WO2017123201A1

WO2017123201A1 - Novel cross protective vaccine compositions for porcine epidemic diarrhea virus

Info

Publication number: WO2017123201A1
Application number: PCT/US2016/012899
Authority: WO
Inventors: Marta Cabana Sumsi; Monica Balasch Sanuy; Laia PLAJA DILMÉ; Alicia Urniza Hostench; Paul J. Dominowski; Jay Gregory Calvert
Original assignee: Zoetis Services Llc
Priority date: 2016-01-11
Filing date: 2016-01-11
Publication date: 2017-07-20
Also published as: EP3402878A1; CA3010977A1

Abstract

The present invention is directed to novel isolates of porcine epidemic diarrhea virus (PEDV) and vaccine compositions made therefrom. The vaccine compositions protect swine from PED disease, and are cross protective against a wide variety of strains, whether variant or prototype, that currently circulate throughout the world. The vaccines are provided in both killed and live virus forms, as derived from strain Calaf14, and suggest specific amino acid modifications to the encoding sequences of all other PEDV strains that may improve their performance as vaccines.. Novel culture methods are also employed to increase reproducible yield of cultured viruses.

Description

Novel Cross Protective Vaccine Compositions for Porcine Epidemic Diarrhea Virus

Field of the Invention

The present invention is directed to novel vaccine compositions that protect swine from disease caused by porcine epidemic diarrhea virus (PEDV). The vaccine compositions are both safe and efficacious, and are based on novel attenuates of PEDV that provide cross protection against a wide variety of PEDV strains, and are safe to use even in live form.

Background of the Invention

Porcine epidemic diarrhea (PED) is highly contagious and is characterized by dehydration, diarrhea, and high mortality in swine, particularly young piglets. The causative agent, porcine epidemic diarrhea virus (PEDV), is a single stranded, positive sense RNA virus identified to the Alphacoronoavirus genus of the family Coronaviridae. PEDV has a total genome size of approximately 28kb and contains 7 open reading frames. Symptoms of PEDV infection are often similar to those caused by transmissible gastroenteritis virus (TGEV), also a member of the Coronaviridae. It should be noted that cross protection between PEDV and TGEV is not generally observed, the overall viral nucleotide sequences being at most about 60% similar.

PED was likely first observed in Europe circa 1970, and the causative virus was subsequently characterized (see for example M. Pensaert et al. Arch. Virol, v. 58, pp 243- 247, 1978 and D. Chasey et al., Res. Vet Sci, v. 25, pp 255-256, 1978). PED disease was generally considered unknown in North America until 2013, at which point widespread outbreaks commenced, and severe economic losses to the swine industry resulted. These prototype North American isolates (year 2013 and thereafter) have remained genetically closely related (i.e. with overall nucleotide identity generally over 99%), and are similar to Asian strains characterized there within a few years prior to the North American outbreaks. PEDV generally grows poorly in culture, and there is a need to identify both particular strains and culture conditions that are appropriate for the culturing of sufficient virus for commercial vaccine preparation. Additionally, there is a need to develop vaccines that provide effective cross protection against known isolates of PEDV, and which are expected to provide effective cross protection against evolving, non-prototype PEDV strains.

Additionally, additional variant strains of PEDV (termed "variant" or "IN DEL" strains, see immediately below) have also been recently identified in North America and Europe, and are among themselves closely related, and are recognizably different from the aforementioned "prototype" North American strains and classical European strains. Such variant strains (based in part on "S" or spike protein sequence) also appeared earlier in Asia, and these Asian isolates are again more similar North American or European variant/INDEI strains than to prototype strains. A well known North American prototype strain, associated with outbreaks of disease beginning in 2013 is USA/Colorado/2013, whose sequence is deposited as GenBank accession No. KF272920, of the NCBI of the United States National Institutes of Health. Bethesda, MD). In this regard, see also A. Vlasova et al. "Distinct Characteristics and Complex Evolution of PEDV Strains, North America, May 2013-February 2014", Emerging Infectious Disease, Vol 20, No. 10, 2014. Original reports from Asia of variant/ INDEL strains include D. S. Song et al., Research in Veterinary Science, v 82, pp. 134-140, 2007; S-J Park et al., Virus Genes, , v 35, pp..55-64, 2007; and further discussion thereof by D. Song et al. (Virus Genes (2012) v 44 pp. 167-175) referring to the DR13 strain, passaged to level 100, and previously licensed in Korea (see also KR patent 0502008).

Accordingly, there is a need to identity both vaccine strains and appropriate vaccine compositions that will be effective against current and emerging worldwide outbreaks of PEDV, thus providing needed cross protection.

Further concerning the recent emergence of variant/ INDEL strains throughout the world, a very early report of such a North American variant strain is PEDV-INDEL (OH851) first isolated by the Ohio Department of Agriculture (see L. Wang et al., Emerg. Infect. Dis., 2014, v. 20, pp. 917-919, see GenBank KJ399978). OH851 is reproduced as SEQ ID NO:7 in the present patent application. Typically, it appears that circulating variant strains

(whether Asian, North American or European) have, as one feature, insertions and deletions in the spike gene (S-INDELS), but such strains nonetheless share about 98-100% identity at a nucleotide level (spike gene, and the overall genome), but such recent isolates only present about 96-97% identity, or lower, at the nucleotide level (spike gene and overall genome) with initial (prototype) North American strains (for example USA/Colorado/2013).

The very first public disclosure of North American S-INDELs may be that of the Iowa State University Veterinary Diagnostic Laboratory, on January 30, 2014, defined such isolates as having having only 93.9-94.6% identity to previously identified USA prototype strains, but being nearly identical (99+%) to each other. Useful insertions and deletions need not be confined to the spike gene. ORF3 modifications (particularly mutations causing truncation of the encoded protein) have been correlated with adaptation to cell culture and reduction of pathogenicity. See S-J. Park etal., Virus Genes, 2008, v 36, pp. 95-104. Others have commented that classification of PEDVs based on ORF3 may be appropriate (J. Zhang et al. Journal of Clinical Microbiology, v. 52(9), pp. 351 1-3514, 2014). T. Oka et al.,

Veterinary Microbiology, 173, pp 258-269 (2014) disclose additional S-INDEL strains, and interestingly, a PEDV strain related instead to prototype virulent strains, but also bearing a large 197 amino acid deletion from the S protein, possibly resulting from passaging.

INDEL-type strains that have recently been reported from Europe include those described by S. Theuns et al. (2015). "Complete genome sequence of a porcine epidemic diarrhea virus from a novel outbreak in Belgium, January 2015." Genome Announcements 3(3), pp. 1-2, May/June 2015; J. Stadler, et al., "Emergence of porcine epidemic diarrhea virus in southern Germany." BMC Veterinary Research, v 11 No.142; pages 1-8, 2015; and. B. Grasland, et al. "Complete genome sequence of a porcine epidemic diarrhea S-Gene Indel strain isolated in France in December 2014." Genome Announcements 3(3), pp. 1-2, May/June 2015.

It should be noted that variant and prototype strains are co- circulating in North America, and elsewhere, and existing populations of strains may result from multiple transmissions across continents or other regions.

Although the so-called variant strains may be less virulent than prototype strains, at least as to some age groups of swine, remaining virulence still makes such viruses unsafe for use in vaccines, if used in live form. Generally to date, attempts to passage prototype strains to avirulence have not been successful, and adequate safety is not achieved after well over 100 passages. The present invention is directed to novel mutant isolates of the variant European strain Calaf14 (see SEQ ID NO:1 herein, and PCT/US 2015/039475 generally, in regard of the wild type) that have been attenuated so that they can be safely administered to swine of all ages, without harm to the animals, and at the same time, are highly immunogenic and cross protective against subsequent challenge of the animals by a wide variety of PEDV strains, including both prototype and variant strains from all Continents. Vaccines from such mutant isolates, whether in killed or live form, are useful to protect swine from PEDV on a worldwide basis. Summary of the Invention

The present invention is directed to novel mutant isolates of Calf 14 PEDV virus that can be safely administered to swine of all ages, without harm to the animals, and at the same time, are highly immunogenic and cross protective against subsequent challenge of the animals by a wide variety of PEDV strains, including both prototype and variant strains.

Vaccines from such mutant isolates, whether in killed or live form, are useful to protect swine from PEDV on a worldwide basis.

The present invention therefore encompasses an vaccine composition comprising inactivated mutant Calaf14 PEDV, one or more adjuvants, and optionally one or more excipients, in an amount effective to elicit production of neutralizing antibodies in swine, with good cross protection against subsequent challenge by both prototype and variant strain PEDV strains that circulate in Nature.. The adjuvant preferably provides an oil-in-water emulsion with additional components. The vaccine compositions of the invention protect swine from infection by PEDV, and are effective in single doses, in two-dose programs, or in vaccination programs involving multiple doses, which may be spread apart by at least a week, and optionally at greater intervals of time, such as one to several months.

The present invention similarly provides vaccine compositions comprising the aforementioned mutant isolates of Calaf14 PEDV, as live vaccines, with our without adjuvants, that are also highly effective and provide good cross protection against subsequent challenge by both prototype and variant strain PEDV strains that circulate in Nature.

It should be noted that depending on the level of epidemic threat in a particular swine population, the vaccine dose program of one, two, or multiple doses may be repeated, from time to time, as a precautionary measure. Additionally, it should be noted that vaccinating a mother sow during pregnancy will provide protection to a young piglet, via maternal transfer of antibodies and T-cells in milk, although such protection may need to be followed up with additional vaccination doses to the piglet. Vaccination of all swine including piglets and adults is contemplated.

Thus, according to the practice of the present invention, there are provided vaccines against PEDV based on inactivated virus and modified live virus

Additionally, the immunogenic composition can comprise other swine antigens, including Escherichia coli and Clostridium perfringens, types A-D, the dosages of which would be equivalent to those found in the commercially-available vaccines, Gletvax® and Litterguard®. The vaccines can contain one or more adjuvants, and optionally one or more excipients, in an amount effective to elicit production of neutralizing antibodies in swine. The adjuvant preferably provides an oil-in-water emulsion with additional components. The immunogenic compositions of the invention protect swine from infection by PEDV are effective in single doses, in two-dose programs, or in vaccination programs involving multiple doses, which may be spread apart by at least a week, and optionally at greater intervals of time, such as one to several months.

Preferably, the PEDV vaccines of the present invention are comprise any of the novel viruses as disclosed herein, such as those encoded by a polynucleotide selected from:

(a) the group consisting of SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6; or

(b) a PEDV virus that is encoded by a nucleotide sequence that is at least 90% identical to one or more of SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 at a full length nucleotide level, as long as said claimed encoding sequence contains a mutant amino acid residue not found in the virus encoded from SEQ ID NO: 1.

More preferably, the PEDV vaccines of the present invention comprise viruses encoded by a nucleotide sequence that is at least 95%, 96%, 97%, 98%, 99% or 99.5% identical to one or more of SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 at a full length nucleotide level, as long as said novel encoding sequence contains a mutant amino acid residue not found in the virus encoded from SEQ ID NO: 1. Preferably, said mutant amino acid residues are selected from the group consisting of.

The vaccines of the present invention are capable of protecting swine from challenge by both variant and prototype strains of PEDV, and preventing or treating symptoms associated with PEDV infection, wherein said protected swine include any of sows, gilts, boars, hogs, and piglets, and wherein achievement of protection is determined by an endpoint selected from the group consisting of prevention or control of any of the PEDV infection symptoms of dehydration, fever, diarrhea, vomiting, poor lactational performance, poor reproduction performance, mortality, and prevention or control of weight loss or failure to gain weight.

As aforementioned, the novel viruses of the vaccine composition may be live or killed, and if killed, a preferred adjuvant is an oil-in-water emulsion, wherein the adjuvant components include Amphigen® and aluminum hydroxide,, nmost preferably, wherein the final concentration of 20% Amphigen is 25% (v/v).

The invention therefor provides a method of protecting swine from challenge against PEDV, comprising administering to the subject a vaccine composition, in an amount sufficient to prevent or treat symptoms associated with PEDV infection, wherein said protected swine include any of sows, gilts, boars, hogs, and piglets, and wherein

achievement of protection is determined by an endpoint selected from the group consisting of prevention or control of any of the PEDV infection symptoms of dehydration, fever, diarrhea, vomiting, poor lactational performance, poor reproduction performance, mortality, and prevention or control of weight loss or failure to gain weight.

Vaccine compositions of the invention are effective in piglets that are 1 day of age or older, in a single or two dose program. Vaccine compositions of the invention are also effective in piglets in a two dose program, wherein the first dose is administered when the piglet is about 1-7 days old, and the second dose is administered when the piglet is 2-5 weeks old. The second does may be optional.

Preferably, the vaccine compositions of the invention have a minimum effective dose is between about 10 and about 10⁶ logi₀TCID_50.

In additional aspects of the invention, the vaccination program provides 2 doses administered to the piglet; and the parent sow, although vaccinated pre-breeding, is not vaccinated pre-farrowing. Additionally, the vaccination program provides 1 dose administered to the piglet; and the parent sow is vaccinated both pre-breeding and pre- farrowing. In additional embodiments, in order to prevent disease in a piglet, there is first administered the vaccine composition to the sow of said piglet, whether pre-farrowing or pre- breeding, following by administering one or more doses of said vaccine composition to said piglet after birth. In order to prevent disease in healthy pigs caused by PEDV, the invention provides that pigs are first vaccinated, followed by annual or pre-farrowing administration of further doses PEDV vaccine.

Brief Description of the Figures

Figure 1 shows a comparison of particular encoding nucleotides in INDEL PEDV strain USA/OH 851/2014 (of the Ohio Department of Agriculture, GenBank KJ399978, "OH 851") to those found in Calaf 14 Passage 0, with any resultant amino acid changes. The Figure then further shows evolution of additional nucleotide changes, and resultant amino acid changes, as Calaf 14 is subsequently passaged from Passage 0 to Passage 60, and then individual clones (A and E) are selected from consensus passage 60. Nucleotides are numbered according to the numbered sequence of PEDV USA/OH851/2014. Note that in Figure 4 shows the ORF1a-1 b gene sequence for Calaf 14, passage 0 Figure 4 shows the ORF1 a-1 b gene sequence for Calaf 14, passage Othe 5' end, and 5 bp at the 3' end. Thus, for example, at line 1 of data herein, position 2929 in OH851 corresponds to Calaf14, passage 0, position 2894. In Calaf14, passage 37 (only), there is a further deletion of "ATA" in the spike gene, relative to passage 11 , and not found in Passage 60, causing downstream nucleotides to be renumbered by 3 nucleotides, thus position 21 ,382, for example, becomes 21 ,379.

Figure 2 shows the alignment of encoded ORF3 proteins of Calaf 14 PEDV Passages 37 (SEQ ID NO:8) and 60 (SEQ ID NO:9).

Figure 3 shows the full nucleotide sequence of ther Passage 60 Calf 14 virus, again noting that approximately 35 bases are missing from the 5' end, and approximately 5 bases are missing from the 3' end, as depicted.

Figure 4 (SEQ ID NO: 10) shows the ORF1 a-1 b gene sequence for Calaf 14, passage 0.

Figure 5 (SEQ ID NO: 11) shows the ORF1 a-1 b gene sequence for Calaf 14, passage 11. Figure 6 (SEQ ID NO: 12) shows the ORF1 a-1 b gene sequence for Calaf 14, passage 37.

Figure 7 (SEQ ID NO: 13) shows the ORF1 a-1 b gene sequence for Calaf 14, passage 60.

Figure 8 (SEQ ID NO: 14) shows the ORF1 a-1 b gene sequence for Calaf14, passage 60 clnA

Figure 9 (SEQ ID NO: 15) shows the ORF1 a-1 b gene sequence for Calaf14, passage 60 clnE

Figure 10 (SEQ ID NO: 16) shows the spike gene sequence for Calaf 14, passage 0.

Figure 11 (SEQ ID NO: 17) shows the spike gene sequence for Calaf 14, passage 11.

Figure 12 (SEQ ID NO: 18) shows the spike gene sequence for Calaf 14, passage 37.

Figure 13 (SEQ ID NO: 19) shows the spike gene sequence for Calaf 14, passage 60.

Figure 14 (SEQ ID NO:20) shows the spike gene sequence for Calaf 14, passage 60, cln A

Figure 15(SEQ ID NO:21) shows the spike gene sequence for Calaf 14, passage 60, cln E Figure 16 (SEQ ID NO:22) shows the ORF3 gene sequence for Calaf 14, passage 0.

Figure 17 (SEQ I D NO:23) shows the ORF3 gene sequence for Calaf 14, passage 11.

Figure 18 (SEQ ID NO:24) shows the ORF3 gene sequence for Calaf 14, passage 37. Figure 19 (SEQ ID NO:25) shows the ORF3 gene sequence for Calaf 14, passage 60.

Figure 20 (SEQ ID NO:26) shows the ORF3 gene sequence for Calaf 14, passage 60, cln A Figure 21 (SEQ ID NO:27) shows the ORF3 gene sequence for Calaf 14, passage 60, cln E Figure 22 (SEQ ID NO:28) shows the E gene for Passage 60.

Figure 23 (SEQ ID NO:29) shows the M gene for Passage 60.

Figure 24 (SEQ ID NO:30) shows the N gene for Passage 60.

Brief Description of the Sequence Listing

SEQ ID NO: 1 provides the DNA sequence encoding PEDV strain Calaf 14, wild type, passage 0.

SEQ ID NO: 2 provides the DNA sequence encoding PEDV strain Calaf 14, passage 11. SEQ ID NO: 3 provides the DNA sequence encoding PEDV strain Calaf 14, passage 37. SEQ ID NO: 4 provides the DNA sequence encoding PEDV strain Calaf 14, passage 60. SEQ ID NO: 5 provides the DNA sequence encoding PEDV strain Calaf 14, passage 60, clone A selected therefrom.

SEQ ID NO: 6 provides the DNA sequence encoding PEDV strain Calaf 14, passage 60, clone E selected therefrom.

SEQ ID NO: 7 provides the DNA sequence encoding PEDV strain OH851.

SEQ ID NO: 8 provides the amino acid sequence of the protein encoded from ORF3, Calaf 14, passage 37.

SEQ ID NO: 9 provides the amino acid sequence of the protein encoded from ORF3, Calaf 14, passage 60.

Detailed Description of the Invention

The present invention provides novel and efficacious vaccines useful to preventing disease caused by PEDV.

Definitions

Vaccines can be made more efficacious by including an appropriate adjuvant in the composition. The term "adjuvant" generally refers to any material that increases the humoral or cellular immune response to an antigen. Adjuvants are used to accomplish two objectives: They slow the release of antigens from the injection site, and they enhance stimulation of the immune system. Traditional vaccines are generally composed of a crude preparation of inactivated or killed or modified live pathogenic microorganisms. The impurities associated with these cultures of pathological microorganisms may act as an adjuvant to enhance the immune response. However, the immunity invoked by vaccines that use homogeneous preparations of pathological microorganisms or purified protein subunits as antigens is often poor. The addition of certain exogenous materials such as an adjuvant therefore becomes necessary. Further, in some cases, synthetic and subunit vaccines may be expensive to produce. Also, in some cases, the pathogen cannot be grown on a commercial scale, and thus, synthetic/subunit vaccines represent the only viable option. The addition of an adjuvant may permit the use of a smaller dose of antigen to stimulate a similar immune response, thereby reducing the production cost of the vaccine. Thus, the effectiveness of some injectable medicinal agents may be significantly increased when the agent is combined with an adjuvant.

Many factors must be taken into consideration in the selection of an adjuvant. An adjuvant should cause a relatively slow rate of release and absorption of the antigen in an efficient manner with minimum toxic, allergenic, irritating, and other undesirable effects to the host. To be desirable, an adjuvant should be non-viricidal, biodegradable, capable of consistently creating a high level of immunity, capable of stimulating cross protection, compatible with multiple antigens, efficacious in multiple species, non-toxic, and safe for the host (eg, no injection site reactions). Other desirable characteristics of an adjuvant are that it is capable of micro-dosing, is dose sparing, has excellent shelf stability, is amenable to drying, can be made oil-free, can exist as either a solid or a liquid, is isotonic, is easily manufactured, and is inexpensive to produce. Finally, it is highly desirable for an adjuvant to be configurable so as to induce either a humoral or cellular immune response or both, depending on the requirements of the vaccination scenario. However, the number of adjuvants that can meet the above requirements is limited. The choice of an adjuvant depends upon the needs for the vaccine, whether it be an increase in the magnitude or function of the antibody response, an increase in cell mediated immune response, an induction of mucosal immunity, or a reduction in antigen dose. A number of adjuvants have been proposed, however, none has been shown to be ideally suited for all vaccines. The first adjuvant reported in the literature was Freund's Complete Adjuvant (FCA) which contains a water-in-oil emulsion and extracts of mycobacterium. Unfortunately, FCA is poorly tolerated and it can cause uncontrolled inflammation. Since the discovery of FCA over 80 years ago efforts have been made to reduce the unwanted side effects of adjuvants.

Some other materials that have been used as adjuvants include metallic oxides (e.g., aluminum hydroxide), alum, inorganic chelates of salts, gelatins, various paraffin-type oils, synthesized resins, alginates, mucoid and polysaccharide compounds, caseinates, and blood-derived substances such as fibrin clots. While these materials are generally efficacious at stimulating the immune system, none has been found to be entirely satisfactory due to adverse effects in the host (e.g., production of sterile abcesses, organ damage,

carcinogenicity, or allergenic responses) or undesirable pharmaceutical properties (e.g., rapid dispersion or poor control of dispersion from the injection site, or swelling of the material).

"Cellular immune response" or "cell mediated immune response" is one mediated by T-lymphocytes or other white blood cells or both, and includes the production of cytokines, chemokines and similar molecules produced by activated T-cells, white blood cells, or both; or a T lymphocyte or other immune cell response that kills an infected cell.

The term "emulsifier" is used broadly in the instant disclosure. It includes substances generally accepted as emulsifiers, e.g., different products of TWEEN® or SPAN® product lines (fatty acid esters of polyethoxylated sorbitol and fatty-acid-substituted sorbitan surfactants, respectively), and different solubility enhancers such as PEG-40 Castor Oil or another PEGylated hydrogenated oil.

"Humoral immune response" refers to one that is mediated by antibodies.

"Immune response" in a subject refers to the development of a humoral immune response, a cellular immune response, or a humoral and a cellular immune response to an antigen. Immune responses can usually be determined using standard immunoassays and neutralization assays, which are known in the art.

"Immunologically protective amount" or "immunologically effective amount" or "effective amount to produce an immune response" of an antigen is an amount effective to induce an immunogenic response in the recipient. The immunogenic response may be sufficient for diagnostic purposes or other testing, or may be adequate to prevent signs or symptoms of disease, including adverse health effects or complications thereof, caused by infection with a disease agent. Either humoral immunity or cell-mediated immunity or both may be induced. The immunogenic response of an animal to an immunogenic composition may be evaluated, e.g., indirectly through measurement of antibody titers, lymphocyte proliferation assays, or directly through monitoring signs and symptoms after challenge with wild type strain, whereas the protective immunity conferred by a vaccine can be evaluated by measuring, e.g., reduction in clinical signs such as mortality, morbidity, temperature number, overall physical condition, and overall health and performance of the subject. The immune response may comprise, without limitation, induction of cellular and/or humoral immunity. "Immunogenic" means evoking an immune or antigenic response. Thus an immunogenic composition would be any composition that induces an immune response.

"Therapeutically effective amount" refers to an amount of an antigen or vaccine that would induce an immune response in a subject receiving the antigen or vaccine which is adequate to prevent or reduce signs or symptoms of disease, including adverse health effects or complications thereof, caused by infection with a pathogen, such as a virus or a bacterium. Humoral immunity or cell-mediated immunity or both humoral and cell-mediated immunity may be induced. The immunogenic response of an animal to a vaccine may be evaluated, e.g., indirectly through measurement of antibody titers, lymphocyte proliferation assays, or directly through monitoring signs and symptoms after challenge with wild type strain. The protective immunity conferred by a vaccine can be evaluated by measuring, e.g., reduction in clinical signs such as mortality, morbidity, temperature number, overall physical condition, and overall health and performance of the subject. The amount of a vaccine that is therapeutically effective may vary depending on the particular adjuvant used, the particular antigen used, or the condition of the subject, and can be determined by one skilled in the art.

"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).

Vaccine & Immunogenic Compositions

The vaccine and immunogenic composition of the present invention induces at least one of a number of humoral and cellular immune responses in a subject swine that has been administered a vaccine composition of the invention. Generally, the vaccine compositions of the invention may be administered to swine of any age, whether male or female, irrespective of reproductive status, and although it is contemplated that a two-dose regimen will be most common, single dose and multiple dose vaccine treatments are also effective in the practice of the invention. A most preferred virus for use according to all aspects of the invention relating to PEDV is that encoded from SEQ ID NOS: 4, 5 and 6.

Such a vaccine protects well against challenge by both prototype and variant strains,, such as North American prototype strain USA/Colorado/2013 (whose sequence is deposited as GenBank accession No. KF272920, of the NCBI of the United States National Institutes of Health. Bethesda, MD), and also numerous other prototype and variant strains, such as similarly deposited Chinese strain AH2012, deposited as GenBank accession No.

KC210145; strain 13-019349, deposited as GenBank accession No. KF267450; strain CH- ZMDZY-1 1 deposited as GenBank accession No. KC196276; strain OH851 (Ohio), GenBank KJ399978; European strain CV777 (see R. Kocherhans et al., Virus Genes, vol 23(2), pp 137-144, 2001 ; strains IA2013-KF452322 and IN2013-KF452323 (see G. Stevenson et al. J. Vet. Diagn. Invest., vol 25, pp.649-654, 2013. Additional strains representative of those against which the current vaccines can protect, include, without limitation, GenBank

Accessions KJ645688 (USA/lowa96/2013); KJ645640 (USA/Oklahoma32/2013); KJ778615 (NPL-PEDv/2013); KJ645647 (USA/Minnesota41/2013); KJ645637 ((USA/Kansas29/2013); KJ645639 (USA/Texas31/2013); KJ645666 (USA/lowa70/2013); KJ645646

(USA/NorthCarolina40/2013); KM189367 (PEDv ON-018); and KJ645669

(USA/Wisconsin74/2013).

The vaccine compositions of the present invention protect against challenge by PEDV generally, including all forms of the virus circulating in Asia, North America, and Europe. Such viruses (against which protection is achieved) can also be identified solely by the amino acid of nucleotide encoding sequences of surface spike protein S, and thus additional isolates against which the present invention is effective include viral coat sequences reported in GenBank (US NIH/NCBI) by their spike protein accessions, to include AID56757.1 ;

AHA38139.1 ; AG058924.1 ; AHA38125.1 ; AIM47748.1 ; AID56895.1 : AID5669.1 : AII20255.1 : AGG34694.1 ; AIE15986.1 ; AHG05730.1 ; AHG05733.1 (all being representative of those having above 99% identity to the USA/Colorado/2013 spike sequence), and further,

AIC82397.1 ; AFL02631.1 ; AHB33810.1 ; AFQ37598.1 ; AGG34691.1 ; AFJ97030.1 ; AFR11479.1 ; and AEW22948.1 (all being representative of those having above 98% identity to the USA/Colorado/2013 spike sequence).

GenBank® is the recognized US-NIH genetic sequence database, comprising an annotated collection of publicly available DNA sequences, and which further incorporates submissions from the European Molecular Biology Laboratory (EMBL) and the DNA

DataBank of Japan (DDBJ), see Nucleic Acids Research, January 2013,v 41 (D1) D36-42 for discussion.

Homologous Sequences and Conservative Amino Acid Changes

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

Biotechnology Information, otherwise known as NCBI, (Bethesda, Maryland, USA) of the United States National Institute of Health). In a specific example for calculations according to the practice of the present invention, reference is made to BLASTP 2.2.6 [Tatusova TA and TL Madden, "BLAST 2 sequences- a new tool for comparing protein and nucleotide sequences." (1999) FEMS Microbiol Lett. 174:247-250.]. Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 0.1 , and the "blosum62" scoring matrix of Henikoff and Henikoff (Proc. Nat. Acad. Sci. USA 89:10915 10919. 1992). The percent identity is then calculated as: Total number of identical matches x 100/ divided by the length of the longer

sequence+number of gaps introduced into the longer sequence to align the two sequences. Preferably, a homologous nucleotide sequence has at least about 90% nucleotide sequence identity, even more preferably at least about 95%, 96%, 97%, 98% and 99% nucleotide sequence identity. Since the genetic code is degenerate, a homologous nucleotide sequence can include any number of "silent" base changes, i.e. nucleotide substitutions that nonetheless encode the same amino acid.

A homologous nucleotide sequence can further contain non-silent mutations, i.e. base substitutions, deletions, or additions resulting in amino acid differences in the encoded polyaminoacid, so long as the sequence remains at least about 90% identical to the polyaminoacid encoded by the first nucleotide sequence or otherwise is useful for practicing the present invention. In this regard, certain conservative amino acid substitutions may be made which are generally recognized not to inactivate overall protein function: such as in regard of positively charged amino acids (and vice versa), lysine, arginine and histidine; in regard of negatively charged amino acids (and vice versa), aspartic acid and glutamic acid; and in regard of certain groups of neutrally charged amino acids (and in all cases, also vice versa), (1) alanine and serine, (2) asparagine, glutamine, and histidine, (3) cysteine and serine, (4) glycine and proline, (5) isoleucine, leucine and valine, (6) methionine, leucine and isoleucine, (7) phenylalanine, methionine, leucine, and tyrosine, (8) serine and threonine, (9) tryptophan and tyrosine, (10) and for example tyrosine, tyrptophan and phenylalanine.

Amino acids can be classified according to physical properties and contribution to secondary and tertiary protein structure. A conservative substitution is recognized in the art as a substitution of one amino acid for another amino acid that has similar properties. Exemplary conservative substitutions may be found in WO 97/09433, page 10, published Mar. 13. 1997 (PCT/GB96/02197, filed Sep. 6, 1996. Alternatively, conservative amino acids can be grouped as described in Lehninger, (Biochemistry, Second Edition; Worth Publishers, Inc. NY:NY (1975), pp. 71-77).

Homologous nucleotide sequences can be determined by comparison of nucleotide sequences, for example by using BLASTN, above. Alternatively, homologous nucleotide sequences can be determined by hybridization under selected conditions. For example, the nucleotide sequence of a second polynucleotide molecule is homologous to SEQ ID NO:1 (or any other particular polynucleotide sequence) if it hybridizes to the complement of SEQ ID NO:1 under moderately stringent conditions, e.g., hybridization to filter-bound DNA in 0.5 M NaHP04, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65°C, and washing in

0.2xSSC/0.1 % SDS at 42°C (see Ausubel et al editors, Protocols in Molecular Biology, Wiley and Sons, 1994, pp. 6.0.3 to 6.4.10), or conditions which will otherwise result in hybridization of sequences that encode a PEDV virus as defined below. Modifications in hybridization conditions can be empirically determined or precisely calculated based on the length and percentage of guanosine/cytosine (GC) base pairing of the probe. The hybridization conditions can be calculated as described in Sambrook, et al., (Eds.), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York (1989), pp. 9.47 to 9.51.

In another embodiment, a second nucleotide sequence is homologous to SEQ ID NO:1 (or any other sequence of the invention) if it hybridizes to the complement of SEQ ID NO:1 under highly stringent conditions, e.g. hybridization to filter-bound DNA in 0.5 M NaHP04, 7% SDS, 1 mM EDTA at 65°C, and washing in 0.1xSSC/0.1 % SDS at 68°C, as is known in the art.

It is furthermore to be understood that the 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.

Preferred Viral isolates

Referring to Figure 1 , it can be seen that important nucleotide changes happen in

ORF1a/b, spike, and ORF3, causing important amino acid changes in the resultant proteins, that provide for the high level of safety achieved for Calaf14 passage 60 (see Example 5 below). It should be noted that 2 clones (A and E, SEQ ID NOS:5 and 6) were also recovered and sequenced from the consensus population (SEQ ID NO:4).

Referring first to spike protein changes, between passages 11 and 37, there is only one stable amino acid change that is then retained in passage 60. The phenylalanine (F) at amino acid position 1269 in spike changes to leucine (L). Between passages 37 and 60, two additional amino acid changes in Spike contribute to attenuation. These are aspartate (D) to tyrosine (Y) at position 262, and asparagine (N) to aspartate (D) to position 1006. It is thus within the practice of the present invention to provide PEDV viruses having these sequence modifications (see SEQ ID NOS 4,5 and 6), or conservative amino acid variants thereof, for example, position 1269 (or the position that corresponds to residue 1269 as would be determined from an appropriate algorithm) could instead be valine, isoleucine, and the like; position 262 tyrosine could be replaced by phenyalanine, valine, .leucine, isoleucine and the like, and asparate at position 1006 could be replaced by by glutamate, for example.

As can also be seen from Figure 1 , there is one amino acid change in ORF1a/1 b of particular note, that helps explain the increased attenuation between passages 37 and 60 (drop from 10% to 0% mortality). It is the A to G nucleotide change at genome position 17, 171 (OH851) or 17, 136 (Calaf14 P37). This results in an E to G (glutamate to glycine) change at amino acid position 5627 of the ORFlab polyprotein. This portion of the polyprotein is proteolytically cleaved to form nsp14, which is known as the Exoribonuclease (ExoN). It is possible that the replacement of a negatively charged amino acid like glutamate with a small non-charged glycine residue (or proline or alanine, for example ) could alter the affinity of the enzyme for its RNA substrate. Alternatively, it could alter the conformation of the enzyme resulting in reduced activity or stability.

In regard of ORF3, it can be seen that a frameshift inducing truncation "ATTA" happens between passages 37 and 60, leading to a shortened expression product. Other amino acid changes noted in Figure 1 are considered less likely to contribute substantially to attenuation since they appear in equally attenuated final variants (for example ORF1 a/1 b position 3319 as T or C, where virulent wild type passage 0 is also C), for example, or where the mutation already arises prior to passage 1 1 , although this passage is not yet substantially attenuated (see, for example, ORF1 a/1 b at position 5122) Nonetheless, such amino acid changes may contribute to the practice of the invention, so that mutant ORF1 a/1 b position 5122 (alanine) may be replaced with glycine, and other relatively small non polar amino acids, for example.

Culturing of Virus

Isolation and propagation of PEDV has been generally difficult. Initial studies using Vero cells for propagation in culture have only been partially effective, and have required a trypsin-containing medium, often with excessive cytopathic effect including cell fusion, synctia formation, and cell detachment (see, for example K. Kusangi et al., J. Vet Med Sci, vol. 54(2), pp.313-318, 1992, and M. Hofmann et al. J. Clinical Microbiology, vol. 26(11), pp2235-2239, 1988). Accordingly, improved passaging methods were developed for the practice of the present invention. General and detailed methods are provided in Examples 1 and 2 below. It should be noted that both USA/Colorado/2013 and Calaf14 can be cultured in Vero cells.

Inactivation of virus

Inactivated or killed viral strains are those which have been inactivated by methods known to those skilled in the art, including treatment with formalin, betapropriolactone (BPL), binary ethyleneimine (BEI), sterilizing radiation, heat, or other such methods.

Adjuvant component

The vaccine compositions of the invention may or may not include adjuvants. In particular, as based on an orally infective virus, the modified live vaccines of the invention may be used adjuvant free, with a sterile carrier. Adjuvants that may be used for oral administration include those based on CT-like immune modulators (rmLT, CT-B, i.e.

recombinant-mutant heat labile toxin of E. coli, Cholera toxin-B subunit); or via encapsulation with polymers and alginates, or with mucoadhesives such as chitosan, or via liposomes. A preferred adjuvanted or non adjuvanted vaccine dose at the minimal protective dose through vaccine release may provide between approximately 10 and approximately 10⁶ logi₀TCID₅₀ of virus per dose, or higher. Adjuvants, if present, may be provided as emulsions, more commonly if non-oral administration is selected, but should not decrease starting titer by more than 0.7 logs (80% reduction.

Immunogenic compositions of the present invention can include one or more well known adjuvants and adjuvant systems. Suitable adjuvants include, but are not limited to, the RIBI adjuvant system (Ribi Inc.; Hamilton, MT); alum; aluminum hydroxide gel; aluminum phosphate; oil-in water emulsions; water-in-oil emulsions such as Freund's complete and incomplete adjuvants; Block copolymer (CytRx; Atlanta, GA); SAF-M (Chiron; Emeryville, CA); AMPHIGEN® adjuvant; killed Bordetella; saponins such as Stimulon™ QS-21

(Antigenics, Framingham, MA. described in U.S. Patent No. 5,057,540, which is hereby incorporated by reference) and particles generated therefrom such as ISCOMS

(immunostimulating complexes), GPI-0100 (Galenica Pharmaceuticals, Inc.; Birmingham, AL) or other saponin fractions; monophosphoryl lipid A (MPL-A); avridine; lipid-amine adjuvant; heat-labile enterotoxin from Escherichia coli (recombinant or otherwise); cholera toxin; and muramyl dipeptide. Also useful is MPL™ (3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton, MT), which is described in U.S. Patent No. 4,912,094, and is hereby incorporated by reference.

Also suitable for use as adjuvants are: synthetic lipid A analogs or aminoalkyl glucosamine phosphate (AGP) compounds, or derivatives or analogs thereof, which are available from Corixa (Hamilton, MT), and which are described in US 6, 1 13,918, hereby incorporated by reference; L121/squalene; D-lactide-polylactide/glycoside; pluronic polyols; muramyl dipeptide; extracts of Mycobacterium tuberculosis; bacterial lipopolysaccharides generally; pertussis toxin (PT); and an E. coli heat-labile toxin (LT), particularly LT-K63, LT- R72, PT-K9/G129; see, e.g., International Patent Publication Nos. WO 93/13302 and WO 92/19265, incorporated herein by reference.

Synthetic polynucleotides, such as oligonucleotides containing CpG motifs (US 6,207,646, hereby incorporated by reference), can also be used as adjuvants for the present invention. CpG oligonucleotides, such as P-class immunostimulatory oligonucleotides, are useful, including E-modified P-class immunostimulatory oligonucleotides.

Sterols can also be useful as adjuvants herein. Those suitable for use can include sitosterols, stigmasterol, ergosterol, ergocalciferol, and cholesterol.

The adjuvant compositions useful in the practice of the invention can generally further include one or more polymers such as, for example, DEAE Dextran, polyethylene glycol, polyacrylic acid, and polymethacrylic acid (e.g., CARBOPOL®). The adjuvant compositions can also further include one or more Th2 stimulants such as, for example, Bay R1005(R) and aluminum.

The adjuvant compositions can additionally or alternatively further include one or more immunomodulatory agents, such as quaternary ammonium compounds (e.g., DDA), interleukins, interferons, or other cytokines. A number of cytokines or lymphokines have been shown to have immune-modulating activity, and thus may be used as adjuvants.

These can include, but are not limited to, the interleukins 1-α, 1-β, 2, 4, 5, 6, 7, 8, 10, 12 (see, e.g., US 5,723,127), 13, 14, 15, 16, 17 and 18 (and its mutant forms), the interferons-a, β and gamma, granulocyte-macrophage colony stimulating factor (see, for example, US 5,078,996, and ATCC Accession Number 39900), macrophage colony stimulating factor, granulocyte colony stimulating factor, GSF, and the tumor necrosis factors a and β. Still other adjuvants useful in this invention include chemokines, including without limitation, MCP-1 , ΜΙΡ-1α, ΜΙΡ-1 β, and RANTES. Adhesion molecules, such as a selectin, e.g., L- selectin, P-selectin, and E-selectin may also be useful as adjuvants. Still other useful adjuvants include, without limitation, a mucin-like molecule, e.g., CD34, GlyCAM-1 and MadCAM-1 ; a member of the integrin family such as LFA-1 , VLA-1 , Mac-1 and p150.95; a member of the immunoglobulin superfamily such as PECAM, ICAMs (e.g., ICAM-1 , ICAM-2 and ICAM-3), CD2 and LFA-3; co-stimulatory molecules such as CD40 and CD40L; growth factors including vascular growth factor, nerve growth factor, fibroblast growth factor, epidermal growth factor, B7.2, PDGF, BL-1 , and vascular endothelial growth factor; receptor molecules including Fas, TNF receptor, Fit, Apo-1 , p55, WSL-1 , DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, and DR6. Still another adjuvant molecule includes Caspase (ICE). See also W098/17799 and W099/43839.

Suitable adjuvants also include, without limitation, MPL™ (3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton, MT), which is described in U.S. Patent No.

4,912,094, which is hereby incorporated by reference. Also suitable for use as adjuvants are synthetic lipid A analogs or aminoalkyl glucosamine phosphate compounds (AGP), or derivatives or analogs thereof, which are available from Corixa (Hamilton, MT), and which are described in United States Patent No. 6, 113,918, which is hereby incorporated by reference. One such AGP is 2-[(R)-3-Tetradecanoyloxytetradecanoylamino] ethyl 2-Deoxy-4- 0-phosphono-3-0-[(R)-3-tetradecanoyoxytetradecanoyl]-2-[(R)-3- tetradecanoyloxytetradecanoyl-amino]-b-D-glucopyranoside, which is also known as 529 (formerly known as RC529). The RC529 adjuvant is formulated as an aqueous form or as a stable emulsion.

Additional adjuvants useful in the practice of the present invention include cholera toxins (CT) and mutants thereof, including those described in published International Patent Application number WO 00/18434 (wherein the glutamic acid at amino acid position 29 is replaced by another amino acid, other than aspartic acid, preferably a histidine). Similar CT toxins or mutants are described in published International Patent Application number WO 02/098368 (wherein the isoleucine at amino acid position 16 is replaced by another amino acid, either alone or in combination with the replacement of the serine at amino acid position 68 by another amino acid; and/or wherein the valine at amino acid position 72 is replaced by another amino acid). Other CT toxins are described in published International Patent Application number WO 02/098369 (wherein the arginine at amino acid position 25 is replaced by another amino acid; and/or an amino acid is inserted at amino acid position 49; and/or two amino acids are inserted at amino acid positions 35 and 36). Said CT toxins or mutant can be included in the immunogenic compositions either as separate entities, or as fusion partners for the polypeptides of the present invention.

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

A preferred adjuvanted may be provided as a 2ML dose in a buffered solution further comprising about 5% (v/v) Rehydragel® (aluminum hydroxide gel) and "20% Amphigen" ® at about 25% final (v/v). Amphigen® is generally described in U.S Patent 5,084,269 and provides de-oiled lecithin (preferably soy) dissolved in a light oil, which is then dispersed into an aqueous solution or suspension of the antigen as an oil-in-water emulsion. Amphigen has been improved according to the protocols of U.S. Patent 6,814,971 (see columns 8-9 thereof) to provide a so-called "20% Amphigen" component for use in the final adjuvanted vaccine compositions of the present invention. Thus, a stock mixture of 10% lecithin and 90% carrier oil (DRAKEOL®, Penreco, Karns City, PA) is diluted 1 : 4 with 0.63% phosphate buffered saline solution, thereby reducing the lecithin and DRAKEOL components to 2% and 18% respectively (i.e. 20% of their original concentrations). Tween 80 and Span 80 surfactants are added to the composition, with representative and preferable final amounts being 5.6% (v/v) Tween 80 and 2.4% (v/v) Span 80, wherein the Span is originally provided in the stock DRAKEOL component, and the Tween is originally provided from the buffered saline component, so that mixture of the saline and DRAKEOL components results in the finally desired surfactant concentrations. Mixture of the DRAKEOL/lecithin and saline solutions can be accomplished using an In-Line Slim Emulsifier apparatus, model 405, Charles Ross and Son, Hauppauge, NY, USA.

The vaccine composition also 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.

As is understood in the art, the order of addition of components can be varied to provide the equivalent final vaccine composition. For example, an appropriate dilution of virus in buffer can be prepared. An appropriate amount of Rehydragel® LV (about 2% aluminum hydroxide content) stock solution can then be added, with blending, in order to permit the desired 5% (v/v) concentration of Rehydragel® LV in the actual final product.

Once prepared, this intermediate stock material is combined with an appropriate amount of

"20% Amphigen" stock (as generally described above, and already containing necessary amounts of Tween 80 and Span 80) to again achieve a final product having 25% (v/v) of

"20% Amphigen". An appropriate amount of 10% merthiolate can finally be added.

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

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

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

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

Typically, the oily phase is present in an amount from 50% to 95% by volume;

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

Non-natural, synthetic emulsifiers suitable for use in the adjuvant formulations of the present invention also include sorbitan-based non-ionic surfactants, e.g. fatty-acid- substituted sorbitan surfactants (commercially available under the name SPAN® or

ARLACEL®), fatty acid esters of polyethoxylated sorbitol (TWEEN®), polyethylene glycol esters of fatty acids from sources such as castor oil (EMULFOR®); polyethoxylated fatty acid (e.g., stearic acid available under the name SIMULSOL® M-53), polyethoxylated

isooctylphenol/formaldehyde polymer (TYLOXAPOL®), polyoxyethylene fatty alcohol ethers (BRIJ®); polyoxyethylene nonphenyl ethers (TRITON® N), polyoxyethylene isooctyl phenyl ethers (TRITON® X). Preferred synthetic surfactants are the surfactants available under the name SPAN® and TWEEN®, such as TWEEN®-80 (Polyoxyethylene (20) sorbitan monooleate) and SPAN®-80 (sorbitan monooleate). Generally speaking, the emulsifier(s) may be present in the vaccine composition in an amount of 0.01 % to 40% by volume, preferably, 0.1 % to 15%, more preferably 2% to 10%.

In an alternative embodiment of the invention, the final vaccine composition contains SP-Oil® and Rehydragel® LV as adjuvants (or other Rehydragel® or Alhydrogel® products), with preferable amounts being about 5-20% SP-Oil (v/v) and about 5-15% Rehydragel LV (v/v), and with 5% and 12%, respectively, being most preferred amounts. In this regard it is understood that % Rehydragel refers to percent dilution from the stock commercial product. (SP-Oil ® is a fluidized oil emulsion with includes a polyoxyethylene-polyoxypropylene block copolymer (Pluronic® L121 , BASF Corporation, squalene, polyoxyethylene sorbitan monooleate (Tween®80, ICI Americas) and a buffered salt solution.) It should be noted that the present invention may also be successfully practiced using wherein the adjuvant component is only Amphigen®.

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

In certain embodiments, TXO adjuvants are prepared as follows:

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

b) The immunostimulatory oligonucleotide, Dextran DEAE and Polyoxyethylene (20) sorbitan monooleate are dissolved in aqueous phase, thus forming the aqueous solution; and c) The aqueous solution is added to the oil solution under continuous homogenization thus forming the adjuvant formulation TXO.

All the adjuvant compositions of the invention can be used with any of the PEDV strains and isolates covered by the present Specification.

Additional adjuvants useful in the practice of the invention include Prezent-A (see generally United States published patent application US20070298053; and "QCDCRT" or "QCDC"-type adjuvants (see generally United States published patent application

US20090324641.

Excipients

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

Dosing

A preferred clinical indication is for treatment, control and prevention in both breeding sows and gilts pre-farrowing, followed by vaccination of piglets. In a representative example (applicable to both sows and gilts), two 2-ML doses of vaccine will be used, although of course, actual volume of the dose is a function of how the vaccine is formulated, with actual dosing amounts ranging from 0.1 to 5ML, taking also into account the size of the animals. Single dose vaccination is also appropriate.

The first dose may be administered as early as pre-breeding to 5-weeks pre- farrowing, with the second dose administered preferably at about 1-3 weeks pre-farrowing. Doses vaccine preferably provide an amount of viral material that corresponds to a TCID₅₀ (tissue culture infective dose) of between about 10⁶ and 10⁸, more preferably between about 10⁷ 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. In a further preferred example, the sow or gilt is vaccinated intramuscularly or orally at 5-weeks pre-farrowing and then 2-weeks pre-farrowing. Under these conditions, a protective immune response can be demonstrated in 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 be via a different route of administration. Although it is preferred to re-vaccinate a mother sow prior to any subsequent farrowings, the vaccine compositions of the invention nonetheless can still provide protection to piglets via ongoing passive transfer of antibodies, even if the mother sow was only vaccinated in association with a previous farrowing.

It should be noted that piglets may then be vaccinated as early as Day 1 of life. For example, piglets can be vaccinated at Day 1 , with or without a booster dose at 3 weeks of age, particularly if the parent sow, although vaccinated pre-breeding, was not vaccinated pre- farrowing. Piglet vaccination may also be effective if the parent sow was previously not 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. It should be noted that the vaccines of the present invention are safe for usein pregnant animals (all trimesters) and neonatal swine. The vaccines of the invention are attenuated to a level of safety (i.e. no mortality, only transient mild clinical signs or signs normal to neonatal swine) that is acceptable for even the most sensitive animals again including neonatal pigs.

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

When provided therapeutically, the vaccine is provided in an effective amount upon the detection of a sign of actual infection. Suitable dose amounts for treatment of an existing infection include between about 10 and about 10⁶ log ₀TCID₅₀ , or higher, of virus per dose (minimum immunizing dose to vaccine release). A composition is said to be

"pharmacologically acceptable" if its administration can be tolerated by a recipient. Such a composition is said to be administered in a "therapeutically or prophylactically effective amount" if the amount administered is physiologically significant.

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

Variation of the dose amounts is well within the practice of the art. Generation of Vero Cells Suitable for Large Scale Virus Production

Viruses of the invention can be conveniently grown in Vero cell stocks that are approved for vaccine production. The following provides a representative method to generate safe and approved cell stock, a vial of Vero cells was subject to additional passaging.. The cells were passed four times in PMEM w/wheat to produce Master Cell Stock (MCS) Lot 834430". The MCS was tested in accordance with 9CFR & EP requirements. The MCS tested satisfactory for sterility, freedom from mycoplasmas, and extraneous agents. Therefore, PF-Vero MCS lot "1834430", is deemed eligible for submission to the Center for Veterinary Biologies Laboratories (CVB-L) for confirmatory testing.

Seed Origin and Passage History is as follows. A Pre-master Cell stock of global Vero cells was previously frozen. For production of the cell stock, the cells were grown in PMEM containing 1 % bovine serum (item # 00-0710-00, BSE compliant) and 3 mM L-glutamine. They were derived from Vero WCS Pass # 136, Lot #071700 MCS+3, 28-Jul-00. The new Pre-master cell stock was frozen at pass # 166, which is MCS+33 from the original global Vero master cell stock. MCS "1833440" was produced from a pre-master Lot All cultures were grown in PMEM w/wheat, 1.0% L-glutamine and 1.0% Bovine Calf serum. 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 degree 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.. 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.

Mycoplasma Testing and Extraneous Testing were accomplished as follows. The

MCS was tested as per 9CFR (028-PUO) and EP 2.6.7. The MCS was found to be free of any Mycoplasma contamination. Extraneous testing was completed as per 9CFR 1 13.52 using NL-BT-2 (Bovine), Vero, NL-ED-5 (Equine), NL-ST-1 (Porcine), NL-DK (Canine), NL-FK (Feline) cells, . The MCS was negative for MGG, CPE and HAd and tested negative by FA for BVD, BRSV, BPV, BAV-1 , BAV-5, Rabies, Reo, BTV, ERV, Equine arteritis, PPV, TGE, PAV, HEV, CD, CPV, FPL and FIP. The MCS was tested by ELISA for FIV and was found to be satisfactory.

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

The invention encompasses methods of preventing PEDV virus infection comprising administering the immunogenic and vaccine compositions of the invention in a swine subject of any age.

When provided therapeutically, the vaccine is provided in an effective amount upon the detection of a symptom of actual infection. A composition is said to be

At least one vaccine or immunogenic composition of the present invention can be administered by any means that achieve the intended purpose, using a pharmaceutical composition as described herein. For example, route of administration of such a composition can be by parenteral, oral, oronasal, intranasal, intratracheal, topical, subcutaneous, intramuscular, transcutaneous, intradermal, intraperitoneal, intraocular, and intravenous administration. In one embodiment of the present invention, the composition is administered by intramuscularly. Parenteral administration can be by bolus injection or by gradual perfusion over time. Any suitable device may be used to administer the compositions, including syringes, droppers, needleless injection devices, patches, and the like. The route and device selected for use will depend on the composition of the adjuvant, the antigen, and the subject, and such are well known to the skilled artisan.

According to the present invention, an "effective amount" of a vaccine or

immunogenic composition is one which is sufficient to achieve a desired biological effect, in this case at least one of cellular or humoral immune response to one or more strains of PEDV. It is understood that the effective dosage will be dependent upon the age, sex, health, and weight of the subject, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired. The ranges of effective doses provided below are not intended to limit the invention and represent examples of dose ranges which may be suitable for administering compositions of the present invention. However, the dosage may be tailored to the individual subject, as is understood and determinable by one of skill in the art, without undue experimentation. Examples

The following examples illustrate only certain and not all embodiments of the invention, and thus, should not be viewed as limiting the scope of the invention. Example 1 : General representative procedures applicable to collection of PEDV virus from tissue samples

Approximately 1 cm of tissue was used for extraction of PEDV virus. The tissue was chopped into fine pieces using a sterile scalpel and sterile scissors in a sterile Petri dish. Work was done in a Bio-safety cabinet to ensure aseptic conditions. 2 ml of sterile PBS was added to the Petri dish to collect tissue and material was transfer to a 15 ml conical tube.

Tissue was homogenized using a Qiagen TissueRuptor at 80% of maximum by pulsing for a total of 30 seconds . Homogenization was performed in an ice bucket to lessen the effect of heat on the PEDV virus. The homogenized material was filtered through a 0.45 uM filter and 60 ul of material was used for RNA isolation and PEDV Q-PCR to confirm the presence of the PEDV virus. The filtered material containing the PEDV virus was further diluted 1 : 10 in sterile PBS and then filtered through a 0.20 uM filter.

The sterile-filtered PEDV homogenate was used to infect confluent mono-layers of Vero 76 cells by transferring 1 ml of filtered material to a T-25 flask containing 2.8E+06 cells planted 3 to 4 days prior. The T-25 flasks of confluent Vero 76 cells were washed 2X with sterile PBS and IX with DMEM media containing 10% TPB, 20ug/ml geneticin and 4ug/ml TPCK trypsin (equivalent to 18.8 USP units/ml). Cells were infected for 1 hour at 37°C and 5%C0₂ in an incubator with gentle swirling every 15 minutes to ensure virus was evenly distributed to all cells. 5 ml of DMEM media containing 10% TPB, 20ug/ml geneticin and 4ug/ml TPCK trypsin (equivalent to 18.8 USP units/ml) was added to flasks and flask were allowed to incubate 2 days. After 2 days, flasks were frozen at -80°C and thawed at 37°C. This material is considered as Passage 1 of the virus. One milliliter of the total volume from the flask was then used for Passage 2 of the virus. The 1 ml of Passage 1 material is used to infect a T-25 flask containing 2.8E+06 cells seeded 3 to 4 days prior. Cells were first washed 2X with sterile PBS and IX with DMEM media containing 10% TPB, 20ug/ml geneticin and 4ug/ml TPCK trypsin (equivalent to 18.8 USP units/ml). Cells were infected for 1 hour at 37°C and 5%C0₂ in an incubator with gentle swirling every 15 minutes to ensure virus was evenly distributed to cells. 5 ml of DMEM media containing 10% TPB, 20ug/ml geneticin and 4ug/ml TPCK trypsin (equivalent to 18.8 USP units/ml) was added to flasks and flask were allowed to incubate for 2 days. This material is Passage 2 of the PEDV virus. Passages are repeated every 2 days until the cells show signs of infection indicated by clusters of cells surrounded by a filmy layer of material and/or a bubble effect on the clustered cells (see Figures 1-3). The appearance of PEDV infected cells was confirmed by a decrease in Ct value in the PEDV Taqman assay. The PEDV-infected cells have a rounded up appearance with a layer of shiny film surrounding the rounded up cells. Example 2: Propagation and Harvest

Plastic flasks or roller bottles are used for growing and expanding cell cultures. Roller bottles or bioreactors will be used for virus propagation. Cells may be washed, to remove serum, prior to inoculation with virus. The virus may be diluted in virus production medium and added directly to the cell monolayer. When bioreactors are used for virus propagation, trypsinized cells will be removed from the roller bottles and a final cell passage grown in uninoculated cell growth medium. Microcarriers for the bioreactors are prepared. The seed virus is diluted to an appropriate volume within a multiplicity of infection (MOI) range of 0.0001 to 10.0

The PED virus causes observable cytopathic effect (CPE). Virus is harvested when viral-induced CPE has reached 50-100% and infected cells have begun sloughing off into the medium (cell monolayer loss exceeding 50%). The roller bottle vessels are removed from the incubator and inspected microscopically for both CPE and evidence of microbial contamination. Following the examination, the antigen fluid is harvested into appropriate sterile containers in an aseptic manner. Bioreactor fluids are examined microscopically for evidence of microbial contamination and for the presence of desired cytopathic effects (CPE).

Following examination, the viral fluids are passed through a≤ 100 micron filter or stainless steel mesh screen to remove microcarriers and harvested into appropriate sterile containers in an aseptic manner. Fluids may be stored at 2°C - 7°C for a maximum of 24 hours until inactivation. The harvested fluids may be used for seed if it is at the proper passage level and has an acceptable infectivity titer. In the case of Calaf14 isolation, the following specific steps were used. PEDV

Calaf14 isolate was obtained from a PEDV positive case detected in a Spanish farm in 2014. Small intestines from 4-day-old piglets displaying clinical features associated with PED, including watery diarrhea and severe dehydration, were collected at necropsy. Individual intestine samples were processed to obtain a clarified intestine homogenate. For that purpose 59 g of intestine sample were suspended in 90 ml PBS (supplemented with

Penicillin, Streptomycin and Gentamicin), disrupted using a tissue homogenizer and subsequently frozen at -80±10°C. Homogenate was thawed at 37±2°C and clarified by centrifugation at 2000rpm for 10 minutes at 4°C. Supernatant was collected, aliquoted and stored frozen at -80±10°C for further uses. Clarified intestine homogenate was found to be PEDV positive and TGEV negative by real-time RT-PCR analysis (Zoetis PEDV N gene- based real-time RT-PCR assay and PEDV-TEGV RT-PCR Rgt kit QIAGEN#283615). Isolate was identified as PEDV Calaf14 and considered passage 0 (P0).

For cell culture and isolation, intestine clarified homogenate was filtered through a 0.45-μηι filter. 1 ml of filtered material was diluted in 10ml of PBS and then filtered again through a 0.22-μηι filter and used as inoculum for virus isolation. Virus isolation was attempted using previously described PEDV cell culture conditions (Pan, Tian et al. 2012, Wicht, Li et al. 2014). Confluent Vero cells in 25-cm2 tissue culture flask were washed twice with PBS and one with the maintenance media-E (MM-E). Maintenance media-E consisted of Eagle's Minimum Essential medium Alpha Modification (MEMa, Gibco) supplemented with 0.3% tryptose phosphate broth (TPB, Gibco), 20 mM HEPES (Gibco) and 15 g/ml trypsin (Dyfco). For the initial infection of cells, 1 ml of inoculum was adsorbed at 37±2°C with + 4-7% C02 for 1 hour. Flasks were replenished with 4ml of maintenance media-E and incubated up to 2 days before being frozen at -80±10°C, thawed, and passaged as as described above. Blind passages were performed from P1 to P4 using 1 ml of previous passage as inoculum and maintenance media-E. Viral replication was verified by real-time RT-PCR analysis each passage. From passage 3 onward, a decrease of Ct value, indicative of an increase of the viral load, demonstrated a productive virus replication.

For subsequent attenuation of Calaf14, Subsequent passages (from P5 onwards) were performed using maintenance media-C (MM-C): Dulbecco's modified Eagle's medium (DMEM, Gibco), supplemented with 0.3% TPB and 10 μg/ml trypsin (DMEMs + 0.30% TPB + 10μg/ml trypsin) as previously described (Pan, Tian et al. 2012, Wicht, Li et al. 2014). From P5 to P42, 25-cm2 tissue culture tissue culture flasks of confluent Vero cells were washed twice with MM-C media and 0.01-1 ml of inoculum (previous passage material) was adsorbed at 37±2°C with + 4-7% C02 for 1 hour. Flasks were replenished with MM-C media and incubated again for a period 1-3 days at 37±2°C with + 4-7% C02. Flasks were frozen, thawed and harvested. Flasks were observed with a light microscope and cytopatic effect (CPE) with syncitia formation started to be evident at passage 6. From P43 to P60 infection was performed as follows: 25-cm2 tissue culture flasks of confluent Vero cells were washed once with MM-C media. Then flasks were filled in with the appropriate volume (5-10 ml) of MM-C media, inoculated with the appropriate volume (0.01-1 ml) of the viral suspension from previous passage without adsorption and incubated for a period 1-3 days at 37±2°C with + 4- 7% C02. When needed, relevant passages (P11 , P37 and P60) viral cultures were scaled up in 150 cm2 or 75 cm2 tissue culture flasks to assure enough volume for attenuation assessment studies. In all the passages, one T-flask was incubated as a cell control culture.

For cloning associated with Passage 60, PEDV P60 was cloned three times by end- point dilution method. The aim is to dilute the virus in order to generate a pure virus stock starting from a single infectious unit. Before virus inoculation 96-well plate of confluent vero cells were washed once with MM-C media. Tenfold serial dilutions of the PEDV in MM-C without serum were performed from 10-1 to 10-8. Each dilution was seeded eleven times (δθμΐ/well of virus dilutions) in a 96-well plate flat bottom. One column of the plate was kept as cell growth control. Plates were incubated at 37°C±2°C, 4-7% C02 during 4-5 days. Each well was observed for virus replication under an inverted microscope. All wells with signs of infection (CPE) were scored as positive. From the highest dilution at which virus replication was detected, the medium from one infected well was harvested. This inoculum was purified twice times more following the same procedure described above. Two clones were finally selected and identify as ClonA and ClonE. From the clones, a 24-well plate of confluent vero cells were infected in order to amplify the virus.

For next generation sequencing, viral RNA from samples P0, P11 , P37, P60, ClonA and ClonE was extracted using the Biosprint technology (QIAGEN) according to the manufacturer's instructions. Cellular DNA was removed by using the Rnase free Dnase set (QIAGEN) and RNA cleanup by using the Rneasy Mini Kit (QIAGEN) following

manufacturer's instructions. Purified RNA was used to obtain the complete genome for each sample by next-generation sequencing (NGS). Samples were sequenced using the lllumina MySeq system at the Service of Genomic and Bioinformatic laboratory in the University Autonoma de Barcelona. Tehcniques generally applicable herein are found in Pan, Y., X. Tian, W. Li, Q. Zhou, D. Wang, Y. Bi, F. Chen and Y. Song (2012). "Isolation and

characterization of a variant porcine epidemic diarrhea virus in China." Virol J 9: 195, and Wicht, O., W. Li, L. Willems, T. J. Meuleman, R. W. Wubbolts, F. J. van Kuppeveld, P. J. Rottier and B. J. Bosch (2014). "Proteolytic activation of the porcine epidemic diarrhea coronavirus spike fusion protein by trypsin in cell culture." J Virol 88(14): 7952-7961. Example 3: Inactivation and Neutralization

Acceptable harvested antigen production fluids will be pooled into suitable

inactivation containers and inactivated using a 5mM binary ethylenimine (BEI) solution. The mixture is cyclized for 60-80 minutes at 36 ± 2°C. Following the addition of inactivant, the antigen will be thoroughly mixed and transferred to an inactivation vessel for the duration of the process (≥48 hours, with agitation). Neutralization of the inactivated antigen fluids will be facilitated through the addition of sterile 1 M Sodium Thiosulfate to a final concentration of approximately 20 mM - 25 mM. Post-inactivated/neutralized antigen production fluids will be tested for sterility and completeness of inactivation and stored at 2-7°C for future use in vaccine serial formulation. Genatamicin can then be used as preservative. This antibiotic will be added at the lot stage. The concentration of gentamicin in the final product will be≤ 30 μg/mL.6.

Example 4: Specific Adjuvant Compositions and Formulations A preferred adjuvanted vaccine composition was assembled as follows. The killed vaccine may provide about 7.8 logi₀TCID₅₀ of killed Calaf14, Passage 60, SEQ ID NO:4, 5 or 6 virus per 2ML dose in a buffered solution further comprising about 5% (v/v) Rehydragel® (aluminum hydroxide gel) and "20% Amphigen" ® at about 25% final (v/v). Doses down to 7.0 logi₀TCID₅₀ of killed virus are also preferred.

Amphigen® is generally described in U.S Patent 5,084,269 and provides de-oiled lecithin (preferably soy) dissolved in a light oil, which is then dispersed into an aqueous solution or suspension of the antigen as an oil-in-water emulsion. Amphigen has been improved according to the protocols of U.S. Patent 6,814,971 (see columns 8-9 thereof) to provide a so-called "20% Amphigen" component for use in the final adjuvanted vaccine compositions of the present invention. Thus, a stock mixture of 10% lecithin and 90% carrier oil (DRAKEOL®, Penreco, Karns City, PA) is diluted 1 : 4 with 0.63% phosphate buffered saline solution, thereby reducing the lecithin and DRAKEOL components to 2% and 18% respectively (i.e. 20% of their original concentrations). Tween 80 and Span 80 surfactants are added to the composition, with representative and preferable final amounts being 5.6% (v/v) Tween 80 and 2.4% (v/v) Span 80, wherein the Span is originally provided in the stock DRAKEOL component, and the Tween is originally provided from the buffered saline component, so that mixture of the saline and DRAKEOL components results in the finally desired surfactant concentrations. Mixture of the DRAKEOL/lecithin and saline solutions was accomplished using an In-Line Slim Emulsifier apparatus, model 405, Charles Ross and Son, Hauppauge, NY, USA.

The vaccine composition also includes Rehydragel® LV (about 2% aluminum hydroxide content in the stock material), as additional adjuvant component (available from Reheis, NJ, USA, and ChemTrade Logistics, USA). With further dilution using 0.63% PBS, the final vaccine composition contains the following compositional amounts: 7.8 logi₀TCID₅₀ of killed virus per 2ML dose; 5% (v/v) Rehydragel® LV; 25% (v/v) of "20% Amphigen", i.e. it is further 4-fold diluted); and 0.01 % (w/v) of merthiolate.

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

Other embodiments and uses of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. All references cited herein, including all publications, U.S. and foreign patents and patent applications, are specifically and entirely incorporated by reference. It is intended that the specification and examples be considered exemplary only with the true scope and spirit of the invention indicated by the following claims.

Example 5: Safety Studies with Live Calaf14 virus, Passages 0, 1 1 , 37, and 60

A safety study was conducted to confirm the safety of Calaf 14 subsequent passages, by challenge of 4-day old, mixed sex pigs, thus Day 0 of challenge is day 4 of life 4 =/- 1 day. Route of infection/ was by esophageal gavage (2ML) using an appropriate tube feed catheter.challenge, of an undiluted cell culture supernatant. Piglets were suckled on their mothers until day 4, and during the study were fed an artificial milk diet, milk having been wiothdrawn for 1-2 hours prior to challenge.

Mortality was determined based on actual deaths and also piglets that must be euthanized per regulatory requirements. Depression, anorexia, and digestive disorders were scored as follows. Depression was scored as normal (active), slightly inactive, pronounced inactivity or severe depression (moribund). Piglets showing any level of depression are quantified below. Anorexia was scored as normal, moderate or severe. Piglets showing any level of anorexia are scored below. Digestive disorders were quantified as low (mild diarrhea), moderate (vomiting, abdominal pain or marked watery diarrhea) and severe (fibrinous or hemorrhagic diarrhea). Piglets showing any level of digestive disorder are scored below. It can thus be seen that Passage 60 substantially achieves safety criteria.

Table 1

Challenge Digestive First First

Passage Mortality Depression Anorexia Shedding

dose disorders detected detected

P1 1 10²²TCID 69% 77% 92% 92% 1 days p.i. 100% 1 days p.i.

P37 10 TCID 10% 50% 50% 100% 2 days p.i. 100% 2 days p.i.

P60 10 TCID 0% 0% 0% 0% NA 60% 6 days p.i.

Claims

1. An isolated Porcine Epidemic Diarrhea Virus (PEDV), wherein said virus is encoded by a polynucleotide selected from:

(b) a PEDV virus that is encoded by a nucleotide sequence that is at least 90% identical to one or more of SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 at a full length nucleotide level, as long as said claimed encoding sequence contains a mutant amino acid residue not found in the virus encoded from SEQ ID NO: 1 , selected from the group consisting of spike protein position 1269 (leucine), spike protein position 262 (tyrosine) and spike protein position 1006 (aspartate), ORF1 a/b polyprotein position 5627 (glycine), and an ORF3 truncation.

2. An isolated Porcine Epidemic Diarrhea Virus (PEDV) according to Claim 1 , wherein said virus is encoded by a a nucleotide sequence that is at least 95%, 96%, 97%, 98%, 99% or 99.5% identical to one or more of SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 at a full length nucleotide level.

3. A vaccine composition comprising the Porcine Epidemic Diarrhea Virus (PEDV) of Claim or Claim 2, and a sterile diluent, wherein said composition, in either adjuvanted or non adjuvanted form, is capable of protecting swine from challenge by both variant and prototype strains of PEDV, and preventing or treating symptoms associated with PEDV infection, wherein said protected swine include any of sows, gilts, boars, hogs, and piglets, and wherein achievement of protection is determined by an endpoint selected from the group consisting of prevention or control of any of the PEDV infection symptoms of dehydration, fever, diarrhea, vomiting, poor lactational performance, poor reproduction performance, mortality, and prevention or control of weight loss or failure to gain weight.

4. The vaccine composition of Claim 3, wherein the virus is killed.

5. The vaccine composition of Claim 3, wherein the virus is live.

6. The vaccine composition of Claim 4, comprising inactivated porcine epidemic diarrhea virus (PEDV), adjuvanted as an oil-in-water emulsion, wherein the adjuvant components include Amphigen® and aluminum hydroxide.

7. The vaccine composition of Claim 6 wherein the final concentration of 20% Amphigen is 25% (v/v).

8. A method of producing a neutralizing antibody response against PEDV in a subject swine comprising administering to the subject the vaccine composition of Claim 3.

9. A method of protecting swine from challenge against PEDV, comprising administering to the subject the vaccine composition of Claim 3, in an amount sufficient to prevent or treat symptoms associated with PEDV infection, wherein said protected swine include any of sows, gilts, boars, hogs, and piglets, and wherein achievement of protection is determined by an endpoint selected from the group consisting of prevention or control of any of the PEDV infection symptoms of dehydration, fever, diarrhea, vomiting, poor lactational performance, poor reproduction performance, mortality, and prevention or control of weight loss or failure to gain weight.

10. The vaccine composition of Claim 3, which is effective in piglets that are 1 day of age or older, in a single or two dose program.

11. The vaccine composition of Claim 10, which is effective in piglets in a two dose program, wherein the first dose is administered when the piglet is about 1-7 days old, and the second dose is administered when the piglet is 2-5 weeks old.

12. The vaccine composition of Claim 3, wherein the minimum effective dose is between about 10 and about 10⁶ logi₀TCID₅₀.

13. 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 of Claim 3 when said piglet is about 1-7 days old, and optionally, administering a second dose of said vaccine when the ;piglet is about 2-5 weeks old.

14. The method of Claim 13, wherein 2 doses are administered to the piglet; and the parent sow, although vaccinated pre-breeding, was not vaccinated pre-farrowing.

15. The method of Claim 13, wherein 1 dose is administered to the piglet; and the parent sow was vaccinated both pre-breeding and pre-farrowing.

16. A method of treating or preventing disease in a piglet caused by PEDV, comprising first administering the vaccine composition of Claim 3 to the sow of said piglet pre-farrowing or pre-breeding; following by administering one or more doses of said vaccine composition to said piglet after birth.

17. A method of preventing disease in healthy pigs caused by PEDV, comprising first vaccinating said pigs with the vaccine composition of Claim 3, followed by annual or pre- farrowing administration of further doses of PEDV vaccine.