# Patent

## Title
Recombinant spike protein subunit based vaccine for porcine epidemic diarrhea virus (PEDV)

## Abstract

The present invention encompasses porcine epidemic diarrhea virus (PEDV) vaccines or compositions. The vaccine or composition may be a vaccine or composition containing PEDV antigens. The invention also encompasses recombinant vectors encoding and expressing PEDV antigens, epitopes or immunogens which can be used to protect porcine animals against PEDV.

## Background
INCORPORATION BY REFERENCE
This application claims priority to provisional application U.S. Ser. No. 62/000,240, filed on 19 May 2014, and incorporated by reference herein in its entirety.


STATEMENT REGARDING SEQUENCE LISTING
The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is Recombinant_PEDV_Paul_Lawrence_ST25. The text file is 192 KB; it was created on 14 May 2015; and it is being submitted electronically via EFS-Web, concurrent with the filing of the specification.
FIELD OF THE INVENTION
The present disclosure relates generally to vaccines and more specifically to a recombinant subunit vaccine to prevent or reduce the chance of infection of pigs by porcine epidemic diarrhea virus (PEDV).
BACKGROUND
Porcine epidemic diarrhea Virus (PEDV) is a severe and highly contagious swine disease. While older pigs have a chance of survival, 80 to 100 percent of the PEDV-infected piglets die within 24 hours of being infected. PEDV spreads primarily through fecal-oral contact (Pospischil et al., 2002; Song and Park, 2012). Once internalized it destroys the inner lining of piglets' intestines, making them incapable of digesting and deriving nutrition from milk and feed (Pospischil et al., 2002). The virus causes diarrhea, vomiting and death from severe dehydration and starvation in piglets. Moreover, the infected piglets shed virus for seven to ten days (Song and Park, 2012).
Porcine Epidemic Diarrhea was first reported as a clinical entity in England in 1971 and was determined to be separate from porcine transmissible gastroenteritis virus (TGEV) (Wood, 1977). The infectious agent was further characterized and identified as a coronavirus-like particle in Belgium in 1978 (Pensaert and de Bouck, 1978). Since then, PEDV has been reported in many European and Asian countries including the Czech Republic, Hungary, Italy, Germany, Spain, Korea, the Philippines, China, Thailand and Japan (Song and Park, 2012; Pospischil et al., 2002). In contrast to infections in Asia, severe PEDV outbreaks with high mortality are rare in Europe. Within Asia, China has seen a large increase in outbreaks since 2010, which has been attributed to the emerging of new strains (Li et al., 2012). In contrast, PEDV has not been detected or reported from Central America or South America countries to date.
PEDV was first reported in the United States in May 2013 in Iowa. Since then, the PEDV has spread rapidly nationwide (The Pig Site, 2013; Promed 2013). The number of confirmed cases of PEDV increased by 296 during March, thus bringing the total number reported to 4,757, since the outbreak according to the U.S. Department of Agriculture's National Animal Health Laboratory Network (NAHLN). Twenty seven U.S states have reported PEDV infection as of March, 2014. However, one case can represent an individual animal or an entire herd at a single site. The hog industry analysts estimate that PEDV has killed approximately 5 million U.S. hogs alone since May 2013. Although highly infectious in pigs, PEDV does not affect humans and is not a food safety risk.
PEDV is a member of the Coronavirinae family and belongs to alphacoronavirus genera. These viruses are enveloped, positive-sense, single-stranded RNA and with a nucleocapsid of helical symmetry of 130 nm in diameter (Pensaert and de Bouck, 1978; Spaan et al., 1988; Kocherhans et al., 2001). Their genomic size ranges from an approximately 26 to 32 Kb, relatively large for an RNA virus. Coronavirus are the largest viruses that are known to infect humans, other mammals, and birds, usually causing subclinical respiratory or gastrointestinal diseases. The PEDV subgenomic mRNAs, which are transcribed from the genome, produce viral protein subunits, such as the spike (S,  ̃180-220 kDa), envelope (E,  ̃8.8 kDa), membrane (M, 27-32 kDa), nucleoprotein (N, 55-58 kDa), and several other proteins of unknown function (Kocherhans et al., 2001; Li et al., 2012).
About two-thirds of the 5′ end of the genome encodes a replicase protein. These proteins are encoded by two slightly overlapping open reading frames (ORF), ORF1a and ORF1b (Bridgen et al., 1988; Kocherhans et al., 2001). These two ORF subunits are connected by a ribosomal frame shift site in all the coronaviruses. This regulates the ratio of the two polypeptides encoded by ORF1a and the read-through product ORF1ab. About 70-80% of the translation products are terminated at the end of ORF1a, and the remaining 20-30% continues to transcribe until the end of ORF1b. The polypeptides are posttranslationally processed by viral encoded proteases (Bridgen et al., 1988; Park et al., 2012; Park et al., 2013). These proteases are encoded within ORF1a and the polymerase-/helicase-function are encoded by ORF1b. The analysis and amino acid alignment of N, M, E, ORF3 and S gene sequences of the highly virulent PEDV strain CV777 shows that PEDV occupies an intermediate position between the two well-characterized members of the group I corona viruses, TGEV and human coronavirus (HCoV-229E) (Pratelli 2011).
The nucleoprotein (N) subunit is a RNA-binding protein, and plays an important role in both virus RNA synthesis and modulating host cell processes. Phosphorylation and dephosphorylation may regulate these processes by exposing various functional motifs (Spencer et al., 2008; Hsieh et al., 2005). The N protein subunit has been implicated in various functions throughout the coronavirus life cycle including encapsulation, packaging, correct folding of the RNA molecule, the deregulation of the host cell cycle (Surjit, et al., 2006; Masters and Sturman, 1990), inhibition of interferon production, up-regulation of COX2 production, up-regulation of AP1 activity, induction of apoptosis, association with host cell proteins, and RNA chaperone activity (Stohlman et al., 1988; Tang et al., 2005; Nelson et al., 2000).
The PEDV E protein subunit is a homooligomer which interacts with the membrane (M) protein subunit in the budding compartment of the host cell, which is located between the endoplasmic reticulum (ER) and the Golgi complex (Duarte et al., 1994; Bridgen et al., 1998). The E protein subunit is a component of the viral envelope that plays a central role in virus morphogenesis and assembly. It also acts as a viroporin, inducing the formation of hydrophilic pores in cellular membranes and is sufficient to form virus-like particles (Madan et al., 2005). The PEDV E protein subunit has no effect on the intestinal epithelial cells (IEC) growth, cell cycle and cyclin-A expression. In contrast, the cells expressing PEDV E protein induce higher levels of IL-8 than control cells (Xu et al., 2013). Studies have shown that PEDV E protein induces ER-stress and activates transcription factor NF-κB, which is responsible for the up-regulation of interleukin 8 (IL-8) and Bcl-2 expression (Liao et al., 2006; Liao et al., 2004; Xu et al., 2013).
The M protein subunit of PEDV is the most abundant component of the viral envelope. In silico analysis of the M protein subunit shows that it consists of a triple-transmembrane segment flanked by a short amino-terminal domain on the exterior of the virion and a long carboxy-tail located inside the virion. The M protein subunit of coronaviruses is indispensable in the assembly process and budding of virions (Zhang et al., 2012). The immune reaction to the M protein of coronaviruses plays an important role in the induction of protection and in mediating the course of the disease (Zhang et al., 2012). Monoclonal antibodies against the M protein subunit of coronaviruses have virus-neutralizing activity in the presence of complement (Qian et al., 2006). Furthermore, the M protein subunit of coronavirus can also stimulate the production of alpha-interferon (α-IFN) which can inhibit viral replication (Xing et al., 2009).
The function of the PEDV ORF3 product subunit remains enigmatic, however computational modeling of PEDV OFR3 protein subunit shows that it may function as an ion channel and regulate virus production (Wang et al., 2012). Small interfering RNA (siRNA) knockdown of ORF3 gene in PEDV infected cells reduces the number of particles released from the cells (Wang et al., 2012). Passing PEDV in cell culture leads to the truncation or loss of ORF3 (Schmitz et al., 1998; Utiger et al., 1995). Homologues of the ORF3 protein subunit are found in all other alphacoronaviruses. The ORF3 protein of hCoV-NL63 was shown to be N-glycosylated at the amino terminus and incorporated into virions. However, deletion of the ORF3 gene from the viral genome had little effect on virus replication in vitro (Donaldson et al., 2008). Similar to other alphacoronaviruses (TGEV and, HCoV-229E) loss of PEDV ORF3 does not affect its replication in vitro (Dijkman et al., 2006; Woods, 2001). Despite a non-essential role in cell culture, the maintenance of the ORF3 gene in alphacoronavirus field strains strongly points to an important role of the ORF3 protein in natural infection in the animal host. Consistently, the loss of virulence of live-attenuated PEDV vaccine strains has been associated with mutations in the ORF3 gene resulting from cell culture adaptation (Song et al., 2007). However, this loss of virulence can also be attributed to concomitant mutations in other genes such as the spike protein gene (Park et al., 2008; Sato et al., 2012). The specific function of the ORF3 protein (and other viral proteins in the 3′ genome region) in PEDV replication and pathogenesis can now be investigated using the reverse genetics system (Li et al., 2013).
The spike protein of the PEDV is a large glycoprotein of  ̃180 to 200 kDa, and belongs to the class I fusion proteins (Bosch et al., 2003). The functional S protein subunit forms a homotrimer on the virion surface. The coronavirus S proteins consists of two subunits and are cleaved by host proteases into the N-terminal S1 subunit and the C-terminal membrane-anchored S2 subunit. The S1 subunit binds to its receptor on the host cell, while the S2 subunit is responsible for fusion activity (Park et al., 2007; de Haan et al., 2004). This cleavage initiates the cell-to-cell fusion and virus entry into cells (Spaan et al., 2008; Simmons et al., 2004). Various proteases are known to be utilized for cleavage of the S protein subunit of each coronavirus. For example, in murine coranavirus mouse hepatitis virus (MHV), the basic amino acid cluster in the middle of the S protein is cleaved by a protease, furin, during its biogenesis. The cleaved S protein subunit is retained on the virion and infected-cell surfaces, inducing cell-to-cell fusion (Spaan et al., 2008). In contrast, S proteins of severe acute respiratory syndrome coronavirus (SARS-CoV), nonfusogenic MHV-2, and HCoV-229E, have no furin recognition site, therefore these S proteins are not cleaved during their biogenesis (Simmons et al., 2004; Matsuyama et al., 2004; Yoshikura et al., 1988; Shirato et al., 2011). These S proteins without a furin recognition site are cleaved by endosomal proteases, such as cathepsins, and other proteases activated by the low-pH environment (Shirato et al., 2011). These coronaviruses, once bound to the receptor, are transported to the endosome, where the S protein subunit is cleaved and activated for fusion, which, in turn, results in the release of the virus genome into the cytoplasm from the endosome (Shirato et al., 2011). Thus, these coranavirus fail to induce syncytia in infected cells, and the S protein on the virion is not in a cleaved form (Shirato et al., 2011). Furthermore, the efficiency of infection of these coronavirus is not influenced by exogenous proteases. Similarly, PEDV has uncleaved S protein and PEDV-infected cells produce syncytia only after treatment with an exogenous protease, features similar to those of the coronavirus described above (Duarte et al., 1994; Durante and Laude, 1994). However, without the exogenous protease treatment, PEDV cannot grow efficiently in vitro (Park et al., 2007; Shirato et al., 2011). This explains the need for protease mediated cleavage of PEDV S protein subunit for virulence and in vitro propagation.
The complete genomic sequences of PEDV isolated from outbreaks in Minnesota and Iowa are available in the GenBank (Colorado, USA: USA/Colorado/2013, accession no. KF272920; 13-019349, accession no. KF267450 and ISU13-19338E-IN-homogenate, accession number KF650370). The genetic and phylogenetic analysis of the three U.S. strains reveals a close relationship with Chinese PEDV strains and possible Chinese origin. The U.S. PEDV strains underwent evolutionary divergence, and are classified into two sublineages. The three emergent U.S. strains are most closely related to a strain isolated in 2012 from Anhui Province in China, which might be the result of multiple recombination events between different genetic lineages or sublineages of PEDV. Molecular clock analysis of the PEDV strain-divergence based on the complete genomic sequences shows an approximately 2 to 3 years' time-frame between the Chinese (December 2010) and the U.S (May 2013) outbreaks [US-USDA, Technical note, PED. Fort Collins (Colo.): USDA; 2013]. The finding that the emergent U.S. PEDV strains share unique genetic features at the 5′-untranslated region with a bat coronavirus provided further support of the evolutionary origin of PEDV from bats and potential cross-species transmission (Graham and Baric 2010; Wang et al., 2014).
All the isolates from recent studies have shown that all PEDV strains in the U.S. are clustered together in one clade within the subgenogroup 2a and are closely related to a strain from China, AH2012 (Sun et al., 2012; Park et al. 2012; Park et al., 2013). However, in February 2014, the Animal Disease Diagnostic Laboratory of the Ohio Department of Agriculture announced that it identified a variant PED strain OH851 which showed 99% and 97% nucleotide identity to PEDVs currently circulating in the United States (Colorado, Iowa, Indiana, and Minnesota) for the whole genome and the full-length spike gene, respectively (Wang et al., 2014). By distinct contrast, the strain OH851 showed only 89% or even lower nucleotide identity to PEDVs currently circulating in the United States in the first 1,170 nt of the S1 region. In that region, nucleotide similarity to that of a PEDV strain from China (CH/HBQX/10, JS120103) was 99%, suggesting that strain OH851 is a new PEDV variant. Phylogenetic analysis of the complete genome indicated that the novel OH851 PEDV is clustered with other strains of PEDV currently circulating in the US, including another strain from Ohio, OH1414. However, phylogenetic analysis of the full-length S gene showed that strain OH851 is clustered with other strains of PEDV from China and most closely related to a PEDV strain from China, CH/HBQX/10, but distantly related to other PEDV strains currently circulating in the US and strain AH2012 (Zheng et al., 2013). These finding strongly suggests that strain OH851 is a variant PEDV. In comparison with the S gene of other strains from the US, the S gene of strain OH851 has 3 deletions (a 1-nt deletion at position 167, a 11-nt deletion at position 176, and a 3-nt deletion at position 416), a 6-nt insertion between positions 474 and 475, and several mutations mainly located in the first 1,170 nt of the S1 region (Zheng et al., 2013).
Due to these sequence deletions, insertion, and mutations the strain OH851 may have been attenuated. Since this strain does not cause severe clinical disease, including death, the novel virus is a potential vaccine candidate that could help protect the US swine industry from the infection caused by the virulent strains of PEDV currently circulating in the US. Furthermore, this analysis also indicates that the US PEDV strains are still evolving.
Over the years numerous PEDV vaccines have been developed and tried without much success. Although vaccines for PEDV exist in China, Japan and South Korea, there is no approved vaccine in the US or Europe (USDA 2013). There are two types of vaccines against PEDV that are currently available in the market—killed or live attenuated. Several Japanese, Chinese and South Korean companies manufacture PEDV vaccines, however, the efficacy and protection by PEDV vaccines is not promising or adequate for the global swine industry. On the other hand, piglets can obtain immunity from their mothers if the sow has an adequate amount of antibodies to pass immunity through colostrum (Geiger et al., 2013). Due to the lack of any efficacious vaccine in the US, one of the common practices followed by the veterinarians to protect the herd is via feedback, which is unacceptable. Recently an alphavirus based PEDV vaccine developed, licensed and distributed by a US company has failed to provide adequate protection.
The available evidence clearly indicates that PEDV is still evolving in the US and there is an immediate need to develop a more effective large scale vaccine. To achieve this, Newport Laboratories Inc., sequenced a PEDV strain isolated from the Midwest region to study its genetics, diversity and develop a subunit or attenuated vaccine.
SUMMARY OF THE INVENTION
In one aspect, the invention is a recombinant subunit vaccine comprising protein of a subunit of porcine epidemic diarrhea virus. The subunit protein may include, for example, protein of the S1 and S2 domains of the S protein gene (i.e., spike or S domain protein) of porcine epidemic diarrhea virus. In another embodiment, the subunit protein may further include the nucleoprotein (N) region of the N subunit gene of porcine epidemic diarrhea virus. In yet another embodiment, the subunit protein may include protein of the E region of the E subunit gene of porcine epidemic diarrhea virus. In yet another embodiment, the subunit protein may include protein of the M region of the M subunit gene of porcine epidemic diarrhea virus. In yet another embodiment, the subunit protein may include protein of the ORF regions of the ORF subunit genes of porcine epidemic diarrhea virus. The above embodiments may further include a suitable adjuvant.
In another aspect, the invention is a recombinant porcine epidemic diarrhea virus protein produced by an expression of a recombinant DNA construct. The construct may include a baculovirus vector and a porcine epidemic diarrhea virus DNA fragment. In one embodiment, the porcine epidemic diarrhea virus DNA fragment is all or part of the nucleoprotein (N) sequence. In another embodiment, the porcine epidemic diarrhea virus DNA fragment is all or part of the E sequence. In yet another embodiment, the porcine epidemic diarrhea virus DNA fragment is all or part of the M sequence. In yet another embodiment, the porcine epidemic diarrhea virus DNA fragment is all or part of the ORF sequence. In yet another embodiment, the porcine epidemic diarrhea virus DNA fragment is all or part of the spike protein (S1 and S2 domains) sequence. Any of the embodiments may further include at least one additional antigen or a combination of PEDV infective or non-infective DNA fragments.
In another aspect, the invention is isolated or purified virus-like particles comprising porcine epidemic diarrhea virus M protein. In one embodiment, the isolated or purified virus-like particles of the porcine epidemic diarrhea virus M protein may be used to create an immunoprotective vaccine with a pharmaceutically acceptable carrier. In another embodiment, the vaccine may include one or more other antigens.
In yet another aspect, the invention is isolated or purified virus-like particles including porcine epidemic diarrhea virus E protein. In one embodiment, the isolated or purified virus-like particles of the porcine epidemic diarrhea virus E protein may be used to create an immunoprotective vaccine with a pharmaceutically acceptable carrier. In another embodiment, the vaccine may include one or more other antigens.



BRIEF DESCRIPTION OF THE DRAWINGS
The figures are aligned with the sequences according to Table 1.









TABLE 1





SEQ ID




NO
TYPE
Description

















1
DNA
5′ UTR nucleotide sequence of Newport Labs




PEDV isolate


2
DNA
Polyprotein nucleotide sequence ORF1a and ORF1b


3
DNA
Spike (S domain) nucleotide sequence


4
DNA
ORF3 Coronavirus NS3b nucleotide sequence


5
DNA
Envelope protein


6
DNA
Reseq19R reverse gap fill primer


7
DNA
Membrane protein nucleotide sequence


8
DNA
Intergenic region B


9
DNA
Nucleoprotein nucleotide sequence


10
DNA
3′ UTR nucleotide sequence of Newport Labs




PEDV isolate


11
protein
ORF1a/ORF1b amino acid sequence


12
protein
Spike (S1 and S2domains) protein amino acid




sequence


13
protein
ORF3 Coronavirus NS3b amino acid sequence


14
protein
Envelope protein amino acid sequence


15
protein
Membrane protein amino acid sequence


16
protein
Nucleoprotein amino acid sequence


17
protein
Truncated and fused S1 and S2 domains of the spike




protein


18
DNA
Codon optimized nucleotide sequence of the




truncated and fused S1 and S2 domains of the spike




protein


19
protein
Codon optimized amino acid sequence of the




truncated and fused S1 and S2 domains of the spike




protein


20
DNA
Final nucleotide sequence for cloning into vector




showing N-terminal gp67 signal peptide sequence


21
protein
Final amino acid sequence showing N-terminal gp67




signal peptide sequence and histidine tag


22
protein
Antigenicity index of a portion of native spike (S1




domain) protein


23
protein
Antigenicity index of a portion of native spike (S1




domain) protein


24
protein
Antigenicity index of a portion of native spike (S1




domain) protein


25
protein
Antigenicity index of a portion of native spike (S1




domain) protein


26
protein
Antigenicity index of a portion of native spike (S1




domain) protein


27
protein
Antigenicity index of a portion of native spike (S1




domain) protein


28
protein
Antigenicity index of a portion of native spike (S1




domain) protein


29
protein
Antigenicity index of a portion of native spike (S1




domain) protein


30
protein
Antigenicity index of a portion of native spike (S1




domain) protein


31
protein
Antigenicity index of a portion of native spike (S1




domain) protein


32
protein
Antigenicity index of a portion of native spike (S1




domain) protein


33
protein
Antigenicity index of a portion of native spike (S1




domain) protein


34
protein
Antigenicity index of a portion of native spike (S1




domain) protein


35
protein
Antigenicity index of a portion of native spike (S1




domain) protein


36
protein
Antigenicity index of a portion of native spike (S1




domain) protein


37
protein
Antigenicity index of a portion of native spike (S1




domain) protein


38
protein
Antigenicity index of a portion of native spike (S1




domain) protein


39
protein
Antigenicity index of a portion of native spike (S1




domain) protein


40
protein
Antigenicity index of a portion of native spike (S1




domain) protein


41
protein
Antigenicity index of a portion of native spike (S1




domain) protein


42
protein
Antigenicity index of a portion of native spike (S1




domain) protein


43
protein
Antigenicity index of a portion of native spike (S1




domain) protein


44
protein
Antigenicity index of a portion of native spike (S1




domain) protein


45
protein
Antigenicity index of a portion of native spike (S1




domain) protein


46
protein
Antigenicity index of a portion of native spike (S1




domain) protein


47
protein
Antigenicity index of a portion of native spike (S1




domain) protein


48
protein
Antigenicity index of a portion of native spike (S1




domain) protein


49
protein
Antigenicity index of a portion of native spike (S1




domain) protein


50
protein
Antigenicity index of a portion of native spike (S1




domain) protein


51
protein
Antigenicity index of a portion of native spike (S1




domain) protein


52
protein
Antigenicity index of a portion of native spike (S1




domain) protein


53
protein
Antigenicity index of a portion of native spike (S1




domain) protein


54
protein
Antigenicity index of a portion of native spike (S1




domain) protein


55
protein
Antigenicity index of a portion of native spike (S2




domain) protein


56
protein
Antigenicity index of a portion of native spike (S2




domain) protein


57
protein
Antigenicity index of a portion of native spike (S2




domain) protein


58
protein
Antigenicity index of a portion of native spike (S2




domain) protein


59
protein
Antigenicity index of a portion of native spike (S2




domain) protein


60
protein
Antigenicity index of a portion of native spike (S2




domain) protein


61
protein
Antigenicity index of a portion of native spike (S2




domain) protein


62
protein
Antigenicity index of a portion of native spike (S2




domain) protein


63
protein
Antigenicity index of a portion of native spike (S2




domain) protein


64
protein
Antigenicity index of a portion of native spike (S2




domain) protein


65
protein
Antigenicity index of a portion of native spike (S2




domain) protein


66
protein
Antigenicity index of a portion of native spike (S2




domain) protein


67
protein
Antigenicity index of a portion of native spike (S2




domain) protein


68
protein
Antigenicity index of a portion of native spike (S2




domain) protein


69
protein
Antigenicity index of a portion of native spike (S2




domain) protein


70
protein
Antigenicity index of a portion of native spike (S2




domain) protein


71
protein
Antigenicity index of a portion of native spike (S2




domain) protein


72
protein
Antigenicity index of a portion of native spike (S2




domain) protein


73
protein
Antigenicity index of a portion of native spike (S2




domain) protein


74
protein
Antigenicity index of a portion of native spike (S2




domain) protein


75
protein
Antigenicity index of a portion of native spike (S2




domain) protein


76
protein
Antigenicity index of a portion of native spike (S2




domain) protein


77
protein
Antigenicity index of a portion of native spike (S2




domain) protein


78
DNA
Original non-truncated non-fused NPL-PEDV spike




(S domain) nucleotide sequence


79
protein
Original non-truncated non-fused NPL-PEDV spike




(S domain) protein sequence


80
DNA
PEDF1 forward primer


81
DNA
PEDF2 forward primer


82
DNA
PEDF3 forward primer


83
DNA
PEDF4 forward primer


84
DNA
PEDF5 forward primer


85
DNA
PEDF6 forward primer


86
DNA
PEDF7 forward primer


87
DNA
PEDF8 forward primer


88
DNA
PEDF9 forward primer


89
DNA
PEDF10 forward primer


90
DNA
PEDF11 forward primer


91
DNA
PEDF12 forward primer


92
DNA
PEDF13 forward primer


93
DNA
PEDF14 forward primer


94
DNA
PEDF15 forward primer


95
DNA
PEDF16 forward primer


96
DNA
PEDF17 forward primer


97
DNA
PED18 forward primer


98
DNA
PEDF19 forward primer


99
DNA
PEDF20 forward primer


100
DNA
PEDF21 forward primer


101
DNA
PEDF22 forward primer


102
DNA
PEDF23 forward primer


103
DNA
PEDF24 forward primer


104
DNA
PEDF25 forward primer


105
DNA
PEDF26 forward primer


106
DNA
PED27 forward primer


107
DNA
PEDF28 forward primer


108
DNA
PEDF29 forward primer


109
DNA
PEDF30 forward primer


110
DNA
PEDF31 forward primer


111
DNA
PEDF32 forward primer


112
DNA
PEDR1 reverse primer


113
DNA
PEDR2 reverse primer


114
DNA
PEDR3 reverse primer


115
DNA
PEDR4 reverse primer


116
DNA
PEDR5 reverse primer


117
DNA
PEDR6 reverse primer


118
DNA
PEDR7 reverse primer


119
DNA
PEDR8 reverse primer


120
DNA
PEDR9 reverse primer


121
DNA
PEDR10 reverse primer


122
DNA
PEDR11 reverse primer


123
DNA
PEDR12 reverse primer


124
DNA
PEDR13 reverse primer


125
DNA
PEDR14 reverse primer


126
DNA
PEDR15 reverse primer


127
DNA
PEDR16 reverse primer


128
DNA
PEDR17 reverse primer


129
DNA
PEDR18 reverse primer


130
DNA
PEDR19 reverse primer


131
DNA
PEDR20 reverse primer


132
DNA
PEDR21 reverse primer


133
DNA
PEDR22 reverse primer


134
DNA
PEDR23 reverse primer


135
DNA
PEDR24 reverse primer


136
DNA
PEDR25 reverse primer


137
DNA
PEDR26 reverse primer


138
DNA
PEDR27 reverse primer


139
DNA
PEDR28 reverse primer


140
DNA
PERR29 reverse primer


141
DNA
PEDR30 reverse primer


142
DNA
PEDR31 reverse primer


143
DNA
PEDR32 reverse primer


144
DNA
Reseq1F forward gap fill primer


145
DNA
Reseq1R reverse gap fill primer


146
DNA
Reseq2F forward gap fill primer


147
DNA
Reseq2R reverse gap fill primer


148
DNA
Reseq3F forward gap fill primer


149
DNA
Reseq3R reverse gap fill primer


150
DNA
Reseq4F forward gap fill primer


151
DNA
Reseq4R reverse gap fill primer


152
DNA
Reseq5F forward gap fill primer


153
DNA
Reseq5R reverse gap fill primer


154
DNA
Reseq6F forward gap fill primer


155
DNA
Reseq6R reverse gap fill primer


156
DNA
Reseq7F forward gap fill primer


157
DNA
Reseq7R reverse gap fill primer


158
DNA
Reseq8F forward gap fill primer


159
DNA
Reseq8R reverse gap fill primer


160
DNA
Reseq9F forward gap fill primer


161
DNA
Reseq9R reverse gap fill primer


162
DNA
Reseq10F forward gap fill primer


163
DNA
Reseq10R reverse gap fill primer


164
DNA
Reseq11F forward gap fill primer


165
DNA
Reseq11R reverse gap fill primer


166
DNA
Reseq12F forward gap fill primer


167
DNA
Reseq12R reverse gap fill primer


168
DNA
Reseq13F forward gap fill primer


169
DNA
Reseq13R reverse gap fill primer


170
DNA
Reseq14F forward gap fill primer


171
DNA
Reseq14R reverse gap fill primer


172
DNA
Reseq15F forward gap fill primer


173
DNA
Reseq15R reverse gap fill primer


174
DNA
Reseq16F forward gap fill primer


175
DNA
Reseq16R reverse gap fill primer


176
DNA
Reseq17F forward gap fill primer


177
DNA
Reseq17R reverse gap fill primer


178
DNA
Reseq18F forward gap fill primer


179
DNA
Reseq18R reverse gap fill primer


180
DNA
Reseq19F forward gap fill primer









A full and enabling description of the present invention is set forth in the remainder of the specification, including reference to the accompanying figures, wherein:
FIGS. 1A-1N are the whole genome sequence of NPL-PED (i.e., Newport Laboratories Porcine Epidemic Diarrhea Virus) with designated open reading frames (ORFs).
FIGS. 1O-1S are the derived amino acid sequence for each individual open reading frame as indicated.
FIGS. 2A-2C are the combined nucleotide and amino acid sequence of the native spike (S domain) isolate.
FIGS. 3A-3M are the antigenicity index of the S domain of NPL-PEDV.
FIGS. 3N-3P illustrate a truncated and full length NPL-PEDV spike (S domain; S1 and S2) amino acid sequence.
FIG. 3Q illustrates truncated and fused S1 and S2 domains of the spike protein of PEDV.
FIGS. 4A-4E are the truncated and fused spike protein nucleotide sequence from 3B codon optimized for an insect cell system. Optimized sequences are shown in gray.
FIGS. 4F-4G are the truncated and fused spike protein from 3C codon optimized for an insect cell system with a gp67 signal peptide at the N-terminus.
FIGS. 5A-5B: Amino acid sequence for subcloning of truncated, fused, codon optimized NPL-PEDV spike (S domain).
FIG. 5C: Nucleotide sequence for subcloning of truncated, fused, codon optimized NPL-PEDV spike (S domain).
FIG. 6A: Overview of steps involved in generating recombinant PEDV spike protein.
FIG. 6B: Cloning of PEDV spike protein gene into transfer vector.
FIG. 6C: Generation of recombinant PEDV spike protein.
FIG. 6D: Western blot analysis of PEDV spike protein.



DETAILED DESCRIPTION OF THE INVENTION
Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms “a”, “an”, and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicate otherwise.
It is noted that in this disclosure and particularly in the claims and/or paragraphs, terms such as “comprises”, “comprised”, “comprising” and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean “includes”, “included”, “including”, and the like; and that terms such as “consisting essentially of” and “consists essentially of” have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention.
The antigenic polypeptides of the invention are capable of protecting against porcine epidemic diarrhea virus (PEDV). That is, they are capable of stimulating an immune response in an animal. By “antigen” or “immunogen” means a substance that induces a specific immune response in a host animal. The antigen of the instant invention is a subunit or portion of an organism; a recombinant vector containing an insert with immunogenic properties; a piece or fragment of DNA capable of inducing an immune response upon presentation to a host animal; a polypeptide, an epitope, a hapten, or any combination thereof.
The term “immunogenic protein, polypeptide, or peptide” as used herein includes polypeptides that are immunologically active in the sense that once administered to the host, it is able to evoke an immune response of the humoral and/or cellular type directed against the protein. A protein fragment according to the invention has at least one epitope or antigenic determinant. An “immunogenic” protein or polypeptide, as used herein, includes the full-length sequence of the protein, analogs thereof, or immunogenic fragments thereof.
As discussed the invention encompasses active fragments and variants of the antigenic polypeptide. Thus, the term “immunogenic protein, polypeptide, or peptide” further contemplates deletions, additions and substitutions to the sequence, so long as the polypeptide functions to produce an immunological response as defined herein. The term “conservative variation” denotes the replacement of an amino acid residue by another biologically similar residue, or the replacement of a nucleotide in a nucleic acid sequence such that the encoded amino acid residue does not change or is another biologically similar residue. In this regard, particularly preferred substitutions will generally be conservative in nature, i.e., those substitutions that take place within a family of amino acids. For example, amino acids are generally divided into four families: (1) acidic—aspartate and glutamate; (2) basic—lysine, arginine, histidine; (3) non-polar—alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar—glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. Examples of conservative variations include the substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another hydrophobic residue, or the substitution of one polar residue for another polar residue, such as the substitution of arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine, and the like; or a similar conservative replacement of an amino acid with a structurally related amino acid that will not have a major effect on the biological activity. Proteins having substantially the same amino acid sequence as the reference molecule but possessing minor amino acid substitutions that do not substantially affect the immunogenicity of the protein are, therefore, within the definition of the reference polypeptide. All of the polypeptides produced by these modifications are included herein. The term “conservative variation” also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid provided that antibodies raised to the substituted polypeptide also immunoreact with the unsubstituted polypeptide.
The term “epitope” refers to the site on an antigen or hapten to which specific B cells and/or T cells respond. The term is also used interchangeably with “antigenic determinant” or “antigenic determinant site”. Antibodies that recognize the same epitope can be identified in a simple immunoassay showing the ability of one antibody to block the binding of another antibody to a target antigen.
An “immunological response” to a composition or vaccine is the development in the host of a cellular and/or antibody-mediated immune response to a composition or vaccine of interest. Usually, an “immunological response” includes but is not limited to one or more of the following effects: the production of antibodies, B cells, helper T cells, and/or cytotoxic T cells, directed specifically to an antigen or antigens included in the composition or vaccine of interest. Preferably, the host will display either a therapeutic or protective immunological response such that resistance to new infection will be enhanced and/or the clinical severity of the disease reduced. Such protection will be demonstrated by either a reduction or lack of symptoms normally displayed by an infected host, a quicker recovery time and/or a lowered viral titer in the infected host.
Synthetic antigens are also included within the definition, for example, polyepitopes, flanking epitopes, and other recombinant or synthetically derived antigens. See, e.g., Bergmann et al., 1993; Bergmann et al., 1996; Suhrbier, 1997; Gardner et al., 1998. Immunogenic fragments, for purposes of the present invention, will usually include at least about 3 amino acids, at least about 5 amino acids, at least about 10-15 amino acids, or about 15-25 amino acids or more amino acids, of the molecule. There is no critical upper limit to the length of the fragment, which could comprise nearly the full-length of the protein sequence, or even a fusion protein comprising at least one epitope of the protein.
Accordingly, a minimum structure of a polynucleotide expressing an epitope is that it has nucleotides encoding an epitope or antigenic determinant of a PEDV polypeptide. A polynucleotide encoding a fragment of a PEDV polypeptide may have a minimum of 15 nucleotides, about 30-45 nucleotides, about 45-75, or at least 57, 87 or 150 consecutive or contiguous nucleotides of the sequence encoding the polypeptide. Epitope determination procedures, such as, generating overlapping peptide libraries (Hemmer et al., 1998), Pepscan (Geysen et al., 1984; Geysen et al., 1985; Van der Zee R. et al., 1989; Geysen, 1990; Multipin® Peptide Synthesis Kits de Chiron) and algorithms (De Groot et al., 1999; PCT/US2004/022605) can be used in the practice of the invention.
The term “nucleic acid” or “polynucleotide” refers to RNA or DNA that is linear or branched, single or double stranded, or a hybrid thereof. The term also encompasses RNA/DNA hybrids. The following are non-limiting examples of polynucleotides: a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, uracyl, other sugars and linking groups such as fluororibose and thiolate, and nucleotide branches. The sequence of nucleotides may be further modified after polymerization, such as by conjugation, with a labeling component. Other types of modifications included in this definition are caps, substitution of one or more of the naturally occurring nucleotides with an analog, and introduction of means for attaching the polynucleotide to proteins, metal ions, labeling components, other polynucleotides or solid support. The polynucleotides can be obtained by chemical synthesis or derived from a microorganism.
The term “gene” is used broadly to refer to any segment of polynucleotide associated with a biological function. Thus, genes include introns and exons as in genomic sequence, or just the coding sequences as in cDNAs and/or the regulatory sequences required for their expression. For example, gene also refers to a nucleic acid fragment that expresses mRNA or functional RNA, or encodes a specific protein, and which includes regulatory sequences.
The invention further comprises a complementary strand to a polynucleotide encoding a PEDV antigen, epitope or immunogen. The complementary strand can be polymeric and of any length, and can contain deoxyribonucleotides, ribonucleotides, and analogs in any combination.
The terms “protein”, “peptide”, “polypeptide” and “polypeptide fragment” are used interchangeably herein to refer to polymers of amino acid residues of any length. The polymer can be linear or branched, it may comprise modified amino acids or amino acid analogs, and it may be interrupted by chemical moieties other than amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling or bioactive component.
An “isolated” biological component (such as a nucleic acid or protein or organelle) refers to a component that has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, for instance, other chromosomal and extra-chromosomal DNA and RNA, proteins, and organelles. Nucleic acids and proteins that have been “isolated” include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant technology as well as chemical synthesis.
The term “purified” as used herein does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified polypeptide preparation is one in which the polypeptide is more enriched than the polypeptide is in its natural environment. That is the polypeptide is separated from cellular components. By “substantially purified” it is intended that such that the polypeptide represents several embodiments at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98%, or more of the cellular components or materials have been removed. Likewise, the polypeptide may be partially purified. By “partially purified” is intended that less than 60% of the cellular components or material is removed. The same applies to polynucleotides. The polypeptides disclosed herein can be purified by any of the means known in the art.
As noted above, the antigenic polypeptides or fragments or variants thereof are PEDV antigenic polypeptides that are produced in insect cells. Fragments and variants of the disclosed polynucleotides and polypeptides encoded thereby are also encompassed by the present invention. By “fragment” is intended a portion of the polynucleotide or a portion of the antigenic amino acid sequence encoded thereby. Fragments of a polynucleotide may encode protein fragments that retain the biological activity of the native protein and hence have immunogenic activity as noted elsewhere herein. Fragments of the polypeptide sequence retain the ability to induce a protective immune response in an animal.
“Variants” is intended to mean substantially similar sequences. For polynucleotides, a variant comprises a deletion and/or addition of one or more nucleotides at one or more sites within the native polynucleotide and/or a substitution of one or more nucleotides at one or more sites in the native polynucleotide. As used herein, a “native” polynucleotide or polypeptide comprises a naturally occurring nucleotide sequence or amino acid sequence, respectively. Variants of a particular polynucleotide of the invention (i.e., the reference polynucleotide) can also be evaluated by comparison of the percent sequence identity between the polypeptide encoded by a variant polynucleotide and the polypeptide encoded by the reference polynucleotide. “Variant” protein is intended to mean a protein derived from the native protein by deletion or addition of one or more amino acids at one or more sites in the native protein and/or substitution of one or more amino acids at one or more sites in the native protein. Variant proteins encompassed by the present invention are biologically active, that is they the ability to elicit an immune response.
As used herein, the term “derivative” or “variant” refers to a polypeptide, or a nucleic acid encoding a polypeptide, that has one or more conservative amino acid variations or other minor modifications such that (1) the corresponding polypeptide has substantially equivalent function when compared to the wild type polypeptide or (2) an antibody raised against the polypeptide is immunoreactive with the wild-type polypeptide. These variants or derivatives include polypeptides having minor modifications of the NPL-PEDV polypeptide primary amino acid sequences that may result in peptides which have substantially equivalent activity as compared to the unmodified counterpart polypeptide. Such modifications may be deliberate, as by site-directed mutagenesis, or may be spontaneous. The term “variant” further contemplates deletions, additions and substitutions to the sequence, so long as the polypeptide functions to produce an immunological response as defined herein.
The term “conservative variation” denotes the replacement of an amino acid residue by another biologically similar residue, or the replacement of a nucleotide in a nucleic acid sequence such that the encoded amino acid residue does not change or is another biologically similar residue. In this regard, particularly preferred substitutions will generally be conservative in nature, as described above.
The polynucleotides of the disclosure include sequences that are degenerate as a result of the genetic code, e.g., optimized codon usage for a specific host. As used herein, “optimized” refers to a polynucleotide that is genetically engineered to increase its expression in a given species. To provide optimized polynucleotides coding for PEDV polypeptides, the DNA sequence of the PEDV gene can be modified to 1) comprise codons preferred by highly expressed genes in a particular species; 2) comprise an A+T or G+C content in nucleotide base composition to that substantially found in said species; 3) form an initiation sequence of said species; or 4) eliminate sequences that cause destabilization, inappropriate polyadenylation, degradation and termination of RNA, or that form secondary structure hairpins or RNA splice sites. Increased expression of PEDV protein in said species can be achieved by utilizing the distribution frequency of codon usage in eukaryotes and prokaryotes, or in a particular species. The term “frequency of preferred codon usage” refers to the preference exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included in the disclosure as long as the amino acid sequence of the PEDV polypeptide encoded by the nucleotide sequence is functionally unchanged.
The invention further encompasses the PEDV polynucleotides contained in a vector molecule or an expression vector and operably linked to a promoter element and optionally to an enhancer. A “vector” refers to a recombinant DNA or RNA plasmid or virus that comprises a heterologous polynucleotide to be delivered to a target cell, either in vitro or in vivo. The heterologous polynucleotide may comprise a sequence of interest for purposes of prevention or therapy, and may optionally be in the form of an expression cassette. As used herein, a vector needs not be capable of replication in the ultimate target cell or subject. The term includes cloning vectors and viral vectors.
The term “recombinant” means a polynucleotide semisynthetic, or synthetic origin which either does not occur in nature or is linked to another polynucleotide in an arrangement not found in nature.
“Heterologous” means derived from a genetically distinct entity from the rest of the entity to which it is being compared. For example, a polynucleotide may be placed by genetic engineering techniques into a plasmid or vector derived from a different source, and is a heterologous polynucleotide. A promoter removed from its native coding sequence and operatively linked to a coding sequence other than the native sequence is a heterologous promoter.
The present invention relates to porcine vaccines or pharmaceutical or immunological compositions which may comprise an effective amount of a recombinant PEDV antigens and a pharmaceutically or veterinarily acceptable carrier, excipient, or vehicle.
The subject matter described herein is directed in part, to compositions and methods related to the PEDV antigen prepared in an insect expression system that was highly immunogenic and protected animals against challenge from PEDV strains.
Newport Laboratories Inc., received small intestine and colon samples from 0-7 day old piglets in May 2013. The piglets were exhibiting vomiting and diarrhea. PEDV is related to transmissible gastroenteritis virus (TGEV) and causes enteric disease clinically indistinguishable from TGEV. There is little to no cross protection afforded by immunity developed to one virus against the other. Similarly, diagnostic tests designed to detect TGEV will not detect PEDV or vice versa. However, we detected PEDV by qPCR using multiplex real-time RT-PCR technique (Kim et al., 2007). In addition, the samples were negative for TGEV or rotavirus group ABC. The samples were then used to extract viral RNA and the complete genome of the NPL-PEDV (i.e., Newport Laboratories Porcine Epidemic Diarrhea Virus). The strain was sequenced using a primer walking technique, assembled and annotated. Complete in silico genome analysis was performed and the spike protein was selected to develop a first generation recombinant vaccine.
PEDV spike protein plays an important role in viral infection and pathogenesis. Mutation of spike protein can cause attenuation and antibodies against spike protein can reduce PEDV infection. In addition, the spike protein gene is highly conserved across pathogenic PEDV strains. Therefore, development of a subunit vaccine containing spike protein may afford broader protection. With this objective a recombinant spike protein gene was codon optimized and synthesized from the NPL-PEDV genome sequence information and expressed in a baculovirus expression system. The next generation of vaccines may include other possible PEDV target antigens such as M and N proteins and possibly portions of ORF1ab. Baculovirus expression systems could also be used to create virus-like particle (VLP) vaccines. This information will also be used to develop attenuated PEDV strains by directed mutagenesis for live vaccination program.
Methods and Results
Isolation of Viral RNA and Whole Genome Sequencing
The intestinal samples were pooled into two sets and were used for total nucleic acid extraction independently. Briefly, 200 μL of PEDV pools 1 and 2 were treated with a DNAse/RNAse cocktail [Epicentre Biotechnologies, WI, USA] using 2 U of enzyme/microliter of suspension after adding MgCl2 to a final concentration of 5 mM. Each sample was incubated for one hour at 37° C. in order to completely digest exogenous host DNA and RNA. After treatment, RNA was purified from the samples using Trizol LS reagent according to the manufacturer's recommendation [Life Technologies, NY, USA]. The precipitated RNA was resuspended in 50 μL of 10 mM Tris buffer and frozen at −80° C. until cDNA conversion. The cDNA conversion was performed using the Maxima H Minus Double-stranded cDNA synthesis kit [Thermo Fisher Scientific, MA, USA] according to the manufacturer's instructions. For each sample 2 μg of input RNA was used in conjunction with random hexamer primers at a 100 pmol concentration. Following cDNA synthesis, samples were treated with RNAse according to the kit instructions to eliminate any residual RNA from the sample preparation. Following treatment samples were immediately frozen and stored at −20° C.
The double stranded DNA was used in polymerase chain reaction using a set of 32 primer pairs spanning the entire PEDV genome. See Table 2.









TABLE 2






Primer name
Primer sequence 5′ → 3′








PEDF1 - SEQ ID NO: 80
ACTTAAAGAGATTTTCTATCTAC


 



PEDF2 - SEQ ID NO: 81
AGGTTGCACGTACTCCAAAGAT


 



PEDF3 - SEQ ID NO: 82
GCATTGGTTAAGCTTGTCAAGG


 



PEDF4 - SEQ ID NO: 83
CTTCAAGTATTATGCCACCAGTG


 



PEDF5 - SEQ ID NO: 84
TGACTTTGCAAGCTATGGAGGAC


 



PEDF6 - SEQ ID NO: 85
GCATGCACCTGAGCTTCTTG


 



PEDF7 - SEQ ID NO: 86
GTTGTAGCTAAGGTTGTACCAAG


 



PEDF8 - SEQ ID NO: 87
ACGTACTGGTATTATATTGCGT


 



PEDF9 - SEQ ID NO: 88
CTTAATGTGCAACCGACAGGTCC


 



PEDF10 - SEQ ID NO: 89
GACAATCCACTTAGTTGTGTGC


 



PEDF11 - SEQ ID NO: 90
CACAGAACACACTTGGCATGTTG


 



PEDF12 - SEQ ID NO: 91
ATGATGGTTCTGCAGCTGGTGT


 



PEDF13 - SEQ ID NO: 92
TGCACAAGGTCTTGTTAACATC


 



PEDF14 - SEQ ID NO: 93
GATGCTGTTAATAATGGTTCTCC


 



PEDF15 - SEQ ID NO: 94
GCCACTGTACGCTTGCAGGCTGG


 



PEDF16 - SEQ ID NO: 95
GAAGACATTCATCGTGTCTATGC


 



PEDF17 - SEQ ID NO: 96
GTGGTTGTATCACTGCTAAAGAGG


 



PED18 - SEQ ID NO: 97
TCGAGCCTGACATTAATAAAGGTC


 



PEDF19 - SEQ ID NO: 98
CACTTGTTATCATATAACGAAG


 



PEDF20 - SEQ ID NO: 99
ACTGTGTCTGAGATGGTCTATGAA


 



PEDF21 - SEQ ID NO: 100
CGTCAGAGCTCGTGCTCCACCAG


 



PEDF22 - SEQ ID NO: 101
ATGATGATACTGAGTGTGACAAG


 



PEDF23 - SEQ ID NO: 102
CAAGTACGGACTTGAAGATTAGC


 



PEDF24 - SEQ ID NO: 103
CTGATATGTATGATGGTAAGATT


 



PEDF25 - SEQ ID NO: 104
CTAGTGGTTACCAGCTTTATTTAC


 



PEDF26 - SEQ ID NO: 105
CCACTGTTTATAAATTCTTGGCTG


 



PED27 - SEQ ID NO: 106
GTCACTAGTGGTGCTGTTTATTC


 



PEDF28 - SEQ ID NO: 107
CTCTGCTATTGGTAATATAACTTC


 



PEDF29 - SEQ ID NO: 108
GTTGACCTTGAGTGGCTCAACCGAG


 



PEDF30 - SEQ ID NO: 109
TGGTCTAGTAGTTAATGTTATAC


 



PEDF31 - SEQ ID NO: 110
GTGGCCGCAAACGGGTGCCATTATC


 



PEDF32 - SEQ ID NO: 111
TAGCGTAGCAGCTTGCTTCGGACC


 



PEDR1 - SEQ ID NO: 112
ATCTTTGGAGTACGTGCAACCT


 



PEDR2 - SEQ ID NO: 113
CCTTGACAAGCTTAACCAATGC


 



PEDR3 - SEQ ID NO: 114
CACTGGTGGCATAATACTTGAAG


 



PEDR4 - SEQ ID NO: 115
GTCCTCCATAGCTTGCAAAGTCA


 



PEDR5 - SEQ ID NO: 116
CAAGAAGCTCAGGTGCATGCTT


 



PEDR6 - SEQ ID NO: 117
CTTGGTACAACCTTAGCTACAAC


 



PEDR7 - SEQ ID NO: 118
ACGCAATATAATACCAGTACGT


 



PEDR8 - SEQ ID NO: 119
GGACCTGTCGGTTGCACATTAAG


 



PEDR9 - SEQ ID NO: 120
GCACACAACTAAGTGGATTGTC


 



PEDR10 - SEQ ID NO: 121
CAACATGCCAAGTGTGTTCTGTG


 



PEDR11 - SEQ ID NO: 122
ACACCAGCTGCAGAACCATCAT


 



PEDR12 - SEQ ID NO: 123
GATGTTAACAAGACCTTGTGCA


 



PEDR13 - SEQ ID NO: 124
GGAGAACCATTATTAACAGCATC


 



PEDR14 - SEQ ID NO: 125
CCAGCCTGCAAGCGTACAGTGGC


 



PEDR15 - SEQ ID NO: 126
GCATAGACACGATGAATGTCTTC


 



PEDR16 - SEQ ID NO: 127
CCTCTTTAGCAGTGTTACAACCAC


 



PEDR17 - SEQ ID NO: 128
GACCTTTATTAATGTCAGGCTCGA


 



PEDR18 - SEQ ID NO: 129
CTTCGTTATATGATAACAAGTG


 



PEDR19 - SEQ ID NO: 130
TCATAGACCATCTCAGACACAGT


 



PEDR20 - SEQ ID NO: 131
CTGGTGGAGCACGAGCTCTGAGC


 



PEDR21 - SEQ ID NO: 132
CTTGTCACACTCAGTATCATCAT


 



PEDR22 - SEQ ID NO: 133
CGTAATCTTCAAGTCCGTACTTG


 



PEDR23 - SEQ ID NO: 134
AAT CTT ACC ATC ATA CAT




ATC AG


 



PEDR24 - SEQ ID NO: 135
GTA AAT AA GCTGGTAACCACT




AG


 



PEDR25 - SEQ ID NO: 136
CAGCCAAGAATTTATAAACAGTGG


 



PEDR26 - SEQ ID NO: 137
GAATAAACAGCACCACTAGTGAC


 



PEDR27 - SEQ ID NO: 138
GAAGTTATATTACCAATAGCAGAG


 



PEDR28 - SEQ ID NO: 139
CTCGGTTGAGCCACTCAAGGTCAAC


 



PERR29 - SEQ ID NO: 140
GTATAACATTAACTACTAGACCA


 



PEDR30 - SEQ ID NO: 141
GATAATGGCACCCGTTTGCGGCCAC


 



PEDR31 - SEQ ID NO: 142
GGTCCGAAGCAAGCTGCTACGCTA


 



PEDR32 - SEQ ID NO: 143
GTGTATCCATATCAACACCGTCAG









The primers were designed based on the consensus-genome sequences of two U.S. PEDV strains—USA/Colorado/2013 (GenBank accession no. KF272920) and 13-019349 (GenBank accession no. KF267450). Primer sets 1-32 were used to amplify segments of the PEDV genome. The letter “F” in the primer name denotes a forward primer. The letter “R” in the primer name denotes a reverse primer. Each reaction was performed using Phire Green Hot-Start II DNA polymerase [Thermo Fisher Scientific, MA, USA] according to the manufacturer's instructions. About 1-10 ng of the DNA template was used in a 50 μl cocktail and the reaction was performed according to the manufacturer's protocol. The amplified products ranging from 900-1000 bp were run on an agarose gel to confirm their size. The PCR cycling conditions were as follows: initial denaturation at 98° C. for 30 sec, followed by 35 cycles: 98° C.—15 sec, 50° C.—15 sec, 72° C.—45 sec and a final extension at 72° C. for 1 min.
The PCR products were purified from each reaction using an IBI Gel/PCR product purification kit [IBI Scientific, IA, USA] according to the manufacturer's recommendation. The final elution was performed using 30 μL of elution buffer. About 15 μL of each purified product was sent to Eurofins/Operon [Eurofins MWG Operon, Ala., USA] for bi-directional sequencing using the amplification primers specific for each product as listed in Table 2.
Sequence Assembly and Analysis
The raw sequence data was assembled using USA/Colorado/2013 (GenBank accession no. KF272920) as reference sequence, using the Geneious assembler [Biomatters LTD., CA, USA]. After assembling the whole NPL-PED genome to the reference genome the gaps and ambiguous regions in NPL-PED genome were closed using a second set of internal primers disclosed in Table 3 following the same PCR and sequencing technique.









TABLE 3






Primer name
Primer sequence 5′ → 3′








Reseq1F - SEQ ID NO: 144
ATCACTGGTCTTAATACAATGTG


 



Reseq1R - SEQ ID NO: 145
CAATACTACCATTGAGTGCTGGTGG


 



Reseq2F - SEQ ID NO: 146
TGCAGAAGTGCTCGAATGATTAC


 



Reseq2R - SEQ ID NO: 147
CTTGTTGAACATCTTCCTGGACAG


 



Reseq3F - SEQ ID NO: 148
TTGTGATTCTTATGGTCCAGG


 



Reseq3R - SEQ ID NO: 149
CTGGCCAACAACGCTGAGTCCAC


 



Reseq4F - SEQ ID NO: 150
CTGCTCTGATTGTTACATCTTGC


 



Reseq4R - SEQ ID NO: 151
TAGCCACAAAAGTAGGAAATCTC


 



Reseq5F - SEQ ID NO: 152
GTTGACTTGCATAACAAGATC


 



Reseq5R - SEQ ID NO: 153
AGCAGTGAATGCATAGCACTTAC


 



Reseq6F - SEQ ID NO: 154
ACAATTGCGATGTTCTTAAGAG


 



Reseq6R - SEQ ID NO: 155
TCCTCACCAAATATATCACTC


 



Reseq7F - SEQ ID NO: 156
CAGACTGTTAAACCTGGCCATTTC


 



Reseq7R - SEQ ID NO: 157
AGGTTGAGCTGTGTCATAGTG


 



Reseq8F - SEQ ID NO: 158
TATGGTTACTTGCGTAAAC


 



Reseq8R - SEQ ID NO: 159
CTCTAACACACCAGCATTAAG


 



Reseq9F - SEQ ID NO: 160
TCTGACTACAGGTTGGCAAATG


 



Reseq9R - SEQ ID NO: 161
GCACTAAGCTAGAATAAGCTTC


 



Reseq10F - SEQ ID NO: 162
TGGATGAGGTCTCTATGTGCAC


 



Reseq10R - SEQ ID NO: 163
CCACAACCCTCATTAGCCTG


 



Reseq11F - SEQ ID NO: 164 
ACTGATCAAGATCTTGCTGTTC


 



Reseq11R - SEQ ID NO: 165
GCTAAGTGATCCCTTGTATC


 



Reseq12F - SEQ ID NO: 166
CTAATGTCAAGACATTGGAGT


 



Reseq12R - SEQ ID NO: 167
TACGACATTGAAAGCAATGTTC


 



Reseq13F - SEQ ID NO: 168
TGGTATATTTACACTAGGAAG


 



Reseq13R - SEQ ID NO: 169
GCAGGAGATCCATATACGTAC


 



Reseq14F - SEQ ID NO: 170
TGCCACTGGATGCCATTATAG


 



Reseq14R - SEQ ID NO: 171
CTAAATAGTGAACACCAATTAAG


 



Reseq15F - SEQ ID NO: 172
TCAACTTGGTACTGTGCTGGC


 



Reseq15R - SEQ ID NO: 173
GACAGTGACACGATCATTATC


 



Reseq16F - SEQ ID NO: 174
GTGAGTTGATTACTGGCACGC


 



Reseq16R - SEQ ID NO: 175
TGTCCTAATACTCATACTAAAG


 



Reseq17F - SEQ ID NO: 176
TCGCTCTGTGGCAGATCTAGTC


 



Reseq17R - SEQ ID NO: 177
TGAGGTGCTGCCTGTACCAGAGAG


 



Reseq18F - SEQ ID NO: 178
CAGATTACATCGATGTTAAC


 



Reseq18R - SEQ ID NO: 179
GACAAGTTAGCAGACTTTGAGAC


 



Reseq19F - SEQ ID NO: 180
GCTGACCTACAGCTGTTGCG


 



Reseq19R - SEQ ID NO: 6
TCATCAACGGGAATAGAACCG









The complete sequence of the two NPL PEDV isolates were assembled as one large contig and annotated. Further global BLAST analysis and alignment was done using the web based software from NCBI, to identify indels/point mutations with reference to US and other PEDV isolates in the GenBank. The whole genome sequence of NPL-PED (FIG. 1A, SEQ ID NOs 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10) contains 28,064 nucleotides (nt), including a 5′ and 3′ UTR. The derived amino acid sequence is also provided (FIG. 1B, SEQ ID NOs: 11, 12, 13, 14, 15 and 16).
The NPL-PEDV genome shares 99% identity with the all the U.S. isolates sequenced to date and many Chinese isolates as well (except for the strain OH851). The top three BLAST hits were against U.S. isolates, USA/Colorado/2013 (KF272920), IA1 (KF468753.1) and Iowa isolate 13-019349 (KF267450.1). NPL-PEDV also shares 99% identity with the Chinese outbreak isolate AH2012 (KC210145). When compared to KF272920, NPL-PEDV has insertions between 20634-20637nt, 25455-25474nt, a nucleotide substitution at 278 (T→C) which falls in the 5′ UTR, an insertion at 9645nt (C) and a deletion at 9648nt.
Analysis of Spike Protein
The spike coding region (S domain) is highly conserved across PEDV strains. The spike protein is composed of two domains: S1 and S2. FIG. 2 shows the amino acid sequence for S1 and S2 (SEQ ID NO: 12) as well as the nucleotide sequence for S1 and S2 (SEQ ID NO: 3). BLAST analysis of the NPL-PEDV strain shows that it shares 99% homology with PEDV isolates from USA and China. Internet based software was used to analyze the antigenicity index (EMBOSS programs). Based on the antigenicity index of the S domain (FIG. 3A), the amino acid sequences showing the highest antigenic index were chosen and fused. The truncated S1 and S2 subunit domains were joined together as indicated in FIG. 3B (SEQ ID NO. 17) and FIG. 3C (SEQ ID NO: 17), deleting intervening amino acids. Deleted regions are indicated with a dash “-” and regions retained for expression are indicated with an asterisk “*” in FIG. 3B. The truncated and fused PEDV spike protein sequence (SEQ ID NO: 17) is shown in FIG. 3C.
The nucleotide sequence of the truncated and fused spike protein was codon optimized for an insect cell system. FIG. 4A shows the original nucleotide sequence of the truncated and fused spike region (S domain, SEQ ID NO: 78) compared with the codon optimized spike region (S domain codon optimized, SEQ ID NO: 18). FIG. 4B shows the original amino acid sequence of the truncated and fused spike region (S domain, SEQ ID NO: 79) compared with the codon optimized spike region (S domain codon optimized, SEQ ID NO: 19).
Gene Synthesis, Cloning into Baculovirus Expression System and Detection of Recombinant Spike
The complete nucleotide sequence of the synthesized spike protein gene is shown in FIG. 5A (SEQ ID NO: 20). The gene was synthesized [GenScript Corporation, NJ, U.S.A] with an engineered 5′ Eco RI site immediately after the signal peptide sequence and an 3′ Hin dIII site to facilitate cloning. This gene was cloned into Bac-to-Bac® Baculovirus Expression System [Life Technologies, NY, U.S.A] according to the manufacturer's instruction and expressed in Sf21, Sf9 or high five cells. FIG. 5B shows the synthesized spike (S domain) amino acid sequence (SEQ ID NO: 21). A C-terminal His tag and an N-terminal signal peptide sequence was engineered to selectively purify the protein from a nickel column and to detect the protein by monoclonal antibody on a Western blot, respectively.
The overall cloning, protein expression and detection is explained in FIGS. 6A-D. FIG. 6A is an overview of the process. The synthetic spike protein gene was generated and cloned into transfer vector as indicated in FIG. 6B. The integrity of the cloned gene was confirmed by bi-directional sequencing. After confirming the sequence the transfer vector was introduced into competent E. coli cells containing a helper plasmid. The E. coli was selected on Luria-Bertani (LB) plates containing 100 μg/ml ampicillin. Transposition mediated by Tn results in the integration of the spike gene into the bacmid. The resultant bacmid DNA was extracted using Qiagen Plasmid preparation kit (Qiagen, Calif., USA) and transfected into Sf9 of Sf21 cells using Cellfectin® reagent following the manufacturer's instructions. Once P1 clones were generated plaque assay and qPCR were used to determine the PFU/ml. Plaque assay was used to isolate single clones (FIG. 6C). The P1 stock was used to generate P2 stock and for confirmation of protein expression Sf9 or Sf2l cells according to the manufacturer's recommendation [Life Technologies, NY, USA]. Once expressed the cell lysate and the supernatant were analyzed for protein expression on a SDS-PAGE gel and transferred onto PVFD membrane. The PVDF membrane was blocked with blocking buffer and probed with mouse anti-His Antibody coupled to HRP (Cat. No. A00612; GenScript Corporation, NJ, USA, FIG. 6D).
The recombinant spike protein was expressed in high five cells and used for vaccination studies in pigs.
In an embodiment, the invention is an immunogenic composition comprising a recombinant polypeptide sequence of the S1 and S2 domains of the S subunit of porcine epidemic diarrhea virus according to SEQ ID NO. 19 and a pharmaceutically or veterinary acceptable vehicle, diluent or excipient. The invention may further include a suitable adjuvant. The adjuvant may be an oil, emulsion, a metal salt (e.g. Al(OH)3), or combinations thereof. In an embodiment, the adjuvant is TRIGEN® or ULTRAGEN® or PrimaVant® (TRIGEN+Quil A), TS6 (described in U.S. Pat. No. 7,371,395 US to Merial), LR4 (described in U.S. Pat. No. 7,691,368, to Merial), or any formulation described in US 2011-0129494 A1 (to Merial).
In another embodiment, the immunogenic composition is a recombinant nucleotide sequence 80% or greater in sequence identity to SEQ ID NO. 18.
In another aspect, the invention is a method of vaccinating a host susceptible to PEDV comprising at least one administration of a recombinant polypeptide sequence of the S1 and S2 domains of the S subunit of porcine epidemic diarrhea virus according to SEQ ID NO. 19 and a pharmaceutically or veterinary acceptable vehicle, diluent or excipient. An adjuvant may also be included.
In another aspect, the invention is a recombinant subunit vaccine for use against porcine epidemic diarrhea virus comprising a porcine epidemic diarrhea virus DNA fragment according to SEQ ID NO. 18 in a baculovirus expression system. In an embodiment, the invention is a recombinant subunit vaccine comprising the amino acid sequence according to SEQ ID NO. 19. In yet another embodiment, the invention is a recombinant subunit vaccine comprising the amino acid sequence according to SEQ ID NO. 20. In yet another embodiment, the invention is a recombinant subunit vaccine comprising the amino acid sequence according to SEQ ID NO. 21. In yet another embodiment, the invention is an immunogenic composition comprising a recombinant polypeptide sequence of the S1 and S2 domains of the S subunit of porcine epidemic diarrhea virus according to SEQ ID NO. 19 and a pharmaceutically or veterinary acceptable vehicle, diluent or excipient and at least one additional antigen associated with a pathogen other than porcine epidemic diarrhea virus.
In yet another embodiment, the invention is an isolated polypeptide sequence, wherein the polypeptide sequence is 80% or greater in sequence identity to SEQ ID NO. 17. In yet another embodiment, the invention is an isolated polypeptide sequence, wherein the polypeptide sequence is 80% or greater in sequence identity to SEQ ID NO:19.
The genome sequence of PEDV can also be used to generate virus-like particles (VLPs) using structural genes or other non-infectious components of PEDV. Examples are virus-like particles made from the E and M subunit genes of PEDV.
The immunological compositions and vaccines according to the invention may comprise or consist essentially of one or more adjuvants. Suitable adjuvants for use in the practice of the present invention are (1) polymers of acrylic or methacrylic acid, maleic anhydride and alkenyl derivative polymers, (2) immunostimulating sequences (ISS), such as oligodeoxyribonucleotide sequences having one or more non-methylated CpG units (Klinman et al., 1996; WO98/16247), (3) an oil in water emulsion, such as the SPT emulsion described on page 147 of “Vaccine Design, The Subunit and Adjuvant Approach” published by M. Powell, M. Newman, Plenum Press 1995, and the emulsion MF59 described on page 183 of the same work, (4) cation lipids containing a quaternary ammonium salt, e.g., DDA (5) cytokines, (6) aluminum hydroxide or aluminum phosphate, (7) saponin or (8) other adjuvants discussed in any document cited and incorporated by reference into the instant application, or (9) any combinations or mixtures thereof.
The oil in water emulsion (3), which is especially appropriate for viral vectors, can be based on: light liquid paraffin oil (European pharmacopoeia type), isoprenoid oil such as squalane, squalene, oil resulting from the oligomerization of alkenes, e.g. isobutene or decene, esters of acids or alcohols having a straight-chain alkyl group, such as vegetable oils, ethyl oleate, propylene glycol, di(caprylate/caprate), glycerol tri(caprylate/caprate) and propylene glycol dioleate, or esters of branched, fatty alcohols or acids, especially isostearic acid esters.
The oil is used in combination with emulsifiers to form an emulsion. The emulsifiers may be nonionic surfactants, such as: esters of on the one hand sorbitan, mannide (e.g. anhydromannitol oleate), glycerol, polyglycerol or propylene glycol and on the other hand oleic, isostearic, ricinoleic or hydroxystearic acids, said esters being optionally ethoxylated, or polyoxypropylene-polyoxyethylene copolymer blocks, such as Pluronic, e.g., L121.
Among the type (1) adjuvant polymers, polymers of crosslinked acrylic or methacrylic acid, e.g., crosslinked by polyalkenyl ethers of sugars or polyalcohols, are appropriate. These compounds are known under the name carbomer (Pharmeuropa, vol. 8, no. 2, June 1996). One skilled in the art can also refer to U.S. Pat. No. 2,909,462, which provides such acrylic polymers crosslinked by a polyhydroxyl compound having at least three hydroxyl groups, or no more than eight such groups, the hydrogen atoms of at least three hydroxyl groups being replaced by unsaturated, aliphatic radicals having at least two carbon atoms. Some radicals are those containing 2 to 4 carbon atoms, e.g., vinyls, allyls and other ethylenically unsaturated groups. The unsaturated radicals can also contain other substituents, such as methyl. Products sold under the name Carbopol (BF Goodrich, Ohio, USA) are especially suitable. They are crosslinked by allyl saccharose or by allyl pentaerythritol. Among them, reference is made to Carbopol 974P, 934P and 971P.
As to the maleic anhydride-alkenyl derivative copolymers, the EMA (Monsanto) are straight-chain or crosslinked ethylene-maleic anhydride copolymers and they are, for example, crosslinked by divinyl ether. Reference is also made to J. Fields et al., 1960.
With regard to structure, the acrylic or methacrylic acid polymers and EMA are formed by basic units having the following formula:




in which:

    
    
        R1 and R2, which can be the same or different, represent H or CH3
        x=0 or 1, preferably x=1
        y=1 or 2, with x+y=2.
    
    


For EMA, x=0 and y=2 and for carbomers x=y=1.
These polymers are soluble in water or physiological salt solution (20 g/l NaCl) and the pH can be adjusted to 7.3 to 7.4, e.g., by soda (NaOH), to provide the adjuvant solution in which the expression vector(s) can be incorporated. The polymer concentration in the final immunological or vaccine composition can range between about 0.01 to about 1.5% w/v, about 0.05 to about 1% w/v, and about 0.1 to about 0.4% w/v.
The cytokine or cytokines (5) can be in protein form in the immunological or vaccine composition, or can be co-expressed in the host with the immunogen or immunogens or epitope(s) thereof. Preference is given to the co-expression of the cytokine or cytokines, either by the same vector as that expressing the immunogen or immunogens or epitope(s) thereof, or by a separate vector thereof.
The invention comprehends preparing such combination compositions; for instance by admixing the active components, advantageously together and with an adjuvant, carrier, cytokine, and/or diluent.
Cytokines that may be used in the present invention include, but are not limited to, granulocyte colony stimulating factor (G-CSF), granulocyte/macrophage colony stimulating factor (GM-CSF), interferon α (IFNγ), interferon β (IFNβ), interferon γ, (IFNγ), interleukin-1α (IL-1α), interleukin-1β (IL-1β), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-11 (IL-11), interleukin-12 (IL-12), tumor necrosis factor α (TNFα), tumor necrosis factor β (TNFβ), and transforming growth factor β (TFGβ). It is understood that cytokines can be co-administered and/or sequentially administered with the immunological or vaccine composition of the present invention. Thus, for instance, the vaccine of the instant invention can also contain an exogenous nucleic acid molecule that expresses in vivo a suitable cytokine, e.g., a cytokine matched to this host to be vaccinated or in which an immunological response is to be elicited (for instance, a porcine cytokine for preparations to be administered to swine).
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## Claims

1. An immunogenic composition comprising a recombinant spike polypeptide of porcine epidemic diarrhea virus comprising SEQ ID NO:19 and a pharmaceutically or veterinary acceptable vehicle, diluent or excipient, and wherein the recombinant spike polypeptide is expressed in a baculovirus expression system.

2. The immunogenic composition of claim further comprising a suitable adjuvant.

3. The immunogenic composition of claim 1, wherein the recombinant spike polypeptide is encoded by a nucleotide comprising the sequence of SEQ ID NO:18.

4. A method of vaccinating a host susceptible to PEDV comprising at least one administration of a vaccine according to claim 2.

5. The composition of claim 2, further comprising at least one additional antigen associated with a pathogen other than porcine epidemic diarrhea virus.