COMPOSITIONS THEREFROM CROSS REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. provisional patent application U.S. Serial No.62/874,094, filed July 15, 2019. The U.S. provisional patent application is herein incorporated by reference in its entirety, including without limitation, the specification, claims, and abstract, as well as any figures, tables, appendices, or drawings thereof. FIELD OF THE INVENTION The present invention is directed to novel immunogenic compositions that protect swine from disease caused by Senecavirus A (SVA). BACKGROUND OF THE INVENTION Senecavirus A causes vesicular disease similar to that of Foot and Mouth Disease, swine vesicular disease, vesicular stomatitis or vesicular exanthema (12). The clinical signs in pigs include vesicles or lesions on snouts and feet (dewclaw, coronary band and sole), anorexia, cutaneous hyperemia, fever, lethargy and lameness (12, 19, 20). Infection with SVA occurs via oronasal route and after incubation period of about 3 to 5 days, lameness and lethargy develops, followed by development of vesicles (19). Commercial vaccine to SVA have not been developed yet. Vaccines used for Foot and Mouth Disease, a related picornavirus, are mostly inactivated, however they lack long term protection, require multiple vaccinations, have short shelf life (22), so the need of novel approach for vaccine development for SVA is essential. SUMMARY OF THE INVENTION

The present invention encompasses immunogenic compositions comprising variant SVA strains. The variant strains are less virulent that traditional SVA strains and may be used, in one embodiment for whole virus, attenuated live vaccines. The novel strains have one or more modifications in nucleic acid sequence from currently known SVA strains and pigs infected with live attenuated serially passaged variant strains of the invention do not cause disease when administered to piglets. Thus, the invention comprises an immunogenic composition, suitable to be used as a vaccine, which comprises a variant SVA strain of the invention, preferably live and attenuated, or an immunogenic fragment thereof, one or more adjuvants, and optionally one or more excipients, in an amount effective to elicit production of neutralizing antibodies in swine. The adjuvant preferably provides an oil-in- water emulsion with additional components. The immunogenic compositions of the invention protect swine from infection by SVA, and are effective in single doses, in two- dose programs, or in vaccination programs involving multiple doses, which may be spread apart by at least a week, and optionally at greater intervals of time, such as one to several months. It should be noted that depending on the level of epidemic threat in a swine population, the vaccine dose program of one, two, or multiple doses may be repeated, from time to time, as a precautionary measure. Additionally, it should be noted that vaccinating a mother sow during pregnancy will provide protection to a young piglet, via maternal transfer of antibodies and T-cells in milk, although such protection may need to be followed up with additional vaccination doses to the piglet. Vaccination of all swine, including piglets and adults is contemplated.

The variant strains include several nucleic acid modifications from traditional SVA strains, including primarily variations in the 5’UTR, also an additional variation was identified in the VP4 coding region. Accordingly, the vaccinating compositions of the present invention are useful to protect swine from disease or challenge by SVA generally, including recent isolates, and other isolates that show homology with SD 15-26 SVA variants. It has surprisingly been found that the variant strain of the invention includes three modifications in the 5’ UTR plus the single silent nucleotidet change in the VP4 coding region that results in a less virulent strain and thus is useful as an attenuated live vaccine. Several additional variants have been introduced to help further distinguish the strains. The modification includes a C to T change at position 28 of the 5’UTR of strain SD 15-26. It expected that the analogous change in other wild type strains will have similar attenuation effects. Additional modifications introduced for identification only include changes from C to T at positions 31 and 32 in the 5’ UTR and a silent change of C to A at position 942 of the VP4 coding region. Accordingly, the vaccinating compositions of the present invention are useful to protect swine from disease or challenge by SVA generally, including recent isolates, and other isolates that show homology SVA SD15-26.

The present invention includes novel nucleotide sequences of SVA, including novel genotypes thereof, all of which are useful in the preparation of vaccines for treating and preventing diseases in swine and other animals. Vaccines provided according to the practice of the invention are effective against multiple swine SVA genotypes and isolates. Diagnostic and therapeutic polyclonal and monoclonal antibodies are also a feature of the present invention, as are infectious clones useful in the propagation of the virus and in the preparation of vaccines. Of importance, there are disclosed vaccines that comprise, as antigen, a whole virus (live or attenuated) or a single antigenic protein of an SVA open reading frame, most particularly from the 5‘UTR, and fragments of the full-length sequence encoding the SVA proteins. The invention also provides for novel variant full length SVA genomes that can replicate efficiently in host animals and tissue culture.

The present invention provides a method of treating or preventing a disease or disorder in an animal caused by infection with Senecavirus A (SVA), including disease states that are directly caused by SVA, and disease states contributed to or potentiated by SVA. Disease states in swine that may be potentiated by SVA, and which may also be treated or prevented according to the practice of the invention, include those caused by or associated with SVA such as Foot and Mouth Disease.

The present invention also includes the option to administer a combination vaccine, that is, a bivalent or multivalent combination of antigens, which may include live, modified live, or inactivated antigens against the non-SVA pathogen, with appropriate choice of adjuvant.

Based in part upon the unique SVA sequences as disclosed herein, the present invention also provides a diagnostic kit for differentiating between porcine animals vaccinated with the above described SVA vaccines and porcine animals infected with field strains of SVA.

Representative embodiments of the invention include an isolated polynucleotide sequence that includes a polynucleotide selected from:

(a) SEQ ID NO: 1, 2, 3, or 4 or a fragment thereof than encodes the SVA VP proteins or a fragment of said protein SVA 5” UTR wherein position 28, 31, and /or 32 is not C; or position 942 of the VP4 coding region with reference to SEQ ID NO: 1; (b) the complement of any sequence in (a);

(c) a polynucleotide that hybridizes with a sequence of (a) or (b) under stringent conditions defined as hybridizing to filter bound DNA in 0.5M NaHPO4, 7% SDS, 1 mM EDTA at 65° C., and washing in 0.1 X SSC/0.1% SDS at 68° C.

(d) a polynucleotide that is at least 70% identical to the polynucleotide of (a) or (b); (e) a polynucleotide that is at least 80% identical to the polynucleotide of (a) or (b); (f) a polynucleotide that is at least 90% identical to the polynucleotide of (a) or (b); and

(g) a polynucleotide that is at least 95% identical to the polynucleotide of (a) or (b) Preferably in combination with a second heterologous sequence.

The invention further provides RNA and DNA molecules, their complements, fragments and vectors and plasmids for the expression of any such RNA or DNA polynucleotides, and for SVA virus that is expressed from such nucleotide sequences, wherein said virus is live, or fully or partially attenuated.

The invention also provides a vaccine that comprises a polynucleotide sequence as aforementioned, and corresponding nucleotide sequences that may function as infectious clones. The invention also includes polynucleotides which encode additional otherwise identical amino acids are replaced by conservative substitutions and further preferably including fusion proteins or other modifications such that the proteins are not naturally occurring.

The invention also provides for novel full length variant SVA genome sequence that can replicate efficiently in host animals and tissue culture, and can be used virus live, preferably attenuated vaccine composition. BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the specification and are included to further demonstrate certain embodiments or various aspects of the invention. In some instances, embodiments of the invention can be best understood by referring to the accompanying drawings in combination with the detailed description presented herein. The description and accompanying drawings may highlight a certain specific example, or a certain aspect of the invention. However, one skilled in the art will understand that portions of the example or aspect may be used in combination with other examples or aspects of the invention.

Figures 1 (A)– (E) depict In vitro characterization of live attenuated vaccine rSVA mSacII. H1299 cells were infected with (A) 0.1 and (B) 10 MOI of wt SVA SD15-26 and rSVA mSacII and virus titer were measured at 2, 4, 8, 12- and 24-hours post-infection. Western blot to detect SVA-VP1 and VP2 protein in (C) wt SVA SD15-26 and (D) rSVA mSacII. (E) Restriction digestion by SacII to show SacII restriction site in wt SVA SD15- 26 but not in rSVA mSacII.

Figures 2 (A)– (F) show attenuation of rSVA mSacII in swine. (A) Presence of lesion on snout and feet when infected with wt SVA SD15-26 but not when infected with rSVA mSacII. (B) Total clinical score in pig’s post-infection. (C) Viremia and virus shedding in (D) oral secretion (E) nasal secretion and (F) rectal swab.

Figures 3 (A) and (B) depict (A) Viral load in tissue 14 days post-infection and (B) Neutralizing antibody titer in both virus

Figure 4 shows Virus protein level in live and inactivated vaccine. Western blot of both live and inactivated vaccine was done, protein in both vaccines compared.

Figures 5 (A)– (F) demonstrate clinical outcome, viremia and shedding in pigs after immunization. Animals were immunized with vaccine or RPMI-1640 (control). (A) Clinical outcome. (B) Total clinical score. (C) Viremia. Virus shedding in (D) oral secretion (E) Nasal secretion and (F) Rectal swab. *a, b, c, d, e, f indicates significant difference between groups Control vs. Inactivated, Control vs. Live IM, Control vs. Live IN, Inactivated vs. Live IM, Inactivated vs. Live IN and Live IM vs. Live IN respectively at p<0.05 (Tukey’s multiple comparison).

Figures 6 (A)– (C) show neutralizing antibody titer post immunization and post challenge. Virus neutralizing antibody titer were measured in various time points post- immunization and post-challenge. Figures 7 (A)– (F) demonstrates Clinical outcome, viremia and shedding in pigs after heterologous SVA challenge. Animals in all groups were challenged with SVA MN15-84-22. (A) Clinical outcome. (B) Total clinical score. (C) Viremia. Virus shedding in (D) oral secretion (E) Nasal secretion and (F) Rectal swab. *a, b, c, d,e,f indicates significant difference between groups Control vs. Inactivated, Control vs. Live IM, Control vs. Live IN, Inactivated vs. Live IM, Inactivated vs. Live IN and Live IM vs. Live IN respectively at p<0.05 (Tukey’s multiple comparison).

Figures 8 (A)– (D) show titers in various tissues

Figures 9 (A)– (C) show Viral load in tissues. Virus load post-challenge in (9A) Tonsil, (9B) Mediastinal lymph node, and (9C) Mesenteric lymph node.

Figures 10 (A)– (C) show titers in various tissues.

Figure 11 is the T7-rSVA-SD15-26-5’UTR (NheI + SfiI) sequence.

Figures 12 (A) and (B) are maps of rSVA_mSacII (A ) is circular and (B) is linear.

DETAILED DESCRIPTION OF THE INVENTION

The following definitions and introductory matters are applicable in the

specification.

The singular terms "a", "an", and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicate otherwise. The word "or" means any one member of a list and also includes any combination of members of that list.

The term "adjuvant" refers to a compound that enhances the effectiveness of the vaccine and may be added to the formulation that includes the immunizing agent.

Adjuvants provide enhanced immune response even after administration of only a single dose of the vaccine. Adjuvants may include, for example, muramyl dipeptides, pyridine, aluminum hydroxide, dimethyldioctadecyl ammonium bromide (DDA), oils, oil-in-water emulsions, saponins, cytokines, and other substances known in the art. Examples of suitable adjuvants are described in U.S. Patent Application Publication No.

US2004/0213817 A1.“Adjuvanted” refers to a composition that incorporates or is combined with an adjuvant.

"Antibodies" refers to polyclonal and monoclonal antibodies, chimeric, and single chain antibodies, as well as Fab fragments, including the products of a Fab or other immunoglobulin expression library. With respect to antibodies, the term, "immunologically specific" refers to antibodies that bind to one or more epitopes of a protein of interest, but which do not substantially recognize and bind other molecules in a sample containing a mixed population of antigenic biological molecules.

An "attenuated" SVA as used herein refers to an SVA which is capable of infecting and/or replicating in a susceptible host but is non-pathogenic or less pathogenic to the susceptible host. For example, the attenuates ed virus may cause no observable/detectable clinical manifestations, or less clinical manifestations, or less severe clinical

manifestations, or exhibit a reduction in virus replication efficiency and/or infectivity, as compared with the related field isolated strains. The clinical manifestations of SVA infection can include, without limitation, vesicles or lesions on snouts and feet (dewclaw, coronary band and sole), anorexia, cutaneous hyperemia, fever, lethargy and lameness.

An "epitope" is an antigenic determinant that is immunologically active in the sense that once administered to the host, it can evoke an immune response of the humoral (B cells) and/or cellular type (T cells). These are chemical groups or peptide sequences on a molecule that are antigenic. An antibody specifically binds an antigenic epitope on a polypeptide. In the animal most antigens will present several or even many antigenic determinants simultaneously. Such a polypeptide may also be qualified as an immunogenic polypeptide and the epitope may be identified as described further.

The term "immunogenic fragment" as used herein refers to a polypeptide or a fragment of a polypeptide, or a nucleotide sequence encoding the same which comprises an allele-specific motif, an epitope or other sequence such that the polypeptide or the fragment will bind an MHC molecule and induce a cytotoxic T lymphocyte ("CTL") response, and/or a B cell response (for example, antibody production), and/or T-helper lymphocyte response, and/or a delayed type hypersensitivity (DTH) response against the antigen from which the immunogenic polypeptide or the immunogenic fragment is derived. A DTH response is an immune reaction in which T cell-dependent macrophage activation and inflammation cause tissue injury. A DTH reaction to the subcutaneous injection of antigen is often used as an assay for cell-mediated immunity.

With the term "induction of an immunoprotective response" is meant a (humoral and/or cellular) immune response that reduces or eliminates one or more of the symptoms of disease, i.e. clinical signs, lesions, bacterial excretion and bacterial replication in tissues in the infected subject compared to a healthy control. Preferably said reduction in symptoms is statistically significant when compared to a control.

An "infectious DNA molecule", for purposes of the present invention, is a DNA molecule that encodes the necessary elements for viral replication, transcription, and translation into a functional virion in a suitable host cell.

The term "isolated" is used to indicate that a cell, peptide or nucleic acid is separated from its native environment. Isolated peptides and nucleic acids may be substantially pure, i.e. essentially free of other substances with which they may bound in nature.

For purposes of the present invention, the nucleotide sequence of a second polynucleotide molecule (either RNA or DNA) is "homologous" to the nucleotide sequence of a first polynucleotide molecule, or has "identity" to said first polynucleotide molecule, where the nucleotide sequence of the second polynucleotide molecule encodes the same polyaminoacid as the nucleotide sequence of the first polynucleotide molecule as based on the degeneracy of the genetic code, or when it encodes a polyaminoacid that is sufficiently similar to the polyaminoacid encoded by the nucleotide sequence of the first polynucleotide molecule so as to be useful in practicing the present invention. Homologous polynucleotide sequences also refer to sense and anti-sense strands, and in all cases to the complement of any such strands. For purposes of the present invention, a polynucleotide molecule is useful in practicing the present invention, and is therefore homologous or has identity, where it can be used as a diagnostic probe to detect the presence of SVA or viral polynucleotide in a fluid or tissue sample of an infected pig, e.g. by standard hybridization or amplification techniques. Generally, the nucleotide sequence of a second polynucleotide molecule is homologous to the nucleotide sequence of a first polynucleotide molecule if it has at least about 70% nucleotide sequence identity to the nucleotide sequence of the first polynucleotide molecule as based on the BLASTN algorithm (National Center for

Biotechnology Information, otherwise known as NCBI, (Bethesda, Md., USA) of the United States National Institute of Health). In a specific example for calculations according to the practice of the present invention, reference is made to BLASTP 2.2.6 [Tatusova TA and TL Madden, "BLAST 2 sequences--a new tool for comparing protein and nucleotide sequences." (1999) FEMS Microbiol Lett.174:247-250.]. Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 0.1, and the "blosum62" scoring matrix of Henikoff and Henikoff (Proc. Nat. Acad. Sci. USA 32589:10915-10919.1992). The percent identity is then calculated as: Total number of identical matches X 100/divided by the length of the longer sequence+number of gaps introduced into the longer sequence to align the two sequences.

Preferably, a homologous nucleotide sequence has at least about 75% nucleotide sequence identity, even more preferably at least about 80%, 85%, 90% and 95% nucleotide sequence identity. Since the genetic code is degenerate, a homologous nucleotide sequence can include any number of "silent" base changes, i.e. nucleotide substitutions that nonetheless encode the same amino acid.

A homologous nucleotide sequence can further contain non-silent mutations, i.e. base substitutions, deletions, or additions resulting in amino acid differences in the encoded polyaminoacid, so long as the sequence remains at least about 70% identical to the polyaminoacid encoded by the first nucleotide sequence or otherwise is useful for practicing the present invention. In this regard, certain conservative amino acid

substitutions may be made which are generally recognized not to inactivate overall protein function: such as in regard of positively charged amino acids (and vice versa), lysine, arginine and histidine; in regard of negatively charged amino acids (and vice versa), aspartic acid and glutamic acid; and in regard of certain groups of neutrally charged amino acids (and in all cases, also vice versa), (1) alanine and serine, (2) asparagine, glutamine, and histidine, (3) cysteine and serine, (4) glycine and proline, (5) isoleucine, leucine and valine, (6) methionine, leucine and isoleucine, (7) phenylalanine, methionine, leucine, and tyrosine, (8) serine and threonine, (9) tryptophan and tyrosine, (10) and for example tyrosine, tyrptophan and phenylalanine.

Homologous nucleotide sequences can be determined by comparison of nucleotide sequences, for example by using BLASTN, above. Alternatively, homologous nucleotide sequences can be determined by hybridization under selected conditions. For example, the nucleotide sequence of a second polynucleotide molecule is homologous to SEQ ID NO:1 (or any other particular polynucleotide sequence) if it hybridizes to the complement of SEQ ID NO:1 under moderately stringent conditions, e.g., hybridization to filter-bound DNA in 0.5 M NaHPO ₄ , 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.2 X SSC/0.1% SDS at 42° C. (see Ausubel et al editors, Protocols in

Molecular Biology, Wiley and Sons, 1994, pp.6.0.3 to 6.4.10), or conditions which will otherwise result in hybridization of sequences that encode a SVA virus as defined below. Modifications in hybridization conditions can be empirically determined or precisely calculated based on the length and percentage of guanosine/cytosine (GC) base pairing of the probe. The hybridization conditions can be calculated as described in Sambrook, et al., (Eds.), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y. (1989), pp.9.47 to 9.51.

In another embodiment, a second nucleotide sequence is homologous to SEQ ID NO: 1 (or any other sequence of the invention) if it hybridizes to the complement of SEQ ID NO: 1 under highly stringent conditions, e.g. hybridization to filter-bound DNA in 0.5 M NaHPO ₄ , 7% SDS, 1 mM EDTA at 65° C., and washing in 0.1 X SSC/0.1% SDS at 68° C., as is known in the art.

"Mammals" include any warm-blooded vertebrates of the Mammalia class, including humans.

A "pharmaceutically acceptable carrier" means any conventional pharmaceutically acceptable carrier, vehicle, or excipient that is used in the art for production and administration of vaccines. Pharmaceutically acceptable carriers are typically non-toxic, inert, solid or liquid carriers.

The terms "porcine" and "swine" are used interchangeably herein and refer to any animal that is a member of the family Suidae such as, for example, a pig.

A "susceptible" host as used herein refers to a cell or an animal that can be infected by SVA. When introduced to a susceptible animal, an attenuated SVA may also induce an immunological response against the SVA or its antigen, and thereby render the animal immunity against SVA infection.

The term "vaccine" refers to an antigenic preparation used to produce immunity to a disease, to prevent or ameliorate the effects of infection. Vaccines are typically prepared using a combination of an immunologically effective amount of an immunogen together with an adjuvant effective for enhancing the immune response of the vaccinated subject against the immunogen.

Vaccine formulations will contain a "therapeutically effective amount" of the active ingredient, that is, an amount capable of eliciting an induction of an immunoprotective response in a subject to which the composition is administered. In the treatment and prevention of SVA disease, for example, a "therapeutically effective amount" would preferably be an amount that enhances resistance of the vaccinated subject to new infection and/or reduces the clinical severity of the disease. Such protection will be demonstrated by either a reduction or lack of symptoms normally displayed by a subject infected with SVA, a quicker recovery time and/or a lowered count of virus particles. Vaccines can be administered prior to infection, as a preventative measure against SVA. Alternatively, vaccines can be administered after the subject already has contracted a disease. Vaccines given after exposure to SVA may be able to attenuate the disease, triggering a superior immune response than the natural infection itself.

Vaccine Formulations/Immunogenic Compositions

The invention also relates to an immunogenic composition, suitable to be used as a vaccine, which comprises a variant SVA strain according to the invention. The

immunogenic compositions according to the invention elicit a specific humoral immune response toward the SVA comprising neutralizing antibodies.

The preferred immunogenic compositions based upon the variant strains disclosed herein can provide live, attenuated viruses which exhibit high immunogenicity while at the same time not producing dangerous pathogenic or lethal effects.

The immunogenic compositions of this invention are not, however, restricted to any particular type or method of preparation. These include, but are not limited to, infectious DNA vaccines (i.e., using plasmids, vectors or other conventional carriers to directly inject DNA into pigs), live vaccines, modified live vaccines, inactivated vaccines, subunit vaccines, attenuated vaccines, genetically engineered vaccines, etc. These vaccines are prepared by standard methods known in the art.

The present invention preferably includes vaccine compositions comprising a live, attenuated variant SVA of the invention and a pharmaceutically acceptable carrier. As used herein, the expression "live, attenuated SVA of the invention" encompasses any live, attenuated SVA strain that includes one or more of the variations described herein. The pharmaceutically acceptable carrier can be, e.g., water, a stabilizer, a preservative, culture medium, or a buffer. Vaccine formulations comprising the attenuated SVA of the invention can be prepared in the form of a suspension or in a lyophilized form or, alternatively, in a frozen form. If frozen, glycerol or other similar agents may be added to enhance stability when frozen. The advantages of live attenuated vaccines, in general, include the presentation of all the relevant immunogenic determinants of an infectious agent in its natural form to the host's immune system, and the need for relatively small amounts of the immunizing agent due to the ability of the agent to multiply in the vaccinated host.

Attenuation of the virus for a live vaccine, so that it is insufficiently pathogenic to substantially harm the vaccinated target animal, may be accomplished by known procedures, including preferably by serial passaging. The following references provide various general methods for attenuation of coronaviruses, and are suitable for attenuation or further attenuation of any of the strains useful in the practice of the present invention: B. Neuman et al., Journal of Virology, vol.79, No.15, pp.9665-9676, 2005; J. Netland et al., Virology, v 399(1), pp.120-128, 2010; Y-P Huang et al.,“Sequence changes of infectious bronchitis virus isolates in the 3’ 7.3 kb of the genome after attenuating passage in embryonated eggs, Avian Pathology, v.36 (1), (Abstract), 2007; and S. Hingley et al., Virology, v.200(1) 1994, pp.1-10; see U.S. Patent 3,914,408; and Ortego et al., Virology, vol.308 (1), pp.13-22, 2003.

Additional genetically engineered vaccines, which are desirable in the present invention, are produced by techniques known in the art. Such techniques involve, but are not limited to, further manipulation of recombinant DNA, modification of or substitutions to the amino acid sequences of the recombinant proteins and the like.

Genetically engineered vaccines based on recombinant DNA technology are made, for instance, by identifying alternative portions of the viral gene encoding proteins responsible for inducing a stronger immune or protective response in pigs (e.g., proteins derived from VP1, VP2, VP3, or VP4, etc.). Various subtypes or isolates of the viral protein genes can be subjected to the DNA-shuffling method. The resulting heterogeneous chimeric viral proteins can be used broad protecting subunit vaccines. Alternatively, such chimeric viral genes or immuno-dominant fragments can be cloned into standard protein expression vectors, such as the baculovirus vector, and used to infect appropriate host cells (see, for example, O'Reilly et al., "Baculovirus Expression Vectors: A Lab Manual," Freeman & Co., 1992). The host cells are cultured, thus expressing the desired vaccine proteins, which can be purified to the desired extent and formulated into a suitable vaccine product.

If the clones retain any undesirable natural abilities of causing disease, it is also possible to pinpoint the nucleotide sequences in the viral genome responsible for any residual virulence, and genetically engineer the virus avirulent through, for example, site- directed mutagenesis. Site-directed mutagenesis is able to add, delete or change one or more nucleotides (see, for instance, Zoller et al., DNA 3:479-488, 1984). An

oligonucleotide is synthesized containing the desired mutation and annealed to a portion of single stranded viral DNA. The hybrid molecule, which results from that procedure, is employed to transform bacteria. Then double-stranded DNA, which is isolated containing the appropriate mutation, is used to produce full-length DNA by ligation to a restriction fragment of the latter that is subsequently transfected into a suitable cell culture. Ligation of the genome into the suitable vector for transfer may be accomplished through any standard technique known to those of ordinary skill in the art. Transfection of the vector into host cells for the production of viral progeny may be done using any of the conventional methods such as calcium-phosphate or DEAE-dextran mediated transfection, electroporation, protoplast fusion and other well-known techniques (e.g., Sambrook et al., "Molecular Cloning: A Laboratory Manual," Cold Spring Harbor Laboratory Press, 1989). The cloned virus then exhibits the desired mutation. Alternatively, two oligonucleotides can be synthesized which contain the appropriate mutation. These may be annealed to form double-stranded DNA that can be inserted in the viral DNA to produce full-length DNA.

An immunologically effective amount of the vaccines of the present invention is administered to a pig in need of protection against viral infection. The immunologically effective amount or the immunogenic amount that inoculates the pig can be easily determined or readily titrated by routine testing. An effective amount is one in which a sufficient immunological response to the vaccine is attained to protect the pig exposed to the SVA virus. Preferably, the pig is protected to an extent in which one to all of the adverse physiological symptoms or effects of the viral disease are significantly reduced, ameliorated or totally prevented.

Vaccines of the present invention can be formulated following accepted convention to include acceptable carriers for animals, such as standard buffers, stabilizers, diluents, preservatives, and/or solubilizers, and can also be formulated to facilitate sustained release. Diluents include water, saline, dextrose, ethanol, glycerol, and the like. Additives for isotonicity include sodium chloride, dextrose, mannitol, sorbitol, and lactose, among others. Stabilizers include albumin, among others. Other suitable vaccine vehicles and additives, including those that are particularly useful in formulating modified live vaccines, are known or will be apparent to those skilled in the art. See, e.g., Remington's

Pharmaceutical Science, 18th ed., 1990, Mack Publishing, which is incorporated herein by reference.

Vaccines of the present invention may further comprise one or more additional immunomodulatory components such as, e.g., an adjuvant or cytokine, among others. Non- limiting examples of adjuvants that can be used in the vaccine of the present invention include the RIBI adjuvant system (Ribi Inc., Hamilton, Mont.), alum, mineral gels such as aluminum hydroxide gel, oil-in-water emulsions, water-in-oil emulsions such as, e.g., Freund's complete and incomplete adjuvants, Block copolymer (CytRx, Atlanta Ga.), QS- 21 (Cambridge Biotech Inc., Cambridge Mass.), SAF-M (Chiron, Emeryville Calif.), AMPHIGEN® adjuvant, saponin, Quil A or other saponin fraction, monophosphoryl lipid A, ionic polysaccharides, and Avridine lipid-amine adjuvant. Non-limiting examples of oil- in-water emulsions useful in the vaccine of the invention include modified SEAM62 and SEAM 1/2 formulations. Modified SEAM62 is an oil-in-water emulsion containing 5% (v/v) squalene (Sigma), 1% (v/v) SPAN® 85 detergent (ICI Surfactants), 0.7% (v/v) TWEEN® 80 detergent (ICI Surfactants), 2.5% (v/v) ethanol, 200 µg/ml Quil A, 100 µg/ml cholesterol, and 0.5% (v/v) lecithin. Modified SEAM 1/2 is an oil-in-water emulsion comprising 5% (v/v) squalene, 1% (v/v) SPAN® 85 detergent, 0.7% (v/v) Tween 80 detergent, 2.5% (v/v) ethanol, 100 µg/ml Quil A, and 50 µg/ml cholesterol. Other immunomodulatory agents that can be included in the vaccine include, e.g., one or more interleukins, interferons, or other known cytokines.

Additional adjuvant systems permit for the combination of both T-helper and B-cell epitopes, resulting in one or more types of covalent T-B epitope linked structures, with may be additionally lipidated, such as those described in WO2006/084319,

WO2004/014957, and WO2004/014956.

In a preferred embodiment of the present invention, ORFI SVA protein, or other SVA proteins or fragments thereof, is formulated with 5% AMPHIGEN® as discussed hereinafter.

Adjuvant components

The vaccine compositions of the invention may or may not include adjuvants. In particular, as based on an orally infective virus, the modified live vaccines of the invention may be used adjuvant free, with a sterile carrier. Adjuvants that may be used for oral administration include those based on CT-like immune modulators (rmLT, CT-B, i.e. recombinant-mutant heat labile toxin of E. coli, Cholera toxin-B subunit); or via encapsulation with polymers and alginates, or with mucoadhesives such as chitosan, or via liposomes. A preferred adjuvanted or non adjuvanted vaccine dose at the minimal protective dose through vaccine release may provide between approximately 10 and approximately 10 ⁶ log ₁₀ TCID ₅₀ of virus per dose, or higher. Adjuvants, if present, may be provided as emulsions, more commonly if non-oral administration is selected, but should not decrease starting titer by more than 0.7 logs (80% reduction.

In one example, adjuvant components are provided from a combination of lecithin in light mineral oil, and also an aluminum hydroxide component. Details concerning the composition and formulation of Amphigen® (as representative lecithin/mineral oil component) are as follows.

A preferred adjuvanted may be provided as a 2ML dose in a buffered solution further comprising about 5% (v/v) Rehydragel® (aluminum hydroxide gel) and“20% Amphigen” ® at about 25% final (v/v). Amphigen® is generally described in U.S Patent 5,084,269 and provides de-oiled lecithin (preferably soy) dissolved in a light oil, which is then dispersed into an aqueous solution or suspension of the antigen as an oil-in-water emulsion. Amphigen has been improved according to the protocols of U.S. Patent 6,814,971 (see columns 8-9 thereof) to provide a so-called“20% Amphigen” component for use in the final adjuvanted vaccine compositions of the present invention. Thus, a stock mixture of 10% lecithin and 90% carrier oil (DRAKEOL®, Penreco, Karns City, PA) is diluted 1: 4 with 0.63% phosphate buffered saline solution, thereby reducing the lecithin and DRAKEOL components to 2% and 18% respectively (i.e.20% of their original concentrations). Tween 80 and Span 80 surfactants are added to the composition, with representative and preferable final amounts being 5.6% (v/v) Tween 80 and 2.4% (v/v) Span 80, wherein the Span is originally provided in the stock DRAKEOL component, and the Tween is originally provided from the buffered saline component, so that mixture of the saline and DRAKEOL components results in the finally desired surfactant

concentrations. Mixture of the DRAKEOL/lecithin and saline solutions can be

accomplished using an In-Line Slim Emulsifier apparatus, model 405, Charles Ross and Son, Hauppauge, NY, USA.

The vaccine composition also may include Rehydragel® LV (about 2% aluminum hydroxide content in the stock material), as additional adjuvant component (available from Reheis, NJ, USA, and ChemTrade Logistics, USA). With further dilution using 0.63% PBS, the final vaccine composition contains the following compositional amounts per 2ML dose; 5% (v/v) Rehydragel® LV; 25% (v/v) of“20% Amphigen”, i.e. it is further 4-fold diluted); and 0.01% (w/v) of merthiolate.

As is understood in the art, the order of addition of components can be varied to provide the equivalent final vaccine composition. For example, an appropriate dilution of virus in buffer can be prepared. An appropriate amount of Rehydragel® LV (about 2% aluminum hydroxide content) stock solution can then be added, with blending, in order to permit the desired 5% (v/v) concentration of Rehydragel® LV in the actual final product. Once prepared, this intermediate stock material is combined with an appropriate amount of “20% Amphigen” stock (as generally described above, and already containing necessary amounts of Tween 80 and Span 80) to again achieve a final product having 25% (v/v) of “20% Amphigen”. An appropriate amount of 10% merthiolate can finally be added.

The vaccinate compositions of the invention permit variation in all of the ingredients, such that the total dose of antigen may be varied preferably by a factor of 100 (up or down) compared to the antigen dose stated above, and most preferably by a factor of 10 or less (up or down),. Similarly, surfactant concentrations (whether Tween or Span) may be varied by up to a factor of 10, independently of each other, or they may be deleted entirely, with replacement by appropriate concentrations of similar materials, as is well understood in the art.

Rehydragel® concentrations in the final product may be varied, first by the use of equivalent materials available from many other manufacturers (i.e. Alhydrogel®,

Brenntag; Denmark), or by use of additional variations in the Rehydragel® line of products such as CG, HPA or HS. Using LV as an example, final useful concentrations thereof including from 0% to 20%, with 2-12% being more preferred, and 4-8% being most preferred, Similarly, the although the final concentration of Amphigen (expressed as % of “20% Amphigen”) is preferably 25%, this amount may vary from 5-50%, preferably 20- 30% and is most preferably about 24-26%.

According to the practice of the invention, the oil used in the adjuvant formulations of the instant invention is preferably a mineral oil. As used herein, the term "mineral oil" refers to a mixture of liquid hydrocarbons obtained from petrolatum via a distillation technique. The term is synonymous with "liquefied paraffin", "liquid petrolatum" and "white mineral oil." The term is also intended to include "light mineral oil," i.e., oil which is similarly obtained by distillation of petrolatum, but which has a slightly lower specific gravity than white mineral oil. See, e.g., Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack Publishing Company, 1990, at pages 788 and 1323). Mineral oil can be obtained from various commercial sources, for example, J. T. Baker

(Phillipsburg, Pa.), USB Corporation (Cleveland, Ohio). Preferred mineral oil is light mineral oil commercially available under the name DRAKEOL®.

Typically, the oily phase is present in an amount from 50% to 95% by volume; preferably, in an amount of greater than 50% to 85%; more preferably, in an amount from greater than 50% to 60%, and more preferably in the amount of greater than 50-52% v/v of the vaccine composition. The oily phase includes oil and emulsifiers (e.g., SPAN® 80, TWEEN® 80 etc), if any such emulsifiers are present.

Non-natural, synthetic emulsifiers suitable for use in the adjuvant formulations of the present invention also include sorbitan-based non-ionic surfactants, e.g. fatty-acid- substituted sorbitan surfactants (commercially available under the name SPAN® or ARLACEL®), fatty acid esters of polyethoxylated sorbitol (TWEEN®), polyethylene glycol esters of fatty acids from sources such as castor oil (EMULFOR®); polyethoxylated fatty acid (e.g., stearic acid available under the name SIMULSOL® M-53),

polyethoxylated isooctylphenol/formaldehyde polymer (TYLOXAPOL®),

polyoxyethylene fatty alcohol ethers (BRIJ®); polyoxyethylene nonphenyl ethers

(TRITON® N), polyoxyethylene isooctylphenyl ethers (TRITON® X). Preferred synthetic surfactants are the surfactants available under the name SPAN® and TWEEN®, such as TWEEN®-80 (Polyoxyethylene (20) sorbitan monooleate) and SPAN®-80 (sorbitan monooleate). Generally speaking, the emulsifier(s) may be present in the vaccine composition in an amount of 0.01% to 40% by volume, preferably, 0.1% to 15%, more preferably 2% to 10%. In an alternative embodiment of the invention, the final vaccine composition contains SP-Oil® and Rehydragel® LV as adjuvants (or other Rehydragel® or

Alhydrogel® products), with preferable amounts being about 5-20% SP-Oil (v/v) and about 5-15% Rehydragel LV (v/v), and with 5% and 12%, respectively, being most preferred amounts. In this regard it is understood that % Rehydragel refers to percent dilution from the stock commercial product. (SP-Oil ® is a fluidized oil emulsion with includes a polyoxyethylene-polyoxypropylene block copolymer (Pluronic® L121, BASF Corporation, squalene, polyoxyethylene sorbitan monooleate (Tween®80, ICI Americas) and a buffered salt solution.)

It should be noted that the present invention may also be successfully practiced using wherein the adjuvant component is only Amphigen®.

In another embodiment of the invention, the final vaccine composition contains TXO as an adjuvant; TXO is generally described in WO 2015/042369. All TXO compositions disclosed therein are useful in the preparation of vaccines of the invention. In TXO, the immunostimulatory oligonucleotide (“T”), preferably an ODN, preferably containing a palindromic sequence, and optionally with a modified backbone, is present in the amount of 0.1 to 5 ug per 50 ul of the vaccine composition (e.g., 0.5– 3 ug per 50 ul of the composition, or more preferably 0.09-0.11 ug per 50 ul of the composition). A preferred species thereof is SEQ ID NO: 8 as listed (page 17) in the WO2015/042369 publication (PCT/US2014/056512). The polycationic carrier (“X”) is present in the amount of 1-20 ug per 50 ul (e.g., 3-10 ug per 50 ul, or about 5 ug per 50 ul). Light mineral oil (“O”) is also a component of the TXO adjuvant.

In certain embodiments, TXO adjuvants are prepared as follows:

a) Sorbitan monooleate, MPL-A and cholesterol are dissolved in light mineral oil. The resulting oil solution is sterile filtered;

b) The immunostimulatory oligonucleotide, Dextran DEAE and Polyoxyethylene (20) sorbitan monooleate are dissolved in aqueous phase, thus forming the aqueous solution; and

c) The aqueous solution is added to the oil solution under continuous homogenization thus forming the adjuvant formulation TXO.

All the adjuvant compositions of the invention can be used with any of the SVA strains and isolates covered by the present Specification. Additional adjuvants useful in the practice of the invention include Prezent-A (see generally United States published patent application US20070298053; and“QCDCRT” or “QCDC”-type adjuvants (see generally United States published patent application

US20090324641.

Excipients

The immunogenic and vaccine compositions of the invention can further comprise pharmaceutically acceptable carriers, excipients and/or stabilizers (see e.g. Remington: The Science and practice of Pharmacy, 2005, Lippincott Williams), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations, and may comprise buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as Mercury((o-carboxyphenyl)thio)ethyl sodium salt (THIOMERSAL), octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3- pentanol; and m-cresol); proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrans; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG), TWEEN or PLURONICS.

Vaccines of the present invention can optionally be formulated for sustained release of the virus, infectious DNA molecule, plasmid, or viral vector of the present invention. Examples of such sustained release formulations include virus, infectious DNA molecule, plasmid, or viral vector in combination with composites of biocompatible polymers, such as, e.g., poly (lactic acid), poly (lactic-co-glycolic acid), methylcellulose, hyaluronic acid, collagen and the like. The structure, selection and use of degradable polymers in drug delivery vehicles have been reviewed in several publications, including A. Domb et al., 1992, Polymers for Advanced Technologies 3: 279-292, which is incorporated herein by reference. Additional guidance in selecting and using polymers in pharmaceutical formulations can be found in texts known in the art, for example M. Chasin and R. Langer (eds), 1990, "Biodegradable Polymers as Drug Delivery Systems" in: Drugs and the Pharmaceutical Sciences, Vol.45, M. Dekker, NY, which is also incorporated herein by reference. Alternatively, or additionally, the virus, plasmid, or viral vector can be microencapsulated to improve administration and efficacy. Methods for

microencapsulating antigens are well-known in the art, and include techniques described, e.g., in U.S. Pat. No.3,137,631; U.S. Pat. No.3,959,457; U.S. Pat. No.4,205,060; U.S. Pat. No.4,606,940; U.S. Pat. No.4,744,933; U.S. Pat. No.5,132,117; and International Patent Publication WO 95/28227, all of which are incorporated herein by reference.

Liposomes can also be used to provide for the sustained release of virus, plasmid, viral protein, or viral vector. Details concerning how to make and use liposomal formulations can be found in, among other places, U.S. Pat. No.4,016,100; U.S. Pat. No. 4,452,747; U.S. Pat. No.4,921,706; U.S. Pat. No.4,927,637; U.S. Pat. No.4,944,948; U.S. Pat. No.5,008,050; and U.S. Pat. No.5,009,956, all of which are incorporated herein by reference.

An effective amount of any of the above-described vaccines can be determined by conventional means, starting with a low dose of virus, viral protein plasmid or viral vector, and then increasing the dosage while monitoring the effects. An effective amount may be obtained after a single administration of a vaccine or after multiple administrations of a vaccine. Known factors can be taken into consideration when determining an optimal dose per animal. These include the species, size, age and general condition of the animal, the presence of other drugs in the animal, and the like. The actual dosage is preferably chosen after consideration of the results from other animal studies.

One method of detecting whether an adequate immune response has been achieved is to determine seroconversion and antibody titer in the animal after vaccination. The timing of vaccination and the number of boosters, if any, will preferably be determined by a doctor or veterinarian based on analysis of all relevant factors, some of which are described above.

The effective dose amount of virus, protein, infectious nucleotide molecule, plasmid, or viral vector, of the present invention can be determined using known techniques, taking into account factors that can be determined by one of ordinary skill in the art such as the weight of the animal to be vaccinated. The dose amount of virus of the present invention in a vaccine of the present invention preferably ranges from about 10 ¹ to about 10 ⁹ pfu (plaque forming units), more preferably from about 10 ² to about 10 ⁸ pfu, and most preferably from about 10 ³ to about 107 pfu. The dose amount of a plasmid of the present invention in a vaccine of the present invention preferably ranges from about 0.1 µg to about 100 mg, more preferably from about 1 µg to about 10 mg, even more preferably from about 10 µg to about 1 mg. The dose amount of an infectious DNA molecule of the present invention in a vaccine of the present invention preferably ranges from about 0.1 µg to about 100 mg, more preferably from about 1 µg to about 10 mg, even more preferably from about 10 µg to about 1 mg. The dose amount of a viral vector of the present invention in a vaccine of the present invention preferably ranges from about 10 ¹ pfu to about 10 ⁹ pfu, more preferably from about 10 ² pfu to about 10 ⁸ pfu, and even more preferably from about 10 ³ to about 10 ⁷ pfu. A suitable dosage size ranges from about 0.5 ml to about 10 ml, and more preferably from about 1 ml to about 5 ml.

Suitable doses for viral protein or peptide vaccines according to the practice of the present invention range generally from 1 to 50 micrograms per dose, or higher amounts as may be determined by standard methods, with the amount of adjuvant to be determined by recognized methods in regard of each such substance. In a preferred example of the invention relating to vaccination of swine, an optimum age target for the animals is between about 1 and 21 days, which at pre-weening, may also correspond with other scheduled vaccinations such as against Mycoplasma hyopneumoniae. Additionally, a preferred schedule of vaccination for breeding sows would include similar doses, with an annual revaccination schedule.

Dosing

A preferred clinical indication is for treatment, control and prevention in both breeding sows and gilts pre-farrowing, followed by vaccination of piglets. In a

representative example (applicable to both sows and gilts), two 2-ML doses of vaccine will be used, although of course, actual volume of the dose is a function of how the vaccine is formulated, with actual dosing amounts ranging from 0.1 to 5ML, taking also into account the size of the animals. Single dose vaccination is also appropriate.

The first dose may be administered as early as pre-breeding to 5-weeks pre- farrowing, with the second dose administered preferably at about 1-3 weeks pre-farrowing. Doses vaccine preferably provide an amount of viral material that corresponds to a TCID ₅₀ (tissue culture infective dose) of between about 10 ⁶ and 10 ⁸ , more preferably between about 10 ⁷ and 10 ^7.5 , and can be further varied, as is recognized in the art. Booster doses can be given two to four weeks prior to any subsequent farrowings. Intramuscular vaccination (all doses) is preferred, although one or more of the doses could be given subcutaneously. Oral administration is also preferred. Vaccination may also be effective in naïve animals, and non-naïve animals as accomplished by planned or natural infections.

In a further preferred example, the sow or gilt is vaccinated intramuscularly or orally at 5-weeks pre-farrowing and then 2-weeks pre-farrowing. Under these conditions, a protective immune response can be demonstrated in SVA-negative vaccinated sows in that they developed antibodies (measured via fluorescent focal neutralization titer from serum samples) with neutralizing activity, and these antibodies were passively transferred to their piglets. The protocols of the invention are also applicable to the treatment of already seropositive sows and gilts, and also piglets and boars. Booster vaccinations can also be given, and these may be via a different route of administration. Although it is preferred to re-vaccinate a mother sow prior to any subsequent farrowings, the vaccine compositions of the invention nonetheless can still provide protection to piglets via ongoing passive transfer of antibodies, even if the mother sow was only vaccinated in association with a previous farrowing.

It should be noted that piglets may then be vaccinated as early as Day 1 of life. For example, piglets can be vaccinated at Day 1, with or without a booster dose at 3 weeks of age, particularly if the parent sow, although vaccinated pre-breeding, was not vaccinated pre-farrowing. Piglet vaccination may also be effective if the parent sow was previously not naïve either due to natural or planned infection. Vaccination of piglets when the mother has neither been previously exposed to the virus, nor vaccinated pre-farrowing may also effective. Boars (typically kept for breeding purposes) should be vaccinated once every 6 months. Variation of the dose amounts is well within the practice of the art. It should be noted that the vaccines of the present invention are safe for use in pregnant animals (all trimesters) and neonatal swine. The vaccines of the invention are attenuated to a level of safety (i.e. no mortality, only transient mild clinical signs or signs normal to neonatal swine) that is acceptable for even the most sensitive animals again including neonatal pigs. Of course, from a standpoint of protecting swine herds both from SVA epidemics and persistent low level SVA occurrence, programs of sustained sow

vaccination are of great importance. It will be appreciated that sows or gilts immunized with SVA MLV will passively transfer immunity to piglets, including SVA-specific IgA, which will protect piglets from SVA associated disease and mortality. Additionally, generally, pigs that are immunized with SVA MLV will have a decrease in amount and/or duration or be protected from shedding SVA in their feces, and further, pigs that are immunized with SVA MLV will be protected from weight loss and failure to gain weight due to SVA, and further, SVA MLV will aid in stopping or controlling the SVA

transmission cycle.

It should also be noted that animals vaccinated with the vaccines of the invention are also immediately safe for human consumption, without any significant slaughter withhold, such as 21 days or less.

When provided therapeutically, the vaccine is provided in an effective amount upon the detection of a sign of actual infection. Suitable dose amounts for treatment of an existing infection include between about 10 and about 10 ⁶ log ₁₀ TCID ₅₀ , or higher, of virus per dose (minimum immunizing dose to vaccine release). A composition is said to be “pharmacologically acceptable” if its administration can be tolerated by a recipient. Such a composition is said to be administered in a“therapeutically or prophylactically effective amount” if the amount administered is physiologically significant.

At least one vaccine or immunogenic composition of the present invention can be administered by any means that achieve the intended purpose, using a pharmaceutical composition as described herein. For example, route of administration of such a composition can be by parenteral, oral, oronasal, intranasal, intratracheal, topical, subcutaneous, intramuscular, transcutaneous, intradermal, intraperitoneal, intraocular, and intravenous administration. In one embodiment of the present invention, the composition is administered by intramuscularly. Parenteral administration can be by bolus injection or by gradual perfusion over time. Any suitable device may be used to administer the compositions, including syringes, droppers, needleless injection devices, patches, and the like. The route and device selected for use will depend on the composition of the adjuvant, the antigen, and the subject, and such are well known to the skilled artisan. Administration that is oral, or alternatively, subcutaneous, is preferred. Oral administration may be direct, via water, or via feed (solid or liquid feed). When provided in liquid form, the vaccine may be lyophilized with reconstitution, provided as a paste, for direct addition to feed (mix in or top dress) or otherwise added to water or liquid feed. Generation of Vero Cells Suitable for Large Scale Virus Production

Viruses of the invention can be conveniently grown in Vero cell stocks that are approved for vaccine production. To generate safe and approved cell stock, a vial of Vero cells was subject to additional passaging. The cells were passed four times in PMEM w/wheat to produce Master Cell Stock (MCS) Lot“1834430”. The MCS was tested in accordance with 9CFR & EP requirements in PGM-Biological Quality Control; Lincoln, NE. The MCS tested satisfactory for sterility, freedom from mycoplasmas, and extraneous agents. Therefore, PF-Vero MCS lot“1834430”, is deemed eligible for submission to the Center for Veterinary Biologics Laboratories (CVB-L) for confirmatory testing.

Seed Origin and Passage History is as follows. A Pre-master Cell stock of global Vero cells was previously frozen. For production of the cell stock, the cells were grown in PMEM (Lincoln item # 00-0779-00) containing 1% bovine serum (item # 00-0710-00, BSE compliant) and 3 mM L-glutamine. They were derived from Vero WCS Pass # 136, Lot #071700 MCS+3, 28-Jul-00. The new Pre-master cell stock was frozen at pass # 166, which is MCS+33 from the original global Vero master cell stock. MCS“1833440” was produced from a pre-Master identified as Vero KZO preMaster, Lot All cultures were grown in PMEM w/wheat, 1.0% L-glutamine and 1.0% Bovine Calf serum. Cells were planted (passage # 167) in 150 cm2 T-Flasks on August 14, 2008. The flasks were incubated in 5.0% CO2 at 36 ^ 1 ^C for 7 days then expanded. (passage # 168) After flasks reached 100% confluency 4 days later, the cultures were passed (#169) into 850 cm2 roller bottles. Rollers were incubated at 36 ^ 1 ^C at 0.125– 0.250 rpm without CO2. The final passage of rollers (#170) was done 4 days later. Cryopreservation was completed by adding 10.0% bovine calf serum and 10.0% dimethyl sulfoxide (DMSO) to the condensed cell suspension on 02Sep08. Vials were labeled as passage level #170. A total of 231 containers containing 4.2 ml were placed into a controlled rate freezer then transferred into liquid nitrogen tank for long term storage at vapor phase. The MCS was produced without the use of antibiotics. All reagents used in MCS production were sourced from Pfizer Global Manufacturing used for licensed antigen production in domestic and global markets. The MCS was produced by Pfizer’s Master Seed Facility, Lincoln, Nebraska. Sterility Testing was as follows. The Master Cell Stock was tested as per 9CFR (026-ST0) and EP 2.6.1 from 29Sep08 to 13Oct08. The MCS was found to be free of bacterial and fungal contamination.

Mycoplasma Testing and Extraneous Testing were accomplished as follows. The MCS was tested as per 9CFR (028-PU0) and EP 2.6.7. The MCS was found to be free of any Mycoplasma contamination. Extraneous testing was completed as per 9CFR 113.52 using NL-BT-2 (Bovine), Vero, NL-ED-5 (Equine), NL-ST-1 (Porcine), NL-DK (Canine), NL-FK (Feline) cells, The MCS was negative for MGG, CPE and HAd and tested negative by FA for BVD, BRSV, BPV, BAV-1, BAV-5, Rabies, Reo, BTV, ERV, Equine arteritis, PPV, TGE, PAV, HEV, CD, CPV, FPL and FIP. The MCS was tested by ELISA for FIV and was found to be satisfactory.

EP extraneous testing was as per 5.2.4 (52-2002). Extraneous testing using Bovine NL-BT-2 and EBK (Primary), Vero, NL-ED-5 (Equine), NL-ST-1 (Porcine), MARC MA 104, NL-DK (Canine) NL-FK (Feline) cells were negative for MGG, CPE, HAd and tested negative by FA for BVD, BPV, BAV-1, BAV-5, Bovine corona, Bovine rotavirus, BHV-3, PI3, IBR, BRSV and BEV-1, Reo, BTV, ERV, Equine arteritis, PPV, PRV, TGE, HEV, PAV, P. rota A1, rota A2, PRRSV, CD, CPI, CAV-2, Measles, C. rota, Rabies, CCV, FP, FCV, FVR, FIP and FeLV.

Polynucleotides of the Invention

Representative embodiments of the invention include an isolated polynucleotide sequence that comprises a polynucleotide of the invention and having a base other than Cat position 28, 31, and/or 32 of the 5’ UTR or the equivalent; and/or base 942 of the VP4 coding region or the equivalent position with reference to SEQ ID NO:1 or a fragment thereof; (b) the complement of any sequence in (a); (c) a polynucleotide that hybridizes with a sequence of (a) or (b) under stringent conditions defined as hybridizing to filter bound DNA in 0.5M NaHPO ₄ , 7% SDS, 1 mM EDTA at 65° C., and washing in 0.1 X SSC/0.1% SDS at 68° C.; (d) a polynucleotide that is at least 70% identical to the polynucleotide of (a) or (b); (e) a polynucleotide that is at least 80% identical to the polynucleotide of (a) or (b); (f) a polynucleotide that is at least 90% identical to the polynucleotide of (a) or (b); and (g) a polynucleotide that is at least 95% identical to the polynucleotide of (a) or (b). In a preferred embodiment the polynucleotide includes a second heterologous polynucleotide sequence. The invention also provides any polypeptide changes which may be associated with these changes as well as conservative substitutions.

Further Genetic Manipulations

The polynucleotide and amino acid sequence information provided by the present invention also makes possible the systematic analysis of the structure and function of the viral genes and their encoded gene products. Knowledge of a polynucleotide encoding a viral gene product of the invention also makes available anti-sense polynucleotides which recognize and hybridize to polynucleotides encoding a polypeptide of the invention, or a fragment thereof. Full length and fragment anti-sense polynucleotides are useful in this respect. The worker of ordinary skill will appreciate that fragment anti-sense molecules of the invention include (i) those which specifically recognize and hybridize to a specific RNA (as determined by sequence comparison of DNA encoding a viral polypeptide of the invention as well as (ii) those which recognize and hybridize to RNA encoding variants of the encoded proteins. Antisense polynucleotides that hybridize to RNA/DNA encoding other SVA peptides are also identifiable through sequence comparison to identify characteristic, or signature sequences for the family of molecules, further of use in the study of antigenic domains in SVA polypeptides, and may also be used to distinguish between infection of a host animal with remotely related non-SVA members of the

Circoviridae.

Guidance for effective codon optimization for enhanced expression in yeast and E. coli for the constructs of the invention is generally known to those of skill in the art. Antibodies

Also contemplated by the present invention are anti-SVA antibodies (e.g., monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, humanized, human, porcine, and CDR-grafted antibodies, including compounds which include CDR sequences which specifically recognize an SVA polypeptide of the invention. The term "specific for" indicates that the variable regions of the antibodies of the invention recognize and bind a SVA polypeptide exclusively (i.e., are able to distinguish a single SVA polypeptide from related polypeptides despite sequence identity, homology, or similarity found in the family of polypeptides), and which are permitted (optionally) to interact with other proteins (for example, S. aureus protein A or other antibodies in ELISA techniques) through interactions with sequences outside the variable region of the antibodies, and in particular, in the constant region of the Ab molecule. Screening assays to determine binding specificity of an antibody of the invention are well known and routinely practiced in the art. For a comprehensive discussion of such assays, see Harlow et al. (Eds), Antibodies A Laboratory Manual; Cold Spring Harbor Laboratory; Cold Spring Harbor, N.Y. (1988), Chapter 6. Antibodies that recognize and bind fragments of the SVA polypeptides of the invention are also contemplated, provided that the antibodies are first and foremost specific for, as defined above, an SVA polypeptide of the invention from which the fragment was derived.

For the purposes of clarity, "antibody" refers to an immunoglobulin molecule that can bind to a specific antigen as the result of an immune response to that antigen.

Immunoglobulins are serum proteins composed of "light" and "heavy" polypeptide chains having "constant" and "variable" regions and are divided into classes (e.g., IgA, IgD, IgE, IgG, and IgM) based on the composition of the constant regions. Antibodies can exist in a variety of forms including, for example, as, Fv, Fab', F(ab') ₂ , as well as in single chains, and include synthetic polypeptides that contain all or part of one or more antibody single chain polypeptide sequences.

Diagnostic Kits

The present invention also provides diagnostic kits. The kit can be valuable for differentiating between porcine animals naturally infected with a field strain of an SVA virus and porcine animals vaccinated with any of the SVA vaccines described herein. The kits can also be of value because animals potentially infected with field strains of SVA virus can be detected prior to the existence of clinical symptoms and removed from the herd or kept in isolation away from naive or vaccinated animals. The kits include reagents for analyzing a sample from a porcine animal for the presence of antibodies to a particular component of a specified SVA virus. Diagnostic kits of the present invention can include as a component a peptide or peptides from the variant SVA strain of the invention which is present in a field strain but not in a vaccine of interest, or vice versa, and selection of such suitable peptide domains is made possible by the extensive amino acid sequencing. As is known in the art, kits of the present invention can alternatively include as a component a peptide which is provided via a fusion protein. The term "fusion peptide" or "fusion protein" for purposes of the present invention means a single polypeptide chain consisting of at least a portion of an SVA virus protein. The following Examples are intended to illustrate the above invention and should not be construed as to narrow its scope. One skilled in the art will readily recognize that the Examples suggest many other ways in which the invention could be practiced. It should be understood that numerous variations and modifications may be made while remaining within the scope of the invention. EXAMPLE 1

Generation and in vitro characterization of recombinant SVA. To study the molecular mechanisms underlying SVA virulence and pathogenesis, we have recently developed a cDNA clone for SVA strain SD15-26. The rSVA was rescued by transfection of in vitro transcribed viral RNA into BHK-21 cells (data not shown), followed by amplification in the highly permissive H1299 cells (data not shown). A unique nt substitution (c®t) was introduced at the 5’UTR of the virus genome (position 28), to differentiate the recombinant from the parental virus.

A second SVA cDNA clone was generated in our laboratory. To facilitate the differentiation of the rSVA virus from the parental wtSVA strain, in this clone, we introduced 3 additional nucleotide changes in the rSVA genome. Two of those changes are located in the 5’UTR (c®t, positions 31 and 32) and the third change consists of a silent nt change (c®a) at position 942 (VP4 coding region) of the rSVA genome (added to remove a SacII restriction endonuclease site) (Fig. 1). A synthetic DNA fragment containing those changes was cloned into the backbone of the rSVA plasmid (virus described above; pBrick- FLSVA-SD15-26) using unique restriction endonucleases (NheI and SfiI). The resultant recombinant SacII mutant virus (rSVASacIIm) was rescued by transfection of in vitro transcribed viral RNA into BHK-21 cells, followed by amplification in the highly permissive H1299 cells. The identity of the rSVA was confirmed by sequencing and restriction digestion with SacII (Fig.1) and the replication properties of the rSVASacIIm were compared to the wtSVA virus in vitro. Notably, multi-step growth curves revealed an impaired replication of rSVASacIIm when compared to wtSVA, as evidenced by significantly lower viral yields in rSVASacIIm-inoculated cells (~1 log after 8 h post-inoculation) (Fig.1A and B).

The pathogenicity of the rSVASacIIm was compared to that of the parental wtSVA strain in pigs. For this, twelve SVA-negative 15-week old finishing pigs (~60 kg) were randomly allocated into two groups (G1: wtSVA, n = 6; and G2: rSVASacIIm, n = 6), inoculated oronasally (5 x 10 ⁸ TCID ₅₀ in 10 ml; 1/2 orally and 1/2 intranasal), and monitored for clinical signs and vesicular lesions for 14 days. Notably, while all pigs inoculated with the wtSVA presented characteristic clinical signs (lethargy, lameness) and lesions (vesicles on the snout and/or foot), none of the rSVASacIIm -inoculated developed overt clinical disease (Fig.2A, B).

The levels of viremia, virus shedding and viral load in tissues were also evaluated. Levels of viremia and virus shedding were significantly lower in rSVASacIIm -inoculated animals than in wtSVA-inoculated animals (data not shown). Additionally, viral load in tissues was markedly reduced in rSVASacIIm inoculated animals when compared to wtSVA-inoculated animals (Fig. 2C-F; Fig. 3). Notably, NA responses were similar in rSVASacIIm and wtSVA-inoculated animals, indicating successful infection of all inoculated pigs (Fig. 3B). Together these results indicate that, while attenuated the rSVASacIIm is highly immunogenic in pigs, thus, representing a promising platform for recombinant live attenuated or inactivated SVA vaccine development. rSVA mSaII is attenuated in swine.

rSVA mSacII retains its immunogenicity in swine.

Clinical outcome, viremia and virus shedding post-immunization with rSVA mSacII. The immunogenicity of live recombinant/attenuated vaccine by two routes and BEI inactivated vaccine were compared in pigs. To ascertain the similar protein content of Live and BEI inactivated vaccine, western blot was done and similar level of VP1 and VP2 proteins were observed (Fig.4).

Twenty-four SVA-negative 28 days old piglets were immunized with vaccine or plain RPMI in case of control as shown in Table 1 and were monitored for clinical signs and vesicular lesions for 35 days. None of the animals presented lesions or any clinical signs (Fig.5A). Clinical scores based on lesion were calculated as previously described (23). As

The levels of viremia in animals of all four groups were assessed in serum: Serum samples collected on days 0, 3, 5, 7, 14 and 21 pi were tested for the presence of SVA RNA using RT-qPCR. SVA was detected in day 3 pi in live IM and live IN groups till day 7 pi. The level of viremia was significantly high (p<0.01) in live vaccine group compared to control and inactivated but there no significant difference (p>0.05) between live IM and live IN group. The viremia was not present in day 14 pi and onward on all groups (Fig. 5C).

Virus shedding was assessed in the oral and nasal secretions and feces on animals of all groups. Oral, nasal and rectal swabs collected on days 0, 3, 5, 7, 14 and 21 pi were examined` ed by RT-qPCR. Virus shedding was detected until day 14 pi on oral swab and day 21 pi on nasal and rectal swab from live IM and live IN groups. No viral genome was detected on control and inactivated group. Live IN group had significantly higher amount of SVA RNA in certain days pi in all secretions (Fig.5D, 5E and 5F).

Immunization with rSVA mSacII provides protection against heterologous SVA strain.

Animals in all groups were challenged with virulent SVA strain SVA MN15-84-22 on day 42 post-immunization (Table 1). They were monitored daily for characteristics clinical signs and clinical scores were calculated. All animals of control group succumbed to illness by day 4 pc. Animals were lame (data not shown) and 4/6 displayed characteristic SVA lesion on feet (Fig.7A). Only 3/6 animals developed lameness (data not shown) and clinical lesion (Fig.7A) in inactivated group. It is interesting to note that none of the animal developed lesion on snout and lesions were mostly confined on coronary band and sole. In both groups, lesion developed on day 4 post-challenge (pc), however, no lameness or lesion were observed on live vaccine group. Peak clinical score was detected in day 6 pc in control and day 9 pc in inactivated group (Fig.7B). Lesions resolved after 13 days pc in inactivated group but was still present in 14 days pc in control. As no gross lesions were observed in both live vaccine groups, clinical scores for these groups remained 0 throughout the remaining experiment (Fig.7B).

The level of viremia was also assessed post-challenge in serum collected on days 0, 3, 7, 10 and 14 pc by RT-qPCR. SVA RNA was detected on day 3 pc on control, inactivated and live IN groups only, where control had significantly higher (p<0.01) genome copy than other two groups (Fig.7C). SVA genome of live IN was detected only on day 3 pc and live IM group only on day 7 pc (Fig.7C). Viremia on control and inactivated group was observed on day 10 pc and not observed afterwards (Fig.7C).

Virus shedding was assessed in oral and nasal secretions and feces in all group on oral, nasal and rectal swabs collected on days 0, 3, 7, 10 and 14 pc by RT-qPCR. Virus shedding was detected until the end of the experiment on day 14 pi in animals of control and inactivated group (Fig.7D, E and F). Virus shedding on Live IM and IN group was significantly less (p<0.01) compared to control and inactivated in both oral and nasal secretion (Fig.7D and E) and no virus shedding was detected in these two live groups in fecal swabs.

Virus load was also investigated in lymphoid tissues using RT-qPCR. Viral load in tonsil, mediastinal and mesenteric lymph nodes are presented in Fig.8 A, B and C. The tonsil presented the highest viral load compared to other tissues. SVA RNA was significantly high (p<0.01) in control compared to other three groups (Fig.8A). No significant difference (p>0.05) in SVA RNA copy number was observed among other three groups (Fig.8A). In mediastinal lymph node, SVA RNA was significantly high (p<0.01) in control compared to Live IM and IN groups (Fig.8B). Similar result was observed in Mesenteric lymph node (Fig.8C). Moreover, SVA RNA was not detected in any animal of Live IN group in mediastinal lymph node (Fig.8C).

The serological responses post-immunization and post-challenge were evaluated by virus neutralization assay (VN). All immunized animal seroconverted and high level of neutralizing antibodies (NA) were observed starting day 3 pi on live IM and IN group and day 14 on inactivated group. Live IM had significantly higher (p<0.01) NA on day 3 and 5 compared to all groups but after day 7 pi Live IN also showed similar NA as IM group (Fig 6). NA in inactivated was lower compared to live vaccinated group post-immunization. There was slight increase in NA in inactivated group on day 28 pi due to booster immunization on day 21 pi. After 7 days post-challenge, there was sharp increase in NA on inactivated and control group, however, NA level on live vaccine group was consistent till the end of experiment. This consistent level of NA in live group probably explains the absence of clinical sign, transient viremia and negligible virus shedding in these groups.

SVA not only causes economic losses but its similarity to FMD which is OIE-listed disease (world wide web at http://oie.int) is of major concern for swine industry. The notification of OIE-listed disease can be deleterious to pig production for any country including investigation, restriction of animal movement, culling of animals, export restriction on animal products (24). Use of SVA as oncolytic agent in human may be beneficial but its association with swine vesicular disease is detrimental. The use of vaccine can help limit the spread of SVA in current endemic population.

Viruses and cells

H1299 cells were purchased from the American Type Culture Collection (ATCC- CRL 5803). Cells were maintained at 37 ^o C with 5% CO ₂ in RPMI-1640 medium (Corning, NY) supplemented with 10% fetal bovine serum (VWR, Chicago, IL) and 2mM L- Glutamine (Corning, NY). Penicillin (100 U/mL) and streptomycin (100 µg/mL) were also added to culture media.

SVA strain SD15-26 was isolated from swine presenting vesicular disease and has been previously characterized in our laboratory (19, 20). SVA strain MN15-84-21 was also isolated from swine presenting vesicular disease and characterized in our laboratory (11). For both virus strain low-passage (passage 4) stocks were prepared, titrated and used in experiments described below.

Generation of rSVA mSacII. Growth curves

Replication kinetics of wt SVA SD15-26 and rSVA mSacII were assessed in vitro. H1299 and PK-15 cells were cultured in six-well plates, inoculated with both virus at a

multiplicity of infection (MOI) of 0.1 (multi-step growth curve) or 10 (single-step growth curve), and har- vested at various time points post-infection (2, 4, 8, 12 and 24 h post- infection). Virus titres were determined for each time point using Spearman and Karber’s method and expressed as TCID ₅₀ /ml.

Western blots

Western blot was done to see any variation in protein level of live and inactivated virus. For both live and inactivated virus, 20µL of sample was taken and mixed with 4× Lamelli Buffer (Bio-Rad, Hercules, CA) containing 5% b-mercaptoethanol and denatured at 95 ^o C for 10 minutes then loaded onto 10% SDS-PAGE gel. Gel was run at 90 volts for 90 minutes and was transferred to nitrocellulose paper and blocked on 5% skim-milk on 1× phosphate-buffered saline (PBS) overnight at 4 ^o C. Nitrocellulose paper was washed 3 times in 1×PBS supplemented with 0.05% Tween 20 (PBST) and incubated with anti-VP1 and anti-VP2 anti-mouse antibody (1:1000) in 0.05% PBST for 2 hours at RT. They were washed again 3 times with 0.05% PBST. IRDye® 800CW Goat anti-Mouse IgG (H+L) (LI-COR Biosciences, Lincoln, NE) was used as secondary antibody (1:15000 on 1% skim-milk on 0.05% PBST) and incubated for 1 hour at RT. It was washed 3 times with PBST 0.05% and observed in LI-COR® Odyssey® Fc Imaging system (LI-COR

Biosciences, Lincoln, NE).

Animal studies.

Pathogenesis experiment

Immunization-challenge experiment

Immunization of SVA vaccines were evaluated in 28 days old 24 pigs at SDSU Animal Resource Wing (ARW). Six animals were kept in each room corresponding to treatment groups, viz: control (non-immunized and receiving plain RPMI 1640), inactivated (receiving BEI inactivated vaccine), Live IM (receiving rSVA mSacII by intramuscular route) and Live IN (receiving rSVA mSacII by intranasal route), and received food and water ad libitum. Strict biosecurity protocols were followed to avoid cross contamination. After one week of acclimatization, immunization was done as presented in Table 1. Inactivation of virus by binary ethylenimine (BEI) was done as previously described (25). The water in oil in water (W/O/W) vaccine was produced by shear-mixing equal volume of oil adjuvant Seppic MONTANIDE ^TM ISA 201 VG (Seppic SA, Paris) with BEI inactivated virus at 31 ^o C in syringes joined by coupler.

Animals were monitored daily for signs and lesions throughout the experiment. Oral, nasal and rectal swabs were collected on days 0, 3, 7, 14, 21, 28- and 35-days post- immunization. Blood was collected on days 0, 3, 5, 7, 14, 21, 28- and 35-days post- immunization. Serum separation by centrifugation and PBMCs isolation by density gradient centrifugation were done as previously described (19, 20).

A heterologous SVA strain (SVA MN15-84-22) was used as challenge virus and animals in all groups were challenged on day 42 post-immunization. Blood and swabs were collected on days 3, 7, 10 and 14 post-challenge and processed and stored as above. All animals were euthanized on day 14 post-challenge at Animal Disease Research and Diagnostic Laboratory (ADRDL), SDSU. Tissues (tonsil, mediastinal and mesenteric lymph nodes) were collected and stored at -80 ^o C or fixed in 10% formalin. Animal experiments were revised and approved by the SDSU Institutional Animal Care and Use Committee (approval number 18-032A).

Real-time PCR.

Nucleic acid was extracted from serum, swabs and tissue samples using cador® Pathogen 96 QIAcube® HT kit (Qiagen). For tissue, approximately 0.5g of each tissue was minced using sterile scalpel, re-suspended in RPMI 1640 medium (10% w/v) and homogenized using stomacher (2 cycles of 60s). Homogenized samples were then centrifuged at 14,000 ×g for 2 minutes at room temperature and 200 µL of cleared supernatant was used for nucleic acid extraction using automated QIAcube HT (Qiagen). Swab samples were vortexed and cleared by centrifugation and 200 µL of supernatant was used for nucleic acid extraction as above. Two hundred uL of serum was also used for nucleic acid extraction. RNA extraction control (Bioline, MA, USA) was also added during nucleic acid extraction for all samples. The presence of SVA RNA in samples were assessed using commercial RT-qPCR reagents (Bioline, MA, USA). Primers and a probe targeting the conserved portion of SVA 3D gene were designed using PrimerQuest Tool (Integrated DNA Technologies Inc., USA). The probe and primers sequence are 5'-/56- /C GG C C/ N/ C CG G GC GC /3 Q/ 3, 5

GAAGCCATGCTCTCCTACTTC 3' and

respectively. RT-qPCR was performed using a SensiFast ^TM Probe Lo-ROX One-Step Kit (Bioline, MA, USA) following manufacturer’s instructions. Amplification and detection were performed with an Applied Biosystems 7500 real time PCR system under following conditions: 10 minutes at 45 ^o C for reverse transcription, 2 minutes at 95 ^o C for polymerase activation and 40 cycles of 5 seconds at 95 ^o C for denaturation and 30 seconds at 60 ^o C for annealing and extension. A standard curve was established by using SVA SD15-26 virus of titer 10 ^7.88 TCID50/ml and preparing 10-fold serial dilutions from 10 ^-1 to 10 ^-10 . Viral genome copy number equivalent to TCID50/ml was calculated based on the standard curve determined using the four- parameter logistic regression model function within MasterPlex Readerfit 2010 software (Hitachi Software Engineering America, Ltd., San Francisco, CA). The amount of viral RNA detected in samples were expressed as log ₁₀ (genome copy number)/mL

Neutralization assays

Neutralizing antibody (NA) response by vaccine and challenge virus were assessed using a virus neutralization assay as previously described (19). NA titers were expressed as log ₂ (reciprocal of highest serum dilution capable of completely inhibiting SVA infection). All assays were performed in triplicate including positive and negative control in all test plates.

Flow cytometry

Statistical analysis

Statistical analysis was performed by analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. Normality were checked before performing any tests. Statistical analysis and data visualization were performed using GraphPAD Prism

8.0.1(244) software (GraphPAD Software Inc., La Jolla, CA).

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EXAMPLE 2

SEQUENCES

While specific embodiments have been described above with reference to the disclosed embodiments and examples, such embodiments are only illustrative and do not limit the scope of the invention. Changes and modifications can be made in accordance with ordinary skill in the art without departing from the invention in its broader aspects as defined in the following claims.

All publications, patents, and patent documents are incorporated by reference herein, as though individually incorporated by reference. The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.

The inventions being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the inventions.