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EP3402878A1 - Nouvelles compositions vaccinales à protection croisée pour le virus de la diarrhée épidémique porcine - Google Patents

Nouvelles compositions vaccinales à protection croisée pour le virus de la diarrhée épidémique porcine

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Publication number
EP3402878A1
EP3402878A1 EP16704501.2A EP16704501A EP3402878A1 EP 3402878 A1 EP3402878 A1 EP 3402878A1 EP 16704501 A EP16704501 A EP 16704501A EP 3402878 A1 EP3402878 A1 EP 3402878A1
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EP
European Patent Office
Prior art keywords
pedv
seq
virus
vaccine composition
dose
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP16704501.2A
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German (de)
English (en)
Inventor
Marta Cabana Sumsi
Monica Balasch Sanuy
Laia PLAJA DILMÉ
Alicia Urniza Hostench
Paul J. Dominowski
Jay Gregory Calvert
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Zoetis Services LLC
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Zoetis Services LLC
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Publication date
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Publication of EP3402878A1 publication Critical patent/EP3402878A1/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20061—Methods of inactivation or attenuation
    • C12N2770/20064—Methods of inactivation or attenuation by serial passage

Definitions

  • the present invention is directed to novel vaccine compositions that protect swine from disease caused by porcine epidemic diarrhea virus (PEDV).
  • the vaccine compositions are both safe and efficacious, and are based on novel attenuates of PEDV that provide cross protection against a wide variety of PEDV strains, and are safe to use even in live form.
  • Porcine epidemic diarrhea is highly contagious and is characterized by dehydration, diarrhea, and high mortality in swine, particularly young piglets.
  • the causative agent, porcine epidemic diarrhea virus (PEDV) is a single stranded, positive sense RNA virus identified to the Alphacoronoavirus genus of the family Coronaviridae.
  • PEDV has a total genome size of approximately 28kb and contains 7 open reading frames. Symptoms of PEDV infection are often similar to those caused by transmissible gastroenteritis virus (TGEV), also a member of the Coronaviridae. It should be noted that cross protection between PEDV and TGEV is not generally observed, the overall viral nucleotide sequences being at most about 60% similar.
  • PED was likely first observed in Europe circa 1970, and the causative virus was subsequently characterized (see for example M. Pensaert et al. Arch. Virol, v. 58, pp 243- 247, 1978 and D. Chasey et al., Res. Vet Sci, v. 25, pp 255-256, 1978). PED disease was generally considered unknown in North America until 2013, at which point widespread outbreaks commenced, and severe economic losses to the swine industry resulted. These prototype North American isolates (year 2013 and thereafter) have remained genetically closely related (i.e. with overall nucleotide identity generally over 99%), and are similar to Asian strains characterized there within a few years prior to the North American outbreaks.
  • PEDV generally grows poorly in culture, and there is a need to identify both particular strains and culture conditions that are appropriate for the culturing of sufficient virus for commercial vaccine preparation. Additionally, there is a need to develop vaccines that provide effective cross protection against known isolates of PEDV, and which are expected to provide effective cross protection against evolving, non-prototype PEDV strains.
  • variant strains of PEDV have also been recently identified in North America and Europe, and are among themselves closely related, and are recognizably different from the aforementioned "prototype” North American strains and classical European strains.
  • variant strains based in part on “S” or spike protein sequence
  • S S or spike protein sequence
  • Asian isolates are again more similar North American or European variant/INDEI strains than to prototype strains.
  • a well known North American prototype strain, associated with outbreaks of disease beginning in 2013 is USA/Colorado/2013, whose sequence is deposited as GenBank accession No. KF272920, of the NCBI of the United States National Institutes of Health. Bethesda, MD).
  • Veterinary Microbiology 173, pp 258-269 (2014) disclose additional S-INDEL strains, and interestingly, a PEDV strain related instead to prototype virulent strains, but also bearing a large 197 amino acid deletion from the S protein, possibly resulting from passaging.
  • INDEL-type strains that have recently been reported from Europe include those described by S. Theuns et al. (2015). "Complete genome sequence of a porcine epidemic diarrhea virus from a novel outbreak in Belgium, January 2015.” Genome Announcements 3(3), pp. 1-2, May/June 2015; J. Stadler, et al., "Emergence of porcine epidemic diarrhea virus in southern Germany.” BMC Veterinary Research, v 11 No.142; pages 1-8, 2015; and. B. Grasland, et al. "Complete genome sequence of a porcine epidemic diarrhea S-Gene Indel strain isolated in France in December 2014.” Genome Announcements 3(3), pp. 1-2, May/June 2015.
  • variant and prototype strains are co- circulating in North America, and elsewhere, and existing populations of strains may result from multiple transmissions across continents or other regions.
  • variant strains may be less virulent than prototype strains, at least as to some age groups of swine, remaining virulence still makes such viruses unsafe for use in vaccines, if used in live form.
  • attempts to passage prototype strains to avirulence have not been successful, and adequate safety is not achieved after well over 100 passages.
  • the present invention is directed to novel mutant isolates of the variant European strain Calaf14 (see SEQ ID NO:1 herein, and PCT/US 2015/039475 generally, in regard of the wild type) that have been attenuated so that they can be safely administered to swine of all ages, without harm to the animals, and at the same time, are highly immunogenic and cross protective against subsequent challenge of the animals by a wide variety of PEDV strains, including both prototype and variant strains from all Continents. Vaccines from such mutant isolates, whether in killed or live form, are useful to protect swine from PEDV on a worldwide basis. Summary of the Invention
  • the present invention is directed to novel mutant isolates of Calf 14 PEDV virus that can be safely administered to swine of all ages, without harm to the animals, and at the same time, are highly immunogenic and cross protective against subsequent challenge of the animals by a wide variety of PEDV strains, including both prototype and variant strains.
  • Vaccines from such mutant isolates are useful to protect swine from PEDV on a worldwide basis.
  • the present invention therefore encompasses an vaccine composition comprising inactivated mutant Calaf14 PEDV, one or more adjuvants, and optionally one or more excipients, in an amount effective to elicit production of neutralizing antibodies in swine, with good cross protection against subsequent challenge by both prototype and variant strain PEDV strains that circulate in Nature.
  • the adjuvant preferably provides an oil-in-water emulsion with additional components.
  • the vaccine compositions of the invention protect swine from infection by PEDV, and are effective in single doses, in two-dose programs, or in vaccination programs involving multiple doses, which may be spread apart by at least a week, and optionally at greater intervals of time, such as one to several months.
  • the present invention similarly provides vaccine compositions comprising the aforementioned mutant isolates of Calaf14 PEDV, as live vaccines, with our without adjuvants, that are also highly effective and provide good cross protection against subsequent challenge by both prototype and variant strain PEDV strains that circulate in Nature.
  • the vaccine dose program of one, two, or multiple doses may be repeated, from time to time, as a precautionary measure. Additionally, it should be noted that vaccinating a mother sow during pregnancy will provide protection to a young piglet, via maternal transfer of antibodies and T-cells in milk, although such protection may need to be followed up with additional vaccination doses to the piglet. Vaccination of all swine including piglets and adults is contemplated.
  • the immunogenic composition can comprise other swine antigens, including Escherichia coli and Clostridium perfringens, types A-D, the dosages of which would be equivalent to those found in the commercially-available vaccines, Gletvax® and Litterguard®.
  • the vaccines can contain one or more adjuvants, and optionally one or more excipients, in an amount effective to elicit production of neutralizing antibodies in swine.
  • the adjuvant preferably provides an oil-in-water emulsion with additional components.
  • the immunogenic compositions of the invention protect swine from infection by PEDV are effective in single doses, in two-dose programs, or in vaccination programs involving multiple doses, which may be spread apart by at least a week, and optionally at greater intervals of time, such as one to several months.
  • the PEDV vaccines of the present invention are comprise any of the novel viruses as disclosed herein, such as those encoded by a polynucleotide selected from:
  • a PEDV virus that is encoded by a nucleotide sequence that is at least 90% identical to one or more of SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 at a full length nucleotide level, as long as said claimed encoding sequence contains a mutant amino acid residue not found in the virus encoded from SEQ ID NO: 1.
  • the PEDV vaccines of the present invention comprise viruses encoded by a nucleotide sequence that is at least 95%, 96%, 97%, 98%, 99% or 99.5% identical to one or more of SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 at a full length nucleotide level, as long as said novel encoding sequence contains a mutant amino acid residue not found in the virus encoded from SEQ ID NO: 1.
  • said mutant amino acid residues are selected from the group consisting of.
  • the vaccines of the present invention are capable of protecting swine from challenge by both variant and prototype strains of PEDV, and preventing or treating symptoms associated with PEDV infection, wherein said protected swine include any of sows, gilts, boars, hogs, and piglets, and wherein achievement of protection is determined by an endpoint selected from the group consisting of prevention or control of any of the PEDV infection symptoms of dehydration, fever, diarrhea, vomiting, poor lactational performance, poor reproduction performance, mortality, and prevention or control of weight loss or failure to gain weight.
  • the novel viruses of the vaccine composition may be live or killed, and if killed, a preferred adjuvant is an oil-in-water emulsion, wherein the adjuvant components include Amphigen® and aluminum hydroxide,, nmost preferably, wherein the final concentration of 20% Amphigen is 25% (v/v).
  • the invention therefor provides a method of protecting swine from challenge against PEDV, comprising administering to the subject a vaccine composition, in an amount sufficient to prevent or treat symptoms associated with PEDV infection, wherein said protected swine include any of sows, gilts, boars, hogs, and piglets, and wherein
  • achievement of protection is determined by an endpoint selected from the group consisting of prevention or control of any of the PEDV infection symptoms of dehydration, fever, diarrhea, vomiting, poor lactational performance, poor reproduction performance, mortality, and prevention or control of weight loss or failure to gain weight.
  • Vaccine compositions of the invention are effective in piglets that are 1 day of age or older, in a single or two dose program. Vaccine compositions of the invention are also effective in piglets in a two dose program, wherein the first dose is administered when the piglet is about 1-7 days old, and the second dose is administered when the piglet is 2-5 weeks old. The second does may be optional.
  • the vaccine compositions of the invention have a minimum effective dose is between about 10 and about 10 6 logi 0 TCID 50.
  • the vaccination program provides 2 doses administered to the piglet; and the parent sow, although vaccinated pre-breeding, is not vaccinated pre-farrowing. Additionally, the vaccination program provides 1 dose administered to the piglet; and the parent sow is vaccinated both pre-breeding and pre- farrowing.
  • in order to prevent disease in a piglet there is first administered the vaccine composition to the sow of said piglet, whether pre-farrowing or pre- breeding, following by administering one or more doses of said vaccine composition to said piglet after birth.
  • the invention provides that pigs are first vaccinated, followed by annual or pre-farrowing administration of further doses PEDV vaccine.
  • Figure 1 shows a comparison of particular encoding nucleotides in INDEL PEDV strain USA/OH 851/2014 (of the Ohio Department of Agriculture, GenBank KJ399978, "OH 851”) to those found in Calaf 14 Passage 0, with any resultant amino acid changes.
  • the Figure then further shows evolution of additional nucleotide changes, and resultant amino acid changes, as Calaf 14 is subsequently passaged from Passage 0 to Passage 60, and then individual clones (A and E) are selected from consensus passage 60. Nucleotides are numbered according to the numbered sequence of PEDV USA/OH851/2014.
  • Figure 4 shows the ORF1a-1 b gene sequence for Calaf 14, passage 0
  • Figure 4 shows the ORF1 a-1 b gene sequence for Calaf 14, passage Othe 5' end, and 5 bp at the 3' end.
  • position 2929 in OH851 corresponds to Calaf14, passage 0, position 2894.
  • passage 37 (only)
  • Figure 2 shows the alignment of encoded ORF3 proteins of Calaf 14 PEDV Passages 37 (SEQ ID NO:8) and 60 (SEQ ID NO:9).
  • Figure 3 shows the full nucleotide sequence of ther Passage 60 Calf 14 virus, again noting that approximately 35 bases are missing from the 5' end, and approximately 5 bases are missing from the 3' end, as depicted.
  • Figure 4 shows the ORF1 a-1 b gene sequence for Calaf 14, passage 0.
  • Figure 5 shows the ORF1 a-1 b gene sequence for Calaf 14, passage 11.
  • Figure 6 shows the ORF1 a-1 b gene sequence for Calaf 14, passage 37.
  • Figure 7 shows the ORF1 a-1 b gene sequence for Calaf 14, passage 60.
  • Figure 8 shows the ORF1 a-1 b gene sequence for Calaf14, passage 60 clnA
  • Figure 9 shows the ORF1 a-1 b gene sequence for Calaf14, passage 60 clnE
  • Figure 10 shows the spike gene sequence for Calaf 14, passage 0.
  • Figure 11 shows the spike gene sequence for Calaf 14, passage 11.
  • Figure 12 shows the spike gene sequence for Calaf 14, passage 37.
  • Figure 13 shows the spike gene sequence for Calaf 14, passage 60.
  • Figure 14 shows the spike gene sequence for Calaf 14, passage 60, cln A
  • Figure 15 shows the spike gene sequence for Calaf 14, passage 60
  • cln E Figure 16 shows the ORF3 gene sequence for Calaf 14, passage 0.
  • Figure 17 shows the ORF3 gene sequence for Calaf 14, passage 11.
  • Figure 18 shows the ORF3 gene sequence for Calaf 14, passage 37.
  • Figure 19 shows the ORF3 gene sequence for Calaf 14, passage 60.
  • Figure 20 shows the ORF3 gene sequence for Calaf 14, passage 60, cln
  • Figure 21 shows the ORF3 gene sequence for Calaf 14, passage 60, cln E Figure 22 (SEQ ID NO:28) shows the E gene for Passage 60.
  • Figure 23 shows the M gene for Passage 60.
  • Figure 24 shows the N gene for Passage 60.
  • SEQ ID NO: 1 provides the DNA sequence encoding PEDV strain Calaf 14, wild type, passage 0.
  • SEQ ID NO: 2 provides the DNA sequence encoding PEDV strain Calaf 14, passage 11.
  • SEQ ID NO: 3 provides the DNA sequence encoding PEDV strain Calaf 14, passage 37.
  • SEQ ID NO: 4 provides the DNA sequence encoding PEDV strain Calaf 14, passage 60.
  • SEQ ID NO: 5 provides the DNA sequence encoding PEDV strain Calaf 14, passage 60, clone A selected therefrom.
  • SEQ ID NO: 6 provides the DNA sequence encoding PEDV strain Calaf 14, passage 60, clone E selected therefrom.
  • SEQ ID NO: 7 provides the DNA sequence encoding PEDV strain OH851.
  • SEQ ID NO: 1 provides the DNA sequence encoding PEDV strain Calaf 14, wild type, passage 0.
  • SEQ ID NO: 8 provides the amino acid sequence of the protein encoded from ORF3, Calaf 14, passage 37.
  • SEQ ID NO: 9 provides the amino acid sequence of the protein encoded from ORF3, Calaf 14, passage 60.
  • the present invention provides novel and efficacious vaccines useful to preventing disease caused by PEDV.
  • Vaccines can be made more efficacious by including an appropriate adjuvant in the composition.
  • adjuvant generally refers to any material that increases the humoral or cellular immune response to an antigen. Adjuvants are used to accomplish two objectives: They slow the release of antigens from the injection site, and they enhance stimulation of the immune system.
  • Traditional vaccines are generally composed of a crude preparation of inactivated or killed or modified live pathogenic microorganisms. The impurities associated with these cultures of pathological microorganisms may act as an adjuvant to enhance the immune response.
  • the immunity invoked by vaccines that use homogeneous preparations of pathological microorganisms or purified protein subunits as antigens is often poor.
  • an adjuvant therefore becomes necessary.
  • synthetic and subunit vaccines may be expensive to produce.
  • the pathogen cannot be grown on a commercial scale, and thus, synthetic/subunit vaccines represent the only viable option.
  • the addition of an adjuvant may permit the use of a smaller dose of antigen to stimulate a similar immune response, thereby reducing the production cost of the vaccine.
  • the effectiveness of some injectable medicinal agents may be significantly increased when the agent is combined with an adjuvant.
  • an adjuvant should cause a relatively slow rate of release and absorption of the antigen in an efficient manner with minimum toxic, allergenic, irritating, and other undesirable effects to the host.
  • an adjuvant should be non-viricidal, biodegradable, capable of consistently creating a high level of immunity, capable of stimulating cross protection, compatible with multiple antigens, efficacious in multiple species, non-toxic, and safe for the host (eg, no injection site reactions).
  • an adjuvant is capable of micro-dosing, is dose sparing, has excellent shelf stability, is amenable to drying, can be made oil-free, can exist as either a solid or a liquid, is isotonic, is easily manufactured, and is inexpensive to produce.
  • an adjuvant it is highly desirable for an adjuvant to be configurable so as to induce either a humoral or cellular immune response or both, depending on the requirements of the vaccination scenario.
  • the number of adjuvants that can meet the above requirements is limited. The choice of an adjuvant depends upon the needs for the vaccine, whether it be an increase in the magnitude or function of the antibody response, an increase in cell mediated immune response, an induction of mucosal immunity, or a reduction in antigen dose.
  • FCA Freund's Complete Adjuvant
  • Some other materials that have been used as adjuvants include metallic oxides (e.g., aluminum hydroxide), alum, inorganic chelates of salts, gelatins, various paraffin-type oils, synthesized resins, alginates, mucoid and polysaccharide compounds, caseinates, and blood-derived substances such as fibrin clots. While these materials are generally efficacious at stimulating the immune system, none has been found to be entirely satisfactory due to adverse effects in the host (e.g., production of sterile abcesses, organ damage,
  • carcinogenicity, or allergenic responses or undesirable pharmaceutical properties (e.g., rapid dispersion or poor control of dispersion from the injection site, or swelling of the material).
  • Cellular immune response or “cell mediated immune response” is one mediated by T-lymphocytes or other white blood cells or both, and includes the production of cytokines, chemokines and similar molecules produced by activated T-cells, white blood cells, or both; or a T lymphocyte or other immune cell response that kills an infected cell.
  • emulsifier is used broadly in the instant disclosure. It includes substances generally accepted as emulsifiers, e.g., different products of TWEEN® or SPAN® product lines (fatty acid esters of polyethoxylated sorbitol and fatty-acid-substituted sorbitan surfactants, respectively), and different solubility enhancers such as PEG-40 Castor Oil or another PEGylated hydrogenated oil.
  • Human immune response refers to one that is mediated by antibodies.
  • Immune response in a subject refers to the development of a humoral immune response, a cellular immune response, or a humoral and a cellular immune response to an antigen. Immune responses can usually be determined using standard immunoassays and neutralization assays, which are known in the art.
  • Immunologically protective amount or “immunologically effective amount” or “effective amount to produce an immune response” of an antigen is an amount effective to induce an immunogenic response in the recipient.
  • the immunogenic response may be sufficient for diagnostic purposes or other testing, or may be adequate to prevent signs or symptoms of disease, including adverse health effects or complications thereof, caused by infection with a disease agent. Either humoral immunity or cell-mediated immunity or both may be induced.
  • the immunogenic response of an animal to an immunogenic composition may be evaluated, e.g., indirectly through measurement of antibody titers, lymphocyte proliferation assays, or directly through monitoring signs and symptoms after challenge with wild type strain, whereas the protective immunity conferred by a vaccine can be evaluated by measuring, e.g., reduction in clinical signs such as mortality, morbidity, temperature number, overall physical condition, and overall health and performance of the subject.
  • the immune response may comprise, without limitation, induction of cellular and/or humoral immunity. "Immunogenic" means evoking an immune or antigenic response.
  • an immunogenic composition would be any composition that induces an immune response.
  • “Therapeutically effective amount” refers to an amount of an antigen or vaccine that would induce an immune response in a subject receiving the antigen or vaccine which is adequate to prevent or reduce signs or symptoms of disease, including adverse health effects or complications thereof, caused by infection with a pathogen, such as a virus or a bacterium. Humoral immunity or cell-mediated immunity or both humoral and cell-mediated immunity may be induced.
  • the immunogenic response of an animal to a vaccine may be evaluated, e.g., indirectly through measurement of antibody titers, lymphocyte proliferation assays, or directly through monitoring signs and symptoms after challenge with wild type strain.
  • the protective immunity conferred by a vaccine can be evaluated by measuring, e.g., reduction in clinical signs such as mortality, morbidity, temperature number, overall physical condition, and overall health and performance of the subject.
  • the amount of a vaccine that is therapeutically effective may vary depending on the particular adjuvant used, the particular antigen used, or the condition of the subject, and can be determined by one skilled in the art.
  • TCID 50 refers to "tissue culture infective dose” and is defined as that dilution of a virus required to infect 50% of a given batch of inoculated cell cultures.
  • Various methods may be used to calculate TCID 50 , including the Spearman-Karber method which is utilized throughout this specification. For a description of the Spearman-Karber method, see B. W. Mahy & H. O. Kangro, Virology Methods Manual, p. 25-46 (1996).
  • the vaccine and immunogenic composition of the present invention induces at least one of a number of humoral and cellular immune responses in a subject swine that has been administered a vaccine composition of the invention.
  • the vaccine compositions of the invention may be administered to swine of any age, whether male or female, irrespective of reproductive status, and although it is contemplated that a two-dose regimen will be most common, single dose and multiple dose vaccine treatments are also effective in the practice of the invention.
  • a most preferred virus for use according to all aspects of the invention relating to PEDV is that encoded from SEQ ID NOS: 4, 5 and 6.
  • Such a vaccine protects well against challenge by both prototype and variant strains, such as North American prototype strain USA/Colorado/2013 (whose sequence is deposited as GenBank accession No. KF272920, of the NCBI of the United States National Institutes of Health. Bethesda, MD), and also numerous other prototype and variant strains, such as similarly deposited Chinese strain AH2012, deposited as GenBank accession No.
  • KJ645688 (USA/lowa96/2013); KJ645640 (USA/Oklahoma32/2013); KJ778615 (NPL-PEDv/2013); KJ645647 (USA/Minnesota41/2013); KJ645637 ((USA/Kansas29/2013); KJ645639 (USA/Texas31/2013); KJ645666 (USA/lowa70/2013); KJ645646
  • the vaccine compositions of the present invention protect against challenge by PEDV generally, including all forms of the virus circulating in Asia, North America, and Europe.
  • viruses can also be identified solely by the amino acid of nucleotide encoding sequences of surface spike protein S, and thus additional isolates against which the present invention is effective include viral coat sequences reported in GenBank (US NIH/NCBI) by their spike protein accessions, to include AID56757.1 ;
  • AIC82397.1 ; AFL02631.1 ; AHB33810.1 ; AFQ37598.1 ; AGG34691.1 ; AFJ97030.1 ; AFR11479.1 ; and AEW22948.1 (all being representative of those having above 98% identity to the USA/Colorado/2013 spike sequence).
  • GenBank® is the recognized US-NIH genetic sequence database, comprising an annotated collection of publicly available DNA sequences, and which further incorporates submissions from the European Molecular Biology Laboratory (EMBL) and the DNA
  • the nucleotide sequence of a second polynucleotide molecule is "homologous" to the nucleotide sequence of a first polynucleotide molecule , or has "identity" to said first polynucleotide molecule, where the nucleotide sequence of the second polynucleotide molecule encodes the same polyaminoacid as the nucleotide sequence of the first polynucleotide molecule as based on the degeneracy of the genetic code, or when it encodes a polyaminoacid that is sufficiently similar to the polyaminoacid encoded by the nucleotide sequence of the first polynucleotide molecule so as to be useful in practicing the present invention.
  • Homologous polynucleotide sequences also refers to sense and anti-sense strands, and in all cases to the complement of any such strands.
  • a polynucleotide molecule is useful in practicing the present invention, and is therefore homologous or has identity, where it can be used as a diagnostic probe to detect the presence of PEDV virus or viral polynucleotide in a fluid or tissue sample of an infected pig, e.g. by standard hybridization or amplification techniques.
  • nucleotide sequence of a second polynucleotide molecule is homologous to the nucleotide sequence of a first polynucleotide molecule if it has at least about 70% nucleotide sequence identity to the nucleotide sequence of the first polynucleotide molecule as based on the BLASTN algorithm (National Center for
  • a homologous nucleotide sequence has at least about 90% nucleotide sequence identity, even more preferably at least about 95%, 96%, 97%, 98% and 99% nucleotide sequence identity. Since the genetic code is degenerate, a homologous nucleotide sequence can include any number of "silent" base changes, i.e. nucleotide substitutions that nonetheless encode the same amino acid.
  • a homologous nucleotide sequence can further contain non-silent mutations, i.e. base substitutions, deletions, or additions resulting in amino acid differences in the encoded polyaminoacid, so long as the sequence remains at least about 90% identical to the polyaminoacid encoded by the first nucleotide sequence or otherwise is useful for practicing the present invention.
  • non-silent mutations i.e. base substitutions, deletions, or additions resulting in amino acid differences in the encoded polyaminoacid, so long as the sequence remains at least about 90% identical to the polyaminoacid encoded by the first nucleotide sequence or otherwise is useful for practicing the present invention.
  • certain conservative amino acid substitutions may be made which are generally recognized not to inactivate overall protein function: such as in regard of positively charged amino acids (and vice versa), lysine, arginine and histidine; in regard of negatively charged amino acids (and vice versa), aspartic acid and glutamic acid; and in regard of certain groups of neutrally charged amino acids (and in all cases, also vice versa), (1) alanine and serine, (2) asparagine, glutamine, and histidine, (3) cysteine and serine, (4) glycine and proline, (5) isoleucine, leucine and valine, (6) methionine, leucine and isoleucine, (7) phenylalanine, methionine, leucine, and tyrosine, (8) serine and threonine, (9) tryptophan and tyrosine, (10) and for example tyrosine, tyrptophan and phenylalanine.
  • Amino acids can be classified according to physical properties and contribution to secondary and tertiary protein structure.
  • a conservative substitution is recognized in the art as a substitution of one amino acid for another amino acid that has similar properties. Exemplary conservative substitutions may be found in WO 97/09433, page 10, published Mar. 13. 1997 (PCT/GB96/02197, filed Sep. 6, 1996.
  • conservative amino acids can be grouped as described in Lehninger, (Biochemistry, Second Edition; Worth Publishers, Inc. NY:NY (1975), pp. 71-77).
  • homologous nucleotide sequences can be determined by comparison of nucleotide sequences, for example by using BLASTN, above.
  • homologous nucleotide sequences can be determined by hybridization under selected conditions.
  • the nucleotide sequence of a second polynucleotide molecule is homologous to SEQ ID NO:1 (or any other particular polynucleotide sequence) if it hybridizes to the complement of SEQ ID NO:1 under moderately stringent conditions, e.g., hybridization to filter-bound DNA in 0.5 M NaHP04, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65°C, and washing in
  • SDS sodium dodecyl sulfate
  • hybridization conditions can be empirically determined or precisely calculated based on the length and percentage of guanosine/cytosine (GC) base pairing of the probe.
  • the hybridization conditions can be calculated as described in Sambrook, et al., (Eds.), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York (1989), pp. 9.47 to 9.51.
  • a second nucleotide sequence is homologous to SEQ ID NO:1 (or any other sequence of the invention) if it hybridizes to the complement of SEQ ID NO:1 under highly stringent conditions, e.g. hybridization to filter-bound DNA in 0.5 M NaHP04, 7% SDS, 1 mM EDTA at 65°C, and washing in 0.1xSSC/0.1 % SDS at 68°C, as is known in the art.
  • isolated polynucleotide molecules and the isolated RNA molecules of the present invention include both synthetic molecules and molecules obtained through recombinant techniques, such as by in vitro cloning and transcription.
  • ORF1a/b spike, and ORF3, causing important amino acid changes in the resultant proteins, that provide for the high level of safety achieved for Calaf14 passage 60 (see Example 5 below).
  • 2 clones A and E, SEQ ID NOS:5 and 6) were also recovered and sequenced from the consensus population (SEQ ID NO:4).
  • spike protein changes between passages 11 and 37, there is only one stable amino acid change that is then retained in passage 60.
  • two additional amino acid changes in Spike contribute to attenuation. These are aspartate (D) to tyrosine (Y) at position 262, and asparagine (N) to aspartate (D) to position 1006.
  • PEDV viruses having these sequence modifications (see SEQ ID NOS 4,5 and 6), or conservative amino acid variants thereof, for example, position 1269 (or the position that corresponds to residue 1269 as would be determined from an appropriate algorithm) could instead be valine, isoleucine, and the like; position 262 tyrosine could be replaced by phenyalanine, valine, .leucine, isoleucine and the like, and asparate at position 1006 could be replaced by by glutamate, for example.
  • Inactivated or killed viral strains are those which have been inactivated by methods known to those skilled in the art, including treatment with formalin, betapropriolactone (BPL), binary ethyleneimine (BEI), sterilizing radiation, heat, or other such methods.
  • BPL betapropriolactone
  • BEI binary ethyleneimine
  • the vaccine compositions of the invention may or may not include adjuvants.
  • the modified live vaccines of the invention may be used adjuvant free, with a sterile carrier.
  • Adjuvants that may be used for oral administration include those based on CT-like immune modulators (rmLT, CT-B, i.e.
  • a preferred adjuvanted or non adjuvanted vaccine dose at the minimal protective dose through vaccine release may provide between approximately 10 and approximately 10 6 logi 0 TCID 50 of virus per dose, or higher.
  • Adjuvants, if present, may be provided as emulsions, more commonly if non-oral administration is selected, but should not decrease starting titer by more than 0.7 logs (80% reduction.
  • Immunogenic compositions of the present invention can include one or more well known adjuvants and adjuvant systems.
  • Suitable adjuvants include, but are not limited to, the RIBI adjuvant system (Ribi Inc.; Hamilton, MT); alum; aluminum hydroxide gel; aluminum phosphate; oil-in water emulsions; water-in-oil emulsions such as Freund's complete and incomplete adjuvants; Block copolymer (CytRx; Atlanta, GA); SAF-M (Chiron; Emeryville, CA); AMPHIGEN® adjuvant; killed Bordetella; saponins such as StimulonTM QS-21
  • MPL-A monophosphoryl lipid A
  • avridine lipid-amine adjuvant
  • heat-labile enterotoxin from Escherichia coli recombinant or otherwise
  • cholera toxin cholera toxin
  • muramyl dipeptide also useful is MPLTM (3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton, MT), which is described in U.S. Patent No. 4,912,094, and is hereby incorporated by reference.
  • lipid A analogs or aminoalkyl glucosamine phosphate (AGP) compounds or derivatives or analogs thereof, which are available from Corixa (Hamilton, MT), and which are described in US 6, 1 13,918, hereby incorporated by reference; L121/squalene; D-lactide-polylactide/glycoside; pluronic polyols; muramyl dipeptide; extracts of Mycobacterium tuberculosis; bacterial lipopolysaccharides generally; pertussis toxin (PT); and an E.
  • AGP aminoalkyl glucosamine phosphate
  • coli heat-labile toxin particularly LT-K63, LT- R72, PT-K9/G129; see, e.g., International Patent Publication Nos. WO 93/13302 and WO 92/19265, incorporated herein by reference.
  • Synthetic polynucleotides such as oligonucleotides containing CpG motifs (US 6,207,646, hereby incorporated by reference), can also be used as adjuvants for the present invention.
  • CpG oligonucleotides such as P-class immunostimulatory oligonucleotides, are useful, including E-modified P-class immunostimulatory oligonucleotides.
  • Sterols can also be useful as adjuvants herein. Those suitable for use can include sitosterols, stigmasterol, ergosterol, ergocalciferol, and cholesterol.
  • the adjuvant compositions useful in the practice of the invention can generally further include one or more polymers such as, for example, DEAE Dextran, polyethylene glycol, polyacrylic acid, and polymethacrylic acid (e.g., CARBOPOL®).
  • the adjuvant compositions can also further include one or more Th2 stimulants such as, for example, Bay R1005(R) and aluminum.
  • the adjuvant compositions can additionally or alternatively further include one or more immunomodulatory agents, such as quaternary ammonium compounds (e.g., DDA), interleukins, interferons, or other cytokines.
  • immunomodulatory agents such as quaternary ammonium compounds (e.g., DDA), interleukins, interferons, or other cytokines.
  • DDA quaternary ammonium compounds
  • interleukins 1- ⁇ , 1- ⁇ , 2, 4, 5, 6, 7, 8, 10, 12 see, e.g., US 5,723,127), 13, 14, 15, 16, 17 and 18 (and its mutant forms
  • the interferons-a, ⁇ and gamma granulocyte-macrophage colony stimulating factor (see, for example, US 5,078,996, and ATCC Accession Number 39900), macrophage colony stimulating factor, granulocyte colony stimulating factor, GSF, and the tumor necrosis factors a and ⁇ .
  • Still other adjuvants useful in this invention include chemokines, including without limitation, MCP-1 , ⁇ -1 ⁇ , ⁇ -1 ⁇ , and RANTES.
  • Adhesion molecules such as a selectin, e.g., L- selectin, P-selectin, and E-selectin may also be useful as adjuvants.
  • Still other useful adjuvants include, without limitation, a mucin-like molecule, e.g., CD34, GlyCAM-1 and MadCAM-1 ; a member of the integrin family such as LFA-1 , VLA-1 , Mac-1 and p150.95; a member of the immunoglobulin superfamily such as PECAM, ICAMs (e.g., ICAM-1 , ICAM-2 and ICAM-3), CD2 and LFA-3; co-stimulatory molecules such as CD40 and CD40L; growth factors including vascular growth factor, nerve growth factor, fibroblast growth factor, epidermal growth factor, B7.2, PDGF, BL-1 , and vascular endothelial growth factor; receptor molecules including Fas, TNF receptor, Fit, Apo-1 , p55, WSL-1 , DR
  • Suitable adjuvants also include, without limitation, MPLTM (3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton, MT), which is described in U.S. Patent No.
  • AGP aminoalkyl glucosamine phosphate compounds
  • AGP is 2-[(R)-3-Tetradecanoyloxytetradecanoylamino] ethyl 2-Deoxy-4- 0-phosphono-3-0-[(R)-3-tetradecanoyoxytetradecanoyl]-2-[(R)-3- tetradecanoyloxytetradecanoyl-amino]-b-D-glucopyranoside, which is also known as 529 (formerly known as RC529).
  • the RC529 adjuvant is formulated as an aqueous form or as a stable emulsion.
  • CT cholera toxins
  • mutants thereof including those described in published International Patent Application number WO 00/18434 (wherein the glutamic acid at amino acid position 29 is replaced by another amino acid, other than aspartic acid, preferably a histidine).
  • CT toxins or mutants are described in published International Patent Application number WO 02/098368 (wherein the isoleucine at amino acid position 16 is replaced by another amino acid, either alone or in combination with the replacement of the serine at amino acid position 68 by another amino acid; and/or wherein the valine at amino acid position 72 is replaced by another amino acid).
  • CT toxins are described in published International Patent Application number WO 02/098369 (wherein the arginine at amino acid position 25 is replaced by another amino acid; and/or an amino acid is inserted at amino acid position 49; and/or two amino acids are inserted at amino acid positions 35 and 36). Said CT toxins or mutant can be included in the immunogenic compositions either as separate entities, or as fusion partners for the polypeptides of the present invention.
  • adjuvant components are provided from a combination of lecithin in light mineral oil, and also an aluminum hydroxide component. Details concerning the composition and formulation of Amphigen® (as representative lecithin/mineral oil component) are as follows.
  • a preferred adjuvanted may be provided as a 2ML dose in a buffered solution further comprising about 5% (v/v) Rehydragel® (aluminum hydroxide gel) and "20% Amphigen" ® at about 25% final (v/v).
  • Amphigen® is generally described in U.S Patent 5,084,269 and provides de-oiled lecithin (preferably soy) dissolved in a light oil, which is then dispersed into an aqueous solution or suspension of the antigen as an oil-in-water emulsion. Amphigen has been improved according to the protocols of U.S.
  • Patent 6,814,971 (see columns 8-9 thereof) to provide a so-called "20% Amphigen" component for use in the final adjuvanted vaccine compositions of the present invention.
  • a stock mixture of 10% lecithin and 90% carrier oil (DRAKEOL®, Penreco, Karns City, PA) is diluted 1 : 4 with 0.63% phosphate buffered saline solution, thereby reducing the lecithin and DRAKEOL components to 2% and 18% respectively (i.e. 20% of their original concentrations).
  • Tween 80 and Span 80 surfactants are added to the composition, with representative and preferable final amounts being 5.6% (v/v) Tween 80 and 2.4% (v/v) Span 80, wherein the Span is originally provided in the stock DRAKEOL component, and the Tween is originally provided from the buffered saline component, so that mixture of the saline and DRAKEOL components results in the finally desired surfactant concentrations.
  • Mixture of the DRAKEOL/lecithin and saline solutions can be accomplished using an In-Line Slim Emulsifier apparatus, model 405, Charles Ross and Son, Hauppauge, NY, USA.
  • the vaccine composition also includes Rehydragel® LV (about 2% aluminum hydroxide content in the stock material), as additional adjuvant component (available from Reheis, NJ, USA, and ChemTrade Logistics, USA). With further dilution using 0.63% PBS, the final vaccine composition contains the following compositional amounts per 2ML dose; 5% (v/v) Rehydragel® LV; 25% (v/v) of "20% Amphigen", i.e. it is further 4-fold diluted); and
  • the order of addition of components can be varied to provide the equivalent final vaccine composition.
  • an appropriate dilution of virus in buffer can be prepared.
  • An appropriate amount of Rehydragel® LV (about 2% aluminum hydroxide content) stock solution can then be added, with blending, in order to permit the desired 5% (v/v) concentration of Rehydragel® LV in the actual final product.
  • the vaccinate compositions of the invention permit variation in all of the ingredients, such that the total dose of antigen may be varied preferably by a factor of 100 (up or down) compared to the antigen dose stated above, and most preferably by a factor of 10 or less (up or down),.
  • surfactant concentrations may be varied by up to a factor of 10, independently of each other, or they may be deleted entirely, with replacement by appropriate concentrations of similar materials, as is well understood in the art.
  • Rehydragel® concentrations in the final product may be varied, first by the use of equivalent materials available from many other manufacturers (i.e. Alhydrogel® .Brenntag; Denmark), or by use of additional variations in the Rehydragel® line of products such as CG, HPA or HS.
  • final useful concentrations thereof including from 0% to 20%, with 2-12% being more preferred, and 4-8% being most preferred,
  • the although the final concentration of Amphigen is preferably 25%, this amount may vary from 5-50%, preferably 20-30% and is most preferably about 24-26%.
  • the oil used in the adjuvant formulations of the instant invention is preferably a mineral oil.
  • mineral oil refers to a mixture of liquid hydrocarbons obtained from petrolatum via a distillation technique.
  • the term is synonymous with "liquefied paraffin", “liquid petrolatum” and “white mineral oil.”
  • the term is also intended to include "light mineral oil,” i.e., oil which is similarly obtained by distillation of petrolatum, but which has a slightly lower specific gravity than white mineral oil. See, e.g., Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack Publishing Company, 1990, at pages 788 and 1323).
  • Mineral oil can be obtained from various commercial sources, for example, J. T. Baker (Phillipsburg, Pa.), USB Corporation
  • Preferred mineral oil is light mineral oil commercially available under the name DRAKEOL®.
  • the oily phase is present in an amount from 50% to 95% by volume;
  • the oily phase includes oil and emulsifiers (e.g., SPAN® 80, TWEEN® 80 etc), if any such emulsifiers are present.
  • Non-natural, synthetic emulsifiers suitable for use in the adjuvant formulations of the present invention also include sorbitan-based non-ionic surfactants, e.g. fatty-acid- substituted sorbitan surfactants (commercially available under the name SPAN® or
  • ARLACEL® fatty acid esters of polyethoxylated sorbitol (TWEEN®), polyethylene glycol esters of fatty acids from sources such as castor oil (EMULFOR®); polyethoxylated fatty acid (e.g., stearic acid available under the name SIMULSOL® M-53), polyethoxylated
  • TYLOXAPOL® polyoxyethylene fatty alcohol ethers
  • BBIJ® polyoxyethylene nonphenyl ethers
  • TWEEN®-80 Polyoxyethylene (20) sorbitan monooleate
  • SPAN®-80 Polyoxyethylene (20) sorbitan monooleate
  • SPAN®-80 sorbitan monooleate
  • the emulsifier(s) may be present in the vaccine composition in an amount of 0.01 % to 40% by volume, preferably, 0.1 % to 15%, more preferably 2% to 10%.
  • the final vaccine composition contains SP-Oil® and Rehydragel® LV as adjuvants (or other Rehydragel® or Alhydrogel® products), with preferable amounts being about 5-20% SP-Oil (v/v) and about 5-15% Rehydragel LV (v/v), and with 5% and 12%, respectively, being most preferred amounts.
  • % Rehydragel refers to percent dilution from the stock commercial product.
  • SP-Oil ® is a fluidized oil emulsion with includes a polyoxyethylene-polyoxypropylene block copolymer (Pluronic® L121 , BASF Corporation, squalene, polyoxyethylene sorbitan monooleate (Tween®80, ICI Americas) and a buffered salt solution.) It should be noted that the present invention may also be successfully practiced using wherein the adjuvant component is only Amphigen®.
  • the final vaccine composition contains TXO as an adjuvant; TXO is generally described in WO 2015/042369. All TXO compositions disclosed therein are useful in the preparation of vaccines of the invention.
  • the immunostimulatory oligonucleotide preferably an ODN, preferably containing a palindromic sequence, and optionally with a modified backbone, is present in the amount of 0.1 to 5 ug per 50 ul of the vaccine composition (e.g., 0.5 - 3 ug per 50 ul of the composition, or more preferably 0.09-0.11 ug per 50 ul of the composition).
  • a preferred species thereof is SEQ ID NO: 8 as listed (page 17) in the WO2015/042369 publication (PCT/US2014/056512).
  • the polycationic carrier (“X") is present in the amount of 1-20 ug per 50 ul (e.g., 3-10 ug per 50 ul, or about 5 ug per 50 ul).
  • Light mineral oil (“O”) is also a component of the TXO adjuvant.
  • TXO adjuvants are prepared as follows:
  • Sorbitan monooleate, MPL-A and cholesterol are dissolved in light mineral oil.
  • the resulting oil solution is sterile filtered;
  • compositions of the invention can be used with any of the PEDV strains and isolates covered by the present Specification.
  • Additional adjuvants useful in the practice of the invention include wax-A (see generally United States published patent application US20070298053; and "QCDCRT” or “QCDC”-type adjuvants (see generally United States published patent application
  • compositions of the invention can be used with any of the PEDV strains and isolates covered by the present Specification.
  • the immunogenic and vaccine compositions of the invention can further comprise pharmaceutically acceptable carriers, excipients and/or stabilizers (see e.g. Remington: The Science and practice of Pharmacy (2005) Lippincott Williams), in the form of lyophilized formulations or aqueous solutions.
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations, and may comprise buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as Mercury((o-carboxyphenyl)thio)ethyl sodium salt (THIOMERSAL), octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine,
  • a preferred clinical indication is for treatment, control and prevention in both breeding sows and gilts pre-farrowing, followed by vaccination of piglets.
  • two 2-ML doses of vaccine will be used, although of course, actual volume of the dose is a function of how the vaccine is formulated, with actual dosing amounts ranging from 0.1 to 5ML, taking also into account the size of the animals. Single dose vaccination is also appropriate.
  • the first dose may be administered as early as pre-breeding to 5-weeks pre- farrowing, with the second dose administered preferably at about 1-3 weeks pre-farrowing.
  • Doses vaccine preferably provide an amount of viral material that corresponds to a TCID 50 (tissue culture infective dose) of between about 10 6 and 10 8 , more preferably between about 10 7 and 10 7 5 , and can be further varied, as is recognized in the art.
  • Booster doses can be given two to four weeks prior to any subsequent farrowings.
  • Intramuscular vaccination (all doses) is preferred, although one or more of the doses could be given subcutaneously. Oral administration is also preferred.
  • Vaccination may also be effective in naive animals, and non-naive animals as accomplished by planned or natural infections.
  • the sow or gilt is vaccinated intramuscularly or orally at 5-weeks pre-farrowing and then 2-weeks pre-farrowing.
  • a protective immune response can be demonstrated in PEDV-negative vaccinated sows in that they developed antibodies (measured via fluorescent focal neutralization titer from serum samples) with neutralizing activity, and these antibodies were passively transferred to their piglets.
  • the protocols of the invention are also applicable to the treatment of already seropositive sows and gilts, and also piglets and boars.
  • Booster vaccinations can also be given and these may be via a different route of administration.
  • the vaccine compositions of the invention nonetheless can still provide protection to piglets via ongoing passive transfer of antibodies, even if the mother sow was only vaccinated in association with a previous farrowing.
  • piglets may then be vaccinated as early as Day 1 of life.
  • piglets can be vaccinated at Day 1 , with or without a booster dose at 3 weeks of age, particularly if the parent sow, although vaccinated pre-breeding, was not vaccinated pre- farrowing.
  • Piglet vaccination may also be effective if the parent sow was previously not naive either due to natural or planned infection. Vaccination of piglets when the mother has neither been previously exposed to the virus, nor vaccinated pre-farrowing may also effective.
  • Boars (typically kept for breeding purposes) should be vaccinated once every 6 months. Variation of the dose amounts is well within the practice of the art.
  • the vaccines of the present invention are safe for usein pregnant animals (all trimesters) and neonatal swine.
  • the vaccines of the invention are attenuated to a level of safety (i.e. no mortality, only transient mild clinical signs or signs normal to neonatal swine) that is acceptable for even the most sensitive animals again including neonatal pigs.
  • animals vaccinated with the vaccines of the invention are also immediately safe for human consumption, without any significant slaughter withhold, such as 21 days or less.
  • the vaccine When provided therapeutically, the vaccine is provided in an effective amount upon the detection of a sign of actual infection.
  • Suitable dose amounts for treatment of an existing infection include between about 10 and about 10 6 log 0 TCID 50 , or higher, of virus per dose (minimum immunizing dose to vaccine release).
  • a composition is said to be
  • composition is said to be administered in a "therapeutically or prophylactically effective amount” if the amount administered is physiologically significant.
  • At least one vaccine or immunogenic composition of the present invention can be administered by any means that achieve the intended purpose, using a pharmaceutical composition as described herein.
  • route of administration of such a composition can be by parenteral, oral, oronasal, intranasal, intratracheal, topical, subcutaneous, intramuscular, transcutaneous, intradermal, intraperitoneal, intraocular, and intravenous administration.
  • the composition is administered by intramuscularly.
  • Parenteral administration can be by bolus injection or by gradual perfusion over time. Any suitable device may be used to administer the compositions, including syringes, droppers, needleless injection devices, patches, and the like.
  • Administration that is oral, or alternatively, subcutaneous, is preferred.
  • Oral administration may be direct, via water, or via feed (solid or liquid feed).
  • the vaccine When provided in liquid form, the vaccine may be lyophilized with reconstitution, pr provided as a paste, for direct addition to feed (mix in or top dress) or otherwise added to water or liquid feed.
  • Viruses of the invention can be conveniently grown in Vero cell stocks that are approved for vaccine production.
  • the following provides a representative method to generate safe and approved cell stock, a vial of Vero cells was subject to additional passaging.. The cells were passed four times in PMEM w/wheat to produce Master Cell Stock (MCS) Lot 834430".
  • MCS Master Cell Stock
  • the MCS was tested in accordance with 9CFR & EP requirements. The MCS tested satisfactory for sterility, freedom from mycoplasmas, and extraneous agents. Therefore, PF-Vero MCS lot "1834430", is deemed eligible for submission to the Center for Veterinary Biologies Laboratories (CVB-L) for confirmatory testing.
  • CVB-L Center for Veterinary Biologies Laboratories
  • Seed Origin and Passage History is as follows.
  • a Pre-master Cell stock of global Vero cells was previously frozen.
  • the cells were grown in PMEM containing 1 % bovine serum (item # 00-0710-00, BSE compliant) and 3 mM L-glutamine. They were derived from Vero WCS Pass # 136, Lot #071700 MCS+3, 28-Jul-00.
  • the new Pre-master cell stock was frozen at pass # 166, which is MCS+33 from the original global Vero master cell stock.
  • MCS "1833440" was produced from a pre-master Lot All cultures were grown in PMEM w/wheat, 1.0% L-glutamine and 1.0% Bovine Calf serum.
  • MCS was tested as per 9CFR (028-PUO) and EP 2.6.7. The MCS was found to be free of any Mycoplasma contamination. Extraneous testing was completed as per 9CFR 1 13.52 using NL-BT-2 (Bovine), Vero, NL-ED-5 (Equine), NL-ST-1 (Porcine), NL-DK (Canine), NL-FK (Feline) cells, .
  • the MCS was negative for MGG, CPE and HAd and tested negative by FA for BVD, BRSV, BPV, BAV-1 , BAV-5, Rabies, Reo, BTV, ERV, Equine arteritis, PPV, TGE, PAV, HEV, CD, CPV, FPL and FIP.
  • the MCS was tested by ELISA for FIV and was found to be satisfactory.
  • the invention encompasses methods of preventing PEDV virus infection comprising administering the immunogenic and vaccine compositions of the invention in a swine subject of any age.
  • the vaccine When provided therapeutically, the vaccine is provided in an effective amount upon the detection of a symptom of actual infection.
  • a composition is said to be
  • composition is said to be administered in a "therapeutically or prophylactically effective amount” if the amount administered is physiologically significant.
  • At least one vaccine or immunogenic composition of the present invention can be administered by any means that achieve the intended purpose, using a pharmaceutical composition as described herein.
  • route of administration of such a composition can be by parenteral, oral, oronasal, intranasal, intratracheal, topical, subcutaneous, intramuscular, transcutaneous, intradermal, intraperitoneal, intraocular, and intravenous administration.
  • the composition is administered by intramuscularly.
  • Parenteral administration can be by bolus injection or by gradual perfusion over time.
  • Any suitable device may be used to administer the compositions, including syringes, droppers, needleless injection devices, patches, and the like. The route and device selected for use will depend on the composition of the adjuvant, the antigen, and the subject, and such are well known to the skilled artisan.
  • immunogenic composition is one which is sufficient to achieve a desired biological effect, in this case at least one of cellular or humoral immune response to one or more strains of PEDV.
  • effective dosage will be dependent upon the age, sex, health, and weight of the subject, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.
  • the ranges of effective doses provided below are not intended to limit the invention and represent examples of dose ranges which may be suitable for administering compositions of the present invention. However, the dosage may be tailored to the individual subject, as is understood and determinable by one of skill in the art, without undue experimentation. Examples
  • Example 1 General representative procedures applicable to collection of PEDV virus from tissue samples
  • tissue was used for extraction of PEDV virus.
  • the tissue was chopped into fine pieces using a sterile scalpel and sterile scissors in a sterile Petri dish. Work was done in a Bio-safety cabinet to ensure aseptic conditions. 2 ml of sterile PBS was added to the Petri dish to collect tissue and material was transfer to a 15 ml conical tube.
  • Tissue was homogenized using a Qiagen TissueRuptor at 80% of maximum by pulsing for a total of 30 seconds . Homogenization was performed in an ice bucket to lessen the effect of heat on the PEDV virus. The homogenized material was filtered through a 0.45 uM filter and 60 ul of material was used for RNA isolation and PEDV Q-PCR to confirm the presence of the PEDV virus. The filtered material containing the PEDV virus was further diluted 1 : 10 in sterile PBS and then filtered through a 0.20 uM filter.
  • the sterile-filtered PEDV homogenate was used to infect confluent mono-layers of Vero 76 cells by transferring 1 ml of filtered material to a T-25 flask containing 2.8E+06 cells planted 3 to 4 days prior.
  • the T-25 flasks of confluent Vero 76 cells were washed 2X with sterile PBS and IX with DMEM media containing 10% TPB, 20ug/ml geneticin and 4ug/ml TPCK trypsin (equivalent to 18.8 USP units/ml). Cells were infected for 1 hour at 37°C and 5%C0 2 in an incubator with gentle swirling every 15 minutes to ensure virus was evenly distributed to all cells.
  • Cells were first washed 2X with sterile PBS and IX with DMEM media containing 10% TPB, 20ug/ml geneticin and 4ug/ml TPCK trypsin (equivalent to 18.8 USP units/ml). Cells were infected for 1 hour at 37°C and 5%C0 2 in an incubator with gentle swirling every 15 minutes to ensure virus was evenly distributed to cells. 5 ml of DMEM media containing 10% TPB, 20ug/ml geneticin and 4ug/ml TPCK trypsin (equivalent to 18.8 USP units/ml) was added to flasks and flask were allowed to incubate for 2 days. This material is Passage 2 of the PEDV virus.
  • Plastic flasks or roller bottles are used for growing and expanding cell cultures. Roller bottles or bioreactors will be used for virus propagation. Cells may be washed, to remove serum, prior to inoculation with virus. The virus may be diluted in virus production medium and added directly to the cell monolayer. When bioreactors are used for virus propagation, trypsinized cells will be removed from the roller bottles and a final cell passage grown in uninoculated cell growth medium. Microcarriers for the bioreactors are prepared. The seed virus is diluted to an appropriate volume within a multiplicity of infection (MOI) range of 0.0001 to 10.0
  • MOI multiplicity of infection
  • the PED virus causes observable cytopathic effect (CPE).
  • CPE cytopathic effect
  • Virus is harvested when viral-induced CPE has reached 50-100% and infected cells have begun sloughing off into the medium (cell monolayer loss exceeding 50%).
  • the roller bottle vessels are removed from the incubator and inspected microscopically for both CPE and evidence of microbial contamination.
  • the antigen fluid is harvested into appropriate sterile containers in an aseptic manner. Bioreactor fluids are examined microscopically for evidence of microbial contamination and for the presence of desired cytopathic effects (CPE).
  • the viral fluids are passed through a ⁇ 100 micron filter or stainless steel mesh screen to remove microcarriers and harvested into appropriate sterile containers in an aseptic manner. Fluids may be stored at 2°C - 7°C for a maximum of 24 hours until inactivation. The harvested fluids may be used for seed if it is at the proper passage level and has an acceptable infectivity titer. In the case of Calaf14 isolation, the following specific steps were used. PEDV
  • Calaf14 isolate was obtained from a PEDV positive case detected in a Spanish farm in 2014.
  • Individual intestine samples were processed to obtain a clarified intestine homogenate. For that purpose 59 g of intestine sample were suspended in 90 ml PBS (supplemented with
  • intestine clarified homogenate was filtered through a 0.45- ⁇ filter. 1 ml of filtered material was diluted in 10ml of PBS and then filtered again through a 0.22- ⁇ filter and used as inoculum for virus isolation. Virus isolation was attempted using previously described PEDV cell culture conditions (Pan, Tian et al. 2012, Wicht, Li et al. 2014). Confluent Vero cells in 25-cm2 tissue culture flask were washed twice with PBS and one with the maintenance media-E (MM-E).
  • MM-E maintenance media-E
  • Maintenance media-E consisted of Eagle's Minimum Essential medium Alpha Modification (MEMa, Gibco) supplemented with 0.3% tryptose phosphate broth (TPB, Gibco), 20 mM HEPES (Gibco) and 15 g/ml trypsin (Dyfco).
  • MEMa Eagle's Minimum Essential medium Alpha Modification
  • TPB tryptose phosphate broth
  • Mibco 20 mM HEPES
  • Dyfco 15 g/ml trypsin
  • Flasks were replenished with 4ml of maintenance media-E and incubated up to 2 days before being frozen at -80 ⁇ 10°C, thawed, and passaged as as described above.
  • MM-C maintenance media-C
  • DMEM Dulbecco's modified Eagle's medium
  • TPB TPB + 10 ⁇ g/ml trypsin
  • PEDV P60 was cloned three times by end- point dilution method. The aim is to dilute the virus in order to generate a pure virus stock starting from a single infectious unit. Before virus inoculation 96-well plate of confluent vero cells were washed once with MM-C media. Tenfold serial dilutions of the PEDV in MM-C without serum were performed from 10-1 to 10-8. Each dilution was seeded eleven times ( ⁇ /well of virus dilutions) in a 96-well plate flat bottom. One column of the plate was kept as cell growth control. Plates were incubated at 37°C ⁇ 2°C, 4-7% C02 during 4-5 days.
  • RNA from samples P0, P11 , P37, P60, ClonA and ClonE was extracted using the Biosprint technology (QIAGEN) according to the manufacturer's instructions.
  • Cellular DNA was removed by using the Rnase free Dnase set (QIAGEN) and RNA cleanup by using the Rneasy Mini Kit (QIAGEN) following
  • NGS next-generation sequencing
  • Samples were sequenced using the lllumina MySeq system at the Service of Genomic and Bioinformatic laboratory in the University Autonoma de Barcelona. Tehcniques generally applicable herein are found in Pan, Y., X. Tian, W. Li, Q. Zhou, D. Wang, Y. Bi, F. Chen and Y. Song (2012). "Isolation and
  • inactivation containers and inactivated using a 5mM binary ethylenimine (BEI) solution The mixture is cyclized for 60-80 minutes at 36 ⁇ 2°C.
  • the antigen will be thoroughly mixed and transferred to an inactivation vessel for the duration of the process ( ⁇ 48 hours, with agitation).
  • Neutralization of the inactivated antigen fluids will be facilitated through the addition of sterile 1 M Sodium Thiosulfate to a final concentration of approximately 20 mM - 25 mM.
  • Post-inactivated/neutralized antigen production fluids will be tested for sterility and completeness of inactivation and stored at 2-7°C for future use in vaccine serial formulation.
  • Genatamicin can then be used as preservative. This antibiotic will be added at the lot stage.
  • the concentration of gentamicin in the final product will be ⁇ 30 ⁇ g/mL.6.
  • Example 4 Specific Adjuvant Compositions and Formulations
  • the killed vaccine may provide about 7.8 logi 0 TCID 50 of killed Calaf14, Passage 60, SEQ ID NO:4, 5 or 6 virus per 2ML dose in a buffered solution further comprising about 5% (v/v) Rehydragel® (aluminum hydroxide gel) and "20% Amphigen" ® at about 25% final (v/v). Doses down to 7.0 logi 0 TCID 50 of killed virus are also preferred.
  • Amphigen® is generally described in U.S Patent 5,084,269 and provides de-oiled lecithin (preferably soy) dissolved in a light oil, which is then dispersed into an aqueous solution or suspension of the antigen as an oil-in-water emulsion.
  • Amphigen has been improved according to the protocols of U.S. Patent 6,814,971 (see columns 8-9 thereof) to provide a so-called "20% Amphigen" component for use in the final adjuvanted vaccine compositions of the present invention.
  • a stock mixture of 10% lecithin and 90% carrier oil (DRAKEOL®, Penreco, Karns City, PA) is diluted 1 : 4 with 0.63% phosphate buffered saline solution, thereby reducing the lecithin and DRAKEOL components to 2% and 18% respectively (i.e. 20% of their original concentrations).
  • Tween 80 and Span 80 surfactants are added to the composition, with representative and preferable final amounts being 5.6% (v/v) Tween 80 and 2.4% (v/v) Span 80, wherein the Span is originally provided in the stock DRAKEOL component, and the Tween is originally provided from the buffered saline component, so that mixture of the saline and DRAKEOL components results in the finally desired surfactant concentrations.
  • Mixture of the DRAKEOL/lecithin and saline solutions was accomplished using an In-Line Slim Emulsifier apparatus, model 405, Charles Ross and Son, Hauppauge, NY, USA.
  • the vaccine composition also includes Rehydragel® LV (about 2% aluminum hydroxide content in the stock material), as additional adjuvant component (available from Reheis, NJ, USA, and ChemTrade Logistics, USA). With further dilution using 0.63% PBS, the final vaccine composition contains the following compositional amounts: 7.8 logi 0 TCID 50 of killed virus per 2ML dose; 5% (v/v) Rehydragel® LV; 25% (v/v) of "20% Amphigen", i.e. it is further 4-fold diluted); and 0.01 % (w/v) of merthiolate.
  • Rehydragel® LV about 2% aluminum hydroxide content in the stock material
  • additional adjuvant component available from Reheis, NJ, USA, and ChemTrade Logistics, USA.
  • the order of addition of components can be varied to provide the equivalent final vaccine composition.
  • an appropriate dilution of killed virus in buffer can be prepared.
  • An appropriate amount of Rehydragel® LV (about 2% aluminum hydroxide content) stock solution can then be added, with blending, in order to permit the desired 5% (v/v) concentration of Rehydragel® LV in the actual final product.
  • this intermediate stock material is combined with an appropriate amount of "20% Amphigen" stock (as generally described above, and already containing necessary amounts of Tween 80 and Span 80) to again achieve a final product having 25% (v/v) of "20% Amphigen".
  • An appropriate amount of 10% merthiolate can finally be added.
  • the vaccinate compositions of the invention permit variation in all of the ingredients, such that the total dose of antigen may be varied preferably by a factor of 100 (up or down) compared to the antigen dose stated above, and most preferably by a factor of 10 or less (up or down),.
  • surfactant concentrations may be varied by up to a factor of 10, independently of each other, or they may be deleted entirely, with replacement by appropriate concentrations of similar materials, as is well understood in the art.
  • Rehydragel® concentrations in the final product may be varied, first by the use of equivalent materials available from many other manufacturers (i.e. Alhydrogel® .Brenntag; Denmark), or by use of additional variations in the Rehydragel® line of products such as CG, HPA or HS.
  • final useful concentrations thereof including from 0% to 20%, with 2-12% being more preferred, and 4-8% being most preferred,
  • the although the final concentration of Amphigen is preferably 25%, this amount may vary from 5-50%, preferably 20-30% and is most preferably about 24-26%.
  • Depression was scored as normal (active), slightly inactive, pronounced inactivity or severe depression (moribund). Piglets showing any level of depression are quantified below. Anorexia was scored as normal, moderate or severe. Piglets showing any level of anorexia are scored below. Digestive disorders were quantified as low (mild diarrhea), moderate (vomiting, abdominal pain or marked watery diarrhea) and severe (fibrinous or hemorrhagic diarrhea). Piglets showing any level of digestive disorder are scored below. It can thus be seen that Passage 60 substantially achieves safety criteria.

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Abstract

La présente invention concerne de nouveaux isolats du virus de la diarrhée épidémique porcine (PEDV) et des compositions vaccinales fabriquées à partir de ces derniers. Les compositions vaccinales protègent les porcs de la maladie PED, et présentent une protection croisée contre une large diversité de souches, qu'elles soient variantes ou prototypes, qui circulent actuellement à travers le monde. Les vaccins sont prévus sous des formes à la fois tuées et vivantes, telles que dérivées de la souche Calaf14, et suggèrent des modifications spécifiques d'acides aminés au niveau des séquences codantes de l'ensemble des autres souches de PEDV qui peuvent améliorer leur performance comme vaccins. De nouveaux procédés de culture sont également utilisés pour accroître le rendement reproductible des virus cultivés.
EP16704501.2A 2016-01-11 2016-01-11 Nouvelles compositions vaccinales à protection croisée pour le virus de la diarrhée épidémique porcine Withdrawn EP3402878A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115074334A (zh) * 2021-03-15 2022-09-20 夏津新希望六和农牧有限公司 猪流行性腹泻病毒株、扩增培养方法、及其制备的疫苗组合物、制备方法和应用

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108060139B (zh) * 2017-08-31 2021-07-20 浙江美保龙生物技术有限公司 一种猪流行性腹泻病毒的分离培养方法
CN107446894A (zh) * 2017-09-15 2017-12-08 天津瑞普生物技术股份有限公司 一种猪流行性腹泻病毒制备方法
KR102879825B1 (ko) * 2018-09-20 2025-11-03 베링거잉겔하임베트메디카게엠베하 변형된 pedv 스파이크 단백질
CN110894216B (zh) * 2019-11-21 2022-03-08 河北农业大学 一种猪流行性腹泻病毒抗原表位肽、单克隆抗体及应用
CN114438040A (zh) * 2020-11-04 2022-05-06 中山大学 一种分泌具有pedv中和活性抗体的单克隆细胞株及其应用
CN114931648B (zh) * 2022-06-15 2024-02-27 金宇保灵生物药品有限公司 一种猪流行性腹泻和猪传染性胃肠炎二联活疫苗耐热保护剂及其制备方法和应用
CN117286162B (zh) * 2023-04-14 2024-08-23 扬州大学 重组猪δ冠状病毒感染性克隆及其构建方法和应用

Family Cites Families (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5078996A (en) 1985-08-16 1992-01-07 Immunex Corporation Activation of macrophage tumoricidal activity by granulocyte-macrophage colony stimulating factor
US5084269A (en) 1986-11-06 1992-01-28 Kullenberg Fred W Adjuvant for dose treatment with antigens
US5057540A (en) 1987-05-29 1991-10-15 Cambridge Biotech Corporation Saponin adjuvant
US4912094B1 (en) 1988-06-29 1994-02-15 Ribi Immunochem Research Inc. Modified lipopolysaccharides and process of preparation
IL101715A (en) 1991-05-02 2005-06-19 Amgen Inc Recombinant dna-derived cholera toxin subunit analogs
WO1993013302A1 (fr) 1991-12-23 1993-07-08 Michael Zoche Moteur avec deshuileur
US5571515A (en) 1994-04-18 1996-11-05 The Wistar Institute Of Anatomy & Biology Compositions and methods for use of IL-12 as an adjuvant
US6207646B1 (en) 1994-07-15 2001-03-27 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
GB9518220D0 (en) 1995-09-06 1995-11-08 Medical Res Council Checkpoint gene
CA2269074A1 (fr) 1996-10-23 1998-04-30 The Trustees Of The University Of Pennsylvania Immunotherapie et vaccins perfectionnes
US6113918A (en) 1997-05-08 2000-09-05 Ribi Immunochem Research, Inc. Aminoalkyl glucosamine phosphate compounds and their use as adjuvants and immunoeffectors
KR100743894B1 (ko) 1998-02-27 2007-07-30 더 트러스티스 오브 더 유니버시티 오브 펜실바니아 백신, 면역치료제 및 그의 이용 방법
DK1117435T3 (da) 1998-09-30 2008-03-17 Wyeth Corp Muteret cholera-holotoxin som adjuvans
AU769539B2 (en) 1999-01-29 2004-01-29 Zoetis Services Llc Adjuvants for use in vaccines
IL159210A0 (en) 2001-06-07 2004-06-01 Wyeth Corp Mutant forms of cholera holotoxin as an adjuvant
JP2005508143A (ja) 2001-06-07 2005-03-31 ワイス・ホールデイングス・コーポレーシヨン アジュバントとしてのコレラホロトキシンの突然変異形
MXPA05002255A (es) 2002-08-26 2005-06-08 Pfizer Prod Inc Vacunas para infecciones del sistema respiratorio y reproductor en ganado.
KR100502008B1 (ko) 2003-04-29 2005-07-18 녹십자수의약품(주) 약독화된 돼지 유행성설사병 바이러스, 그를 포함하는면역원성 조성물 및 상기 바이러스를 검출하는 방법
NZ621834A (en) 2008-06-27 2015-10-30 Zoetis Services Llc Novel adjuvant compositions
CN104152578B (zh) * 2013-06-03 2016-03-30 中国农业科学院上海兽医研究所 猪流行性腹泻病毒rt-pcr鉴别诊断试剂盒及其应用
NZ757210A (en) 2013-09-19 2022-12-23 Zoetis Services Llc Oil-based adjuvants
SI3166634T1 (sl) * 2014-07-11 2021-11-30 Zoetis Services Llc Sestave novih cepiv za virus prašičje epidemične diareje

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115074334A (zh) * 2021-03-15 2022-09-20 夏津新希望六和农牧有限公司 猪流行性腹泻病毒株、扩增培养方法、及其制备的疫苗组合物、制备方法和应用
CN115074334B (zh) * 2021-03-15 2024-01-19 夏津新希望六和农牧有限公司 猪流行性腹泻病毒株、扩增培养方法、及其制备的疫苗组合物、制备方法和应用

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