WO2016138421A1 - Porcine epidemic diarrhea virus strains and immunogenic compositions therefrom - Google Patents
Porcine epidemic diarrhea virus strains and immunogenic compositions therefrom Download PDFInfo
- Publication number
- WO2016138421A1 WO2016138421A1 PCT/US2016/019846 US2016019846W WO2016138421A1 WO 2016138421 A1 WO2016138421 A1 WO 2016138421A1 US 2016019846 W US2016019846 W US 2016019846W WO 2016138421 A1 WO2016138421 A1 WO 2016138421A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- pedv
- seq
- immediately
- virus
- protein
- Prior art date
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
- A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/12—Antidiarrhoeals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
- C07K14/08—RNA viruses
- C07K14/165—Coronaviridae, e.g. avian infectious bronchitis virus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
-
- 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
Definitions
- the present invention is directed to novel immunogenic compositions that protect swine from disease caused by porcine epidemic diarrhea virus (PEDV).
- PEDV porcine epidemic diarrhea virus
- 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 belonging to the Alphacoronavirus genus of the family
- 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) and porcine deltacoronavirus (PDCoV), both of which are also members 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.
- TGEV transmissible gastroenteritis virus
- PDCoV porcine deltacoronavirus
- PEDV was not identified in North America until 2013, at which point widespread outbreaks commenced, and severe economic losses to the swine industry resulted.
- the virus appeared in multiple, widely distributed sow herds within days, and it has spread to at least 32 states. Producers can expect losses of up to 100% in naive neonatal piglets.
- Present recommendations for management of infection include implementation of strict biosecurity and/or intentional exposure of the whole herd to PEDV to accomplish immunity.
- PEDV remains as a significant threat to the global swine industry.
- PEDV generally grows poorly in culture, and there is a need to identify both particular strains 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 PEDV strains worldwide.
- the present invention encompasses immunogenic compositions comprising variant PEDV strains passaged from prototype strain and from indel strains.
- the U.S. PEDV S-INDEL-variant strains are genetically different from the U.S. PEDV prototype- like strains, including characteristic insertions and deletions in the spike protein SI domain region, particularly the first 1170 bases thereof.
- the insertions and deletions include 3 deletions (a 1-nt deletion at position 167, an 11-nt deletion at position 176, and a 3-nt deletion at position 416), a 6-nt insertion between positions 474 and 475, and several other mutations mainly located in the first 1,170 nucleotides of the SI region.
- PEDV S- INDEL-variant strains are less virulent than the U.S. PEDV prototype strains and may be used, in one embodiment or whole virus, as attenuated live vaccines. Pigs infected with live attenuated serially passaged S-INDEL-variant strains of the invention do not cause disease when administered to piglets and, importantly, exhibited cross protection against challenge with either the virulent prototype PEDV strains or the S-INDEL-variant strains.
- the invention comprises an immunogenic composition, suitable to be used as a vaccine, which comprises a S-INDEL-variant PEDV strain of the invention, preferably live and attenuated, or an immunogenic fragment thereof, one or more adjuvants, and optionally one or more excipients, in an amount effective to elicit production of neutralizing antibodies in swine.
- the adjuvant preferably provides an oil-in-water emulsion with additional components.
- the immunogenic compositions of the invention protect swine from infection by 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 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 colostrum and 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 U.S. PEDV S-INDEL-variant strain of the invention provides cross reactivity/protection against other PEDV strains and it is expected that protection will be conferred against European strains generally, and more particularly against the recently emerging isolates in Europe that had very high genetic similarity (>99%) to the U.S. S-INDEL-variant strains at the whole genome sequence
- the vaccinating compositions of the present invention are useful to protect swine from disease or challenge by European strains of PEDV generally, including recent isolates.
- the present invention includes novel nucleotide and amino acid sequences of PEDV, including novel genotypes thereof, all of which are useful in the preparation of vaccines for treating and preventing diseases in swine and other animals.
- Vaccines provided according to the practice of the invention are effective against multiple swine PEDV genotypes and isolates. Diagnostic and therapeutic polyclonal and monoclonal antibodies are also a feature of the present invention, as are infectious clones useful in the propagation of the virus and in the preparation of vaccines.
- vaccines that comprise, as antigen, a whole virus (live or attenuated) or an single antigenic protein of a PEDV open reading frame, most particularly from the first 2.2 kb of the spike gene, more particularly the SI domain, and also fragments of the full length sequence encoding the PEDV proteins.
- the invention also provides the full length genomic sequences of PEDV strains at different passages in cell culture that can replicate efficiently in host animals and tissue culture.
- the present invention provides a method of treating or preventing a disease or disorder in an animal caused by infection with PEDV, including disease states that are directly caused by PEDV, and disease states contributed to or potentiated by PEDV.
- Disease states in swine that may be potentiated by PEDV, and which may also be treated or prevented according to the practice of the invention, include those caused by or associated with Transmissible Gastroenteritis virus, PHEV, and PRCV.
- the present invention also includes the option to administer a combination vaccine, that is, a bivalent or multivalent combination of antigens, which may include live, modified live, or inactivated antigens against the non-PEDV pathogen, with appropriate choice of adjuvant.
- a combination vaccine that is, a bivalent or multivalent combination of antigens, which may include live, modified live, or inactivated antigens against the non-PEDV pathogen, with appropriate choice of adjuvant.
- the present invention also provides a diagnostic kit for differentiating between porcine animals vaccinated with the above described PEDV vaccines and porcine animals infected with field strains of PEDV.
- Representative embodiments of the invention include an isolated polynucleotide sequence that includes a genomic polynucleotide which encodes variant PEDV proteins which are attenuated and may be used as an immunogenic composition.
- This can include whole genome sequences selected from the group consisting of:
- the invention further provides RNA and DNA molecules, their complements, fragments and vectors and plasmids for the expression of any such RNA or DNA polynucleotides, and for PEDV virus that is expressed from such nucleotide sequences, wherein said virus is live, or fully or partially attenuated.
- the invention also provides a vaccine that comprises a polynucleotide sequence as aforementioned, and corresponding nucleotide sequences that may function as infectious clones.
- the invention also includes nucleic acid ORFs which encode variant proteins from PEDV as reflected in Table 1 herewith. Table 1 variations are described with reference to PEDV USA/IL20697/2014 passage 5.
- the invention further provides nucleic acid sequences and resultant protein variants that have amino acid substitutions and which reduce virulence, cause attenuation and allow the compositions to be used safely as immunogenic compositions and as vaccines.
- polypeptide vaccines wherein the antigen is defined by (a) the spike protein; or (b) an amino acid sequence that is at least 90 percent identical thereto; or (c) an arginine rich region thereof.
- the invention includes vaccine compositions comprising a a live attenuated variant strain porcine epidemic diarrhea virus (PEDV), and
- PEDV live attenuated variant strain porcine epidemic diarrhea virus
- composition capable of protecting swine from challenge by both variant and prototype strains of PEDV and preventing or treating one or more of symptoms associated with PEDV infection, 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, wherein said strain encodes proteins with a substitution of the following amino acids and their conservative variants and proteins with the specific substitutions and 99% homology to the remainder of the sequences:
- polyprotein la/lb (SEQ ID NO: 46): P at position 551 (P immediately after DEDAT);
- spike protein(SEQ ID NO:47) P at position 1009 (P immediately after IGNIT), H at position 973 (H immediately after ALPFS)., L at position 48 (L immediately after APAVV), V at position 345 (A immediately after TNLSF);
- ORF 3 deletion of I at position 144 (I immediately after
- nucleocapsid protein H at position 27 (H immediately after LRVTN).
- Spike protein (SEQ ID NO:47): P at position 1009 (P immediately after
- IGNIT IGNIT
- H at position 973 H immediately after ALPFS.
- L at position 48 L immediately after APAVV
- ORF 3 SEQ IDNO: 48: deletion of I at position 144 (I immediately after YDGKS), deletion of 174-189 (immediately after LYLAI), LTANPL at position 138-143 (immediately after NGKAA), and,
- nucleocapsid protein H at position 27 (H immediately after LRVTN).
- spike protein SEQ ID NO:47: K at position 633 (K immediately after TPKPL); ORF 3 (SEQ IDNO:48): deletion of L at position 189 (L immediately after TANPL).
- ORF 3 (SEQ IDNO:48): deletion of L at position 189 (L immediately after TANPL).
- ORF 3 (SEQ IDNO:48): deletion of L at position 189 (L immediately after TANPL).
- ORF 3 (SEQ ID NO:48): deletion of 138-144(immediately after NGKAA) deletion of L at 189 (L immediately after TANPL),
- Nucleocapsid (SEQ ID NO:51) H at position 27 (H immediately after LRVTN).
- polyprotein la/lb SEQ ID NO:46: M at position 1591 (M immediately after VVKVS), S at position 5087 (S immediately after YLFST), S at position 6138 (S immediately after WQTFS); spike protein(SEQ ID NO:47): R at position 884 (R immediately after VYDPA), A at position 959 (A immediately after LIGGM), D at position 1225 (D immediately after IESLV) ;
- ORF 3 (SEQ IDNO:48): deletion of Y at position 8 (Y immediately after LGLFO), deletion of 138-144 (immediately after NGKAA), and deletion of 174 to 189 189
- polyprotein la/lb SEQ ID NO:46: M at position 1591 (M immediately after VVKVS), S at position 5087 (S immediately after YLFST), S at position 6138 (S immediately after WQTFS);
- spike protein (SEQ ID NO:47): R at position 884 (immediately after VYDPA), A at position 959 (immediately after LIGGM), H at position 973 (H immediately after ALPFS), D at position 1225 (D immediately after IESLV);
- ORF 3 (SEQ IDNO:48): deletion of Y at position 8 (Y immediately after LGLFO), deletion of 138-144 (immediately after NGKAA), and deletion of 174 to 189 (immediately after LYLAI),
- spike protein (SEQ ID NO:47): L at position 48 (immediately after APAVV), V at position 345 (immediately after TNLSF); T at position 1272 (T immediately after DVFNA) envelope protein (SEQ IDNO:49) S at position 62 (S immediately after YRVYK); nucleocapsid protein (SEQ I D NO:51): H at position 27 (H immediately after LRVTN).
- the invention further includes passages and amino acid substitution from passaging the prototype virus, deposited at Genbank® Accesssion KF650371 Passage 3. Variations in amino acids are reflected the following:
- polyprotein la/lb (SEQ IDNO:52): V at 814, A at 1076, F at 1564, 1 at 1896, H at 2310 Y at 2600, F at 3247, V at 3473, or R at 3522;
- spike protein SEQ IDNO:54: N at 257, 1 at 326, F at 375, Y at 491, R at 881, R at 888 F at 1277, T at 1339, or L at 1358;
- ORF 3 (SEQ ID NO: 55) stop at 39 or later;
- Envelope protein (SEQ ID NO:56) position I at 69;
- nucleocapsid protein SEQ ID NO: 58 position L at 141, Q at 418, N at 424, or I at 439.
- any PEDV passages described herein wherein said modifications include introducing mutations of another passage (Prototype, F DEL lineage 1, or INDEL lineage 2 and combinations thereof) described herein using known methods according to one of skill in the art, for example, homologous recombination.
- GenBank® is the recognized United States-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 DataBank of Japan (DDBJ), see Nucleic Acids Research, January 2013,v 41(D1) D36-42 for discussion.
- EMBL European Molecular Biology Laboratory
- DDBJ DNA DataBank of Japan
- Figure 1 shows a phylogenetic analysis of the PEDV SI portion nucleotide sequences from clinical cases in January 2014 when our group first identified U.S. PEDV S-INDEL-variant strain.
- the tree was constructed using the distance-based neighbor- joining method of the software MEGA 5.2. Bootstrap analysis was carried out on 1,000 replicate data sets, and values are indicated adjacent to the branching points.
- the newly identified U.S. PEDV S-INDEL-variant strains (ISU cases 1-5) are indicated with solid circles that had sequences different from PEDV identified in US in 2013 (U.S. PEDV prototype strain)which are indicated with triangles.
- Figure 2 shows phylogenetic trees based on the SI portion sequences and the whole genome sequences. See the attachment.
- the US prototype-like strains have been detected in South Korea, Canada and Mexico; the US variant-INDEL-like strains have been detected in South Korea, Mexico and Germany.
- Figure 3A shows the SI sequence of the strain of the invention (SEQ ID NO: 1).
- Figure 3B shows the whole genome sequence of the S-INDEL-variant PEDV cell culture isolate 2014020697-P5 passage 5, lineage 1 (SEQ ID NO:8).
- Figure 4 is an alignment comparing the spike gene sequences of the PEDV US S- INDEL-variant isolate 2014020697-P5 passage 5, linage 1 (SEQ ID NO:2) to the PEDV US prototype isolates 2013019338-P3 passage 3 (SEQ ID NO:3), 2013022038-P3 passage 3 (SEQ ID NO:4), 2013035140-P3 passage 3 (SEQ ID NO: 5), 2013049379-P3 passage 3 (SEQ ID NO:6), and 2013049469-P1 passage 1 (SEQ ID NO:7), identical bases are indicated with dots.
- Figure 5 is a comparison of the whole genome of PEDV US variant isolate
- 2014020697-P5 passage 5 lineage 1 (SEQ ID NO:8) to the PEDV US prototype isolates 2013019338-P3 passage 3 (SEQ ID NO: 9), 2013022038-P3 passage 3 (SEQ ID NO: 10), 2013035140-P3 passage 3 (SEQ ID NO: 11), 2013049379-P3 passage 3 (SEQ ID NO: 12), and 2013049469-P1 passage 1 (SEQ ID NO: 13), identical bases are indicated with dots.
- Figures 6A-6D are the phylogenetic analysis of the full-length genome and SI portion nucleotide sequences.
- Three U.S. prototype PEDV isolates obtained in this study USA/NC35140/2013, USA/IA49379/2013 and USA/NC49469/2013) and two isolates previously isolated but evaluated in this study (U.S. PEDV prototype isolate
- USA/F 19338/2013 and U.S. S-INDEL-variant isolate USA/IL20697 are indicated with bullet points or triangle.
- Forty-five PEDV sequences selected from GenBank® were also included for analysis.
- the trees were constructed using the distance-based neighbor-joining method (6A and 6C) and the maximum likelihood method (Fig. 6B and Fig. 6D) of the software MEGA6.
- the U.S. prototype-like PEDVs are shown in blue color fonts with clade 1 and clade 2 indicated.
- the U.S. S-INDEL-variant-like PEDVs are shown in red color fonts.
- Figure 7A-7D show the clinical assessment and virus shedding of 5-day-old pigs inoculated with U.S. PEDV prototype and S-INDEL-variant isolates.
- Figure 7A shows the average diarrhea scores over the course of 7 days post inoculation (DPI).
- Figure 7B shows the average daily weight gain (ADG).
- ADG average daily weight gain
- Statistical analyses on ADG were performed among groups from (-1) to 3 DPI and from (-1) to 7 DPI, respectively.
- Virus shedding in rectal swabs are shown in Figure 7C and in sera in Figure 7D of inoculated pigs was determined by a quantitative PEDV N gene-based real-time RT-PCR.
- virus titers LoglOGenomic copies/ml
- virus titers were mean virus titers of all available pigs (both PCR- positive and negative pigs) in each group. Standard error bars are shown in each figure.
- viremia levels among the groups were statistically analyzed at 3 DPI and 7 DPI, respectively. Labels without the same letters indicate significant differences, for example A and B have significant difference but A and AB have no significant differences.
- Figure 8A-8B show the examination of gross lesions of inoculated pigs at 3 DPI and 7 DPI necropsies. Average scores of contents of small intestine, cecum and colon are shown in Figure 8 A. Average scores of lesions of small intestine, cecum and colon are shown in Figure 8B. The scoring criteria were described in Materials and Methods
- Example 2 Statistical analyses were performed on various inoculated groups, but each time on one tissue type and on one time point. Labels without the same letters indicate significant differences.
- Figure 9A-9T show representative microscopic lesions and IHC staining of inoculated pigs necropsied at 3 DPI. Hematoxylin and eosin-stained tissue sections of small intestine (ileum) from different inoculation group (Fig. 9A-9E).
- FIG. 9F-9J Immunohistochemistry-stained tissue sections of ileum (Fig. 9F-9J), cecum (Fig. 9K-90), and colon (Fig. 9P-9T) from different inoculation group. All images are in lOOx magnification.
- Figure 10A-10D shows the mean villus height ( ⁇ ), crypt depth ( ⁇ ), villus/crypt ratio, and immunohistochemistry scores of pigs necropsied at 3 DPI.
- Statistical analyses were performed on various inoculated groups, but each time on one tissue type. Labels without the same letters indicate significant differences.
- Figure 1 1 A-l ID shows the mean villus height ( ⁇ ), crypt depth ( ⁇ ), villus/crypt ratio, and immunohistochemistry scores of pigs necropsied at 7 DPI. Statistical analyses were performed on various inoculated groups, but each time on one tissue type. Labels without the same letters indicate significant differences.
- FIG 12 is a schematic diagrams of PEDV genome organization and putative functions of viral proteins.
- the PEDV entire genome organization is depicted (top).
- the 5' leader, ORFs la and lb encoding replicase polyproteins, spike (S), ORF3, envelope (E), membrane (M) and nucleocapsid (N) genes are shown, with the ribosomal frameshift site indicated.
- Predicted cleavage products (nspl -nspl 6) of the replicase polyproteins and putative functional domains are depicted (bottom). Nspl may possibly suppress the innate immune response and host protein synthesis. The exact function of nsp2 remains unclear.
- the PL2pro is also an interferon antagonist.
- Nsp3, nsp4 and nsp6 contain transmembrane domains (TM) and function as membrane anchor proteins.
- Nsp7 and nsp8 form a hexadecameric complex with a central channel for binding to the double-stranded RNA and initiating RNA synthesis.
- Nsp9 is a single-stranded RNA-binding protein (RBP).
- NsplO is a co-factor (activator) of nspl4 and nspl6.
- the nspl4 has 3'-5' exonuclease (ExoN) at the N-terminal and 7-methyl transferase (7MT) at the C-terminal.
- the nspl 6 acts as the 2'-0- methyltransferase (2'-0-MT).
- the interactions between nsplO, nspl4 and nspl6 plays a crucial role in replication fidelity and methylation of the virus.
- the nspl 1 function is not fully understood yet.
- Nspl 2 is the viral RNA-dependent RNA polymerase (RdRP).
- RdRP viral RNA-dependent RNA polymerase
- Nspl 3 harbors a zinc-binding domain (ZBD) and viral helicase.
- ZBD zinc-binding domain
- Nspl 3 also has NTPase and RNA 5' triphosphatase activity.
- Nspl 5 contains a motif known as nidoviral uridylate-specific endoribonuclease (NendoU).
- the S protein functions as the virus attachment protein interacting with the cell receptor. Additionally, the S protein is postulated to harbor neutralization epitopes and is also associated with viral virulence/attenuation.
- the M protein and E protein play a pivotal role in viral assembly.
- the N protein binds to the viral genome RNA and packs into the nucleocapsid.
- the N protein has also been shown to antagonize interferon- ⁇ production.
- the accessory protein encoded by ORF3 may be associated with cell culture adaptation and may also have an influence on cell cycle and subcellular structure.
- Figure 13 is an image showing IFA antibody testing of antisera against the U.S. PEDV prototype and S-INDEL-variant strains. The average IFA antibody titers are shown at the top and the number of IFA antibody positive samples is shown at the bottom.
- Pro antisera antisera collected from the U.S. PEDV prototype strain-inoculated pigs
- Var antisera antisera collected from the U.S. PEDV S-INDEL-variant strain-inoculated pigs
- Neg antisera antisera collected from negative control pigs
- Pro IFA the U.S. PEDV prototype strain-based IFA
- Var IFA the U.S.
- PEDV S-INDEL-variant strain-based IFA Figure 14A-14C show testing of antisera against the U.S. PEDV prototype and S- INDEL-variant strains by ProWV ELISA (Fig. 14 A), ProSl ELISA (Fig. 14B) and VarSl ELISA (Fig. 14C).
- the solid black line indicates the S/P ratio above which the sample was positive; the dot black line indicates the S/P ratio below which the sample was negative; samples with S/P ratios between the solid and dot black line were suspect.
- ProWV ELISA the U.S. PEDV prototype strain whole virus-based ELISA;
- ProSl ELISA the U.S. PEDV prototype strain SI -based ELISA; VarSl ELISA: the U.S. PEDV S- INDEL-variant strain S 1 -based ELISA.
- Figure 15 shows virus neutralization antibody testing of antisera against the U.S. PEDV prototype and S-INDEL-variant strains. The average VN antibody titers are shown at the top and the number of VN antibody positive samples is shown at the bottom.
- Pro VN the U.S. PEDV prototype strain-based VN
- Var VN the U.S. PEDV S-INDEL-variant strain-based VN.
- Figure 16 show a table comparing the nucleotide and amino acid changes of U.S. prototype PEDV isolate USA/IN19338/2013 during serial passages in cell culture.
- Figure 17 shows the growth curve of the U.S. PEDV prototype isolate
- Figure 18 is a graph showing virus shedding in rectal swabs of various inoculation groups during 0-7 days post inoculation (DPI). Different letters indicate significant differences.
- Figure 19 shows small intestine microscopic lesion severity scores in various groups of piglets inoculated with the PEDV USA/IN 19338/2013 P7, P25, P50, P75 and PI 00 at 3 days post inoculation. Labels without the same letters indicate significant differences, for example A and B have significant differences but A and AB have no significant differences.
- Figure 20 shows the average villus-height-to-crypt-depth ratios of small intestines in various groups of piglets inoculated with the PEDV USA/IN 19338/2013 P7, P25, P50, P75 and PI 00 at 3 days post inoculation. Different letters indicate significant differences.
- Figure 21 shows the average immunohistochemistry scores in small intestines, ceca and colons in various groups of piglets inoculated with the PEDV USA/IN19338/2013 P7, P25, P50, P75 and PI 00 at 3 days post inoculation.
- Figure 22A-22C shows sequences according to exemplary embodiments of the invention.
- Figure 22A shows the sequence of 2014020697-P8R1 passage 8, lineage 2 ORFla/lb polyprotein (SEQ ID NO: 17); 2014020697-P8R1 passage 8, lineage 2 spike protein (SEQ ID NO: 18), 2014020697-P8R1 passage 8, lineage 2 ORF 3 protein
- FIG. 19 shows the 2014020697-P18R1 passage 18, lineage 2 clone G8b
- ORFla/lb polyprotein (SEQ ID NO: 23), 2014020697-P18R1 passage 18, lineage 2 clone G8b spike protein (SEQ ID NO: 24), 2014020697-P18R1 passage 18, lineage 2 clone G8b ORF3 protein (truncated) (SEQ ID NO: 25), 2014020697-P18R1 passage 18, lineage 2 clone G8b envelope protein (SEQ ID NO: 26), 2014020697-P18R1 passage 18, lineage 2 clone G8b membrane protein (SEQ ID NO: 27) and 2014020697-P18R1 passage 18, lineage 2 clone G8b nucleocapsid protein (SEQ ID NO: 28).
- Figure 22C is 2014020697- P18R1 passage 18, lineage 2 clone F6a ORFla/lb polyprotein (SEQ ID NO: 29);
- Figure 23A shows the genomic nucleotide sequence for 2014020697-P8R1 passage 8, lineage 2 (SEQ ID NO:35)
- Figure 23B shows the nucleotide sequence for 2014020697-P18R1 passage 18, lineage 2 clone G8b complete genome (SEQ ID NO: 36)
- Figure 23C shows 2014020697-P18R1 passage 18, lineage 2 clone F6a complete genome nucleotide sequence (SEQ ID NO: 37).
- Figure 24 is a chart showing the INDEL sequence comparison for various passages.
- Figure 25 is the genomic nucleotide sequence of 2014020697-P45 passage 45, lineage 1 (SEQ ID NO: 39).
- Figure 26 is a comparison of the genomic nucleotide sequences of 2014020697-P5 passages 5, lineage 1 (P5) (SEQ ID NO:8), 2014020697-P7R1 passage 7, lineage 2 (SEQ ID NO: 15), 2014020697-P8R1 passage 8, lineage 2 (SEQ ID NO:35), 2014020697-P18R1 passage 18, lineage 2 clone G8b (SEQ ID NO: 36), 2014020697-P18R1 passage 18, lineage 2 clone F6a (SEQ ID NO:37) and 2014020697-P45 passage 45, lineage 1 (SEQ ID NO:39).
- Figure 27 shows sequences according to exemplary embodiments of the invention.
- polyprotein SEQ ID NO: 40
- 2014020697-P45 passage 45 lineage 1 spike protein
- 2014020697-P45 passage 45 lineage 1 ORF3 protein (SEQ ID NO:42);
- 2014020697-P45 passage 45 lineage 1 envelope protein (SEQ ID NO:43); 2014020697- P45 passage 45, lineage 1 membrane protein (SEQ ID NO:44); and 2014020697-P45 passage 45, lineage 1 nucleocapsid protein (SEQ ID NO:45) are shown.
- Figure 28A-28B shows fecal virus shedding titers.
- Fig. 28A shows fecal virus shedding titers on each time point of each group with upper standard error bars shown.
- Fig. 28B shows statistic analysis of overall virus shedding titer in rectal swabs on D0-D28 and D28-D56.
- D0-D28 the N/N, N/V and N/P groups received the same inoculum and were analyzed as the same treatment, similarly for the V/V and V/P groups, and P/V and P/P groups. Different letters indicate statistically significant difference in virus shedding amount between groups.
- Figure 29A-29B Macro-pathological scores.
- Fig. 29A is the group mean macro- pathological scores at D4 (4 days post the 1 st inoculation) with the standard error bars shown.
- Fig. 29B is the group mean macro-pathological scores at D34 (6 days post the challenge at D28) with the standard error bars shown.
- Small intestine, cecum, and colon content scores as well as the gross lesion scores of small intestine, cecum and colon organs were shown. Different letters indicate statistically significant differences observed with the same parameter between groups. Yellow highlighted are statistics of V-strain challenged pigs, and green highlighted are statistics of P-strain challenged pigs.
- Figure 30A-30B shows the histopathology measurement at D4.
- Fig. 30A shows the group mean of villus height/crypt depth ratios of distal jejunum and ileum with the standard error bars shown.
- Fig. 30B shows the group mean of PEDV IHC scores in distal jejunum, ileum, cecum, and colon with the standard error bars shown. Different letters indicate statistically significant differences observed in the same tissue between groups.
- Figure 31 A- 3 IB shows the histopathology measurement at D34.
- Fig. 31 A show the group mean of villus height/crypt depth ratios of distal jejunum and ileum with the standard error bars shown.
- Fig. 3 IB shows the group mean of PEDV IHC scores in distal jejunum, ileum, cecum, and colon with the standard error bars shown. Different letters indicate statistically significant differences observed in the same tissue between groups. Yellow highlighted are statistics of V-strain challenged pigs, and green highlighted are statistics of P-strain challenged pigs.
- Figure 32A-232B shows the serum IFA antibody titers against either P-strain virus or V-strain virus.
- Fig. 32A shows the group mean of serum IFA titers against P-strain virus with the standard error bars shown.
- Fig. 32B shows the group mean of serum IFA titers against V-strain virus with the standard error bars shown.
- Figure 33 A-33B shows the serum VN antibody titers against either P-strain virus or V-strain virus.
- Fig. 33 A shows the group mean of serum VN titers against P-strain virus with the standard error bars shown.
- Fig. 33B shows the group mean of serum VN titers against V-strain virus with the standard error bars shown.
- adjuvant refers to a compound that enhances the effectiveness of the vaccine, and may be added to the formulation that includes the immunizing agent.
- Adjuvants provide enhanced immune response even after administration of only a single dose of the vaccine.
- Adjuvants may include, for example, muramyl dipeptides, pyridine, aluminum hydroxide, dimethyldioctadecyl ammonium bromide (DDA), oils, oil-in-water emulsions, saponins, cytokines, and other substances known in the art. Examples of suitable adjuvants are described in U.S. Patent Application Publication No.
- Adjuvanted refers to a composition that incorporates or is combined with an adjuvant.
- Antibodies refers to polyclonal and monoclonal antibodies, chimeric, and single chain antibodies, as well as Fab fragments, including the products of a Fab or other immunoglobulin expression library.
- immunoglobulin expression library the term, “immunologically specific” refers to antibodies that bind to one or more epitopes of a protein of interest, but which do not substantially recognize and bind other molecules in a sample containing a mixed population of antigenic biological molecules.
- an "attenuated" PEDV as used herein refers to a PEDV which is capable of infecting and/or replicating in a susceptible host, but is non-pathogenic or less-pathogenic to the susceptible host.
- the attenuated virus may cause no
- the clinical manifestations of PEDV infection can include, without limitation, clinical diarrhea, vomiting, lethargy, loss of condition and dehydration.
- An “epitope” is an antigenic determinant that is immunologically active in the sense that once administered to the host, it is able to evoke an immune response of the humoral (B cells) and/or cellular type (T cells). These are particular chemical groups or peptide sequences on a molecule that are antigenic.
- An antibody specifically binds a particular antigenic epitope on a polypeptide. In the animal most antigens will present several or even many antigenic determinants simultaneously. Such a polypeptide may also be qualified as an immunogenic polypeptide and the epitope may be identified as described further.
- immunogenic fragment refers to a polypeptide or a fragment of a polypeptide, or a nucleotide sequence encoding the same which comprises an allele-specific motif, an epitope or other sequence such that the polypeptide or the fragment will bind an MHC molecule and induce a cytotoxic T lymphocyte ("CTL") response, and/or a B cell response (for example, antibody production), and/or T-helper lymphocyte response, and/or a delayed type hypersensitivity (DTH) response against the antigen from which the immunogenic polypeptide or the immunogenic fragment is derived.
- CTL cytotoxic T lymphocyte
- B cell response for example, antibody production
- T-helper lymphocyte response for example, T-helper lymphocyte response
- DTH delayed type hypersensitivity
- an immunoprotective response is meant a (humoral and/or cellular) immune response that reduces or eliminates one or more of the symptoms of disease, i.e. clinical signs, lesions, bacterial excretion and bacterial replication in tissues in the infected subject compared to a healthy control.
- said reduction in symptoms is statistically significant when compared to a control.
- infectious DNA molecule is a DNA molecule that encodes the necessary elements for viral replication, transcription, and translation into a functional virion in a suitable host cell.
- isolated is used to indicate that a cell, peptide or nucleic acid is separated from its native environment. Isolated peptides and nucleic acids may be substantially pure, i.e. essentially free of other substances with which they may bound in nature.
- 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 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 75% nucleotide sequence identity, even more preferably at least about 80%, 85%, 90% and 95% nucleotide sequence identity. Since the genetic code is degenerate, a homologous nucleotide sequence can include any number of "silent" base changes, i.e. nucleotide substitutions that nonetheless encode the same amino acid.
- a homologous nucleotide sequence can further contain non-silent mutations, i.e. base substitutions, deletions, or additions resulting in amino acid differences in the encoded polyaminoacid, so long as the sequence remains at least about 70% identical to the polyaminoacid encoded by the first nucleotide sequence or otherwise is useful for practicing the present invention.
- non-silent mutations i.e. base substitutions, deletions, or additions resulting in amino acid differences in the encoded polyaminoacid, so long as the sequence remains at least about 70% identical to the polyaminoacid encoded by the first nucleotide sequence or otherwise is useful for practicing the present invention.
- certain conservative amino acid i.e. base substitutions, deletions, or additions resulting in amino acid differences in the encoded polyaminoacid
- 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
- 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: l (or any other particular polynucleotide sequence) if it hybridizes to the complement of SEQ ID NO: l under moderately stringent conditions, e.g., hybridization to filter-bound DNA in 0.5 M NaHP0 4 , 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C, and washing in 0.2 X SSC/0.1% SDS at 42° C. (see Ausubel et al editors, Protocols in
- hybridization conditions can be empirically determined or precisely calculated based on the length and percentage of guanosine/cytosine (GC) base pairing of the probe.
- the hybridization conditions can be calculated as described in Sambrook, et al., (Eds.), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y. (1989), pp. 9.47 to 9.51.
- 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 NaHP0 4 , 7% SDS, 1 mM EDTA at 65° C, and washing in 0.1 X SSC/0.1% SDS at 68° C, as is known in the art.
- 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.
- the vaccine-capable attenuated PEDV viruses of the present invention contain substantial deletions of ORF3 protein, resulting from attenuating mutations in the ORF3 nucleotide sequence, that cause substantial internal deletions and/or most typically the appearance of truncated translation resultant from frameshifting and the appearance of stop codons. It should therefore be noted that within the practice of the present invention, alignment and percent identity calculations and stated identity results (whether or nucleotide or amino acid sequences) can be calculated with or without reference to deleted ORF3 sequences.
- mammals include any warm-blooded vertebrates of the Mammalia class, including humans.
- a “pharmaceutically acceptable carrier” means any conventional pharmaceutically acceptable carrier, vehicle, or excipient that is used in the art for production and administration of vaccines.
- Pharmaceutically acceptable carriers are typically non-toxic, inert, solid or liquid carriers.
- porcine and “swine” are used interchangeably herein and refer to any animal that is a member of the family Suidae such as, for example, a pig.
- a "susceptible" host as used herein refers to a cell or an animal that can be infected by PEDV. When introduced to a susceptible animal, an attenuated PEDV may also induce an immunological response against the PEDV or its antigen, and thereby render the animal immunity against PEDV infection.
- vaccine refers to an antigenic preparation used to produce immunity to a disease, in order to prevent or ameliorate the effects of infection.
- Vaccines are typically prepared using a combination of an immunologically effective amount of an immunogen together with an adjuvant effective for enhancing the immune response of the vaccinated subject against the immunogen.
- Vaccine formulations will contain a "therapeutically effective amount" of the active ingredient, that is, an amount capable of eliciting an induction of an immunoprotective response in a subject to which the composition is administered.
- a "therapeutically effective amount” would preferably be an amount that enhances resistance of the vaccinated subject to new infection and/or reduces the clinical severity of the disease.
- Vaccines can be administered prior to infection, as a preventative measure against PEDV.
- vaccines can be administered after the subject already has contracted a disease.
- Vaccines given after exposure to PEDV may be able to attenuate the disease, triggering a superior immune response than the natural infection itself.
- the invention also relates to an immunogenic composition, suitable to be used as a vaccine, which comprises a variant PEDV strain according to the invention.
- the immunogenic compositions according to the invention elicit a specific humoral immune response toward the PEDV comprising neutralizing antibodies.
- the preferred immunogenic compositions based upon the variant strains disclosed herein can provide live, attenuated viruses which exhibit high immunogenicity while at the same time not producing dangerous pathogenic or lethal effects.
- the immunogenic compositions of this invention are not, however, restricted to any particular type or method of preparation. These include, but are not limited to, infectious DNA vaccines (i.e., using plasmids, vectors or other conventional carriers to directly inject DNA into pigs), live vaccines, modified live vaccines, inactivated vaccines, subunit vaccines, attenuated vaccines, genetically engineered vaccines, etc. These vaccines are prepared by standard methods known in the art.
- the present invention preferably includes vaccine compositions comprising a live, attenuated variant PEDV of the invention and a pharmaceutically acceptable carrier.
- live, attenuated PEDV of the invention encompasses any live, attenuated PEDV strain that includes one or more of the variations described herein.
- the pharmaceutically acceptable carrier can be, e.g., water, a stabilizer, a preservative, culture medium, or a buffer.
- Vaccine formulations comprising the attenuated PEDV of the invention can be prepared in the form of a suspension or in a lyophilized form or, alternatively, in a frozen form. If frozen, glycerol or other similar agents may be added to enhance stability when frozen.
- live attenuated vaccines in general, include the presentation of all the relevant immunogenic determinants of an infectious agent in its natural form to the host's immune system, and the need for relatively small amounts of the immunizing agent due to the ability of the agent to multiply in the vaccinated host.
- Attenuation of the virus for a live vaccine may be accomplished by known procedures, including preferably by serial passaging.
- the following references provide various general methods for attenuation of coronaviruses, and are suitable for attenuation or further attenuation of any of the strains useful in the practice of the present invention: B. Neuman et al., Journal of Virology, vol. 79, No. 15, pp. 9665-9676, 2005; J. Netland et al., Virology, v 399(1), pp.
- Additional genetically engineered vaccines which are desirable in the present invention, are produced by techniques known in the art. Such techniques involve, but are not limited to, further manipulation of recombinant DNA, modification of or substitutions to the amino acid sequences of the recombinant proteins and the like.
- Genetically engineered vaccines based on recombinant DNA technology are made, for instance, by identifying alternative portions of the viral gene encoding proteins responsible for inducing a stronger immune or protective response in pigs (e.g., proteins derived from M, GP2, GP3, GP4, or GP5, etc.).
- proteins derived from M, GP2, GP3, GP4, or GP5, etc. e.g., proteins derived from M, GP2, GP3, GP4, or GP5, etc.
- Various subtypes or isolates of the viral protein genes can be subjected to the DNA-shuffling method.
- the resulting heterogeneous chimeric viral proteins can be used broad protecting subunit vaccines.
- such chimeric viral genes or immuno-dominant fragments can be cloned into standard protein expression vectors, such as the baculovirus vector, and used to infect appropriate host cells (see, for example, O'Reilly et al., "Baculovirus Expression Vectors: A Lab Manual,” Freeman & Co., 1992).
- the host cells are cultured, thus expressing the desired vaccine proteins, which can be purified to the desired extent and formulated into a suitable vaccine product.
- clones retain any undesirable natural abilities of causing disease, it is also possible to pinpoint the nucleotide sequences in the viral genome responsible for any residual virulence, and genetically engineer the virus avirulent through, for example, site- directed mutagenesis.
- Site-directed mutagenesis is able to add, delete or change one or more nucleotides (see, for instance, Zoller et al., DNA 3 :479-488, 1984).
- oligonucleotide is synthesized containing the desired mutation and annealed to a portion of single stranded viral DNA.
- the hybrid molecule which results from that procedure, is employed to transform bacteria.
- double-stranded DNA which is isolated containing the appropriate mutation, is used to produce full-length DNA by ligation to a restriction fragment of the latter that is subsequently transfected into a suitable cell culture. Ligation of the genome into the suitable vector for transfer may be accomplished through any standard technique known to those of ordinary skill in the art.
- Transfection of the vector into host cells for the production of viral progeny may be done using any of the conventional methods such as calcium-phosphate or DEAE-dextran mediated transfection, electroporation, protoplast fusion and other well-known techniques (e.g., Sambrook et al., "Molecular Cloning: A Laboratory Manual," Cold Spring Harbor Laboratory Press, 1989).
- the cloned virus then exhibits the desired mutation.
- two oligonucleotides can be synthesized which contain the appropriate mutation. These may be annealed to form double-stranded DNA that can be inserted in the viral DNA to produce full-length DNA.
- An immunologically effective amount of the vaccines of the present invention is administered to a pig in need of protection against viral infection.
- the immunologically effective amount or the immunogenic amount that inoculates the pig can be easily determined or readily titrated by routine testing.
- An effective amount is one in which a sufficient immunological response to the vaccine is attained to protect the pig exposed to the PEDV virus.
- the pig is protected to an extent in which one to all of the adverse physiological symptoms or effects of the viral disease are significantly reduced, ameliorated or totally prevented.
- Vaccines of the present invention can be formulated following accepted convention to include acceptable carriers for animals, such as standard buffers, stabilizers, diluents, preservatives, and/or solubilizers, and can also be formulated to facilitate sustained release.
- Diluents include water, saline, dextrose, ethanol, glycerol, and the like.
- Additives for isotonicity include sodium chloride, dextrose, mannitol, sorbitol, and lactose, among others.
- Stabilizers include albumin, among others.
- Other suitable vaccine vehicles and additives, including those that are particularly useful in formulating modified live vaccines, are known or will be apparent to those skilled in the art. See, e.g., Remington's
- Vaccines of the present invention may further comprise one or more additional immunomodulatory components such as, e.g., an adjuvant or cytokine, among others.
- adjuvants that can be used in the vaccine of the present invention include the RIBI adjuvant system (Ribi Inc., Hamilton, Mont.), alum, mineral gels such as aluminum hydroxide gel, oil-in-water emulsions, water-in-oil emulsions such as, e.g., Freund's complete and incomplete adjuvants, Block copolymer (CytRx, Atlanta Ga.), QS- 21 (Cambridge Biotech Inc., Cambridge Mass.), SAF-M (Chiron, Emeryville Calif), AMPHIGEN® adjuvant, saponin, Quil A or other saponin fraction, monophosphoryl lipid A, ionic polysaccharides, and Avridine lipid-amine adjuvant.
- RIBI adjuvant system Rost, Hamilton, Mont.
- mineral gels
- Non-limiting examples of oil- in-water emulsions useful in the vaccine of the invention include modified SEAM62 and SEAM 1/2 formulations.
- Modified SEAM62 is an oil-in-water emulsion containing 5% (v/v) squalene (Sigma), 1% (v/v) SPAN® 85 detergent (ICI Surfactants), 0.7% (v/v) TWEEN® 80 detergent (ICI Surfactants), 2.5% (v/v) ethanol, 200 ⁇ Quil A, 100 ⁇ g/ml cholesterol, and 0.5% (v/v) lecithin.
- Modified SEAM 1/2 is an oil-in-water emulsion comprising 5% (v/v) squalene, 1% (v/v) SPAN® 85 detergent, 0.7% (v/v) Tween 80 detergent, 2.5% (v/v) ethanol, 100 ⁇ g/ml Quil A, and 50 ⁇ g/ml cholesterol.
- Other immunomodulatory agents that can be included in the vaccine include, e.g., one or more interleukins, interferons, or other known cytokines.
- Additional adjuvant systems permit for the combination of both T-helper and B-cell epitopes, resulting in one or more types of covalent T-B epitope linked structures, with may be additionally lipidated, such as those described in WO2006/084319,
- ORFI PEDV protein or other PEDV proteins or fragments thereof, is formulated with 5% AMPHIGEN® as discussed hereinafter.
- 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. recombinant-mutant heat labile toxin of E. coli, Cholera toxin-B subunit); or via encapsulation with polymers and alginates, or with mucoadhesives such as chitosan, or via liposomes.
- a preferred adjuvanted or non adjuvanted vaccine dose at the minimal protective dose through vaccine release may provide between approximately 10 and approximately 10 6 logioTCIDso of virus per dose, or higher.
- TCID50 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 TCID50, 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).
- 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).
- 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
- 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 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 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
- 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®
- 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).
- 011 can be obtained from various commercial sources, for example, J. T. Baker
- 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; preferably, in an amount of greater than 50% to 85%; more preferably, in an amount from greater than 50% to 60%, and more preferably in the amount of greater than 50-52%) v/v of the vaccine composition.
- the oily phase includes oil and emulsifiers (e.g., SPAN® 80, TWEEN® 80 etc), if any such emulsifiers are present.
- Non-natural, synthetic emulsifiers suitable for use in the adjuvant formulations of the present invention also include sorbitan-based non-ionic surfactants, e.g. fatty-acid- substituted sorbitan surfactants (commercially available under the name SPAN® or ARLACEL®), fatty acid esters of polyethoxylated sorbitol (TWEEN®), polyethylene glycol esters of fatty acids from sources such as castor oil (EMULFOR®); polyethoxylated fatty acid (e.g., stearic acid available under the name SEVIULSOL® M-53),
- 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 (EMUL
- 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
- 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.
- 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.
- 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,
- Vaccines of the present invention can optionally be formulated for sustained release of the virus, infectious DNA molecule, plasmid, or viral vector of the present invention.
- sustained release formulations include virus, infectious DNA molecule, plasmid, or viral vector in combination with composites of biocompatible polymers, such as, e.g., poly (lactic acid), poly (lactic-co-glycolic acid), methylcellulose, hyaluronic acid, collagen and the like.
- biocompatible polymers such as, e.g., poly (lactic acid), poly (lactic-co-glycolic acid), methylcellulose, hyaluronic acid, collagen and the like.
- the virus, plasmid, or viral vector can be microencapsulated to improve administration and efficacy.
- microencapsulating antigens are well-known in the art, and include techniques described, e.g., in U.S. Pat. No. 3,137,631; U.S. Pat. No. 3,959,457; U.S. Pat. No. 4,205,060; U.S. Pat. No. 4,606,940; U.S. Pat. No. 4,744,933; U.S. Pat. No. 5,132, 117; and International Patent Publication WO 95/28227, all of which are incorporated herein by reference.
- Liposomes can also be used to provide for the sustained release of virus, plasmid, viral protein, or viral vector. Details concerning how to make and use liposomal formulations can be found in, among other places, U.S. Pat. No. 4,016,100; U.S. Pat. No. 4,452,747; U.S. Pat. No. 4,921,706; U.S. Pat. No. 4,927,637; U.S. Pat. No. 4,944,948; U.S. Pat. No. 5,008,050; and U.S. Pat. No. 5,009,956, all of which are incorporated herein by reference.
- An effective amount of any of the above-described vaccines can be determined by conventional means, starting with a low dose of virus, viral protein plasmid or viral vector, and then increasing the dosage while monitoring the effects.
- An effective amount may be obtained after a single administration of a vaccine or after multiple administrations of a vaccine.
- Known factors can be taken into consideration when determining an optimal dose per animal. These include the species, size, age and general condition of the animal, the presence of other drugs in the animal, and the like.
- the actual dosage is preferably chosen after consideration of the results from other animal studies.
- One method of detecting whether an adequate immune response has been achieved is to determine seroconversion and antibody titer in the animal after vaccination.
- the timing of vaccination and the number of boosters, if any, will preferably be determined by a doctor or veterinarian based on analysis of all relevant factors, some of which are described above.
- the effective dose amount of virus, protein, infectious nucleotide molecule, plasmid, or viral vector, of the present invention can be determined using known techniques, taking into account factors that can be determined by one of ordinary skill in the art such as the weight of the animal to be vaccinated.
- the dose amount of virus of the present invention in a vaccine of the present invention preferably ranges from about 10 1 to about 10 9 pfu (plaque forming units), more preferably from about 10 2 to about 10 8 pfu, and most preferably from about 10 3 to about 107 pfu.
- the dose amount of a plasmid of the present invention in a vaccine of the present invention preferably ranges from about 0.1 ⁇ g to about 100 mg, more preferably from about 1 ⁇ g to about 10 mg, even more preferably from about 10 ⁇ g to about 1 mg.
- the dose amount of an infectious DNA molecule of the present invention in a vaccine of the present invention preferably ranges from about 0.1 ⁇ g to about 100 mg, more preferably from about 1 ⁇ g to about 10 mg, even more preferably from about 10 ⁇ g to about 1 mg.
- the dose amount of a viral vector of the present invention in a vaccine of the present invention preferably ranges from about 10 1 pfu to about 10 9 pfu, more preferably from about 10 2 pfu to about 10 8 pfu, and even more preferably from about 10 3 to about 10 7 pfu.
- a suitable dosage size ranges from about 0.5 ml to about 10 ml, and more preferably from about 1 ml to about 5 ml.
- Suitable doses for viral protein or peptide vaccines range generally from 1 to 50 micrograms per dose, or higher amounts as may be determined by standard methods, with the amount of adjuvant to be determined by recognized methods in regard of each such substance.
- an optimum age target for the animals is between about 1 and 21 days, which at pre-weening, may also correspond with other scheduled vaccinations such as against Mycoplasma hyopneumoniae.
- a preferred schedule of vaccination for breeding sows would include similar doses, with an annual revaccination schedule.
- a preferred clinical indication is for treatment, control and prevention in both breeding sows and gilts pre-farrowing, followed by vaccination of piglets.
- 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 TCID50 (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 use in pregnant animals (all trimesters) and neonatal swine.
- the vaccines of the invention are attenuated to a level of safety (i.e. no mortality, only transient mild clinical signs or signs normal to neonatal swine) that is acceptable for even the most sensitive animals again including neonatal pigs.
- levels of safety i.e. no mortality, only transient mild clinical signs or signs normal to neonatal swine
- programs of sustained sow vaccination are of great importance.
- sows or gilts immunized with PEDV MLV will passively transfer immunity to piglets, including PEDV-specific IgA, which will protect piglets from PEDV associated disease and mortality.
- pigs that are immunized with PEDV MLV will have a decrease in amount and/or duration or be protected from shedding PEDV in their feces, and further, pigs that are immunized with PEDV MLV will be protected from weight loss and failure to gain weight due to PEDV, and further, PEDV MLV will aid in stopping or controlling the PEDV transmission cycle.
- 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 logio TCID50, or higher, of virus per dose (minimum immunizing dose to vaccine release).
- a composition is said to be "pharmacologically acceptable” if its administration can be tolerated by a recipient. Such a composition is said to be administered in a "therapeutically or prophylactically effective amount” if the amount administered is physiologically significant.
- At least one vaccine or immunogenic composition of the present invention can be administered by any means that achieve the intended purpose, using a pharmaceutical composition as described herein.
- 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.
- 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.
- a vial of Vero cells was subject to additional passaging.
- the cells were passed four times in PMEM w/wheat to produce Master Cell Stock (MCS) Lot "1834430".
- MCS Master Cell Stock
- the MCS was tested in accordance with 9CFR & EP requirements in PGM-Biological Quality Control; Lincoln, E.
- 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 (Lincoln item # 00-0779-00) containing 1% bovine serum (item # 00-0710-00, BSE compliant) and 3 mM L-glutamine. They were derived from Vero WCS Pass # 136, Lot #071700 MCS+3, 28-Jul-OO.
- the new Pre-master cell stock was frozen at pass # 166, which is MCS+33 from the original global Vero master cell stock.
- MCS "1833440" was produced from a pre-Master identified as Vero KZO preMaster, Lot All cultures were grown in PMEM w/wheat, 1.0% L-glutamine and 1.0% Bovine Calf serum. Cells were planted (passage # 167) in 150 cm2 T-Flasks on August 14, 2008. The flasks were incubated in 5.0% C02 at 36 1 C for 7 days then expanded, (passage # 168) After flasks reached 100% confluency 4 days later, the cultures were passed (#169) into 850 cm2 roller bottles. Rollers were incubated at 36 1 C at 0.125 - 0.250 rpm without C02. The final passage of rollers (#170) was done 4 days later.
- Cryopreservation was completed by adding 10.0% bovine calf serum and 10.0% dimethyl sulfoxide (DMSO) to the condensed cell suspension on 02Sep08. Vials were labeled as passage level #170. A total of 231 containers containing 4.2 ml were placed into a controlled rate freezer then transferred into liquid nitrogen tank for long term storage at vapor phase. The MCS was produced without the use of antibiotics. All reagents used in MCS production were sourced from Pfizer Global Manufacturing used for licensed antigen production in domestic and global markets. The MCS was produced by Pfizer' s Master Seed Facility, Lincoln, Iowa.
- Sterility Testing was as follows. The Master Cell Stock was tested as per 9CFR (026-STO) and EP 2.6.1 from 29Sep08 to 13Oct08. The MCS was found to be free of bacteria and fungal contamination.
- 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 113.52 using NL-BT-2 (Bovine), Vero, NL-ED-5 (Equine), NL-ST-1 (Porcine), NL-DK
- 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.
- Representative embodiments of the invention include an isolated polynucleotide sequence that comprises a polynucleotide selected from the group consisting of: (a) (SEQ ID NOs: 1, 2, 3, 8, 15, 35, 36, 37, 39, and/or 59-77); or a fragment thereof than encodes, ORFla/lb polyproteins, the PEDV spike protein preferably domain 1, ORF3 protein, envelope protein, membrane protein, nucleocapsid protein or a fragment(s) of said proteins; (b) the complement of any sequence in (a); (c) a polynucleotide that hybridizes with a sequence of (a) or (b) under stringent conditions defined as hybridizing to filter bound DNA in 0.5M NaHP0 4 , 7% SDS, 1 mM EDTA at 65° C, and washing in 0.1 X SSC/0.1% SDS at 68° C; (d) a polynucleotide that is at least 70% identical to the polynucleotide of (a
- the invention also provides a polypeptide encoded by any of the open reading frames of the genotype of SEQ ID NOs: 1, 2, 3, 8, 15, 35, 36, 37, 39, 59-78, combinations thereof, or a polypeptide that is at least 90% identical thereto, domains thereof, or to a fragment thereof, including the option that additional otherwise identical amino acids are replaced by conservative substitutions.
- the invention also provides a polypeptide encoded by any of the open reading frames of the variant PEDV strain of the invention, preferably the spike protein, or more preferably spike protein SI domain, or a polypeptide that is at least 90% identical thereto, or to a fragment thereof, including the option that additional otherwise identical amino acids are replaced by conservative substitutions.
- the polypeptide is expressed from the first 1170 nucleotides of the SI region of spike protein of the variant PEDV strain of the invention.
- PEDV polypeptide- based vaccines wherein the antigen is defined by: a protein encoded by an open reading frames of SEQ ID NOs: 1, 2, 3, 8, 15, 35, 36, 37, 39, 59-77, combinations thereof, or an immunogenic fragment thereof.
- Further embodiments include an amino acid sequence where the antigen is: encoded by the nucleotides of the SEQ ID NOs: 23-34, 40-58.
- the polynucleotide and amino acid sequence information provided by the present invention also makes possible the systematic analysis of the structure and function of the viral genes and their encoded gene products.
- Knowledge of a polynucleotide encoding a viral gene product of the invention also makes available anti-sense polynucleotides which recognize and hybridize to polynucleotides encoding a polypeptide of the invention, or a fragment thereof. Full length and fragment anti-sense polynucleotides are useful in this respect.
- fragment anti-sense molecules of the invention include (i) those which specifically recognize and hybridize to a specific RNA (as determined by sequence comparison of DNA encoding a viral polypeptide of the invention as well as (ii) those which recognize and hybridize to RNA encoding variants of the encoded proteins.
- Antisense polynucleotides that hybridize to RNA/DNA encoding other PEDV peptides are also identifiable through sequence comparison to identify characteristic, or signature sequences for the family of molecules, further of use in the study of antigenic domains in PEDV polypeptides, and may also be used to distinguish between infection of a host animal with remotely related non-PEDV members of the Circoviridae.
- anti-PEDV antibodies e.g., monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, humanized, human, porcine, and CDR-grafted antibodies, including compounds which include CDR sequences which specifically recognize a PEDV polypeptide of the invention.
- the term "specific for” indicates that the variable regions of the antibodies of the invention recognize and bind a PEDV polypeptide exclusively (i.e., are able to distinguish a single PEDV polypeptide from related polypeptides despite sequence identity, homology, or similarity found in the family of polypeptides), and which are permitted (optionally) to interact with other proteins (for example, S.
- aureus protein A or other antibodies in ELISA techniques through interactions with sequences outside the variable region of the antibodies, and in particular, in the constant region of the Ab molecule.
- Screening assays to determine binding specificity of an antibody of the invention are well known and routinely practiced in the art. For a comprehensive discussion of such assays, see Harlow et al. (Eds), Antibodies A Laboratory Manual; Cold Spring Harbor Laboratory; Cold Spring Harbor, N.Y. (1988), Chapter 6.
- Antibodies that recognize and bind fragments of the PEDV polypeptides of the invention are also contemplated, provided that the antibodies are first and foremost specific for, as defined above, a PEDV polypeptide of the invention from which the fragment was derived.
- antibody refers to an immunoglobulin molecule that can bind to a specific antigen as the result of an immune response to that antigen.
- Immunoglobulins are serum proteins composed of "light” and “heavy” polypeptide chains having "constant” and “variable” regions and are divided into classes (e.g., IgA, IgD, IgE, IgG, and IgM) based on the composition of the constant regions.
- Antibodies can exist in a variety of forms including, for example, as, Fv, Fab', F(ab') 2, as well as in single chains, and include synthetic polypeptides that contain all or part of one or more antibody single chain polypeptide sequences.
- the present invention also provides diagnostic kits.
- the kit can be valuable for differentiating between porcine animals naturally infected with a field strain of a PEDV virus and porcine animals vaccinated with any of the PEDV vaccines described herein.
- the kits can also be of value because animals potentially infected with field strains of PEDV virus can be detected prior to the existence of clinical symptoms and removed from the herd, or kept in isolation away from naive or vaccinated animals.
- the kits include reagents for analyzing a sample from a porcine animal for the presence of antibodies to a particular component of a specified PEDV virus.
- kits of the present invention can include as a component a peptide or peptides from the variant PEDV strain of the invention which is present in a field strain but not in a vaccine of interest, or vice versa, and selection of such suitable peptide domains is made possible by the extensive amino acid sequencing.
- kits of the present invention can alternatively include as a component a peptide which is provided via a fusion protein.
- fusion peptide or “fusion protein” for purposes of the present invention means a single polypeptide chain consisting of at least a portion of a PEDV virus protein, preferably of ORF1, or ORF3and a heterologous peptide or protein.
- PEDV SI first 2.2 kb portion of the spike gene sequencing was undertaken to help determine the genetic relatedness and molecular epidemiology of PEDV in US swine.
- PEDV SI sequences from 10 cases are similar to each other and to the PEDV strains identified in US swine in 2013 (99.1-100% nucleotide identities).
- the PEDV SI sequences from the other 5 cases only have 93.9- 94.6% nucleotide identities to the PEDV strains previously identified in US swine in 2013.
- the SI gene of this new virus isolate has been determined and is reported herein as SEQ ID NO: 1. Based on the data currently available, it appears unlikely that this strain is a mutant evolved from PEDV previously identified in US swine.
- the PEDV real-time RT- PCR offered at ISU VDL is targeting the nucleocapsid (N) gene.
- N nucleocapsid
- the N-gene is known to be a conserved portion of the PEDV genome.
- the PEDV N-gene real-time RT- PCR being conducted at the ISU VDL seems to be readily detecting these new PEDVs.
- the full-length N gene sequences of the new PEDVs have been determined and were similar to the PEDVs previously identified in the US.
- Figure 2 shows phylogenetic trees based on the SI portion sequences and the whole genome sequences. See the attachment.
- the US prototype-like strains have been detected in South Korea, Canada and Mexico; the US variant-INDEL-like strains have been detected in South Korea, Mexico and Germany.
- At least two genetically different porcine epidemic diarrhea virus (PEDV) strains have been identified in the United States: U.S. PEDV prototype and S-INDEL- variant strains.
- the objective of this study was to compare the pathogenicity differences of the U.S. PEDV prototype and S-INDEL-variant strains in conventional neonatal piglets under experimental infections.
- Fifty PEDV-negative 5-day-old pigs were divided into 5 groups of 10 pigs each and were inoculated orogastrically with three U.S.
- PEDV prototype isolates (IN19338/2013-P7, NC35140/2013-P7, and NC49469/2013-P7), an S-INDEL-variant isolate (2014020697-P7), and virus-negative culture medium, respectively, with virus titers of 10 4 TCID50/ml, 10 ml per pig. All three PEDV prototype isolates tested in this study, regardless of their phylogenetic clades, had similar pathogenicity and caused severe enteric disease in 5-day-old pigs as evidenced by clinical signs, fecal virus shedding, and gross and histopathological lesions. Compared to pigs inoculated with the three U.S.
- PEDV prototype isolates pigs inoculated with the S-INDEL-variant isolate had significantly diminished clinical signs, virus shedding in feces, gross lesions in small intestines, ceca and colons, histopathological lesions in small intestines and immunohistochemistry staining in ileum.
- the U.S. PEDV prototype and the S-INDEL-variant strains induced similar viremia levels in inoculated pigs.
- Whole genome sequences of the PEDV prototype and S-INDEL-variant strains were determined but the molecular basis of virulence differences between these PEDV strains remains to be elucidated using reverse genetics approach. The present study provides a strong foundation to the understanding of the molecular mechanisms which contribute to PEDV virulence and vaccine attenuation.
- USA/NC35140/2013-P7, USA/IA49379/2013 -P7, and USA NC49469/2013-P7 were determined and compared to those of the previously described U.S. PEDV prototype isolate USA/IN19338/2013 and U.S. PEDV S-INDEL-variant isolate USA/IL20697/2014 with results summarized in Table 3.
- Table 3 Schematic diagrams of PEDV genome organization and putative functions of viral proteins are described in Figure 12. The prototype isolates USA/TNI 9338/2013 -P7, USA/NC35140/2013-P7, USA/IA49379/2013 -P7 and
- USA/NC49469/2013-P7 all had a genome of 28,038 nucleotides in length and had 99.75- 99.91% nucleotide (nt) identity (26-69 nt differences) to each other at the whole genome level.
- the spike genes of these prototype isolates all had 4,161 nucleotides in length and had 99.54-99.88% nt identity (5-19 nt differences) to each other.
- the S-INDEL-variant isolate 2014020697-P7 had a genome of 28,029 nucleotides in length and had 99.08-99.22% nt identity (220-259 nt differences) at the whole genome level to the four prototype isolates evaluated in this study.
- the S gene of the variant isolate 2014020697-P7 had three characteristic deletions (a 1-nt deletion of G at position 167, an 11-nt deletion of AGGGTGTCAAT at positions 176-186, and a 3-nt deletion of ATA at positions 416-418) and one insertion (a 6-nt insertion of CAGGAT between positions 474 and 475).
- Fig. 6 Phylogenetic analyses of the PEDV isolates described in this study and 45 PEDV reference sequences are provided in Fig. 6.
- the U.S. PEDV prototype-like strains clustered together, which can be further divided into clade 1 and clade 2; however, the U.S. PEDV S-F DEL-variant-like strains clustered separately.
- the U.S. PEDV prototype-like strains also clustered into clade 1 and clade 2;
- the S-FNDEL-variant-like strains formed a separate sublineage within clade 2.
- the phylogenetic clusters in the SI sequence-based neighbor-joining tree Fig. 6C and the maximum likelihood tree Fig. 6D were similar.
- the U.S. PEDV prototype-like strains clustered together which can be further divided into clade 1 and clade 2, similar to the whole genome sequence-based neighbor-joining tree Fig. 6A; the U.S.
- PEDV S-FNDEL-variant-like strains formed a separate branch that was more closely related to some classical PEDV isolates such as Europe/CV777, South Korea/SM98, and China/SD- M which have the same pattern of insertions and deletions in the S gene as the U.S. PEDV S- FNDEL-variant-like strains.
- the prototype isolates FN19338 and IA49379 belong to the clade 1 and the isolate NC35140 belongs to the clade 2 regardless of the whole genome-based trees Fig. 6A, 6B or the SI -based trees Fig. 6C, 6D.
- the prototype isolate NC49469 belongs to the clade 1 in the whole genome-based trees Fig. 6A, 6B but belongs to the clade 2 in the SI -based trees Fig. 6C, 6D.
- Three prototype isolates USA/FN 19338/2013 -P7 (SEQ ID NO:59)
- ORF nucleotide (IN19338, IA49379, NC49469, and NC35140)
- ORFlab (293- 16-50 (0.08- 9-16 (0.13- 64-96 (0.31- 7-19 (0.10- lab polyprotein (6781)
- nsp3 Gly896-Gly2516 5-22 (0.10- 3-9 (0.18- 6-24 (0.12- 1-10 (0.06-
- nsp6 Ser3300- 0-2 (0- 0-1 (0-
- nspl4 Asn5547- 0-3 (0- 0-1 (0- 1-2 (0.06- 0-1 (0-
- Gln6141 (595) 0.17%) 0.17%) 0.11%) 0.17%) nspl5: Gly6142- 0-2 (0- 0-2 (0- 8-9 (0.79- 1-2 (0.29-
- Nucleotides are numbered according to the sequence of the U.S. PEDV prototype isolate USA/IN19338/2013 (GenBank accession number KF650371).
- nt nucleotide
- amino acid (aa) differences were calculated at each gene or
- PEDV-inoculated pigs From (-1) to 3 DPI, PEDV-inoculated pigs (G1-G4) had significantly lower average daily gain (ADG, P ⁇ 0.0001) compared to pigs in G5 (negative control), but there were no significant differences in ADG (P-values ranged from 0.089-1) among G1-G4 (Fig. 7B).
- G1-G3 prototype isolates
- G4 variant isolate
- PEDV RNA was detected in rectal swab samples from all pigs in G1-G3 (prototype isolates) at 1 DPI until the end of the study.
- G4 variant isolate
- PEDV RNA was detected in rectal swabs from 5/10, 8/10, 10/10, 5/5, 5/5, 3/5, and 2/5 pigs at 1, 2, 3, 4, 5, 6 and 7 DPI, respectively.
- the average genomic copies per ml of virus shed in rectal swabs are summarized in Fig. 7C.
- Pigs in G3 had the highest level of fecal virus shedding at 1-2 DPI (approximately 109 genomic copies/ml with Ct 16) and the fecal virus shedding gradually declined to
- PEDV RNA was detected in semm samples from all pigs in G1-G4 necropsied at 3 DPI with average Ct values of 33.2 (Gl), 30.5 (G2), 31.9 (G3) and 28.4 (G4). There were no significant differences between average PEDV genomic copies in sera of G1-G4 at 3 DPI [P- values ranged from 0.077-0.646, Fig. 7D. At 7 DPI, PEDV RNA was detected in semm samples from 3-4 out of 5 pigs in G1-G4 with average Ct values (only on PCR-positive pigs) of 33.5 (Gl), 34.2 (G2), 37.4 (G3) and 35.8 (G4). The average genomic copies of PEDV in sera of G4 (variant isolate) had no significant difference (P-values ranged from 0.050-0.717) compared to those in G1-G3 (prototype isolates) at 7 DPI Fig. 7D.
- Vims distributions in tissues are summarized in Table 5.
- 3 DPI regardless of Gl, G2, G3 or G4, average PEDV RNA concentrations in ileums, ceca, colons and mesenteric lymph nodes were higher than the concentrations in other tissues within the same inoculation group.
- viral RNA concentrations in cecum and colon of G4 variant isolate
- viral RNA concentrations in other tissues of G4 were similar to those in the corresponding tissues of G1-G3; the same types of tissues in G1-G3 had similar levels of viral RNA.
- Data at 7 DPI overall supported similar conclusions to 3 DPI except that viral genomic copies in cecum and colon were much lower than the genomic copies in ileum and mesenteric lymph node in G4.
- DPI days post inoculation
- MLN mesenteric lymph node
- Ct cycle threshold
- Mean Ct was mean Ct value of PCR-positive pigs ( pigs with PCR Ct ⁇ 45). * Genomic copies/ml was geometric mean of genomic copies per milliter of all pigs (both PCR-positive and negative pigs). Staistical analyses were performed on the same tissue types for G1-G4 groups and different letters indicate significant differences.
- Severe lesions consistent with viral enteritis were observed in small intestinal sections (duodenum, jejunum, and ileum) of all pigs in G1-G3 (prototype isolates) necropsied at 3 DPI and 7 DPI.
- Mild microscopic lesions consistent with viral enteritis were observed in small intestinal sections of pigs in G4 (variant isolate) necropsied at 3 DPI; however, microscopic lesions were not remarkable in small intestinal sections of pigs in G4 necropsied at 7 DPI.
- the average villus heights, crypt depths, and villus/crypt ratios of small intestine sections at 3 DPI were overall similar across the three groups G1-G3 inoculated with the prototype isolates (Fig. 10).
- the average crypt depths of all small intestinal sections at 3 DPI were not significantly different between G4 (variant isolate) and G5 (negative control); however, pigs in G4 had significantly decreased average villus heights and lower average villus/crypt ratios in duodenum, middle and distal jejunum and ileum at 3 DPI compared to pigs in G5 (Fig. 10).
- the average villus heights and villus/crypt ratios of small intestinal sections were overall similar across the three groups G1-G3 Fig. 1 lA-1 ID. Pigs in G1-G3 overall had significantly decreased average villus heights and lower average villus/crypt ratios in small intestinal sections than pigs in G4 and G5 at 7 DPI (Fig. 11 A, 11C). Interestingly, the average villus heights at 7 DPI were either not significantly different between G4 (variant isolate) and G5 (negative control) or were significantly higher in G4 than G5 (Fig. 11 A).
- the average villus/crypt ratios at 7 DPI were not significantly different in proximal, middle and distal jejunum, and ileum between G4 and G5 although the average villus/crypt ratios in duodenum were significantly different between G4 and G5 (Fig. IOC). Comparison of average crypt depths at 7 DPI is presented in Fig. 1 IB. The average crypt depths were similar in all small intestinal sections between the prototype isolate-inoculated groups G2 and G3; both G2 and G3 had significantly longer crypt depths compared to the negative control group G5. Another prototype-isolate inoculated group Gl had the average crypt depth values that were between the negative control group G5 and the prototype isolate-inoculated groups G2 and G3.
- the variant isolate-inoculated group G4 had significantly increased average crypt depths in duodenum, proximal and distal jejunums compared to the negative control group G5, while G4 had similar average crypt depths to Gl, G2, and G3 in most of the small intestinal sections.
- PEDV IHC staining was only performed on serial sections of ileum.
- PEDV S-F DEL-variant isolate 2014020697-P7 had significantly diminished clinical signs, virus shedding in feces, gross lesions in small intestines, ceca and colons, histopathological lesions in small intestines, and IHC scores in ileum, compared to three U.S. PEDV prototype isolates USA/IN 19338/2013 -P7, USA/NC35140/2013-P7 and USA/NC49469/2013-P7.
- Recent experimental studies by other groups also demonstrated that S-INDEL PEDVs overall had lower pathogenicity compared to the U.S. prototype strains in 3-4 day old pigs or 1-week-old pigs (Lin et al., 2015a; Yamamoto et al., 2015).
- S-INDEL PEDV pathogenicity can be age-dependent. Further investigations of pathogenicity of S-F DEL PEDV variants in weaned pigs, finisher pigs, gilts and sows are warranted.
- PEDV is an enteropathogenic coronavirus that infects the villous enterocytes, resulting in villous atrophy and malabsorptive diarrhea.
- Some quantities of PEDV could be taken into the blood stream through mechanisms not-fully-understood. But PEDV is not believed to actively replicate in blood and viremia levels may not necessarily correlate to virulence/pathogenicity.
- PEDV RNA In the current study, high levels of PEDV RNA were detected in small intestine, cecum, colon and mesenteric lymph nodes while low levels of PEDV RNA were detected in non-enteric tissues (tonsil, heart, lung, liver, spleen, kidney, and muscle) from pigs inoculated with either prototype or S-INDEL-variant PEDV. Previous studies indicated that PEDV viral antigen (U.S.
- PEDV IHC staining was only performed on ileum, cecum and colon of inoculated pigs.
- PEDV IHC staining was observed in 100% ileums, 60-100% ceca, and 40-100%) colons at 3 DPI.
- the average IHC scores in ileums were significantly lower in the variant isolate-inoculated pigs than in the prototype isolates-inoculated pigs, consistent with observations on gross pathology and histopathological lesions of small intestines.
- prototype isolate-inoculated groups had longer average crypt depth than the negative control group, suggesting that crypt elongation continued to replace the damaged villus enterocytes but the villus epithelium has not recovered back to normal.
- the average crypt depths of some G4 (variant isolate) sections of small intestine were significantly longer than G5 (negative control) at 7 DPI, suggesting that elongation of crypts occurred later in G4 compared to the prototype isolate-inoculated groups G1-G3.
- the proliferated crypts eventually recovered the destroyed villus enterocytes apparent at 3 DPI in G4. IHC staining also supported these observations.
- PEDV prototype and S-INDEL-variant strains can provide homologous and heterologous protection against two virus strains in a weaned pig model.
- U.S. PEDV S-INDEL-variant strain is less virulent than U.S. PEDV prototype strains in neonatal pigs.
- the striking sequence differences between U.S. prototype and S-INDEL-variant PEDVs are located in the spike gene especially the SI portion.
- the sequence differences in the spike gene may be partially responsible for the virulence differences between U.S.
- PEDV isolates used in this study were confirmed negative for porcine deltacoronavirus (PDCoV), transmissible gastroenteritis virus (TGEV), and porcine rotaviruses (groups A, B, & C), porcine reproductive and respiratory syndrome virus, and porcine circovirus.
- PDCoV porcine deltacoronavirus
- TGEV transmissible gastroenteritis virus
- porcine rotaviruses groups A, B, & C
- porcine reproductive and respiratory syndrome virus porcine circovirus.
- Pigs were blocked by weight and then randomly divided into 5 groups of 10 pigs each, one group per room on a solid floor. Pigs were fed a mixture of Esbilac (Hampshire, IL) liquid milk replacer and yogurt and had free access to water. After 1-day acclimation (piglets were 6 days old), pigs in groups 1 through 5 (G1-G5) were orogastrically inoculated with three U.S. PEDV prototype isolates USA/FN 19338/2013 -P7 (Gl), USA NC35140/2013-P7 (G2), USA/NC49469/2013-P7 (G3), one U.S.
- S-F DEL-variant isolate 2014020697-P7 (G4), or virus-negative culture medium (G5), respectively (lOml/pig; all viruses were at the 7th passage in cell culture with the titer of 104 TCID50/ml) (Table 4).
- Body weights were recorded prior to inoculation and then at 3 and 7 days post inoculation (DPI).
- the average daily gain (ADG) was calculated on pigs from (-1) to 3 DPI and (-1) to 7 DPI.
- Serum samples were collected at 0, 3, and 7 DPI.
- Rectal swabs were collected daily from each pig from 0 DPI to necropsy and were submerged into 1ml PBS immediately after collection. Five pigs from each group were randomly selected for necropsy at 3 DPI, and the remaining pigs were necropsied at 7 DPI.
- Tissue lesions were categorized as normal, thin-walled, and/or gas-distended.
- the presence of thin-walled intestines or gas- distended organs was numerated as 1 point, respectively; the presence of both thin- walled and gas-distended was numerated as 2 points.
- rectal swabs collected at 3 DPI and 7 DPI before necropsy were tested for PDCoV, TGEV, and porcine rotaviruses (groups A, B &C) by virus-specific PCRs and for hemolytic E. coli and
- Salmonella spp. by routine bacterial cultures at ISU VDL.
- Viral RNA was extracted from rectal swabs, serum, and 10% tissue homogenates as previously described (Chen et al., 2014). Five ⁇ of each RNA template was used in PCR setup in a 25 ⁇ total reaction using Path-IDTM Multiplex One-Step RT-PCR Kit (Thermo Fisher Scientific). The primers, probes and in vitro transcribed RNA used to generate standard curves of a quantitative PEDV N gene-based real-time RT-PCR had been described (Lowe et al., 2014; Madson et al., 2014; Thomas et al., 2015). Based on standard curves, virus concentration in the unit of genomic copies/ml in tested samples was calculated. The mean cycle threshold (Ct) values were calculated based on PCR positive samples, and the mean virus concentrations were calculated based on all pigs within the group (both PCR positive and negative pigs).
- Ct mean cycle threshold
- Tonsil, heart, lung, liver, spleen, kidney, mesenteric lymph node, stomach, duodenum, proximal jejunum, middle jejunum, distal jejunum, ileum, cecum, and colon tissues were fixed in 10% formalin, embedded, sectioned, and stained with hematoxylin and eosin (H&E) and examined by a veterinary pathologist blinded to individual animal identifications and treatment groups.
- Villus lengths and crypt depths were measured from three representative villi and crypts of duodenum, proximal jejunum, middle jejunum, distal jejunum, and ileum, using a computerized image system following previously described procedures (Madson et al., 2014).
- Villus-height-to-crypt-depth (villus/crypt) ratio of each tissue was calculated as the quotient of the average villus length divided by the average crypt depth.
- GLIMMIX Generalized linear mixed model was used for all statistical comparisons with Statistical Analysis System (SAS) version 9.3 (SAS institute, Cary, NC). P-value ⁇ 0.05 was defined as statistically significant. P-values of overall fecal viral shedding level
- BioEdit a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acids Symp Ser 41, 95-98.
- At least two genetically different porcine epidemic diarrhea virus (PEDV) strains have been identified in the United States (U.S. PEDV prototype and S -INDEL- variant strains).
- the current serological assays offered at veterinary diagnostic laboratories for detection of PEDV-specific antibody are based on the U.S. PEDV prototype strain.
- the objectives of this study were: 1) isolate the U.S. PEDV S -INDEL- variant strain in cell culture; 2) generate antisera against the U.S. PEDV prototype and S-INDEL-variant strains by experimentally infecting weaned pigs; 3) determine if the various PEDV serological assays could detect antibodies against the U.S. PEDV S-INDEL-variant strain and vice versa.
- PEDV S-INDEL-variant strain was isolated in cell culture in this study. Three groups of PEDV-negative, 3 -week-old pigs (five pigs per group) were inoculated orally with a U.S. PEDV prototype isolate (previously isolated in our lab), an S-INDEL- variant isolate or virus-negative culture medium.
- Serum samples collected at 0, 7, 14, 21 and 28 days post inoculation were evaluated by the following PEDV serological assays: 1) indirect fluorescent antibody (IF A) assays using the prototype and S-INDEL-variant strains as indicator viruses; 2) virus neutralization (VN) tests against the prototype and S-INDEL- variant viruses; 3) PEDV prototype strain whole virus based ELISA; 4) PEDV prototype strain SI -based ELISA; and 5) PEDV S-INDEL-variant strain SI -based ELISA.
- IF A indirect fluorescent antibody
- VN virus neutralization
- the positive antisera against the prototype strain reacted to and neutralized both prototype and S-INDEL-variant viruses, and the positive antisera against the S-INDEL-variant strain also reacted to and neutralized both prototype and S-INDEL-variant viruses, as examined by IF A antibody assays and VN tests.
- Antibodies against the two PEDV strains could be detected by all three ELISAs although detection rates varied to some degree.
- Porcine epidemic diarrhea caused by porcine epidemic diarrhea virus (PEDV) was first recorded in England in the early 1970s and has since spread to other European and Asian countries [1]. In North America, PEDV was detected for the first time in the United States (U.S.) in April 2013 [2] and subsequently PEDV was reported in
- PEDV is an enveloped, single-stranded, positive-sense RNA virus belonging to the order Nidovirale, the family Coronaviridae, subfamily
- Coronavirinae genus Alphacoronavirus [5].
- the PEDV genome is approximately 28 kb in length and includes ORFla and ORFlb encoding the replicase polyproteins and other opening reading frames (ORFs) encoding four structural proteins [spike (S), envelope (E), membrane (M), and nucleocapsid (N)] and one nonstructural protein NS3B (encoded by ORF3) [1].
- the PEDV Minnesotal88 was genetically very closely related to the U.S. PEDV prototype strains and it is arguable whether it should be called a 'third' strain of PEDV in U.S.
- the PEDV PC 177 and Minnesotal88 are probably the mutants of the U.S. PEDV prototype strains. Therefore, there are at least two genetically different PEDV strains currently circulating in U.S. swine: U.S. PEDV prototype strain and S-INDEL-variant strain.
- the U.S. PEDV prototype strains have been successfully isolated and propagated in cell culture by several groups [7, 10, 12, 13].
- a number of serological assays including an indirect fluorescent antibody (IF A) assay, a virus neutralization (VN) test, a whole virus- based enzyme-linked immunosorbent assay (ELISA), a recombinant SI protein-based ELISA, and recombinant nucleocapsid protein-based ELIS As, have been developed for the detection of PEDV-specific antibodies [14-18]. All of these serological assays are based on the U.S. PEDV prototype strains.
- PEDV IF A antibody assays using the prototype and S-INDEL-variant strains as indicator viruses, respectively
- ELIS As PEDV prototype strain whole virus- based ELISA, PEDV prototype strain SI -based ELISA, and PEDV S-INDEL-variant strain SI -based ELISA
- VN tests using the prototype and S-INDEL-variant strains as indicator viruses were conducted to evaluate the in vitro cross-neutralization of two U.S. strains.
- INDEL-variant strain one small intestine homogenate (with PEDV N gene-based real-time RT-PCR cycle threshold (Ct) value of 16.1) [17, 19] from a pig located in Illinois was inoculated orogastrically into three PEDV-nai ' ve weaned pigs at 3 weeks of age (10 ml per pig).
- the homogenate used for inoculation was confirmed negative for transmissible gastrointestinal virus (TGEV), porcine rotavirus groups A, B, C and porcine
- PDCoV deltacoronavirus
- Rectal swabs and feces were collected from each inoculated pig twice a day and tested by the PEDV realtime RT-PCR on the same day. Once the RT-PCR Ct values of the rectal swabs were ⁇ 15, the pig was euthanized and necropsied within 24 hours. Small intestine tissues and cecum contents were collected for attempting virus isolation in cell culture as previously described [7]. This animal study was performed according to the procedures approved by the Iowa State University Institutional Animal Care and Use Committee (IACUC, approval number 3-14-7766-S).
- IACUC Iowa State University Institutional Animal Care and Use Committee
- the whole genome sequence of the U.S. PEDV S-INDEL-variant strain cell culture isolate USA/IL20697/2014 obtained in this study was determined by next-generation sequencing (NGS) technology using an Illumina MiSeq platform as described previously [7].
- NGS next-generation sequencing
- the PEDV SI portion sequences of the isolate USA/IL20697/2014 and the clinical sample from which the virus isolate was derived were determined by Sanger sequencing following the previously described procedures [7].
- PEDV S-INDEL-variant cell culture isolate USA/IL20697/2014-P7 (SEQ ID NO:62) (Var group), and virus-negative culture medium (Neg group), respectively, with virus titers of 104 TCID50/ml, 10 ml per pig.
- Rectal swabs were collected from all pigs daily between 0 and 7 DPI, and then at 10, 14, 21 and 28 DPI, and tested by a PEDV N gene-based quantitative real-time RT-PCR [20] to confirm infection.
- Serum samples were collected from all pigs at 0, 7, 14, 21 and 28 days post inoculation (DPI) for cross-reactivity and cross- neutralization evaluations.
- one pig antiserum against the European PEDV CV777 strain one pig antiserum against the TGEV Purdue strain, one pig antiserum against the porcine heamagglutinating encephalomyelitis virus (PHEV), one pig antiserum against the porcine respiratory coronavirus (PRCV), and one pig antiserum against PDCoV were included in this study for evaluations.
- Antisera against PEDV CV777, TGEV Purdue, and PHEV strains were purchased from National Veterinary Service Laboratory, Ames, IA.
- Antisera against PRCV and PDCoV were positive control sera obtained from the ISU VDL.
- IFA Indirect fluorescent antibody
- the U.S. PEDV prototype strain whole virus-based indirect ELISA (ProWV ELISA) was developed and validated at the ISU VDL for detection of PEDV-specific IgG antibody [15, 16]. All serum samples were tested by this ProWV ELISA following the procedures that had been described in detail [20]. The sample-to-positive (S/P) ratio of >0.8 was considered antibody positive, an S/P ratio between 0.6-0.8 was considered suspect, and an S/P ratio ⁇ 0.6 was considered negative.
- PEDV S-INDEL-variant strain was codon optimized and synthesized with the addition of a 5' Kozac sequence, a 5' eukaryotic signal sequence, and a 3' 6x-His tag by GeneArt® Gene Synthesis (Thermo Fisher Scientific, Waltham, MA, USA).
- the resultant 2,358 base pair DNA fragment was cloned into a Zoetis proprietary eukaryotic expression vector
- the authenticity and orientation of the insert in the recombinant plasmid was confirmed by sequencing.
- the recombinant plasmid was transiently transfected into human embryonic kidney (HEK) 293 cells using a Zoetis proprietary PEI transfection method. At 7 days post-transfection, culture supernatants were harvested and filter sterilized.
- the recombinant protein was purified via Ni-NTA Purification System (Thermo Fisher Scientific). The optimum antigen concentration and the optimum serum dilutions for the VarSl ELISA were determined using a checkerboard titration.
- Polystyrene 96-well microtitration plates (Nunc®, Thermo Fisher Scientific) were coated ( ⁇ per well) with PEDV variant SI protein and incubated overnight at 4°C. After 5 washes with PBS, the plates were blocked (300 ⁇ 1 ⁇ 11) with PBS containing 1% bovine serum albumin (Jackson ImmunoResearch Inc., West Grove, PA, USA) for 2 h at 25°C. Plates were dried at 37°C for 4 h and stored at 4°C in a sealed bag with desiccant packs until use. Serum samples were diluted 1 :50 and added to the coated plates ( ⁇ /well). Plates were incubated at 25°C for 1 h and then washed 5 times with PBS.
- Serum samples were tested by a U.S. PEDV prototype strain-based VN (Pro VN) and a U.S. PEDV S-INDEL-variant strain-based VN (Var VN) following the previously described procedures [20].
- the PEDV prototype isolate USA/IN19338/2013 was used as the indicator virus in the Pro VN assay and the S-INDEL-variant isolate
- the Log2 (IF A titer/ 10) of the Pro antisera and the Var antisera tested by Pro IF A and Var IFA were analyzed in a generalized linear mixed model (GLIMMIX). Days post inoculation and antigen were used as independent variables, and pig ID and the interaction of pig ID and antigen were set as random effects.
- the Log2 (VN titer) of the Pro antisera and the Var antisera tested by Pro VN and Var VN were analyzed in a similar way.
- ELISA antigen, pig ID and DPI were used as independent variables. All statistical analyses were performed with Statistical Analysis System (SAS) version 9.3 (SAS institute, Cary, NC, USA), with p value ⁇ 0.05 considered significantly different.
- SAS Statistical Analysis System
- Virus isolation was first attempted on 68 clinical samples received at the ISU VDL that tested positive for the U.S. PEDV S-INDEL-variant strain but virus isolation attempts in cell culture were unsuccessful. Subsequently a PEDV S-INDEL-variant strain-positive intestine homogenate was used to inoculate three 3-week-old pigs.
- the rectal swab of one pig had a PEDV RT-PCR Ct ⁇ 15 at 2 DPI and the pig was euthanized and necropsied at 3 DPI.
- the rectal swabs of the other two pigs had PEDV RT-PCR Ct ⁇ 15 at 3 DPI and both pigs were euthanized and necropsied at 4 DPI.
- the small intestine tissues and cecum contents collected at necropsy were used to attempt virus isolation in Vero cells.
- the U.S. PEDV S-INDEL-variant strain was successfully isolated from small intestine homogenates and cecum contents collected from all 3 pigs.
- Typical PEDV cytopathic effects including syncytial body formation and cell detachment were observed and the virus growth was confirmed by immunofluorescence staining using PEDV-specific monoclonal antibody SD6-29.
- U.S. PEDV S-INDEL-variant isolate designated as USA/IL20697/2014 was selected for further propagation and characterization. This isolate was serially passed in Vero cells and the infectious titers ranged from 103-105 TCID50/ml for the first ten passages.
- the whole genome sequences of the isolate 2014020697-P5 passage 5, linage 1 (SEQ ID NO:8) had 99.3-99.9% nucleotide identity to other U.S. PEDV S-INDEL-variant sequences available in GenBank.
- the SI sequences of the USA/IL20697/2014 cell culture isolate P5 had 99.8% nucleotide identity (only 4 nucleotide differences) to the original intestine homogenate from which the virus isolate was derived.
- the USA/IL20697/2014 isolate was tested at the ISU VDL and confirmed negative for TGEV, PRCV, PDCoV, porcine rotavirus A, B, C, influenza A virus, porcine reproductive and respiratory syndrome virus, and porcine circovirus 2 by virus-specific PCRs. Generation of antisera against the U.S. prototype and S-INDEL-variant PEDVs.
- the variant strain-based IFA (Var IFA) gave similar results on the Pro antisera except that one serum collected at 14 DPI was negative by the Var IFA assay.
- the positive Pro antisera overall reacted better to the Pro IFA assay than to the Var IFA assay, with 1.4 log2 higher titer on average (Fig. 13).
- the positive Var antisera reacted similarly to both Pro IFA and Var IFA assays, with less than 0.1 log2 titer differences on average (Fig. 13).
- the antisera collected from the negative control group were antibody negative by both PEDV Pro IFA and Var IFA assays throughout the study.
- the pig antiserum against the European PEDV CV777 strain had similar antibody titers by the Pro IFA assay (titer 320) and by the Var IFA assay (titer 160).
- the antisera against TGEV Purdue, PHEV, PDCoV, and PRCV viruses were all negative by both PEDV Pro IFA and Var IFA assays.
- the Pro antisera collected at 0 and 7 DPI were all antibody negative by ProWV ELISA, ProSl ELISA, and VarSl ELISA.
- the Pro antisera collected at 21 and 28 DPI were all positive by three ELISAs. When comparing the total number of positive Pro antisera at 14, 21 and 28 DPI by each ELISA, there were no significant differences among three ELISAs to detect antibody against the U.S. PEDV prototype strain.
- the Var antisera collected at 0 and 7 DPI were antibody negative by all three ELISAs, with the exception of one serum at 7 DPI that was in the suspect range by the ProSl ELISA (Fig. 14).
- the Var antisera collected at 14, 21 and 28 DPI had variable numbers of positive, suspect and negative results by three ELISAs (Fig. 14).
- the ProWV ELISA detected 14 sera as antibody positive, 1 as suspect, and 10 as negative; the ProSl ELISA detected 8 sera as positive, 5 as suspect, and 12 as negative; the VarSl ELISA detected 12 sera as positive, 3 as suspect, and 10 as negative.
- the antisera collected from the negative control group (Neg antisera) were antibody negative by all three PEDV ELISAs throughout the study period 0-28 DPI.
- the pig antiserum against the European PEDV CV777 strain was antibody positive by all three PEDV ELISAs.
- the antisera against TGEV Purdue, PHEV, PDCoV, and PRCV viruses were all negative by three PEDV ELISAs.
- VN antibodies were detected as early as 7 DPI in sera of most of the pigs inoculated with either a prototype strain or an S-INDEL-variant strain, regardless of testing by Pro VN or Var VN assays.
- Serum samples collected at 14, 21 and 28 DPI from all pigs inoculated with PEDV prototype strain or S-INDEL-variant strain were VN antibody positive by both Pro VN and Var VN assays.
- the positive Pro antisera had similar VN antibody titers by the Pro VN and Var VN assays and there was no significant difference between the two assays.
- VN antibody titers of the positive Pro antisera tested by the homologous Pro VN assay were, on average, 0.8 log2 higher than the VN antibody titers of the positive Var antisera tested by the homologous Var VN assay (Fig. 15).
- the antisera collected from the negative control group were antibody negative by both Pro VN and Var VN assays throughout the study period 0-28 DPI.
- the pig antiserum against the European PEDV CV777 strain was antibody positive by the Pro VN assay (titer 64) and by the Var VN assay (titer 16).
- Purdue, PHEV, PDCoV, and PRCV viruses were all negative by both PEDV Pro VN and Var VN assays.
- the ProWV ELISA and ProSl ELISA have been previously developed and validated to detect PEDV-specific antibodies [14-16].
- a PEDV VarSl ELISA was developed in this study. However, this VarSl ELISA was only validated using a limited number of field antisera against the U.S. PEDV S-INDEL-variant strain before testing the experimentally generated antisera in this study. Further validation of this VarSl ELISA using large number of serum samples would be needed to determine the performance of this assay. All three PEDV ELISAs reacted with the Pro antisera and Var antisera. The three ELISAs detected Pro antisera similarly. However, it appeared that the ProSl ELISA used in this study was not as efficient as the ProWV ELISA and the VarSl ELISA to detect the antibodies against the U.S. PEDV S-INDEL-variant strain under the conditions of this study.
- the antibodies against the U.S. prototype strain and the antibodies against the U.S. S- INDEL-variant strain neutralized both virus strains to similar titers.
- the U.S. PEDV prototype strain-based VN tests currently run in the laboratories can be used to detect antibodies against both U.S. PEDV strains.
- the distinct genetic differences between the U.S. prototype and S-INDEL-variant PEDVs are located in the SI region (nucleotides 1-2214 corresponding to aa 1-738, according to positions in the prototype strain USA/IN19338/2013, GenBank accession number KF650371), especially the N-terminal region of the S gene (nucleotides 1-1170 corresponding to aa 1-390) whereas the remaining portions of the genomes are relatively conserved between the two U.S. strains [4, 8, 10].
- the PEDV prototype strain SI protein used for the ProSl ELISA and the PEDV S-INDEL-variant strain SI protein used for the VarSl ELISA had 92% amino acid identity.
- the reported PEDV neutralizing epitopes are located in the S protein amino acid residues 499-638, 744-759, 756-771, and 1368-1374 [1, 21].
- the protein sequences in these locations harboring the neutralizing epitopes are conserved between the U.S. prototype and S-INDEL-variant PEDVs. This can explain why the antibodies against the two PEDV strains were able to cross-neutralize two virus strains.
- the ProSl and VarSl ELISAs were developed using the recombinant PEDV SI proteins (aa 1-738). Although the U.S. prototype and S-INDEL-variant PEDVs have considerable differences in aa 1-390, the two strains still have some common epitopes in this region.
- the recombinant SI proteins of two PEDV strains have relatively conserved sequences from aa 390-738 including the neutralizing epitopes in this region.
- the IFA antibody assay and ProWV ELISA are supposed to detect antibodies against multiple antigenic proteins of PEDV and thus they are expected to detect antibodies against both U.S. prototype and S-INDEL-variant PEDVs.
- the nucleocapsid protein is rather conserved among PEDVs, it is expected that the nucleocapsid protein- based ELISAs should detect antibodies against both U.S.
- PEDV strains Applicants also included one pig antiserum against the classical European PEDV CV777 strain for evaluation and the PEDV CV777 antibody was detected by all serological assays evaluated in this study. However, antisera against TGEV Purdue, PHEV, PDCoV and PRCV had no cross-reactivity with PEDV serological assays evaluated in this study.
- Huang YW Dickerman AW, Pineyro P, Li L, Fang L, Kiehne R, Opriessnig T, Meng XJ. 2013. Origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the United States. MBio 4:e00737-00713.
- Gerber PF Gerber PF, Gong Q, Huang YW, Wang C, Holtkamp D, Opriessnig T. 2014.
- the present invention provides a major achievement, the provision of modified live vaccines, based on INDEL-type variants (a further example of which being OH851, as first described by the Ohio Department of Agriculture, L. Wang et al., Emerg. Infect. Dis., 2014, v. 20, pp. 917-919) that are effective against the current spectrum of worldwide epidemics, and particularly are cross protective against so-called prototype viruses (an example being well known USA/Colorado/2013, the sequence of which is available under GenBank Accession No. KF272920), all as generally described above, which have caused devastating losses in swine herds.
- INDEL-type variants a further example of which being OH851, as first described by the Ohio Department of Agriculture, L. Wang et al., Emerg. Infect. Dis., 2014, v. 20, pp. 917-919
- prototype viruses an example being well known USA/Colorado/2013, the sequence of which is available under GenBank Accession No. KF272920
- vaccines of the present invention also accord substantial flexibility as to dosing, and timing of dosing, being effective for administration, for example, to sows, both before and during pregnancy, and for administration to piglets, including to those born of naive sows. Effectiveness as to boars is also provided.
- such vaccines in both adjuvanted and non-adjuvanted forms, provide protection to sows, gilts, piglets, hogs and boars against challenge with PEDV, including prototype and emerging INDEL-type isolates.
- the present vaccines will therefore provide previously unattainable protective benefits, to include the achievement successful (1) vaccination of pregnant sows and gilts for protection, prevention, aid in prevention, or aid in control against disease caused by PEDV (anorexia, weight loss, dehydration, fever, diarrhea, vomiting, poor lactational performance, poor reproduction performance); (2) vaccination of pregnant sows and gilts for protection, prevention, aid in prevention, or aid in control against disease and mortality caused by PEDV in piglets; (3) vaccination of swine 1 day of age or older for protection, prevention, aid in prevention, or aid in control against disease caused by PEDV; (4) booster vaccinations to be administered to sows prior to subsequent farrowings or annually; (5) that healthy swine may be vaccinated with the PEDV MLV and then boostered with PEDV inactivated vaccine; and (6) generally for the protection of swine against weight loss or failure to gain weight caused by PEDV, all and the like.
- PEDV anorexia, weight loss, dehydration, fever, diarrhea, vomiting, poor lactation
- PEDV PEDV
- a killed vaccine a RNA particle vaccine
- Pathogenic changes of selected viruses were evaluated in a neonatal pig model. Sixty piglets (5-day-old), free of PED virus and antibody, were randomly divided into 6 groups with 10 pigs per group. Pigs were fed with milk replacer. Groups 1 through 5 (Gl-5) were orally inoculated with PEDV USA/IN 19338/2013 at the passages 7 (SEQ ID NO:59), 25 (SEQ ID NO:71), 50 (SEQ ID NO:72), 75 (SEQ ID NO:74) and 100 (SEQ ID NO:75), respectively (10 4 TCID50/ml, 10 ml/pig) and Group 6 (G6) received the same volume of virus-free culture media as negative control (Table 6). Clinical observations were recorded.
- Rectal swabs were collected at arrival and daily and tested by a PEDV nucleocapsid gene- based quantitative real-time RT-PCR14.
- Five pigs from each group were necropsied at 3 and 7 days post inoculation (DPI), respectively.
- DPI inoculation
- Gross and microscopic lesions of small intestine, cecum and colon were examined and immunohistochemistry (IHC) staining was performed.
- Whole genome sequences of the viruses in original tissue homogenate as well as at the passages P3, P7, P9, P25, P50, P65, P75 and PI 00 were determined using next-generation sequencing technology [13].
- the negative control piglets remained negative throughout the study period. Piglets inoculated with P7, P25, P50, P75, or PI 00 virus all had watery diarrhea at 1-4 DPI and mild to moderate diarrhea at 5-7 DPI. Piglets inoculated with P7, P25, P50, P75, or PI 00 virus all shed virus in rectal swabs with a trend that piglets inoculated with higher passage virus shed lower amount of viruses (Fig. 18). Specifically, virus shedding levels were: P7 > P25 > P50, P75, PI 00 > Neg Ctrl (> means significantly higher; but no significant differences among P50, P75 and PI 00 groups).
- the villus-height-to-crypt-depth ratios were: P7, P25, P50 ⁇ P75, PI 00 ⁇ Neg Ctrl in jejunum and P7, P25, P50 ⁇ P75, PI 00, Neg Ctrl in ileum at 3 DPI (Fig. 20).
- the IHC staining in jejunum was: P7, P25, P50, P75 > P100 > Neg Ctrl
- the IHC staining in ileum was: P7, P25, P50 > P75, P100, Neg Ctrl; there were no significant differences of IHC scores between various viruses in cecum or colon (Fig. 21). Differences between groups at 7 DPI were not very apparent.
- nucleotide and deduced amino acid changes during serial passages up to P100 are mainly located in replicase nonstructural protein (nsp) 2, nsp3, nsp4, nsp5, nsp6, nspl5, spike (S), ORF3, envelope (E), membrane (M), and nucleocaspid (N) proteins.
- nsp replicase nonstructural protein
- nsp3 replicase nonstructural protein
- E envelope
- M membrane
- N nucleocaspid
- amino acid changes at 15 positions may potentially be associated with virus attenuation during serial passages in cell culture. These 15 positions include: ppla protein 1564Ser>Phe, 1896Thr>Ile, 2600Asn>Tyr,
- sequence alignment techniques are well known so that any amino acid position in a PEDV protein can be mapped to the appropriate amino acid position in other isolates.
- Preferred algorithms and alignment programs for this purpose include ClustalX version 2.0 (Larkin et al, Bioinformatics 23, 2947-2948 (2007)), BioEdit version 7.0.4.1 (Hall, Nucl Acids Symp Ser 41, 95-98 (1999)), and Lasergene software suite (DNASTAR Inc, Madison WI), which includes the Clustal W algorithm for multiple sequence alignment to align sequences and compare sequence similarity and differences.
- Figure 16 shows that the following amino acid changes are among those preferred for generating properly attenuated PEDV isolates, which provide for vaccine efficacy while exhibiting substantial safety: from ORF1 a and b, amino acid positions 814 (Val), 1076 (Val), 1564 (Phe), 1896 (Ilu), 2310 (His), 2600 (Tyr), 3247 (Phe), 3473 (Val), 3522 (Arg); from the Spike gene, amino acid positions 257 (Asn), 326 (lie), 375 (Phe), 491 (Tyr), 881 (Arg), 888 (Arg), 1277 (Phe), 1339 (Thr), 1358 (Leu); from ORF3, corresponding to amino acid position 39 or thereafter, any nucleotide change that provided a stop codon; from genome region E, 69 (He) for the encoded envelope protein; for genome region M, 208 (Thr) for the encoded membrane protein; and for genome region N, 141 (Leu), 418 (Glu), 424 (Asp) and 4
- Figure 24 and Tables 1 and 2 provide certain information concerning amino acid changes that correspond to attenuates of a variant strain USA/IL20697/2014.
- the variant isolate USA/IL20697/2014 was serially passed in cell culture in two independent lineages.
- the virus was passed in cell culture up to 60 th passage (P60); the viruses at different passages were named as 2014020697-P1, 2014020697-P2,
- 2014020697-P3...2014020697-P60 whole genome sequences of the viruses 2014020697-P3 (SEQ ID NO:63), 2014020697-P5 (SEQ ID NO:8), 2014020697-P7 (SEQ ID NO:62), 2014020697-P18 (SEQ ID NO:64), 2014020697-P30 (SEQ ID NO:65), 2014020697-P45 (SEQ ID NO: 39), and 2014020697-P60 (SED ID NO: 66) were determined and compared.
- the virus was serially passed in cell culture and the viruses at some passages were also plaque (colony) purified.
- whole genome sequences were determined and compared for the viruses 2014020697-P3R1 (SEQ ID NO:67), 2014020697-P5R1 (SEQ ID NO:68), 2014020697-P7R1 (SEQ ID NO: 15), 2014020697- P8R1 (SEQ ID NO:35), 2014020697-P18R1 clone 89G8b (SEQ ID NO:36), 2014020697- P18R1 clone 94F6a (SEQ ID NO: 37), and 2014020697-P18R1 clone 92F6a (SED ID NO:37) (it should be noted that P18R1 94F6a and P18R1 92F6a (SEQ ID NO:37) are duplicates of the same clone run for verification purposes, see Tables 1 and 2).
- ORFla/lb at encoded amino acid position 551 provides leucine.
- leucine appears to be almost invariant in PEDV isolates (whether prototype or variant-INDEL) at this position, in that leucine was found to represent residue 551 in approximately 500 randomly selected compared published sequences.
- 2014020697-P18R1 F6a (SEQ ID NO:37), and 2014020697-P18R1 G8b (SEQ ID NO:36), both show remarkable levels of safety and efficacy, it is noted that the 2014020697-P18R1 F6a does provide a further enhancement in both safety and efficacy, making it a highly commercializable material.
- 2014020697-P18R1 F6a provides proline at position 551, an amino acid well known to disrupt or define boundaries between peptide secondary structural domain types, and thus the contribution of Pro 551 to the final phenotype would be expected.
- Glycine may also be substituted for proline at this position in any PEDV strain, in order to improve vaccine safety. It is therefore an embodiment of the invention to provide proline or glycine at this locus generally, in all genomes of PEDV, which are being modified or selected for to provide safe and efficacious vaccines, whether from prototype or variant (INDEL) strains. It should also be noted that the proline/glycine replacement can also be made immediately adjacent to position 1009, such as within about 2-3 amino acid residues thereof, upstream or downstream.
- An additional feature of many of the viruses of the invention that are useful as vaccines is the expression therein of only a truncated ORF3 protein, typically caused by a frameshift mutation in the ORF3 reading frame (see Table 2) and therefore the appearance of a stop codon, or alternatively, the appearance of other features (such as a deleted amino acid, again see Table 2 ) resulting in a partially or wholly inoperative ORF3 protein.
- ORF3 deletions and/or frameshifts in ORF3 protein appear to be associated with adaptation to tissue culture conditions, and therefore it is likely that ORF3 is necessary for overcoming host immune defense, becoming unnecessary under in vitro culture conditions. As such, ORF3 deletions are important for vaccine viruses, contributing to their safety for use in swine.
- Table 2 the truncated ORF3 protein expressed from 2014020697-P18R1 G8b and 2014020697-P18R1 F6a (SEQ ID NO:31), it is noted (Table 2) that this mutation has already been successfully achieved by passage P8R1 (see SEQ ID NO: 18), with the resultant protein sequence being:
- vaccine viruses of the invention include those having SEQ ID NO: 18 as resultant ORF3 protein, or any fragment thereof (10, 20, 30, 40, 50, 60 amino acid residues, and the like), or any fragment of full length ORF3 protein that is less able to inhibit host immune function.
- the objective of the study was to determine the safety and cross protection of specific attenuate passages of PEDV isolates derived from the variant (INDEL) strain of PED, USA/IL/2014-20697, when administered orally to piglets at 3 (+/- 2) days of age (Day 0) and Day 21 followed by a virulent PEDv challenge at Day 35 (+/- 2).
- Safety was determined by the incidence of mortality and clinical signs related to PEDv post inoculation at Day 0 and 21.
- Indication of cross-protection was determined by the incidence of mortality and clinical signs related to virulent PEDv post challenge.
- the tested viruses are those encoded from DNA sequences: (a) SEQ ID NO:36 (designated Clone G6b, which remains unchanged between passages 18 and 19 thereof); (b) SEQ ID NO:37 (designated Clone F6, which remains unchanged between passages 18 and 19 thereof); and (c) from a different set of passages from the USA/IL/2014-2069 ancestor,
- Passage 38 (SEQ ID NO:78) thereof. It should be noted that SEQ ID NOS 36 and 37 differ from each other only in that encoded amino acid position 551 of polyprotein la/lb, is either L or P at position 551 (see immediately after DAT in SEQ ID NO:46).
- TCID50 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.
- 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 actual challenge material (having a concentration of 1x105 TCID50/ 5 ML dose) is closely related to USA/Colorado/2013, GenBank Accession No. KF272920, being specifically a contemporary North American epidemic isolate from the University of Minnesota Veterinary Diagnostic Laboratory, Accession D13-031630, from a farm in Iowa, live material.
- the sample is negative for rotavirus A, B, and C, TGE, Clostridium difficile toxin and Clostridium perfringens.
- PCR assessment of fecal shedding was performed by RTqPCR analysis wherein a reported value of 35 is negative (equal to a control) and values less than 35 are positive for shed virus. The number reported is the number of cycles used for detection, with 35 being the maximum at which non-detection is declared. Protocol followed is generally as otherwise recited in the specification, and is similarly achieved as follows. Fecal swabs are collected in 3 milliliters of DMEM media and stored at -80°C until use. Samples are thawed at 4°C and mixed by vortexing for 5 seconds. One milliliter of sample is removed from tube and placed in a 96-well block and centrifuged at 3200 rpm for 10 minutes to sediment fecal particulate material.
- Cycle threshold values were determined by RT-QPCR analysis using the Path-ID Multiplex One-Step RT-PCR kit (Applied Biosystems/Life Technologies) and PEDV N-gene primers and probe.
- PEDV Forward Primer PEDV N gene-F 5 ' -GAATTCCCAAGGGCGAAAAT-3 ' @ [ 1 OOuM]
- Reverse Primer PEDV N gene-R 5'- TTTTCGAC AAATTCCGC ATCT-3 ' @ [lOOuM]
- PEDV Probe 6FAM 5'-CGTAGCAGCTTGCTTCGGACCCA 3' TAMRA.
- a standard curve is generated with the PEDV N-gene PCR amplicon and Ct values are used to determine a copy number value for each sample based on the standard curve.
- fecal shedding was measured via PCR. Referencing the "35" control value (no shedding), it can be seen that all clones protected the piglets compared to a challenged control that received only media as vaccination. It should be noted that protection was greater for Clone F6a than for clone G8b, consistent with the completely unique proline residue present in this clone (position 551 for Orfla/b protein).
- the vaccines incorporating the SEQ ID NO:36 and 37 viruses are useful irrespective of whether the mother sow is seropositive or seronegative prior to birth of the piglet, and that colostrum derived maternal antibody does not prevent the present vaccines from being effective, irrespective of whether the mother sow was previously infected with a wild strain, a vaccine strain (including being infected thereby from the piglet), or was vaccinated with a live or killed vaccine, in either case one or more times, including pre- breeding and pre-farrowing.
- a second dose to the piglet can also be given, if desired by the producer.
- representative examples of vaccination programs useful in the practice of the invention include: a method of treating or preventing disease in a piglet caused by PEDV, comprising administering to said piglet a first dose of the vaccine composition when said piglet is about 1-7 days old, further or optionally, administering a second dose of said vaccine when the piglet is about 2-5 weeks old.
- the parent sow can be vaccinated pre- breeding or pre-farrowing in the alternative, or at both said time points, irrespective or whether the mother sow was already seropositive (such was from previous infection).
- the mother sow is seronegative
- the objective of the present study was thus to determine the safety and cross protection of two passaged PEDV isolates (SEQ ID NOS: 36 and 37) derived from the variant (INDEL) strain of PED, USA/IL/2014-20697, when administered orally to piglets at 1 days of age (Day 0) followed by a virulent PEDv challenge at Day 21 (22 days of age).
- Safety will be determined by the incidence of mortality and clinical signs related to PEDv post inoculation.
- Indication of cross-protection was determined by the incidence of mortality and clinical signs and intestinal lesions related to virulent PEDv post challenge.
- Vaccination doses are lxlO 4 TCID 50 using 2ML per oral doses.
- TCID50 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 TCID50, 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).
- Piglets within each group were further assigned to one of three outcomes, sacrificed for necropsy on Day 6, sacrificed for necropsy on Day 28, or the piglets complete the study and are reported out to Day 40.
- the actual challenge material (having a concentration of lxlO 5 TCID50/ 5 ML dose) is closely related to USA/Colorado/2013, GenBank Accession No. KF272920, being specifically a contemporary North American epidemic isolate from the University of Minnesota Veterinary Diagnostic Laboratory, Accession D13-031630, from a farm in
- the sample is negative for rotavirus A, B, and C, TGE, Clostridium difficile toxin and Clostridium perfringens.
- fecal shedding scores in the Table below. Dates below are calculated from Day 0 (i.e. 1 day of age, the date of vaccination with G8b or F6a attenuated virus).
- PCR assessment of fecal shedding was performed by RTqPCR analysis wherein a reported value of 40 is negative (equal to a control) and values less than 40 are positive for shed virus. The number reported is the number of cycles used for detection, with 40 being the maximum at which non-detection is declared. Protocol followed is generally as otherwise recited in the specification, and is similarly achieved as follows. Fecal swabs are collected in 3 milliliters of DMEM media and stored at -80°C until use. Samples are thawed at 4°C and mixed by vortexing for 5 seconds. One milliliter of sample is removed from tube and placed in a 96-well block and centrifuged at 3200 rpm for 10 minutes to sediment fecal particulate material.
- Cycle threshold values were determined by RT-QPCR analysis using the Path-ID Multiplex One-Step RT-PCR kit (Applied Biosystems/Life Technologies) and PEDV N-gene primers and probe.
- PEDV Forward Primer PEDV N gene-F 5 ' -GAATTCCC AAGGGCGAAAAT-3 ' @ [lOOuM]
- Reverse Primer PEDV N gene-R 5'- TTTTCGAC AAATTCCGC ATCT-3 ' @ [lOOuM]
- PEDV Probe 6FAM 5'-CGTAGCAGCTTGCTTCGGACCCA 3' TAMRA.
- a standard curve is generated with the PEDV N-gene PCR amplicon and Ct values are used to determine a copy number value for each sample based on the standard curve.
- PEDV Porcine epidemic diarrhea virus
- Eighty-five 3 -week-old pigs were purchased from a conventional breeding farm and delivered to the Iowa State University Laboratory Animal Resources facilities. All pigs were intramuscularly injected with a dose of Excede® upon arrival and confirmed negative for PEDV, porcine deltacoronavirus, and transmissible gastroenteritis virus by virus-specific PCRs on rectal swabs and negative for PEDV antibody by a virus-specific indirect fluorescent antibody (IF A) assay on serum samples.
- IF A indirect fluorescent antibody
- Pigs were blocked by weight and then randomly divided into 7 groups with 15 or 10 pigs per group (Table 11). Pigs were orogastrically inoculated with virus-negative culture medium (N), PEDV prototype isolate USA/IN19338/2013-P7 (P-prototype strain), or PEDV S-INDEL-variant isolate USA/IL20697/2014-P7R1 (V-variant strain) at Day 0 (DO) followed by challenge at D28. Inoculation at each point used 10ml of inoculum per pig, containing 104 TCID50/ml for virus inocula.
- N virus-negative culture medium
- PEDV prototype isolate USA/IN19338/2013-P7 P-prototype strain
- PEDV S-INDEL-variant isolate USA/IL20697/2014-P7R1 V-variant strain
- Inocula 10 4 TCID 5 o/ml, 10 ml per pig for either the Prototype or the Variant isolates; or 10 ml culture medium per pig for Neg Control.
- Serum samples were collected at DO, 7, 14, 21, 28, 35, 42, 49 and 56 and tested by indirect fluorescent antibody (IF A) assay and virus neutralization (VN) assay using two PEDV strains as indicator viruses, respectively.
- IF A indirect fluorescent antibody
- VN virus neutralization
- Small intestines were collected for histopathology evaluation and immunohistochemistry (IHC) staining.
- GLIMMIX Generalized linear mixed
- the Neg control pigs did not shed any virus in rectal swabs during D0-D28 as tested by quantitative PEDV rRT-PCR.
- Fecal shedding in the P-strain-inoculated groups reached the peak level at D4 and then gradually declined.
- fecal shedding in the V-strain-inoculated groups gradually increased in the first a few days and reached the peak level at D7.
- the N/P and N/N group did not have diarrhea while the N/V group developed watery diarrhea during D33-D38 (5-10 days post 2nd inoculation). No any virus was detected from the N/N group in rectal swabs from D28 to D56.
- Five pigs necropsied from the N/N, N/V and N/P groups at D34 (6 days post the 2nd inoculation) were compared to evaluate the macro- and micro-pathological lesions caused by the P-strain and V-strain in 7-week-old pigs.
- Fecal virus shedding was compared among 7 groups after the 2nd inoculation at D28 (Fig 1).
- V/V and P/V groups some minor macro-pathological changes were only observed in the N/V groups but were not apparent in the V/V and P/V groups.
- the average content scores and organ lesions in small intestines were not significantly different among N/V, V/V and P/V groups. But the average content scores in ceca and colons as well as the average lesion scores in colons were significantly lower in the V/V and P/V groups than the N/V group (Fig 29B).
- V/V and P/V groups had numerically higher average VH/CD ratios in distal jejunums and ileums than the N/V group; however, significant differences were only observed between the N/V and V/V groups in distal jejunum and ileum VH/CD ratios as well as between the V/V and P/V groups in distal jejunum VH/CD ratios (Fig 31 A).
- the V/V and P/V groups both had significantly lower average IHC scores in distal jejunums and ileums than the N/V group (Fig 3 IB).
- U.S. PEDV prototype strain achieved higher levels of fecal virus shedding than S-INDEL-variant strain in 3-week-old pigs but the opposite was observed in 7-week-old pigs. However, this observation needs to be further confirmed to determine optimal ages at which Prototype and INDEL vaccines provide peak efficacy in older pigs.
- U.S. PEDV prototype strain provided protection against challenge with both homologous and heterologous strains in weaned pigs;
- U.S. PEDV S-INDEL-variant strain provided protection against homologous strain challenge and at least partial protection against heterologous (prototype) strain challenge in weaned pigs.
- SEQ ID NO: 10 Whole genome 2013022038-P3
- SEQ ID NO: 12 Whole genome 2013049379-P3
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Virology (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Communicable Diseases (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Epidemiology (AREA)
- Immunology (AREA)
- Mycology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Oncology (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Pulmonology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
Claims
Priority Applications (8)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201680025487.6A CN108064165B (en) | 2015-02-27 | 2016-02-26 | Porcine epidemic diarrhea virus strains and immunogenic compositions produced therefrom |
AU2016225119A AU2016225119B2 (en) | 2015-02-27 | 2016-02-26 | Porcine Epidemic Diarrhea Virus strains and immunogenic compositions therefrom |
JP2017544865A JP6824177B2 (en) | 2015-02-27 | 2016-02-26 | Porcine epidemic diarrhea virus strain and immunogenic composition obtained from it |
CA2977980A CA2977980C (en) | 2015-02-27 | 2016-02-26 | Porcine epidemic diarrhea virus strains and immunogenic compositions therefrom |
US15/556,446 US11058763B2 (en) | 2015-02-27 | 2016-02-26 | Porcine epidemic diarrhea virus strains and immunogenic compositions therefrom |
EP16756474.9A EP3261668A4 (en) | 2015-02-27 | 2016-02-26 | Porcine epidemic diarrhea virus strains and immunogenic compositions therefrom |
HK18106191.4A HK1246658A1 (en) | 2015-02-27 | 2018-05-14 | Porcine epidemic diarrhea virus strains and immunogenic compositions therefrom |
US17/021,261 US12005113B2 (en) | 2015-02-27 | 2020-09-15 | Porcine epidemic diarrhea virus strains and immunogenic compositions therefrom |
Applications Claiming Priority (8)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201562121955P | 2015-02-27 | 2015-02-27 | |
US62/121,955 | 2015-02-27 | ||
US201562209119P | 2015-08-24 | 2015-08-24 | |
US62/209,119 | 2015-08-24 | ||
US201562250961P | 2015-11-04 | 2015-11-04 | |
US62/250,961 | 2015-11-04 | ||
US201662276022P | 2016-01-07 | 2016-01-07 | |
US62/276,022 | 2016-01-07 |
Related Child Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US15/556,446 A-371-Of-International US11058763B2 (en) | 2015-02-27 | 2016-02-26 | Porcine epidemic diarrhea virus strains and immunogenic compositions therefrom |
US17/021,261 Continuation US12005113B2 (en) | 2015-02-27 | 2020-09-15 | Porcine epidemic diarrhea virus strains and immunogenic compositions therefrom |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2016138421A1 true WO2016138421A1 (en) | 2016-09-01 |
Family
ID=56789331
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2016/019846 WO2016138421A1 (en) | 2015-02-27 | 2016-02-26 | Porcine epidemic diarrhea virus strains and immunogenic compositions therefrom |
Country Status (9)
Country | Link |
---|---|
US (2) | US11058763B2 (en) |
EP (1) | EP3261668A4 (en) |
JP (2) | JP6824177B2 (en) |
CN (2) | CN113769079A (en) |
AU (1) | AU2016225119B2 (en) |
CA (1) | CA2977980C (en) |
HK (1) | HK1246658A1 (en) |
TW (1) | TWI619812B (en) |
WO (1) | WO2016138421A1 (en) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US10034934B2 (en) | 2014-03-11 | 2018-07-31 | Regents Of The University Of Minnesota | Porcine epidemic diarrhea virus vaccines and methods of use thereof |
CN108586609A (en) * | 2018-05-24 | 2018-09-28 | 青岛博隆基因工程有限公司 | A kind of monoclonal antibody of porcine epidemic diarrhea resisting virus and application |
WO2018188639A1 (en) * | 2017-04-13 | 2018-10-18 | 浙江海隆生物科技有限公司 | Porcine epidemic diarrhea virus s protein, subunit vaccine thereof, and preparation method for and application of the subunit vaccine |
KR20200121447A (en) * | 2019-04-16 | 2020-10-26 | 주식회사 바이오앱 | Vaccine composition for preventing porcine epidemic diarrhea and manufacturing method thereof |
Families Citing this family (31)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CA2954632C (en) * | 2014-07-11 | 2022-08-30 | Zoetis Services Llc | Novel vaccine compositions for porcine epidemic diarrhea virus and porcine deltacoronavirus |
WO2016138421A1 (en) * | 2015-02-27 | 2016-09-01 | Iowa State University Research Foundation, Inc. | Porcine epidemic diarrhea virus strains and immunogenic compositions therefrom |
CN108822191B (en) | 2017-04-13 | 2023-07-07 | 浙江海隆生物科技有限公司 | Porcine epidemic diarrhea virus S protein and subunit vaccine thereof, and preparation method and application thereof |
CN108315490B (en) * | 2018-04-20 | 2022-03-11 | 咸阳职业技术学院 | RT-PCR (reverse transcription-polymerase chain reaction) diagnostic kit for porcine acute diarrhea syndrome coronavirus and delta coronavirus and detection method thereof |
TWI744654B (en) * | 2018-06-25 | 2021-11-01 | 中央研究院 | Baculovirus and composition for detection and preventing porcine epidemic diarrhea virus infection |
US11013796B2 (en) | 2018-08-30 | 2021-05-25 | Iowa State University Research Foundation, Inc. | Porcine parainfluenza virus type 1 isolates and immunogenic compositions therefrom |
KR20210062015A (en) * | 2018-09-20 | 2021-05-28 | 베링거잉겔하임베트메디카게엠베하 | Modified PEDV Spike Protein |
CN109439799A (en) * | 2018-11-06 | 2019-03-08 | 江西农业大学 | It is a kind of for detecting the sleeve type PCR primer and kit of pig acute diarrhea coronavirus |
US11413347B2 (en) | 2019-09-02 | 2022-08-16 | Reber Genetics Co., Ltd. | Vaccine composition and method of preventing porcine epidemic diarrhea virus infection in swine |
TWI703983B (en) * | 2019-09-25 | 2020-09-11 | 台灣糖業股份有限公司 | Porcine epidemic diarrhea virus ts isolate and uses of the same |
AU2021313130A1 (en) | 2020-07-20 | 2023-02-09 | Enanta Pharmaceuticals, Inc. | Functionalized peptides as antiviral agents |
CN112175086B (en) * | 2020-10-13 | 2022-01-21 | 江西农业大学 | Monoclonal antibody of anti-porcine epidemic diarrhea virus nsp13 protein and application |
CN114438040A (en) * | 2020-11-04 | 2022-05-06 | 中山大学 | Monoclonal cell strain secreting antibody with PEDV (porcine epidemic diarrhea Virus) neutralizing activity and application thereof |
US11352363B1 (en) | 2020-11-23 | 2022-06-07 | Enanta Pharmaceuticals, Inc. | Spiropyrrolidine derived antiviral agents |
US11384090B2 (en) | 2020-11-23 | 2022-07-12 | Enanta Pharmaceuticals, Inc. | Spiropyrrolidine derived antiviral agents |
KR20230124583A (en) | 2020-11-23 | 2023-08-25 | 이난타 파마슈티칼스, 인코포레이티드 | Novel spiropyrrolidine-derived antiviral agent |
CN112680421B (en) * | 2021-02-01 | 2022-09-13 | 中国农业科学院哈尔滨兽医研究所(中国动物卫生与流行病学中心哈尔滨分中心) | Monoclonal antibody with broad spectrum neutralizing porcine epidemic diarrhea virus and application thereof |
WO2022235605A1 (en) | 2021-05-04 | 2022-11-10 | Enanta Pharmaceuticals, Inc. | Novel macrocyclic antiviral agents |
US11319325B1 (en) | 2021-05-11 | 2022-05-03 | Enanta Pharmaceuticals, Inc. | Macrocyclic spiropyrrolidine derived antiviral agents |
CN113633763B (en) * | 2021-06-28 | 2023-04-28 | 南华大学 | Novel coronavirus S1-E vaccine and preparation method thereof |
US11339170B1 (en) | 2021-07-23 | 2022-05-24 | Enanta Pharmaceuticals, Inc. | Spiropyrrolidine derived antiviral agents |
CN113527477B (en) * | 2021-07-23 | 2023-05-23 | 河南农业大学 | Swine-derived anti-PDCoV-N protein scFv, expression vector, construction method and application thereof |
US11325916B1 (en) | 2021-07-29 | 2022-05-10 | Enanta Pharmaceuticals, Inc. | Spiropyrrolidine derived antiviral agents |
CN113583141B (en) * | 2021-08-04 | 2023-03-03 | 江西农业大学 | Swine epidemic diarrhea virus Nsp9 protein, fusion protein containing Nsp9 protein, and preparation method and application thereof |
WO2023086350A1 (en) | 2021-11-12 | 2023-05-19 | Enanta Pharmaceuticals, Inc. | Alkyne-containing antiviral agents |
US11912714B2 (en) | 2021-11-12 | 2024-02-27 | Enanta Pharmaceuticals, Inc. | Spiropyrrolidine derived antiviral agents |
WO2023086400A1 (en) | 2021-11-12 | 2023-05-19 | Enanta Pharmaceuticals, Inc. | Novel spiropyrrolidine derived antiviral agents |
US11993600B2 (en) | 2021-12-08 | 2024-05-28 | Enanta Pharmaceuticals, Inc. | Saturated spirocyclics as antiviral agents |
CN114773458A (en) * | 2022-04-19 | 2022-07-22 | 军事科学院军事医学研究院军事兽医研究所 | Polyclonal antibody of porcine epidemic diarrhea virus Nsp15 protein and preparation method thereof |
CN117143207B (en) * | 2023-09-05 | 2024-03-12 | 湖南派智生物科技有限公司 | Recombinant epitope protein, encoding gene, recombinant plasmid, preparation method, engineering bacteria and application |
CN118147173A (en) * | 2024-05-11 | 2024-06-07 | 华南农业大学 | MRNA based on porcine epidemic diarrhea virus S protein gene, application and vaccine |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103861098A (en) * | 2014-03-07 | 2014-06-18 | 青岛易邦生物工程有限公司 | Bivalent vaccine for preventing porcine epidemic diarrhea and Escherichia coli |
CN104262488A (en) * | 2014-09-24 | 2015-01-07 | 普莱柯生物工程股份有限公司 | Preparation and application of fusion protein and vaccine composition thereof |
WO2015153425A1 (en) * | 2014-04-03 | 2015-10-08 | Boehringer Ingelheim Vetmedica, Inc. | Porcine epidemic diarrhea virus vaccine |
Family Cites Families (15)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5084269A (en) | 1986-11-06 | 1992-01-28 | Kullenberg Fred W | Adjuvant for dose treatment with antigens |
MX9301736A (en) | 1992-03-30 | 1994-01-31 | Smithkline Beecham Corp | PASTEURELLA HAEMOLYTICA TYPE A-1 BACTERINE-TOXOID VACCINE. |
AU769539B2 (en) | 1999-01-29 | 2004-01-29 | Zoetis Services Llc | Adjuvants for use in vaccines |
US6974577B2 (en) | 2001-02-04 | 2005-12-13 | Novartris Ag | Inactivated bovine scours vaccines, processes and method of preventing bovine scours |
EP1613346B8 (en) | 2003-04-04 | 2013-01-09 | Pah Usa 15 Llc | Microfluidized oil-in-water emulsions and vaccine compositions |
US8057993B2 (en) * | 2003-04-26 | 2011-11-15 | Ibis Biosciences, Inc. | Methods for identification of coronaviruses |
KR101484469B1 (en) | 2010-10-26 | 2015-01-21 | 주식회사 중앙백신연구소 | Mixed vaccine comprising the whole virus of pcv-2 for preventing porcine circovirus associated diseases |
CN104152578B (en) | 2013-06-03 | 2016-03-30 | 中国农业科学院上海兽医研究所 | Porcine epidemic diarrhea virus RT-PCR differential diagnosis kit and application thereof |
EP3116539B1 (en) * | 2014-03-11 | 2018-10-10 | Regents of the University of Minnesota | Porcine epidemic diarrhea virus vaccines and methods of use thereof |
US9713639B2 (en) | 2014-05-19 | 2017-07-25 | Merial, Inc. | Recombinant spike protein subunit based vaccine for porcine epidemic diarrhea virus (PEDV) |
WO2016007955A1 (en) * | 2014-07-11 | 2016-01-14 | Merial, Inc. | Inactivated vaccine for porcine epidemic diarrhea virus (pedv) |
CA2954632C (en) | 2014-07-11 | 2022-08-30 | Zoetis Services Llc | Novel vaccine compositions for porcine epidemic diarrhea virus and porcine deltacoronavirus |
CN107075487A (en) | 2014-08-08 | 2017-08-18 | 乌得勒支大学控股有限公司 | The Porcine epidemic diarrhea virus of mutation is used for the purposes in vaccine |
CN104383528B (en) | 2014-10-24 | 2016-08-24 | 江苏省农业科学院 | Pig epidemic diarrhea inactivated vaccine and preparation method thereof |
WO2016138421A1 (en) * | 2015-02-27 | 2016-09-01 | Iowa State University Research Foundation, Inc. | Porcine epidemic diarrhea virus strains and immunogenic compositions therefrom |
-
2016
- 2016-02-26 WO PCT/US2016/019846 patent/WO2016138421A1/en active Application Filing
- 2016-02-26 US US15/556,446 patent/US11058763B2/en active Active
- 2016-02-26 CN CN202110533880.9A patent/CN113769079A/en active Pending
- 2016-02-26 EP EP16756474.9A patent/EP3261668A4/en not_active Withdrawn
- 2016-02-26 JP JP2017544865A patent/JP6824177B2/en active Active
- 2016-02-26 AU AU2016225119A patent/AU2016225119B2/en active Active
- 2016-02-26 CA CA2977980A patent/CA2977980C/en active Active
- 2016-02-26 CN CN201680025487.6A patent/CN108064165B/en active Active
- 2016-03-01 TW TW105106129A patent/TWI619812B/en active
-
2018
- 2018-05-14 HK HK18106191.4A patent/HK1246658A1/en unknown
-
2020
- 2020-09-15 US US17/021,261 patent/US12005113B2/en active Active
-
2021
- 2021-01-08 JP JP2021002183A patent/JP2021072798A/en active Pending
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103861098A (en) * | 2014-03-07 | 2014-06-18 | 青岛易邦生物工程有限公司 | Bivalent vaccine for preventing porcine epidemic diarrhea and Escherichia coli |
WO2015153425A1 (en) * | 2014-04-03 | 2015-10-08 | Boehringer Ingelheim Vetmedica, Inc. | Porcine epidemic diarrhea virus vaccine |
CN104262488A (en) * | 2014-09-24 | 2015-01-07 | 普莱柯生物工程股份有限公司 | Preparation and application of fusion protein and vaccine composition thereof |
Non-Patent Citations (4)
Title |
---|
CHEN ET AL.: "Evaluation of serological cross-reactivity and cross-neutralization between the United States porcine epidemic diarrhea virus prototype and S-INDEL-variant strains", BMC VETERINARY RESEARCH, vol. 12, 5 April 2016 (2016-04-05), pages 1 - 10, XP055477488 * |
See also references of EP3261668A4 * |
WANG ET AL.: "Porcine Epidemic Diarrhea Virus Variants with High Pathogenicity, China", EMERGING INFECTIOUS DISEASES, vol. 19, no. 12, 1 December 2013 (2013-12-01), pages 2048 - 2049, XP055477486 * |
ZHANG, Y. ET AL.: "New Variant of Porcine Epidemic Diarrhea Virus, United States , 2014", EMERGING INFECTIOUS DISEASES, vol. 20, no. 5, 1 May 2014 (2014-05-01), pages 917 - 919, XP055165576, DOI: doi:10.3201/eid2005.140195 * |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US10034934B2 (en) | 2014-03-11 | 2018-07-31 | Regents Of The University Of Minnesota | Porcine epidemic diarrhea virus vaccines and methods of use thereof |
WO2018188639A1 (en) * | 2017-04-13 | 2018-10-18 | 浙江海隆生物科技有限公司 | Porcine epidemic diarrhea virus s protein, subunit vaccine thereof, and preparation method for and application of the subunit vaccine |
CN108586609A (en) * | 2018-05-24 | 2018-09-28 | 青岛博隆基因工程有限公司 | A kind of monoclonal antibody of porcine epidemic diarrhea resisting virus and application |
CN108586609B (en) * | 2018-05-24 | 2021-10-08 | 青岛博隆基因工程有限公司 | Monoclonal antibody for resisting porcine epidemic diarrhea virus and application |
KR20200121447A (en) * | 2019-04-16 | 2020-10-26 | 주식회사 바이오앱 | Vaccine composition for preventing porcine epidemic diarrhea and manufacturing method thereof |
KR102213745B1 (en) | 2019-04-16 | 2021-02-09 | 주식회사 바이오앱 | Vaccine composition for preventing porcine epidemic diarrhea and manufacturing method thereof |
Also Published As
Publication number | Publication date |
---|---|
HK1246658A1 (en) | 2018-09-14 |
AU2016225119B2 (en) | 2021-06-03 |
TWI619812B (en) | 2018-04-01 |
US12005113B2 (en) | 2024-06-11 |
CN108064165A (en) | 2018-05-22 |
EP3261668A4 (en) | 2018-10-24 |
CA2977980A1 (en) | 2016-09-01 |
CN113769079A (en) | 2021-12-10 |
JP2018507691A (en) | 2018-03-22 |
US20220088179A1 (en) | 2022-03-24 |
EP3261668A1 (en) | 2018-01-03 |
US20180064804A1 (en) | 2018-03-08 |
JP6824177B2 (en) | 2021-02-03 |
TW201641689A (en) | 2016-12-01 |
CA2977980C (en) | 2022-04-26 |
CN108064165B (en) | 2022-06-14 |
US11058763B2 (en) | 2021-07-13 |
AU2016225119A1 (en) | 2017-09-07 |
JP2021072798A (en) | 2021-05-13 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US12005113B2 (en) | Porcine epidemic diarrhea virus strains and immunogenic compositions therefrom | |
JP5890469B2 (en) | Highly pathogenic pig breeding / respiratory syndrome (HPPRRS) vaccine | |
Liu et al. | A newly isolated Chinese virulent genotype GIIb porcine epidemic diarrhea virus strain: biological characteristics, pathogenicity and immune protective effects as an inactivated vaccine candidate | |
US9579373B2 (en) | Porcine reproductive and respiratory syndrome virus, compositions, vaccine and methods of use | |
JP7129978B2 (en) | Effective pre-weaning vaccination against European strains of porcine reproductive and respiratory syndrome (PRRS) virus | |
US11124777B2 (en) | Attenuated porcine sapelovirus strain and immunogenic compositions therefrom | |
US20160008457A1 (en) | Inactivated Vaccine for Porcine Epidemic Diarrhea Virus (PEDV) | |
US20220249650A1 (en) | Senecavirus a virus strains and immunogenic compositions therefrom | |
RU2817873C2 (en) | Prime-boost vaccination schedule | |
US20220023414A1 (en) | Prime-Boost Vaccination Regimen |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 16756474 Country of ref document: EP Kind code of ref document: A1 |
|
ENP | Entry into the national phase |
Ref document number: 2017544865 Country of ref document: JP Kind code of ref document: A |
|
ENP | Entry into the national phase |
Ref document number: 2977980 Country of ref document: CA |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
ENP | Entry into the national phase |
Ref document number: 2016225119 Country of ref document: AU Date of ref document: 20160226 Kind code of ref document: A |
|
WWE | Wipo information: entry into national phase |
Ref document number: 15556446 Country of ref document: US |
|
REEP | Request for entry into the european phase |
Ref document number: 2016756474 Country of ref document: EP |