EA045363B1 - VACCINE AGAINST PORGINE PARVOVIRUS - Google Patents

Description

List of sequences

This patent application contains sequence listings in accordance with 37 C.F.R. 1.821-1.825. Accordingly, the sequence listing appended to this application is incorporated by reference in its entirety.

State of the art

Porcine parvovirus is an autonomous replicating virus of the family Parvovirinae of the genus Protoparvovirus within the family Parvoviridae, containing a single-stranded DNA molecule of approximately 5100 nucleotides (Cotmore et al., 2014: Arch Virol.: 159(5): 1239-1247; Molitor et al., 1984: Virology: 137(2): 241-54). Only the minus strand of DNA is packaged into virions. The viral genome encodes three capsid proteins (VP1, VP2, VP3) and one non-structural protein (NS1). The parvovirus capsid has a diameter of approximately 22-25 nanometers and is composed of VP1 and VP2 subunits. These proteins are derived from alternately spliced variants of the same RNA molecule and thus partially overlap in sequence. In addition, porcine parvovirus exhibits high levels of sequence similarity to feline panleukopenia virus, canine parvovirus, and rodent parvovirus (Ranz et al., 1989: J. gen. Virol: 70: 2541-2553).

Although there are differences in strains of porcine parvovirus, some being highly pathogenic and others less pathogenic or not at all pathogenic, when the virus becomes established or becomes endemic in a given country, pathogenic strains appear to spread throughout the population.

Porcine parvovirus (PPV) infection is a common cause of reproductive failure in pig production worldwide. Serological studies demonstrate that porcine parvovirus is common in all pig-producing regions of the world, with up to 80% of animals showing seroconversion.

Porcine parvovirus (PPV) causes reproductive failure in pigs, leading to death and intrauterine mummification, stillbirth and other manifestations of reproductive failure in pregnant sows. (Joo & Johnson. 1976. Veterinary Bulletin 46, 653-660; Mengeling. 1978. J. Am. Vet. Med. Assoc. 172, 1291-1294).

PPV causes reproductive failure when susceptible (non-immune) pigs and sows become infected during pregnancy. This is the only time the virus causes illness. Infection of pigs occurs due to consumption of the virus in food or inhalation. PPV then circulates in the circulatory system and, in pregnant pigs, crosses the placenta and infects developing embryos and fetuses. After natural infection, active immunity develops, which can last throughout the life of the pig. If active immunity is acquired before pregnancy, developing piglets are not at risk. At birth, piglets acquire maternal immunity in the sow's colostrum, and this maternal immunity persists until 20 weeks of age. The higher the level of active immunity in a sow, the more maternal immunity is passed on to her piglets. Natural PPV infection may subsequently occur.

The disease caused by PPV in piglets is often called SMEDI (an acronym for stillbirth, mummification, embryonic death and infertility). If infection occurs between days 0-30 of gestation, it can result in embryonic mortality, resulting in reduced litter size. The most obvious feature that occurs as a result of infection at 30-70 days of pregnancy is the birth of mummified piglets. Mummification is the process of sterile digestion of tissue from piglets that die in the uterus after skeletal hardening begins. PPV infection is also associated with stillbirth and weak piglets if infection occurs late in pregnancy. Miscarriage can also result from PPV infection, but is not a common clinical feature of this disease. In general, PPV infection reduces the number of piglets born per sow per year.

Currently available PPV vaccines are produced by growing the native virus in primary porcine-derived cells or in stable cell lines. The infectious virus is then isolated and inactivated with chemical agents to ultimately produce a whole cell killed antiviral vaccine. However, such processes for growing native infectious virus are problematic from a biosecurity and safety perspective. Thus, there is a need for recombinant PPV vaccines.

Subunit vaccines based on recombinant proteins may have poor immunogenicity due to misfolding of the target protein or improper presentation to the immune system. In addition, whole cell killed vaccines present all antigens of the native virus, whereas a subunit vaccine is limited to a specific amino acid sequence.

Recombinant PPV vaccines have already been described in the prior art, but to date only whole cell killed vaccines have been commercialized. Thus, it appears that optimal recombinant subunit vaccines against PPV have not yet been developed to demonstrate efficacy and safety. Described to date

- 1 045363 At this point, recombinant subunit PPV vaccines have not been tested or monitored in laboratory challenge experiments. The recombinant subunit PPV vaccines evaluated did not perform as well as whole cell PPV vaccines, or the recombinant subunit PPV vaccines were not safe (showed adverse reactions). Thus, there remains a need for recombinant subunit PPV vaccines that are highly effective and safe.

Field isolates of porcine parvovirus (PPV) have been identified that are genetically and antigenically distinct from vaccine strains. PPV genotype 2 virus, PPV-27a, is highly virulent in pregnant pigs following challenge, as demonstrated by the high fetal mortality of sows infected with PPV-27a (85%) compared to sows infected with other PPV strains, e.g. -NADL-2. However, currently available commercially available PPV vaccines are based on inactivated whole virus preparations of genotype 1 PPV strains isolated about 30 years ago (Jozwik et al 2009; Journal of General Virology; 90; 2437-2441). Thus, there is a need for new vaccines that protect against new highly virulent pathogenic strains of PPV that are more consistent with PPV in this area.

Other sources of prior art include the following.

EP 0 551 449 A1 discloses a method for producing a VP2 subunit vaccine against porcine parvovirus.

In the publication Cadar D et al. (Infection, Genetics and Evolution 2012, 12: 1163-1171) describes the phylogeny and evolutionary genetics of porcine parvovirus in wild pigs.

In the publication Streck AF et al. (Journal of General Virology 2011, 92: 2628-2636) describes a high rate of viral evolution in the capsid protein of porcine parvovirus.

WO 88/02026 concerns empty viral capsid vaccines.

In Martinez C et al. (Vaccine 1992, 10(10): 684-690) discloses the production of empty porcine parvovirus capsids with high immunogenic activity.

In Xu F et al. (Applied and Environmental Microbiology 2007, 73(21): 7041-7047) describes the induction of immune responses in mice after intragastric administration of the bacterium Lactobacillus casei, which produces the porcine parvovirus VP2 protein.

Moreover, there is a need for new and improved vaccines against highly pathogenic PPV strains that provide broader protection against different (heterologous) PPV strains (cross protection).

Detailed Description of the Invention

Before describing aspects of the present invention, it should be noted that as used in the specification and the accompanying claims, the singular form of the singular also includes references to the plural unless the context clearly indicates otherwise. Thus, for example, a reference to an antigen includes a plurality of antigens, a reference to a virus is a reference to one or more viruses and their equivalents known to those skilled in the art, and so on. Unless otherwise defined, all technical and scientific terms used by the authors have their meanings as commonly understood by those skilled in the art to which the present invention relates. Although any methods and materials similar or equivalent to those described by the authors may be used in the practice or testing of the present invention, preferred methods, devices and materials are described herein. All publications mentioned by the authors are incorporated herein by reference for the purpose of describing and disclosing the cell lines, vectors and methodologies reported in the publications that may be used in connection with the invention. Nothing contained in this specification should be construed as an admission that an invention is not entitled to be opposed to such disclosure by virtue of an earlier invention.

The present invention solves the problems inherent in the prior art and provides a clear advance over the prior art. In general, the present invention provides porcine parvovirus (PPV) viral protein 2 (VP2) having:

at amino acid position 228 there is a glutamic acid residue or a glutamate residue; and/or at amino acid position 414 a serine residue, and/or at amino acid position 419 a glutamine residue; and/or at amino acid position 436 a threonine residue; wherein the numbering of amino acid positions refers to the amino acid sequence of wild type PPV VP2.

It is an advantage that the experimental data provided by the present invention reveal that the PPV VP2 subunit vaccine according to the present invention is safe and effective for the prevention of PPV viremia and fetal infection. In addition, the experimental data provided by the present invention reveal that the vaccine in accordance with the present invention has a broad spectrum of protection as the vaccine protects against heterologous North American as well as heterologous European challenge strains of PPV.

- 2 045363

It is an advantage that the experimental data provided by the present invention reveals that the PPV VP2 subunit vaccine according to the present invention is as effective as the whole killed virus, and this effect was obtained unexpectedly. However, extensive inactivation processes (which are necessary to inactivate native PPVs when preparing whole killed virus vaccines) can be avoided by using a recombinant subunit vaccine consisting of PPV VP2.

The term porcine parvovirus or PPV is well known among those skilled in the art. However, porcine parvovirus is an autonomous replicating virus of the parvovirus genus within the family Parvoviridae, containing a single-stranded DNA molecule. The viral genome encodes three capsid proteins (VP1, VP2, VP3) and one non-structural protein (NS1). The disease caused by PPV in piglets is often called SMEDI (an acronym for stillbirth, mummification, embryonic death and infertility). The term porcine parvovirus covers all possible strains, genotypes, phenotypes and serotypes of porcine parvovirus.

The term viral protein 2 or VP2 refers to the capsid protein VP2 of porcine parvovirus. The term viral protein 2 or VP2 is well known among those skilled in the art.

The terms protein, amino acid and polypeptide are used interchangeably. The term protein refers to a sequence of amino acids consisting of naturally occurring amino acids as well as their derivatives. Natural amino acids are well known among experts in the field and are described in standard biochemistry textbooks. Within an amino acid sequence, amino acid residues are connected by peptide bonds. In addition, the two ends of the amino acid sequence are indicated as the carboxyl terminus (C-terminus) and the amino terminus (N-terminus). The term protein covers essentially purified proteins or protein preparations, further including other proteins. In addition, the term also refers to protein fragments. In addition, it includes chemically modified proteins. Such modifications may be artificial modifications or natural modifications such as phosphorylation, glycosylation, myristylation, and the like.

The term wherein the numbering of amino acid positions refers to the amino acid sequence of the wild-type PPV VP2 refers to the numbering of amino acid positions related to the amino acid sequence of the full-length wild-type PPV VP2 protein. Preferably, the amino position numbering herein is indicated with reference to the wild-type PPV VP2 protein sequence having 579 amino acid residues, including the methionine residue at (N-terminal) amino acid position 1. The term wherein the amino position numbering refers to the wild-type PPV VP2 amino acid sequence encompasses PPV Wild-type VP2 as exemplified in SEQ ID NO: 1 (PPV 27a VP2).

In one aspect of the present invention, the PPV VP2 also has:

at amino acid position 25 isoleucine residue; and/or at amino acid position 36 a serine residue; and/or at amino acid position 37 an isoleucine residue.

Thus, the present invention provides porcine parvovirus (PPV) viral protein 2 (VP2) having:

at amino acid position 228 there is a glutamic acid residue or a glutamate residue; and/or at amino acid position 414 a serine residue; and/or at amino acid position 419 a glutamine residue, and/or at amino acid position 436 a threonine residue; wherein the numbering of amino acid positions refers to the amino acid sequence of wild type PPV VP2.

PPV VP2 also features:

at amino acid position 25 isoleucine residue; and/or at amino acid position 36 a serine residue; and/or at amino acid position 37 an isoleucine residue.

In one aspect of the present invention, the numbering of amino acid positions refers to the amino acid sequence shown in SEQ ID NO: 1. Thus, the present invention provides porcine parvovirus (PPV) viral protein 2 (VP2) having:

at amino acid position 228 there is a glutamic acid residue or a glutamate residue; and/or at amino acid position 414 a serine residue; and/or at amino acid position 419 a glutamine residue; and/or at amino acid position 436, a threonine residue, wherein the amino acid position numbering refers to the amino acid sequence of the wild type PPV VP2, and the amino acid position numbering refers to the amino acid sequence shown in SEQ ID NO: 1.

Thus, the present invention provides porcine parvovirus (PPV) viral protein 2 (VP2) having:

at amino acid position 228 there is a glutamic acid residue or a glutamate residue; and/or at amino acid position 414 a serine residue, and/or at amino acid position 419 a glutamine residue; and/or

- 3 045363 at amino acid position 436 is a threonine residue, the numbering of amino acid positions referring to the amino acid sequence shown in SEQ ID NO: 1.

In one aspect of the present invention, PPV VP2 is recombinant PPV VP2.

The term recombinant as used herein refers specifically to a protein molecule that is expressed from a recombinant DNA molecule, such as a polypeptide generated using recombinant DNA technologies. An example of such technologies involves where the DNA encoding the protein to be expressed (eg PPV VP2) is inserted into a suitable expression vector, preferably a baculovirus expression vector, which in turn is used for transfection or, in the case of a baculovirus expression vector, for infection of a cell -host to obtain a protein or polypeptide encoded by DNA. The term recombinant PPV VP2 as used herein thus specifically refers to a protein molecule that is expressed from a recombinant DNA molecule.

In one aspect of the present invention, PPV VP2 is a recombinant baculovirus-expressed PPV VP2.

The term baculovirus or baculovirus system is well known among those skilled in the art. Additionally, the term baculovirus is explained in more detail below.

In one aspect of the present invention, the above PPV VP2 includes or consists of an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 5-16.

In one aspect of the present invention, the above PPV VP2 includes or consists of an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3% , at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identical sequences with the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 5-16. Thus, the present invention provides porcine parvovirus (PPV) viral protein 2 (VP2) having:

at amino acid position 228 there is a glutamic acid residue or a glutamate residue; and/or at amino acid position 414 a serine residue; and/or at amino acid position 419 a glutamine residue; and/or at amino acid position 436, a threonine residue, wherein the amino acid position numbering refers to the amino acid sequence of the wild type PPV VP2, and wherein said PPV VP2 includes or consists of an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99, 7%, at least 99.8%, or at least 99.9% sequence identity with the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 5-16.

In one aspect of the present invention, the above PPV VP2 includes or consists of the amino acid sequence of SEQ ID NO: 1, or SEQ ID NO: 2 or SEQ ID NO: 5-16 or includes or consists of any fragment that has at least 210, at least 250 or at least 300 contiguous amino acid residues from SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 5-16. Thus, the present invention provides porcine parvovirus (PPV) viral protein 2 (VP2) having:

at amino acid position 228 there is a glutamic acid residue or a glutamate residue; and/or at amino acid position 414 a serine residue; and/or at amino acid position 419 a glutamine residue; and/or at amino acid position 436, a threonine residue, wherein the amino acid position numbering refers to the amino acid sequence of the wild type PPV VP2, and wherein said PPV VP2 includes or consists of the amino acid sequence SEQ ID NO: 1, or SEQ ID NO: 2, or SEQ ID NO : 5-16 or includes or consists of any fragment that has at least 210, at least 250 or at least 300 contiguous amino acid residues from SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 5- 16.

In one aspect of the present invention, the above PPV VP2 includes or consists of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 5-16. Thus, the present invention provides porcine parvovirus (PPV) viral protein 2 (VP2) having:

at amino acid position 228 there is a glutamic acid residue or a glutamate residue; and/or at amino acid position 414 a serine residue; and/or at amino acid position 419 a glutamine residue; and/or at amino acid position 436 a threonine residue, and the numbering of amino acid positions

- 4045363 refers to the amino acid sequence of wild type PPV VP2, and wherein said PPV VP2 includes or consists of the amino acid sequence of SEQ ID NO: 1, or SEQ ID NO: 2, or SEQ ID NO: 5-16.

In one aspect of the present invention, the aforementioned PPV VP2 is encoded by a nucleotide sequence encoding an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 5-16.

In one aspect of the present invention, the above PPV VP2 is encoded by a nucleotide sequence encoding the amino acid sequence of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% , at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, according to at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity with amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 5-16. Thus, the present invention provides porcine parvovirus (PPV) viral protein 2 (VP2) having:

at amino acid position 228 there is a glutamic acid residue or a glutamate residue; and/or at amino acid position 414 a serine residue; and/or at amino acid position 419 a glutamine residue; and/or at amino acid position 436, a threonine residue, wherein the numbering of amino acid positions refers to the amino acid sequence of the wild type PPV VP2, and the above PPV VP2 is encoded by a nucleotide sequence encoding an amino acid sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99, 7%, at least 99.8%, or at least 99.9% sequence identity with the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 5-16.

In one aspect of the present invention, the above PPV VP2 is encoded by a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 5-16. Thus, the present invention provides porcine parvovirus (PPV) viral protein 2 (VP2) having:

at amino acid position 228 there is a glutamic acid residue or a glutamate residue; and/or at amino acid position 414 a serine residue; and/or at amino acid position 419 a glutamine residue; and/or at amino acid position 436, a threonine residue, wherein the numbering of amino acid positions refers to the amino acid sequence of the wild type PPV VP2, and the above-mentioned PPV VP2 is encoded by a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 5-16.

SEQ ID NO: 4 is the codon-optimized nucleotide sequence of PPV 27a VP2, which has been further modified to include two ClaI restriction enzyme sites (amino acid position 25 contains an isoleucine residue, amino acid position 36 contains a serine residue, amino acid position position 37 is an isoleucine residue) to be adjacent to the VP2 coding region, which consists of glycine repeats. However, ClaI restriction enzyme sites were included so as not to interrupt the VP2 coding region. SEQ ID NO: 2 is the protein sequence corresponding to SEQ ID NO: 4. SEQ ID NO: 3 is the codon-optimized nucleotide sequence of PPV 27a VP2 (without ClaI restriction enzyme sites). SEQ ID NO: 1 represents the protein sequence corresponding to SEQ ID NO: 3. SEQ ID NO: 5-16 represents other PPV VP2 protein sequences with or without ClaI restriction enzyme sites (SEQ ID NO: 5-10) NO: 11-16).

The terms nucleic acid or nucleic acid sequence or nucleotide sequence or polynucleotide are used interchangeably herein and refer to polynucleotides, including DNA molecules, RNA molecules, cDNA molecules or derivatives. The term covers single-stranded as well as double-stranded polynucleotides. Nucleic acid in accordance with the present invention includes isolated polynucleotides (ie, isolated from the natural environment) and genetically modified forms. Also included are chemically modified polynucleotides, including naturally occurring modified polynucleotides, such as glycosylated or methylated polynucleotides, or artificially modified polynucleotides, such as biotinylated polynucleotides. Additionally, the terms nucleic acid and polynucleotide are used interchangeably and refer to any nucleic acid. The terms nucleic acid and polynucleotide also specifically include nucleic acids consisting of the base

- 5 045363 ions that differ from biological bases (adenine, guanine, thymine, cytosine and uracil).

The term identity or sequence identity is known to those skilled in the art and refers to the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, i.e. a control sequence and a given sequence to be compared with the control sequence. Sequence identity is determined by comparing a given sequence to a reference sequence after optimal sequence alignment to obtain the highest degree of sequence similarity determined by comparison between strands of such sequences. After such an alignment, sequence identity is established based on the comparison of corresponding positions, for example, sequences are identical at a particular position if that position, nucleotides or amino acid residues are identical. The total number of such identical positions is then divided by the total number of nucleotides or residues in the reference sequence to determine the percentage of sequence identity. Sequence identities are readily calculated by known methods including, but not limited to, those described in Computational Molecular Biology, Lesk, A. N., ed., Oxford University Press, New York (1988), Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology, von Heinge, G., Academic Press (1987); Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York (1991); and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988), the ideas of which are incorporated herein by reference. Preferred methods for determining sequence identity are designed to obtain the closest match between the sequences tested. Methods for determining sequence identity are codified in publicly available computer programs that determine sequence identity between given sequences. Examples of such programs include, but are not limited to, the GCG software package (Devereux, J., et al., Nucleic Acids Research, 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., J Molec Biol 215: 403-410 (1990) The BLASTX program is commercially available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCVI NLM NIH Bethesda, MD 20894, Altschul, S. F. et al. al., J. Molec. Biol., 215: 403-410 (1990), the ideas of which are incorporated herein by reference.) These programs optimally align sequences using default deletion penalties to obtain the highest level of sequence identity between a given and control sequences. By way of illustration, in the case of a polynucleotide having a nucleotide sequence that has at least, for example, 85%, preferably 90%, even more preferably 95% sequence identity with a control nucleotide sequence, it is intended that the nucleotide sequence of the polynucleotide is identical to the control sequence, except that the polynucleotide sequence may include up to 15, preferably up to 10, even more preferably up to 5 point mutations for every 100 nucleotides of the control nucleotide sequence. In other words, in a polynucleotide having a nucleotide sequence that has at least 85%, preferably 90%, even more preferably 95% identity with a reference nucleotide sequence, up to 15%, preferably up to 10%, even more preferably up to 5% nucleotides in the control sequence may be deleted or replaced by another nucleotide, or a certain amount of nucleotides up to 15%, preferably up to 10%, even more preferably up to 5% of the total number of nucleotides in the control sequence may be inserted into the control sequence. These control sequence mutations may occur at the 5' or 3' terminal positions of the control nucleotide sequence or anywhere between these terminal positions, either as individual insertions among nucleotides in the control sequence, or by one or more contiguous groups within the control sequence. Likewise, in the case of a polypeptide having a given amino acid sequence that has at least, for example, 85%, preferably 90%, even more preferably 95% sequence identity with a control amino acid sequence, it is intended that the given amino acid sequence of the polypeptide is identical to the control sequence, except that a given polypeptide sequence may include up to 15, preferably up to 10, even more preferably up to 5 amino acid changes for every 100 amino acids of the reference amino acid sequence. In other words, to obtain a given polypeptide sequence that has at least 85%, preferably 90%, even more preferably 95% sequence identity with a reference amino acid sequence, up to 15%, preferably up to 10%, even more preferably up to 5% amino acid residues in the control sequence may be deleted or replaced by another amino acid, or a certain amount of amino acids up to 15%, preferably up to 10%, even more preferably up to 5% of the total amino acid residues in the control sequence may be inserted into the control sequence. These changes control

- 6 045363 of the amino acid sequence may occur at the amino or carboxyl terminal positions of the control amino acid sequence or at any other location between these terminal positions, or individual insertions among residues in the control sequence, or one or more contiguous groups within the control sequence. Preferably, positions of residues that are not identical are distinguished by conservative amino acid substitutions. However, conservative substitutions are not included as hits when determining sequence identity.

The terms identity, sequence identity, and percentage identity are used interchangeably herein. In terms of this invention, it is found that to determine the percentage identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for the purpose of optimal comparison (for example, gaps may be included in the sequence of the first amino acid or nucleic acid for optimal alignment for the purpose of comparison with the second amino acid or nucleic acid sequence). The amino acids or nucleotide residues at the corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid or nucleotide residue as the corresponding position in the second sequence, the molecules at that position are identical. The percentage identity between two sequences depends on the number of identical positions shared by the sequences [i.e. % identity = number of identical positions / total number of positions (i.e. overlapping positions) x 100]. Preferably the two sequences are of the same length.

Sequence comparison is carried out along the entire length of the two sequences being compared or along the length of a fragment of two sequences. Typically, comparisons are made over the entire length of the two sequences being compared. However, sequence identity can also be determined by a region, for example, of twenty, fifty, one hundred or more amino acid residues.

Those skilled in the art will recognize that several different computer programs are available for determining homology between two sequences. For example, comparing sequences and determining the percentage of identity between two sequences is carried out using a mathematical algorithm. In a preferred embodiment, the percentage identity between two amino acid or nucleic acid sequences is determined using the Needleman-Wunsch algorithm [J. Mol. Biol. (48): 444-453 (1970)], included in the GAP program in the Accelrys GCG software package (available at http://www.accelrys.com/products/gcg/), using a Blosum 62 matrix or a PAM250 matrix, and a penalty for opening a gap of 16, 14, 12, 10, 8, 6 or 4 and a penalty for extending a gap of 1, 2, 3, 4, 5 or 6. Those skilled in the art will appreciate that all of these different parameters produce slightly different results, but the overall percent identity of the two sequences does not change significantly when applying different algorithms.

Protein sequences or nucleic acid sequences in accordance with the present invention can also be used as a search sequence to perform searches in public databases, for example, to identify other members of the family or related sequences. Such searches are performed using the programs BLASTN and BLASTP (version 2.0) as published by Altschul, et al. (1990) J. Mol. Biol. 215:403-10. A BLAST protein search is performed using the BLASTP program, weight =50, word length =3, to obtain amino acid sequences homologous to protein molecules in accordance with the invention. Gapped BLAST is used to obtain gap alignments for comparison purposes, as described in Altschul et al. (1997) Nucleic Acids Res. 25(17): 3389-3402. When using the BLAST and Gapped BLAST programs, the default parameters of the corresponding programs (for example, BLASTP and BLASTN) are used. See the National Center for Biotechnology Information home page at http://www.ncbi.nlm.nih.gov/.

In addition, the present invention provides an immunogenic composition comprising PPV VP2, as described by the authors. Thus, the present invention provides an immunogenic composition comprising porcine parvovirus (PPV) viral protein 2 (VP2) having:

at amino acid position 228 there is a glutamic acid residue or a glutamate residue; and/or at amino acid position 414 a serine residue; and/or at amino acid position 419 a glutamine residue; and/or at amino acid position 436 a threonine residue, wherein the numbering of amino acid positions refers to the amino acid sequence of the wild type PPV VP2.

The term immunogenic composition refers to a composition that includes at least one antigen that elicits an immune response in the host to which the immunogenic composition is administered. Such an immune response may be a cellular and/or antibody-mediated immune response to the immunogenic composition of the invention. Preferably, the immunogenic composition induces an immune response, more preferably providing protective immunity against one or more clinical signs of PPV infection. The owner is also described as

- 7 045363 subject. Preferably, any of the hosts or subjects described or mentioned is a pig.

Typically, the immune response includes, but is not limited to, one or more of the following effects: production or activation of antibodies, B cells, helper T cells, suppressor T cells and/or cytotoxic T cells and/or gamma delta T -cells specifically directed to the antigen or antigens included in the immunogenic composition in accordance with the invention. Preferably, the host should demonstrate a protective immune response or therapeutic response.

A protective immune response or protective immunity is demonstrated through a reduction or absence of clinical signs typically found in infected hosts, faster recovery and/or a reduced duration of infectivity, or a reduced titer of the pathogen in the tissues or body fluids or feces of the infected host.

In the event that the host demonstrates a protective immune response, such as increased resistance to a new infection and/or reduced clinical severity of the disease, the immunogenic composition is described as a vaccine.

In one aspect of the present invention, the immunogenic composition is formulated for administration in a single dose.

The volume of a single dose is determined in other sections of this description.

The immunogenic composition is preferably administered locally or systemically. Suitable conventionally used routes of administration are oral or parenteral administration, such as intranasal, intravenous, intramuscular, intraperitoneal, subcutaneous, and inhalation. However, depending on the nature and mode of action of the compound, the immunogenic composition may also be administered by other routes. However, in the most preferred embodiment, the immunogenic composition is administered intramuscularly.

In one aspect of the present invention, the immunogenic composition is administered intramuscularly.

In one aspect of the present invention, the immunogenic composition is safe for pigs and/or sows during pregnancy and lactation.

In one aspect of the present invention, the immunogenic composition is safe for pigs and/or sows after 30 days of pregnancy, preferably after 40 days of pregnancy.

In one aspect of the present invention, the immunogenic composition also includes a pharmaceutically acceptable carrier.

The term pharmaceutically acceptable carrier includes any and all solvents, dispersants, coatings, stabilizers, diluents, preservatives, antimicrobial and antifungal agents, isotonic agents, adsorption retarding agents, adjuvants, immunostimulants, and combinations thereof.

Diluents may include water, saline, dextrose, ethanol, glycerin, and the like. Isotonic agents may include, but are not limited to, sodium chloride, dextrose, mannitol, sorbitol, and lactose. Stabilizers include, but are not limited to, albumin and alkali metal salts of ethylenediaminetetraacetic acid.

In one aspect of the present invention, the pharmaceutically acceptable carrier is a carbomer.

Preferably, the immunogenic composition may also include one or more other immunomodulators, such as, for example, interleukins, interferons or other cytokines. The amount and concentration of adjuvants and additives used in the context of the present invention can be easily determined by one skilled in the art.

In some aspects, the immunogenic composition of the present invention contains an adjuvant. Adjuvants as used herein may include aluminum hydroxide and aluminum phosphate, saponins e.g. Quil A, QS-21 (Cambridge Biotech Inc., Cambridge MA), GPI-0100 (Galenica Pharmaceuticals, Inc., Birmingham, AL), water emulsion in oil, oil-in-water emulsion, water-in-oil-in-water emulsion. The emulsion, in particular, can be based on light liquid vaseline oil (according to the requirements of the European Pharmacopoeia); isoprenoid oil such as squalane or squalene; oils obtained from the oligomerization of alkenes, in particular isobutene or decene; esters of acids or alcohols that contain a linear alkyl group, more particularly vegetable oils, ethyl oleate, propylene glycol di-(caprylate/caprate), glyceryl tri-(caprylate/caprate) or propylene glycol dioleate; esters of branched fatty acids or alcohols, in particular isostearic acid esters. The oil is used in combination with emulsifiers to form an emulsion. The emulsifiers are preferably non-ionic surfactants, in particular esters of sorbitol, mannide (eg anhydromannitolate), glycol, polyglycerol, propylene glycol and oleic, isostearic, ricinoleic or hydroxystearic acid, which are optionally ethoxylated, and polyoxypropylene-polyoxyethylene block copolymers, in in particular products like Pluronic, especially L121. See Hunter et al., The Theory and Practical Application of Adjuvants (Ed. Stewart-Tull, D.E.S.), JohnWiley and Sons, NY, pp. 51-94 (1995) and Todd et al., Vaccine 15: 564-570 (1997). Typical adjuvan

- 8 045363 these are the SPT emulsion described on page 147 of Vaccine Design, The Subunit and Adjuvant Approach, edited by M. Powell and M. Newman, Plenum Press, 1995, and the MF59 emulsion, described on page 183 of the same book.

Another example of an adjuvant may be a compound selected from polymers of acrylic or methacrylic acid and copolymers of maleic anhydride and an alkenyl derivative. The optimal adjuvant compounds are polymers of acrylic or methacrylic acid, cross-linked, in particular, with polyalkenyl esters of sugars or polyhydric alcohols. These compounds are known as carbomers (Phameuropa Vol. 8, No. 2, June 1996). Those skilled in the art may also refer to US Patent No. 2,909,462, which describes such acrylic polymers crosslinked with a polyhydroxylated compound having at least 3 hydroxyl groups, preferably no more than 8, wherein the hydrogen atoms of at least three hydroxyls are replaced by unsaturated aliphatic radicals having at least 2 carbon atoms. Preferred radicals are those containing from 2 to 4 carbon atoms, such as vinyls, allyls and other ethylenically unsaturated groups. Unsaturated radicals themselves may contain other substituents, such as methyl. Particularly suitable are the products sold under the name Carbopol; (BF Goodrich, Ohio, USA). They are cross-linked with allyl sucrose or allyl pentaerythritol. Among them we can mention Carbopol 974P, 934P and 971P. The most preferred is the use of Carbopol 971P. Copolymers of maleic anhydride and an alkenyl derivative include EMA copolymers (Monsanto), which are copolymers of maleic anhydride and ethylene. Dissolution of these polymers in water results in the formation of an acid solution that must be neutralized, preferably to a physiological pH, to obtain an adjuvant solution into which the immunogenic, immunological or vaccine composition itself is included.

Other suitable adjuvants include, but are not limited to, RIBI adjuvant system (Ribi Inc.), block copolymer (CytRx, Atlanta GA), SAF-M (Chiron, Emeryville CA), monophosphoryl lipid A adjuvant auridin lipid amine, heat labile enterotoxin from E. coli (recombinant or otherwise), cholera toxin, IMS 1314 or muramyl dipeptide, or natural or recombinant cytokines or analogs thereof or endogenous cytokine release stimulators, etc.

It is expected that the adjuvant may be added in an amount of from about 100 μg to about 10 mg per dose, preferably in an amount from about 100 μg to about 10 mg per dose, more preferably in an amount from about 500 μg to about 5 mg per dose, further more preferably in an amount of from about 750 μg to about 2.5 mg per dose, and most preferably in an amount from about 1 mg per dose. Alternatively, the adjuvant may be at a concentration of from about 0.01 to 50%, preferably at a concentration from about 2 to 30%, more preferably at a concentration from about 5 to 25%, even more preferably at a concentration from about 7 to 22 %, and most preferably in a concentration of 10 to 20% by volume of the finished product.

In one aspect of the present invention, the immunogenic composition includes from about 0.1 to 50 μg of PPV VP2 antigen. Preferably, the immunogenic composition comprises from about 0.2 μg and 40 μg, more preferably from about 0.3 to 30 μg, more preferably from about 0.4 to 20 μg, and even more preferably from about 0.5 to 10 μg , with the most preferred amount being 0.5, 0.75, 1, 1.25, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 μg of PPV VP2 antigen.

In one aspect of the present invention, the immunogenic composition is a vaccine.

The term vaccine has already been described elsewhere in this description. However, in the event that the host exhibits a protective immune response, for example, to increase resistance to a new infection and/or reduce the clinical severity of the disease, the immunogenic composition is described as a vaccine.

In one aspect of the present invention, the immunogenic composition protects against homologous and/or heterologous challenge. It is an advantage that the experimental data provided by the present invention reveals that the vaccine of the present invention has a broad spectrum of protection as the vaccine protects against heterologous North American as well as heterologous European challenge strains.

The terms protect and prevent and prevent are used interchangeably in this application. These terms are defined elsewhere in this description.

In one aspect of the present invention, the immunogenic composition protects against challenge with North American and/or European isolates.

The term North American and European isolates is well known among those skilled in the art. The term North American and/or European isolates covers all isolates that have been or will be isolated in North America and Europe.

In one aspect of the present invention, the immunogenic composition provides cross-immunity against North American and/or European isolates.

- 9 045363

In one aspect of the present invention, the immunogenic composition is effective for treating and/or preventing clinical signs caused by PPV infection in a subject in need thereof. The terms treatment and/or prevention, clinical signs and needs are defined elsewhere in this description.

In addition, the present invention provides a polynucleotide comprising a nucleotide sequence that encodes PPV VP2, as described by the authors.

In addition, the present invention provides a polynucleotide comprising a nucleotide sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% of the sequence with the nucleotide sequence SEQ ID NO: 3 or SEQ ID NO: 4.

The present invention further provides a polynucleotide comprising or consisting of the nucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 4.

In addition, the present invention provides a vector including a polynucleotide as described by the authors.

In one aspect of the present invention, the vector is an expression vector.

In one aspect of the present invention, the vector is a baculovirus.

The term vector is well known among those skilled in the art. The term vector, as known to those skilled in the art, refers to a polynucleotide construct, typically a plasmid or virus, that is used to transfer genetic material into a host cell. Vectors can be, for example, viruses, plasmids, cosmids or phages. The vector in the context of the present invention may consist of DNA or RNA. In some embodiments, the vector consists of DNA. An expression vector is a vector capable of directing the expression of a protein encoded by one or more genes carried by the vector when present in an appropriate environment. Vectors are preferably capable of self-replication. Typically, an expression vector includes a transcription promoter, a gene, and a transcription terminator. Gene expression is typically under the control of a promoter, and the gene is considered to be operably linked to the promoter.

As used herein, the term operably linked is used to describe the relationship between regulatory elements and a gene or coding region thereof. Typically, gene expression is carried out under the control of one or more regulatory elements, which include, among others, constitutive or inducible promoters, tissue-specific regulatory elements and enhancers. A gene or coding region is considered to be operably linked or operably linked or operably associated with regulatory elements in the sense that the gene or coding region is under the control or influence of the regulatory element. For example, a promoter is considered to be operably linked by a coding sequence if the promoter transcribes or expresses the coding sequence.

Vectors and methods for preparing and/or using expression vectors (or recombinants) may be identical or similar to those disclosed in: US Pat. Nos. 4,603,112, 4,769,330, 5,174,993, 5,505,941, 5,338,683, 5,494,807, 4,722,848, 5,942, 235, 5,364,773, 5,762,938, 5,770,212 , 5,942,235, 382,425, PCT publications WO 94/16716, WO 96/39491, WO 95/30018; Paoletti, Applications of pox virus vectors to vaccination: An update, PNAS USA 93: 11349-11353, October 1996; Moss, Genetically engineered poxviruses for recombinant gene expression, vaccination, and safety, PNAS USA 93: 11341-11348, October 1996; Smith et al., US Patent No. 4,745,051 (recombinant baculovirus); Richardson, S. D. (Editor), Methods in Molecular Biology 39, Baculovirus Expression Protocols (1995 Humana Press Inc.); Smith et al., Production of Human Beta Interferon in Insect Cells Infected with a Baculovirus Expression Vector, Molecular and Cellular Biology, December, 1983, Vol. 3, No. 12, p. 2156-2165; Pennock et al., Strong and Regulated Expression of Escherichia coli B-Galactosidase in Infect Cells with a Baculovirus vector, Molecular and Cellular Biology March 1984, Vol. 4, No. 3, p. 406; EPAO 370 573; U.S. application no. 920.197, filed Oct. 16, 1986; EP patent publication No. 0265785; US Patent No. 4,769,331 (recombinant herpesvirus); Roizman, The function of herpes simplex virus genes: A primer for genetic engineering of novel vectors, PNAS USA 93: 11307-11312, October 1996; Andreansky et al., The application of genetically engineered herpes simplex viruses to the treatment of experimental brain tumors, PNAS USA 93: 11313-11318, October 1996; Robertson et al., Epstein-Barr virus vectors for gene delivery to B lymphocyte, PNAS USA 93: 11334-11340, October 1996; Frolov et al., Alphavirus-based expression vectors: Strategies and application, PNAS USA 93: 11371-11377, October 1996; Kitson et al., J. Virol. 65, 3068-3075, 1991; U.S. Pat. Nos. 5,591,439, 5,552,143; WO 98/00166; US Accepted Applications, ser. Nos. 08/675,556, and 08/675,566, both filed July 3, 1996 (recombinant adenovirus); publications Grunhaus et al., 1992, Adenovirus as cloning vectors, Seminars in Virology (Vol. 3) p. 237-52, 1993; Ballay et al. EMBO Journal, vol. 4, p. 3861-65, Graham, Tibtech 8, 85-87, April, 1990; Prevec et al., J. Gen. Virol. 70, 42434; PCT WO 91/11525; Feigner et al. (1994), J. Biol. Chem. 269, 2550 - 2561, Science, 259: 1745-49, 1993; and McClements et al., Immunization with

- 10 045363

DNA vaccines encoding glycoprotein D or glycoprotein B, alone or in combination, induces protective immunity in animal models of herpes simplex virus-2 disease, PNAS USA 93: 11414-11420, October 1996; and US Patent Nos. 5,591,639, 5,589,466, and 5,580,859, as well as WO 90/11092, WO 93/19183, WO 94/21797, WO 95/11307, WO 95/20660; Tang et al., Nature, and Furth et al., Analytical Biochemistry regarding DNA expression vectors, etc. See also WO 98/33510; Ju et al., Diabetologia, 41: 736-739, 1998 (lentiviral expression system); Sanford et al., U.S. Pat. No. 4,945,050; Fischbachet al. (Intracel); wO 90/01543; Robinson et al., Seminars in Immunology vol. 9, pp. 271-283 (1997), (DNA vector systems); Szoka et al., US Patent No. 4,394,448 (method of inserting DNA into living cells); McCormick et al., US Patent No. 5,677,178 (use of cytopathic viruses); and US Patent No. 5,928,913 (vectors for gene delivery); as well as other documents mentioned by the authors.

The terms regulatory element and expression control element are used interchangeably and refer to nucleic acid molecules that affect the expression of an operably linked coding sequence in a particular host organism. These terms are used in a broad sense to cover all elements that promote or regulate transcription, including promoters, essential elements required for the fundamental interaction of RNA polymerase and transcription factors, upstream elements, enhancers, and response elements. Typical regulatory elements in prokaryotes are promoters, operator sequences, and ribosome binding sites. Regulatory elements used in eukaryotic cells may include, but are not limited to, transcriptional and translational control sequences such as promoters, enhancers, splicing signals, polyadenylation signals, terminators, protein degradation signals, internal ribosome entry element (IRES), 2A sequences, and the like, which provide and/or regulate the expression of the coding sequence and/or the formation of the encoded polypeptide in the host cell.

As used herein, the term promoter means a nucleotide sequence that allows RNA polymerase to bind and directs transcription of a gene. As a rule, the promoter is located in the 5' non-coding region of the gene, close to the transcription initiation site of the gene. Sequence elements within promoters that perform transcription initiation functions are often characterized by consensus nucleotide sequences. Examples of promoters include, but are not limited to, promoters from bacteria, yeast, plants, viruses and mammals (including humans). A promoter can be inducible, repressible and/or constitutive. Inducible promoters initiate increased levels of transcription from DNA under their control in response to certain changes in culture conditions, such as changes in temperature.

As used herein, the term enhancer refers to a type of regulatory element that can increase transcriptional efficiency, regardless of the distance or orientation of the enhancer relative to the transcription start site.

Formation of the viral vector can be accomplished using any suitable engineering techniques generally known to those skilled in the art, including, but not limited to, standard restriction endonuclease digestion, ligation, transformation, plasmid purification, and DNA sequencing techniques, for example, as described in Sambrook et al. (Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, N.Y. (1989)).

The term baculovirus is well known among those skilled in the art. However, as used herein, baculovirus specifically means a system for producing a desired protein in insect cells using a recombinant baculovirus vector for expressing said protein. A baculovirus expression system generally includes all elements necessary to achieve expression of a recombinant protein in insect cells, and typically includes engineering a baculovirus vector to express the desired protein, introducing the engineered baculovirus vector into insect cells, culturing insect cells containing the engineered baculovirus vector, in a suitable growth medium so that the desired protein is expressed, and isolation of the protein. Typically, baculovirus vector engineering involves the construction and isolation of recombinant baculoviruses in which the coding sequence for a selected gene is inserted downstream of a promoter for a minor viral gene, with most currently used baculovirus expression systems based on the California alfalfa nuclear polyhedrosis virus (AcMNPV) sequence ( (Virology 202 (2), 586-605 (1994), NCBI Accession No.: NC_001623) Baculovirus expression systems are well known among those skilled in the art and have been described, for example, in Baculovirus Expression Vectors: A Laboratory Manual, David R. O 'Reilly, Lois Miller, Verne Luckow, Oxford Univ. Press (1994), The Baculovirus Expression System: A Laboratory Guide, Linda A. King, R. D. Possee, Chapman & Hall (1992) A typical non-limiting example of a baculovirus system for the production of a recombinant protein is described, for example, in document WO 2006/072065 A2.

Preferred baculovirus vectors include a baculovirus such as BaculoGold (BD Biosciences Pharmingen, San Diego, Calif.) or DiamondBac (Sigma Aldrich), particularly provided that

- 11 045363 producing cells are insect cells. Although the baculovirus expression system is preferred, those skilled in the art will appreciate that other expression systems may be useful within the meaning of the present invention.

In addition, the present invention provides a cell including a polynucleotide or vector, as described by the authors. Preferably the vector is a baculovirus.

The term cell is well known among those skilled in the art. The term cell includes a eukaryotic cell, such as an animal cell, a protozoan cell, a plant cell, or a fungal cell. Preferably, the eukaryotic cell is a mammalian cell, such as CHO, BHK or COS, or a fungal cell, such as Saccharomyces cerevisiae, or an insect cell, such as Sf9.

In one aspect of the present invention, the cell is an insect cell.

Insect cell, as used herein, means a cell or cell culture taken from a species of insect. Of particular interest from the point of view of the present invention are insect cells taken from the species Spodoptera frugiperda and Trichoplusia ni.

Preferably, the insect cell, as mentioned by the authors, is a Spodoptera frugiperda (Sf) cell or a cell from a cell line taken from Spodoptera frugiperda, more preferably selected from the group consisting of an Sf9 cell and an Sf+ cell. Accordingly, the insect cells mentioned by the authors are preferably Spodoptera frugiperda (Sf) cells or cells from a cell line taken from Spodoptera frugiperda, more preferably selected from the group consisting of Sf9 cells and Sf+ cells.

In one aspect of the present invention, the insect cell is selected from the group consisting of Sf9 cells and Sf+ cells.

In addition, the present invention provides a virus-like particle including PPV VP2, as described by the authors.

The term virus-like particle (VLP) covers the non-replicating, empty viral envelope of a virus. VLPs typically consist of one or more viral proteins, which include, but are not limited to, proteins referred to as capsids, coating, surface, and/or envelope proteins, or particulate polypeptides derived from these proteins. VLPs can be formed spontaneously after recombinant expression of the protein in an appropriate expression system. The presence of VLPs following recombinant expression of viral proteins is detected using conventional techniques known to those skilled in the art, such as electron microscopy, x-ray crystallography, and the like. See, for example, Baker et al., Biophys. J. (1991) 60: 1445-1456; Hagensee et al., J. Virol. (1994) 68:4503–4505. For example, cryoelectron microscopy is performed on vitrified aqueous samples of a given VLP preparation and images are recorded under appropriate exposure conditions.

The term virus-like particle (VLP) also covers VLPs consisting of multiple VP2 PPVs.

In one aspect of the present invention, the virus-like particle consists of a plurality of PPV VP2, as described by the authors.

In addition, the present invention provides a method for producing PPV VP2, as described by the authors, comprising transfecting a cell with a vector, as described by the authors.

In addition, the present invention provides a method for producing PPV VP2, as described by the authors, comprising transfecting a cell, preferably an insect cell, with a baculovirus, as described by the authors.

The compositions, if desired, may be presented in a package or dosing device, which may contain one or more unit dosage forms containing the active ingredient. The packaging may include, for example, metal or plastic film, such as a blister pack. The package or dosing device may be accompanied by instructions for administration, preferably for administration to animals, in particular pigs. The container(s) may be accompanied by a note in the form prescribed by the governmental authority regulating the manufacture, use or sale of pharmaceutical or biological products, which note shall reflect the authority's approval for the manufacture, use or sale for the purpose of administration to humans.

Thus, the present invention provides a kit comprising PPV VP2 or an immunogenic composition as described by the authors.

In one aspect of the present invention, the kit also includes instructions for treating and/or preventing diseases in pigs.

In one aspect of the present invention, the kit also includes instructions for treating and/or preventing PPV infections.

In another aspect of the present invention, the PPV virus in accordance with the present invention was inactivated, resulting in a whole inactivated virus with viral protein 2 (VP2), as described by the authors.

Thus, the present invention also relates to inactivated porcine parvovirus

- 12 045363 (PPV), including viral protein 2 (VP2), which has:

at amino acid position 228 there is a glutamic acid residue or a glutamate residue; and/or at amino acid position 414 a serine residue; and/or at amino acid position 419 a glutamine residue; and/or at amino acid position 436 a threonine residue, wherein the numbering of amino acid positions refers to the amino acid sequence of the wild type PPV VP2.

In addition, the present invention also provides an inactivated porcine parvovirus (PPV) comprising viral protein 2 (VP2) that includes or consists of an amino acid sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least at least 99.8%, or at least 99.9% sequence identity with the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 5-16.

In addition, the present invention also relates to an immunogenic composition comprising an inactivated porcine parvovirus (PPV) including viral protein 2 (VP2), as described by the authors.

Thus, the present invention also relates to an immunogenic composition comprising an inactivated porcine parvovirus (PPV) comprising viral protein 2 (VP2) which has:

at amino acid position 228 there is a glutamic acid residue or a glutamate residue; and/or at amino acid position 414 a serine residue; and/or at amino acid position 419 a glutamine residue; and/or at amino acid position 436 a threonine residue, wherein the numbering of amino acid positions refers to the amino acid sequence of the wild type PPV VP2.

Thus, the present invention also provides an immunogenic composition comprising an inactivated porcine parvovirus (PPV) comprising viral protein 2 (VP2) that comprises or consists of an amino acid sequence that has at least 90%, at least 91%, at least at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99 ,1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7 %, at least 99.8%, or at least 99.9% sequence identity with the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 5-16.

From the point of view of the present invention, any conventional inactivation method can be used. Thus, inactivation can be accomplished using chemical and/or physical treatment methods known to those skilled in the art. Preferred inactivation methods include the addition of cyclized binary ethyleneamine (BEI), including the addition of a solution of 2-bromoethyleneamine hydrobromide (BEA) that has been cyclized to binary ethyleneamine (BEI). Preferred additional chemical inactivation agents include, but are not limited to, Triton X-100, sodium deoxycholate, cetyltrimethylammonium bromide, β-propiolactone, thimerosal, phenol, and formaldehyde (formalin). However, inactivation may also include a neutralization step. Preferred neutralizing agents include, but are not limited to, sodium thiosulfate, sodium bisulfate, and the like.

Preferred formalin inactivation conditions include a formaldehyde concentration of about 0.02% (v/v) to 2.0% (v/v), more preferably from about 0.1% (v/v) to 1.0% (v/v), even more preferably from about 0.15% (v/v) to 0.8% (v/v), even more preferably from about 0. 16% (v/v) to 0.6% (v/v), and most preferably from about 0.2% (v/v) to 0.4% (v/v) . Incubation time depends on PPV resistance. Typically, the inactivation process is continued until no PPV growth is detectable in the appropriate culture system.

Preferably, the inactivated PPV according to the present invention is inactivated with formaldehyde, preferably using the concentration described above.

The inactivated PPV of the invention can be incorporated into liposomes using known techniques such as those described in Nature 1974, 252, 252-254 or Journal of Immunology 1978, 120, 1109-13. In another embodiment of the invention, the inactivated PPV in accordance with the invention can be conjugated to suitable biological compounds, such as polysaccharides, peptides, proteins, etc., or a combination thereof.

The present invention also relates to the use of:

PPV VP2 as described by the authors;

an immunogenic composition as described by the authors;

polynucleotide as described by the authors;

vector, as described by the authors;

- 13 045363 cells, as described by the authors;

baculovirus as described by the authors; and/or virus-like particles, as described by the authors for the preparation of a medicament, preferably a vaccine.

The present invention also relates to the use of PPV VP2, as described by the authors, or an immunogenic composition, as described by the authors, for the preparation of a medicament for the treatment and/or prevention of PPV infection, the reduction, prevention and/or treatment of clinical signs caused by PPV infection, and/or for treatment and/or prevention of disease caused by PPV infection.

In addition, the present invention provides a method of immunizing a subject, comprising administering to such subject an immunogenic composition as described by the inventors.

The term immunization refers to active immunization by administering an immunogenic composition to a subject to be immunized such that an immune response is elicited against an antigen included in such immunogenic composition.

Preferably, immunization results in a reduction in the incidence of PPV infection in the herd and/or a reduction in the severity of clinical signs caused by or associated with a particular PPV infection.

In addition, immunization of a subject in need thereof with the immunogenic compositions provided by the present invention results in preventing the subject from contracting a PPV infection. In an even more preferred embodiment, the immunization results in an effective, long-lasting immunological response against PPV infection. It should be understood that the above period of time should be greater than 1 month, preferably greater than 2 months, more preferably greater than 3 months, more preferably greater than 4 months, more preferably greater than 5 months, more preferably greater than 6 months. It should be understood that immunization may not be effective in all immunized subjects. However, the term requires that a significant proportion of subjects in the herd be effectively immunized.

Preferably, in the context of the present invention, a herd of subjects is assumed in which, under normal conditions, i.e. without immunization, clinical signs caused by or associated with PPV infection appear. Whether herd subjects are effectively immunized can be determined without undue effort on the part of those skilled in the art. Preferably, immunization is considered effective if clinical signs are present in at least 33%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, even more preferably in at least 95%, most preferably 100% of subjects in a given herd are reduced in frequency or severity by at least 10%, more preferably by at least 20%, even more preferably by at least 30%, even more preferably by at least 40%, even more preferably by at least 50%, even more preferably by at least 60%, even more preferably by at least 70%, even more preferably by at least 80%, even more preferably at least 90%, even more preferably at least 95%, most preferably 100%, compared to subjects who were either not immunized or were immunized with an immunogenic composition that were present before the present invention, but were subsequently infected with a specific PPV.

In addition, the present invention provides a method of treating and/or preventing clinical signs caused by PPV infection in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an immunogenic composition as described by the inventors.

Advantageously, as shown in the examples section of this specification, the immunogenic composition provided is effective in treating and/or preventing clinical signs caused by PPV infection in a subject in need thereof.

The term treatment and/or prevention refers to reducing the incidence of a particular PPV infection in a herd and/or reducing the severity of clinical signs caused by or associated with a particular PPV infection. Thus, the term treatment and/or prevention also refers to reducing the number of animals in a herd infected with a particular PPV (= reducing the incidence of a particular PPV infection) and/or reducing the severity of clinical signs typically associated with or caused by PPV infection in a group of animals. which received an effective amount of the immunogenic composition provided in accordance with the invention, compared with a group of animals that did not receive such an immunogenic composition.

Treatment and/or prophylaxis typically involves administering an effective amount of an immunogenic composition in accordance with the present invention to a subject or herd of subjects that requires or benefits from such treatment/prophylaxis. The term treatment refers to the administration of an effective amount of an immunogenic composition after the subject or at least some of the animals in the herd have already been infected with such PPV and such animals have already exhibited some of the clinical signs caused by such inoculation.

- 14 045363 PPV infection or related to it. The term prophylaxis refers to the administration to a subject, prior to any infection of such subject, of a PPV, or to a situation where at least such animal, or none of the animals in a group of animals, exhibits any clinical signs caused by or associated with infection by such PPV. The terms prophylaxis and prevention are used interchangeably in this application.

The term effective amount as used herein means, among other things, an amount of antigen that produces or is capable of eliciting an immune response in a subject. Such an effective amount is capable of reducing the incidence of a particular PPV infection in the herd and/or reducing the severity of clinical signs of a particular PPV infection.

Preferably, clinical signs are reduced in frequency or severity by at least 10%, more preferably by at least 20%, even more preferably by at least 30%, even more preferably by at least 40%, even more preferably - by at least 50%, even more preferably by at least 60%, even more preferably by at least 70%, even more preferably by at least 80%, even more preferably by at least 90% , even more preferably at least 95%, most preferably 100%, compared to subjects who received no treatment or received an immunogenic composition that was available before the present invention, but were subsequently infected with a particular PPV.

The term clinical signs as used herein refers to whether a subject has signs of PPV infection. Clinical signs of infection depend on the pathogen chosen. Examples of such clinical signs include, but are not limited to, transient leukopenia and reproductive failure, characterized by infection and death of the embryo and/or fetus, or a combination thereof. Examples of directly observable clinical signs include reduced litter size, increased mummification of the embryo or fetus per brood, autolysis of the embryo or fetus, reduced embryo or fetal size, reduced embryo or fetal weight, etc., or combinations thereof. Other examples of such clinical signs include, but are not limited to, increased viremia, increased viral load in target tissues and blood, increased transmission/spread of PPV to littermates, etc., or combinations thereof.

Preferably, clinical signs that are reduced in incidence or severity in a treated subject compared to subjects who are either untreated or treated with an immunogenic composition that was available prior to the present invention but are subsequently infected with a particular PPV relate to transient leukopenia and reproductive failure, characterized by infection and death of the embryo and/or fetus, or a combination thereof.

The term needy or required as used herein means that the administration/treatment is associated with the promotion or improvement of health status or clinical signs or any other beneficial medical effect on the health of animals (including their embryos or fetuses) that receive the immunogenic composition in accordance with the present invention .

The term decrease or reduced or lessen or reduce are used interchangeably in this application. The term reduction means that the clinical sign is reduced by at least 10%, more preferably by at least 20%, even more preferably by at least 30%, even more preferably by at least 40%, even more preferably by at least 50%, even more preferably by at least 60%, even more preferably by at least 70%, even more preferably by at least 80%, even more preferably by at least 90% , even more preferably - at least 95% more preferably - 100% compared to subjects not subjected to treatment (immunization), but subsequently infected with a particular PPV.

In addition, the present invention provides a method of reducing reproductive failure in a subject compared to a non-immunized control subject of the same species, the method comprising administering to the subject a therapeutically effective amount of an immunogenic composition as described by the inventors.

Advantageously, as shown in the examples section of this specification, the immunogenic composition provided was effective in reducing reproductive failure.

In addition, the present invention provides a method of reducing embryonic and fetal mortality in a subject compared to a non-immunized control subject of the same species, the method comprising administering to the subject a therapeutically effective amount of an immunogenic composition as described by the inventors.

Advantageously, as shown in the examples section of this specification, the immunogenic composition provided has been effective in reducing embryonic and fetal mortality.

In addition, the present invention provides a method for actively immunizing breeding pigs (sows and gilts) to protect embryos and fetuses from porcine parvovirus infection, and can

- 15 045363 sob includes administering to such pigs (sows and gilts) a therapeutically effective amount of an immunogenic composition as described by the authors.

In one aspect of the present invention, said subject is selected from the group consisting of pigs, cattle, cats or dogs.

Preferably, said subject is a pig. It should be understood that pig refers to both female and male animals. Semen may contain PPV, so females and males of breeding animals are covered by the definition of pig. Thus, the definition of pig includes male animals, such as boars, as well as female animals, such as gilts and sows.

The term pig as used herein preferably refers to a pig before and during its first gestation/pregnancy. In contrast, the term sow in the context of this description preferably refers to a pig after its first farrow - as a positive result of its first gestation/pregnancy.

In one aspect of the present invention, said subject is a pig, preferably a gilt and/or a sow.

In one aspect of the present invention, the immunogenic composition is administered once.

It should be understood that a single dose is administered only once.

The dose volume per subject depends on the method of vaccination and the age of the subject. Preferably, the unit dose has a total volume of from about 0.2 to 2.5 ml, more preferably from about 0.2 to 2.0 ml, even more preferably from about 0.2 to 1.75 ml, even more preferably - from about 0.2 to 1.5 ml, even more preferably from about 0.4 to 1.25 ml, even more preferably from about 0.4 to 1.0 ml, with a single dose of 0.5 being most preferred or 1.0 ml. Most preferably, a single dose has a total volume of 0.5, 1, 1.5 or 2 ml.

In one aspect of the present invention, the immunogenic composition is administered in two or more doses.

As shown in the examples section of this specification, the provided immunogenic composition was effective after two doses were administered to a subject in need thereof.

However, the immunogenic composition may be administered in two or more doses, with the first dose administered before the second (booster) dose. Preferably, the second dose is administered at least 15 days after the first dose. More preferably, the second dose is administered between 15 and 40 days after the first dose. Even more preferably, the second dose is administered at least 17 days after the first dose. Even more preferably, the second dose is administered between 17 and 30 days after the first dose. Even more preferably, the second dose is administered at least 19 days after the first dose. Even more preferably, the second dose is administered between 19 and 25 days after the first dose. Most preferably, the second dose is administered at least 21 days after the first dose. Even more preferably, the second dose is administered approximately 21 days after the first dose or 21 days after the first dose. In a preferred aspect of the two-daily administration regimen, both the first and second doses of the immunogenic composition are administered in equal amounts. Preferably, each dose is administered in the preferred amount indicated above, with the most preferred dose being 1 ml or 2 ml for the first and second dose. In addition to the first and second dose regimen, an alternative embodiment includes additional subsequent doses. For example, in these aspects, a third, fourth, or fifth dose may be administered. Preferably, the subsequent third, fourth and fifth doses in this regimen are administered in the same amount as the first dose, with a time interval between doses corresponding to the time between the aforementioned first and second doses.

The dose volume per subject depends on the method of vaccination and the age of the subject. Preferably, the total volume is from about 0.2 to 5 ml, more preferably from about 0.5 to 3.0 ml, even more preferably from about 1.0 to 2.5 ml, even more preferably from about 1. 0 to 2.0 ml. The most preferred volume is 1, 1.5, 2 or 2.5 ml per dose.

The immunogenic composition is preferably administered locally or systemically. Suitable conventionally used routes of administration are oral or parenteral administration, such as intranasal, intravenous, intradermal, transdermal, intramuscular, intraperitoneal, subcutaneous, and inhalation. However, depending on the nature and mode of action of the compound, the immunogenic composition may also be administered by other routes. For example, such routes include intradermal, intravenous, intravascular, intraarterial, intraperitoneal, intrathecal, intratracheal, intradermal, intracardiac, intralobar, intracerebral, intrapulmonary, rectal, and vaginal. However, in a more preferred embodiment, the immunogenic composition is administered subcutaneously or intramuscularly. In the most preferred embodiment, the immunogenic composition is administered intramuscularly.

In one aspect of the present invention, the above immunogenic composition is administered intramuscularly.

In one aspect of the present invention, the above immunogenic composition is administered

- 16 045363 gilts and/or sows.

Preferably, the immunogenic composition is administered to gilts and/or sows at least 3 months of age, more preferably at least 4 months of age, most preferably at least 5 months of age.

In one aspect of the present invention, the immunogenic composition is administered to gilts and/or sows at least 3 months of age.

In one aspect of the present invention, the above immunogenic composition is administered to gilts and/or sows prior to pregnancy.

In a two-dose regimen, the second dose of the above immunogenic composition is preferably administered to gilts and/or sows 2, 3, 4 or 5 weeks before mating/insemination, most preferably approximately 3 weeks before mating/insemination. Preferably, the first dose of the above immunogenic composition is administered to gilts and/or sows 2, 3, 4, 5 or 6 weeks before the second dose, most preferably approximately 3 weeks before the second dose. However, following a two-dose regimen, gilts and/or sows are preferably re-vaccinated every 3, 4, 5, 6, 7 or 8 months, most preferably approximately every 6 months.

In one aspect of the present invention, the above immunogenic composition is administered to gilts and/or sows during pregnancy and lactation.

In one aspect of the present invention, the immunogenic composition is safe for pigs and/or sows during pregnancy and lactation.

In one aspect of the present invention, the immunogenic composition is safe for pigs and/or sows after 30 days of pregnancy, preferably after 40 days of pregnancy.

Preferably, the immunogenic composition according to the present invention comprises from 0.1 to 50 μg, preferably from 0.25 to 25 μg, more preferably from 0.5 to 12.5 μg, even more preferably from 0.5 to 5 μg , most preferably 0.5 to 2 μg of PPV VP2 antigen. More preferably, the immunogenic composition of the present invention includes the PPV VP2 antigen of the present invention in an amount of about 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2 .25, 2.5, 2.75, 3, 3.5, 4, 4.5 or 5 mcg.

In one aspect of the present invention, the immunogenic composition includes from 0.1 to 50 μg of PPV VP2 antigen, preferably from 0.5 and 10 μg of PPV VP2 antigen.

In one aspect of the present invention, the immunogenic composition protects against homologous and/or heterologous challenges.

In one aspect of the present invention, the immunogenic composition protects against challenge with North American and/or European isolates.

In one aspect of the present invention, the immunogenic composition provides cross-immunity against North American and/or European isolates.

In one aspect of the present invention, the above method provides an improvement in an efficacy parameter selected from the group consisting of: reduced transient leukopenia and reproductive failure, characterized by infection and death of the embryo and/or fetus, or a combination thereof, compared to a non-immunized control subject groups of the same type.

In one aspect of the present invention, the above method results in an improvement in a performance parameter selected from the group consisting of: reduced litter size, increased embryo or fetal mummification per brood, embryo or fetal autolysis, reduced embryo or fetal size, reduced embryo weight, or fetus, increased viremia, increased viral load in target tissues and blood, increased transmission/spread of PPV to littermates, or combinations thereof, compared to a non-immunized control subject of the same species.

Embodiments

The following points are also described by the authors:

1. Viral protein 2 (VP2) of porcine parvovirus (PPV), having:

at amino acid position 228 there is a glutamic acid residue or a glutamate residue; and/or at amino acid position 414 a serine residue; and/or at amino acid position 419 a glutamine residue; and/or at amino acid position 436 a threonine residue, wherein the numbering of amino acid positions refers to the amino acid sequence of the wild type PPV VP2.

2. PPV VP2 according to paragraph 1, characterized in that PPV VP2 also has:

at amino acid position 25 isoleucine residue; and/or at amino acid position 36 a serine residue; and/or at amino acid position 37 an isoleucine residue.

3. PPV VP2 according to claim 1 or 2, characterized in that the numbering of amino acid positions refers to the amino acid sequence shown in SEQ ID NO: 1.

- 17 045363

4. PPV VP2 according to any of points 1 to 3, characterized in that PPV VP2 is a recombinant PPV VP2.

5. PPV VP2 according to any of paragraphs 1 to 4, characterized in that PPV VP2 is a PPV VP2 expressed by a recombinant baculovirus.

6. PPV VP2 according to any one of claims 1 to 5, wherein said PPV VP2 includes or consists of an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 5-16.

7. PPV VP2 according to any one of paragraphs 1 to 6, characterized in that the above PPV VP2 includes or consists of an amino acid sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99, 8%, or at least 99.9% sequence identity with the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 5-16.

8. PPV VP2 according to any one of paragraphs 1 to 6, characterized in that the above PPV VP2 includes or consists of the amino acid sequence of SEQ ID NO: 1, or SEQ ID NO: 2 or SEQ ID NO: 5-16 or includes or consists of from any fragment that has at least 210, at least 250, or at least 300 contiguous amino acid residues of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 5-16.

9. PPV VP2 according to any one of claims 1 to 6, wherein said PPV VP2 includes or consists of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 5-16.

10. PPV VP2 according to any one of paragraphs 1 to 9, characterized in that the above PPV VP2 is encoded by a nucleotide sequence encoding an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 5-16.

11. PPV VP2 according to any one of paragraphs 1 to 10, characterized in that the above PPV VP2 is encoded by a nucleotide sequence encoding an amino acid sequence that has at least 90%, at least 91%, at least 92%, at least at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99 .8%, or at least 99.9% sequence identity with the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 5-16.

12. PPV VP2 according to any one of paragraphs 1 to 11, characterized in that the above PPV VP2 is encoded by a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 5-16.

13. An immunogenic composition comprising PPV VP2 according to any of paragraphs 1 to 12.

14. The immunogenic composition according to claim 13, characterized in that the immunogenic composition is formulated for administration in a single dose.

15. Immunogenic composition according to paragraphs 13 or 14, characterized in that the immunogenic composition is administered intramuscularly.

16. Immunogenic composition according to any of paragraphs 13 to 15, characterized in that the immunogenic composition is safe for pigs and/or sows during pregnancy and lactation.

17. The immunogenic composition according to any one of paragraphs 13 to 16, characterized in that the immunogenic composition is safe for pigs and/or sows after 30 days of pregnancy, preferably after 40 days of pregnancy.

18. The immunogenic composition according to any one of claims 13 to 17, characterized in that the immunogenic composition also includes a pharmaceutically acceptable carrier.

19. The immunogenic composition according to claim 18, characterized in that the pharmaceutically acceptable carrier is a carbomer.

20. The immunogenic composition according to any one of paragraphs 13 to 19, characterized in that the immunogenic composition includes from 0.1 to 50 μg of PPV VP2 antigen, preferably from 0.5 and 10 μg of PPV VP2 antigen.

21. The immunogenic composition according to any of paragraphs 13 to 20, characterized in that the immunogenic composition is a vaccine.

22. An immunogenic composition according to any of paragraphs 13 to 21, characterized in that the immunogenic composition protects against homologous and/or heterologous challenge infection.

23. Immunogenic composition according to any of paragraphs 13 to 22, characterized in that the immuno

- 18 045363 gene composition protects against provocative infection with North American and/or European isolates.

24. The immunogenic composition according to any one of paragraphs 13 to 23, characterized in that the immunogenic composition provides cross-immunity against North American and/or European isolates.

25. The immunogenic composition according to any one of claims 13 to 24, wherein the immunogenic composition is effective for treating and/or preventing clinical signs caused by PPV infection in a subject in need thereof.

26. A polynucleotide comprising a sequence that encodes PPV VP2 according to any of items 1 to 12.

27. A polynucleotide comprising a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96 %, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4 %, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity with the nucleotide sequence SEQ ID NO : 3 or SEQ ID NO: 4.

28. A polynucleotide comprising or consisting of the nucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 4.

29. A vector comprising a polynucleotide according to any of paragraphs 26 to 28.

30. The vector according to paragraph 29, characterized in that the vector is an expression vector.

31. The vector according to paragraph 29 or 30, characterized in that the vector is a baculovirus.

32. A cell comprising a polynucleotide according to any of paragraphs 26 to 28 or a vector according to any of paragraphs 29 to 31.

33. The cell according to claim 32, characterized in that the cell is an insect cell.

34. The insect cell according to claim 33, characterized in that the insect cell is selected from the group that includes Sf9 cells and Sf+ cells.

35. A virus-like particle, including PPV VP2 according to any of paragraphs 1 to 12.

36. The virus-like particle according to item 35, characterized in that the virus-like particle consists of a plurality of PPV VP2 according to any of items 1 to 12.

37. A method for producing PPV VP2 according to any of paragraphs 1 to 12, including transfecting a cell with a vector according to any of paragraphs 29 to 31.

38. The method for producing PPV VP2 according to any of paragraphs 1 to 12, including transfecting a cell, preferably an insect cell, with a baculovirus according to paragraph 31.

39. A kit comprising PPV VP2 or an immunogenic composition according to any of paragraphs 1 to 25.

40. The kit according to item 39, characterized in that the kit also includes instructions for the treatment and/or prevention of diseases in pigs.

41. The kit according to paragraph 39, characterized in that the kit also includes instructions for the treatment and/or prevention of PPV infection.

42. Application:

PPV VP2 for any of points 1 to 12;

the immunogenic composition according to any one of paragraphs 13 to 25;

a polynucleotide according to any one of paragraphs 26 to 28;

vector for any of points from 29 to 31;

cells according to any of paragraphs 32 to 34;

baculovirus according to paragraph 31; and/or virus-like particles according to paragraph 35 or 36;

for the preparation of a medicine, preferably a vaccine.

43. Use of PPV VP2 according to any of paragraphs 1 to 12 or an immunogenic composition according to any of paragraphs 13 to 25 for the preparation of a medicament for the treatment and/or prevention of PPV infection, reduction, prevention and/or treatment of clinical signs caused by PPV infection, and/or for the treatment and/or prevention of disease caused by PPV infection.

44. A method of immunizing a subject, comprising administering to such subject an immunogenic composition according to any one of claims 13 to 25.

45. A method of treating and/or preventing clinical signs caused by PPV infection in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the immunogenic composition of any one of claims 13 to 25.

46. A method of reducing reproductive failure in a subject compared to a non-immunized control subject of the same species, the method comprising administering to the subject a therapeutically effective amount of the immunogenic composition of any one of claims 13 to 25.

47. A method for reducing embryonic and fetal mortality in a subject compared to a non-immune subject

- 19 045363 a neutralized control group of the same species, the method comprising administering to the subject a therapeutically effective amount of the immunogenic composition of any one of claims 13 to 25.

48. The method according to any one of paragraphs 44 to 47, characterized in that the above-mentioned subject is selected from the list including pigs, cattle, cats and dogs.

49. Method according to any one of paragraphs 44 to 48, characterized in that said subject is a pig, preferably a gilt and/or a sow.

50. The method according to any of paragraphs 44 to 49, characterized in that the immunogenic composition is administered once.

51. The method according to any one of paragraphs 44 to 49, characterized in that the immunogenic composition is administered in two or more doses.

52. The method according to any of paragraphs 44 to 51, characterized in that the immunogenic composition is administered intramuscularly.

53. The method according to any of paragraphs 44 to 52, characterized in that the immunogenic composition is administered to gilts and/or sows.

54. The method according to any of paragraphs 44 to 53, characterized in that the immunogenic composition is administered to gilts and/or sows of at least 3 months of age.

55. The method according to any of paragraphs 44 to 54, characterized in that the immunogenic composition is administered to gilts and/or sows before pregnancy.

56. The method according to any of paragraphs 44 to 54, characterized in that the immunogenic composition is administered to gilts and/or sows during pregnancy and lactation.

57. The method according to any of paragraphs 44 to 56, characterized in that the immunogenic composition is safe for pigs and/or sows during pregnancy and lactation.

58. The method according to any one of paragraphs 44 to 57, characterized in that the immunogenic composition is safe for pigs and/or sows after 30 days of pregnancy, preferably after 40 days of pregnancy.

59. The method according to any one of paragraphs 44 to 58, characterized in that the immunogenic composition includes between 0.1 and 50 μg of PPV VP2 antigen, preferably between 0.5 and 10 μg of PPV VP2 antigen.

60. The method according to any of paragraphs 44 to 59, characterized in that the immunogenic composition protects against homologous and/or heterologous challenge infection.

61. The method according to any of paragraphs 44 to 60, characterized in that the immunogenic composition protects against challenge infection with North American and/or European isolates.

62. The method according to any of paragraphs 44 to 61, characterized in that the immunogenic composition provides cross-immunity against North American and/or European isolates.

63. The method according to any of paragraphs 44 to 62, characterized in that as a result of the above method, an improvement in the effectiveness parameter selected from the group includes: reduced transient leukopenia and reproductive failure, characterized by infection and death of the embryo and/or fetus , or a combination thereof, compared with a non-immunized control subject of the same species.

64. An immunogenic composition for use according to any of paragraphs 44 to 63, characterized in that as a result of the above-mentioned method, an improvement in an efficiency parameter selected from the group that includes: reduced litter size, increased mummification of the embryo or fetus per brood, autolysis is ensured embryo or fetus, reduced embryo or fetal size, reduced embryo or fetal weight, increased viremia, increased viral load in target tissues and blood, increased transmission/spread of PPV to littermates, or combinations thereof, compared to a non-immunized control subject of the same kind.

65. The immunogenic composition according to any one of claims 13 to 25 for use in a method of immunizing a subject, comprising administering the above-mentioned immunogenic composition to such subject.

66. The immunogenic composition of any one of claims 13 to 25 for use in a method of treating or preventing clinical signs caused by PPV infection in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of said immunogenic composition.

67. The immunogenic composition of any one of claims 13 to 25 for use in a method of reducing reproductive failure in a subject compared to a non-immunized control subject of the same species, the method comprising administering to the subject a therapeutically effective amount of said immunogenic composition.

68. The immunogenic composition of any one of claims 13 to 25 for use in a method of reducing embryonic and fetal mortality in a subject compared to a non-immunized control subject of the same species, the method comprising administering to the subject a therapeutically effective amount of said immunogenic composition.

69. An immunogenic composition for use according to any one of paragraphs 65 to 68, characterized

- 20 045363 in that the above-mentioned subject is selected from a list including pigs, cattle, cats and dogs.

70. An immunogenic composition for use according to any one of paragraphs 65 to 69, characterized in that said subject is a pig, preferably a gilt and/or a sow.

71. An immunogenic composition for use according to any of paragraphs 65 to 70, characterized in that the immunogenic composition is administered once.

72. An immunogenic composition for use according to any one of paragraphs 65 to 70, characterized in that the immunogenic composition is administered in two or more doses.

73. An immunogenic composition for use according to any of paragraphs 65 to 72, characterized in that the immunogenic composition is administered intramuscularly.

74. An immunogenic composition for use according to any of paragraphs 65 to 73, characterized in that the immunogenic composition is administered to gilts and/or sows.

75. An immunogenic composition for use according to any of paragraphs 65 to 74, characterized in that the immunogenic composition is administered to gilts and/or sows of at least 3 months of age.

76. An immunogenic composition for use according to any of paragraphs 65 to 75, characterized in that the immunogenic composition is administered to gilts and/or sows before pregnancy.

77. An immunogenic composition for use according to any of paragraphs 65 to 75, characterized in that the immunogenic composition is administered to gilts and/or sows during pregnancy and lactation.

78. An immunogenic composition for use according to any of paragraphs 65 to 77, characterized in that the immunogenic composition is safe for pigs and/or sows during pregnancy and lactation.

79. An immunogenic composition for use according to any one of paragraphs 65 to 78, characterized in that the immunogenic composition is safe for pigs and/or sows after 30 days of pregnancy, preferably after 40 days of pregnancy.

80. An immunogenic composition for use according to any one of paragraphs 65 to 78, characterized in that the immunogenic composition includes between 0.1 μg and 50 μg of PPV VP2 antigen, preferably between 0.5 and 10 μg of PPV VP2 antigen.

81. An immunogenic composition for use according to any of paragraphs 65 to 80, characterized in that the immunogenic composition protects against homologous and/or heterologous challenge infection.

82. An immunogenic composition for use according to any one of paragraphs 65 to 81, characterized in that the immunogenic composition protects against challenge infection with North American and/or European isolates.

83. An immunogenic composition for use according to any of paragraphs 65 to 82, characterized in that the immunogenic composition provides cross-immunity against North American and/or European isolates.

84. An immunogenic composition for use according to any of paragraphs 65 to 83, characterized in that as a result of the above-mentioned method, an improvement in an efficiency parameter selected from the group includes: reduced transient leukopenia and reproductive failure, characterized by infection and death of the embryo, and /or fetus, or a combination thereof, compared with a non-immunized control subject of the same species.

85. An immunogenic composition for use according to any of paragraphs 65 to 84, characterized in that as a result of the above-mentioned method, an improvement in an efficiency parameter selected from the group that includes: reduced litter size, increased mummification of the embryo or fetus per brood, autolysis embryo or fetus, reduced embryo or fetal size, reduced embryo or fetal weight, increased viremia, increased viral load in target tissues and blood, increased transmission/spread of PPV to littermates, or combinations thereof, compared to a non-immunized control subject of the same kind.

86. A method of actively immunizing breeding pigs (sows and gilts) to protect embryos and fetuses from porcine parvovirus infection, the method comprising administering to such pigs (sows and gilts) a therapeutically effective amount of the immunogenic composition according to paragraphs 13 to 25.

87. An immunogenic composition according to any one of claims 13 to 25 for use in a method of actively immunizing breeding pigs (sows and gilts) to protect embryos and fetuses from porcine parvovirus infection, the method comprising administering to such pigs (sows and gilts) a therapeutically effective amount of the above immunogenic composition.

- 21 045363

Examples

The following examples are presented below to illustrate specific embodiments of the present invention. These examples are illustrative only and should not be construed as limiting the scope or principles underlying the present invention.

Example 1.

Preparation of subunit vaccine against PPV.

The PPV VP2 antigen is selected for expression in baculovirus-infected insect cells based on the German isolate PPV 27a. The nucleotide sequence of porcine parvovirus (PPV) 27a VP2 was obtained from Genbank under accession number AY684871.1. The PPV 27a VP2 coding region is reverse translated and codon optimized for Drosophila (SEQ ID NO: 4 and SEQ ID NO: 3). The codon-optimized PPV 27a VP2 gene was chemically synthesized at Integrated DNA Technologies. The PPV 27a gene is then subcloned into the baculovirus transfer vector pVL1393 and co-transfected with the linearized DiamondBac® baculovirus backbone into Sf9 insect cells to generate a recombinant baculovirus containing the PPV 27a VP2 gene under the control of a polyhedrin promoter.

When expressed in insect cells, Sf9 PPV VP2 self-organizes into a nonenveloped VLP (data not shown).

The PPV VP2 antigen is supplemented with carbomer as an adjuvant (Carbopol).

Example 2.

Early stage clinical trial of a PPV vaccine.

In all animal studies, animals are in good health and nutritional status before the start of the study. Before the randomization procedure, a medical examination is performed. Non-medicated food is used throughout the study. The diet is selected according to the age, condition and species of the test animal in accordance with the standard operating procedure approved for the center. Water was provided ad libitum throughout the study.

The purpose of this challenge study is to provide a proof-of-concept for dose-finding a pre-breeding subunit vaccine against porcine parvovirus (PPV) (see Example 1). Gilts are vaccinated and inseminated before challenge with a live virulent PPV isolate (PPV 002346-5; North American strain) after approximately 40 days of gestation (dG). Fruits are assessed for PPV infection at approximately 90 dG.

The study plan is described in Table. 1.

Table 1

Study plan

Treatment Vaccination Insemination Pregnancy assessment Provocation Opening ΤΙ Negative control 2 ml on D0 right, neck IM AND 2 ml for D21 lev. neck IM D34-D42 D71 6 ml at D80 (~40dG) PPV 0023465 right, neck IM and IN D129/130 (~90dG) T2 PPV 10 mcg TK Positive control (whole cell inactivated PPV) NTX No Not applicable Not applicable D79 (39dG)

NTX = no treatment/challenge control; IN = intranasal; IM = intramuscular; dG = days of pregnancy.

Sixty-seven gilts from a previously PPV-tested herd with no history of reproductive disease or PPV vaccination were used. Gilts are randomized into 6 treatment groups (T) n=9, combined into 3 pens, receiving vaccination on D0 and stimulation on D21: T1 NC (negative control receiving water for injection), T2 PPV 10 μg, T3 PC (positive control ; whole, inactivated porcine parvovirus (PPV), Erysipelothrix rhusiopathiae, Leptospira canicola, L. grippotyphosa, L. hardjo, L. icterohaemorrhagiae and L. Pomona; mass produced; used in accordance with the manufacturer's instructions). Include untreated control (NTX) gilts, one per pen. Following vaccination, gilts are synchronized (by administering Matrix®; altrenogest, Intervet Schering-Plough Animal Health; as labeled for 14 consecutive days, D18 to D31) and then mated between D35 and D42. Fifty-four of the 67 gilts became pregnant. At D80 (approximately 40 dG), NTX gilts are necropsied and the remaining gilts are inoculated with 6 ml of PPV strain PPV002346-5 (North American strain) at 4.25 log ₁₀ TCID ₅₀ per dose (2 ml intramuscularly and 2 ml in each nostril intranasally). Gilts were bled weekly, except during synchronization and mating periods (D35–D70). Serological testing is performed on serum samples collected at D0, D7, D14, D21, D28 and D73; Serology and polymerase chain reaction (PCR) testing (as described in Jozwik et al. 2009; Journal of General Virology, 90, 2437-2441) for viremia is performed

- 22 045363 on serum samples taken at D80, D87, D94, D101, D108, D115, D122 and D128. Gilts are necropsied at D129 or D130 (approximately 90 dG). At autopsy, each reproductive tract is removed and the position of the fetus in the uterus, the condition of the fetus, its size and weight are recorded. Thoracic lavage and lung samples from each fetus are collected. Thoracic wash samples are collected under aseptic conditions from each fetus. In general, 3 ml of sterile PBS is injected into the chest cavity using a sterile needle and syringe. The liquid is aspirated back into the syringe and injected into an appropriate sterile SST (serum separator tube) of suitable size. Thoracic wash samples are tested for the presence of PPV using PCR and for the presence of PPV antibody using hemagglutination inhibition (HI). Lung tissue is stored frozen.

Gilt viremia (PPV).

All gilts were negative for PPV viremia before challenge on D0, D73 (data not shown), and D80 (Table 2). All negative controls are viremic on D87, and 4/7 are viremic on D94.

Post-vaccination T3 gilts seroconvert after booster vaccination. T2 exhibits a serological response to initial vaccination and remains seropositive after booster vaccination. T1 control gilts remain serologically negative for PPV until challenge. After challenge, all negative control gilts are viremic at D87 (seven days after challenge). One T3 pig is viremic on D87. All other gilts are not viremic at these time points (see Table 2).

NTX gilts remained serologically negative and their fetuses were PPV negative by PCR on thoracic wash samples.

table 2

Frequency distribution of PPV-positive gilts (PCR) given a PPV challenge dose after 40 days of gestation (dG) on D80

Treatment/description Study day (dG = days of pregnancy) D80 D87 D94 D101 D108 D115 D122 D128 dG 40 dG 47 dG 54 dG 61 dG68 dG 75 dG 82 dG 89 T1 Negative control 0/7 7/7 4/7 0/7 0/7 0/7 0/7 0/7 T2 10 mcg PPV 0/8 0/8 0/8 0/8 0/8 0/8 0/8 0/8 TK Positive control (whole cell inactivated PPV) 0/9 1/9 0/9 0/9 0/9 0/8 0/8 0/8 NTX No 0/3 ΝΑ ΝΑ N.A. N.A. N.A. N.A. N.A.

NA = not applicable.

Fruit results.

All NTX fetuses were considered normal by autopsy at D80 (Table 3). At final necropsy on D129 and D130, 22.5% of T1 fetuses (negative control) are normal, while 98.39% of T3 fetuses and 97.62% of T2 fetuses are normal. The mean size and weight of T1 (negative control) fruits are 11.5 cm and 168.8 g, respectively, while the mean size and weight of T2 fruits are 17.5 cm and 590.1 g, respectively.

All T4 (NTX) fetuses are negative for PPV by PCR on thoracic wash samples (see Table 3). PPV infection was confirmed in 67/80 T1 negative control fetuses (83.75%). Sixty-two of the 67 negative control fetuses confirmed to be infected with PPV were mummies. In contrast, PPV infection is confirmed in only 0.79% of T2 fetuses.

Based on the defining efficacy parameters as stated in the European Pharmacopoeia (Monograph 01/2008: 0965), it can be concluded that all vaccines [including the positive control (whole cell inactivated PPV)] meet the criteria for protection against infection (>80% of fetuses negative on PPV).

- 23 045363

Table 3

Litter details: number, size, weight and condition of fetuses and laboratory confirmation of PPV infection (PCR on thoracic wash samples)

Treatment T1 T2 TK T4 Description NC PPV 10 mcg Positive control (whole cell inactivated PPV) NTX* #pigs 5 8 9 3 General # fruits 80 126 124 44 Avg. litter size 16.0 15.8 13.8 14.7 Fetal condition: Mummies 62 3 2 0 Normal 18 123 122 44 % normal 22.50 97.62 98.39 100.0 Average size (cm) I.5 17.5 17.8 6.0 Average weight (g) 168.8 590.1 580.3 I,9 Laboratory confirmation of PPV infection: #PPV+ fruits 67 1 3 0 % positive 83.75 0.79 2.42 0.0 % protected 16.25 99.21 97.58 *Dissection of NTX fetuses after 50 days of gestation NC = Negative Control

Conclusion.

The PPV vaccine of the present invention has demonstrated protection of fetuses following challenge with a virulent heterologous PPV. The study results show that the vaccine is safe when administered in the prenatal period and is effective in significantly reducing viremia and transplacental infection of fetuses. In addition, the vaccine has been demonstrated to protect against the heterologous North American strain of PPV for challenge. In addition, the PPV VP2 subunit protein has been demonstrated to be as effective as the whole killed virus.

Example 3.

Establishment of the minimum immunizing dose of the ppv vaccine - protected against the heterologous North American strain of PPV.

The purpose of this challenge study is to establish the minimum immunizing dose (MID) for porcine parvovirus (PPV) vaccine. Gilts are given a challenge dose of a live virulent PPV serotype 1 isolate (PPV 002346-5) after approximately 40 days of gestation (dG). The vaccine is considered effective if >80% of fetuses in the vaccinated group are PPV negative after challenge. Confirmatory parameters include fetal size, weight and condition, the viremia status of the gilts after challenge, and the serological status of the gilts.

Gilts (with no history of reproductive disease or PPV vaccination) are randomly assigned to treatment groups: T01 = negative control (placebo product (PMP) matched) and T02 = 1.0 μg PPV/2 ml dose). No treatment/challenge (NTX) gilts are randomly assigned to pens as general health controls.

Gilts receive 2 ml of the appropriate drug intramuscularly on D0 and D21. After vaccination, gilts are mated between D37 and D50 and then assessed for pregnancy status at D74. On D81, pregnant gilts are given a challenge dose of 6.77 log ₁₀ TCID50/6 ml PPV serotype 1 intramuscularly and intranasally. Gilts are bled weekly, except during synchronization and mating periods (D36-D73). Hemagglutination inhibition (HI) assays are performed on serum samples collected on D7, D14, D21, D28, and D35; HI and polymerase chain reaction (PCR) (see Example 2) for viremia are performed on serum samples collected at D-3, D74, D80, D88, D95, D102 and D127. Gilts are necropsied at D128 and D129 (approximately 90 dG). At necropsy, the reproductive tract of each sow is removed and the position of the fetus in the uterus, the condition of the fetus, its size and weight are recorded. Thoracic wash samples (see Example 2) are collected from each fetus and tested for the presence of PPV using PCR.

Gilt viremia (PPV).

The vaccines are considered safe as animals show no abnormal body temperature 24 or 48 hours after vaccination, no abnormal local reactions due to the vaccine, and no clinical signs associated with vaccination (data not shown).

All gilts are negative for PPV viremia before vaccination, before D74 challenge and before D80 challenge. Thus, after vaccination, clinical signs associated with the administration of the vaccine are not observed. On D88, all ten T01 gilts are viremic and all vaccinated gilts

- 24 045363 are negative. All other blood samples taken at D95, D102 and D127 are negative for PPV viremia for all experimental groups (Table 4).

Table 4

Frequency distribution of PPV-positive gilts (PCR) given a challenge dose of PPV after ~40 days of gestation (dG) on D81_____________

Treatment/description Study day / days of pregnancy (dG) D74 D80 D88 D95 D102 D127 dG 32 dG 38 dG 46 dG 53 dG 60 dG 85 T01 Negative control 0/12 0/12 10/10* 0/10 0/10 0/10 T02 PPV (1.0 mcg/dose) 0/12 0/12 0/12 0/12 0/12 0/12

* 2 gilts were diagnosed as not pregnant and were therefore excluded from the group.

Fruit results.

At final necropsy on D128 and D129, 38% of the T01 (negative control) fetuses were normal, while 95% of the vaccinated group were normal. The mean size and weight of T01 (negative control) fruits were 14.4 cm and 245.9 g, respectively, while the mean size and weight of fruits in vaccinated pigs were 19.3 cm and 550 g, respectively (Table 5). Thus, the vaccinated group meets the criteria for protection against PPV infection, since according to the defining parameter for effectiveness against PPV established in Ph. Eur. 01/2008:0965, >80% of fetuses in the experimental group should be PPV negative.

PPV infection was confirmed in 113/146 negative control fetuses (T01) (77%). However, PPV infection in the vaccinated group (T02) is only 10%.

Table 5

Litter details: number, size, weight and condition of fetuses, and laboratory confirmation of PPV infection by PCR on thoracic wash samples)

Treatment T01 T02 Description Negative control PPV 1µg #pigs 10 eleven Total number of fruits 146 148 Average litter size 14.6 13.5 Fetal condition: #necrotic. (%) 9 (6%) 0 (0%) #mummies(%) 82 (56%) 8 (5%) # normal (%) 55 (38%) 140 (95%) Average size (cm) 14.4 19.3 Average weight (g) 245.9 550.0 Laboratory confirmation of PPV infection: # fruits from position thorac. prom. (%) 113 (77%) 15 (10%) % protected 90% # = quantity, % = percentage

Conclusion.

The PPV VP2 subunit vaccine of the present invention demonstrates protection of fetuses following challenge with a virulent heterologous PPV. The results of this study reveal that the vaccine is safe and effective in preventing viremia in gilts and PPV infection in fetuses when administered with just 1 μg of PPV VP2 subunit vaccine. In addition, the vaccine has been demonstrated to protect against a heterologous North American challenge strain.

Example 4.

Establishment of the minimum immunizing dose of the ppv vaccine - protected against the heterologous European strain of PPV.

The purpose of this study is to evaluate the onset of immunity provided by the porcine parvovirus vaccine (also called PPV vaccine or PPV VP2). In addition, safety and efficacy are assessed using a randomized, blinded, negative control study design involving vaccination and challenge.

The pigs are randomly divided into three groups. In groups 1 and 2, pigs are vaccinated twice, at three-week intervals (on D0 and D21). The second dose is administered three weeks before mating. All drugs are administered intramuscularly (IM) in a volume of 2 ml. Group 2 receives the PPV vaccine, while Group 1 is a placebo group receiving sterile diluent as a control product, and Group 3 serves as a strict control, without any treatment.

- 25 045363

The pigs are synchronized for estrus and three weeks after the second vaccination are artificially inseminated. Pregnant animals on D84 between days 39 and 42 of gestation receive a challenge dose of a virulent, heterologous PPV strain.

At D132-135, after approximately 90 days of gestation, the gilts are sacrificed, necropsied, and the fetuses examined.

Table 6 Study plan

Group 1st treatment D0 (2 ml in right. side neck IM) 2nd treatment D21 (2 ml per left. side neck IM) Provocation D84 6.0 Logi ₀ TCID ₅₀ /6 ml) dose (~40dG) 2 ml right, neck IM and 2 ml in each nostril intranasally Opening 1 (Negative control) Control product Control product Heb. strain PPV 401/09 (198669) D132 - D135 2 PPV (1 mcg/dose) PPV (1 mcg/dose) Heb. strain PPV 401/09 (198669) D132 - D135 3(Strict control) - - Without provocation D83

Assessment of PPV viremia in gilts before and after challenge using PCR.

All animals are negative for PPV by PCR for D-6 and D-1 before vaccination, which meets the inclusion criteria. After vaccination, all animals in the strict control and challenge groups are negative for prechallenge PPV antigen, and thus prechallenge PPV infection can be ruled out.

Viremia is examined on days 7 (D90), 14 (D97) and 21 (D104) after challenge and on the day of autopsy. After provocation, viremia is not detected in vaccinated animals; viremia occurs only in unvaccinated control animals.

At D90, (7 days after challenge) already 95% of unvaccinated control animals are positive for PPV. On D97, 60% of these animals were still positive, whereas on D104, all animals tested were PPV negative. In contrast, in the vaccinated group, all animals tested for D90, D97 or D104 are negative for PPV.

Table 7

Number of animals with viremia after challenge

7 days after provocation (D90) 14 days after provocation (D97) 21 days after provocation (D104) Control 19/20 (95%) 12/20 (60%) 0/20 PPV 0/20 0/20 0/20

Fruit results.

The percentage of fetuses infected with PPV was 91.4% in the control group, but only 4.3% in the PPV group (see Table 8).

Table 8

Percentage of positive fetuses per group and litter size

Group N Number of pigs N Number of fruits N Number of positions fruits % PPV positive (PCR) of fetuses FOR treatment regimen ¹ N Average litter size Min. % positive fruits per brood Max. % positive fruits per brood Control 19 269 246 91.4 14.2 57 100 PPV 19 231 10 4.3 12.2 0 20

¹ Number of PPV-positive fetuses/Number of fruits per group. N is the total number.

Assessment of the condition of the fruits.

All fetuses were assessed for their condition and divided into three categories: normal, mummified and autolyzed.

The majority of mummified and autolyzed fetuses are found in the control group. Only 39.8% of fetuses in this group are in normal condition, while in the vaccinated groups 97.4% (PPV group) of fetuses are in normal condition (see Table 9).

- 26 045363

Table 9 Fetal condition

Group Fetal condition I% normal] ι% autolyzed] % mummified] IN (total)] Control 39.8% 12.3% 48.0% 269 PPV 97.4% 0.9% 1.7% 231

Conclusion.

The PPV vaccine of the present invention demonstrates fetal protection following challenge with a virulent heterologous PPV, indicating that the vaccine is safe and effective in preventing PPV viremia and fetal infection using as little as 1 μg of vaccine. In addition, the vaccine has also been demonstrated to protect against the heterologous European strain for PPV challenge. Thus, the vaccine has a broad spectrum of protection as the vaccine protects against heterologous North American as well as heterologous European challenge strains.

All compositions and methods that are disclosed and claimed by the authors can be obtained and performed without unnecessary experiments in the light of the presented description. Although the compositions and methods of the present invention have been described in terms of preferred embodiments, those skilled in the art will appreciate that changes can be made to the compositions and methods and to the steps or sequence of steps of the method described by the inventors without departing from the spirit, spirit, and scope of the invention. More specifically, it will be apparent that certain agents that are chemically and physiologically related can substitute for the agents described herein in achieving the same or similar results. All such substitutions and modifications apparent to those skilled in the art are intended to be encompassed within the spirit, scope and concept of the invention as defined by the claims below.

List of sequences < 110> Boehringer Ingelheim Veterinary Medicine GmbH < 120> VACCINE AGAINST PORK PARVOVIRUS < 130> 01-3228 < 160>16 < 170> PatentIn version 3.5 < 210>1 < 211>579 < 212> PRT < 213> Porcine parvovirus < 400>1

Met Ser Glu Asn Val Glu Gln His 15

Leu Ser Ala Thr Gly Asn Glu Ser 20

Gly Arg Gly Ala Gly Gly Val Gly 3540

Gln Thr Glu Phe Gln Tyr Leu Gly 5055

His Ala Ser Arg Leu Ile His Leu 6570

Lys Arg Ile His Val Leu Asn Ser 85

Val Gln Asp Asp Ala His Thr Gln 100

Asp Ala Asn Ala Trp Gly Val Trp 115120

Asn Pro Ile Asn Ala Gly Thr Glu 1015

Gly Gly Gly Gly Gly Gly Gly Gly 2530

Val Ser Thr Gly Ser Phe Asn Asn 45

Glu Gly Leu Val Arg Ile Thr Ala 60

Asn Met Pro Glu His Glu Thr Tyr 7580

Glu Ser Gly Val Ala Gly Gln Met 9095

Met Val Thr Pro Trp Ser Leu Ile 105110

Phe Asn Pro Ala Asp Trp Gln Leu 125

- 27 045363

Ile Ser 130 Asn Asn Met Thr Glu 135 Ile Asn Leu Val Ser 140 Phe Glu Gln Glu Ile 145 Phe Asn Val Val Leu 150 Lys Thr Ile Thr Glu 155 Ser Ala Thr Ser Pro 160 Pro Thr Lys Ile Tyr 165 Asn Asn Asp Leu Thr 170 Ala Ser Leu Met Val 175 Ala Leu Asp Thr Asn 180 Asn Thr Leu Pro Tyr 185 Thr Pro Ala Ala Pro 190 Arg Ser Glu Thr Leu 195 Gly Phe Tyr Pro Trp 200 Leu Pro Thr Lys Pro 205 Thr Gln Tyr Arg Tyr 210 Tyr Leu Ser Cys Thr 215 Arg Asn Leu Asn Pro 220 Pro Thr Tyr Thr Gly 225 Gln Ser Glu Gln Ile 230 Thr Asp Ser Ile Gln 235 Thr Gly Leu His Ser 240 Asp Ile Met Phe Tyr 245 Thr Ile Glu Asn Ala 250 Val Pro Ile His Leu 255 Leu Arg Thr Gly Asp 260 Glu Phe Ser Thr Gly 265 Ile Tyr His Phe Asp 270 Thr Lys Pro Leu Lys 275 Leu Thr His Ser Trp 280 Gln Thr Asn Arg Ser 285 Leu Gly Leu Pro Pro 290 Lys Leu Leu Thr Glu 295 Pro Thr Thr Glu Gly 300 Asp Gln His Pro Gly 305 Thr Leu Pro Ala Ala 310 Asn Thr Arg Lys Gly 315 Tyr His Gln Thr Ile 320 Asn Asn Ser Tyr Thr 325 Glu Ala Thr Ala Ile 330 Arg Pro Ala Gln Val 335 Gly Tyr Asn Thr Pro 340 Tyr Met Asn Phe Glu 345 Tyr Ser Asn Gly Gly 350 Pro Phe Leu Thr Pro 355 Ile Val Pro Thr Ala 360 Asp Thr Gln Tyr Asn 365 Asp Asp Glu Pro Asn 370 Gly Ala Ile Arg Phe 375 Thr Met Gly Tyr Gln 380 His Gly Gln Leu Thr 385 Thr Ser Ser Gln Glu 390 Leu Glu Arg Tyr Thr 395 Phe Asn Pro Gln Ser 400 Lys Cys Gly Arg Ala 405 Pro Lys Gln Gln Phe 410 Asn Gln Gln Ser Pro 415 Leu Asn Leu Gln Asn 420 Thr Asn Asn Gly Thr 425 Leu Leu Pro Ser Asp 430 Pro Ile Gly Gly Lys 435 Thr Asn Met His Phe 440 Met Asn Thr Leu Asn 445 Thr Tyr Gly Pro Leu 450 Thr Ala Leu Asn Asn 455 Thr Ala Pro Val Phe 460 Pro Asn Gly Gln

- 28 045363

Ile Trp Asp Lys Glu 465

Thr Ala Pro Phe Val 485

Lys Ile Ala Pro Asn 500

Gln Pro Arg Ile Ile 515

Thr Phe Thr Ala Lys 530

Gln His Thr Thr Thr 545

Ile Gly Gly Ile Lys

565

Lys Leu Tyr

Leu Asp Thr Asp Leu 470

Cys Lys Asn Asn Pro 490

Leu Thr Asp Asp Phe 505

Thr Tyr Ser Asn Phe 520

Met Arg Ser Ser Asn 535

Ala Glu Asn Ile Gly 550

Met Phe Pro Glu Tyr 570

Lys Pro Arg Leu His 475

Pro Gly Gln Leu Phe 495

Asn Ala Asp Ser Pro 510

Trp Trp Lys Gly Thr 525

Met Trp Asn Pro Ile 540

Asn Tyr Ile Pro Thr 555

Ser Gln Leu Ile Pro

575

Val 480

Val

Gln

Leu

Gln

Asn 560

Arg

<210> <211> <212> <213> <400> Met Ser 1 2 579 PRT Porcine parvovirus 2 Glu Asn Val Glu Gln 5 His Asn Pro 10 Ile Asn Ala Gly Thr 15 Leu Ser Ala Thr 20 Gly Asn Glu Ser Ile 25 Gly Gly Gly Gly Gly 30 Gly Gly Arg Gly 35 Ser Ile Gly Val Gly 40 Val Ser Thr Gly Ser 45 Phe Asn Gln Thr 50 Glu Phe Gln Tyr Leu 55 Gly Glu Gly Leu Val 60 Arg Ile Thr His 65 Ala Ser Arg Leu Ile 70 His Leu Asn Met Pro 75 Glu His Glu Thr Lys Arg Ile His Val 85 Leu Asn Ser Glu Ser 90 Gly Val Ala Gly Gln 95 Val Gln Asp Asp 100 Ala His Thr Gln Met 105 Val Thr Pro Trp Ser 110 Leu Asp Ala Asn 115 Ala Trp Gly Val Trp 120 Phe Asn Pro Ala Asp 125 Trp Gln Ile Ser 130 Asn Asn Met Thr Glu 135 Ile Asn Leu Val Ser 140 Phe Glu Gln Ile 145 Phe Asn Val Val Leu 150 Lys Thr Ile Thr Glu 155 Ser Ala Thr Ser Pro Thr Lys Ile Tyr 165 Asn Asn Asp Leu Thr 170 Ala Ser Leu Met Val 175 Leu Asp Thr Asn Asn Thr Leu Pro Tyr Thr Pro Ala Ala Pro Arg

Glu

Gly

Asn

Ala

Tyr 80

Met

Ile

Leu

Glu

Pro 160

Ala

Ser

- 29 045363

180

185

190

Glu Thr Leu Gly Phe 195

Arg Tyr Tyr Leu Ser 210

Gly Gln Ser Glu Gln 225

Asp Ile Met Phe Tyr 245

Arg Thr Gly Asp Glu 260

Pro Leu Lys Leu Thr 275

Pro Pro Lys Leu Leu 290

Gly Thr Leu Pro Ala 305

Asn Asn Ser Tyr Thr 325

Tyr Asn Thr Pro Tyr 340

Leu Thr Pro Ile Val 355

Pro Asn Gly Ala Ile 370

Thr Thr Ser Ser Gln 385

Lys Cys Gly Arg Ala 405

Asn Leu Gln Asn Thr 420

Gly Gly Lys Thr Asn 435

Pro Leu Thr Ala Leu 450

Ile Trp Asp Lys Glu 465

Thr Ala Pro Phe Val 485

Lys Ile Ala Pro Asn 500

Tyr Pro Trp Leu Pro 200

Cys Thr Arg Asn Leu 215

Ile Thr Asp Ser Ile 230

Thr Ile Glu Asn Ala 250

Phe Ser Thr Gly Ile 265

His Ser Trp Gln Thr 280

Thr Glu Pro Thr Thr 295

Ala Asn Thr Arg Lys 310

Glu Ala Thr Ala Ile 330

Met Asn Phe Glu Tyr 345

Pro Thr Ala Asp Thr 360

Arg Phe Thr Met Gly 375

Glu Leu Glu Arg Tyr 390

Pro Lys Gln Gln Phe 410

Asn Asn Gly Thr Leu 425

Met His Phe Met Asn 440

Asn Asn Thr Ala Pro 455

Leu Asp Thr Asp Leu 470

Cys Lys Asn Asn Pro 490

Leu Thr Asp Asp Phe 505

Thr Lys Pro Thr Gln 205

Asn Pro Pro Thr Tyr 220

Gln Thr Gly Leu His 235

Val Pro Ile His Leu 255

Tyr His Phe Asp Thr 270

Asn Arg Ser Leu Gly 285

Glu Gly Asp Gln His 300

Gly Tyr His Gln Thr 315

Arg Pro Ala Gln Val 335

Ser Asn Gly Gly Pro 350

Gln Tyr Asn Asp Asp 365

Tyr Gln His Gly Gln 380

Thr Phe Asn Pro Gln 395

Asn Gln Gln Ser Pro 415

Leu Pro Ser Asp Pro 430

Thr Leu Asn Thr Tyr 445

Val Phe Pro Asn Gly 460

Lys Pro Arg Leu His 475

Pro Gly Gln Leu Phe 495

Asn Ala Asp Ser Pro 510

Tyr

Thr

Ser 240

Leu

Lys

Leu

Pro

Ile 320

Gly

Phe

Glu

Leu

Ser 400

Leu

Ile

Gly

Gln

Val 480

Val

Gln

- 30 045363

Gln Pro Arg Ile Ile Thr Tyr Ser Asn Phe Trp Trp Lys Gly Thr Leu

515 520 525

Thr Phe 530

Gln His 545

Thr Ala

Thr Thr

Lys Met

Thr Ala

550

Arg Ser 535

Glu Asn

Ser Asn

Ile Gly

Met Trp 540

Asn Tyr 555

Asn Pro

Ile Pro

Ile Gln

Thr Asn

560

Ile Gly Gly Ile Lys Met Phe Pro Glu Tyr Ser Gln Leu Ile Pro Arg

565 570575

Lys Leu Tyr <210>3 <211>1760 <212> DNA <213> Porcine parvovirus <400>3 ggatccgcca ccatgtccga gaacgtggag cagcacaacc cgataaacgc aggcacagag ctgtcggcga ctggcaatga gagcggaggc ggaggcggcg gaggaggtgg acgcggcgca gg cggagtgg gcgtttcgac cggaagcttc aataatcaaa ccgagtttca gtacctgggc gagggtttgg tgcggattac ggctcacgcg tcccgactga tacatctcaa tatgccggag catgagacct acaagcgtat ccatgtcctg aactcggaat cgggcgtcgc cggtcagatg gtccaagatg atgctcatac tcagatggtg acaccctgga gcttgataga tgccaacgca tggggcgtgt ggttcaaccc tgcggattgg cagctgataa gcaataacat gacagaaatc aatttggtta gtttgagca agagatattt aat gtcgtgc tgaaaaccat cacagagagc gccacgagcc ccccgacgaa gatttacaat aacgacctga cggcgtcctt gatggtcgcc ttggacacaa ataacaccct cccgtacacc cccgcggccc cccgcagcga gaccctgggc ttttatccct ggctgcccac caagccaacg cagtatcgct actacct gag ttgtacacga aatttgaatc cgccgacata cactggtcag tcggagcaga tcacggacag cattcaaacg ggcctgcact ccgatatcat gttttacacg atagagaacg cagtacccat ccacctgctg cgtacgggag atgagttctc gaccggtatc tatcattttg acacaaaacc cttgaaattg acgcacagtt ggcaaaccaa tcgctcgctg ggcttgcccc caaagttgtt gacggaaccc accaccgagg gtgaccaaca cccaggcact ctccccgcag caaatacccg caagggctat catcaaacga tcaacaatag ctataccgag gctaccgcca ttcggccagc acaggtggga tacaacacac cttacatgaa ctttgaatac tccaacggcg gcccgttcct gaccccgata gttccgaccg ccgacactca gtacaacgat gacgagccga acggcgccat caggtttacc atgggctatc agcac ggtca attgacaact tcgtcgcaag aactggaacg ctatacattc aaccctcaga gtaagtgtgg ccgggcaccc aaacaacagt tcaaccagca atccccactg aacctgcaga ataccaacaa tggcacgctg ctgccatccg atcccattgg aggaaagacc aacatgcatt tcatgaacac gctgaataca tacggaccac tgaccgccct gaacaatacc gcacccgtct tccctaatgg ccagatctgg gataaagagc tggatacgga cctgaagccc cgactccacg tgactgcgcc cttt gtgtgc aaaaataacc caccgggaca gttgttcgtc aaaatagccc ccaacttgac cgacgacttc aatgcagaca gccctcagca gccgcgaatc atcacctatt cgaacttctg gtggaagggc acgctgactt tcacggctaa gatgcgctcg agcaatatgt ggaacccaat ccagcaacat acca caaccg ctgaaaatat tggcaattac atccctacga atataggcgg aataaagatg tttccggagt attcccagct cattccacgc

120

180

240

300

360

420

480

540

600

660

720

780

840

900

960

1020

1080

1140

1200

1260

1320

1380

1440

1500

1560

1620

1680

1740 aagctgtatt aagcggccgc

1760 <210>4 <211>1760 <212> DNA <213> Porcine parvovirus <400>4 ggatccgcca ccatgtccga gaacgtggag cagcacaacc cgataaacgc aggcacagag ctgtcggcga ctggcaatga atcgatcggc ggaggcggcg gaggaggtgg acgcggatcg atcggagtgg gcgtttcgac cggaagcttc aataatcaaa ccgagtttca gtacctgggc gagggtttgg tgcggattac ggctcacgcg tcccgactga tacatctcaa tatgccggag catgagacct acaagcgtat ccatgtcctg aactcggaat cgggcgtcgc cggtcagatg

120

180

240

300

- 31 045363 gtccaagatg tggggcgtgt aatttggtta gccacgagcc ttggacacaa ttttatccct aatttgaatc ggcctgcact cgtacgggag acgcacagtt accaccgagg catcaaacga tacaacacac gttccgaccg atgggctatc aaccctcaga atgctcatac ggttcaaccc gtttcgagca ccccgacgaa ataacaccct ggctgcccac cgccgacata ccgatatcat atgagttctc ggcaaaccaa gtgaccaaca tcaacaatag cttacatgaa ccgacactca agcacggtca gtaagtgtgg tcagatggtg tgcggattgg agagatattt gatttacaat cccgtacacc caagccaac g cactggtcag gttttacacg gaccggtatc tcgctcgctg cccaggcact ctataccgag ctttgaatac gtacaacgat attgacaact ccgggcaccc acaccctgga cagctgataa aatgtcgtgc aacgacctga cccgcggccc cagtatcgct tcggagcaga atagagaacg tatcattttg ggcttgcccc ctccccgcag gctaccgcca tccaacggcg gacgagccga tcgtcgcaag aaacaacagt gcttgataga gcaataacat tga aaaccat cggcgtcctt cccgcagcga actacctgag tcacggacag cagtacccat acacaaaacc caaagttgtt caaatacccg ttcggccagc gcccgttcct acggcgccat aactggaacg tcaaccagca tgccaacgca gacagaaatc cacagagagc gatggtcgcc gaccctgggc ttgtacacga cattcaaacg ccacctgctg cttgaaattg gacggaaccc caagggctat acaggtggga gaccccgata caggtttacc ctatacattc atccccactg

360

420

480

540

600

660

720

780

840

900

960

1020

1080

1140

1200

1260 aacctgcaga aacatgcatt gcacccgtct cgactccacg aaaatagccc atcacctatt agcaatatgt atccctacga aagctgtatt ataccaacaa tcatgaacac tccctaatgg tgactgcgcc ccaacttgac cgaacttctg ggaacccaat atataggcgg aagcggccgc tggcacgctg gctgaata ca ccagatctgg ctttgtgtgc cgacgacttc gtggaagggc ccagcaacat aataaagatg ctgccatccg tacggaccac gataaagagc aaaaataacc aatgcagaca acgctgactt accacaaccg tttccggagt atcccattgg tgaccgccct tggatacgga caccgggaca gccctcagca tcacggcta a ctgaaaatat attcccagct aggaaagacc gaacaatacc cctgaagccc gttgttcgtc gccgcgaatc gatgcgctcg tggcaattac cattccacgc

1320

1380

1440

1500

1560

1620

1680

1740

1760 <210>5 <211>579 <212> PRT <213> Porcine parvovirus <400>5

Met Ser Glu Asn Val Glu Gln His Asn Pro Ile Asn Ala Gly ThrGlu

5 1015

Leu Ser Ala Thr Gly Asn Glu Ser Ile Gly Gly Gly Gly Gly GlyGly

2530

Gly Arg Gly Ser Ile Gly Val Gly Val Ser Thr Gly Ser Phe Asn Asn 35 4045

Gln Thr Glu Phe Gln Tyr Leu Gly Glu Gly Leu Val Arg Ile Thr Ala 50 5560

His Ala Ser Arg Leu Ile His Leu Asn Met Pro Glu His Glu ThrTyr

70 7580

Lys Arg Ile His Val Leu Asn Ser Glu Ser Gly Val Ala Gly GlnMet

9095

Val Gln Asp Asp Ala His Thr Gln Met Val Thr Pro Trp Ser Leu Ile 100 105110

Asp Ala Asn Ala Trp Gly Val Trp Phe Asn Pro Ala Asp Trp Gln Leu 115 120125

Ile Ser Asn Asn Met Thr Glu Ile Asn Leu Val Ser Phe Glu Gln Ala 130 135140

Ile Phe Asn Val Val Leu Lys Thr Ile Thr Glu Ser Ala Thr Ser Pro 145 150 155160

- 32 045363

Pro Thr Lys Ile Tyr 165 Asn Asn Asp Leu Thr 170 Ala Ser Leu Met Val 175 Ala Leu Asp Thr Asn 180 Asn Thr Leu Pro Tyr 185 Thr Pro Ala Ala Pro 190 Arg Ser Glu Thr Leu 195 Gly Phe Tyr Pro Trp 200 Leu Pro Thr Lys Pro 205 Thr Gln Tyr Arg Tyr 210 Tyr Leu Ser Cys Ile 215 Arg Asn Leu Asn Pro 220 Pro Thr Tyr Thr Gly 225 Gln Ser Glu Gln Ile 230 Thr Asp Ser Ile Gln 235 Thr Gly Leu His Ser 240 Asp Ile Met Phe Tyr 245 Thr Ile Glu Asn Ala 250 Val Pro Ile His Leu 255 Leu Arg Thr Gly Asp 260 Glu Phe Ser Thr Gly 265 Ile Tyr His Phe Asp 270 Thr Lys Pro Leu Lys 275 Leu Thr His Ser Trp 280 Gln Thr Asn Arg Ser 285 Leu Gly Leu Pro Pro 290 Lys Leu Leu Thr Glu 295 Pro Thr Thr Glu Gly 300 Asp Gln His Pro Gly 305 Thr Leu Pro Ala Ala 310 Asn Thr Arg Lys Gly 315 Tyr His Gln Thr Thr 320 Asn Asn Ser Tyr Thr 325 Glu Ala Thr Ala Ile 330 Arg Pro Ala Gln Val 335 Gly Tyr Asn Thr Pro 340 Tyr Met Asn Phe Glu 345 Tyr Ser Asn Gly Gly 350 Pro Phe Leu Thr Pro 355 Ile Val Pro Thr Ala 360 Asp Thr Gln Tyr Asn 365 Asp Asp Glu Pro Asn 370 Gly Ala Ile Arg Phe 375 Thr Met Asp Tyr Gln 380 His Gly Gln Leu Thr 385 Thr Ser Ser Gln Glu 390 Leu Glu Arg Tyr Thr 395 Phe Asn Pro Gln Ser 400 Lys Cys Gly Arg Ala 405 Pro Lys Gln Gln Phe 410 Asn Gln Gln Ser Pro 415 Leu Asn Leu Gln Asn 420 Thr Asn Asn Gly Thr 425 Leu Leu Pro Ser Asp 430 Pro Ile Gly Gly Lys 435 Thr Asn Met His Phe 440 Met Asn Thr Leu Asn 445 Thr Tyr Gly Pro Leu 450 Thr Ala Leu Asn Asn 455 Thr Ala Pro Val Phe 460 Pro Asn Gly Gln Ile 465 Trp Asp Lys Glu Leu 470 Asp Thr Asp Leu Lys 475 Pro Arg Leu His Val 480 Thr Ala Pro Phe Val 485 Cys Lys Asn Asn Pro 490 Pro Gly Gln Leu Phe 495 Val

- 33 045363

Lys Ile Ala Pro Asn Leu Thr Asp 500

Gln Pro Arg Ile Ile Thr Tyr Ser 515520

Thr Phe Thr Ala Lys Met Arg Ser 530535

Gln His Thr Thr Thr Ala Glu Asn

545550

Ile Gly Gly Ile Arg Met Phe Pro 565

Lys Leu Tyr

Asp Phe Asn Ala Asp Ser Pro Gln 505510

Asn Phe Trp Trp Lys Gly Thr Leu 525

Ser Asn Met Trp Asn Pro Ile Gln 540

Ile Gly Asn Tyr Ile Pro Thr Asn 555 560

Glu Tyr Ser Gln Leu Ile Pro Arg 570 575 <210>6 <211>579 <212> PRT <213> Porcine parvovirus <400>6

Met Ser Glu Asn Val Glu Gln His 15

Leu Ser Ala Thr Gly Asn Glu Ser 20

Gly Arg Gly Ser Ile Gly Val Gly 3540

Gln Thr Glu Phe Gln Tyr Leu Gly 5055

His Ala Ser Arg Leu Ile His Leu 6570

Lys Arg Ile His Val Leu Asn Ser 85

Val Gln Asp Asp Ala His Thr Gln 100

Asp Ala Asn Ala Trp Gly Val Trp 115120

Ile Ser Asn Asn Met Thr Glu Ile 130135

Ile Phe Asn Val Val Leu Lys Thr 145150

Pro Thr Lys Ile Tyr Asn Asn Asp 165

Leu Asp Thr Asn Asn Thr Leu Pro 180

Glu Thr Leu Gly Phe Tyr Pro Trp

195200

Arg Tyr Tyr Leu Ser Cys Thr Arg

Asn Pro Ile Asn Ala Gly Thr Glu 1015

Ile Gly Gly Gly Gly Gly Gly Gly 2530

Val Ser Thr Gly Ser Phe Asn Asn 45

Glu Gly Leu Val Arg Ile Thr Ala 60

Asn Met Pro Glu His Glu Thr Tyr 7580

Glu Ser Gly Val Ala Gly Gln Met 9095

Met Val Thr Pro Trp Ser Leu Ile 105110

Phe Asn Pro Ala Asp Trp Gln Leu 125

Asn Leu Val Ser Phe Glu Gln Glu 140

Ile Thr Glu Ser Ala Thr Ser Pro 155160

Leu Thr Ala Ser Leu Met Val Ala 170175

Tyr Thr Pro Ala Ala Pro Arg Ser 185190

Leu Pro Thr Lys Pro Thr Gln Tyr 205

Asn Leu Asn Pro Pro Thr Tyr Thr

- 34 045363

210 215 220

Gly 225 Gln Ser Glu Gln Ile 230 Thr Asp Ser Ile Gln 235 Thr Gly Leu His Ser 240 Asp Ile Met Phe Tyr 245 Thr Ile Glu Asn Ala 250 Val Pro Ile His Leu 255 Leu Arg Thr Gly Asp 260 Glu Phe Ser Thr Gly 265 Ile Tyr His Phe Asp 270 Thr Lys Pro Leu Lys 275 Leu Thr His Ser Trp 280 Gln Thr Asn Arg Ser 285 Leu Gly Leu Pro Pro 290 Lys Leu Leu Thr Glu 295 Pro Thr Thr Glu Gly 300 Asp Gln His Pro Gly 305 Thr Leu Pro Ala Ala 310 Asn Thr Arg Lys Gly 315 Tyr His Gln Thr Ile 320 Asn Asn Ser Tyr Thr 325 Glu Ala Thr Ala Ile 330 Arg Pro Ala Gln Val 335 Gly Tyr Asn Thr Pro Tyr Met Asn Phe Glu Tyr Ser Asn Gly Gly Pro Phe 340 345 350 Leu Thr Pro 355 Ile Val Pro Thr Ala 360 Asp Thr Gln Tyr Asn 365 Asp Asp Glu Pro Asn 370 Gly Ala Ile Arg Phe 375 Thr Met Gly Tyr Gln 380 His Gly Gln Leu Thr 385 Thr Ser Ser Gln Glu 390 Leu Glu Arg Tyr Thr 395 Phe Asn Pro Gln Ser 400 Lys Cys Gly Arg Ala 405 Pro Lys Gln Gln Phe 410 Asn Gln Gln Ser Pro 415 Leu Asn Leu Gln Asn 420 Thr Asn Asn Gly Thr 425 Leu Leu Pro Ser Asp 430 Pro Ile Gly Gly Lys 435 Thr Asn Met His Phe 440 Met Asn Thr Leu Asn 445 Thr Tyr Gly Pro Leu 450 Thr Ala Leu Asn Asn 455 Thr Ala Pro Val Phe 460 Pro Asn Gly Gln Ile 465 Trp Asp Lys Glu Leu 470 Asp Thr Asp Leu Lys 475 Pro Arg Leu His Val 480 Thr Ala Pro Phe Val 485 Cys Lys Asn Asn Pro 490 Pro Gly Gln Leu Phe 495 Val Lys Ile Ala Pro 500 Asn Leu Thr Asp Asp 505 Phe Asn Ala Asp Ser 510 Pro Gln Gln Pro Arg 515 Ile Ile Thr Tyr Ser 520 Asn Phe Trp Trp Lys 525 Gly Thr Leu Thr Phe Thr Ala Lys Met Arg Ser Ser Asn Met Trp Asn Pro Ile Gln

530 535 540

- 35 045363

Gln His Thr Thr Thr Ala Glu Asn

545 550

Ile Gly Gly Ile Lys Met Phe Pro

565

Ile Gly Asn Tyr Ile Pro Thr

555

Glu Tyr Ser Gln Leu Ile Pro

570 575

Asn 560

Arg

Lys Leu Tyr <210>7 <211>579 <212> PRT <213> Porcine parvovirus <400>7

Met Ser Glu Asn Val Glu Gln 15

Leu Ser Ala Thr Gly Asn Glu 20

His Asn Pro Ile Asn Ala Gly Thr

15

Ser Ile Gly Gly Gly Gly Gly Gly

thirty

Gly Arg Gly Ser Ile 35

Gln Thr Glu Phe Gln 50

His Ala Ser Arg Leu 65

Lys Arg Ile His Val 85

Val Gln Asp Asp Ala 100

Asp Ala Asn Ala Trp 115

Ile Ser Asn Asn Met 130

Ile Phe Asn Val Val 145

Pro Thr Lys Ile Tyr 165

Leu Asp Thr Asn Asn 180

Glu Thr Leu Gly Phe 195

Arg Tyr Tyr Leu Ser 210

Gly Gln Ser Glu Gln 225

Asp Ile Met Phe Tyr

245

Arg Thr Gly Asp Glu

Gly Val Gly Val Ser 40

Tyr Leu Gly Glu Gly 55

Ile His Leu Asn Met 70

Leu Asn Ser Glu Ser 90

His Thr Gln Met Val 105

Gly Val Trp Phe Asn 120

Thr Glu Ile Asn Leu 135

Leu Lys Thr Ile Thr 150

Asn Asn Asp Leu Thr

170

Thr Leu Pro Tyr Thr 185

Tyr Pro Trp Leu Pro 200

Cys Thr Arg Asn Leu 215

Ile Thr Asp Ser Ile 230

Thr Ile Glu Asn Ala

250

Phe Ser Thr Gly Ile

Thr Gly Ser Phe Asn 45

Leu Val Arg Ile Thr 60

Pro Glu His Glu Thr 75

Gly Val Ala Gly Gln 95

Thr Pro Trp Ser Leu 110

Pro Ala Asp Trp Gln 125

Val Ser Phe Glu Gln 140

Glu Ser Ala Thr Ser 155

Ala Ser Leu Met Val

175

Pro Ala Ala Pro Arg 190

Thr Lys Pro Thr Gln 205

Asn Pro Pro Thr Tyr 220

Gln Thr Gly Leu His 235

Val Pro Ile His Leu

255

Tyr His Phe Asp Thr

Glu

Gly

Asn

Ala

Tyr 80

Met

Ile

Leu

Glu

Pro 160

Ala

Ser

Tyr

Thr

Ser 240

Leu

Lys

- 36 045363

260 265 270 Pro Leu Lys 275 Leu Thr His Ser Trp 280 Gln Thr Asn Arg Ser 285 Leu Gly Leu Pro Pro 290 Lys Leu Leu Thr Glu 295 Pro Thr Thr Glu Gly 300 Asp Gln His Pro Gly 305 Thr Leu Pro Ala Ala 310 Asn Thr Arg Lys Gly 315 Tyr His Gln Thr Ile 320 Asn Asn Ser Tyr Thr 325 Glu Ala Thr Ala Ile 330 Arg Pro Ala Gln Val 335 Gly Tyr Asn Thr Pro 340 Tyr Met Asn Phe Glu 345 Tyr Ser Asn Gly Gly 350 Pro Phe Phe Ile Pro 355 Ile Val Pro Thr Ala 360 Asp Thr Gln Tyr Asn 365 Asp Asp Glu Pro Asn 370 Gly Gly Ile Arg Phe 375 Thr Met Gly Tyr Gln 380 His Gly Gln Leu Ile 385 Thr Ser Ser Gln Glu 390 Val Glu Arg Tyr Thr 395 Phe Asn Pro Gln Arg 400 Lys Cys Gly Arg Gly 405 Ala Lys Gln Gln Phe 410 Asn Gln Gln Ser Pro 415 Leu Asn Ile Gln Asn 420 Thr Asn Asn Gly Thr 425 Ile Leu Pro Ser Asp 430 Pro Ile Gly Gly Lys 435 Thr Asn Met His Phe 440 Met Asn Thr Pro Asn 445 Thr Tyr Gly Pro Leu 450 Thr Ala Val Asn Asn 455 Thr Ala Pro Val Phe 460 Pro Asn Gly Gln Ile 465 Trp Asp Lys Glu Leu 470 Asp Thr Asp Leu Lys 475 Pro Arg Leu His Val 480 Thr Ala Pro Phe Val 485 Cys Lys Asn Asn Pro 490 Pro Gly Gln Leu Phe 495 Val Lys Ile Ala Pro 500 Asn Leu Thr Asp Asp 505 Phe Asn Ala Asp Ser 510 Pro Gln Gln Pro Arg 515 Ile Ile Thr Tyr Ser 520 Asn Phe Trp Trp Lys 525 Gly Thr Leu Thr Phe 530 Thr Ala Lys Met Arg 535 Ser Ser Asn Met Trp 540 Asn Pro Ile Gln Gln 545 His Thr Thr Thr Ala 550 Glu Asn Ile Gly Asn 555 Tyr Ile Pro Thr Asn 560 Ile Gly Gly Ile Lys 565 Met Phe Pro Glu Tyr 570 Ser Gln Leu Ile Pro 575 Arg

Lys Leu Tyr <210> 8

- 37 045363 <211> 579 <212> PRT <213> Porcine parvovirus <400> 8

Met 1 Ser Glu Asn Val 5 Glu Gln His Asn Pro 10 Ile Asn Ala Gly Thr 15 Glu Leu Ser Ala Thr 20 Gly Asn Glu Ser Ile 25 Gly Gly Gly Gly Gly 30 Gly Gly Gly Arg Gly 35 Ser Ile Gly Val Gly 40 Val Ser Thr Gly Ser 45 Phe Asn Asn Gln Thr 50 Glu Phe Gln Tyr Leu 55 Gly Glu Gly Leu Val 60 Arg Ile Thr Ala His 65 Ala Ser Arg Leu Ile 70 His Leu Asn Met Pro 75 Glu His Glu Thr Tyr 80 Lys Arg Ile His Val 85 Leu Asn Ser Glu Ser 90 Gly Val Ala Gly Gln 95 Met Val Gln Asp Asp 100 Ala His Thr Gln Met 105 Val Thr Pro Trp Ser 110 Leu Ile Asp Ala Asn 115 Ala Trp Gly Val Trp 120 Phe Asn Pro Ala Asp 125 Trp Gln Leu Ile Ser 130 Asn Asn Met Thr Glu 135 Ile Asn Leu Val Ser 140 Phe Glu Gln Ala Ile 145 Phe Asn Val Val Leu 150 Lys Thr Ile Thr Glu 155 Ser Ala Thr Ser Pro 160 Pro Thr Lys Ile Tyr 165 Asn Asn Asp Leu Thr 170 Ala Ser Leu Met Val 175 Ala Leu His Thr Asn 180 Asn Thr Leu Pro Tyr 185 Thr Pro Ala Ala Pro 190 Arg Ser Glu Thr Leu 195 Gly Phe Tyr Pro Trp 200 Leu Pro Thr Lys Pro 205 Thr Gln Tyr Arg Tyr 210 Tyr Leu Ser Cys Ile 215 Arg Asn Leu Asn Pro 220 Pro Thr Tyr Thr Gly 225 Gln Ser Glu Gln Ile 230 Thr Asp Ser Ile Gln 235 Thr Gly Leu His Ser 240 Asp Ile Met Phe Tyr 245 Thr Ile Glu Asn Ala 250 Val Pro Ile His Leu 255 Leu Arg Thr Gly Asp 260 Glu Phe Ser Thr Gly 265 Ile Tyr His Phe Asp 270 Thr Lys Pro Leu Lys 275 Leu Thr His Ser Trp 280 Gln Thr Asn Arg Ser 285 Leu Gly Leu Pro Pro 290 Lys Leu Leu Thr Glu 295 Pro Thr Thr Glu Gly 300 Asp Gln His Pro Gly 305 Thr Leu Pro Ala Ala 310 Asn Thr Arg Lys Gly 315 Tyr His Gln Thr Ile 320

- 38 045363

Asn Asn Ser Tyr Thr 325 Glu Ala Thr Ala Ile 330 Arg Pro Ala Gln Val 335 Gly Tyr Asn Thr Pro 340 Tyr Met Asn Phe Glu 345 Tyr Ser Asn Gly Gly 350 Pro Phe Leu Thr Pro 355 Ile Val Pro Thr Ala 360 Asp Thr Gln Tyr Asn 365 Asp Asp Glu Pro Asn 370 Gly Ala Ile Arg Phe 375 Thr Met Asp Tyr Gln 380 His Gly His Leu Thr 385 Thr Ser Ser Gln Glu 390 Leu Glu Arg Tyr Thr 395 Phe Asn Pro Gln Ser 400 Lys Cys Gly Arg Thr 405 Pro Lys Gln Gln Phe 410 Asn Gln Gln Ser Pro 415 Leu Asn Leu Gln Asn 420 Thr Asn Asn Gly Thr 425 Leu Leu Pro Ser Asp 430 Pro Ile Gly Gly Lys 435 Thr Asn Met His Phe 440 Met Asn Thr Leu Asn 445 Thr Tyr Gly Pro Leu 450 Thr Ala Leu Asn Asn 455 Thr Ala Pro Val Phe 460 Pro Asn Gly Gln Ile 465 Trp Asp Lys Glu Leu 470 Asp Thr Asp Leu Lys 475 Pro Arg Leu His Val 480 Thr Ala Pro Phe Val 485 Cys Lys Asn Asn Pro 490 Pro Gly Gln Leu Phe 495 Val Lys Ile Ala Pro 500 Asn Leu Thr Asp Asp 505 Phe Asn Ala Asp Ser 510 Pro Gln Gln Pro Arg 515 Ile Ile Thr Tyr Ser 520 Asn Phe Trp Trp Lys 525 Gly Thr Leu Thr Phe 530 Thr Ala Lys Met Arg 535 Ser Ser Asn Met Trp 540 Asn Pro Ile Gln Gln 545 His Thr Thr Thr Ala 550 Glu Asn Ile Gly Lys 555 Tyr Ile Pro Thr Asn 560 Ile Gly Gly Ile Lys 565 Met Phe Pro Glu Tyr 570 Ser Gln Leu Ile Pro 575 Arg

Lys Leu Tyr <210>9 <211>579 <212> PRT <213> Porcine parvovirus <400>9

Met Ser Glu Asn Val Glu Gln 15

Leu Ser Ala Thr Gly Asn Glu 20

His Asn Pro Ile Asn Ala Gly Thr Glu 10 15

Ser Ile Gly Gly Gly Gly Gly Gly Gly 25 30

- 39 045363

Gly Arg Gly 35 Ser Ile Gly Val Gly 40 Val Ser Thr Gly Ser 45 Phe Asn Asn Gln Thr 50 Glu Phe Gln Tyr Leu 55 Gly Glu Gly Leu Val 60 Arg Ile Thr Ala His 65 Ala Ser Arg Leu Ile 70 His Leu Asn Met Pro 75 Glu His Glu Thr Tyr 80 Lys Arg Ile His Val 85 Leu Asn Ser Glu Ser 90 Gly Val Ala Gly Gln 95 Met Val Gln Asp Asp 100 Ala His Thr Gln Met 105 Val Thr Pro Trp Ser 110 Leu Ile Asp Ala Asn 115 Ala Trp Gly Val Trp 120 Phe Asn Pro Ala Asp 125 Trp Gln Leu Ile Ser 130 Asn Asn Met Thr Glu 135 Ile Asn Leu Val Ser 140 Phe Glu Gln Glu Ile 145 Phe Asn Val Val Leu 150 Lys Thr Ile Thr Glu 155 Ser Ala Thr Ser Pro 160 Pro Thr Lys Ile Tyr 165 Asn Asn Asp Leu Thr 170 Ala Ser Leu Met Val 175 Ala Leu Asp Thr Asn 180 Asn Thr Leu Pro Tyr 185 Thr Pro Ala Ala Pro 190 Arg Ser Glu Thr Leu 195 Gly Phe Tyr Pro Trp 200 Leu Pro Thr Lys Pro 205 Thr Gln Tyr Arg Tyr 210 Tyr Leu Ser Cys Thr 215 Arg Asn Leu Asn Pro 220 Pro Thr Tyr Thr Gly 225 Gln Ser Glu Gln Ile 230 Thr Asp Ser Ile Gln 235 Thr Gly Leu His Ser 240 Asp Ile Met Phe Tyr 245 Thr Ile Glu Asn Ala 250 Val Pro Ile His Leu 255 Leu Arg Thr Gly Asp 260 Glu Phe Ser Thr Gly 265 Ile Tyr His Phe Asp 270 Thr Lys Pro Leu Lys 275 Leu Thr His Ser Trp 280 Gln Thr Asn Arg Ser 285 Leu Gly Leu Pro Pro 290 Lys Leu Leu Thr Glu 295 Pro Thr Thr Glu Gly 300 Asp Gln His Pro Gly 305 Thr Leu Pro Ala Ala 310 Asn Thr Arg Lys Gly 315 Tyr His Gln Thr Ile 320 Asn Asn Ser Tyr Thr 325 Glu Ala Thr Ala Ile 330 Arg Pro Ala Gln Val 335 Gly Tyr Asn Thr Pro 340 Tyr Met Asn Phe Glu 345 Tyr Ser Asn Gly Gly 350 Pro Phe Leu Thr Pro 355 Ile Val Pro Thr Ala 360 Asp Thr Gln Tyr Asn 365 Asp Asp Glu

- 40 045363

Pro Asn 370 Gly Ala Ile Arg Phe 375 Thr Met Gly Tyr Gln 380 His Gly Gln Leu Thr 385 Thr Ser Ser Gln Glu 390 Leu Glu Arg Tyr Thr 395 Phe Asn Pro Gln Ser 400 Lys Cys Gly Arg Ala 405 Pro Lys Gln Gln Phe 410 Asn Gln Gln Ser Pro 415 Leu Asn Leu Gln Asn 420 Thr Asn Asn Gly Thr 425 Leu Leu Pro Ser Asp 430 Pro Ile Gly Gly Lys 435 Thr Asn Met His Phe 440 Met Ser Thr Leu Asn 445 Thr Tyr Gly Pro Leu 450 Thr Ala Leu Asn Asn 455 Thr Ala Pro Val Phe 460 Pro Asn Gly Gln Ile 465 Trp Asp Lys Glu Leu 470 Asp Thr Asp Leu Lys 475 Pro Arg Leu His Val 480 Thr Ala Pro Phe Val 485 Cys Lys Asn Asn Pro 490 Pro Gly Gln Leu Phe 495 Val Lys Ile Ala Pro 500 Asn Leu Thr Asp Asp 505 Phe Asn Ala Asp Ser 510 Pro Gln Gln Pro Arg 515 Ile Ile Thr Tyr Ser 520 Asn Phe Trp Trp Lys 525 Gly Thr Leu Thr Phe 530 Thr Ala Lys Met Arg 535 Ser Ser Asn Met Trp 540 Asn Pro Ile Gln Gln 545 His Thr Thr Thr Ala 550 Glu Asn Ile Gly Asn 555 Tyr Ile Pro Thr Asn 560 Ile Gly Gly Ile Lys 565 Met Phe Pro Glu Tyr 570 Ser Gln Leu Ile Pro 575 Arg

Lys Leu Tyr <210>10 <211>579 <212> PRT <213> Porcine parvovirus <400>10

Met Ser Glu Asn Val Glu Gln 15

Leu Ser Ala Thr Gly Asn Glu 20

Gly Arg Gly Ser Ile Gly Val 35

Gln Thr Glu Phe Gln Tyr Leu 50 55

His Ala Ser Arg Leu Ile His 65 70

His Asn Pro Ile Asn Ala Gly Thr Glu 1015

Ser Ile Gly Gly Gly Gly Gly Gly Gly 2530

Gly Val Ser Thr Gly Ser Phe Asn Asn 4045

Gly Glu Gly Leu Val Arg Ile Thr Ala 60

Leu Asn Met Pro Glu His Glu Thr Tyr 7580

- 41 045363

Lys Arg Ile His Val 85 Leu Asn Ser Glu Ser 90 Gly Val Ala Gly Gln 95 Met Val Gln Asp Asp 100 Ala His Thr Gln Met 105 Val Thr Pro Trp Ser 110 Leu Ile Asp Ala Asn 115 Ala Trp Gly Val Trp 120 Phe Asn Pro Ala Asp 125 Trp Gln Leu Ile Ser 130 Asn Asn Met Thr Glu 135 Ile Asn Leu Val Ser 140 Phe Glu Gln Glu Ile 145 Phe Asn Val Val Leu 150 Lys Thr Ile Thr Glu 155 Ser Ala Thr Ser Pro 160 Pro Thr Lys Ile Tyr 165 Asn Asn Asp Leu Thr 170 Ala Ser Leu Met Val 175 Ala Leu Asp Thr Asn 180 Asn Thr Leu Pro Tyr 185 Thr Pro Ala Ala Pro 190 Arg Ser Glu Thr Leu 195 Gly Phe Tyr Pro Trp 200 Leu Pro Thr Lys Pro 205 Thr Gln Tyr Arg Tyr 210 Tyr Leu Ser Cys Thr 215 Arg Asn Leu Asn Pro 220 Pro Thr Tyr Thr Gly 225 Gln Ser Glu Gln Ile 230 Thr Asp Ser Ile Gln 235 Thr Gly Leu His Ser 240 Asp Ile Met Phe Tyr 245 Thr Ile Glu Asn Ala 250 Val Pro Ile His Leu 255 Leu Arg Thr Gly Asp 260 Glu Phe Ser Thr Gly 265 Ile Tyr His Phe Asp 270 Thr Lys Pro Leu Lys 275 Leu Thr His Ser Trp 280 Gln Thr Asn Arg Ser 285 Leu Gly Leu Pro Pro 290 Lys Leu Leu Thr Glu 295 Pro Thr Thr Glu Gly 300 Asp Gln His Pro Gly 305 Thr Leu Pro Ala Ala 310 Asn Thr Arg Lys Gly 315 Tyr His Gln Thr Thr 320 Asn Asn Ser Tyr Thr 325 Glu Ala Thr Ala Ile 330 Arg Pro Ala Gln Val 335 Gly Tyr Asn Thr Pro 340 Tyr Met Asn Phe Glu 345 Tyr Ser Asn Gly Gly 350 Pro Phe Leu Thr Pro 355 Ile Val Pro Thr Ala 360 Asp Thr Gln Tyr Asn 365 Asp Asp Glu Pro Asn 370 Gly Ala Ile Arg Phe 375 Thr Met Gly Tyr Gln 380 His Gly Gln Leu Thr 385 Thr Ser Ser Gln Glu 390 Leu Glu Arg Tyr Thr 395 Phe Asn Pro Gln Ser 400 Lys Cys Gly Arg Ala Pro Lys Gln Gln Phe Asn Gln Gln Ser Pro Leu

405 410 415

- 42 045363

Asn Leu Gln Asn 420 Thr Asn Asn Gly Thr 425 Leu Leu Pro Ser Asp 430 Pro Ile Gly Gly Lys 435 Thr Asn Met His Phe 440 Met Asn Thr Leu Asn 445 Thr Tyr Gly Pro Leu 450 Thr Ala Leu Asn Asn 455 Thr Ala Pro Val Phe 460 Pro Asn Gly Gln Ile 465 Trp Asp Lys Glu Leu 470 Asp Thr Asp Leu Lys 475 Pro Arg Leu His Val 480 Thr Ala Pro Phe Val 485 Cys Lys Asn Asn Pro 490 Pro Gly Gln Leu Phe 495 Val Lys Ile Ala Pro 500 Asn Leu Thr Asp Asp 505 Phe Asn Ala Asp Ser 510 Pro Gln Gln Pro Arg 515 Ile Ile Thr Tyr Ser 520 Asn Phe Trp Trp Lys 525 Gly Thr Leu Thr Phe 530 Thr Ala Lys Met Arg 535 Ser Ser Asn Met Trp 540 Asn Pro Ile Gln Gln 545 His Thr Thr Thr Ala 550 Glu Asn Ile Gly Asn 555 Tyr Ile Pro Thr Asn 560 Ile Gly Gly Ile Lys 565 Met Phe Pro Glu Tyr 570 Ser Gln Leu Ile Pro 575 Arg Lys Leu Tyr

<210> 11 <211> 579 <212> PRT <213> Porcine parvovirus

<400> Met Ser 1 eleven Glu Asn Val 5 Glu Gln His Asn Pro 10 Ile Asn Ala Gly Thr 15 Glu Leu Ser Ala Thr 20 Gly Asn Glu Ser Gly 25 Gly Gly Gly Gly Gly 30 Gly Gly Gly Arg Gly 35 Ala Gly Gly Val Gly 40 Val Ser Thr Gly Ser 45 Phe Asn Asn Gln Thr 50 Glu Phe Gln Tyr Leu 55 Gly Glu Gly Leu Val 60 Arg Ile Thr Ala His 65 Ala Ser Arg Leu Ile 70 His Leu Asn Met Pro 75 Glu His Glu Thr Tyr 80 Lys Arg Ile His Val 85 Leu Asn Ser Glu Ser 90 Gly Val Ala Gly Gln 95 Met Val Gln Asp Asp 100 Ala His Thr Gln Met 105 Val Thr Pro Trp Ser 110 Leu Ile Asp Ala Asn 115 Ala Trp Gly Val Trp 120 Phe Asn Pro Ala Asp 125 Trp Gln Leu

- 43 045363

Ile

Ile 145

Pro

Leu

Glu

Arg

Gly 225

Asp

Arg

Pro

Pro

Gly 305

Asn

Tyr

Leu

Pro

Thr 385

Lys

Asn

Gly

Pro

Ser 130

Phe

Thr

Asp

Thr

Tyr 210

Gln

Ile

Thr

Leu

Pro 290

Thr

Asn

Asn

Thr

Asn 370

Thr

Cys

Leu

Gly

Leu 450

Asn

Asn

Lys

Thr

Leu 195

Tyr

Ser

Met

Gly

Lys 275

Lys

Leu

Ser

Thr

Pro 355

Gly

Ser

Gly

Gln

Lys 435

Thr

Asn

Val

Ile

Asn 180

Gly

Leu

Glu

Phe

Asp 260

Leu

Leu

Pro

Tyr

Pro 340

Ile

Ala

Ser

Arg

Asn 420

Thr

Ala

Met

Val

Tyr 165

Asn

Phe

Ser

Gln

Tyr 245

Glu

Thr

Leu

Ala

Thr 325

Tyr

Val

Ile

Gln

Ala 405

Thr

Asn

Leu

Thr

Leu 150

Asn

Thr

Tyr

Cys

Ile 230

Thr

Phe

His

Thr

Ala 310

Glu

Met

Pro

Arg

Glu 390

Pro

Asn

Met

Asn

Glu 135

Lys

Asn

Leu

Pro

Ile 215

Thr

Ile

Ser

Ser

Glu 295

Asn

Ala

Asn

Thr

Phe 375

Leu

Lys

Asn

His

Asn 455

Ile

Thr

Asp

Pro

Trp 200

Arg

Asp

Glu

Thr

Trp 280

Pro

Thr

Thr

Phe

Ala 360

Thr

Glu

Gln

Gly

Phe 440

Thr

Asn Leu Val Ser 140 Phe Glu Gln Ala Ile Thr Glu 155 Ser Ala Thr Ser Pro 160 Leu Thr 170 Ala Ser Leu Met Val 175 Ala Tyr 185 Thr Pro Ala Ala Pro 190 Arg Ser Leu Pro Thr Lys Pro 205 Thr Gln Tyr Asn Leu Asn Pro 220 Pro Thr Tyr Thr Ser Ile Gln 235 Thr Gly Leu His Ser 240 Asn Ala 250 Val Pro Ile His Leu 255 Leu Gly 265 Ile Tyr His Phe Asp 270 Thr Lys Gln Thr Asn Arg Ser 285 Leu Gly Leu Thr Thr Glu Gly 300 Asp Gln His Pro Arg Lys Gly 315 Tyr His Gln Thr Thr 320 Ala Ile 330 Arg Pro Ala Gln Val 335 Gly Glu 345 Tyr Ser Asn Gly Gly 350 Pro Phe Asp Thr Gln Tyr Asn 365 Asp Asp Glu Met Asp Tyr Gln 380 His Gly Gln Leu Arg Tyr Thr 395 Phe Asn Pro Gln Ser 400 Gln Phe 410 Asn Gln Gln Ser Pro 415 Leu Thr 425 Leu Leu Pro Ser Asp 430 Pro Ile Met Asn Thr Leu Asn 445 Thr Tyr Gly Ala Pro Val Phe Pro Asn Gly Gln

460

- 44 045363

Ile 465 Trp Asp Lys Glu Leu 470 Asp Thr Asp Leu Lys 475 Pro Arg Leu His Thr Ala Pro Phe Val 485 Cys Lys Asn Asn Pro 490 Pro Gly Gln Leu Phe 495 Lys Ile Ala Pro 500 Asn Leu Thr Asp Asp 505 Phe Asn Ala Asp Ser 510 Pro Gln Pro Arg 515 Ile Ile Thr Tyr Ser 520 Asn Phe Trp Trp Lys 525 Gly Thr Thr Phe 530 Thr Ala Lys Met Arg 535 Ser Ser Asn Met Trp 540 Asn Pro Ile Gln 545 His Thr Thr Thr Ala 550 Glu Asn Ile Gly Asn 555 Tyr Ile Pro Thr Ile Gly Gly Ile Arg 565 Met Phe Pro Glu Tyr 570 Ser Gln Leu Ile Pro 575 Lys Leu Tyr

Val 480

Val

Gln

Leu

Gln

Asn 560

Arg <210>12 <211>579 <212> PRT <213> Porcine parvovirus <400>12

Met Ser Glu Asn Val Glu Gln 15

Leu Ser Ala Thr Gly Asn Glu 20

His Asn Pro Ile Asn Ala Gly Thr

15

Ser Gly Gly Gly Gly Gly Gly Gly

thirty

Gly Arg Gly Ala Gly 35

Gln Thr Glu Phe Gln 50

His Ala Ser Arg Leu 65

Lys Arg Ile His Val 85

Val Gln Asp Asp Ala 100

Asp Ala Asn Ala Trp 115

Ile Ser Asn Asn Met 130

Ile Phe Asn Val Val 145

Pro Thr Lys Ile Tyr

165

Gly Val Gly Val Ser 40

Tyr Leu Gly Glu Gly 55

Ile His Leu Asn Met 70

Leu Asn Ser Glu Ser 90

His Thr Gln Met Val 105

Gly Val Trp Phe Asn 120

Thr Glu Ile Asn Leu 135

Leu Lys Thr Ile Thr 150

Asn Asn Asp Leu Thr

170

Thr Gly Ser Phe Asn 45

Leu Val Arg Ile Thr 60

Pro Glu His Glu Thr 75

Gly Val Ala Gly Gln 95

Thr Pro Trp Ser Leu 110

Pro Ala Asp Trp Gln 125

Val Ser Phe Glu Gln 140

Glu Ser Ala Thr Ser 155

Ala Ser Leu Met Val

175

Glu

Gly

Asn

Ala

Tyr 80

Met

Ile

Leu

Glu

Pro 160

Ala

- 45 045363

Leu

Glu

Arg

Gly 225

Asp

Arg

Pro

Pro

Gly 305

Asn

Tyr

Leu

Pro

Thr 385

Lys

Asn

Gly

Pro

Ile 465

Thr

Lys

Asp Thr Asn Asn Thr 180

Thr Leu Gly Phe Tyr 195

Tyr Tyr Leu Ser Cys 210

Gln Ser Glu Gln Ile 230

Ile Met Phe Tyr Thr 245

Thr Gly Asp Glu Phe 260

Leu Lys Leu Thr His 275

Pro Lys Leu Leu Thr 290

Thr Leu Pro Ala Ala

310

Asn Ser Tyr Thr Glu 325

Asn Thr Pro Tyr Met 340

Thr Pro Ile Val Pro 355

Asn Gly Ala Ile Arg 370

Thr Ser Ser Gln Glu 390

Cys Gly Arg Ala Pro 405

Leu Gln Asn Thr Asn 420

Gly Lys Thr Asn Met 435

Leu Thr Ala Leu Asn

450

Trp Asp Lys Glu Leu 470

Ala Pro Phe Val Cys 485

Ile Ala Pro Asn Leu 500

Leu Pro Tyr Thr Pro 185

Pro Trp Leu Pro Thr 200

Thr Arg Asn Leu Asn 215

Thr Asp Ser Ile Gln 235

Ile Glu Asn Ala Val 250

Ser Thr Gly Ile Tyr 265

Ser Trp Gln Thr Asn 280

Glu Pro Thr Thr Glu 295

Asn Thr Arg Lys Gly 315

Ala Thr Ala Ile Arg 330

Asn Phe Glu Tyr Ser 345

Thr Ala Asp Thr Gln 360

Phe Thr Met Gly Tyr 375

Leu Glu Arg Tyr Thr 395

Lys Gln Gln Phe Asn 410

Asn Gly Thr Leu Leu 425

His Phe Met Asn Thr 440

Asn Thr Ala Pro Val

455

Asp Thr Asp Leu Lys 475

Lys Asn Asn Pro Pro 490

Thr Asp Asp Phe Asn 505

Ala Ala Pro Arg Ser 190

Lys Pro Thr Gln Tyr 205

Pro Pro Thr Tyr Thr 220

Thr Gly Leu His Ser 240

Pro Ile His Leu Leu 255

His Phe Asp Thr Lys 270

Arg Ser Leu Gly Leu 285

Gly Asp Gln His Pro 300

Tyr His Gln Thr Ile 320

Pro Ala Gln Val Gly 335

Asn Gly Gly Pro Phe 350

Tyr Asn Asp Asp Glu 365

Gln His Gly Gln Leu 380

Phe Asn Pro Gln Ser 400

Gln Gln Ser Pro Leu 415

Pro Ser Asp Pro Ile 430

Leu Asn Thr Tyr Gly 445

Phe Pro Asn Gly Gln

460

Pro Arg Leu His Val 480

Gly Gln Leu Phe Val 495

Ala Asp Ser Pro Gln 510

- 46 045363

Gln Pro Arg Ile Ile Thr Tyr Ser Asn Phe Trp 515520

Thr Phe Thr Ala Lys Met Arg Ser Ser Asn Met 530535

Gln His Thr Thr Thr Ala Glu Asn Ile Gly Asn

545 550555

Ile Gly Gly Ile Lys Met Phe Pro Glu Tyr Ser 565570

Trp

Trp 540

Tyr

Lys 525

Asn

Ile

Gly

Pro

Pro

Thr

Ile

Thr

Gln Leu Ile Pro

575

Leu

Gln

Asn 560

Arg

Lys Leu Tyr <210>13 <211>579 <212> PRT <213> Porcine parvovirus <400>13

Met Ser Glu Asn Val Glu Gln 15

Leu Ser Ala Thr Gly Asn Glu 20

Gly Arg Gly Ala Gly Gly Val 35

His Asn Pro 10 Ile Asn Ala Gly Thr 15 Ser Gly 25 Gly Gly Gly Gly Gly 30 Gly Gly 40 Val Ser Thr Gly Ser 45 Phe Asn

Gln Thr Glu Phe Gln 50

His Ala Ser Arg Leu 65

Lys Arg Ile His Val 85

Val Gln Asp Asp Ala 100

Asp Ala Asn Ala Trp 115

Ile Ser Asn Asn Met 130

Ile Phe Asn Val Val 145

Pro Thr Lys Ile Tyr 165

Leu Asp Thr Asn Asn 180

Glu Thr Leu Gly Phe 195

Arg Tyr Tyr Leu Ser 210

Tyr Leu Gly Glu Gly 55

Ile His Leu Asn Met 70

Leu Asn Ser Glu Ser 90

His Thr Gln Met Val 105

Gly Val Trp Phe Asn 120

Thr Glu Ile Asn Leu 135

Leu Lys Thr Ile Thr 150

Asn Asn Asp Leu Thr 170

Thr Leu Pro Tyr Thr 185

Tyr Pro Trp Leu Pro 200

Cys Thr Arg Asn Leu 215

Leu Val Arg Ile Thr 60

Pro Glu His Glu Thr 75

Gly Val Ala Gly Gln 95

Thr Pro Trp Ser Leu 110

Pro Ala Asp Trp Gln 125

Val Ser Phe Glu Gln 140

Glu Ser Ala Thr Ser 155

Ala Ser Leu Met Val

175

Pro Ala Ala Pro Arg 190

Thr Lys Pro Thr Gln 205

Asn Pro Pro Thr Tyr 220

Glu

Gly

Asn

Ala

Tyr 80

Met

Ile

Leu

Glu

Pro 160

Ala

Ser

Tyr

Thr

- 47 045363

Gly 225 Gln Ser Glu Gln Ile 230 Thr Asp Ser Ile Gln 235 Thr Gly Leu His Ser 240 Asp Ile Met Phe Tyr 245 Thr Ile Glu Asn Ala 250 Val Pro Ile His Leu 255 Leu Arg Thr Gly Asp 260 Glu Phe Ser Thr Gly 265 Ile Tyr His Phe Asp 270 Thr Lys Pro Leu Lys 275 Leu Thr His Ser Trp 280 Gln Thr Asn Arg Ser 285 Leu Gly Leu Pro Pro 290 Lys Leu Leu Thr Glu 295 Pro Thr Thr Glu Gly 300 Asp Gln His Pro Gly 305 Thr Leu Pro Ala Ala 310 Asn Thr Arg Lys Gly 315 Tyr His Gln Thr Ile 320 Asn Asn Ser Tyr Thr 325 Glu Ala Thr Ala Ile 330 Arg Pro Ala Gln Val 335 Gly Tyr Asn Thr Pro 340 Tyr Met Asn Phe Glu 345 Tyr Ser Asn Gly Gly 350 Pro Phe Phe Ile Pro 355 Ile Val Pro Thr Ala 360 Asp Thr Gln Tyr Asn 365 Asp Asp Glu Pro Asn 370 Gly Gly Ile Arg Phe 375 Thr Met Gly Tyr Gln 380 His Gly Gln Leu Ile 385 Thr Ser Ser Gln Glu 390 Val Glu Arg Tyr Thr 395 Phe Asn Pro Gln Arg 400 Lys Cys Gly Arg Gly 405 Ala Lys Gln Gln Phe 410 Asn Gln Gln Ser Pro 415 Leu Asn Ile Gln Asn 420 Thr Asn Asn Gly Thr 425 Ile Leu Pro Ser Asp 430 Pro Ile Gly Gly Lys 435 Thr Asn Met His Phe 440 Met Asn Thr Pro Asn 445 Thr Tyr Gly Pro Leu 450 Thr Ala Val Asn Asn 455 Thr Ala Pro Val Phe 460 Pro Asn Gly Gln Ile 465 Trp Asp Lys Glu Leu 470 Asp Thr Asp Leu Lys 475 Pro Arg Leu His Val 480 Thr Ala Pro Phe Val 485 Cys Lys Asn Asn Pro 490 Pro Gly Gln Leu Phe 495 Val Lys Ile Ala Pro 500 Asn Leu Thr Asp Asp 505 Phe Asn Ala Asp Ser 510 Pro Gln Gln Pro Arg 515 Ile Ile Thr Tyr Ser 520 Asn Phe Trp Trp Lys 525 Gly Thr Leu Thr Phe 530 Thr Ala Lys Met Arg 535 Ser Ser Asn Met Trp 540 Asn Pro Ile Gln Gln 545 His Thr Thr Thr Ala 550 Glu Asn Ile Gly Asn 555 Tyr Ile Pro Thr Asn 560

- 48 045363

Ile Gly Gly Ile Lys Met Phe Pro

565

Glu Tyr Ser Gln Leu Ile Pro

570 575

Arg

Lys Leu Tyr <210>14 <211>579 <212> PRT <213> Porcine parvovirus <400>14

Met Ser Glu Asn Val Glu Gln 15

Leu Ser Ala Thr Gly Asn Glu 20

Gly Arg Gly Ala Gly Gly Val 35

His Asn Pro Ile Asn Ala Gly Thr 1015

Ser Gly Gly Gly Gly Gly Gly Gly 2530

Gly Val Ser Thr Gly Ser Phe Asn 4045

Gln Thr Glu Phe Gln 50

His Ala Ser Arg Leu 65

Lys Arg Ile His Val 85

Val Gln Asp Asp Ala 100

Asp Ala Asn Ala Trp 115

Ile Ser Asn Asn Met 130

Ile Phe Asn Val Val 145

Pro Thr Lys Ile Tyr 165

Leu His Thr Asn Asn 180

Glu Thr Leu Gly Phe 195

Arg Tyr Tyr Leu Ser 210

Gly Gln Ser Glu Gln 225

Asp Ile Met Phe Tyr 245

Arg Thr Gly Asp Glu 260

Tyr Leu Gly Glu Gly 55

Ile His Leu Asn Met 70

Leu Asn Ser Glu Ser 90

His Thr Gln Met Val 105

Gly Val Trp Phe Asn 120

Thr Glu Ile Asn Leu 135

Leu Lys Thr Ile Thr 150

Asn Asn Asp Leu Thr 170

Thr Leu Pro Tyr Thr 185

Tyr Pro Trp Leu Pro 200

Cys Ile Arg Asn Leu 215

Ile Thr Asp Ser Ile 230

Thr Ile Glu Asn Ala

250

Phe Ser Thr Gly Ile 265

Leu Val Arg Ile Thr 60

Pro Glu His Glu Thr 75

Gly Val Ala Gly Gln 95

Thr Pro Trp Ser Leu 110

Pro Ala Asp Trp Gln 125

Val Ser Phe Glu Gln 140

Glu Ser Ala Thr Ser 155

Ala Ser Leu Met Val

175

Pro Ala Ala Pro Arg 190

Thr Lys Pro Thr Gln 205

Asn Pro Pro Thr Tyr 220

Gln Thr Gly Leu His 235

Val Pro Ile His Leu

255

Tyr His Phe Asp Thr 270

Glu

Gly

Asn

Ala

Tyr 80

Met

Ile

Leu

Ala

Pro 160

Ala

Ser

Tyr

Thr

Ser 240

Leu

Lys

- 49 045363

Pro Leu Lys 275 Leu Thr His Ser Trp 280 Gln Thr Asn Arg Ser 285 Leu Gly Leu Pro Pro 290 Lys Leu Leu Thr Glu 295 Pro Thr Thr Glu Gly 300 Asp Gln His Pro Gly 305 Thr Leu Pro Ala Ala 310 Asn Thr Arg Lys Gly 315 Tyr His Gln Thr Ile 320 Asn Asn Ser Tyr Thr 325 Glu Ala Thr Ala Ile 330 Arg Pro Ala Gln Val 335 Gly Tyr Asn Thr Pro 340 Tyr Met Asn Phe Glu 345 Tyr Ser Asn Gly Gly 350 Pro Phe Leu Thr Pro 355 Ile Val Pro Thr Ala 360 Asp Thr Gln Tyr Asn 365 Asp Asp Glu Pro Asn 370 Gly Ala Ile Arg Phe 375 Thr Met Asp Tyr Gln 380 His Gly His Leu Thr 385 Thr Ser Ser Gln Glu 390 Leu Glu Arg Tyr Thr 395 Phe Asn Pro Gln Ser 400 Lys Cys Gly Arg Thr 405 Pro Lys Gln Gln Phe 410 Asn Gln Gln Ser Pro 415 Leu Asn Leu Gln Asn 420 Thr Asn Asn Gly Thr 425 Leu Leu Pro Ser Asp 430 Pro Ile Gly Gly Lys 435 Thr Asn Met His Phe 440 Met Asn Thr Leu Asn 445 Thr Tyr Gly Pro Leu 450 Thr Ala Leu Asn Asn 455 Thr Ala Pro Val Phe 460 Pro Asn Gly Gln Ile 465 Trp Asp Lys Glu Leu 470 Asp Thr Asp Leu Lys 475 Pro Arg Leu His Val 480 Thr Ala Pro Phe Val 485 Cys Lys Asn Asn Pro 490 Pro Gly Gln Leu Phe 495 Val Lys Ile Ala Pro 500 Asn Leu Thr Asp Asp 505 Phe Asn Ala Asp Ser 510 Pro Gln Gln Pro Arg 515 Ile Ile Thr Tyr Ser 520 Asn Phe Trp Trp Lys 525 Gly Thr Leu Thr Phe 530 Thr Ala Lys Met Arg 535 Ser Ser Asn Met Trp 540 Asn Pro Ile Gln Gln 545 His Thr Thr Thr Ala 550 Glu Asn Ile Gly Lys 555 Tyr Ile Pro Thr Asn 560 Ile Gly Gly Ile Lys 565 Met Phe Pro Glu Tyr 570 Ser Gln Leu Ile Pro 575 Arg

Lys Leu Tyr <210> 15 <211> 579 <212> PRT

- 50 045363 <213> Porcine parvovirus <400> 15

Met 1 Ser Glu Asn Val 5 Glu Gln His Asn Pro 10 Ile Asn Ala Gly Thr 15 Glu Leu Ser Ala Thr 20 Gly Asn Glu Ser Gly 25 Gly Gly Gly Gly Gly 30 Gly Gly Gly Arg Gly 35 Ala Gly Gly Val Gly 40 Val Ser Thr Gly Ser 45 Phe Asn Asn Gln Thr 50 Glu Phe Gln Tyr Leu 55 Gly Glu Gly Leu Val 60 Arg Ile Thr Ala His 65 Ala Ser Arg Leu Ile 70 His Leu Asn Met Pro 75 Glu His Glu Thr Tyr 80 Lys Arg Ile His Val 85 Leu Asn Ser Glu Ser 90 Gly Val Ala Gly Gln 95 Met Val Gln Asp Asp 100 Ala His Thr Gln Met 105 Val Thr Pro Trp Ser 110 Leu Ile Asp Ala Asn 115 Ala Trp Gly Val Trp 120 Phe Asn Pro Ala Asp 125 Trp Gln Leu Ile Ser 130 Asn Asn Met Thr Glu 135 Ile Asn Leu Val Ser 140 Phe Glu Gln Glu Ile 145 Phe Asn Val Val Leu 150 Lys Thr Ile Thr Glu 155 Ser Ala Thr Ser Pro 160 Pro Thr Lys Ile Tyr 165 Asn Asn Asp Leu Thr 170 Ala Ser Leu Met Val 175 Ala Leu Asp Thr Asn 180 Asn Thr Leu Pro Tyr 185 Thr Pro Ala Ala Pro 190 Arg Ser Glu Thr Leu 195 Gly Phe Tyr Pro Trp 200 Leu Pro Thr Lys Pro 205 Thr Gln Tyr Arg Tyr 210 Tyr Leu Ser Cys Thr 215 Arg Asn Leu Asn Pro 220 Pro Thr Tyr Thr Gly 225 Gln Ser Glu Gln Ile 230 Thr Asp Ser Ile Gln 235 Thr Gly Leu His Ser 240 Asp Ile Met Phe Tyr 245 Thr Ile Glu Asn Ala 250 Val Pro Ile His Leu 255 Leu Arg Thr Gly Asp 260 Glu Phe Ser Thr Gly 265 Ile Tyr His Phe Asp 270 Thr Lys Pro Leu Lys 275 Leu Thr His Ser Trp 280 Gln Thr Asn Arg Ser 285 Leu Gly Leu Pro Pro 290 Lys Leu Leu Thr Glu 295 Pro Thr Thr Glu Gly 300 Asp Gln His Pro Gly 305 Thr Leu Pro Ala Ala 310 Asn Thr Arg Lys Gly 315 Tyr His Gln Thr Ile 320 Asn Asn Ser Tyr Thr Glu Ala Thr Ala Ile Arg Pro Ala Gln Val Gly

- 51 045363

325 330 335 Tyr Asn Thr Pro 340 Tyr Met Asn Phe Glu 345 Tyr Ser Asn Gly Gly 350 Pro Phe Leu Thr Pro 355 Ile Val Pro Thr Ala 360 Asp Thr Gln Tyr Asn 365 Asp Asp Glu Pro Asn 370 Gly Ala Ile Arg Phe 375 Thr Met Gly Tyr Gln 380 His Gly Gln Leu Thr 385 Thr Ser Ser Gln Glu 390 Leu Glu Arg Tyr Thr 395 Phe Asn Pro Gln Ser 400 Lys Cys Gly Arg Ala 405 Pro Lys Gln Gln Phe 410 Asn Gln Gln Ser Pro 415 Leu Asn Leu Gln Asn 420 Thr Asn Asn Gly Thr 425 Leu Leu Pro Ser Asp 430 Pro Ile Gly Gly Lys 435 Thr Asn Met His Phe 440 Met Ser Thr Leu Asn 445 Thr Tyr Gly Pro Leu 450 Thr Ala Leu Asn Asn 455 Thr Ala Pro Val Phe 460 Pro Asn Gly Gln Ile 465 Trp Asp Lys Glu Leu 470 Asp Thr Asp Leu Lys 475 Pro Arg Leu His Val 480 Thr Ala Pro Phe Val 485 Cys Lys Asn Asn Pro 490 Pro Gly Gln Leu Phe 495 Val Lys Ile Ala Pro 500 Asn Leu Thr Asp Asp 505 Phe Asn Ala Asp Ser 510 Pro Gln Gln Pro Arg 515 Ile Ile Thr Tyr Ser 520 Asn Phe Trp Trp Lys 525 Gly Thr Leu Thr Phe 530 Thr Ala Lys Met Arg 535 Ser Ser Asn Met Trp 540 Asn Pro Ile Gln Gln 545 His Thr Thr Thr Ala 550 Glu Asn Ile Gly Asn 555 Tyr Ile Pro Thr Asn 560 Ile Gly Gly Ile Lys 565 Met Phe Pro Glu Tyr 570 Ser Gln Leu Ile Pro 575 Arg

Lys Leu Tyr <210>16 <211>579 <212> PRT <213> Porcine parvovirus <400>16

Met Ser Glu Asn Val Glu Gln 15

Leu Ser Ala Thr Gly Asn Glu 20

Gly Arg Gly Ala Gly Gly Val 35

His Asn Pro Ile Asn Ala Gly Thr Glu 1015

Ser Gly Gly Gly Gly Gly Gly Gly Gly 2530

Gly Val Ser Thr Gly Ser Phe Asn Asn 4045

- 52 045363

Gln Thr 50 Glu Phe Gln Tyr Leu 55 Gly Glu Gly Leu Val 60 Arg Ile Thr Ala His 65 Ala Ser Arg Leu Ile 70 His Leu Asn Met Pro 75 Glu His Glu Thr Tyr 80 Lys Arg Ile His Val 85 Leu Asn Ser Glu Ser 90 Gly Val Ala Gly Gln 95 Met Val Gln Asp Asp 100 Ala His Thr Gln Met 105 Val Thr Pro Trp Ser 110 Leu Ile Asp Ala Asn 115 Ala Trp Gly Val Trp 120 Phe Asn Pro Ala Asp 125 Trp Gln Leu Ile Ser 130 Asn Asn Met Thr Glu 135 Ile Asn Leu Val Ser 140 Phe Glu Gln Glu Ile 145 Phe Asn Val Val Leu 150 Lys Thr Ile Thr Glu 155 Ser Ala Thr Ser Pro 160 Pro Thr Lys Ile Tyr 165 Asn Asn Asp Leu Thr 170 Ala Ser Leu Met Val 175 Ala Leu Asp Thr Asn 180 Asn Thr Leu Pro Tyr 185 Thr Pro Ala Ala Pro 190 Arg Ser Glu Thr Leu 195 Gly Phe Tyr Pro Trp 200 Leu Pro Thr Lys Pro 205 Thr Gln Tyr Arg Tyr 210 Tyr Leu Ser Cys Thr 215 Arg Asn Leu Asn Pro 220 Pro Thr Tyr Thr Gly 225 Gln Ser Glu Gln Ile 230 Thr Asp Ser Ile Gln 235 Thr Gly Leu His Ser 240 Asp Ile Met Phe Tyr 245 Thr Ile Glu Asn Ala 250 Val Pro Ile His Leu 255 Leu Arg Thr Gly Asp 260 Glu Phe Ser Thr Gly 265 Ile Tyr His Phe Asp 270 Thr Lys Pro Leu Lys 275 Leu Thr His Ser Trp 280 Gln Thr Asn Arg Ser 285 Leu Gly Leu Pro Pro 290 Lys Leu Leu Thr Glu 295 Pro Thr Thr Glu Gly 300 Asp Gln His Pro Gly 305 Thr Leu Pro Ala Ala 310 Asn Thr Arg Lys Gly 315 Tyr His Gln Thr Thr 320 Asn Asn Ser Tyr Thr 325 Glu Ala Thr Ala Ile 330 Arg Pro Ala Gln Val 335 Gly Tyr Asn Thr Pro 340 Tyr Met Asn Phe Glu 345 Tyr Ser Asn Gly Gly 350 Pro Phe Leu Thr Pro 355 Ile Val Pro Thr Ala 360 Asp Thr Gln Tyr Asn 365 Asp Asp Glu Pro Asn Gly Ala Ile Arg Phe Thr Met Gly Tyr Gln His Gly Gln Leu

370 375 380

- 53 045363

Thr Thr Ser Ser Gln Glu Leu Glu

385 390

Lys Cys Gly Arg Ala Pro Lys Gln 405

Asn Leu Gln Asn Thr Asn Asn Gly 420

Gly Gly Lys Thr Asn Met His Phe 435440

Pro Leu Thr Ala Leu Asn Asn Thr 450455

Ile Trp Asp Lys Glu Leu Asp Thr 465470

Arg Tyr Thr Phe Asn Pro Gln Ser 395400

Gln Phe Asn Gln Gln Ser Pro Leu 410415

Thr Leu Leu Pro Ser Asp Pro Ile 425430

Met Asn Thr Leu Asn Thr Tyr Gly 445

Ala Pro Val Phe Pro Asn Gly Gln 460

Asp Leu Lys Pro Arg Leu His Val 475 480

Thr Ala Pro Phe Val Cys Lys Asn 485

Lys Ile Ala Pro Asn Leu Thr Asp 500

Gln Pro Arg Ile Thr Tyr Ser 515520

Thr Phe Thr Ala Lys Met Arg Ser 530535

Gln His Thr Thr Thr Ala Glu Asn

545550

Ile Gly Gly Ile Lys Met Phe Pro 565

Asn Pro Pro Gly Gln Leu Phe Val 490495

Asp Phe Asn Ala Asp Ser Pro Gln 505510

Asn Phe Trp Trp Lys Gly Thr Leu 525

Ser Asn Met Trp Asn Pro Ile Gln 540

Ile Gly Asn Tyr Ile Pro Thr Asn 555 560

Glu Tyr Ser Gln Leu Ile Pro Arg 570 575

Lys Leu Tyr

Claims (20)

1. Viral protein 2 (VP2) of porcine parvovirus (PPV), containing: at amino acid position 25 isoleucine residue; and at amino acid position 36, a serine residue; and at amino acid position 37, an isoleucine residue; and at amino acid position 228, a glutamic acid residue or glutamate residue; and at amino acid position 414 there is a serine residue; and at amino acid position 419 there is a glutamine residue; and at amino acid position 436 there is a threonine residue; and wherein the amino acid position numbering refers to the amino acid sequence of wild-type PPV VP2, wherein the amino acid sequence of wild-type PPV VP2 refers to the amino acid sequence shown in SEQ ID NO: 1. 2. PPV VP2 according to claim 1, characterized in that the above-mentioned PPV VP2: I) includes or consists of an amino acid sequence that has at least 90% identity with the amino acid sequence of SEQ ID NO: 2; or II) encoded by a nucleotide sequence encoding an amino acid sequence - 54 045363 identity that has at least 90% identity with the amino acid sequence of SEQ ID NO: 2. 3. Immunogenic composition, including PPV VP2 according to any of claims 1, 2. 4. The immunogenic composition according to claim 3, wherein the immunogenic composition also includes a pharmaceutically acceptable carrier, the pharmaceutically acceptable carrier being preferably a carbomer. 5. Immunogenic composition according to claim 3 or 4, characterized in that the immunogenic composition is a vaccine. 6. A polynucleotide comprising a sequence that encodes PPV VP2 according to any one of claims 1, 2. 7. A vector comprising a polynucleotide according to claim 6. 8. The vector according to claim 7, where the vector is an expression vector, preferably a baculovirus. 9. A cell comprising a polynucleotide according to claim 6 or a vector according to claim 7 or 8. 10. The cell of claim 9, wherein the cell is an insect cell, more preferably an insect cell selected from the group consisting of SI9 cells, Sf+ cells. 11. Virus-like particle, including PPV VP2 at any of the pi. 1.2. 12. A method for producing PPV VP2 according to any of claims 1, 2, including transfecting a cell with a vector according to claim 7 or 8. 13. The production method according to claim 12, where the cell is an insect cell, preferably an insect cell selected from the group consisting of Sf9 cells, Sf+ cells. 14. Use of PPV VP2 according to any of claims 1, 2 as a medicine. 15. Use according to claim 14, where the medicinal product is a vaccine. 16. Application of a virus-like particle according to i. 11 as a medicine. 17. Use according to claim 16, where the medicinal product is a vaccine. 18. Use of an immunogenic composition according to any one of claims 3-5 in a method of treating and/or preventing PPV infection and/or in a method of reducing, preventing and/or treating clinical signs caused by PPV infection in a subject in need, the method comprising administering to the subject therapeutically an effective amount of the immunogenic composition according to any one of claims 3 to 5, wherein preferably such clinical signs caused by PPV infection are selected from the group consisting of transient leukopenia and reproductive failure characterized by embryonic infection and fetal death, decreased litter size, increased embryo mummification, or fetus in each litter, autolysis of the embryo or fetus, decreased embryo or fetal size, decreased embryo or fetal weight, increased viremia, increased viral load in target tissues and blood, increased transmission/spread of PPV among conspecifics, or a combination thereof; and wherein the subject is a mumps, and wherein preferably the immunogenic composition is safe for the mumps from day 30 of pregnancy, more preferably from day 40 of pregnancy. 19. Use of an immunogenic composition according to any one of claims 3 to 5 in a method of treating and/or preventing clinical signs caused by a PPV infection in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an immunogenic composition according to any one of claims 3 -5, wherein the subject is a mumps, and wherein preferably the immunogenic composition is safe for the mumps from day 30 of pregnancy, more preferably from day 40 of pregnancy. 20. The use of an immunogenic composition according to any one of claims 3-5 in a method of reducing reproductive failure in a subject compared to a subject of a non-immunized control group of the same species, the method comprising administering to the subject a therapeutically effective amount of an immunogenic composition according to any one of claims 3-5, wherein the subject is a mumps, and wherein preferably the immunogenic composition is safe for the mumps from day 30 of pregnancy, more preferably from day 40 of pregnancy.