRUS TYPE 2 (PCV2) IMMUNOGENIC COMPOSITION, incorporated by reference in its entirety herein.

[0004] SEQUENCE LISTING

[0005] The following application contains a sequence listing submitted electronically as a Standard ST.26 compliant XML file entitled "PCV2_Seq List_59572.xml," created on December 4, 2024, as 20,480 bytes in size, the entire contents of which are incorporated by reference herein.

[0006] BACKGROUND OF THE INVENTION

[0007] Field of the Invention

[0008] The present invention relates to Porcine circovirus type 2 (PCV2) immunogenic compositions and uses thereof.

[0009] Description of Related Art

[0010] Porcine circovirus type 2 (PCV2) is the primary causative agent of several syndromes collectively known as porcine circovirus-associated disease (PCVAD). According to the American Association of Swine Veterinarians (AASV), PCVAD can be subclinical or include one or more clinical manifestations including multisystemic disease with weight loss and high mortality, respiratory disease, porcine dermatologic and nephropathy syndrome, enteric signs including diarrhea, and reproductive disorders on an individual or herd basis. Although PCV2 is required to cause the characteristic lymphoid depletion of PCVAD, many strains likely require a cofactor to cause the full spectrum of clinical signs associated with PCVAD. Coinfection with several other viral and / or bacterial pathogens has been shown to cause an increase in incidence and a markedly more severe clinical course of disease.

[0011] In or about 2006, the first USDA fully licensed PCV2 vaccine was licensed for use in pigs 4 weeks of age or older. This genetically engineered chimeric vaccine was created by inserting the immunogenic capsid protein of PCV2 into the genetic backbone of a nonpathogenic PCV1 isolate, cultured in vitro, inactivated, mixed with an oil adjuvant, and bottled for injection. Soon thereafter, recombinant vaccines utilizing the baculovirus-insect system (BICS), first described in 1983, that heterologously produce the Capsid protein of PCV2 were licensed by the USDA and became market dominant. Since the early 2010s, little has changed with PCV2 vaccines. Currently, all PCV2 vaccines are injected. Killed whole virus vaccines are available, but historically have been used in Europe and countries other than the USA, have a risk of ineffective inactivation during manufacturing, posing a risk to the herds that are injected. Genetically engineered and BICS-based vaccines, while safer due to the lack of replicative PCV2 nucleic acids and the inability of baculovirus to actively infect mammals, are injected, thus they are only capable of inducing systemic immunity in the host. As the primary route of PCV2 transmission is by oro-nasal contact with infected oro-nasal fluids and / or feces, induction of the mucosal immune system is needed to protect hosts more fully from this respiratory virus and intramuscularly injection does not activate this lineage of immunity. Vaccination at any one site of the mucosa, i.e., ocular, nasopharyngeal, respiratory, oral, gastrointestinal, and genitourinary mucosae, induces a system wide mucosal antigen specific secretory IgA, systemic antigen specific serum IgG, and antigen-specific T-cell responses, which are capable of transiting and patrolling all tissues to aid in the prevention and fighting of pathogens.

[0012] SUMMARY OF THE INVENTION

[0013] The present disclosure is broadly concerned with a recombinant virus-like particle (VLP) based PCV2 vaccine that is produced in Bacillus subtilis and configured specifically for mucosal administration to a susceptible host animal (susceptible to PVC2 infection). VLPs are referred to as being “virus-like” because the expression constructs assemble into particles having a similar morphology to the actual virion itself, in this case the PCV2 virion. In one or more embodiments, the present disclosure is broadly concerned with an orally delivered PCV2 vaccine for swine that is produced in B. subtilis fermentation conditions. Currently, all PCV2 vaccines are injected, which is labor-intensive and involves manual handling and injection of individual animals. An orally delivered vaccine has many benefits, with two primary benefits being induction of mucosal immunity, which is important for respiratory virus defenses such as PCV2, and ease of mass application via the barn’s drinking water system. Further, this invention utilizes virus-like particles. As established in the literature, use of VLPs induces non-linear, conformational epitope neutralizing antibodies, which are critical to preventing and / or controlling PCV2 infection. Further, a second linear epitope may be included in the vaccine formulation, which targets the amino terminal of the PCV2 Capsid monomer and may interfere with viral Capsid self-assembly during active PCV2 infection. Additionally, this invention uses a bacterium, B. subtilis, to produce the recombinant VLPs and linear epitope antigens, which is unique from other vaccines. Finally, the PCV2 Capsid sequence illustrated in the working examples pertains to PCV2d, one of the more recent PCV2 sub-type to evolve, making it genetically more similar to most of the current circulating strains.

[0014] BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The patent or application fde contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0016] Fig. 1A is a plasmid map for the VLP expression construct using the pHTIO vector used in the Examples.

[0017] Fig. IB is a plasmid map for the linear express construct using the pHTIO vector used in the Examples.

[0018] Fig. 2 A is a plasmid map for the VLP expression construct using the pBiotech-Opt plasmid.

[0019] Fig. 2B is a plasmid map for the linear express construct using the pBiotech-Opt plasmid.

[0020] Fig. 3 A is a graph of the mean OD450 ELISA values for orally vaccinated birds (Oral / Oral).

[0021] Fig. 3B is a graph of the mean OD450 ELISA values for intramuscularly vaccinated birds (IM / IM).

[0022] Fig. 4A is a graph of the mean OD450 ELISA for orally vaccinated birds (Oral / Oral).

[0023] Fig. 4B is a graph of the mean OD450 ELISA values for intramuscularly vaccinated birds (IM / IM).

[0024] Fig. 5 is a graph of fluorescent focus neutralization (FFN) titer results with Log2 calculation and analysis.

[0025] Fig. 6 shows graphs of weight gain of the animals in the pig study.

[0026] Fig. 7 shows a graph of Log2 serum neutralizing antibody titers against PCV2d analysed by FFN.

[0027] Fig. 8 shows graphs of quantitation of serum PCV2 viremia and positive / negative analysis of serum in a subset of pigs. DETAILED DESCRIPTION

[0028] The present invention is concerned with Porcine Circovirus Type 2 (PCV2) immunogenic compositions. The compositions can be used for vaccination / immunization in a subject to induce an immune response against PCV2 infection and / or reduce disease associated with PCV2 infection in swine. The compositions comprise immunogenic agents against PCV2, preferably recombinantly produced immunogenic agents, and in preferred embodiments, at least one immunogenic agent in the fomi of a recombinantly produced virus-like particle (VLP). As used herein, an “immunogenic agent” refers to an agent used to stimulate the immune system of a subject, so that one or more functions of the immune system are increased generally, and preferably, with some level of specificity directed towards the immunogenic agent. An immunogenic agent may comprise an antigen (e.g., a peptide, polypeptide, or protein), a nucleic acid encoding an antigen (e.g., an expression vector), and / or a recombinant cell producing or presenting an antigen or cellular component, which can stimulate a host immune system to make a secretory, humoral, and / or cellular immune response, preferably a secretory, humoral, and / or cellular immune response specific to that antigen. The antigens may be full-length proteins or portions thereof. It is well established that immune system recognition of many proteins is based on a relatively small number of amino acids, often referred to as the (linear) epitope, which may be only 8 to 10 amino acids in length. Additionally, the immune response may recognize one or more conformational epitopes, which are comprised of discontinuous amino acid sequences that are brought together by protein folding in its native state. VLPs are structures that morphologically resemble a virus but are devoid of the genetic material required for viral replication and active infection. Specifically, in the context of PCV2, academic experimental research has shown conclusively that conformational epitopes on intact whole virus or VLPs are important to produce an effective neutralizing immune response. In the case of the PCV2 VLPs described herein, the inventive VLPs consist of a viral capsid structure devoid of replicative viral nucleic acids.

[0029] Immunogenic agents according to the various embodiments of the invention are preferably recombinantly produced, where nucleic acids encoding for target antigenic sequence(s) are introduced into a suitable expression vector for expression in a suitable host cell system. The recombinant PCV2 VLPs described herein are typically formed by translation of sequences encoding one or more antigenic proteins. The produced antigenic capsid proteins selfassemble into VLPs. Unless otherwise indicated by the context, the present disclosure uses the term “produced” when referring to protein translation to further indicate that a functional protein has been made. Terms related to “expression” are generally used when referring to DNA or RNA synthesis, but may be inclusive of DNA or RNA synthesis as well as subsequent downstream production of the expression products, i.e., production of the protein.

[0030] Additional immunogenic agents against PCV2 can be included in the compositions, including individually produced proteins, polypeptides, antigens, or fragments thereof derived from the virus, that do not assemble into VLPs or other secondary structures and / or which may be presented on or in association with a recombinant host cell. Embodiments described herein, for example, include known or experimentally derived linear epitope products of PCV2 antigenic agents for co-presentation to the immune system with the VLPs. For example, exemplified herein are linear fusion proteins comprising one or more PCV2 antigenic epitopes, translationally attached at one or both ends of an immune potentiating sequence (e.g., adjuvant), such as a heat shock protein sequence or fragment. These can be secreted into the cell culture medium or remain retained within the host bacterial cell.

[0031] In one or more embodiments, the produced fusion proteins are retained in the host bacterial cells, which act as secondary vehicles for delivery of the antigenic agents to the subject through the gastrointestinal tract. Multiple copies of the same antigen or multiple antigens from different proteins may be included in the vaccine vector.

[0032] Expression Vectors

[0033] In one or more embodiments, expression constructs are designed and synthesized to comprise the sequences encoding the PCV2 polypeptides desired as the immunogenic agents in the composition. Polynucleotide sequences coding for molecules (structural and / or antigen polypeptides) that form and / or are incorporated into the VLPs or are otherwise expressed can be codon optimized for expression in the target host cell. In one or more embodiments, the expression constructs comprise coding sequences (nucleotide sequences) encoding for one or more PCV2 capsid proteins, preferably a synthetic open reading frame (ORF) that encodes a consensus PCV2 capsid protein. In one or more embodiments, the expression constructs comprise nucleotide sequences encoding for antigens derived from PCV2d subtype capsid proteins. In one or more embodiments, the expression constructs comprise nucleotide sequences encoding for monomeric PCV2 capsid proteins, which are configured to self-assemble, after translation, into VLPs. In one or more embodiments, the expression constructs comprise nucleotide sequences encoding for one or more linear epitopes derived from the PCV2 virus capsid protein. Once the constructs are synthesized, they can be cloned into any suitable vector, plasmid, or replicon for expression. The expression vector(s) typically contain(s) coding sequences and expression control elements which allow expression of the coding regions in a suitable host. The control elements generally include one or more of a promoter, enhancer, ribosomal binding site(s), exon, intron, splicing sites, translation initiation codon, translation stop codon(s), and transcription termination sequences, and / or an insertion site for introducing the insert into the vector. In one or more embodiments, the expression vector further comprises a secretion signal, preferably one optimized for the host cell system, to direct secretion of the expression products from the recombinant cell and into the cell culture media / supernatant. In one or more embodiments the expression vector further comprises a Gram-positive bacterial secretion signal.

[0034] In one or more embodiments, the expression vector further comprises a sequence encoding for an immune system adjuvant or immunostimulatory molecule, which can be an agonist or antagonist, meaning that the expression product triggers activation or inactivation (as the case may be) of a desired downstream signaling pathway in the immune system of the subject, and as such, can be used to enhance the immune response induced by the immunogenic composition. In one or more embodiments, the expression vector comprises a sequence encoding for a TLR4 agonist, heat shock protein, or other immunogenic carrier protein such as CRM 197 or members of the RTX superfamily of toxins and the like, which are co-expressed with the immunogenic sequences.

[0035] It will be apparent that a vector may contain one or more sequences as described herein. For example, a single vector may carry sequences encoding all the proteins found in the VLP. Alternatively, multiple vectors may be used (e.g., multiple constructs, each encoding a single polypeptide-encoding sequence or multiple constructs, each encoding one or more polypeptide - encoding sequences). In embodiments in which a single vector comprises multiple polypeptide- encoding sequences, the sequences may be operably linked to the same or different transcriptional control elements (e.g., promoters) within the same vector. In one or more embodiments, the expression vector may include a promoter that is host specific.

[0036] Recombinant Production

[0037] The expression vector(s) are then used to transform a suitable host cell for recombinant expression of the immunogenic agents by the host. Many suitable expression systems are commercially available. Depending on the expression system and the selected host, the immunogenic agents of the invention are produced by proliferating host cells that are transformed with the expression vectors described above under conditions in which the immunogenic agents of interest are expressed, whereby the particle-forming polypeptides are produced and VLPs can be formed. The selection of the appropriate growth conditions is within the skill of the art. Suitable host cells include, but are not limited to, bacterial hosts such as E. coli, Bacillus spp. , Lactobacillus spp., and Streptococcus spp.

[0038] Bacillus spp. is a preferred bacterial platform to produce vectored vaccines according to the invention. Bacillus spp. Gram-positive bacteria host and several species, including Bacillus subtilis, are considered by the Food and Drug Administration (FDA) as being a generally safe microorganism (GRAS) that is free of exotoxins and endotoxins, with a very comprehensive record of safe oral consumption. In addition, it has established itself as a robust cell host and an efficient technological platform for the expression of bioactive and high-yield protein production, with the potential for scaling in a bioreactor. Researchers have shown oral Bacillus vaccine vectors expressing recombinant foreign antigens to stimulate systemic, mucosal, humoral, and cell- mediated immune responses against heterologous antigens. Furthermore, Bacillus, as the vector, contributes probiotic properties to the immunogenic composition, which increase the health of the subject stimulating the innate immune response through the toll-like receptor pathways, fortify the gastrointestinal system by enhancing the production of tight junction repair proteins, and down regulate the inflammatory response in the subject typically caused by pathogenic infection.

[0039] Bacilli, particularly B. subtilis, make suitable bacterial vaccine vectors for expressing and producing heterologous polypeptides. In addition, endogenous bacterial genes may be mutated or attenuated to create bacterial vectors with low to no pathogenesis to the infected or immunized subject, while maintaining immunogenicity.

[0040] The ability of the Bacillus spp. to survive the gastrointestinal tract of the host and give rise to a mucosal immune response is documented. Oral vaccines using a Bacillus spp. vector produce a strong mucosal immune response and are generally easy to administer to both animals and humans. As demonstrated herein, B. subtilis effectively manufactures recombinant antigens that generate a strong immune response in host subjects. A Bacillus strain that could be used for effective mucosal, e.g., oral, vaccination would provide a vector that could be used to readily and repeatedly vaccinate a subject against PCV2 infection.

[0041] In one or more embodiments, recombinant expression vectors are transformed into B. subtilis by electroporation or natural transformation protocols found within the skill of the art creating the recombinant B. subtilis stains as bacterial vaccine vectors. Recombinant strains transformed with either the VLP or linear expression constructs are separately, and individually cultured, and recombinant protein production is activated or induced with an appropriate trigger (e.g., chemical, temperature, or light) depending on the inducible promoter used in the expression vector. For example, when the expression vector is under the control of the lac operon, recombinant protein production can be activated by adding isopropyl P-D-l- thiogalactopyranoside (IPTG) to the culture. Likewise, a variety of inducible promoters can be activated by cumic acid.

[0042] The recombinant bacterial strains are cultured under appropriate conditions to allow for heterologous expression of the immunogenic antigens. In the case of the VLP vector, the translated monomers self-assemble are secreted into the culture supernatant and self-assemble into VLPs in the culture media. In the case of the linear vector, the linear antigenic epitope may or may not be secreted into the culture media and is dependent on the presence of a suitable secretion signal coding region within the expression vector upstream of the antigen coding sequence. Cultures are inactivated with formaldehyde and individually, or in combination, mixed with a suitable delivery vehicle for administration.

[0043] Compositions

[0044] Immunogenic compositions are described herein which comprise recombinantly produced immunogenic agents, as described herein, dispersed in a suitable delivery vehicle or carrier, which can be used to elicit an immune response against PCV2 infection when administered to a subject. The immunogenic compositions comprise a therapeutically effective amount of PCV2 immunogenic agent dispersed or suspended in a pharmaceutically acceptable carrier. In one or more embodiments, the immunogenic compositions will comprise a mixture of two or more PCV2 immunogenic agents, preferably at least one immunogenic agent that is a VLP and at least one immunogenic agent that is a linear epitope.

[0045] A pharmaceutically acceptable carrier is any carrier suitable for in vivo administration. As used herein, the term “pharmaceutically acceptable” means not biologically or otherwise undesirable, in that it can be administered to a subject without excessive toxicity, irritation, or allergic response, and does not cause unacceptable biological effects or interact in a deleterious manner with any of the other components of the composition in which it is contained. A pharmaceutically-acceptable carrier would be selected to minimize any degradation of the immunogenic agent and to minimize any adverse side effects in the subject. Pharmaceutically- acceptable ingredients include those acceptable for veterinary use as well as human pharmaceutical use. For example, pharmaceutically-acceptable carriers suitable for mucosal administration include aqueous solutions such as, water, buffered solutions, commercially available proprietarily formulated carriers and adjuvants, naturally occurring mono, di-, and polysaccharides, carbohydrates, sugars, polymers, proteins, and the like, including any of the following including mixtures thereof: celluloses, derivatives thereof, and microcrystalline forms thereof such as methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose, and the like; other naturally derived polysaccharides and polymers such as sodium alginate, gelatin, chitosan, collagen, hyaluronic acid, dextran, pullulan, alginic acid, pectic acid, phytic acid and phytin; mono or oligosaccharides such as D-mannitol, sorbitol, glucose, lactose, fructose, inositol, sucrose, amylose, and the like; dextrins such as a-, P~ or y-cyclodextrin, dimethyl-P-cyclodextrin, dextrin; native starches and their derivatives such as hydroxyethyl or hydroxypropyl starch and carboxymethyl starch; as well as gums such as gum arabic, tragacanth gum and glucomannan. Suitable carriers can further include bacterial cell culture fluids, media, and nutrients from recombinant production, as well as proteins, such as serum proteins, casein, albumin, and the like.

[0046] Additional components of the compositions may suitably include excipients such as stabilizers, preservatives, diluents, emulsifiers, and lubricants. For oral delivery, flavoring agents or palatability enhancers can also be included in the compositions. Other ingredients may be included in the composition, such as adjuvants, other active agents, preservatives, buffering agents (e.g., histidine), salts, and other pharmaceutically-acceptable ingredients. In one or more embodiments, the pharmaceutically-acceptable carrier comprises a combination of one or more of the above-described vehicles, and preferably is configured for example, in the case of methyl cellulose to form a gelled or hydrogel matrix as a stabilizing vehicle for delivering the immunogenic agents and further for enhancing mucosal delivery (e.g., for protecting the immunogenic agents against premature gastrointestinal degradation).

[0047] In one or more embodiments, the immunogenic composition is substantially free of any separately added adjuvants. That is, the only adjuvant or immunostimulatory molecules present in the composition are those described above which have been recombinantly co-expressed with the PCV2 antigenic components. In one or more embodiments, the immunogenic composition comprises at least one separately added adjuvant. Any adjuvant suitable for oral delivery in animal health can be used, including, without limitation, mucoadhesive encapsulating adjuvants, polymer- or carbomer-based adjuvants, oily adjuvants, and the like. The term “adjuvant” refers to any additional substance besides the PCV2 antigenic agents themselves that are coadministered with the antigens to enhance or increase the immune response to the antigenic agents, as compared to administering the same antigenic agent dosage without such adjuvant. For example, adjuvants can be used to increase the duration and persistence of the immune response and / or decrease the effective dosage or number of dosages of the antigenic agent to induce the same immune response and / or decrease the antigenic load per dosage that is needed for therapeutic efficacy. In any of the embodiments, the compositions comprise a therapeutically effective amount of the immunogenic agents distributed in the carrier.

[0048] As used herein, a “therapeutically effective” amount refers to the amount that will elicit the biological or medical response of a tissue, system, or subject that is being sought by a researcher or clinician, such as to elicit some desired therapeutic or prophylactic effect as against the disease or condition. In the present case, the effect is elicited against PCV2 infection by priming or stimulating an immune response specific to PCV2. One of skill in the art recognizes that an amount may be considered therapeutically “effective” even if the disease, condition, or symptom is not totally eradicated or prevented, but it or its symptoms and / or effects are improved or alleviated partially in the subject.

[0049] In one or more embodiments, a therapeutically effective dose is a minimum dose of intact PCV2 VLPs that is at least equal to 1 x 103TCIDso of PCV2 VLPs per mL of vaccine as quantified via ELISA using a live PCV2 virus as a reference standard, preferably greater than or equal to 1 x 104TCIDso of PCV2 VLPs per mL of vaccine as quantified via ELISA using a live PCV2 virus as a reference standard, and more preferably, has an equivalency of greater than or equal to 1 x 105TCIDso of PCV2 VLPs per mL of vaccine as quantified via ELISA using a live PCV2 virus as a reference standard.

[0050] In one or more embodiments, a therapeutically effective dose is a minimum dose of linear epitope of at least 0.5 pg / mL of vaccine.

[0051] In one or more embodiments, each dose of vaccine is a 2-mL unit dosage form of the immunogenic composition. As described below, preferably the immunogenic composition is administered as two dosages (4 mL total) with an intervening interval of time between the first and second dosage, e.g., using a prime and boost regimen exemplified herein.

[0052] Treatment / Immunization

[0053] The immunogenic compositions are useful for immunization or vaccination against PCV2 infection and PCVAD. Immunization or vaccination refers to the process of inducing a high level of antibody and / or cellular immune response in a subject as compared to an unvaccinated control, that is directed against a pathogen or antigen to which the subject has been exposed. Thus, described herein are vaccination methods or methods of stimulating an immune response against PCV2 infection, so as to inhibit, reduce, or even prevent symptoms of PCV2 infection and / or PCVAD. Also described herein are methods of enhancing the immune response against PCV2 infection in a subject as compared to an unvaccinated control. Also described herein are methods for aiding in the prevention or reduction of one or more of clinical symptoms associated with PCV2 infection and / or PCVAD as compared to an unvaccinated control.

[0054] The methods generally comprise administering to the subject an immunogenic agent as described herein in a therapeutically effective amount to stimulate or enhance the immune response of the subject when subsequently exposed to PCV2. In one or more embodiments, the methods comprise administering the immunogenic compositions to a host animal susceptible to PCV2 in a therapeutically effective amount. Exemplary host animals include swine.

[0055] The immunogenic composition can be delivered intramuscularly, subcutaneously, intradermally, or intravenously using a needle and syringe, but is preferably delivered mucosally, such as orally via the drinking water or directly by oral gavage or via intranasal administration such as a nasal spray or drip. Vaccination at any one site of the mucosa, i.e., ocular, nasopharyngeal, respiratory, oral, gastrointestinal, and genitourinary mucosae, induces a system wide mucosal antigen specific secretory IgA, systemic antigen specific serum IgG, and antigenspecific T-cell responses, which are capable of transiting and patrolling all tissues to aid in the prevention and fighting of pathogens.

[0056] The immunogenic composition may be administered by one or more dosages, which dosages can be administered, by the same or different route, to achieve the desired prophylactic or therapeutic effect. The immunogenic composition can also be administered using a prime and boost regimen. In one or more embodiments, the composition is administered as two dosages with an interval of between from about 5 days to about 21 days between the first and second dosages (preferably from about 10 to about 14 days from the day of the first dosage and the second dosage) for a total of two dosages over the course of one to three weeks. The dosing interval can be extended if desired and the skilled person can optimize it for the therapeutic dose present in each unit dosage form. In some embodiments, the methods described herein are useful for eliciting an immune response against PCV2 infection.

[0057] Such an “immune response” includes, for example, the production or activation of antibodies, B cells and / or the various T cells, directed to PCV2. The immune response will be demonstrated by a lack of observable clinical symptoms, or reduction of clinical symptoms normally displayed by an infected animal, faster recovery times from infection, maintenance of body weight and / or weight gain and appetite, reduced duration or amount of viral shedding, and the like. Accordingly, vaccinated animals will display resistance to new infection (or observable signs of infection) or reduced severity of infection, as compared to unvaccinated animals. “Reducing” the incidence, severity, and / or duration of clinical symptoms and / or viral shedding, means reducing the number of infected animals in a group, reducing, or eliminating the number of animals exhibiting clinical signs of infection, or reducing the severity of any clinical signs that are present in the animals, in comparison to wild-type infection in unvaccinated animals.

[0058] In one or more embodiments, enhancing an immune response includes, but is not limited to, enhancing antibody responses. Advantageously, the immunogenic compositions of the invention specifically generate in the subject anti -PC V2 antibodies. In one or more embodiments, the IgA response is enhanced, more particularly, the mucosal secretory IgA response is enhanced after administration of the vaccine vector as compared to a control. The control may be the same subject prior to administration of the vector or a comparable subject that is unvaccinated. The IgA response may be increased as much as two-times, three-times, four- times, five-times or more as compared to the response of a control subject. In one or more embodiments, the serum IgY or IgG response is enhanced. In one or more embodiments, administration of the immunogenic agent results in production of serum neutralizing antibodies that recognize PCV2 VLPs and neutralize PCV2d subtype in a live-virus neutralization assay.

[0059] The enhanced immune response may also result in a reduction of the ability of PVC2 to grow or replicate and colonize the subject after administration of the immunogenic agents described herein. Such a reduction may be tested by challenging a subject administered the immunogenic agent with PCV2 and monitoring the symptoms of infection of the subject as compared to a control subject.

[0060] In some embodiments, the immunogenic composition can be provided in unit dosage form in a suitable container. The term “unit dosage form” refers to a physically discrete unit suitable as a unitary dosage for human or animal use. Each unit dosage form may contain a predetermined amount of the immunogenic agents (and / or other active agents) in the carrier calculated to produce the desired effect. In other embodiments, the immunogenic composition can be provided separate from the carrier (e.g., in its own vial, ampule, sachet, or other suitable container) for on-site mixing before administration to a subject.

[0061] A kit comprising the immunogenic composition is also disclosed herein. The kit further comprises instructions for administering the immunogenic composition to a subject. The recombinant immunogenic agents can be provided as part of a dosage unit, already dispersed in a pharmaceutically-acceptable carrier, or it can be provided separately from the carrier (e.g., along with culture media). The kit can further comprise instructions for preparing the immunogenic agents for administration to a subject.

[0062] As used herein, the term “vaccine” is sometimes used interchangeably to refer to an immunogenic composition capable of eliciting partial or complete immunogenic protection against a disease or condition in the subject to which it has been administered. Although vaccines are generally considered prophylactic, the vaccines or immunogenic composition may be used for therapeutic treatment of a disease or a condition. Compositions according to the embodiments disclosed herein are useful in treating viral infection from PCV2 in a subject (e.g., swine) and / or preventing or reducing clinical symptoms of infection. Such clinical symptoms include respiratory distress, fever (above 40 °C, 104 °F), anorexia, lethargy. Thus, embodiments described herein have therapeutic and / or prophylactic uses, and in particular can be used for prophylactic treatment of a viral infection from PCV2. In general, the compositions are administered prophylactically, that is, before the subject demonstrates detectable clinical signs of an infection, such that the subject develops an adaptive immune response to infection by the virus. As such, the methods are useful for preventing the development of observable clinical symptoms from viral infection, and / or reducing the incidence or severity of clinical symptoms, and / or effects of the infection, and / or reducing the duration of the infection / symptoms / effects, and / or reducing the amount and / or duration viral shedding / viremia, as compared with unvaccinated control animals. Thus, the composition may only partially prevent and / or lessen the extent of morbidity due to the viral infection (i.e., reduce the severity of the symptoms and / or effects of the infection, and / or reduce the duration of the infection / symptoms / effects), as compared with unvaccinated control animals. Yet, the composition is still considered to treat or “prevent” the target infection or disease, even though it is not 100% effective.

[0063] Additional advantages of the various embodiments of the invention will be apparent to those skilled in the art upon review of the disclosure herein and the working examples below. It will be appreciated that the various embodiments described herein are not necessarily mutually exclusive unless otherwise indicated herein. For example, a feature described or depicted in one embodiment may also be included in other embodiments, but is not necessarily included. Thus, the present invention encompasses a variety of combinations and / or integrations of the specific embodiments described herein. As used herein, the phrase "and / or," when used in a list of two or more items, means that any one of the listed items can be employed by itself or any combination of two or more of the listed items can be employed. For example, if a composition is described as containing or excluding components A, B, and / or C, the composition can contain or exclude A alone; B alone; C alone; A and B in combination; A and C in combination; B and C in combination; or A, B, and C in combination.

[0064] The present description also uses numerical ranges to quantify certain parameters relating to various embodiments of the invention. It should be understood that when numerical ranges are provided, such ranges are to be construed as providing literal support for claim limitations that only recite the lower value of the range as well as claim limitations that only recite the upper value of the range. For example, a disclosed numerical range of about 10 to about 100 provides literal support for a claim reciting "greater than about 10" (with no upper bounds) and a claim reciting "less than about 100" (with no lower bounds).

[0065] EXAMPLES

[0066] The following examples set forth methods in accordance with the invention. It is to be understood, however, that these examples are provided by way of illustration and nothing therein should be taken as a limitation upon the overall scope of the invention.

[0067] EXAMPLE 1

[0068] Recombinant Vectors

[0069] Two recombinant expression constructs (synthetic DNA sequences (SEQ ID NO:7 and 9)) were designed in silica by the inventors from wild type sequences, and then codon optimized and synthesized in vitro by Genscript, Inc. Wild-type PCV2 capsid protein (Genbank AFU91520.1) was used as the starting point, see SEQ ID NO: 10.

[0070] The first codon optimized construct PCV2-VLP (SEQ ID NO: 1) constitutes a synthetic open reading frame (ORF) that encodes a consensus PCV2 Capsid protein, namely the PCV2d Capsid Open Reading Frame (ORF) (SEQ ID NO: 3 (PCV2-VLP)), with a SecY Gram-positive secretion signal at the amino terminus (SEQ ID NO:6). Further, the TLR4-agonist, APPHALS (SEQ ID NO:8), was encoded in the flexible CD-loop of the PCV2 Capsid protein. The expression construct encodes for the full sequence as shown in SEQ ID NO: 5; however, the processed and secreted PCV2-VLP will have the SEQ ID NO:3, as the secretion signal sequence is cleaved from the expressed protein sequence during processing. Construct Design for Bacillus to make VLPs -

[0071] SEQ ID NO:5 Pel-PCV2 Cap (Protein sequence: PCV2-VLP monomeric protein (capsid with SecY secretion signal at N-terminus))

[0072] MKKVMLATALFLGLTPAGANAADLTYPRRRYRRRRHRPRSHLGQILRRRPWLVHPRHR

[0073] YRWRRKNGIFNTRLSRTFGYTVKATTVRTPSWAVDMMRFNINDFLPPGGGAPPHALSS

[0074] NPLTVPFEYYRIRKVKVEFWPCSPITQGDRGVGSTAVVLDDNFVTKANALTYDPYVNYS SRHTIPQPFSYHSRYFTPKPVLDRTIDYFQPNNKRNQLWLRLQTTANVDHVGLGTAFEN SKYDQDYNIRVTMYVQFREFNLKDPPLKPK* *

[0075] The second construct PCV2-lin (SEQ ID NO:2) 2 (PCV2-lin)) encodes linear epitopes derived from a conserved sequence of the PCV2 Capsid protein (SEQ ID NO: 4). The second component consists of two linear epitopes derived from the PCV2 virus Capsid protein synthetically coupled to an immunostimulating / immunopotentiating molecule, swine HSP70 (Hspa2) (SEQ ID NO: 11). The second fraction also contains the same TL4-agonist, APPHALS (SEQ ID NO;8), at the extreme amino terminus with the construct order as follows: APPHALS (agonist, SEQ ID NO:8)— Capsid / Epitopel— Capsid / Epitope2 — HSP70 / Immunomolecule (see SEQ ID NO:9, codon optimized SEQ ID NO:2 and SEQ ID NO:4):

[0076] SEQ ID NO:4 - PCV2-lin, including AAY (CTL linker) and KK (B cell epitope linker) for epitopes in bold

[0077] MAPPHALSEAAAKAAYRPWLVHPRHRYAAYGKKGVLDDNFVTKANALTYDPYVNYS SGKKGGGGSARGPAIGIDLGTTYSCVGVFQHGKVEIIANDQGNRTTPSYVAFTDTERLIG DAAKNQVAMNPTNTIFDAKRLIGRKFEDATVQSDMKHWPFRVVSEGGKPKVQVEYKG EIKTFFPEEIS SM VLTKMKEI AE A YLGGK VQ S A VITVP A YFND SQRQ ATKD AGTITGLNV LRIINEPTAAAIAYGLDKKGCAGGEKNVLIFDLGGGTFDVSILTIEDGIFEVKSTAGDTHL GGEDFDNRMVSHLAEEFKRKHKKDIGPNKRAVRRLRTACERAKRTLSSSTQASIEIDSL

[0078] YEGVDFYTSITRARFEELNADLFRGTLEPVEKALRDAKLDKGQIQEIVLVGGSTRIPKIQK LLQDFFNGKELNKSINPDEAVAYGAAVQAAILIGDKSENVQDLLLLDVTPLSLGIETAGG VMTPLIKRNTTIPTKQTQTFTT YSDNQ S S VL VQ VYEGERAMTKDNNLLGKFDLTGIPP AP RGVPQIEVTFDIDANGILNVTAADKSTGKENKITITNDKGRLSKDDIDRMVQEAERYKSE DEANRDRVAAKNAVESYTYNIKQTVEDEKLRGKISEQDKNKILDKCQEVINWLDRNQM

[0079] AEKDEYEHKQKELERVCNPIISKLYQGGPGGGGGTGASGGPTIEEVD

[0080] Each expression construct was subsequently cloned into the E. coli-B. subtilis shuttle vector, pHTIO by Genscript, Inc. at the unique BarnHl and Xba\ restriction sites in the pHTIO vector. These new expression vectors were termed, pPCV2-VLP and pPCV2-lin. Plasmid maps are shown in the Figures.

[0081] EXAMPLE 2

[0082] Vaccine Production

[0083] Each vector prepared as in Example 1 was individually electroporated into B. subtilis SLL11. Transformants were selected on tryptic soy agar with chloramphenicol (5 pg / ml, TSACm), creating the recombinant Bacillus subtilis stains, Bs-PCV2-VLP, and Bs-PCV2-lin, respectively, which were frozen in 12% glycerol stocks. The recombinant proteins are then individually produced during fermentation of pPCV2-VLP or pPCV2-lin at between 28 and 37 °C and induction with isopropyl 0-D-1 -thiogalactopyranoside (IPTG) at concentration ranging from 0.1 to 0.5 mM. In pPCV2-VLP, the secretion signal in the expression vector directs the monomeric PCV2 Capsid proteins to the secretion pathway apparatus of the host Gram-positive bacteria and during secretion from B. subtilis, the secretion signal is cleaved. Once in the culture supernatant the monomeric Capsid proteins self-assemble into a PCV2 virus-like particle (VLP). The culture(s) is inactivated with formalin. Additionally, the same ORF gene sequences were cloned into a proprietary plasmid, pBiotech, whereby, production of the proteins is under the control of a cumic acid inducible promoter system (Figures 2A and 2B). The same process of verification and selection on TSA containing tetracycline (5 pg / mL, TSATe) was used to select transformants, new transformed strains were labeled Bs-PCV2-VLP-BT and Bs-PCV2-lin-BT.

[0084] Three colonies from each transformation reaction were selected and inoculated into tryptic soy broth with chloramphenicol or tetracycline (5 pg / ml, TSBCmor TSBTC), cultured overnight at 37 °C with agitation. The following day, 1 mL of each culture was mixed with 150 pl sterile glycerol (12% v / vf) and frozen at -80 °C for storage and subsequent analyses.

[0085] The recombinant strains were evaluated for growth kinetics and protein production. Bs- PCV2-Cap was inoculated into 10 ml of TSBCmand cultured overnight at 37°C with agitation. The next day the culture was transferred to a 500 mL Erlenmeyer flask containing 90 ml of TSBCm. It was incubated at 37°C for 8 hours with orbital agitation at 150 rpm. Every hour, samples were taken to determine the optical density at % = 600 nm (OD600). Growth was compared to a control strain and data was recorded and graphed (not shown). At 3 hours of incubation IPTG was added to the culture a final concentration of 0.5 mM to induce recombinant protein production. Induction of recombinant protein production slightly slowed the log phase growth of the strain, but the final concentration of the cells is equal between the recombinant strain and control strain.

[0086] VLP production in the Bs-PCV2-VLP strain was assessed by Dot-Blot. Bs-PCV2-VLP was cultured similarly to above, but with the modification of a 20 mL culture in a 50 mL conical vial. Between 6- and 7-hours post-inoculation (3 to 4 hours post-addition of IPTG), the culture was subjected to centrifugation (4000 rpm, 20 min 4 °C). The pellet was separated from the culture supernatant by transferring the latter to a new 50 ml falcon tube. Subsequently separate serial dilutions of each culture supernatant were prepared by serially diluting the supernatant 1 :2 with sterile PBS buffer (see figure below). Then, 10 pl of each dilution was dotted onto 0.22 pm nitrocellulose membrane and allowed to airdry for approximately 30 minutes. All three colonies secreted PCV2 Capsid protein into the supernatant (data not shown). One colony had detection of the Capsid protein at a greater dilution, suggesting a greater amount of Capsid protein was produced in that culture. This colony will be used to make vaccine in further analyses of the antigen. EXAMPLE 3

[0087] Evaluation of Immunogenicity of Bs-PCV2-VLP Vaccine

[0088] To produce the test vaccines, the inactivated culture(s) from Bs-PCV2-VLP was mixed with a naturally occurring polysaccharide, methyl cellulose (encapsulation media), which acts as the vehicle and protectant for the antigen(s), and stored at 4 °C until use.

[0089] 1. Vaccination groups and routes of vaccine administration

[0090] Oral administration: Ten chickens were vaccinated (0.2 mL / bird / dose) on day 3 and day 17 of life. Serum samples and intestinal mucosa scrapings were collected from humanely euthanized birds on day 28 of life.

[0091] Intramuscular (IM) administration: Ten chickens were intramuscularly vaccinated (0.2 mL / bird / dose) on day 20 and day 35 of life. Serum samples and intestinal mucosa scrapings were collected from humanely euthanized birds on day 45 of life.

[0092] Five control birds were left unvaccinated and used as a pooled experimental negative control. Serum and intestinal mucosal samples were collected on the same day as 3.1.2.2.

[0093] 2. Analysis of anti-Bs-PCV2-Cap serum IgY and secretory IgA in broiler chickens

[0094] Serum IgY evaluation

[0095] An overnight culture of Bs-PCV2-Cap was diluted 1 : 10 into fresh TSB with 5 pg / mL of chloramphenicol and cultured at 37 °C and agitation (225 RPM). After three hours of incubation, IPTG was added to a final concentration of 0.5 mM, and the culture was incubated another five hours. Subsequently, formaldehyde (0.3% v / vf) was added to the culture to inactivate the culture and it was stored at 4 °C. The following day, 1 ml of the suspension was subjected to mechanical disruption (0.5 gram 0.1mm silica beads, 4 cycles at maximum speed (Benchmark Bug Buster™ of 40 seconds with intervals of 4 minutes on ice).

[0096] A portion of the disrupted sample was diluted 1 :80 in bicarbonate buffer and then 100 pl of this matrix was plated in each well of a 96-well NUNC Maxisorp microtiter plate. The plate was incubated at 4 °C overnight. The following day the wells were washed thrice with washing buffer (TBS-Tween-20 0.05%, TBS-T).

[0097] Following washing, each well was loaded with 200 pl of blocking solution (Fortis Lifesciences, Cat. # El 04) and incubated for 1 hour at room temperate.

[0098] Individual serum samples and the pooled negative control serum from each treatment group were initially diluted 1 :20 in sterile PBS and then serially diluted 2-fold in sterile PBS to 1 / 640, then 100 pl of each dilution was loaded into a well and incubated at room temperature for 1 hour. Subsequently, each well was washed thrice with washing buffer. Secondary antibody: 100 .1 of an anti-chicken IgY H+L-HRP antibody (Fortis Lifesciences catalog A30-107) diluted 1 :40,000 was loaded into each well and incubated at room temperature for 1 hour followed by washing thrice with wash buffer.

[0099] Detection of the ELISA reaction was detected by adding 100 pl of 1-Step TMB solution (Fortis Lifesciences, Cat. #E102) to each well. The reaction was allowed to proceed for 3 minutes at room temperature and then 100 pl of 0.16 N sulfuric acid was added to each well to stop the reaction. The stopped ELISA reaction was read in a Biotek microtiter plate reader at % = 450 nm. The OD450 readings from treated birds within each treatment group were averaged and graphed against the pooled untreated negative control serum.

[0100] Fig. 3A shows the graphical representation of the mean Serum IgY OD450 ELISA values for orally vaccinated birds (Oral / Oral). Fig. 3B shows the graphical representation of the mean Serum IgY OD450 ELISA values for intramuscularly vaccinated birds (IM / IM) vaccinated with two doses of the experimental Bs-PCV2-VLP vaccine.

[0101] As shown in the data, both routes of vaccination induce serum anti-Bs-PCV2-VLP IgYs, but the parenteral route induces a greater total ant-Bs-PCV2-VLP number of IgYs compared to the oral route of administration.

[0102] Secretory IgA (slgA) evaluation

[0103] An overnight culture of Bs-PCV2-Cap was diluted 1 : 10 into fresh TSB with 5 pg / mL of chloramphenicol and cultured at 37 °C and agitation (225 RPM). After three hours of incubation, IPTG was added to a final concentration of 0.5 mM, and the culture was incubated another five hours. Subsequently, formaldehyde (0.3% v / vf) was added to the culture to inactivate the culture and it was stored at 4 °C. The following day, 1 ml of the suspension was subjected to mechanical disruption (0.5 gram 0.1mm silica beads, 4 cycles at maximum speed (Benchmark Bug Buster™ of 40 seconds with intervals of 4 minutes on ice).

[0104] A portion of the disrupted sample was diluted 1 :80 in bicarbonate buffer and then 100 pl of this matrix was plated in each well of a 96-well NUNC Maxisorp microtiter plate. The plate was incubated at 4 °C overnight. The following day the wells were washed thrice with washing buffer (TBS-Tween-20 0.05%, TBS-T).

[0105] Following washing, each well was loaded with 200 pl of blocking solution (Fortis Lifesciences, Cat. # El 04) and incubated for 1 hour at room temperate.

[0106] Individual mucosa samples and the pooled negative control mucosa from each treatment group were initially diluted 1 :2 in sterile PBS and then serially diluted 2-fold in sterile PBS to 1 / 64, then 100 pl of each dilution was loaded into a well and incubated at room temperature for 1 hour. Subsequently, each well was washed thrice with washing buffer.

[0107] Secondary antibody: 100 JJ.1 of an anti-chicken IgA-HRP antibody (Fortis Lifesciences catalog A30-103A) diluted 1 :40,000 was loaded into each well and incubated at room temperature for 1 hour followed by washing thrice with wash buffer.

[0108] Detection of the ELISA reaction was detected by adding 100 pl of 1-Step TMB solution (Fortis Lifesciences, Cat. E102) to each well. The reaction was allowed to proceed for 3 minutes at room temperature and then 100 pl of 0.16 N sulfuric acid was added to each well to stop the reaction. The stopped ELISA reaction was read in a Biotek microtiter plate reader at = 450 nm. The OD450 readings from treated birds within each treatment group were averaged and graphed against the pooled untreated negative control serum (See results).

[0109] Fig. 4A shows the graphical representation of the mean Secretory IgA OD450 ELISA values for orally vaccinated birds (Oral / Oral) vaccinated with two doses of the experimental Bs- PCV2-VLP vaccine. Fig. 4B shows the graphical representation of the mean secretory IgA (slgA) OD450 ELISA values for intramuscularly vaccinated birds (IM / IM) vaccinated with two doses of the experimental Bs-PCV2-VLP vaccine.

[0110] As can be seen from the data, both parenteral and oral administration promoted similar slgA values having maximum differences up to dilution 1 / 8.

[0111] 3. Determination of the chicken serum anti-PCV2 of neutralizing antibodies in serum samples from animals vaccinated with PCV2 VLPs produced in chickens

[0112] Analysis of chicken serum was carried out by the Iowa State University Veterinary Diagnostic Lab. Samples that generate titers with a numerical value are considered positive for neutralizing antibody detection to the stated agent at the reported dilution of the tested sample. The (>) symbol would indicate the sample is positive for antibody detection at the highest sample dilution tested. The (<) symbol would indicate the sample antibody level is below the detection sensitivity of this assay at the beginning sample dilution, therefore negative at this dilution. PCV2d isolate was used in PCV2 FFN testing. Table: Sample identification and FFN titer results with Log2 calculation

[0113] Fig. 5 is graphical and statistical analysis of Log2 FFN converted titers.

[0114] As shown in the data, two doses of the experimental Bs-PCV2-VLP vaccine given orally induced anti-PCV2 serum neutralizing antibodies (SN) in all birds, while two doses of the vaccine administered IM only produced SN in 60% of the birds. Of the positive birds, the 2xOral mean SN titer (mean Log2 = 4.3) was 2.3 times greater than the SN titer positive birds (mean Log2 = 3) in the 2xIM group.

[0115] 4. Conclusion

[0116] Overall, the Bacillus subtilis produces recombinant PCV2 Capsid monomers which are secreted into the culture media and spontaneously assemble into a PCV2 VLP. Further, vaccination of broiler chickens indicates that serum IgY and serum neutralizing antibodies are produced that recognize the PCV2 VLP and neutralize PCV2d in a live-virus neutralization assay. Further, mucosal secretory IgA is produced by the vaccine, which is important for preventing infection of respiratory viruses, such as PCV2.

[0117] EXAMPLE 4

[0118] Evaluation of a two recombinant Porcine Circovirus 2d (PCV2d) Vaccines against PCV2 infection in commercial swine

[0119] A trial conducted in pigs to determine if two recombinant vaccines produced in Bacillus subtilis induce serum neutralizing antibodies and reduce serum viraemia. The tested vaccines were either: 1) a monovalent PCV2 VLP (PCV2-VLP) and / or 2) bivalent PCV2-VLP and PCV2 Cap linear epitope (PCV2-lin) vaccine. The experimental vaccines were evaluated in a randomized and blinded challenge study. The vaccines were given orally to the animals.

[0120] Materials and Methods

[0121] Table . Trial Design

[0122] PCV2d Challenge

[0123] The circovirus challenge was sourced from the University of Iowa Veterinary Diagnostic Laboratory and identified as PCV2d isolate USA / NC 24897 / 2016. Each pig was given 3 ml (1.5 ml per nostril) intranasally via a syringe / nasal cannula with a target challenge dose of 1 x IO3TCID50 / ml.

[0124] Animals

[0125] The pigs were a mixture of male and female animals. Animals were 18-21 days of age at delivery. Piglets were examined and only those found to be clinically healthy were enrolled in the study.

[0126] Piglets were treated with 0.5 ml Excede at weaning. No antibiotics were in the feed. Pig body weights were taken on Day 0 (weaning), Day 35 (day of challenge) and Day 49 (necropsy), but body weights were not used in any decision criteria. Pigs were analyzed for both PCV2d PCR and antibody (4-dilution IF A).

[0127] There were 45 pigs in the study. Animals were housed in a partial slatted wean to finish barn. Feed was provided ad libitum.

[0128] Randomization / Treatment Groups:

[0129] The pigs were distributed into 15 pens of 3 pigs per pen. The mixed gender pigs were ear- tagged, weighed, and randomized by weight. Pigs were randomized by litter to take into account any potential PCV2 litter effect. All three pigs per pen were from the same litter and represent each of the 3 treatments.

[0130] Pigs were observed once daily to determine health status and condition of facility pens from Day 0 to Day 49 on a GHO (general health obs) data collection form.

[0131] Moderate (Score 2) diarrhea occurred in many pigs the week post-weaning and were injected with enroll oxacin.

[0132] Results and Discussion

[0133] There was little to no evidence of clinical coughing or septicemia associated with the PCV2d challenge. On day 49, all remaining pigs were weighed, necropsied, and serum was collected.

[0134] Weight gain:

[0135] The mean day 0 weight of the experimental group was within 0.4 lbs of each other. Animals vaccinated with the PCV2-VLP and PCV2-VLP / lin vaccines had numerically greater total weight gain and numerically better ADG calculations throughout the trial period. The data is not statistically significant because the range and standard deviation of all groups, while consistent across groups, was wide. For example, the standard deviation among all groups varied between 8.75 to 14.1 lbs. (3.96 to 6.4 kg) and the coefficient of variation ranged between 11.2% to 14.6% in the groups. Therefore, the SD was greater than the differences in the mean between the groups. It’s important to note, a large data set is often needed to statistically identify small gains in weight gain.

[0136] As shown in Fig. 6, Vaccinated animals had better weight gain: (A) the weight of individual animals was measured on study days, 0, 35, and 49 and the data represents the mean and standard deviation (SD) of each experimental group. (B) is the average daily gain (ADG) of each experimental group was calculated by summing the weight of animals from individual groups and dividing it by the numbers of days being analyzed. In the event an animal died in the analyzed study period, the data of the animal was removed from this calculation. The promising data point for the vaccine treated animals is they maintained a numerically greater final weight and maintained that weight through a viremic period with a better ADG after inoculation with PCV2d virus (d35-d49), suggesting the vaccine may help alleviate growth performance depression often observed when pigs become clinically or sub-clinically infected with PCV2.

[0137] PCV2d serum neutralizing antibody titers:

[0138] Serum samples were collected from all animals within each study group at d 35 (day of challenge) and d 49 (necropsy). Samples were submitted to the ISU-VDL to determine the greatest dilution of each serum sample to neutralize a PCV2d isolate infection of PK15 cells in vitro (FFN assay). Log2 serum neutralizing antibody titers against PCV2d are shown in Fig. 7. Data represents the mean (line) and SD (T bars) of all pigs within each treatment group. The box represents the lowest and highest dilution within each group. Many of the animals in the saline control group appear to have detectable PCV2 neutralizing serum antibodies even though piglets were screened for PCV2 infection prior to enrollment. As PCV2 is pervasive within swine farms, detection is not completely surprising. Importantly, after challenging with PCV2d, the Mono- VLP and Bivalent- VLP / lin vaccinated animals had statistically significant increases in their neutralizing antibody titers (d49 vs d35) compared to the saline control group, which had decreased nAb titers at d 49 vs. d 35.

[0139] These data suggest the two vaccines used in an oral prime / boost vaccination strategy induce significant neutralizing antibodies titers, which are sustained throughout infection. Contrastingly, in the unvaccinated control group, the animals were unable to maintain high titer of nAbs, therefore; a decrease in nAb titer is detected, possibly due to significant viral replication and the nAbs being “mopped” up by the large amount of virus in the serum.

[0140] PCF2 quantitative PCR (qPCR) of serum samples:

[0141] Using Excel, half the pens were randomly chosen for PCV2 quantitation by qPCR from serum samples on d42, d45 and d49 of the trial. The data represents an equal number of pigs from each treatment group. The results are shown in Fig. 8A and B. As shown in Fig. 8, Quantitation of serum PCV2 viremia and positive / negative analysis of serum in a subset of pigs: A) Quantitation of PCV2 genome copies by qPCR and expressed as a Ct value. The qPCR was conducted at the ISU-VDL. B) Conversion of qPCR data from A to a positive or negative presentation. A Ct value > 37 is considered “negative” by the VDL and any Ct value <37 is considered “positive” for PCV2 genomic RNA. Data was analyzed using 3x2 Chi-squared test in Prism 10.1.0.

[0142] In the saline control sub-group, all the pigs became viremic by d 49 and had the lowest Ct (highest PCV2 genome copies) at d 49 indicating greater viral replication in those pigs. Whereas, in the two vaccinated groups -30% of the pigs analyzed in this subset remained PCV2 negative out to d 49. The Mono-VLP group has the highest Ct (lowest PCV2 genomic copies) values at d 42 and d 45, but both vaccinated groups had the same mean at d 49, suggesting the vaccine is partially capable of controlling PCV2 replication.

[0143] Both experimental vaccines were able to keep -30% of vaccinated and challenged pigs PCV2 viremia-free, and overall, those pigs had numerical higher Ct values, strongly suggesting the vaccine helps limit PCV2 viral replication compared to pigs that are unvaccinated. Combined with the nAb titer and weight data, both experimental vaccines show modest efficacy in preventing PCV2-associated weight loss and controlling viral replication.

[0144] Conclusions

[0145] The nasal inoculation of commercial swine pre-screened for PCV2 exposure did result in serum viremia over the course of the 14-day observation study. The general disease and infection, on average, was mild across all groups.

[0146] Experimentally vaccinated groups finished the trial at a numerical higher final weight, on average, and had better numerical ADG as measured across the entirety of the trial (d 0-49), prior to challenge (d 0-35) and during the observation period (d 35-49). Macro- and micro-scopic histopathological analysis did not identify severe disease or differences between the groups and the same was true for PCV2 IHC tissue analysis (data not shown).

[0147] Even though all piglets were screened for prior PCV2 exposure by PCR and total anti- PCV2 Ab ELISA, some control pigs appear to maintain high levels of nAb titers, which may have impacted vaccination response in treated pigs.

[0148] A possible explanation for the differences in nAb titers and serum viremia prevention could be antigen dose. Each pig received 2 mL of vaccines per dose (4 mL total), but the amount of VLP in each dose was not initially quantified. Thus, it’s possible the dose of VLPs need to be increased in the vaccine for full immune reaction. The dose used in this study was based on the CFU count of Bacillus, 5 x 107CFU / mL, but since the VLPs are secreted and in the supernatant, the quantity likely does not directly correlate with Bacillus CFU counts. A dose determination assay was conducted in Example 5 using live PCV2 as a reference standard to better understand dosing. Notably, however, the vaccines, even at the lower dosages, appear to promote weight gain and there is still evidence for serum neutralizing antibody induction and modest control of serum viremia.

[0149] EXAMPLE 5

[0150] PCV2 VLP dose determination ELISA

[0151] ELISA Method

[0152] A culture supernatant of live PCV2 sub-type d (PCV2d) virus was purchased from Iowa State University Veterinary Diagnostics Laboratory (ISU-VDL). The 50% tissue culture infectious dose (TCID50), defined as the value which represents the amount of virus dilution required to induce cytopathic effects in 50% of wells containing the inoculated cell culture after a defined period of time, was determined to be ~3 x 105TCIDso for the culture received.

[0153] Using the culture supernatant material, 10-fold dilutions of the material were diluted in bicarbonate buffer and 100 pl of each dilution were plated in duplicate in a 96-well Nunc Maxisorp ELISA plate. Further, the induced Bacillus PCV2 VLP culture was diluted 10-fold in bicarbonate buffer and 100 pl of each dilution was plated in duplicate in a 96-well Nunc Maxisorp ELISA plate as well. Three technical replicates of these dilutions were analyzed. The plate was incubated for ~16 hours at 4 °C on an ELISA plate rocker. The following day, the wells were emptied, and each well was washed with 200 pl of TBST (Tris-buffer saline, 0.05 Tween-20) three times, subsequently the addition of 100 pl of blocking buffer (TBST with 1% casein) was loaded into the wells and the plate was incubated for 1 hour at room temperature on an ELISA plate rocker. The wells were emptied, and each well was loaded with 100 pl of diluted mouse anti-PCV2 monoclonal antibody (1 :750 in TBST-0.1% casein, RTI, Inc Brookings, SD, Cat #PCV-2-A) and the plate was incubated for 1 hour at room temperature on an ELISA plate rocker. The wells were then emptied and washed three times with TBST and then loaded with diluted anti-mouse antibody conjugated to HRP (anti-mouse HRP, 0.5 pl / ml in TSBT-0.1% casein, Bethyl Laboratories Catalog # A90-116B) and the plate was incubated for 1 hour at room temperature on an ELISA plate rocker. The plate was emptied, and the wells were washed three times with TBST, then each well was loaded with 100 pl of room temperature SureBlue™ TMB 1-C Microwell peroxidase substrate (SeraCare, Cat#5120-0075) and incubated at 37 °C for 10 minutes. The HRP-substrate reaction was stopped by the addition of 100 pl of stop solution (0.16 M sulfuric acid) to each well. The optical density of the color reaction of each well was read at = 450 nm (OD450) in a TECAN Infinite Pro 200 plate reader.

[0154] Results

[0155] Using the above method a linear standard curve WAS created, and the linear equation was used to estimate the TCID50 of the Bacillus PCV2 VLP culture. The estimated minimum dose of intact PCV2 VLPs utilized was determined to be equivalent to 1 x 103TCID50 of VLP using the live PCV2 virus as the reference standard.

[0156] SEQUENCES

[0157] The sequences used in the foregoing work are below for reference, and included in the sequence listing text file.

[0158] SEQ ID NO:1 - DNA sequence 1 : PCV2-VLP (codon optimized): ATGAAGAAAGTTATGCTGGCGACCGCGCTGTTCCTGGGTCTGACCCCGGCGGGTGCG AACGCGGCGGACCTGACCTACCCGCGTCGTCGTTACCGTCGTCGTCGTCACCGTCCG CGTAGCCACCTGGGTCAGATCCTGCGTCGTCGTCCGTGGCTGGTGCACCCGCGTCAC CGTTATCGTTGGCGTCGTAAGAACGGCATTTTCAACACCCGTCTGAGCCGTACCTTT GGTTACACCGTGAAAGCGACCACCGTTCGTACCCCGAGCTGGGCGGTTGACATGAT GCGTTTCAACATCAACGATTTTCTGCCGCCGGGTGGCGGTGCGCCGCCGCACGCGCT GAGCAGCAACCCGCTGACCGTGCCGTTCGAGTACTATCGTATCCGTAAGGTGAAAGT TGAATTTTGGCCGTGCAGCCCGATTACCCAAGGTGACCGTGGCGTGGGTAGCACCGC GGTGGTTCTGGACGATAACTTTGTTACCAAGGCGAACGCGCTGACCTACGATCCGTA TGTGAACTACAGCAGCCGTCACACCATCCCGCAGCCGTTCAGCTATCACAGCCGTTA CTTTACCCCGAAGCCGGTTCTGGACCGTACCATTGATTATTTCCAACCGAACAACAA ACGTAACCAGCTGTGGCTGCGTCTGCAAACCACCGCGAACGTGGACCACGTTGGCCT GGGTACCGCGTTTGAGAACAGCAAGTATGACCAGGATTACAACATTCGTGTGACCA TGTACGTTCAATTCCGTGAATTTAACCTGAAAGATCCGCCGCTGAAGCCGAAATAAT AA

[0159] SEQ ID NO:2 - DNA sequence 2: PCV2-lin (codon optimized):

[0160] ATGGCTCCGCCGCACGCGCTGAGCGAGGCGGCGGCGAAAGCGGCGTATCGTCCGTG GCTGGTGCACCCGCGTCACCGTTATGCGGCGTATGGTAAGAAAGGCGTTCTGGACG ATAACTTCGTGACCAAAGCGAACGCGCTGACCTACGACCCGTATGTTAACTACAGCA GCGGTAAGAAAGGTGGCGGTGGCAGCGCGCGTGGTCCGGCGATTGGTATTGACCTG GGTACCACCTATAGCTGCGTGGGCGTTTTCCAACACGGCAAGGTGGAAATCATTGCG AACGATCAGGGTAACCGTACCACCCCGAGCTACGTTGCGTTTACCGACACCGAGCGT

[0161] CTGATCGGCGATGCGGCGAAAAACCAAGTGGCGATGAACCCGACCAACACCATCTT

[0162] CGATGCGAAGCGTCTGATTGGTCGTAAATTTGAAGACGCGACCGTTCAAAGCGATAT

[0163] GAAGCACTGGCCGTTCCGTGTGGTTAGCGAGGGTGGCAAGCCGAAAGTGCAGGTTG

[0164] AGTATAAGGGCGAAATCAAAACCTTCTTTCCGGAGGAAATTAGCAGCATGGTGCTG

[0165] ACCAAGATGAAAGAGATTGCGGAAGCGTATCTGGGTGGCAAGGTTCAGAGCGCGGT

[0166] TATCACCGTGCCGGCGTACTTTAACGACAGCCAGCGTCAAGCGACCAAAGATGCGG

[0167] GCACCATTACCGGTCTGAACGTGCTGCGTATCATTAACGAACCGACCGCGGCGGCG

[0168] ATTGCGTATGGCCTGGACAAGAAAGGTTGCGCGGGTGGCGAGAAGAACGTTCTGAT

[0169] TTTCGACCTGGGTGGCGGTACCTTTGATGTGAGCATCCTGACCATTGAGGACGGCAT

[0170] CTTCGAAGTTAAAAGCACCGCGGGTGATACCCACCTGGGCGGTGAAGACTTCGATA

[0171] ACCGTATGGTTAGCCACCTGGCGGAGGAATTTAAGCGTAAACACAAGAAAGACATC

[0172] GGTCCGAACAAGCGTGCGGTTCGTCGTCTGCGTACCGCGTGCGAGCGTGCGAAACG

[0173] TACCCTGAGCAGCAGCACCCAGGCGAGCATCGAGATTGACAGCCTGTATGAAGGCG

[0174] TGGATTTTTACACCAGCATCACCCGTGCGCGTTTCGAGGAACTGAACGCGGACCTGT

[0175] TCCGTGGTACCCTGGAGCCGGTTGAAAAGGCGCTGCGTGACGCGAAGCTGGATAAA

[0176] GGCCAGATCCAAGAAATTGTGCTGGTTGGCGGTAGCACCCGTATCCCGAAGATTCA

[0177] GAAACTGCTGCAAGACTTCTTTAACGGCAAGGAGCTGAACAAAAGCATTAACCCGG

[0178] ATGAAGCGGTTGCGTATGGTGCGGCGGTGCAAGCGGCGATCCTGATTGGTGACAAA

[0179] AGCGAAAACGTTCAGGACCTGCTGCTGCTGGATGTGACCCCGCTGAGCCTGGGTATT

[0180] GAGACCGCGGGCGGTGTTATGACCCCGCTGATCAAGCGTAACACCACCATTCCGAC

[0181] CAAACAGACCCAAACCTTCACCACCTATAGCGATAACCAGAGCAGCGTGCTGGTTC

[0182] AAGTGTACGAGGGTGAACGTGCGATGACCAAGGACAACAACCTGCTGGGCAAATTT

[0183] GATCTGACCGGTATCCCGCCGGCTCCGCGTGGTGTGCCGCAGATCGAAGTGACCTTT

[0184] GACATCGATGCGAACGGCATTCTGAACGTGACCGCGGCGGACAAGAGCACCGGCAA

[0185] GGAGAACAAAATCACCATTACCAACGACAAGGGCCGTCTGAGCAAAGACGATATTG

[0186] ATCGTATGGTTCAAGAGGCGGAACGTTACAAAAGCGAGGACGAAGCGAACCGTGAT

[0187] CGTGTTGCGGCGAAGAACGCGGTGGAGAGCTACACCTATAACATCAAACAGACCGT

[0188] GGAGGATGAAAAGCTGCGTGGTAAAATCAGCGAGCAGGACAAGAACAAAATTCTG

[0189] GATAAGTGCCAAGAAGTTATCAACTGGCTGGACCGTAACCAGATGGCGGAGAAAGA

[0190] TGAGTATGAACACAAGCAAAAAGAGCTGGAACGTGTGTGCAACCCGATCATTAGCA

[0191] AACTGTACCAGGGCGGTCCGGGCGGTGGCGGTGGCACCGGTGCGAGCGGTGGCCCG

[0192] ACCATCGAGGAAGTTGAC SEQ ID NO: 3 - Processed and secreled VCN2-\'LP monomer from Bacillus self-assembles into VI P:

[0193] ADLTYPRRRYRRRRHRPRSHLGQILRRRPWLVHPRHRYRWRRKNGIFNTRLSRTFGYTV

[0194] KATTVRTPSWAVDMMRFNINDFLPPGGGAPPHALSSNPLTVPFEYYRIRKVKVEFWPCS

[0195] PITQGDRGVGSTAVVLDDNFVTKANALTYDPYVNYSSRHTIPQPFSYHSRYFTPKPVLDR TIDYFQPNNKRNQLWLRLQTTANVDHVGLGTAFENSKYDQDYNIRVTMYVQFREFNLK DPPLKPK

[0196] SEQ ID NO: 4 - Protein sequence : PCV2-lin:

[0197] MAPPHALSEAAAKAAYRPWLVHPRHRYAAYGKKGVLDDNFVTKANALTYDPYVNYS

[0198] SGKKGGGGSARGPAIGIDLGTTYSCVGVFQHGKVEIIANDQGNRTTPSYVAFTDTERLIG

[0199] DAAKNQVAMNPTNTIFDAKRLIGRKFEDATVQSDMKHWPFRVVSEGGKPKVQVEYKG

[0200] EIKTFFPEEISSMVLTKMKEIAEAYLGGKVQSAVITVPAYFNDSQRQATKDAGTITGLNV LRIINEPTAAAIAYGLDKKGCAGGEKNVLIFDLGGGTFDVSILTIEDGIFEVKSTAGDTHL

[0201] GGEDFDNRMVSHLAEEFKRKHKKDIGPNKRAVRRLRTACERAKRTLSSSTQASIEIDSLY

[0202] EGVDFYTSITRARFEELNADLFRGTLEPVEKALRDAKLDKGQIQEIVLVGGSTRIPKIQKL

[0203] LQDFFNGKELNKSINPDEAVAYGAAVQAAILIGDKSENVQDLLLLDVTPLSLGIETAGGV

[0204] MTPLIKRNTTIPTKQTQTFTTYSDNQSSVLVQVYEGERAMTKDNNLLGKFDLTG1PPAPR GVPQIEVTFDIDANGILNVTAADKSTGKENKITITNDKGRLSKDDIDRMVQEAERYKSED EANRDRVAAKNAVESYTYNIKQTVEDEKLRGKISEQDKNKILDKCQEVINWLDRNQMA EKDEYEHKQKELERVCNPIISKLYQGGPGGGGGTGASGGPTIEEVD

[0205] SEQ ID NO:5 - Pel-PCV2 Cap (Protein sequence: PCV2-VLP monomeric protein (capsid with SecY secretion signal at N-terminus))

[0206] MKKVMLATALFLGLTPAGANAADLTYPRRRYRRRRHRPRSHLGQILRRRPWLVHPRHR

[0207] YRWRRKNGIFNTRLSRTFGYTVKATTVRTPSWAVDMMRFNINDFLPPGGGAPPHALSS

[0208] NPLTVPFEYYRIRKVKVEFWPCSPITQGDRGVGSTAVVLDDNFVTKANALTYDPYVNYS SRHTIPQPFSYHSRYFTPKPVLDRTIDYFQPNNKRNQLWLRLQTTANVDHVGLGTAFEN SKYDQDYNIRVTMYVQFREFNLKDPPLKPK* *

[0209] SEQ ID NO:6 - Pel secretion signal

[0210] MKKVMLATALFLGLTPAGANAADL SEQ ID NO: 7 - Pel-PCV2 Cap (in silica designed construct)

[0211] ATGAAAAAAGTGATGCTGGCAACTGCGCTGTTTTTAGGTTTGACGCCGGCAGGGGCC

[0212] AATGCCGCTGACCTGACGTATCCGCGGAGACGCTACAGAAGGCGCAGACACCGGCC

[0213] TCGTTCACATTTAGGACAAATCCTTCGACGGCGTCCATGGCTTGTTCATCCGCGTCAC

[0214] AGATACAGGTGGAGAAGAAAAAACGGGATTTTTAATACAAGATTAAGCCGCACATT

[0215] CGGCTATACGGTGAAGGCAACGACAGTAAGAACCCCAAGCTGGGCTGTGGATATGA

[0216] TGAGATTCAATATTAATGATTTTTTGCCGCCCGGAGGAGGTGCGCCTCCGCATGCTT

[0217] TGTCGTCCAATCCGCTTACCGTTCCGTTTGAATATTACCGGATACGAAAAGTCAAGG

[0218] TAGAGTTTTGGCCGTGCTCTCCAATCACACAAGGAGACCGCGGCGTCGGCAGTACG

[0219] GCCGTCGTGCTGGATGATAACTTTGTGACAAAAGCGAATGCGCTTACATATGACCCC

[0220] TATGTCAATTACAGCTCTCGCCACACAATTCCTCAGCCTTTTTCTTATCATTCACGTT

[0221] ATTTCACTCCAAAACCTGTTCTCGACCGGACCATTGATTACTTTCAGCCGAACAATA

[0222] AACGAAACCAGCTGTGGCTCCGTCTGCAAACAACAGCAAACGTTGATCATGTCGGT

[0223] CTTGGCACTGCTTTCGAAAACTCAAAATATGACCAGGATTATAATATCCGCGTAACG

[0224] ATGTATGTTCAATTTCGTGAATTTAACTTAAAAGATCCGCCTTTGAAGCCAAAGTAA TGA

[0225] SEQ ID NO:8 - TLR4-agonist

[0226] APPEALS

[0227] SEQ ID NO:9 (PCV2-lin) (in silica designed construct)

[0228] ATGGCACCGCCTCATGCGCTGTCAGAAGCCGCTGCAAAAGCTGCATACCGGCCATG

[0229] GCTTGTACACCCGAGACATCGCTATGCCGCCTACGGCAAAAAAGGGGTTCTGGATG

[0230] ACAACTTTGTCACGAAGGCCAATGCTCTGACTTATGACCCATATGTCAATTATTCGT

[0231] CAGGAAAAAAAGGCGGTGGAGGCTCGGCGAGGGGCCCCGCTATCGGAATCGATTTA

[0232] GGTACAACGTACTCGTGTGTTGGCGTGTTTCAGCATGGGAAAGTCGAAATCATCGCA

[0233] AATGATCAGGGCAATCGGACCACTCCAAGCTACGTGGCTTTTACCGATACAGAAAG

[0234] GCTGATCGGAGATGCCGCCAAAAATCAGGTTGCGATGAATCCGACTAACACAATAT

[0235] TTGACGCTAAGCGGTTAATCGGGCGTAAATTCGAGGACGCCACAGTTCAATCAGAC

[0236] ATGAAACATTGGCCGTTCCGCGTCGTTTCCGAAGGGGGCAAACCGAAAGTACAGGT

[0237] CGAGTACAAAGGAGAGATCAAAACTTTCTTTCCAGAAGAAATTTCTTCTATGGTACT TCATTACCGTTCCAGCCTACTTCAACGACTCCCAAAGACAGGCTACAAAGGACGCAG

[0238] GGACGATCACAGGCTTAAACGTGCTGAGAATCATTAATGAACCTACGGCGGCAGCA

[0239] ATTGCGTATGGCTTAGATAAAAAGGGGTGCGCTGGCGGAGAAAAAAACGTTCTTAT

[0240] TTTCGATCTAGGAGGCGGAACATTTGATGTGAGCATCCTGACAATTGAAGATGGCAT

[0241] TTTTGAAGTGAAATCTACAGCCGGAGATACGCATTTGGGCGGAGAGGATTTCGACA

[0242] ACAGAATGGTCAGCCATCTGGCAGAAGAATTTAAACGAAAGCACAAAAAAGATATA

[0243] GGTCCGAATAAGCGTGCAGTACGCAGACTTCGCACAGCATGTGAAAGAGCAAAACG

[0244] CACTTTGTCAAGCAGCACACAGGCTAGTATAGAGATTGACTCTCTGTACGAGGGCGT

[0245] CGATTTTTATACCTCAATAACACGCGCCCGTTTTGAAGAATTGAATGCTGATTTATTC

[0246] CGTGGTACGTTAGAGCCTGTAGAAAAAGCATTGAGGGATGCCAAACTGGACAAGGG

[0247] ACAAATTCAAGAAATTGTGCTTGTAGGTGGTTCAACTAGAATACCTAAGATTCAGAA

[0248] ATTGCTGCAAGATTTTTTTAACGGTAAAGAGCTTAATAAAAGCATCAACCCCGATGA

[0249] GGCCGTGGCATATGGAGCGGCTGTTCAGGCGGCAATTCTCATTGGAGACAAATCTG

[0250] AAAATGTTCAGGATCTTCTGCTTCTTGATGTCACGCCTCTCAGCCTAGGGATCGAAA

[0251] CGGCAGGGGGTGTGATGACTCCTTTAATTAAGCGAAATACAACCATTCCCACCAAAC

[0252] AGACGCAAACCTTTACAACATATTCCGATAACCAATCCTCGGTCCTCGTTCAGGTAT

[0253] ACGAAGGTGAACGAGCCATGACAAAAGACAACAATCTTCTCGGAAAGTTTGATTTG

[0254] ACGGGGATTCCGCCGGCACCTAGGGGCGTGCCGCAAATCGAGGTAACGTTTGACAT

[0255] CGATGCGAACGGAATTTTAAACGTTACGGCGGCGGACAAGTCAACAGGCAAAGAAA

[0256] ACAAGATCACGATTACAAATGATAAAGGGCGTCTTTCTAAAGATGATATTGATAGA

[0257] ATGGTTCAAGAGGCGGAACGTTATAAATCAGAGGACGAGGCAAATCGGGATCGCGT

[0258] GGCGGCGAAAAATGCTGTTGAATCATATACGTATAATATTAAGCAAACGGTCGAGG

[0259] ATGAAAAGTTACGGGGTAAAATTAGTGAACAAGATAAAAATAAGATTCTTGACAAA

[0260] TGCCAGGAAGTCATTAACTGGCTGGACCGGAACCAAATGGCTGAGAAAGATGAATA

[0261] TGAACACAAACAAAAAGAGCTTGAAAGAGTCTGCAATCCGATCATATCCAAGCTCT

[0262] ATCAGGGCGGCCCGGGCGGCGGCGGAGGAACTGGGGCAAGTGGGGGACCGACAAT CGAAGAAGTAGAT

[0263] SEQ ID NO: 10 - GenBank AFU91520.1, capsid protein [Porcine circovirus 2]

[0264] MTYPRRRFRRRRHRPRSHLGQILRRRPWLVELPRHRYRWRRKNGIFNTRLSRTIGYTVKK

[0265] TTVRTPSWNVDMMRFNINDFLPPGGGSNPLTVPFEYYRIRKVKVEFWPCSPITQGDRGV

[0266] GSTAVILDDNFVTKANALTYDPYVNYSSRHTITQPFSYHSRYFTPKPVLDRTIDYFQPNN

[0267] KRNQLWLRLQTTGNVDHVGLGTAFENSIYDQDYNIRITMYVQFREFNLKDPPLNPK SEQ ID NO: 11 - Heat shock protein 70-2 Genbank QWE78713.1 [S s- scrofa}

[0268] MSARGPAIGIDLGTTYSCVGVFQHGKVEIIANDQGNRTTPSYVAFTDTERLIGDAAKNQ

[0269] VAMNPTNTIFDAKRLIGRKFEDATVQSDMKHWPFRVVSEGGKPKVQVEYKGEIKTFFPE EISSMVLTKMKEIAEAYLGGKVQSAVITVPAYFNDSQRQATKDAGTITGLNVLRIINEPT

[0270] AAAIAYGLDKKGCAGGEKNVLIFDLGGGTFDVSILTIEDGIFEVKSTAGDTHLGGEDFDN RMVSHLAEEFKRKHKKDIGPNKRAVRRLRTACERAKRTLSSSTQASIEIDSLYEGVDFYT SITRARFEELNADLFRGTLEPVEKALRDAKLDKGQIQEIVLVGGSTRIPKIQKLLQDFFNG KELNKSINPDEAVAYGAAVQAAILIGDKSENVQDLLLLDVTPLSLGIETAGGVMTPLIKR NTTIPTKQTQTFTTYSDNQSSVLVQVYEGERAMTKDNNLLGKFDLTGIPPAPRGVPQIEV TFDIDANGILNVTAADKSTGKENKITITNDKGRLSKDDIDRMVQEAERYKSEDEANRDR VAAKNAVESYTYNIKQTVEDEKLRGKISEQDKNKILDKCQEVINWLDRNQMAEKDEYE HKQKELERVCNPIISKLYQGGPGGGGGTGASGGPTIEEVD

Claims

CLAIMS:

1. A bacterial vaccine vector for stimulating an immune response against PCV2 infection in a host animal susceptible to infection by PCV2, the vaccine vector comprising a recombinant Bacillus bacterium comprising a nucleic acid encoding a conserved PCV2 capsid protein, which is configured to self-assemble, after expression, into a non-infectious PCV2 virus-like particle to stimulate an immune response against PCV2 infection in said host animal susceptible to infection by PCV2.

2. The bacterial vaccine vector of claim 1, wherein said recombinant Bacillus bacterium is Bacillus subtilis.

3. The bacterial vaccine vector of claim 1, wherein said recombinant Bacillus bacterium comprises an expression vector comprising said nucleic acid encoding for said conserved PCV2 capsid protein.

4. The bacterial vaccine vector of claim 3, wherein said expression vector further comprises an inducible promoter for inducing transcription of said nucleic acid.

5. The bacterial vaccine vector of claim 3 or 4, wherein said expression vector further comprises a Gram-positive bacterial secretion signal.

6. The bacterial vaccine vector of claim 3, 4, or 5, wherein said expression vector further comprises a nucleotide sequence encoding for an immunostimulatory molecule as an adjuvant to enhance said immune response stimulated by said PCV2 virus-like particle.

7. The bacterial vaccine vector of claim 3, 4, 5, or 6, wherein said expression vector further comprises a nucleotide sequence encoding for one or more neutralizing epitopes.

8. The bacterial vaccine vector of claims 1-7, wherein said conserved PCV2 capsid protein comprises SEQ ID N0:3:ADLTYPRRRYRRRRHRPRSHLGQILRRRPWLVHPRHRYRWRRKNGIFNTRLSRTFGYTV KATTVRTPSWAVDMMRFNINDFLPPGGGAPPHALSSNPLTVPFEYYRIRKVKVEFWPCSPITQGDRGVGSTAVVLDDNFVTKANALTYDPYVNYSSRHTIPQPFSYHSRYFTPKPVLDR TIDYFQPNNKRNQLWLRLQTTANVDHVGLGTAFENSKYDQDYNIRVTMYVQFREFNLK DPPLKPK, or an immunogenic fragment thereof, or a homolog thereof having at least 80% sequence identity, preferably at least 90% sequence identity, more preferably, at least 95% sequence identity.

9. A cell culture comprising cell culture media, a bacterial vaccine vector of claims 1-8 and further comprising said non-infectious PCV2 virus-like particles secreted into said cell culture media, wherein said bacterial vaccine vector is inactivated.

10. An immunogenic composition comprising a bacterial vaccine vector of claims 1-8 or said cell culture of claim 9, dispersed in a pharmaceutically acceptable carrier.

11. The immunogenic composition of claim 10, further comprising an additional bacterial vaccine vector comprising a nucleic acid encoding for a linear PCV2 epitope.

12. The immunogenic composition of claim 11, wherein said additional bacterial vaccine vector comprises an expression vector comprising said nucleic acid encoding for said linear PCV2 epitope.

13. The immunogenic composition of claim 12, wherein said expression vector encodes for at least two different linear PCV2 epitopes.

14. The immunogenic composition of claim 12, wherein said expression vector further comprises a nucleotide sequence encoding for an immunostimulatory molecule as an adjuvant to enhance said immune response, wherein said immunostimulatory molecule is a toll-like receptor agonist and / or a heat shock protein.

15. The immunogenic composition of claims 10-14, wherein said pharmaceutically acceptable carrier is configured for mucosal administration.

16. The immunogenic composition of claims 10-15, wherein said pharmaceuticallyacceptable carrier is water, buffered solutions, naturally occurring mono, di-, and polysaccharides, carbohydrates, sugars, polymers, proteins, and combinations thereof.

17. The immunogenic composition of claims 10-16, wherein said pharmaceutically acceptable carrier is selected from the group consisting of celluloses, derivatives thereof, and microcrystalline forms thereof; other naturally derived polysaccharides and polymers; mono or oligosaccharides; dextrins; native starches and their derivatives; gums; bacterial cell culture fluids, media, and nutrients; and serum proteins, casein, albumin, and mixtures of any of the foregoing.

18. A method of stimulating an immune response against PCV2 infection, so as to inhibit, reduce, or even prevent symptoms of PCV2 infection and / or PCVAD in a host animal susceptible to PCV2 infection, the method comprising: administering a therapeutically effective amount of said bacterial vaccine vector of claims 1-8, said inactivated cell culture of claim 9, or said immunogenic composition of claims 10-17 to a host animal in need thereof, wherein said host animal has inhibited, reduced, or prevented symptoms of PCV2 infection and / or PCVAD as compared to an unvaccinated control.

19. The method of claim 18, wherein said bacterial vaccine vector, inactivated cell culture, or immunogenic composition is administered mucosally to said host animal.

20. The method of claim 19, wherein said bacterial vaccine vector, inactivated cell culture, or immunogenic composition is administered orally or nasally.

21. The method of claims 18-20, wherein said bacterial vaccine vector, inactivated cell culture, or immunogenic composition is administered as a first dosage of said bacterial vaccine vector, inactivated cell culture, or immunogenic composition.

22. The method of claim 21, further comprising administering a second dosage of said bacterial vaccine vector, inactivated cell culture, or immunogenic composition to said host animal as a booster dosage.

23. The method of claim 18-22, wherein said host animal displays an enhanced immune response against PCV2 infection.

24. The method of claim 23, wherein said enhanced immune response comprises detectable IgY / IgG and / or IgA antibodies.

25. The method of claims 23 or 24, wherein said enhanced immune response comprises one or more responses selected from detectable levels of neutralizing antibodies and / or anti-PCV2 antibodies.

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