A live attenuated pseudorabies virus vaccine for pigs containing a deletion of gene UL23

Abstract

The present disclosure provides an attenuated porcine herpesvirus 1 (pseudorabies virus) whose TK, gI, and gE genes have been modified relative to the parent field strain, such that the resulting virus is safe and effective for use as a live vaccine to protect porcine animals from challenge with virulent pseudorabies virus.

Description

[Technical Field]

[0001] The present invention is generally in the field of vaccines against pseudorabies virus. [Background technology]

[0002] Pseudorabies virus (PRV) is a disease that infects many species of animals worldwide. PRV infection is variously called infectious bulbar paralysis, Aujeszky's disease, and mad itch. Clinical signs of PRV infection include abortion, high mortality in sows, and coughing, sneezing, fever, constipation, depression, seizures, ataxia, circling, and excessive salivation in piglets and adult sows. Mortality approaches 100% in piglets under one month of age but is less than 10% in pigs aged one to six months. Pregnant pigs may reabsorb their fetuses or give birth to mummified, stillborn, or weak piglets. In cattle, symptoms include intense itching, followed by neurological signs and death. In dogs, symptoms include intense itching, jaw and pharyngeal paralysis, howling, and death. Any infected secondary host generally survives only two to three days. The itching or itch is considered a phantom limb sensation because the virus has never been found at the site of the scratch.

[0003] Infections are known in important livestock such as pigs, cattle, dogs, cats, sheep, rats, and mink. The host range is very wide, including most mammals and, experimentally, at least many species of birds (for a detailed list of hosts, see D.P. Gustafson, "Pseudorabies," Diseases of Swine, 5th ed., A.D. Leman et al., eds., (1981)). However, adult pigs and occasionally rats do not die from the disease and are therefore carriers. However, for other species, the disease is fatal.

[0004] Swine populations are particularly susceptible to PRV infection. Adult pigs rarely show symptoms or die from the disease, but piglets become acutely ill upon infection and often die within 24 to 48 hours, usually without specific clinical signs (T.C. Jones and R.D. Hunt, Veterinary Pathology, 5th ed., Lea & Febiger (1983)).

[0005] PRV is a herpesvirus. The PRV genome is characterized by two unique regions (UL and US), which are flanked by internal and terminal repeats (IRS and TRS, respectively). The sequence and gene arrangement of the entire PRV genome are known, and a map of potential transcription mechanisms has been established, well supported by experimental data. Recombination between inverted repeats can generate two possible isomers of the genome with the US region in opposite orientations. The functions of 70 different genes have been identified. For a general discussion of PRV biology and its mechanism of action, see Pomeranz et al., Microbiol. And Mol. Biol. Reviews 205, Sept., 462-500.

[0006] PRV vaccines have been produced by various techniques, and vaccination has been practiced in endemic areas of Europe for over 15 years. Although vaccination has reduced losses, vaccination maintains the virus in the environment. Vaccinated animals exposed to virulent virus may survive the infection and then shed more virulent virus. Thus, vaccinated animals may harbor a latent infection that can recur. (See DP Gustafson, supra.)

[0007] Live attenuated and inactivated vaccines against PRV are commercially available in the United States and are approved by the USDA (see C.E. Aronson, ed., Veterinary Pharmaceuticals & Biologicals, (1983)).

[0008] Live attenuated and inactivated vaccines against PRV are commercially available in the United States and approved by the USDA (see C.E. Aronson, ed., Veterinary Pharmaceuticals & Biologicals, (1983)). Nevertheless, there remains a need for new PRV vaccines, particularly live attenuated vaccines that are safe and effective. Summary of the Invention

[0009] The present application provides a method for protecting pigs from one or more symptoms of PRV infection caused by infectious PRV, the method comprising administering to pigs a vaccine comprising a modified live pseudorabies virus comprising a genome of SEQ ID NO: 3, or a nucleic acid sequence at least 95% identical thereto, wherein the modified live pseudorabies virus comprises a deletion of UL23 gene nucleotides 480-846, and the infectious PRV is of a genotype I or genotype II lineage.

[0010] In certain embodiments, the genome of the modified live pseudorabies virus comprises a deletion of the gE gene and the gI gene. In certain embodiments, the US1, US2, and US9 genes in the genome of the modified live PRV are unmodified. In a most preferred embodiment, the genome of the modified live PRV is SEQ ID NO: 3.

[0011] In certain embodiments, a single dose of the vaccine comprises 10 2 ~10 7 TCID 50 Preferably, the vaccine is administered intramuscularly. In some embodiments, the PRV symptoms that the vaccine protects against may be selected from the group consisting of viral shedding, coughing, sneezing, fever, constipation, depression, seizures, ataxia, circling, and excessive salivation.

[0012] In preferred embodiments, at least 80%, or at least 90%, or at least 95%, or 100% of pigs are protected from PRV symptoms when exposed to the infectious PRV within 6 months of vaccination with the modified live pseudorabies vaccine disclosed herein.

[0013] In certain embodiments, the vaccinated pig is a piglet that may be at least 3 weeks old, or at least 7 weeks old, or older.

[0014] In other embodiments, the vaccinated pig is a sow. In certain embodiments, the sow is pregnant. In more specific embodiments, the sow is 2-3 months pregnant. In a most preferred embodiment, administration of a live pseudorabies virus vaccine disclosed herein to a pregnant sow does not affect the farrowing rate, abortion rate, or survival rate of the offspring of the pregnant sow. DETAILED DESCRIPTION OF THE INVENTION

[0015] The following definitions and introductions are applicable herein.

[0016] The singular forms "a," "an," and "the" include plural referents unless the context dictates otherwise. Similarly, the word "or" is intended to include "and" unless the context dictates otherwise. The word "or" means any one member of a particular list and also includes any combination of members of that list.

[0017] The term "adjuvant" refers to a compound that enhances the effectiveness of a vaccine and can be added to a formulation containing an immunizing agent. Adjuvants provide an enhanced immune response even after administration of only a single dose of vaccine. Adjuvants can include, for example, muramyl dipeptide, pyridine, aluminum hydroxide, dimethyldioctadecylammonium bromide (DDA), oil, oil-in-water emulsion, saponin, cytokines, and other substances known in the art. Examples of suitable adjuvants are described in U.S. Patent Application Publication No. 2004 / 0213817A1. "Adjuvanted" refers to a composition that incorporates or is combined with an adjuvant.

[0018] "Antibody" refers to polyclonal and monoclonal antibodies, chimeric and single-chain antibodies, and Fab fragments, including the products of a Fab or other immunoglobulin expression library. With respect to an antibody, the term "immunologically specific" refers to an antibody that binds to one or more epitopes of a protein of interest but does not substantially recognize or bind other molecules in a sample containing a mixed population of antigenic biomolecules.

[0019] As used herein, "attenuated" PRV refers to a PRV that can infect and / or replicate in a susceptible host but is non-pathogenic or less pathogenic to the susceptible host. For example, an attenuated virus may not cause observable / detectable clinical pathology, less clinical pathology, or less severe clinical pathology, or may exhibit reduced viral replication efficiency and / or infectivity, compared to a relevant field isolate. Clinical symptoms of PRV infection may include, but are not limited to, coughing, sneezing, fever, constipation, depression, seizures, ataxia, circling, and excessive salivation in piglets and adult pigs.

[0020] An "epitope" is an antigenic determinant that is immunologically active, in the sense that it can elicit a humoral (B cell) and / or cellular (T cell) immune response when administered to a host. These are specific chemical groups or peptide sequences on a molecule that are antigenic. Antibodies specifically bind to particular antigenic epitopes on a polypeptide. In animals, most antigens will simultaneously present several or even many antigenic determinants. Such polypeptides can also be qualified as immunogenic polypeptides, and epitopes can be identified as further described.

[0021] For purposes of the present invention, the nucleotide sequence of a second polynucleotide molecule (either RNA or DNA) is "identical" to the nucleotide sequence of a first polynucleotide molecule if the nucleotide sequence of the second polynucleotide molecule encodes the same polyamino acids as the nucleotide sequence of the first polynucleotide molecule based on the degeneracy of the genetic code, or if the nucleotide sequence of the second polynucleotide molecule encodes polyamino acids that are sufficiently similar to the polyamino acids encoded by the nucleotide sequence of the first polynucleotide molecule. Generally, the nucleotide sequence of a second polynucleotide molecule is identical to the nucleotide sequence of a first polynucleotide molecule if the nucleotide sequence of the second polynucleotide molecule has at least about 85% nucleotide sequence identity to the nucleotide sequence of the first polynucleotide molecule based on the BLASTN algorithm (National Center for Biotechnology Information, otherwise known as NCBI, United States National Institutes of Health, Bethesda, Md., USA). A specific example for calculations in accordance with the practice of the present invention is BLASTP 2.2.6 [Tatusova TA and TL Madden, "BLAST 2 sequences—a new tool for comparing protein and nucleotide sequences." (1999) FEMS Microbiol Lett. 174:247-250.] Briefly, two amino acid sequences are aligned to optimize the alignment score using a gap opening penalty of 10, a gap extension penalty of 0.1, and the "blosum62" scoring matrix of Henikoff and Henikoff (Proc. Nat. Acad. Sci. USA 325 89:10915-10919, 1992). The percent identity is then calculated as follows: total number of perfect matches × 100 divided by the length of the longer sequence plus the number of gaps introduced into the longer sequence to align the two sequences.

[0022] The term "isolated" is used to indicate that a cell, peptide, or nucleic acid is free from its native environment. Isolated peptides and nucleic acids can be substantially pure, i.e., essentially free from other materials with which they may be found in nature.

[0023] The phrase "lacking functional protein" means that the amount and / or activity of the protein encoded by the modified gene is reduced by at least 95% compared to the protein encoded by the unmodified gene. In certain embodiments, the amount and / or activity of the protein encoded by the modified gene is reduced by at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.5%, or at least 99.9%. In certain embodiments, the amount and / or activity of the protein encoded by the modified gene is completely eliminated.

[0024] "Pharmaceutically acceptable carrier" means any conventional pharmaceutically acceptable carrier, vehicle, or excipient used in the art for the production and administration of vaccines. Pharmaceutically acceptable carriers are typically non-toxic, inert, solid, or liquid carriers.

[0025] The terms "porcine" and "swine" are used interchangeably herein and refer to any animal that is a member of the Suidae family, such as a pig.

[0026] As used herein, a "susceptible" host refers to a cell or animal that can be infected by PEDV. When introduced into a susceptible animal, the attenuated PEDV can also induce an immunological response against PEDV or its antigens, thereby immunizing the animal against PEDV infection.

[0027] The term "vaccine" refers to an antigenic preparation used to generate immunity against disease in order to prevent or reduce the effects of infectious disease. Vaccines are typically prepared using an immunologically effective amount of a combination of immunogens together with an adjuvant effective to enhance the immune response of the vaccinated subject to the immunogens.

[0028] The vaccine formulation will contain a "therapeutically effective amount" of the active ingredient, i.e., an amount capable of inducing an immunoprotective response in the subject to which the composition is administered. In the treatment and prevention of PEDV disease, for example, a "therapeutically effective amount" is preferably an amount that enhances the vaccinated subject's resistance to new infection and / or reduces the clinical severity of the disease. Such protection will be demonstrated by either a reduction or elimination of symptoms typically exhibited by subjects infected with PRV, a faster recovery time, and / or a reduced number of viral particles. The vaccine can be administered before infection as a preventative measure against PRV. Alternatively, the vaccine can be administered after the subject has already contracted the disease. A vaccine given after exposure to PRV can alleviate the disease and induce a superior immune response than natural infection itself.

[0029] The present disclosure provides attenuated strains of PRV that are safe and effective when used in vaccines and protect pigs from challenge with virulent PRV strains. In certain embodiments, the attenuated strains of PRV contain modifications in the thymidine kinase (TK), glycoprotein I (gI), and glycoprotein E (gE) genes relative to the parent field strain.

[0030] Suitable parent strains include, but are not limited to, FS18 (SEQ ID NO: 1), strain JS2012 (SEQ ID NO: 2), strain TJ (GenBank Accession No. KJ789182), strain HeN1 (GenBank Accession No. KP098534), strain HLJ8 (GenBank Accession No. KT824771), strain HN1201 (GenBank Accession No. KP722022). Other suitable strains are those encoded by a nucleotide sequence that is at least 85% identical (i.e., at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.2% identical, at least 99.4% identical, at least 99.6% identical, at least 99.8% identical, at least 99.9% identical) to full-length SEQ ID NO:1 or SEQ ID NO:2.

[0031] In certain embodiments, the parent strain is at least 85% identical to SEQ ID NO: 1 or 2, as described in the previous paragraph, and at least half (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%) of the differing bases result in codons encoding similar amino acids, i.e., conservative amino acid substitutions. It is recognized that certain such conservative amino acid substitutions generally do not inactivate overall protein function. For example, for positively charged amino acids (and vice versa), lysine, arginine, and histidine; for negatively charged amino acids (and vice versa), aspartic acid and glutamic acid; and for certain groups of neutrally charged amino acids (and vice versa in all cases), (1) alanine and serine, (2) asparagine, glutamine, and histidine, (3) cysteine ​​and serine, (4) glycine and proline, (5) isoleucine, leucine, and valine, (6) methionine, leucine, and isoleucine, (7) phenylalanine, methionine, leucine, and tyrosine, (8) serine and threonine, (9) tryptophan and tyrosine, (10) and, for example, tyrosine, tryptophan, and phenylalanine. Amino acids can be classified according to their physical properties and contribution to secondary and tertiary protein structure. Thus, a conservative substitution is recognized in the art as the substitution of one amino acid for another amino acid with similar properties, and exemplary conservative substitutions can be found in WO97 / 09433, published March 13, 1997, p. 10 (PCT / GB96 / 02197, filed September 6, 1996). Alternatively, conservative amino acids can be grouped as described in Lehninger, (Biochemistry, Second Edition; Worth Publishers, Inc. NY: NY (1975), pp. 71-77). Protein sequences can be aligned using multiple sequence alignment in both Vector NTI Advance 11.5 and CLUSTAL 2.1.As used herein, the recitation of a particular amino acid or nucleotide sequence is intended to encompass all silent variations of the nucleic acid sequence, and any and all conservatively modified variations of the amino acid sequence.

[0032] The sequences and functions of the gI, gE, and TK proteins of pseudorabies virus are known. Thymidine kinase is encoded by the UL23 gene and is involved in nucleotide synthesis. Glycoproteins I and E are virion proteins encoded by the US7 and US8 genes, respectively. Pomeranz et al. disclose that gI and gE are complexed with each other. For purposes of this disclosure, the UL23, US7, and US8 genes may be referred to as the "TK gene," the "gI gene," and the "gE gene," respectively.

[0033] In certain embodiments, the attenuated strain of PRV further comprises a modified version of one or more (i.e., one, two, or all three) of the US1, US2, and US9 genes. These genes encode the RSp40 / ICP22, 11K, and 28K proteins, respectively. In certain embodiments, at least one of the US2 and US9 genes is unmodified. Thus, for example, the virus can comprise an unmodified US2, a modified US9, and a modified or unmodified US1. Alternatively, the virus can comprise an unmodified US9, a modified US2, and a modified or unmodified US1.

[0034] In certain other embodiments, in the attenuated strains of PRV of the invention, the US1, US2, and US9 genes are unmodified.

[0035] Pomeranz et al. have reported that US1 encodes the RSp40 / ICP22 protein. The function of RSp40 / ICP22 in PRV is currently unknown, but Pomeranz has reported that its HSV-1 homolog acts as a regulator of gene expression. US2 encodes a protein present in the viral tegument. US9 encodes an envelope protein that is involved in protein sorting in axons and functions as a type II tail-anchored membrane protein.

[0036] Modifications in the genes encoding the TK, gI, and gE proteins, and optional modifications in the US1, US2, and US9 genes, result in viruses lacking functional proteins expressed by these genes.

[0037] For example, in certain embodiments, viruses of the present invention lacking a functional gE protein can be prepared by a number of means. For example, a stop codon may be introduced into the proximal portion of the ORF encoding the gE protein. In different embodiments, the stop codon may be introduced after 10 or fewer N-terminal amino acids, for example, 9, 8, 7, 6, 5, 4, 3, or 2 N-terminal amino acids. In other embodiments, the transcription start site may be altered so that transcription does not initiate. In yet other embodiments, all of the nucleotides in the ORF encoding the gE protein are deleted.

[0038] The viruses of the invention also lack a functional gI protein. In certain embodiments, at least nucleotides 269 through 1101 are deleted from the 1101-nucleotide ORF encoding the gI protein. In certain embodiments, the deletion begins upstream of nucleotide 269, e.g., nucleotides 250, 200, 150, 100, 50, or even further upstream. In certain embodiments, all 1101 nucleotides are deleted. In certain embodiments, a stop codon is introduced at or upstream of position 269 without introducing a frameshift mutation.

[0039] The virus of the present invention also has a modified US23 gene encoding a TK protein. The UL23 gene has a 963-nucleotide-long ORF. In certain embodiments, this 963-nucleotide-long ORF lacks at least one (or at least two, or at least three, or all four) subsequences selected from the sequences defined by nucleotides 526-607, 480-846, 280-723, and 364-615 of the 963-nucleotide-long ORF. Of course, longer deletions, such as a deletion defined by positions 280-846 incorporating all four subsequences, or a deletion defined by positions 300-650 including two subsequences, can also be present. Alternatively, all 963 nucleotides of the ORF can be deleted. Alternatively, a stop codon can be introduced (without causing a frameshift) upstream of position 526, upstream of position 364, upstream of position 480, upstream of position 280, etc. Mutation of the transcription start site is also possible in certain embodiments.

[0040] Optional modifications to any one of the US1, US2, and US9 genes preferably cause the resulting virus to lack the protein encoded by the modified gene. Suitable mutations include gene deletions, insertions, substitutions, etc. As described above, frameshift mutations can be introduced, thus producing proteins with minimal similarity to the proteins encoded by the unmodified gene. In-frame mutations can include the introduction of a stop codon into the proximal portion of the gene (e.g., within the N-terminal 20, 15, 10, 5, or 3 amino acids), or mutations in the transcription start site so that the corresponding ORF is not transcribed.

[0041] In certain embodiments, an attenuated virus of the invention has a genome that is at least 85% identical to SEQ ID NO: 1 or 2 and has the following modifications: a) 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%, or 100%) of the ORFs encoding gI protein; b) 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%, or 100%) of the ORFs encoding gE protein; c) The ORF encoding the TK protein lacks at least one (i.e., at least two, at least three, or all four) of the subsequences defined by nucleotides at positions 526-607, 480-846, 280-723, and 364-615 of this 963-nucleotide long ORF.

[0042] In certain other embodiments, the modified live virus is encoded by SEQ ID NO:3 or a sequence at least 85% identical thereto, with the proviso that the viral-encoding sequence includes modifications to the UL23 gene (encoding TK), the US7 gene (encoding gI), and the US8 gene (encoding gE). Some modified live viruses according to this embodiment of the invention may include unmodified US1, US2, and US9 genes. Some other modified live viruses according to this embodiment of the invention further include optional modifications to the US1, US2, and US9 genes, as described above, such as a modified US2, an unmodified US9, and a modified or unmodified US1, or a modified US9, an unmodified US2, and a modified or unmodified US1. In another embodiment, all three of these genes (US1, US2, US9) are modified.

[0043] Methods for modifying genes so that the resulting virus lacks the functional proteins encoded by these genes are well known. These methods include, but are not limited to, complete or partial deletion, frameshift mutation, nucleotide exchange, or insertion. For example, one can modify the promoter that regulates the gene or the transcription start site. Alternatively (or additionally), one can insert a mutation that results in a premature stop codon in the coding sequence. Other suitable methods are within the expertise of those skilled in the art.

[0044] The above-mentioned modifications can be introduced into the viral genome by a number of methods, including, but not limited to, targeted mutagenesis and homologous recombination.

[0045] The first step of this technique involves constructing a recombinant DNA molecule for recombination with PrV genomic DNA. Such a recombinant DNA molecule can be derived from any suitable plasmid, cosmid, or phage, with a plasmid being most preferred, and contains a fragment of PrV DNA containing the DNA of a portion of the PrV genome as defined above. The DNA sequence of the portion of the PrV genome as defined above is preferably flanked by PrV nucleic acid sequences of an appropriate length, e.g., 50-3000 bp, to allow in vivo homologous recombination with the viral PrV genome to occur.

[0046] The recombinant DNA molecules thus obtained are suitable for introducing mutations into the PrV genome.

[0047] Cells, such as pig kidney cells or Vero cells, can then be transfected with PrV DNA or infected with wild-type PrV in the presence of the recombinant DNA molecule as described above, thereby allowing recombination to occur between sequences in the recombinant DNA molecule and corresponding sequences in the PrV genome.

[0048] Recombination can also be induced by cotransfecting cells with a nucleic acid sequence containing the mutated sequence flanked by appropriate adjacent PrV sequences that do not contain the plasmid sequence. Recombinant virus progeny can then be generated in cell culture and selected, for example, genotypically or phenotypically. Another possibility is to detect the absence of the polypeptide encoded by the nucleic acid sequence in which the mutation is localized. Similarly, the presence of the polypeptide encoded by the inserted heterologous nucleic acid sequence can be detected. Recombinant viruses can also be reliably selected based on resistance to compounds such as neomycin, gentamicin, or mycophenolic acid.

[0049] The selected recombinant PrV can be grown on a large scale in cell culture, after which the recombinant PrV-containing material or the heterologous polypeptide expressed by the PrV can be harvested.

[0050] Alternatively or additionally to recombinant DNA techniques, viruses of the invention may be prepared using cell culture passage in combination with clonal enrichment and clonal selection. For example, clones with deletions in one of the genes, e.g., gI or gE, may be selected for further propagation, and it is known that TK natural gene deletion mutants can result from viral replication defects.

[0051] Suitable cell lines include, but are not limited to, the porcine testicular cell line ST, the porcine kidney cell line PK-15 or MRS-2, the rabbit kidney cell line RK, the African green monkey kidney cell line Vero, the monkey embryonic kidney epithelial cell line Marc-145, the bovine kidney cell line MDBK, the bovine testicular cell line BT, chicken embryo fibroblasts (CEF), and the baby hamster kidney cell line BHK-21. In a preferred embodiment, the suitable cell line is Vero (ATCC CCL-81).

[0052] An immunologically effective amount of the vaccine of the present invention is administered to pigs in need of protection against viral infection. The immunologically effective or immunogenic amount to be inoculated into pigs can be easily determined or easily titrated by routine testing. An effective amount is an amount that achieves a sufficient immunological response to the vaccine to protect pigs exposed to the PRV virus. Preferably, pigs are protected to the extent that one or all of the adverse physiological symptoms or effects of the viral disease are significantly reduced, alleviated, or completely prevented.

[0053] The vaccines of the present invention can be formulated according to accepted practice to include veterinary-acceptable carriers, such as standard buffers, stabilizers, diluents, preservatives, and / or solubilizers, and can also be formulated to promote sustained release. Diluents include water, saline, dextrose, ethanol, glycerol, and the like. Additives for isotonicity include, among others, sodium chloride, dextrose, mannitol, sorbitol, and lactose. Stabilizers include, among others, albumin. Other suitable vaccine vehicles and additives, including those particularly useful in formulating modified live vaccines, will be known or apparent to those skilled in the art. See, for example, Remington's Pharmaceutical Science, 18th ed., 1990, Mack Publishing, incorporated herein by reference.

[0054] The vaccines of the present invention can be non-adjuvanted. Alternatively, the vaccines of the present invention can further comprise one or more additional immunomodulatory components, such as, for example, an adjuvant or cytokine, among others. Non-limiting examples of adjuvants that can be used in the vaccines of the present invention include the RIBI adjuvant system (Ribi Inc., Hamilton, Mont.), alum, mineral gels such as aluminum hydroxide gel, oil-in-water emulsions, water-in-oil emulsions such as Freund's complete and incomplete adjuvants, block copolymers (CytRx, Atlanta, Ga.), QS-21 (Cambridge Biotech Inc., Cambridge, Mass.), SAF-M (Chiron, Emeryville, Calif.), AMPHIGEN® adjuvant, saponin, Quil A or other saponin fractions, monophosphoryl lipid A, ionic polysaccharides, and Avridine lipid-amine adjuvants. Non-limiting examples of oil-in-water emulsions useful in the vaccines of the present invention include modified SEAM62 and SEAM1 / 2 formulations. Modified SEAM62 is an oil-in-water emulsion containing 5% (v / v) squalene (Sigma), 1% (v / v) SPAN® 85 detergent (ICI surfactant), 0.7% (v / v) TWEEN® 80 detergent (ICI surfactant), 2.5% (v / v) ethanol, 200 μg / ml Quil A, 100 μg / ml cholesterol, and 0.5% (v / v) lecithin. Modified SEAM1 / 2 is an oil-in-water emulsion containing 5% (v / v) squalene, 1% (v / v) SPAN® 85 detergent, 0.7% (v / v) Tween 80 detergent, 2.5% (v / v) ethanol, 100 μg / ml Quil A, and 50 μg / ml cholesterol. Other immunomodulatory agents that can be included in the vaccine include, for example, one or more interleukins, interferons, or other known cytokines.

[0055] Additional adjuvant systems allow for the combination of both T helper and B cell epitopes, resulting in one or more types of covalently linked TB epitope structures that may additionally be lipidated, such as those described in WO2006 / 084319, WO2004 / 014957, and WO2004 / 014956.

[0056] In a preferred embodiment of the present invention, the ORFI PEDV protein, or other PEDV proteins or fragments thereof, as discussed below, is formulated in 5% AMPHIGEN®.

[0057] Adjuvant components The vaccine compositions of the present invention may or may not contain an adjuvant. Specifically, as based on orally infectious viruses, the modified live vaccines of the present invention can be used without an adjuvant, with a sterile carrier. Adjuvants that can be used for oral administration include those based on CT-like immunomodulators (rmLT, CT-B, i.e., recombinant mutant heat-labile toxin of E. coli, cholera toxin-B subunit), or via encapsulation with a polymer and a mucoadhesive such as alginate or chitosan, or via liposomes. A preferred adjuvanted or unadjuvanted vaccine dose with a minimum protective dose through vaccine release is approximately 10 to approximately 10 per dose. 6 log 10 TCID 50 TCID 50 " refers to the "tissue culture infectious dose" and is defined as the dilution of virus required to infect 50% of a given batch of inoculated cell cultures. TCID, including the Spearman-Karber method utilized throughout this specification, 50Various methods can be used to calculate the log(s) of the starting titer. For a description of the Spearman-Karber method, see B.W. Mahy & H.O. Kangro, Virology Methods Manual, pp. 25-46 (1996). If present, the adjuvant, more commonly provided as an emulsion if parenteral administration is selected, must not reduce the starting titer by more than 0.7 log (an 80% reduction).

[0058] In one example, the adjuvant component is provided by a combination of lecithin in light mineral oil, and also an aluminum hydroxide component. Details regarding the composition and formulation of AMPHIGEN® (as a representative lecithin / mineral oil component) are as follows:

[0059] A preferred adjuvanted product may be provided as a 2 mL dose in a buffered solution further comprising about 5% (v / v) REHYDRAGEL® (aluminum hydroxide gel) and "20% AMPHIGEN®" to about 25% final (v / v). AMPHIGEN® is generally described in U.S. Pat. No. 5,084,269 and provides deoiled lecithin (preferably soybean) dissolved in light mineral oil, which is then dispersed in an aqueous solution or suspension of antigen as an oil-in-water emulsion. Amphigen has been improved according to the protocol of U.S. Pat. No. 6,814,971 (see columns 8-9 thereof) to provide the so-called "20% Amphigen" component for use in the final adjuvanted vaccine composition of the present invention. Thus, a stock mixture of 10% lecithin and 90% carrier oil (DRAKEOL®, Penreco, Karns City, Pa.) is diluted 1:4 with 0.63% phosphate-buffered saline solution, thereby reducing the lecithin and DRAKEOL® components to 2% and 18%, respectively (i.e., 20% of their original concentrations). Tween 80 and Span 80 surfactants are added to this composition; typical and preferred final amounts are 5.6% (v / v) TWEEN® 80 and 2.4% (v / v) SPAN® 80, with SPAN® provided first in the stock DRAKEOL® component and TWEEN® provided first in the buffered saline component, such that the mixture of saline and DRAKEOL® components results in the final desired surfactant concentrations. The mixture of DRAKEOL® / lecithin and saline solution can be achieved using an In-Line Slim Emulsifier device, model 405 (Charles Ross and Son, Hauppauge, NY, USA).

[0060] The vaccine composition also contains REHYDRAGEL® LV (approximately 2% aluminum hydroxide content in stock material) as an additional adjuvant component (available from Reheis, NJ, USA, and ChemTrade Logistics, USA). Upon further dilution using 0.63% PBS, the final vaccine composition contains the following amounts of components per 2 mL dose: 5% (v / v) REHYDRAGEL® LV, 25% (v / v) "20% Amphigen" (i.e., further diluted 4-fold), and 0.01% (w / v) merthiolate.

[0061] As is understood in the art, the order of component addition can be varied to provide a comparable final vaccine composition. For example, an appropriate dilution of virus in buffer can be prepared. Then, while blending, an appropriate amount of REHYDRAGEL® LV (approximately 2% aluminum hydroxide content) stock solution can be added to achieve the desired 5% (v / v) concentration of REHYDRAGEL® LV in the actual final product. Once prepared, this intermediate stock material can be combined with an appropriate amount of "20% Amphigen" stock (which generally already contains the necessary amounts of Tween 80 and Span 80, as described above) to again achieve a final product with 25% (v / v) "20% Amphigen." Finally, an appropriate amount of 10% merthiolate can be added.

[0062] The vaccination compositions of the present invention allow for variation in all of the formulation components so that the total antigen dose can preferably vary by 100-fold (up or down) compared to the antigen doses described above, and most preferably by no more than 10-fold (up or down). Similarly, surfactant concentrations (whether TWEEN® or SPAN®) may be varied up to 10-fold, independently of each other, or they may be omitted entirely, replaced by similar materials of appropriate concentrations, as is well understood in the art.

[0063] The REHYDRAGEL® concentration in the final product can be varied by first using equivalent materials available from many other manufacturers (i.e., ALHYDROGEL®, Brenntag; Denmark) or by using additional variations in the REHYDRAGEL® product line, such as CG, HPA, or HS. Using LV as an example, its final useful concentrations include 0%-20%, with 2-12% being more preferred and 4-8% being most preferred. Similarly, the final concentration of AMPHIGEN® (expressed as a % of "20% Amphigen") is preferably 25%, but this amount can vary from 5-50%, preferably 20-30%, and most preferably about 24-26%.

[0064] In accordance with the practice of the present invention, the oil used in the adjuvant formulation of the present invention is preferably mineral oil. As used herein, the term "mineral oil" refers to a mixture of liquid hydrocarbons obtained from petrolatum via distillation techniques. This term is synonymous with "liquid paraffin," "liquid petrolatum," and "white mineral oil." This term is also intended to include "light mineral oil," i.e., an oil similarly obtained by distillation of petrolatum but having a slightly lower specific gravity than white mineral oil. See, for example, Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack Publishing Company, 1990, pages 788 and 1323). Mineral oil can be obtained from various commercial sources, such as JT Baker (Phillipsburg, Pa.) and USB Corporation (Cleveland, Ohio). A preferred mineral oil is light mineral oil, commercially available under the name DRAKEOL®.

[0065] The immunogenic and vaccine compositions of the invention can further comprise pharmaceutically acceptable carriers, excipients, and / or stabilizers in the form of lyophilized formulations or aqueous solutions (see, e.g., Remington: The Science and Practice of Pharmacy, 2005, Lippincott Williams). Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as mercuric((o-carboxyphenyl)thio)ethyl sodium salt (THIOMERSAL®), octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl, or benzyl alcohol, alkylparabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); It may include proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextran; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming couples such as sodium; ionic metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG), TWEEN®, or PLURONICS®.

[0066] The vaccine of the present invention can optionally be formulated for sustained release of the virus, infectious DNA molecule, plasmid, or viral vector of the present invention. Examples of such sustained release formulations include a virus, infectious DNA molecule, plasmid, or viral vector combined with a biocompatible polymer complex, such as poly(lactic acid), poly(lactic-co-glycolic acid), methylcellulose, hyaluronic acid, collagen, etc. The structure, selection, and use of degradable polymers in drug delivery vehicles have been reviewed in several publications, including A. Domb et al., 1992, Polymers for Advanced Technologies 3:279-292 (incorporated herein by reference). Additional guidance on the selection and use of polymers in pharmaceutical formulations can be found in texts known in the art, such as M. Chasin and R. Langer (eds), 1990, "Biodegradable Polymers as Drug Delivery Systems" in Drugs and the Pharmaceutical Sciences, Vol. 45, M. Dekker, NY, also incorporated herein by reference. Alternatively, or in addition, virus, plasmid, or viral vector can be microencapsulated to improve administration and efficacy.Methods for microencapsulating antigens are well known in the art, and include, for example, the techniques described in U.S. Patent No. 3,137,631, U.S. Patent No. 3,959,457, U.S. Patent No. 4,205,060, U.S. Patent No. 4,606,940, U.S. Patent No. 4,744,933, U.S. Patent No. 5,132,117, and International Patent Publication No. 95 / 28227, all of which are incorporated herein by reference.

[0067] Liposome can also be used to provide the sustained release of virus, plasmid, viral protein or viral vector.The details of the method of making and using liposome preparations can be found in, among others, United States Patent No. 4,016,100, United States Patent No. 4,452,747, United States Patent No. 4,921,706, United States Patent No. 4,927,637, United States Patent No. 4,944,948, United States Patent No. 5,008,050 and United States Patent No. 5,009,956, all of which are incorporated herein by reference.

[0068] The effective amount of any of the above-mentioned vaccines can be determined by conventional means, starting with a low dose of virus, viral protein plasmid, or viral vector, and then increasing the dosage while monitoring the effect. An effective amount may be obtained after a single administration of the vaccine, or after multiple administrations of the vaccine. Known factors can be considered when determining the optimal dose per animal. These include the species, size, age, and general condition of the animal, the presence of other drugs in the animal, etc. The actual dosage is preferably selected after considering the results from other animal studies.

[0069] One way to detect whether an adequate immune response has been achieved is to determine seroconversion and antibody titers in animals after vaccination. The timing of vaccinations and the number of boosters, if any, are preferably determined by a physician or veterinarian based on an analysis of all relevant factors, some of which are described above.

[0070] In a preferred embodiment of the present invention relating to vaccination of pigs, the optimal age target for the animals is approximately 1-21 days, which may also accommodate other scheduled vaccinations, such as against Mycoplasma hyopneumoniae or porcine reproductive and respiratory syndrome virus. Additionally, a preferred vaccination schedule for breeding sows involves similar doses and includes an annual revaccination schedule.

[0071] dosage In a particularly preferred embodiment, disclosed is a method for protecting pigs from one or more symptoms of PRV infection caused by infectious PRV, the method comprising administering to pigs a vaccine comprising a modified live pseudorabies virus comprising a genome of SEQ ID NO: 3, or a nucleic acid sequence at least 95% identical thereto, wherein the modified live pseudorabies virus comprises a deletion of UL23 gene nucleotides 480-846, and wherein the infectious PRV is of a genotype I or genotype II lineage.

[0072] The actual volume of the dose is a function of how the vaccine is formulated, and actual doses range from 0.05 to 5 ml, taking into account the size of the animal. Single-dose vaccination is also appropriate. The amount of pseudorabies virus in the vaccine is 10 per dose. 2 TCID 50 ~10 8 TCID 50 , preferably 10 per dose 2 ~10 7 TCID 50 , or more preferably 10 per dose 3.5 ~10 5.5 TCID 50 The route of administration can vary, with the preferred route of administration of the vaccine being intramuscular.

[0073] In a different embodiment, the one or more symptoms are selected from the group of viral shedding, coughing, sneezing, fever, constipation, depression, seizures, ataxia, circling, and excessive salivation.

[0074] The modified live pseudorabies virus can cross-protect against a wide range of infectious PRV strains, such as, for example, PRV strain FS21PF1115 or PRV strain AV25 or PRV strain Rong A.

[0075] The preferred clinical indication is for the pre-farrowing treatment, control, and prophylaxis of both breeding sows and gilts, followed by vaccination of piglets. In a typical example (applicable to both sows and gilts), a single dose of vaccine is used, although of course, two-dose vaccination regimens are also envisioned if desired. In certain embodiments, at least 80% of pigs are protected from PRV symptoms when exposed to the infectious PRV within six months of vaccination with the vaccine containing the modified live pseudorabies virus.

[0076] It should be noted that piglets may then be vaccinated as early as day 1 of age. For example, piglets can be vaccinated on day 1, with or without a booster dose at 3 weeks of age, especially if the sow was vaccinated before breeding but not before farrowing. Piglet vaccination can also be effective if the sow was not previously vaccinated, either due to natural or deliberate infection. Piglet vaccination can also be effective when the mother has either not been previously exposed to the virus or has not been vaccinated before farrowing.

[0077] In other embodiments, the pig is a piglet between 3 weeks and about 7 weeks old, e.g., a 3 week old piglet, a 4 week old piglet, a 5 week old piglet, a 6 week old piglet, or a 7 week old piglet. In older piglets, e.g., 7 week old piglets, the vaccine provides 100% protection.

[0078] In another series of embodiments, the pig is a sow, preferably a pregnant sow. Most preferably, the pregnant sow is 2-3 months pregnant. When administered to a pregnant sow, the vaccines described herein do not affect the farrowing rate, abortion rate, or survival rate of the pregnant sow's offspring.

[0079] Wild boars (typically kept for breeding purposes) need to be vaccinated once every six months. Dosage variations are desirable within the practice of the art. It should be noted that the vaccines of the present invention are safe for use in pregnant animals (all three trimesters) and newborn pigs. The vaccines of the present invention are attenuated to a level of safety acceptable for even the most sensitive animals, including newborn pigs (i.e., no mortality, transient mild clinical signs, or only normal signs in newborn pigs). Naturally, a sustained sow vaccination program is crucial in protecting pig herds from both PRV epidemics and persistent low-level PRV outbreaks. It will be appreciated that sows or gilts immunized with PRV MLVs passively transfer immunity to piglets, including PRV-specific IgA, which protects piglets from PRV-associated disease and mortality. Additionally, pigs immunized with PRV MLVs will generally be protected from shedding PRV in their feces at a reduced amount and / or duration, and further, pigs immunized with PRV MLVs will be protected from clinical signs of PRV, including, but not limited to, mortality, reproductive, neurological, and respiratory pathology, and further, PRV MLVs will help stop or control the PEDV transmission cycle.

[0080] It is also noted that animals vaccinated with the vaccine of the present invention are immediately safe for human consumption without any significant slaughter hold, such as 21 days or less.

[0081] When provided therapeutically, the vaccine is provided in an amount effective upon detection of symptoms of actual infection. A composition is said to be "pharmacologically acceptable" if its administration can be tolerated by a recipient. Such a composition is said to be administered in a "therapeutically or prophylactically effective amount" if the amount administered is physiologically significant.

[0082] Pharmaceutical compositions as described herein can be used to administer at least one vaccine or immunogenic composition of the present invention by any means that achieves the intended purpose. For example, the administration route of such compositions can be parenteral, oral, oronasal, intranasal, intratracheal, topical, subcutaneous, intramuscular, transdermal, intradermal, intraperitoneal, intraocular, and intravenous. In one embodiment of the present invention, the composition is administered intramuscularly. Parenteral administration can be by bolus injection or by gradual perfusion over time. The composition can be administered using any suitable device, including a syringe, a dropper, a needleless injection device, a patch, etc. The route and device selected for use depend on the adjuvant, antigen, and composition of interest, and are well known to those skilled in the art. Oral or subcutaneous administration is preferred. Oral administration can be directly, through water, or through feed (solid or liquid feed). When provided in liquid form, the vaccine may be reconstituted and freeze-dried or may be provided as a paste for direct addition to feed (mixed in or poured over) or otherwise added to water or liquid feed.

[0083] The following examples are intended to illustrate the above invention and should not be construed as narrowing its scope. Those skilled in the art will readily recognize that the examples suggest many other ways in which the invention can be practiced. It should be understood that numerous variations and modifications can be made while remaining within the scope of the invention.

[0084] Example 1 Piglets that were negative for both PRV antigen and antibody were assigned to groups with seven piglets in each group. The piglets were provided with free access to commercial feed and water.

[0085] Strain M1707 (containing SEQ ID NO:3 and lacking nucleotides 480-846 of the UL23 gene encoding TK) was formulated in MEM, gelatin, NZ amine, glutamine, sucrose, dextran 40, lactose, sorbitol, and penicillin-streptomycin.

[0086] A second group of pigs was vaccinated with strain Bartha K61. A third group of pigs was vaccinated with DMEM. The vaccination method was intramuscular injection in the neck. The inoculum volume for all treatment groups was 1 mL per piglet. After inoculation, clinical observations were performed daily, including measuring the rectal temperature of the pigs. [Table 1]

[0087] Data show that F35 lab-grade strain M1707 produced >10 genotypes in 3-4 week old piglets (7 pigs treated) and in 7 week old target age piglets (14 pigs treated). 6.5 TCID 50 Treatment of pigs with three different batches of the lab product was shown to be safe. All pigs, including the control pigs, had normal body temperatures and showed no clinical signs within the 14-day observation period.

[0088] Example 2 Piglets that were negative for both PRV antigen and antibody were assigned to groups with seven piglets in each group. The piglets were provided with free access to commercial feed and water.

[0089] Three lots of PRV strain M1707 were prepared: Lot A, Lot B, and Lot C. The virus was formulated in MEM, gelatin, NZ amine, glutamine, sucrose, dextran 40, lactose, sorbitol, and penicillin-streptomycin.

[0090] On day 0, 10 per dose 5.0 TCID 50Pigs in groups T01 to T03 were treated with intramuscular injections of one of the preparations from the lot in an amount of 100 ml. T04 pigs were vaccinated with the Bartha K61 commercial vaccine, T05 pigs with commercial vaccine C, and T06 pigs with DMEM. After vaccination, the rectal temperatures of the pigs were measured daily. Clinical observations revealed that all pigs had normal body temperatures, good appetites, normal mental states, no respiratory or digestive symptoms, and no neurological symptoms within the 21-day observation period. The three vaccinated groups and the control group were treated with 2 ml (10 ml) of strain FS21PF1115 on day 21. 5.0 TCID 50 ) was challenged intranasally.

[0091] Protection was determined by the severity (or absence) of symptoms. Results showed that all pigs in groups T01 / T02 / T03 / T05 were protected, with one pig in group T04 being affected. In the control group (T06), 0 of 5 pigs were protected, and all of them were confirmed to be affected. [Table 2]

[0092] On day 0, 10 per dose 5.0 TCID 50 T01-T03 pigs were treated with intramuscular injection of one formulation from the lot in an amount of 10 ml. T04-T05 pigs were treated with intramuscular injection of 2 ml per dose. The T06 control group was treated with DMEM only. After inoculation, the rectal temperatures of the pigs were measured daily. Clinical observations showed that all pigs had normal body temperatures, good appetites, normal mental states, no respiratory or digestive symptoms, and no neurological symptoms within the 21-day observation period. All groups were inoculated with strain FS21PF1115, 2 ml (10 5.0 TCID 50 ) was challenged intranasally.

[0093] Protection was determined by the severity (or absence) of symptoms. In the three groups immunized with strain M1707, 15 of 15 pigs were protected. In the group immunized with strain Bartha K61, 4 of 5 pigs were protected. In the group immunized with strain C, 4 of 5 pigs were protected. In the control group, 0 of 5 pigs were protected. [Table 3]

[0094] Example 3 Piglets negative for both PRV antigen and antibody were assigned to four groups with five piglets in each group. The piglets were provided with free access to commercial feed and water.

[0095] PRV strain 1707 was formulated in MEM, gelatin, NZ amine, glutamine, sucrose, dextran 40, lactose, sorbitol, and penicillin-streptomycin. Each vaccinated pig received 10 doses. 5.0 TCID 50 The control group was treated with DMEM. The formulation was administered by intramuscular injection. Six months after vaccination, the three vaccinated groups and the control group received 2 mL (10 6.0 TCID 50 ) was challenged intranasally. [Table 4]

[0096] Duration of immunity was determined by the severity (or absence) of symptoms. Results showed that 15 of 15 pigs were protected and none became ill in the three vaccination / challenge groups. In the control group, 0 of 5 pigs were deemed protected and 5 became ill.

[0097] Example 4 Sows 2-3 months pregnant (negative for PRV antigen and antibody) were divided into two groups with five sows in each group. The sows were provided with commercial feed and unlimited drinking water.

[0098] Group 1 sows were inoculated with a mixture prepared by strain M1707 (containing SEQ ID NO:3 and lacking nucleotides 480-846 of the UL23 gene encoding TK) along with MEM, gelatin, NZ amine, glutamine, sucrose, dextran 40, lactose, sorbitol, and penicillin-streptomycin. Group 2 pigs were inoculated with DMEM. The inoculation method was intramuscular injection into the neck.

[0099] After inoculation, the rectal temperatures of the test sows were measured and recorded daily. Clinical observations were conducted for 14 days, including appetite, mental state, respiratory condition, and other specific clinical symptoms caused by the pseudorabies virus. Before the sows' expected farrowing date, it was important to observe whether they had reproductive disorders such as abortion, stillbirth, and mummification. When the sows gave birth, the presence or absence of vulnerable, stillborn, or mummified babies and their corresponding numbers were recorded. The farrowing status and survival of the piglets were also recorded within two weeks of farrowing.

[0100] The results showed that after immunization, all pigs had normal body temperature, good appetite, normal mental state, no respiratory or digestive symptoms, and no neurological symptoms during the 14-day observation period. No abortions were observed.

[0101] On the day of farrowing, the sow litters, healthy litters, vulnerable litters, aborted fetuses, and stillbirths were counted, and the healthy litter rate was calculated. The average healthy litter rate for the immunized group (T01 group) was 86.0%. The average healthy rate for the control group (T02 group) was 91.9%. Statistical results showed that there was no significant difference in farrowing between immunized and control sows. [Table 5]

[0102] Example 5 Piglets (negative for PRV antigen and antibody) were assigned to five groups with five piglets in each group. The piglets were provided with commercial feed and unlimited drinking water. Pigs in groups 1 to 4 were inoculated with a mixture of M1707 strain (containing SEQ ID NO: 3 and lacking nucleotides 480 to 846 of the UL23 gene encoding TK) along with MEM, gelatin, NZ amine, glutamine, sucrose, dextran 40, lactose, sorbitol, and penicillin-streptomycin. Pigs in group 5 were inoculated with DMEM. The inoculation method was intramuscular injection into the neck. [Table 6]

[0103] On day 0, groups T01 to T04 were each administered 10 5.0 TCID 50 , 10 4.0 TCID 50 , 10 3.0 TCID 50 , and 10 2.0 TCID 50 The control group, T05, was inoculated with DMEM alone. After inoculation, the rectal temperatures of the pigs were measured daily. Clinical observations showed that all pigs had normal body temperatures, good appetites, normal mental states, no respiratory or digestive symptoms, and no neurological symptoms during the 21-day observation period. On the 21st day, 2 mL (10 5.0 TCID 50 The FS21PF1115 virulent strain of PRV (containing FS21PF1115) was used to challenge the T01 / T02 / T03 / T04 groups intranasally. The results show that in groups T01 / T02 / T03 / T04, all of them were protected with over 4 / 5 protection. In the control group, 0 of 5 pigs were protected and all of them were infected. [Table 7]

[0104] Example 6 Piglets (negative for PRV antigen and antibody) were assigned to two groups with five piglets in each group. The piglets were provided with commercial feed and unlimited drinking water. Group 1 pigs were inoculated with a mixture prepared by the M1707 strain (containing SEQ ID NO: 3 and lacking nucleotides 480-846 of the UL23 gene encoding TK) along with MEM, gelatin, NZ amine, glutamine, sucrose, dextran 40, lactose, sorbitol, and penicillin-streptomycin. Group 2 pigs were inoculated with DMEM. The inoculation method was intramuscular injection into the neck. [Table 8]

[0105] On day 0, group T01 received an intramuscular injection of 1 ml at a dose of 10 TCID. The control group T02 was inoculated with DMEM alone. On day 21 after inoculation, 2 ml (10 7.0 TCID 50 The inoculated and control groups were challenged intranasally with a classical virulent strain of PRV (Rong A strain) containing PRV. Results showed that in group T01, 5 / 5 piglets were protected. In the control group, 0 of 5 piglets were protected, and all of them became ill. [Table 9]

[0106] These data indicate that the modified live PRV strain M1707 is capable of broad cross-protection.

[0107] Example 7 PRV antigen- and antibody-negative sheep were divided into two groups with five sheep in each group. The sheep were provided with commercial feed and unlimited drinking water. Group 1 sheep were inoculated with a mixture prepared by the M1707 strain (containing SEQ ID NO: 3 and lacking nucleotides 480-846 of the UL23 gene encoding TK) along with MEM, gelatin, NZ amine, glutamine, sucrose, dextran 40, lactose, sorbitol, and penicillin-streptomycin. Group 2 sheep were inoculated with DMEM. The inoculation method was intramuscular injection into the neck. [Table 10]

[0108] On day 0, the T01 group received 10 6.0 TCID 50 The control group, T02, received 1 ml of the immunized sheep intramuscular injection at a dose of 0.01 mg / kg. The control group, T02, received DMEM alone. After inoculation, the sheep's rectal temperature was measured and recorded regularly every day, and several indicators, including appetite, mental state, respiratory condition, and specific clinical symptoms caused by pseudorabies virus, were clinically observed for 14 days. The results showed that after immunization, all sheep had normal body temperature, good appetite, normal mental state, no respiratory or digestive symptoms, and no neurological symptoms during the 14-day observation period.

Claims

1. 1. A method for protecting pigs from one or more symptoms of PRV infection caused by infectious PRV, said method comprising administering to said pig a vaccine comprising a modified live pseudorabies virus comprising the genome of SEQ ID NO:3, or a nucleic acid sequence which is at least 95% identical thereto, wherein said modified live pseudorabies virus comprises a deletion of UL23 gene nucleotides 480-846, and said infectious PRV is of a genotype I or genotype II lineage.

2. 2. The method of claim 1, wherein the genome of the modified live pseudorabies virus comprises a deletion of the gE gene and the gI gene.

3. 3. The method of claim 1 or 2, wherein the US1, US2, and US9 genes are unmodified in the genome of the modified live PRV.

4. 2. The method of claim 1, wherein the genome of the modified live PRV is SEQ ID NO:

3.

5. The method of any one of claims 1 to 4, wherein the infectious PRV is of the genotype II lineage.

6. The method of any one of claims 1 to 4, wherein the infectious PRV is of the genotype I lineage.

7. A single dose of the vaccine is 10 ２ ~10 ７ T.C.I.D. ５０ The method according to any one of claims 1 to 6, comprising:

8. 8. The method of any one of claims 1 to 7, wherein the one or more symptoms are selected from the group of viral shedding, coughing, sneezing, fever, constipation, depression, seizures, ataxia, circling, and excessive salivation.

9. The method of any one of claims 1 to 8, wherein the vaccine is administered intramuscularly.

10. The method of claims 1 to 9, wherein the infectious PRV is PRV strain FS21PF1115 or PRV strain AV25.

11. The infectious PRV is ５．０ T.C.I.D. ５０ Or 10 ６．０ T.C.I.D. ５０ PRV strain FS21PF1115 in an amount of 10 ７．０ T.C.I.D. ５０ 11. The method of claim 10, wherein the amount of PRV strain AV25 is

12. 12. The method of any one of claims 1 to 11, wherein at least 80% of the pigs are protected from PRV symptoms when exposed to the infectious PRV within 6 months of vaccination with the vaccine comprising the modified live pseudorabies virus.

13. The method according to any one of claims 1 to 12, wherein the pig is a piglet.

14. 14. The method of claim 13, wherein the piglet is at least 3 weeks old.

15. 14. The method of claim 13, wherein the piglet is at least 7 weeks old.

16. 16. The method of claim 15, wherein 100% of the pigs are protected.

17. The method according to any one of claims 1 to 12, wherein the pig is a sow.

18. 18. The method of claim 17, wherein the sow is pregnant.

19. 19. The method of claim 18, wherein the sow is 2 to 3 months pregnant.

20. 20. The method of claim 18 or 19, wherein said administration of said vaccine does not affect farrowing rate, abortion rate, or survival rate of offspring of said pregnant sow.

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