# Patent

## Title
N-linked glycosylation alteration in E1 glycoprotein of classical swine fever virus and novel classical swine fever virus vaccine

## Abstract

E1, along with Erns and E2 is one of the three envelope glycoproteins of Classical Swine Fever Virus (CSFV). Our previous studies indicated that glycosylation status of either E2 or Erns strongly influence viral virulence in swine. Here, we have investigated the role of E1 glycosylation of highly virulent CSFV strain Brescia during infection in the natural host. The three putative glycosylation sites in E1 were modified by site directed mutagenesis of a CSFV Brescia infectious clone (BICv). A panel of virus mutants was obtained and used to investigate whether the removal of putative glycosylation sites in the E1 glycoprotein would affect viral virulence/pathogenesis in swine. We observed that rescue of viable virus was completely impaired by removal of all three putative glycosylation sites in E1. Single mutations of each of the E1 glycosylation sites showed that CSFV amino acid N594 (E1.N3 virus), as well the combined mutation of N500 and N513 (E1.N1N2 virus) resulted in BICv attenuation. Infection of either E1.N1N2 or E1.N3 viruses were able to efficiently protected swine from challenge with virulent BICv at 3 and 28 days post-infection. These results, along with those demonstrating the role of glycosylation of Erns and E2, suggest that manipulation of the pattern of glycosylation could be a useful tool for development of CSF live-attenuated vaccines.

## Background
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to the characterization of the role that glycosylation of the transmembrane glycoprotein E1 of highly virulent Classical Swine Fever Virus (CSFV) strain Brescia plays during infection in the natural host and to the utilization of a strategy for manipulating the pattern of glycosylation for particular E1 glycosylation sites in order to alter CSFV virulence, providing a useful tool in the design and development of CSF live-attenuated vaccines.
2. Description of the Relevant Art
Classical swine fever (CSF) is a highly contagious disease of swine. The etiological agent, CSF virus (CSFV), is a small, enveloped virus with a positive, single-stranded RNA genome and, along with Bovine Viral Diarrhea Virus (BVDV) and Border Disease Virus (BDV), is classified as a member of the genus Pestivirus within the family Flaviridae (Becher et al. 2003. Virology 311: 96-104). The 12.5 kb CSFV genome contains a single open reading frame that encodes a 3898-amino-acid polyprotein and ultimately yields 11 to 12 final cleavage products (NH2—Npro-C-Erns-E1-E2-p7-NS2-NS3-NS4A-NS4B—NS5A-NS5B—COOH) through co- and post-translational processing of the polyprotein by cellular and viral proteases (Rice, C. M. 1996. In: Fundamental Virology, 3rd edition, Knipe et al., eds., Lippincott Raven, Philadelphia, Pa., pages 931-959). Structural components of the CSFV virion include the capsid (C) protein and glycoproteins Erns, E1, and E2. E1 and E2 are anchored to the envelope at their carboxyl termini and Ems loosely associates with the viral envelope (Slater-Handshy et al. 2004. Virology 319: 36-48; Weiland et al. 1990. J. Virol. 64: 3563-3569; Weiland et al. 1999. J. Gen. Virol. 80: 1157-1165). E1 and E2 are type I transmembrane proteins with an N-terminal ectodomain and a C-terminal hydrophobic anchor (Thiel et al. 1991. J. Virol. 65: 4705-4712). E1 has been implicated (Wang et al. 2004. Virology 330: 332-341), along with Erns and E2 (Hulst et al. 1997. J. Gen Virol. 78: 2779-2787), in viral adsorption to host cells. Importantly, modifications introduced into these glycoproteins appear to have an important effect on CSFV virulence (Meyers et al. 1999. J. Virol. 73: 10224-10235; Risatti et al. 2005a. J. Virol. 79: 3787-3796; Risatti et al. 2005. Virology 355: 94-101; Risatti et al. 2005b. Virology 343: 116-127; Van Gennip et al. 2004. J. Virol. 78: 8812-8823).
Glycosylation is one of the most common types of protein modifications. N-linked oligosaccharides are added to specific asparagine residues in the context of the consensus sequence Asn-X-Ser/Thr (Kornfeld and Kornfeld. 1985. Annu. Rev. Biochem. 54: 631-664). According to a glycosylation analysis algorithm (Retrieved from the Internet: cbs.dtu.dk/services/), E1 of the CSFV strain Brescia has three putative N-linked glycosylation sites although this is not confirmed by experimental evidence. Predicted E1 glycosylation sites (at CSFV amino acid residue position N500, N513 and N594) are highly conserved among CSFV isolates and two of them (N513 and N594) are also conserved in other Pestiviruses. However, the significance of viral envelope protein glycosylation in virus replication, pathogenesis, and virulence in the natural host is not completely defined. It has just been recently described that specific removal of certain putative glycosylation sites in Erns and E2 significantly alters the virulence of highly virulent Brescia strain in swine (Fernandez Sainz et al. 2008. Virology (370:122-129); Risatti et al. 2007. J. Vinci. 81: 924-933).
Strategies for controlling disease in the event of a CSFV outbreak include the production of rationally designed live attenuated vaccine CSFV strains. Thus, the effect of modification of glycosylation sites of other of the CSFV virion glycoproteins need to be evaluated. Here, we report the effects of modification of particular predicted E1 glycosylation sites. We used oligonucleotide site-directed mutagenesis of the E1 gene of the highly virulent CSFV strain Brescia to construct a panel of glycosylation mutants. These mutants were evaluated to determine whether the removal of each of these glycosylation sites in the E1 glycoprotein could affect viral infectivity and virulence in swine.
SUMMARY OF THE INVENTION
We have discovered glycosylation sites within the classical swine fever virus (CSFV) E1 glycoprotein where modification of the sites results in CSFV having novel virulence determinants.
In accordance with this discovery, it is an object of the invention to provide a recombinant CSFV comprising DNA encoding a modified CSFV E1 glycoprotein wherein specific glycosylation sites within E1 have been mutated resulting in an alteration in the site, i.e., the formerly glycosylated amino acid being altered and replaced by a non-glycosylated amino acid.
It is also an object of the invention to provide an isolated polynucleotide molecule comprising a genetically modified DNA sequence encoding a genetically modified infectious RNA molecule encoding a genetically modified CSFV. The CSFV is genetically modified such that when it infects a porcine animal it is unable to produce CSFV in the animal and it is able to elicit an effective immunoprotective response against infection by a CSFV in the animal. Mutated sequences or sequences homologous thereto contain a mutation that renders the encoded CSFV attenuated and able to elicit an effective immunoprotective response against infection by a CSFV in the animal.
It is additionally an object of the invention to provide an isolated infectious RNA molecule encoded by the isolated polynucleotide molecule recited above, and isolated infectious RNA molecules homologous thereto, which isolated infectious RNA molecules each encode a genetically modified CSFV, disabled in its ability to produce CSF.
An added object of the invention is to provide immunogenic compositions comprising a viable recombinant CSFV comprising a modified CSFV E1 glycoprotein displaying a glycosylation pattern different from that of the non-mutated E1 glycoprotein.
An additional object of the invention is to provide a rationally designed live attenuated CSFV vaccine which lessens severity of CSF disease when challenged with virulent Brescia CSFV wherein said vaccine comprises an altered glycosylation pattern as compared to that of the infectious, non-mutated virus.
Another object of the invention is to provide a rationally designed live attenuated CSFV vaccine effective to protect an animal from clinical CSF disease when challenged with virulent Brescia CSFV wherein said vaccine comprises an altered glycosylation pattern as compared to that of the infectious, non-mutated virus.
A further object of the invention is to provide a marker vaccine which allows a serological distinction between vaccinated animals and animals infected with CSFV.
A still further object of the invention is to provide a method for making a genetically modified CSFV, which method comprises mutating an infectious cDNA sequence, transforming the modified DNA into a modified infectious RNA molecule encoding a modified CSFV, and rescuing the genetically modified CSFV there from subsequent to said mutation.
Yet another object of the invention is to provide a method for protecting an animal against CSF by administering an effective amount of rationally designed live attenuated CSFV vaccine.
An additional object of the invention is to provide a method for delaying onset or severity of CSF in an animal by administering an effective amount of rationally designed live attenuated CSFV vaccine.
Other objects and advantages of this invention will become readily apparent from the ensuing description.



BRIEF DESCRIPTION OF THE DRAWINGS
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the U.S. Patent and Trademark Office upon request and payment of the necessary fee.
FIG. 1A is a schematic representation of glycosylation mutants of Classical Swine Fever Virus E1 protein, generated by site-directed mutagenesis of a cDNA full-length clone PBIC (Terpstra et al. 1990. Dtsch Tierarztl Wochenschr 97: 77-79). Wild type E1 glycoprotein shown at the top. Y: putative glycosylation sites. Mutants were named with an N (N-linked glycosylation) followed by a number that represents the relative position of putative glycosylation sites within E1 amino acid sequence (500, 513, 594). Relative virus yield is final point virus yield as proportion of final end point (72 hours post-infection) virus yield of parental BICv. FIG. 1B) shows the in vitro growth characteristics of E1 glycosylation mutants and parental BICv. Primary swine macrophage cell cultures were infected (MOI=0.01) with each of the mutants or BICv and virus yield was titrated at times post infection in SK6 cells. Data represent means and standard deviations from two independent experiments. Sensitivity of virus detection: >1.8 TCID50/ml. FIG. 1C shows plaque formation of E1 glycosylation mutants and BICv. SK6 monolayers were infected, overlaid with 0.5% agarose and incubated at 37° C. for 3 days. Plates were fixed with 50% (vol/vol) ethanol-acetone and stained by immunohistochemistry with mAb WH303.
FIGS. 2A, 2B, and 2C depict the virus titers and FIGS. 2D and 2E, the hematological data, in nasal swabs, tonsil scrapings, and blood from animals infected with CSFV E1 glycosylation mutants or parental BICv. Peripheral white blood cell and platelet counts are expressed as numbers/ul of blood. Data represent means and standard deviations from at least two animals. Sensitivity of virus detection: >1.8 TCID50/ml.



DETAILED DESCRIPTION OF THE INVENTION
Virus glycoproteins are crucial in key steps of the virus cycle such as attachment to host cell receptors, entry, assembly of newly produced viral progeny, and exit. In vivo viral glycoproteins have been shown to influence infectivity (Ansari et al. 2006. J. Virol. 80: 3994-4004), virulence (Hulse et al. 2004. J. Virol. 78: 9954-9964; Panda of al. 2004. J. Virol. 78: 4965-4975), and host immune response (Abe of al. 2004. J. Virol. 78: 9605-9611). Added oligosaccharides confer proper function to viral glycoproteins since alteration of those glycosylation sites have shown dramatic consequences for viruses affecting protein folding (Herbert et al. 1997. J. Cell Biol. 139: 613-623; Kornfeld, supra; Shi et al. 2005. J. Virol. 79: 13725-13734; Shi and Elliott. 2004. J. Virol. 78: 5414-5422; Slater-Handshy, supra) and protein active conformation (Meunier et al. 1999. J. Gen. Virol. 80: 887-896). In this study, we analyzed glycosylation of the CSFV E1 glycoprotein and evaluated its effect on the virulence of CSFV in swine. DNA encoding CSFV strain Brescia E1 glycoprotein contains 3 N-linked putative glycosylation sites (Retrieved from the Internet: cbs.dtu.dk/services/) (Moorman et al. 1990. Vet. Microbiol. 23: 185-191). Sequence analysis of CSFV E1 glycoprotein showed that 3 of the N-linked glycosylation sites are highly conserved in CSV strains CSFV and two of them (N513 and N594) also among BVDV type I and II, and BDV strains (data not shown), implying an important role for these sites in all Pestiviruses. However, very little is known about the role of glycosylation on the function of Pestivirus glycoproteins. All putative glycosylation sites in E1 were modified by site-directed mutagenesis using a full-length cDNA infectious clone of virulent strain Brescia as the target sequence. Here, we showed that some of these sites have a major role in virulence and protection; some of the sites seem to be critical for the production of viable virus.
Cleavage and glycosylation patterns of the hemagglutinin gene of H5 avian influenza viruses have been shown to affect pathogenicity in chickens (Deshpande et al. 1987. Proc. Natl. Acad. Sci. USA 84: 36-40; Horimoto and Kawaoka. 1994. J. Virol. 68: 3120-3128). More recently it has been shown that glycosylation patterns of the neuraminidase gene of highly pathogenic H5N1 avian flu viruses are important for increased virulence in chickens (Hulse, supra). The mechanisms by which these patterns affect avian flu virulence are unknown. Similarly, a single mutation (E1.N3v) or multiple mutations (E1.N1N2v) within E1 resulted in attenuated viruses with restricted in vivo replication ability (see Table 2). Unlike the acute fatal disease induced by BICv, infections caused by these mutants were sub-clinical in swine and characterized by decreased viral replication in target organs and reduced virus shedding. Interestingly, mutants E1.N1v, and E1.N2v retained the same capability of causing severe disease in swine as parental BICv, showing that in vivo E1 functions are retained and not influenced by the lack of glycans at positions N500 and N513. As with avian flu, the genetic basis and the molecular mechanisms underlying CSFV virulence remain unknown.
As shown in this study, single mutations of E1 putative glycosylation sites have no effect on in vitro or in vivo infectivity of CSFV, with the exception of residue N594 in the E1.N3v mutant. However, when multiple site mutations were introduced in E1, we observed that any multiple mutations involving residue N594 (E1.N1N2, E1.N1N3 or E1.N1N2N3) render non-viable viruses (data not shown).
Production and manipulation of the isolated polynucleotide molecules described herein are within the skill in the art and can be carried out according to recombinant techniques described, among other places, in Sambrook et al. 1989. Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Innis et al. (eds). 1995. PCR Strategies, Academic Press, Inc., San Diego, which are incorporated herein by reference.
The subject invention provides isolated polynucleotide molecules comprising genetically modified DNA sequences that encode genetically modified infectious RNA molecules that encode genetically modified Classical Swine Fever Viruses (CSFVs).
In particular, the subject invention provides an isolated polynucleotide molecule comprising a genetically modified DNA sequence encoding a genetically modified infectious RNA molecule that encodes a genetically modified CSFV, wherein said DNA sequences are SEQ ID NO:1. (E1.N1N2) and SEQ ID NO: 2 (E1.N3) or sequences homologous thereto encoding the mutated viruses. Said DNA sequences encode infectious RNA molecules that are the RNA genomes of the mutated CSF viruses E1.N1N2 and E1.N3, respectively.
It is understood that terms herein referring to nucleic acid molecules such as “isolated polynucleotide molecule” and “nucleotide sequence include both DNA and RNA molecules and include both single-stranded and double-stranded molecules whether it is natural or synthetic origin.
For example, SEQ ID NO:1 is a DNA sequence corresponding to the genetically modified RNA genome of a genetically modified CSFV. Thus, a DNA sequence complementary to the DNA sequence set forth in SEQ ID NO:1 is a template for, i.e. is complementary to or “encodes”, the RNA genome of the SF virus (i.e., RNA that encodes the CSFV).
Furthermore, when reference is made herein to sequences homologous to a sequence in the Sequence Listing, it is to be understood that sequences are homologous to a sequence corresponding to the sequence in the Sequence Listing and to a sequence complementary to the sequence in the Sequence Listing.
An “infectious RNA molecule”, for purposes of the present invention, is an RNA molecule that encodes the necessary elements for viral replication, transcription, and translation into a functional virion in a suitable host cell, provided, if necessary, with a peptide or peptides that compensate for any genetic modifications, e.g. sequence deletions, in the RNA molecule.
An “isolated infectious RNA molecule” refers to a composition of matter comprising the aforementioned infectious RNA molecule purified to any detectable degree from its naturally occurring state, if such RNA molecule does indeed occur in nature. Likewise, an “isolated polynucleotide molecule” refers to a composition of matter comprising a polynucleotide molecule of the present invention purified to any detectable degree from its naturally occurring state, if any.
For purposes of the present invention, two DNA sequences are substantially homologous when at least 80% (preferably at least 85% and most preferably 90%) of the nucleotides match over the defined length of the sequence using algorithms such as CLUSTRAL or PILEUP. Sequences that are substantially homologous can be identified in a Southern hybridization experiment under stringent conditions as is known in the art. See, for example, Sambrook et al., supra. Sambrook et al. describe highly stringent conditions as a hybridization temperature 5-10° C. below the Tm of a perfectly matched target and probe; thus, sequences that are “substantially homologous” would hybridize under such conditions.
As used herein, “substantially similar” refers to nucleic acid fragments wherein changes in one or more nucleotide bases results in substitution of one or more amino acids, but do not affect the functional properties of the polypeptide encoded by the nucleotide sequence. “Substantially similar” also refers to modifications of the nucleic acid fragments of the instant invention such as deletion or insertion of nucleotides that do not substantially affect the functional properties of the resulting transcript. It is therefore understood that the invention encompasses more than the specific exemplary nucleotide or amino acid sequences and includes functional equivalents thereof. Alterations in a nucleic acid fragment that result in the production of a chemically equivalent amino acid at a given site, but do not affect the functional properties of the encoded polypeptide, are well known in the art. Thus, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, changes which result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also be expected to produce a functionally equivalent product. Nucleotide changes which result in alteration of the N-terminal and C-terminal portions of the polypeptide molecule would also not be expected to alter the activity of the polypeptide. Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity of the encoded products. A method of selecting an isolated polynucleotide that affects the level of expression of a polypeptide in a virus or in a host cell (eukaryotic, such as plant, yeast, fungi, or algae; prokaryotic, such as bacteria) may comprise the steps of: constructing an isolated polynucleotide of the present invention or an isolated chimeric gene of the present invention; introducing the isolated polynucleotide or the isolated chimeric gene into a host cell; measuring the level of a polypeptide in the host cell containing the isolated polynucleotide; and comparing the level of a polypeptide in the host cell containing the isolated polynucleotide with the level of a polypeptide in a host cell that does not contain the isolated polynucleotide.
Moreover, substantially similar nucleic acid fragments may also be characterized by their ability to hybridize. Estimates of such homology are provided by either DNA-DNA or DNA-RNA hybridization under conditions of stringency as is well understood by those skilled in the art (1985. Nucleic Acid Hybridization, Hames and Higgins, Eds., IRL Press, Oxford, U.K.). Stringency conditions can be adjusted to screen for moderately similar fragments, such as homologous sequences from distantly related organisms, to highly similar fragments, such as genes that duplicate functional enzymes from closely related organisms.
Thus, isolated sequences that encode a modified CSFV E1 glycopeptide (E1.N1N2 or E1.N3) and which hybridize under stringent conditions, as described herein, to the modified CSFV E1 sequences disclosed herein, i.e., SEQ ID NO:1 (E1.N1N2) or SEQ ID NO: 2 (E1.N3) or to fragments thereof, are encompassed by the present invention. Fragments of a nucleotide sequence that are useful as hybridization probes may not encode fragment proteins retaining biological activity.
Substantially similar nucleic acid fragments of the instant invention may also be characterized by the percent identity of the amino acid sequences that they encode to the amino acid sequences disclosed herein, as determined by algorithms commonly employed by those skilled in this art.
Methods of alignment of sequences for comparison are well known in the art. Thus, the determination of percent identity between any two sequences can be accomplished using a mathematical algorithm. Non-limiting examples of such mathematical algorithms are the algorithm of Myers and Miller (1988. CABIOS 4:11-17), the local homology algorithm of Smith et al. (1981. Adv. Appl. Math. 2:482); the homology alignment algorithm of Needleman and Wunsch (1970. J. Mol. Biol. 48:443-453); the search-for-similarity-method of Pearson and Lipman (1988. Proc. Natl. Acad. Sci. 85:2444-2448; the algorithm of Karlin and Altschul (1990. Proc. Natl. Acad. Sci. USA 87:2264), modified as in Karlin and Altschul (1993. Proc. Natl. Acad. Sci. USA 90:5873-5877).
Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine sequence identity. Such implementations include, but are not limited to: CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, Calif.); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Version 8 (available from Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis., USA). Alignments using these programs can be performed using the default parameters.
As used herein, “sequence identity” or “identity” in the context of two nucleic acid or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins, it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule.
As used herein, “percentage of sequence identity” means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.
As used herein, “reference sequence” is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.
The term “substantial identity” of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 80% sequence identity, preferably at least 85%, more preferably at least 90%, most preferably at least 95% sequence identity compared to a reference sequence using one of the alignment programs described using standard parameters. One of skill in the art will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like. Substantial identity of amino acid sequences for these purposes normally means sequence identity of at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95%. Preferably, optimal alignment is conducted using the homology alignment algorithm of Needleman et al. (1970. J. Mol. Biol. 48:443).
Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. However, stringent conditions encompass temperatures in the range of about 1° C. to about 20° C., depending upon the desired degree of stringency as otherwise qualified herein.
A “substantial portion” of an amino acid or nucleotide sequence comprises an amino acid or a nucleotide sequence that is sufficient to afford putative identification of the protein or gene that the amino acid or nucleotide sequence comprises. Amino acid and nucleotide sequences can be evaluated either manually by one skilled in the art, or by using computer-based sequence comparison and identification tools that employ algorithms such as BLAST. In general, a sequence of ten or more contiguous amino acids or thirty or more contiguous nucleotides is necessary in order to putatively identify a polypeptide or nucleic acid sequence as homologous to a known protein or gene. Moreover, with respect to nucleotide sequences, gene-specific oligonucleotide probes comprising 30 or more contiguous nucleotides may be used in sequence-dependent methods of gene identification and isolation. In addition, short oligonucleotides of 12 or more nucleotides may be use as amplification primers in PCR in order to obtain a particular nucleic acid fragment comprising the primers. Accordingly, a “substantial portion” of a nucleotide sequence comprises a nucleotide sequence that will afford specific identification and/or isolation of a nucleic acid fragment comprising the sequence. The skilled artisan, having the benefit of the sequences as reported herein, may now use all or a substantial portion of the disclosed sequences for purposes known to those skilled in this art. Accordingly, the instant invention comprises the complete sequences as reported in the accompanying Sequence Listing, as well as substantial portions at those sequences as defined above.
By “variants” substantially similar sequences are intended. For nucleotide sequences, conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of one of the CSFV E1 glycoproteins of the invention. Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR), a technique used for the amplification of specific DNA segments. Generally, variants of a particular nucleotide sequence of the invention will have generally at least about 90%, preferably at least about 95% and more preferably at least about 98% sequence identity to that particular nucleotide sequence as determined by sequence alignment programs described elsewhere herein.
By “variant protein” a protein derived from the native protein by deletion (so-called truncation) or addition of one or more amino acids to the N-terminal and/or C-terminal end of the native protein; deletion or addition of one or more amino acids at one or more sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein is intended. Variant proteins encompassed by the present invention are biologically active, that is they possess the desired biological activity, that is, a modified CSFV E1 glycoprotein activity, i.e., E1.N1N2 or E1.N3 glycoprotein activity as described herein. Such variants may result from, for example, genetic polymorphism or from human manipulation. Biologically active variants of a modified CSFV E1 glycoprotein of the invention, i.e., E1.N1N2 or E1.N3, will have at least about 90%, preferably at least about 95%, and more preferably at least about 98% sequence identity to the amino acid sequence for the native protein as determined by sequence alignment programs described elsewhere herein. A biologically active variant of a protein of the invention may differ from that protein by as few as 1-15 amino acid residues, or even 1 amino acid residue.
The polypeptides of the invention may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Novel proteins having properties of interest may be created by combining elements and fragments of proteins of the present invention, as well as with other proteins. Methods for such manipulations are generally known in the art. Thus, the genes and nucleotide sequences of the invention include both the naturally occurring sequences as well as mutant forms. Likewise, the proteins of the invention encompass naturally occurring proteins as well as variations and modified forms thereof. Such variants will continue to possess the desired modified CSFV E1 glycoprotein activity, i.e., E1.N1N2 or E1.N3 glycoprotein activity. Obviously, the mutations that will be made in the DNA encoding the variant must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure.
The deletions, insertions, and substitutions of the protein sequences encompassed herein are not expected to produce radical changes in the characteristics of the protein. However, when it is difficult to predict the exact effect of the substitution, deletion, or insertion in advance of doing so, one skilled in the art will appreciate that the effect will be evaluated by routine screening assays where the effects of modified CSFV E1 glycoprotein, i.e., E1.N1N2 or E1.N3 glycoprotein activity, can be observed.
“Codon degeneracy” refers to divergence in the genetic code permitting variation of the nucleotide sequence without affecting the amino acid sequence of an encoded polypeptide. Accordingly, the instant invention relates to any nucleic acid fragment comprising a nucleotide sequence that encodes all or a substantial portion of the amino acid sequences set forth herein.
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.
As used herein, the term “CSF” encompasses disease symptoms in swine caused by a CSFV infection. Examples of such symptoms include, but are not limited to, anorexia, depression, fever, purple skin discoloration, staggering gait, diarrhea and cough. As used herein, a CSFV that is “unable to produce CSF” refers to a virus that can infect a pig, but which does not produce any disease symptoms normally associated with a CSF infection in the pig, or produces such symptoms, but to a lesser degree, or produces a fewer number of such symptoms, or both.
The terms “porcine” and “swine” are used interchangeably herein and refer to any animal that is a member of the family Suidae such as, for example, a pig. “Mammals” include any warm-blooded vertebrates of the Mammalia class, including humans.
The terms “classical swine fever virus” and “CSFV”, as used herein, unless otherwise indicated, mean any strain of CSF viruses.
The term “open reading frame”, or “ORF”, as used herein, means the minimal nucleotide sequence required to encode a particular CSFV protein without an intervening stop codon.
Terms such as “suitable host cell” and “appropriate host cell”, unless otherwise indicated, refer to cells into which RNA molecules (or isolated polynucleotide molecules or viral vectors comprising DNA sequences encoding such RNA molecules) of the present invention can be transformed or transfected. “Suitable host cells” for transfection with such RNA molecules, isolated polynucleotide molecules, or viral vectors, include mammalian, particularly porcine cells, and are described in further detail below.
A “functional virion” is a virus particle that is able to enter a cell capable of hosting a CSFV, and express genes of its particular RNA genome (either an unmodified genome or a genetically modified genome as described herein) within the cell. Cells capable of hosting a CSFV include swine kidney cells (SK6) and primary porcine macrophage cell cultures. Other mammalian cells, especially other porcine cells, may also serve as suitable host cells for CSF virions.
The isolated polynucleotide molecules of the present invention encode CSF viruses that can be used to prepare live attenuated vaccines using art-recognized methods for protecting swine from infection by a CSFV, as described in further detail below. Furthermore, these isolated polynucleotide molecules are useful because they can be mutated using molecular biology techniques to encode genetically-modified CSF viruses useful, inter alia, as vaccines for protecting swine from CSF infection. Such genetically-modified CSF viruses, as well as vaccines comprising them, are described in further detail below.
Accordingly, the subject invention further provides a method for making a genetically modified CSFV, which method comprises mutating the DNA sequence encoding an infectious RNA molecule which encodes the CSFV as described above, and expressing the genetically modified CSFV using a suitable expression system. A CSFV, either wild-type or genetically modified, can be expressed from an isolated polynucleotide molecule using suitable expression systems generally known in the art, examples of which are described in this application. For example, the isolated polynucleotide molecule can be in the form of a plasmid capable of expressing the encoded virus in a suitable host cell in vitro.
The term “genetically modified”, as used herein and unless otherwise indicated, means genetically mutated, i.e. having one or more nucleotides replaced, deleted and/or added. Polynucleotide molecules can be genetically mutated using recombinant techniques known to those of ordinary skill in the art, including by site-directed mutagenesis, or by random mutagenesis such as by exposure to chemical mutagens or to radiation, as known in the art.
The subject invention further provides an isolated polynucleotide molecule comprising a DNA sequence encoding an infectious RNA molecule which encodes a genetically modified CSFV that is unable to produce CSF in a porcine animal, wherein the DNA sequence encoding the infectious RNA molecule encoding said modified CSFV is SEQ ID NO:1 or SEQ ID NO: 2 or a sequences homologous thereto, contains one or more mutations that genetically disable the encoded CSFV in its ability to produce CSF. “Genetically disabled” means that the CSFV is unable to produce CSF in a swine animal infected therewith.
In one embodiment, the genetically modified CSFV disabled in its ability to cause CSF is able to elicit an effective immunoprotective response against infection by a CSFV in a swine animal. Accordingly, the subject invention also provides an isolated polynucleotide molecule comprising a DNA sequence encoding an infectious RNA molecule which encodes a CSFV that is genetically modified such that when it infects a porcine animal it: a) is unable to produce CSF in the animal, and b) is able to elicit an effective immunoprotective response against infection by a CSFV in the animal, wherein the DNA sequence encoding said modified CSFV is SEQ ID NO:1 (E1.N1N2) or SEQ ID NO: 2 (E1.N3) or sequences homologous thereto, contains one or more mutations that genetically disable the encoded CSFV in its ability to produce CSF.
The term “immune response” for purposes of this invention means the production of antibodies and/or cells (such as T lymphocytes) that are directed against, or assist in the decomposition or inhibition of, a particular antigenic epitope or particular antigenic epitopes. The phrases “an effective immunoprotective response”, “immunoprotection”, and like terms, for purposes of the present invention, mean an immune response that is directed against one or more antigenic epitopes of a pathogen so as to protect against infection by the pathogen in a vaccinated animal. For purposes of the present invention, protection against infection by a pathogen includes not only the absolute prevention of infection, but also any detectable reduction in the degree or rate of infection by a pathogen, or any detectable reduction in the severity of the disease or any symptom or condition resulting from infection by the pathogen in the vaccinated animal as compared to an unvaccinated infected animal. An effective immunoprotective response can be induced in animals that have not previously been infected with the pathogen and/or are not infected with the pathogen at the time of vaccination. An effective immunoprotective response can also be induced in an animal already infected with the pathogen at the time of vaccination.
An “antigenic epitope” is, unless otherwise indicated, a molecule that is able to elicit an immune response in a particular animal or species. Antigenic epitopes are proteinaceous molecules, i.e. polypeptide sequences, optionally comprising non-protein groups such as carbohydrate moieties and/or lipid moieties.
The genetically modified CSF viruses encoded by the above-described isolated polynucleotide molecules are, in one embodiment, able to elicit an effective immunoprotective response against infection by a CSFV. Such genetically modified CSF viruses are preferably able to elicit an effective immunoprotective response against any strain of CSF viruses.
In one embodiment, the mutation or mutations in the isolated polynucleotide molecule encoding the genetically disabled CSFV are non-silent and occur in one or more open reading frames of the nucleotide sequence encoding the CSFV.
As used herein, unless otherwise indicated, “coding regions” refer to those sequences of RNA from which CSFV proteins are expressed, and also refer to cDNA that encodes such RNA sequences. Likewise, “ORFs” refer both to RNA sequences that encode CSFV proteins and to cDNA sequence encoding such RNA sequences.
Determining suitable locations for a mutation or mutations that will encode a CSFV that is genetically disabled so that it is unable to produce CSF yet remains able to elicit an effective immunoprotective response against infection by a CSFV can be made based on SEQ ID NO:1 and SEQ ID NO: 2 provided herein. One of ordinary skill can refer to the sequence of the infectious cDNA clone of CSFV provided by this invention, make sequence changes which will result in a mutation altering the glycosylation pattern of the glycoprotein, and test the viruses encoded thereby both for their ability to produce CSF in swine, and to elicit an effective immunoprotective response against infection by a CSFV. In so doing, one of ordinary skill can refer to techniques known in the art and also those described and/or exemplified herein.
For example, an ORF of the sequence encoding the infectious RNA molecule encoding the CSFV can be mutated and the resulting genetically modified CSFV tested for its ability to cause CSF.
In a further preferred embodiment, an antigenic epitope of the genetically modified CSFV of the present invention is a detectable antigenic epitope. Such isolated polynucleotide molecules and the CSF viruses they encode are useful, inter alia, for studying CSF infections in swine, determining successfully vaccinated swine, and/or for distinguishing vaccinated swine from swine infected by a wild-type CSFV. Preferably, such isolated polynucleotide molecules further contain one or more mutations that genetically disable the encoded CSFV in its ability to produce CSF, and more preferably are able to elicit an effective immunoprotective response in a porcine animal against infection by a CSFV.
Antigenic epitopes that are detectable, and the sequences that encode them, are known in the art. Techniques for detecting such antigenic epitopes are also known in the art and include serological detection of antibody specific to the heterologous antigenic epitope by means of, for example, Western blot, ELISA, or fluorescently labeled antibodies capable of binding to the antibodies specific to the heterologous antigenic epitope. Techniques for serological detection useful in practicing the present invention can be found in texts recognized in the art, such as Coligan, J. E., et al. (eds), 1998, Current Protocols in Immunology, John Willey & Sons, Inc., which is hereby incorporated by reference in its entirety. Alternatively, the antigenic epitope itself can be detected by, for example, contacting samples that potentially comprise the antigenic epitope with fluorescently-labeled antibodies or radioactively-labeled antibodies that specifically bind to the antigenic epitopes.
The present invention further provides an isolated polynucleotide molecule comprising a DNA sequence encoding an infectious RNA molecule which encodes a genetically modified CSFV that detectably lacks a CSFV antigenic epitope, wherein the DNA sequence encoding the RNA molecule encoding the modified CSFV is SEQ ID NO:1 (E1.N1.N2) or SEQ ID NO: 2 (E1.N3) or sequences homologous thereto, except that it lacks one or more nucleotide sequences encoding a detectable CSFV antigenic epitope. Such isolated polynucleotide molecules are useful for distinguishing between swine infected with a recombinant CSFV of the present invention and swine infected with a wild-type CSFV. For example, animals vaccinated with killed, live or attenuated CSFV encoded by such an isolated polynucleotide molecule can be distinguished from animals infected with wild-type CSF based on the absence of antibodies specific to the missing antigenic epitope, or based on the absence of the antigenic epitope itself; If antibodies specific to the missing antigenic epitope, or if the antigenic epitope itself, are detected in the animal, then the animal was exposed to and infected by a wild-type CSFV. Means for detecting antigenic epitopes and antibodies specific thereto are known in the art, as discussed above. Preferably, such an isolated polynucleotide molecule further contains one or more mutations that genetically disable the encoded CSFV in its ability to produce CSF. More preferably, the encoded virus remains able to elicit an effective immunoprotective response against infection by a CSFV.
Vaccines of the present invention can be formulated following accepted convention to include acceptable carriers for animals, including humans (if applicable), such as standard buffers, stabilizers, diluents, preservatives, and/or solubilizers, and can also be formulated to facilitate sustained release. Diluents include water, saline, dextrose, ethanol, glycerol, and the like. Additives for isotonicity include sodium chloride, dextrose, mannitol, sorbitol, and lactose, among others. Stabilizers include albumin, among others. Other suitable vaccine vehicles and additives, including those that are particularly useful in formulating modified live vaccines, are known or will be apparent to those skilled in the art. See, e.g., Remington's Pharmaceutical Science, 18th ed., 1990, Mack Publishing, which is incorporated herein by reference.
Vaccines of the present invention can further comprise one or more additional immunomodulatory components such as, e.g., an adjuvant or cytokine, among others. Non-limiting examples of adjuvants that can be used in the vaccine of the present invention include the RIBI adjuvant system (Ribi Inc., Hamilton, Mont.), alum, mineral gels such as aluminum hydroxide gel, oil-in-water emulsions, water-in-oil emulsions such as, e.g., Freund's complete and incomplete adjuvants, Block copolymer (CytRx, Atlanta Ga.), QS-21 (Cambridge Biotech Inc., Cambridge Mass.), SAF-M (Chiron, Emeryville Calif.), AMPHIGENTM adjuvant, saponin, Quil A or other saponin fraction, monophosphoryl lipid A, and Avridine lipid-amine adjuvant. Non-limiting examples of oil-in-water emulsions useful in the vaccine of the invention include modified SEAM62 and SEAM 1⁄2 formulations. Modified SEAM62 is an oil-in-water emulsion containing 5% (v/v) squalene (Sigma), 1% (v/v) SPANTM 85 detergent (ICI Surfactants), 0.7% (v/v) TWEENTM 80 detergent (ICI Surfactants), 2.5% (v/v) ethanol, 200 μg/ml Quil A, 100 μg/ml cholesterol, and 0.5% (v/v) lecithin. Modified SEAM 1⁄2 is an oil-in-water emulsion comprising 5% (v/v) squalene, 1% (v/v) SPANTM 85 detergent, 0.7% (v/v) Tween 80 detergent, 2.5% (v/v) ethanol, 100 μg/ml Quil A, and 50 μg/ml cholesterol. Other immunomodulatory agents that can be included in the vaccine include, e.g., one or more interleukins, interferons, or other known cytokines.
Vaccines of the present invention can optionally be formulated for sustained release of the virus, infectious RNA molecule, plasmid, or viral vector of the present invention. Examples of such sustained release formulations include virus, infectious RNA molecule, plasmid, or viral vector in combination with composites of biocompatible polymers, such as, e.g., poly(lactic acid), poly(lactic-co-glycolic acid), methylcellulose, hyaluronic acid, collagen and the like. The structure, selection and use of degradable polymers in drug delivery vehicles have been reviewed in several publications, including Domb et al. 1992. Polymers for Advanced Technologies 3: 279-292, which is incorporated herein by reference. Additional guidance in selecting and using polymers in pharmaceutical formulations can be found in texts known in the art, for example M. Chasin and R. Langer (eds), 1990, “Biodegradable Polymers as Drug Delivery Systems” in: Drugs and the Pharmaceutical Sciences, Vol. 45, M. Dekker, NY, which is also incorporated herein by reference. Alternatively, or additionally, the virus, plasmid, or viral vector can be microencapsulated to improve administration and efficacy. Methods for microencapsulating antigens are well-known in the art, and include techniques described, e.g., in U.S. Pat. No. 3,137,631; U.S. Pat. No. 3,959,457; U.S. Pat. No. 4,205,060; U.S. Pat. No. 4,606,940; U.S. Pat. No. 4,744,933; U.S. Pat. No. 5,132,117; and International Patent Publication WO 95/28227, all of which are incorporated herein by reference.
Liposomes can also be used to provide for the sustained release of virus, plasmid, or viral vector. Details concerning how to make and use liposomal formulations can be found in, among other places, U.S. Pat. No. 4,016,100; U.S. Pat. No. 4,452,747; U.S. Pat. No. 4,921,706; U.S. Pat. No. 4,927,637; U.S. Pat. No. 4,944,948; U.S. Pat. No. 5,008,050; and U.S. Pat. No. 5,009,956, all of which are incorporated herein by reference.
An effective amount of any of the above-described vaccines can be determined by conventional means, starting with a low dose of virus, plasmid or viral vector, and then increasing the dosage while monitoring the effects. An effective amount may be obtained after a single administration of a vaccine or after multiple administrations of a vaccine. Known factors can be taken into consideration when determining an optimal dose per animal. These include the species, size, age and general condition of the animal, the presence of other drugs in the animal, and the like. The actual dosage is preferably chosen after consideration of the results from other animal studies.
One method of detecting whether an adequate immune response has been achieved is to determine seroconversion and antibody titer in the animal after vaccination. The timing of vaccination and the number of boosters, if any, will preferably be determined by a doctor or veterinarian based on analysis of all relevant factors, some of which are described above.
The effective dose amount of virus, infectious RNA molecule, plasmid, or viral vector, of the present invention can be determined using known techniques, taking into account factors that can be determined by one of ordinary skill in the art such as the weight of the animal to be vaccinated. The dose amount of virus of the present invention in a vaccine of the present invention preferably ranges from about 101 to about 109 pfu (plaque forming units), more preferably from about 102 to about 108 pfu, and most preferably from about 103 to about 107 pfu. The dose amount of a plasmid of the present invention in a vaccine of the present invention preferably ranges from about 0.1 g to about 100 mg, more preferably from about 1 μg to about 10 mg, even more preferably from about 10 μg to about 1 mg. The dose amount of an infectious RNA molecule of the present invention in a vaccine of the present invention preferably ranges from about 0.1 μg to about 100 mg, more preferably from about 1 μg to about 10 mg, even more preferably from about 10 μg to about 1 mg. The dose amount of a viral vector of the present invention in a vaccine of the present invention preferably ranges from about 101 pfu to about 109 pfu, more preferably from about 102 pfu to about 108 pfu, and even more preferably from about 103 to about 107 pfu. A suitable dosage size ranges from about 0.5 ml to about 10 ml, and more preferably from about 1 ml to about 5 ml.
In summary, our studies determined that individual N-linked glycosylation in glycoprotein E1 sites are not essential for viral particle formation or virus infectivity in cultured swine macrophages or the natural host, with one individual site, N594, involved in attenuation of the virulent parental virus. This study also showed that in the context of two or more putative glycosylation site modifications, residue N594 is critical for virus viability. The effective protective immunity elicited by E1.N3v and E1.N1N2v suggests that glycosylation of E1 could be modified for the development of live-attenuated vaccines. An improved understanding of the genetic basis of virus virulence and host range will permit future rational design of efficacious biological tools for controlling CSF.
EXAMPLES
Having now generally described this invention, the same will be better understood by reference to certain specific examples, which are included herein only to further illustrate the invention and are not intended to limit the scope of the invention as defined by the claims.
Example 1
Viruses and Cell Cultures
Swine kidney cells (SK6) (Terpstra et al., supra) free of Bovine Viral Diarrhea Virus (BVDV) were cultured in Dulbecco's Minimal Essential Medium (DMEM, GIBCO, Grand Island, N.Y.) with 10% fetal calf serum (FCS, Atlas Biologicals, Fort Collins, Colo.). CSFV Brescia strain (obtained from the Animal and Plant Health Inspection Service, Plum Island Animal Disease Center) was propagated in SK6 cells and used for an infectious cDNA clone (Risatti et al. 2005a, supra). Growth kinetics were assessed on primary swine macrophage cell cultures prepared as described by Zsak et al. (1996. J. Virol. 70: 8865-8871). Titration of CSFV from clinical samples was performed using SK6 cells in 96-well plates (Costar, Cambridge, Mass.). Viral infectivity was detected, after 4 days in culture, by an immunoperoxidase assay using the CSFV monoclonal antibodies WH303 (Edwards et al. 1991. Vet Microbiol. 29:101-108) and the Vectastain ABC kit (Vector Laboratories, Burlingame, Calif.). Titers were calculated using the method of Reed and Muench (1938. American J. Hygiene 27: 493-497) and expressed as TCID50/ml. As performed, test sensitivity was ≧1.8 TCID50/ml. Plaque assays were performed using SK6 cells in 6-well plates (Costar). SK6 monolayers were infected, overlaid with 0.5% agarose and incubated at 37° C. for 3 days. Plates were fixed with 50% (vol/vol) ethanol-acetone and stained by immunohistochemistry with mAb WH303.
Example 2
Construction of CSFV Glycosylation Mutants
A full-length infectious clone of the virulent Brescia isolate (pBIC) (Risatti of et al. 2005a, supra) was used as a template in which N-linked glycosylation sites in the E1 glycoprotein were mutated. Glycosylation sites were predicted using analysis tools from the Center for Biological Sequence Analysis (Retrieved from the Internet: cbs.dtu.dk/services/). Mutations were introduced by site-directed mutagenesis using the QuickChange XL Site-Directed Mutagenesis kit (Stratagene, Cedar Creek, Tex.) performed per manufacturer's instructions and using the following primers (only forward primer sequences are shown); E1.N1v: TATGCCCTATCACCT TATTGTGCTGTGACAAGCAAAATAGGGTAC (SEQ ID NO:5); E1.N2v: GGGTACATA TGGTACACTAACGCCTGTACCCCGGCTTGCCTCCCC (SEQ ID NO:6); E1.N3v: GAAGGCTGTGACACA AACCAGCTGGCTTTAACAGTGGAACTCAGGACT (SEQ ID NO:7).
Example 3
In Vitro Rescue of CSFV Brescia and Glycosylation Mutants
Full-length genomic clones were linearized with SrfI and in vitro transcribed using the T7 Megascript system (Ambion, Austin, Tex.). RNA was precipitated with LiCl and transfected into SK6 cells by electroporation at 500 volts, 720 ohms, 100 watts with a BTX 630 electroporator (BTX, San Diego, Calif.). Cells were seeded in 12-well plates and incubated for 4 days at 37EC and 5% CO2. Virus was detected by immunoperoxidase staining as described above, and stocks of rescued viruses were stored at −70EC.
Infectious RNA was in vitro transcribed from full-length infectious clones of the CSFV Brescia strain or a set of glycosylation mutants (Table 1, FIG. 1) and used to transfect SK6 cells. Mutants referred to as E1.N1, E1.N2, E1.N3 represent each of three putative glycosylation sites starting from the N terminus of E1 (Table 1), whereas multiple mutants are represented by combinations of indicated sites (FIG. 1A). Viruses were rescued from transfected cells by day 4 post-transfection. Nucleotide sequences of the rescued virus genomes were identical to parental DNA plasmids, confirming that only mutations at predicted glycosylation sites were reflected in rescued viruses.







TABLE 1







Set of CSFV E1 glycosylation mutant viruses constructed.












Wild-Type
Mutant




E1 Position
Sequence
Sequence
Codon Change
Mutant





500
NVTS
AVTS
AAT → GCT
E1.N1


513
NCTP
ACTP
AAC → GCC
E1.N2


594
NLTV
ALTV
AAT → GCT
E1.N3


500/513
NVTS/NCTP
AVTS/ACTP
AAT → GCT/
E1.N1N2





AAC → GCC


513/594
NCTP/NLTV
ACTP/ALTV
AAC → GCC/
E1.N2N3





AAT → GCT


500/594
NVTS/NLTV
AVTS/ALTV
AAT → GCT/
E1.N1N3





AAT → GCT


500/513/594
NVTS/NCTP/NLTV
AVTS/ACTP/ALTV
AAT → GCT/
E1.N1N2N3





AAC → GCC/





AAT → GCT









Example 4
DNA Sequencing and Analysis
Full-length infectious clones and in vitro rescued viruses were completely sequenced with CSFV specific primers by the dideoxynucleotide chain-termination method (Sanger et al. 1977. Proc. Natl. Acad. Sci. USA 74: 5463-5467). Viruses recovered from infected animals were sequenced in the mutated area. Sequencing reactions were prepared with the Dye Terminator Cycle Sequencing Kit. (Applied Biosystems, Foster City, Calif.). Reaction products were sequenced on a PRISM 3730xl automated DNA Sequencer (Applied Biosystems). Sequence data were assembled with the Phrap software program (http://www.phrap.org), with confirmatory assemblies performed using CAP3 (Huang et al. 1999. Genome Res. 9: 868-877). The final DNA consensus sequence represented an average five-fold redundancy at each base position. Sequence comparisons were conducted using BioEdit software (Retrieved from the Internet: mbio.ncsu.edu/BioEdit/bioedit.html).
The DNA sequence encoding a modified CSFV E1 glycoprotein, i.e., E1.N1N2 is identified by SEC) ID NO: 1. The DNA sequence encoding a modified CSFV E1 glycoprotein, i.e., E1.N3 is identified by SEQ ID NO: 2. The glycoproteins encoded by these DNA molecules are identified by SEQ ID NOs: 3 and 4, respectively.
Example 5
In Vitro and In Vivo Analysis of Glycosylation Mutants
In vitro growth characteristics of mutant viruses E1.N1v, E1.N2v, E.1N3v and E1.N1N2v were evaluated relative to parental BICv in a multistep growth curve (FIG. 1B). Primary porcine macrophage cell cultures were infected at a multiplicity of infection (MOI) of 0.1 TCID50 per cell. Virus was adsorbed for 1 h (time zero), and samples were collected at times post-infection through 72 h.
All single glycosylation site mutants exhibited titers approximately an order lower than those corresponding to BICv. Additionally, when viruses were tested for their plaque size in SK6 cells, E1.N3v exhibited a noticeable reduction in plaque size relative to BICv (FIG. 1C). Interestingly, some viruses were not rescued from SK-6 cells transfected with RNA transcribed from full-length cDNA clones carrying multiple glycosylation site mutations (E1.N1N2N3, E1.N1N3 and E1.N2N3) that included substitutions at the E1.N3 position (N594).
To examine the effect of E1 glycosylation on CSFV virulence, and establish the impact of mutations at individual glycosylation sites in swine virulence, individual mutants were intranasally inoculated with 105 TCID50 and monitored for clinical disease relative to the parental virus. Swine used in all animal studies were 10 to 12 weeks old, forty-pound commercial bred pigs. For screening, 10 pigs were randomly allocated into 5 groups of 2 animals each, and pigs in each group were inoculated with one of the single glycosylation mutants, E1.N1N2v or BICv. Clinical signs (anorexia, depression, fever, purple skin discoloration, staggering gait, diarrhea and cough) and changes in body temperature were recorded daily throughout the experiment and scored as previously described (Mittelholzer et al. 2000. Vet. Microbiol. 74: 293-308).
BICv exhibited a characteristic virulent phenotype (Table 2). Animals infected with E1.N3v survived the infection and remained normal throughout the observation period (21 days). All animals infected with E1.N1v and E1.N2v presented clinical signs of CSF starting 5 to 8 DPI, with clinical presentation and severity similar to those observed in animals inoculated with BICv. White blood cell and platelet counts dropped by 4 to 6 DPI in animals inoculated with E1.N1 and E1.N2v, and BICv and kept declining until death, while a transient decrease was observed in animals inoculated with E1.N1v (FIG. 2). Since E1.N1v and E1.N2v were as virulent as the wild type BICv it was interesting to assess the influence on viral virulence of the simultaneous removal of both glycosylation sites. Two animals were infected with E1.N1N2v under the same conditions above described. Infected animals remained normal throughout the observation period (21 days) along with a transient decrease in the hematological (Table 2 and FIG. 2).







TABLE 2







Swine survival and fever response following infection with CSFV E1


glycosylation mutants and parental BICv.













Mean time to
Mean time of
Mean time of



Survivor/
death: Days
fever onset:
fever duration:


Virus
total
∀SD
Days ∀SD
Days ∀SD





BICv
 6/6*
8.2 (0.9)
3.4 (1.1)
6.5 (0.9)


E1.N1v
0/2
5.5 (0.7)
  2 (0.0)
3.5 (0.7)


E1.N2v
0/2
9.5 (2.1)
  3 (0.0)
6.5 (2.1)


E1.N3v
 6/6*
—
—
—


E1.N1N2v
 6/6*
—
—
—









The capability of E1.N3v and E1.N1N2v to establish a systemic infection in intranasally inoculated animals was compared with that of virulent parental virus BICv. To assess the effect of the E1.N3v and E1.N1N2v mutation on virus shedding and distribution in different organs during infection, pigs were randomly allocated into 3 groups of 9 animals each and intranasally inoculated (see above) with E1.N3v, E1.N1N2v or BICv. One pig per group was sacrificed at 2, 4, 6, 8 and 12 DPI. Blood, nasal swabs and tonsil scraping samples were obtained from pigs at necropsy. Tissue samples (tonsil, mandibular lymph node, spleen and kidney) were snap-frozen in liquid nitrogen for subsequent virus titration. The remaining 4 pigs in each room were monitored to check for appearance of clinical signs during a 21-day period.
Virus shedding and viremia in E1.N3v and E1.N1N2v inoculated animals was undetectable while values in E1.N1v, E1.N2v were 1.5-2.5 logs below of those of BICv infected swine depending on the time post-infection (FIG. 2). In all cases partial nucleotide sequences of E2 protein from viruses recovered from infected animals were identical to those of stock viruses used for inoculation (data not shown).
Titers measured in tissue samples are shown in Table 3. In vivo replication of E1.N3v and E1.N1N2v were transient in tonsils with titers reduced up to 102 to 105, depending on the time post-infection, relative to those of BICv. Differences between E1.N3v and E1.N1N2v and BICv virus titers were also observed in mandibular lymph nodes, spleen, and kidney, indicating a limited capability of E1.N3v and E1.N1N2v to spread within the host.







TABLE 3







Titers of virus in tissues after intranasal inoculation


with mutant E1.N1N2v, E1.N3v or parental BICv.









Log10TCID50/g in:















Mandibular




Virus
DPI
Tonsil
Lymph Node
Spleen
Kidney















E1.N1N2v
2
n.d.*
2.63
1.97
n.d.



4
4.47
n.d.
n.d.
1.97



6
3.63
n.d.
2.13
2.47



8
2.8
n.d.
n.d.
n.d.



12
n.d.
n.d.
n.d.
n.d.


E1.N3v
2
2.3
n.d.
2.13
2.8



4
1.97
n.d.
1.97
n.d.



6
2.3
n.d.
2.47
2.47



8
7.8
n.d.
1.97
n.d.



12
n.d.
n.d.
1.97
2.13


BICv
2
3.12
1.97
n.d.
n.d.



4
7.13
3.8
2.97
2.8



6
6.8
6.13
6.13
5.13



8
7.13
4.97
7.13
5.47



12
D#
D
D
D





*n.d. (not detectable): virus titers equal or less than 1.8 TCID50 (log10).


# D, animals in this group were all dead by this time point.






Example 6
Immunization, Challenge, and Clinical Analysis
For protection studies, 18 pigs were randomly allocated into 5 groups of 4 animals each. Pigs in groups 1 and 2 were inoculated with E1.N1N2v, pigs in groups 3 and 4 were inoculated with E1.N3v and pigs in group 5 were mock infected. At 3 DPI (groups 1 and 3) or 28 DPI (groups 2 and 4), animals were challenged with BICv along with animals in group 5. Clinical signs and body temperature were recorded daily throughout the experiment as described above. Blood, serum, nasal swabs and tonsil scrapings were collected at times post-challenge, with blood obtained from the anterior vena cava in EDTA-containing tubes (Vacutainer) for total and differential white blood cell counts. Total and differential white blood cell and platelet counts were obtained using a Beckman Coulter ACT (Beckman, Coulter, Calif.).
The limited in vivo replication kinetics of E1.N3v and E1.N1N2v is similar to that observed with CSICv (Risatti et al. 2005a, supra), a CSFV vaccine strain. However, restricted viral in vivo replication could also impair protection against wild-type virus infection. Thus, the ability of E1.N3v and E1.N1N2v to induce protection against virulent BICv was assessed in early and late vaccination-exposure experiments.
Mock-vaccinated control pig groups receiving BICv only (n=2) developed anorexia, depression, and fever by 4 days post-challenge (DPC), and a marked reduction of circulating leukocytes and platelets by 4 DPC (data not shown), and died or were euthanized in extremis by 9 DPC (Table 4). Notably, E1.N3v and E1.N1N2v induced complete protection by 3 DPI. All pigs survived infection and remained clinically normal, without significant changes in their hematological values (data not shown). Pigs challenged at 28 days post N1v infection were also protected, remaining clinically normal, without alterations of hematological profiles (data not shown).







TABLE 4







Detection of virus in nasal swabs, tonsil scrapings,


and blood samples obtained after challenge of E1.N1N2v-


or E1.N3v-vaccinated animals with virulent BICv.









Days Post-Challenge















Challenge










Group
Sample
0
4
6
8
12
14
21





E1.N1N2v
Nasal
0/4*
1/4 (1.9)
1/4 (2.5)
1/4 (3.1)
0/4
0/4
0/4


3DPI
Tonsil
0/4
1/2 (2.1)
1/4 (2.8)
2/4 (2.7)
0/4
0/4
0/4



Blood
0/4
1/2 (2.9)#
3/4 (4)
1/4 (4.8)
0/4
0/4
0/4


E1.N1N2v
Nasal
0/4
0/4
0/4
0/4
0/4
0/4
0/4


28DPI
Tonsil
0/4
0/4
0/4
0/4
0/4
0/4
0/4



Blood
0/4
0/4
0/4
0/4
0/4
0/4
0/4


E1.N3v
Nasal
0/4
0/4
0/4
0/4
0/4
0/4
0/4


3DPI
Tonsil
0/4
0/4
0/4
0/4
0/4
0/4
0/4



Blood
0/4
0/4
0/4
0/4
0/4
0/4
0/4


E1.N3v
Nasal
0/4*
0/4
0/4
0/4
0/4
0/4
0/4


28DPI
Tonsil
0/4
0/4
0/4
0/4
0/4
0/4
0/4



Blood
0/4
0/4
0/4
0/4
0/4
0/4
0/4


Control
Nasal
0/2
0/2
2/2 (2.1)
2/2 (5.1)
D


3DPI
Tonsil
0/2
1/2 (2.1)
2/2 (2.7)
2/2 (4.1)



Blood
0/2
1/2 (2.2)
2/2 (6.4)
2/2 (7.2)


Control
Nasal
0/2
0/2
1/2 (2.1)
2/2 (3.6)
D


28DPI
Tonsil
0/2
1/2 (1.9)
2/2 (4.2)
2/2 (3.6)



Blood
0/2
1/2 (2.1)
2/2 (5.1)
2/2 (7.1)





*Number of animals positive for isolated virus over total number of challenged animals.


#Number in parentheses indicates average virus titers expressed as log10 TCID50/ml for four animals.


D Animals in this group were all dead by this time point.






Viremia and virus shedding of vaccinated-exposed animals was examined at 4, 6, 8, 14 and 21 DPC (Table 4). As expected, in mock-vaccinated control animals, viremia was observed by 4 DPC, with virus titers remaining high by 8 DPC (approximately 107 TCID50/ml). Furthermore, virus was detected in nasal swabs and tonsil scrapings of these animals after 4 DPC. Conversely, viremia was detected by 4 DPC in all clinical samples of one of the four E1.N1N2v-infected animals challenged at 3 DPI, while no virus was detected in any sample from E1.N3v-infected animals at any time post challenge (Table 4). Virus was not detected in clinical samples obtained from any E1.N3v- or E1.N1N2v-infected pigs challenged at 28 DPI. Therefore, even though E1.N3v and E1.N1N2v showed a limited in vivo growth, a solid protection was induced shortly after vaccination.
All publications and patents mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.
The foregoing description and certain representative embodiments and details of the invention have been presented for purposes of illustration and description of the invention. It is not intended to be exhaustive or to limit the invention to the precise forms disclosed. It will be apparent to practitioners skilled in this art that modifications and variations may be made therein without departing from the scope of the invention.

## Claims

1. An isolated polynucleotide molecule comprising a DNA sequence encoding an infectious RNA molecule that encodes a genetically modified classical swine fever virus (CSFV) mutant of a highly pathogenic native Brescia strain, wherein said CSFV mutant encodes a genetically modified E1 glycoprotein set forth in SEQ ID NO:4 that said genetic modified E1 gene has a mutated amino acid asparagine in position 594 to alanine, said altered amino acid having a glycosylation pattern different from the amino acid present in position 594 of said highly pathogenic native Brescia strain, wherein said genetically modified CSFV mutant is attenuated and unable to produce a pathogenic CSFV infection in a porcine animal.

2. An isolated recombinant classical swine fever virus comprising the RNA molecule according to claim 1, said RNA molecule encodes a genetically modified mutated CSFV E1 glycoprotein having a sequence identified by SEQ ID NO: 4.

3. The isolated polynucleotide molecule of claim 1, wherein said DNA sequence is SEQ ID NO: 2 or its complement thereof.

4. A CSF vaccine comprising a genetically modified CSFV mutant that does not produce CSF disease in swine, wherein said virus is encoded by the polynucleotide of claim 1.

5. A genetically modified CSFV mutant, wherein the CSF virus is encoded by the isolated polynucleotide molecule of claim 1.

6. A vaccine for protecting a porcine animal against infection by a CSFV, which vaccine comprises (a) a genetically modified CSFV encoded by an infectious RNA molecule encoded by the polynucleotide molecule according to claim 1, (b) said infectious RNA molecule of claim 1, wherein the vaccine is in an effective amount to produce immunoprotection against a CSFV infection; and a carrier acceptable for veterinary use.

7. A method of producing an attenuated recombinant classical swine fever virus comprising DNA encoding a modified CSFV E1glycoprotein, comprising:
(a) mutating a region of the E1 gene of the highly pathogenic strain Brescia, wherein said region encodes the modified CSFV E1 glycoprotein set forth in SEQ ID NO: 4, and whereby mutations in said DNA result in a change in the glycosylation pattern characteristic of CSFV E1 glycoprotein; and
(b) achieving attenuation of CSFV as a result of such modification.

8. A method for generating a genetically modified CSFV, which method comprises transfecting a host cell with an infectious RNA molecule that encodes the genetically modified CSFV mutant according to claim 1 and obtaining the genetically modified CSFV mutant generated by the transfected host cell.

9. A rationally designed live attenuated CSF vaccine comprising a recombinant classical swine fever virus according to claim 2.

10. A method of immunizing an animal against CSF, comprising administering to said animal a vaccine comprising a recombinant classical swine fever virus according to claim 2.

11. A method of protecting an animal against CSF, comprising administering to said animal an effective amount of the vaccine of claim 9 effective to protect said animal from clinical CSF.