MX2008003046A - Methods and compositions for vaccination of animals with prrsv antigens with improved immunogenicity - Google Patents
Methods and compositions for vaccination of animals with prrsv antigens with improved immunogenicityInfo
- Publication number
- MX2008003046A MX2008003046A MXMX/A/2008/003046A MX2008003046A MX2008003046A MX 2008003046 A MX2008003046 A MX 2008003046A MX 2008003046 A MX2008003046 A MX 2008003046A MX 2008003046 A MX2008003046 A MX 2008003046A
- Authority
- MX
- Mexico
- Prior art keywords
- prrsv
- asparagine
- seq
- protein
- linked glycosylation
- Prior art date
Links
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Abstract
Pigs challenged with hypoglycosylated variants of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) major surface protein GP5 exhibited increased production of PRRSV-neutralizing antibodies relative to the levels of neurtalizing antibodies produced by pigs immunized with wild type (wt) or glycosylated GP5. This invention provides for methods of obtaining improved immune responses in pigs to PRRSV, compositions useful for obtaining the improved immune responses as well as isolated polynucleotides that encode hypoglycosylated variants of PRRSV major surface protein GP5.
Description
METHODS AND COMPOSITIONS FOR VACCINATION OF ANIMALS WITH ANTIGENS OF THE PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUSES WITH IMPROVED IMMUNOGENICITY
CROSS REFERENCE
This application claims priority of Provisional Patent Application No. 60 / 712,357, filed on August 30, 2005.
Declaration regarding Research or Development with
Federal Sponsorship This invention was made with the support of the Government under a National Research Initiative Competitive Grant # 2004-01576 (Concession of Competence and Initiative for National Research # 2004-01576) of the U.S. Department of Agriculture and under a COBRE program of the National Institute of Health of the National Center for Research Resources Project # P20RR15636 (Project # P20RR15636 of the National Center of Resources for Research). The government has certain rights over this invention.
BACKGROUND OF THE INVENTION
TECHNICAL FIELD
This invention relates generally to compositions comprising Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) antigens with improved immunogenicity and methods for their use. The compositions and methods described herein result in improved immunogenic responses of pigs to the PRRSV antigens, thus providing improved protection of the pigs against infection by PRRSV.
TECHNICAL BACKGROUND
PRRSV is an economically important pathogen that affects pigs. Infection of sows and sows with PRRSV can result in reproductive failure. PRRSV also causes respiratory diseases in pigs of all ages. It is possible to vaccinate pigs to protect them from infection by PRRSV. However, currently commercially available vaccines (most of which are live attenuated vaccines) are somewhat ineffective and therefore should be improved. The full immunological mechanisms of protection against PRRSV are unclear; however, it has
It was clearly shown that PRRSV neutralizing antibodies are central to this protection. Unfortunately, PRRSV itself (in its wild type form) or current live vaccines derived therefrom have little ability to induce virus-specific neutralizing antibodies in a timely manner and at effective (i.e., protective) levels. U.S. Patent No. 6,500,662, "Infectious cDNA clone of North American Virus of Porcine Reproductive and Respiratory Syndrome (PRRS) virus and use thereof (by Cálvert et al, December 31, 2002) describes the development of a cDNA clone of US PRRSV and its use as a vaccine However, U.S. Patent No. 6,500,662 does not disclose PRRSV vaccines comprising hypoglycosylated antigens of PRRSV In another study, GP5 protein sequences (or ORF5 protein) were compared. ) of various strains of the North American PRRSV with each other and with the isolated strain of the representative European PRRSV as the strain Lelystad, revealing that the glycosylation sites linked to N in Aspargina 44 (N44) and Aspargina 51 (N51) of the consensus sequence of GP5 were conserved in all the strains isolated from the PRRSV examined (Pirzadeh et al., Can. J. Vet Res., 1998, 62: 170-177), however, the glycosylation site in N located in Aspargina 31 ( N 31) of the GP5 consensus sequence was absent in certain strains isolated from the North American PRRSV and absent in the isolated Lelystad strain of the European PRRSV. The recombinant GST-GP5 fusion proteins from four (4) strains of PRRSV
North American and the Lelystad strain were produced in E. coli as insoluble, renatured inclusion bodies, and used as immunogens in rabbits. Said recombinant proteins produced in E. coli retained but did not glycosylate their native N glycosylation sites. It has also been shown that inoculation of pigs with a DNA vaccine comprising a CMV fusion promoter to the GP5 gene of the isolated strain of the North American PRRSV 1AF-Klop provides protection in animals immunized against the PRRSV test (Pirzadeh and Dea, 1998, J. General Virology, 79, 989-999). This isolated strain of the particular GP5 gene encodes a GP5 protein that contains the Aspargin N31, N44 and N51 residues that are presumably osylated when expressed in pigs immunized with the DNA vaccine. Vaccination of pigs with GST-GP5 produced in E. coli, which retains but does not osylate the N osylation sites native to the GP5 gene of strain 1AF-Klop, did not protect the lungs of the pigs tested with the virus. European infectious PRRSV clones containing mutations resulting in the expression of hypoosylated proteins of PRRSV have also been described (Wissink et al., 2004, J. Gen. Viral 85: 3715-23). This particular reference reports that PRRSV containing mutations in the Residue of Aspargina 53 (N53) of the GP protein (5) of the Lelystad strain of PRRSV that prevents N-linked osylation of that site is infectious and can produce infectious particles of the virus of the Lelystad strain of PRRSV. In contrast, PRRSV that contains mutations in the N46 of the
GP protein (5) from the Lelystad strain of PRRSV that prevent N-linked glycosylation of that site is non-infectious and does not produce infectious particles of the Lelystad strain of PRRSV strain. Wissink et al. speculate that glycan sites in N in the GP2a protein of the European PRRSV, and, by analogy, the N53 site of the GP5 protein, can act at many different levels in the natural host including receptor interactions or immune protection. In viruses other than PRRSV, glycan residues have been implicated in a variety of functions. N-linked glycosylation, in general, is important for the replication, recognition, and biological activity of proteins (Helenius, A. and M. Aebi., Annu Rev. Biochem. 73: 1019-1049, 2004. Williams , DB and Glycoconj J., 12: iii-iv, 1995; Zhang, et al., Glycobiology 14: 1229-46, 2004). In many enveloped viruses, the enveloping proteins are modified by the addition of sugar portions and the N-linked glycosylation of the enveloping protein exerts various viral glycoprotein functions such as receptor binding, membrane fusion, penetration into the cells, and virus budding (Braakman, I. and E. van Anken, Traffic 1: 533-9, 2000, Doms et al., Virology 193: 545-62, 1993). Recent studies have demonstrated the role of N-linked glycosylation of the Hantaan virus glycoprotein in protein replication and intracellular trafficking (Shi, X. and RM Elliott, J. Virol. 78: 5414-22, 2004) as well as in the biological activity and antigenicity of the haemagglutinin protein (HA) of the influenza virus (Abe, Y., et al., J. Virol. 78: 9605-11, 2004). Moreover, it has become evident that the glycosylation of enveloping viral proteins is a
Main mechanism for immune viral evasion and persistence used by numerous different enveloped viruses to escape, block or minimize the response of neutralizing antibodies to the virus. Examples of this effect have been reported for the SIV (Reitter, JN et al., Nat. Med. 4: 679-84, 1998) and HIV-1 (Wei, X. et al., Nature 422: 307-12, 2003), HBV (Lee, J. et al .. Biochem. Biophys., Res. Commun. 303: 427-32, 2003), influenza (Skehel, JJ et al. al., Proc. Nati, Acad. Sci. USA 81: 1779-83, 1984) and the LDV arterioveus (Chen, Z. et al., Virology 266: 88-98, 2000).
BRIEF DESCRIPTION OF THE INVENTION
It is in view of the above problems that the present invention was developed. It has been demonstrated that hypoglycosylated variants of a PRRSV major surface GP5 protein increased the level of PRRSV-neutralizing antibodies in immunized pigs relative to the levels of neutralizing antibodies produced by pigs immunized with the wild type (wt) or GP5 glycosylated This invention first provides a method for obtaining an improved immune response in a pig to an antigen of the Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), which comprises the administration of a composition comprising a polynucleotide encoding a hypoglycosylated variant of the polypeptide of the GP5 of PRRSV in
wherein at least one N-linked glycosylation site corresponding to asparagine 34 or asparagine 51 is inactivated in a reference GP5 protein of SEQ ID NO: 1. In certain embodiments of the method, at least one N-linked glycosylation site corresponding to asparagine 34 or asparagine 51 is inactivated in SEQ ID NO: 1. This polynucleotide may comprise an infectious RNA or PRRSV DNA molecule encoding an infectious PRRSV RNA molecule. The infectious PRRSV RNA molecule is a derivative of the North American PRRSV or a derivative of the European PRRSV. Alternatively, this polynucleotide may comprise a DNA molecule wherein an active promoter in mammalian cells is operably linked to said polynucleotide encoding a hypoglycosylated GP5 protein. This promoter in certain preferred embodiments of the invention is a CMV promoter (for its acronym in English). Alternatively, this polynucleotide can be a viral vector. Representative viral vectors that may be used include vaccinia virus vectors, herpes simplex viral vectors, adenovirus vectors, alphavirus vectors, and TGEV vectors (for its acronym in English). A variety of types and sources of PRRSV sequences can be used to obtain the polynucleotide encoding a hypoglycosylated variant of the PRRSV GP5 polypeptide wherein at least one N-linked glycosylation site corresponding to asparagine 34 or asparagine 51 is inactivated. a reference GP5 protein of SEQ ID NO: 1. In certain embodiments of the invention, a polynucleotide that encodes a
Hypoglycosylated polypeptide variant of GP5 of PRRSV is obtained by direct synthesis, mutagenesis of a nucleotide sequence of GP5 of the isolated strain of the North American PRRSV or mutagenesis of a nucleotide sequence of the consensus American PRRSV GP5. The nucleotide sequence of GP5 of the isolated strain of the North American PRRSV can be selected from the group of nucleotides encoding the GP5 proteins of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 , SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13 The nucleotide sequence of GP5 of the isolated strain of the North American PRRSV encodes a consensual GP5 protein that is at least 85% identical to SEQ ID NO: 14. In other embodiments of the invention, the polynucleotide encoding a hypoglycosylated polypeptide variant of PRRSV GP5 is obtained by direct synthesis, mutagenesis of a GP5 nucleotide sequence of the isolated strain of the European PRRSV or mutagenesis of a consensual European PRRSV GP5 nucleotide sequence. The GP5 nucleotide sequence of the isolated strain of the European PRRSV encodes a GP5 protein that is at least 85% identical to SEQ ID NO: 15. A variety of N-linked glycosylation site inactivation methods used for N can be used. effectively practice the method of this invention. A preferred method of inactivation of an N-linked glycosylation site corresponding to asparagine 51 is to replace the asparagine codon with a codon encoding an amino acid
different from asparagine. This replacement codon can encode an alanine or a glutamine residue. In more preferred embodiments, both of said N-linked glycosylation sites corresponding to asparagine 34 and asparagine 51 are inactivated in a reference GP5 protein of SEQ ID NO: 1. In other embodiments, the N-linked glycosylation site corresponding to asparagine 34 is inactivated. This N-linked glycosylation site of asparagine 34 can be inactivated by replacing a codon encoding said asparagine 34 with a codon encoding an amino acid other than asparagine. The codon encoding another amino acid can encode an alanine or a glutamine residue. In other preferred embodiments, both N-linked glycosylation sites corresponding to asparagine 34 and asparagine 51 in a reference GP5 protein can be inactivated by replacing codons encoding asparagine 34 and asparagine 51 with codons encoding an amino acid other than asparagine. . Both codons encoding asparagine 34 and asparagine 51 can be replaced with codons that encode either an alanine residue or a glutamine residue to inactivate those glycosylation sites. Alternatively, the codons can be replaced with codons that encode an alanine residue to inactivate both glycosylation sites. Alternatively, one of the N-linked glycosylation sites is inactivated by replacing a codon encoding said asparagine 34 or said asparagine 51 with a codon encoding an amino acid other than asparagine while the other site linked with N glycosylation is inactivated by other techniques. .
In preferred embodiments, the method employs an infectious PRRSV RNA molecule that is a North American PRRSV derivative encoding a hypoglycosylated variant protein of the PRRSV GP5 polypeptide wherein at least one N-linked glycosylation site corresponding to asparagine 34 or asparagine 51 in a reference GP5 protein of SEQ ID NO: 1. In more preferred embodiments, both of said N-linked glycosylation sites corresponding to asparagine "34 and asparagine 51 are inactivated in a reference protein GP5 of SEQ ID NO: 1. Both of the N-linked glycosylation sites corresponding to asparagine 34 and asparagine 51 in a reference GP5 protein can be inactivated by replacing codons encoding asparagine 34 and asparagine 51 with codons encoding a different amino acid of Asparagine Both codons that code for asparagine 34 and asparagine 51 can be replaced with codons that encode Any of an alanine or a glutamine residue to inactivate those glycosylation sites. Alternatively, both codons can be replaced with codons that encode an alanine residue to inactivate that glycosylation site. To practice this method, the composition containing the polynucleotide is administered by subcutaneous injection, intravenous injection, intradermal injection, parenteral injection, intramuscular injection, needle free injection, electroporation, oral delivery, intranasal delivery, oronasal delivery, or any combination thereof. same. The composition
administered may further comprise a therapeutically acceptable vehicle. This therapeutically acceptable carrier is selected from the group consisting of a protein, a buffer, a surfactant, and a polyethylene glycol polymer, or any combination thereof. The composition administered may further comprise an adjuvant. This adjuvant can be aluminum hydroxide, Quil A, a suspension of alumina gel, mineral oils, glycerides, fatty acids, fatty acid byproducts, mycobacteria, and CpG oligodeoxynucleotides, or any combination thereof. The composition administered may also comprise a second adjuvant such as interleukin 1 (IL-1), IL-2, IL-4, IL-5, IL-6, IL-12, gamma interferon (g-IFN), necrosis factor cellular, MDP (muramyl dipeptide) immune stimulating complexes (ISCOM), and liposomes. The improved immune response of a pig to a PRRSV antigen can comprise an increased production of the PRRSV neutralizing antibodies by said pig. Increased production of PRRSV neutralizing antibodies is typically observed with pig immunization by the methods and compositions of this invention. The improved immune response can be obtained in a sow, a first sow, a boar, or a piglet. The invention also provides a method for obtaining an improved immune response in a pig to an antigen of the Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), which comprises
administration of a composition comprising a hypoglycosylated variant of the PRRSV GP5 polypeptide wherein at least one N-linked glycosylation site corresponding to asparagine 34 or asparagine 51 is inactivated in a reference GP5 protein of SEQ ID NO: 1 to said pig. The hypoglycosylated variant protein of the PRRSV GP5 polypeptide can be produced by the same polynucleotides used in the methods previously described in bacterial, yeast, or mammalian expression systems. The present invention also provides compositions comprising a polynucleotide encoding a hypoglycosylated variant of the North American PRRSV GP5 polypeptide wherein at least one N-linked glycosylation site corresponding to asparagine 51 is inactivated in a reference GP5 protein of the SEQ. ID NO: 1, and a therapeutically acceptable vehicle. In certain embodiments, the compositions comprise polynucleotides wherein both N-linked glycosylation sites corresponding to asparagine 34 and asparagine 51 are inactivated in SEQ ID NO: 1. In preferred embodiments, this composition can comprise any of an infectious RNA molecule or an infectious DNA molecule of the North American PRRSV that encodes an infectious RNA molecule of the North American PRRSV. In other embodiments, the polynucleotide comprises a DNA molecule wherein an active promoter in mammalian cells is operably linked to said polynucleotide encoding said hypoglycosylated variant of the GP5 polypeptide of the
North American PRRSV. In certain preferred embodiments, this promoter is a CMV promoter. In still other embodiments, the polynucleotide in the composition comprises a viral vector. Viral vectors that can be used in the composition can be any of a vaccine virus vector, a herpes simplex viral vector, an adenovirus vector, an alphavirus vector and a TGEV vector. In the polynucleotides of the composition, an N-linked glycosylation site is inactivated by replacing a codon encoding said asparagine 51 with a codon encoding an amino acid other than asparagine. The codon encoding a different amino acid than asparagine encodes an alanine or a glutamine residue. In other embodiments of this composition, an additional N-linked glycosylation site is inactivated by replacing a codon encoding asparagine 34 with a codon that encodes an amino acid other than asparagine. This codon encoding another amino acid can encode an alanine or a glutamine residue. The preferred compositions are thus provided by this application, the preferred compositions comprise polynucleotides encoding a hypoglycosylated variant protein of the North American PRRSV GP5 polypeptide wherein both of said N-linked glycosylation sites corresponding to asparagine 34 and asparagine 51 are inactivated in a GP5 protein. of North American reference of SEQ ID NO: 1. Both of said N-linked glycosylation sites can be inactivated by replacing the codons encoding said asparagine 34 and said asparagine 51 with codons that
they encode an amino acid different from asparagine. These codons that encode an amino acid other than asparagine can encode either an alanine residue or a glutamine residue. The therapeutically acceptable carrier used in the composition may be a protein, a buffer, a surfactant, and a polyethylene glycol polymer, or any combination thereof. The composition further comprises at least one adjuvant. This adjuvant can be aluminum hydroxide, Quil A, a suspension of alumina gel, mineral oils, glycerides, fatty acids, fatty acid byproducts, mycobacteria, and CpG oligodeoxynucleotides, or any combination thereof. The composition may further comprise a second adjuvant selected from the group consisting of interleukin 1 (IL-1), IL-2, IL-4, IL-5, IL-6, IL-12, gamma interferon (g-IFN), cell necrosis factor, MDP (muramyl dipeptide) immune stimulating complexes (ISCOM), and liposomes. Also provided by the invention is a composition comprising a hypoglycosylated variant of the North American PRRSV GP5 polypeptide wherein at least one N-linked glycosylation site corresponding to asparagine 51 is inactivated in a reference GP5 protein of SEQ ID NO. : 1 and a therapeutically acceptable vehicle. In preferred embodiments, the N-linked glycosylation sites corresponding to both of asparagine 34 or asparagine 51 are inactivated in a reference GP5 protein of SEQ ID NO: 1. The hypoglycosylated variant protein of the North American PRRSV GP5 polypeptide may
produced by the same polynucleotides used in the methods previously described in bacterial, yeast, or mammalian expression systems. The present invention also provides isolated polynucleotides encoding a hypoglycosylated variant of the North American PRRSV GP5 polypeptide wherein at least one N-linked glycosylation site corresponding to asparagine 51 is inactivated in a reference GP5 protein of SEQ ID NO: 1. In certain embodiments, polynucleotides are provided wherein both N-linked glycosylation sites corresponding to asparagine 34 and asparagine 51 are inactivated in SEQ ID NO: 1. In preferred embodiments, this isolated polynucleotide may comprise any of an RNA infection molecule. or an infectious DNA molecule of the North American PRRSV that encodes an infectious molecule of North American PRRSV RNA. In other embodiments, the isolated polynucleotide comprises a DNA molecule wherein an active promoter in mammalian cells is operably linked to said polynucleotide encoding said hypoglycosylated variant of the GP5 polypeptide of the North American PRRSV. In certain preferred embodiments, this promoter is a CMV promoter. In still other embodiments, the isolated polynucleotide comprises a viral vector. Viral vectors that can be used in the composition can be any of a vaccine virus vector, a herpes simplex viral vector, an adenovirus vector, an alphavirus vector, and a TGEV vector.
In the isolated polynucleotides an N-linked glycosylation site is inactivated by replacing a codon encoding said asparagine 51 with a codon encoding an amino acid other than asparagine. The codon encoding an amino acid other than asparagine encodes an alanine or a glutamine residue. In other preferred embodiments, an additional N-linked glycosylation site is inactivated by replacing a codon encoding asparagine 34 with a codon encoding an amino acid other than asparagine. This codon encoding another amino acid can encode an alanine or a glutamine residue. The preferred polynucleotides encoding a hypoglycosylated variant protein of the North American PRRSV GP5 polypeptide wherein both are inactivated are then provided. of said glycosylation sites corresponding to asparagine 34 and asparagine 51 in a North American reference GP5 protein of SEQ ID NO: 1. Both of said N-linked glycosylation sites can be inactivated by replacing the codons encoding said asparagine 34 and said asparagine 51 with codons encoding an amino acid other than asparagine. These codons that encode an amino acid other than asparagine can encode either an alanine residue or a glutamine residue. The invention also provides an isolated polypeptide which is a hypoglycosylated variant of the North American PRRSV GP5 polypeptide wherein at least one N-linked glycosylation site corresponding to asparagine 51 is inactivated in a GP5 protein of
reference of SEQ ID NO: 1. The hypoglycosylated variant protein of the isolated American PRRSV GP5 polypeptide can be produced by the same polynucleotides used in the methods previously described in bacterial, yeast, or mammalian expression systems and purified by chromatography or other techniques. Further features and advantages of the present invention, as well as the structure and operation of various embodiments of the present invention, are described in detail in the following with reference to the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
The accompanying drawings, which are incorporated in and forming a part of the specification, illustrate the embodiments of the present invention and together with the description, serve to explain the principles of intention. In the drawings: Figures 1A and 1B illustrate the transient expression of the GP5 and M proteins of the PRRVS. A. Diagram of the bicistronic construction showing the transcribed regions of GP5 and M that flank the IRES (IE) of the encephalomyocarditis virus. The transcribed regions are under the control of the T7 RNA polymerase promoter (black rectangle) immediately present in the 5 'direction of the transcribed region of GP5. The flexed arrow shows the position and direction of transcription by the polymerase
T7 RNA from the vector. B. Expression of GP5 and M proteins in cells transfected with the bicistronic vector. Simulated transfected cells (lane 1) or transfected plasmid cells (lanes 2-7) were radiolabeled as described in Materials and Methods, immunoprecipitated with anti-GP5 antibody (lanes 1-5) or anti-M antibody (lanes 6-) 7). The immunoprecipitated proteins were untreated (-) (bands 1, 2, 6, and 7) or were treated with Endo H (band 3), PNGase F (band 4) and analyzed by electrophoresis. Lane 5 contains immunoprecipitated proteins from transfected cells treated (+) with tunicamycin. The mobilities of proteins with relative molecular mass (Mr) in kilodaltons are shown. Figures 2A-2B and Table A illustrate glycosylation analysis of WT-GP5 and its mutants using a bicistronic plasmid. A. A schematic of a bicistronic vector and GP5 of PRRSV are shown with the three putative glycosylation sites at amino acid positions 34, 44 and 51. B. Various mutants used in the present study. C. Expression of the wt GP5 and mutant and its sensitivity to Endo H. The experiment was developed as described in the legend of Figures 1A and 1B, the proteins were immunoprecipitated with anti-GP5 antibody, digested with Endo H (+) or they were undigested (-) and analyzed by electrophoresis. The mutant GP5 proteins are shown by arrowheads. On the right is the mobility of proteins with relative molecular mass (Mr) in Kilodaltons.
Figures 3A-3C illustrate the characterization of mutant viruses encoding mutant GP5. A. Growth kinetics of a wt stage (FL-12) and various mutant PRRSVs in MARC-145 cells. Cells in six-well plates were infected with PRRSV at an MOI of 3, culture supernatants were collected at indicated times after infection and virus titers were determined. The average values with standard deviation (error bars) of three independent experiments are shown. B. Plate morphology of mutant virus "Open arrows and arrowheads show plaques that are less clear C. Transcomplementation to recover mutant PRRSV Quantitative analysis of mutant virus recovery from cells expressing wt GP5 protein The average virus yield of three independent experiments with standard deviation (represented by error bars) is shown, Figures 4A-4B illustrate an examination of GP5 incorporated in mutant virions and synthesized in cells infected with mutant virus. sedimentarion the radiolabeled virions of culture supernatants of infected cells, the GP5 protein was immunoprecipitated, treated with (+) or without (-) Endo H and analyzed by electrophoresis. The GP5 with and without digestion of Endo H in bands 1 and 2 is shown by the white brackets. B. Cells infected with various mutant viruses were radiolabelled GP5 was immunoprecipitated, treated with (+) or without (-) Endo H and analyzed by electrophoresis. GP5 with and without digestion of Endo H in bands 2 and 3 shows by the white brackets. The mobilities of proteins with relative molecular mass (Mr) in kilodaltons are shown on the right side of each panel. Figure 5 illustrates an alignment of the n-terminus amino acid sequences of GP5 of the North American PRRSV with the reference n-terminus sequence of GP5 of the North American PRRSV (SEQ ID NO: 1, strain NVSL 97-7895). The first 60 amino acids of the N-terminus of an alignment of the 200 amino acid proteins are shown. The N-linked glycosylation sites of asparagine 34"NSS" and asparagine 51"NGT" are shown in bold. Other N-linked glycosylation sites located between residues 29 and 35 of the reference GP5 protein are underlined. Figure 6 illustrates an alignment of the N-terminus amino acid sequence of GP5 of the European PRRSV with the reference n-terminus sequence of GP5 of the North American PRRSV (SEQ ID NO: 1, strain NVSL 97-7895). An alignment of the entire GP5 protein of approximately 200 amino acids is shown in which the N-linked glycosylation site of asparagine 51"NGT" in the proteins (ie, asparagine 53 in SEQ ID NO: 15) is shown in bold. ).
DETAILED DESCRIPTION OF THE INVENTION
Definitions "Acceptable vehicle", as used herein, refers to a vehicle that is not harmful to the other ingredients of the composition and is not harmful to the material to which it is to be applied. "Therapeutically acceptable vehicle" refers to a vehicle that is not harmful to the other ingredients of the composition and is not harmful to humans or other animals recipients thereof. In the context of the other ingredients of the composition, "no-harm" means that the vehicle will not react with or degrade the other ingredients or otherwise interfere with its effectiveness. The interference with the efficacy of an ingredient does not cover a mere dilution of the ingredient. In the context of the animal, "not harmful" means that the vehicle is not harmful or lethal to the plant or animal. "Adjuvant", as used herein, refers to any material used in conjunction with an antigen that increases the ability of that antigen to induce an immune response. "Administration", as used herein, refers to any means for providing a polynucleotide, a polypeptide or a composition thereof to a subject. Non-limiting examples of means of administration include subcutaneous injection, intravenous injection, intradermal injection, parenteral injection, intramuscular injection,
water-free injection, electroporation, oral delivery, intranasal delivery, oronasal delivery, or any combination thereof. "Antigen", as used herein, refers to any entity that induces an immune response in a host. "Consensus sequence", as used herein, refers to an amino acid sequence, DNA or RNA created by aligning two or more homologous sequences and deriving a new sequence representing the amino acid sequence, DNA or Common RNA; "Hypoglycosylated variant of PRRSV GP5 polypeptide", as used herein, refers to GP5 proteins of PRRSV wherein the original or non-variant amino acid sequence comprising one or more N-linked glycosylation sites has changed to reduce the number of glycosylation sites linked to N in the resulting GP5 variant protein. Under this definition, expression of an original or non-variant GP5 protein in E.coli to produce a GP5 protein with the original GP5 sequence containing the same number of glycosylation sites will not result in the production of a hypoglycosylated variant of the the GP5 of the PRRSV. "Immune response", as used herein, refers to the production of antibodies and / or cells (such as T lymphocytes) that bind, degrade or otherwise inhibit a particular antigen. Related phrases such as "an improved immune response" refer to the use of methods and compositions that result in any measurable improvement in
the response of a host immunized to an antigen. For example, measurable enhancements of an immune response include, but are not limited to, an increased production of neutralizing antibodies (ie, increased titres of antibodies) relative to the levels of production observed in control animals that have been immunized with antigens that lack the structural modifications that provide an improved immune response. "Infectious RNA molecule" refers to an RNA molecule that encodes all the elements necessary for the production of a functional virion when introduced into a permissive host cell. "Infectious clone", as used herein, refers to a DNA molecule that encodes an infectious RNA molecule. "North American PRRSV", as used herein, refers to any PRRSV comprising polynucleotide sequences associated with a strain isolated from the North American PRRSV, such as, but not limited to, strain NVSL 97-7895 (Truong et al, Virology, 325: 308-319 and references contained therein) or strains lAF-Klop, MLV, ATCC VR-2332, ATCC VR-2385, IAF-BAJ, IAF-DESR, IAF-CM, IAF 93-653, IAF 93-2616, IAF 94-3182, IAF 94-287 described in Pirzadeh et al. Dog. J. Vet Res, 62: 170-177 and contained therein). For this invention, the PRRSV comprising the polynucleotide sequences associated with a strain isolated from the North American PRRSV are PRRSV containing polynucleotide sequences in
wherein the coding region of GP5 encodes a polypeptide having at least 85% identity of the protein sequence for SEQ ID NO: 1. "European PRRSV", as used herein, refers to any PRRSV comprising sequences of polynucleotides associated with a strain isolated from the North American PRRSV, such as, but not limited to, the Lelystad strain (Wissink et al., J. Gen. Virol. 85: 3715, 2004 and references contained therein). For this invention, PRRSV comprising polynucleotide sequences associated with an isolated strain of the European PRRSV are PRRSV containing polynucleotide sequences wherein the coding region of GP5 encodes a polypeptide having at least 85% identity of the sequence of protein for SEQ ID NO: 15. "Percent identity", as used herein, refers to the number of elements (i.e., amino acids or nucleotides) in a sequence that are identical within a defined length of two optimally aligned segments of DNA, RNA or protein. . To calculate the "percent identity", the number of identical elements is divided by the total number of elements in the defined length of the aligned segments and multiplied by 100. When the identity percentage is used with reference to proteins it is understood that certain amino acid residues may not be identical but are nevertheless conservative substitutions of amino acids that reflect substitutions of amino acid residues with similar chemical properties (eg, acid or basic, hydrophobic or hydrophilic, donor residues or hydrogen bond acceptors). Said
Substitutions may not change the functional properties of the molecule. Accordingly, the percent identity of the protein sequences can be increased to count as conservative substitutions.
Introduction Glycoprotein 5 (GP5) of porcine reproductive and respiratory syndrome virus (PRRSV) is the most abundant envelope glycoprotein and a major inducer of neutralizing antibodies in vivo. Three putative glycosylation sites linked to N (N34, N44, and N51) are located in the ectodomain where there is also a major neutralization epitope. To determine which of these putative glycosylation sites is used in the PRRSV life cycle and the role of the glycan moieties in the induction of neutralizing antibodies, a panel of GP5 mutants was generated. they contained substitutions for single and multiple amino acids at those sites. The transient expression of the wild-type (wt) as well as the mutant proteins and subsequent biochemical studies revealed that the mature GP5 contains sugar-type higher sugar portions at all three sites. These mutations were subsequently incorporated into a full-length cDNA clone to recover the infectious PRRSV. The results showed that mutations involving the N44 residue did not result in the production of infectious progeny, indicating that N44 is the most critical amino acid residue for viral infectivity. The viruses that carry the mutations in N34, N51, and N 34/51 grew to valuations
lower than PRRSV wt and exhibited a reduced cytopathic effect in MARC 145 cells. In serum neutralization assays, mutant viruses exhibited an increased sensitivity to neutralization by PRRSV-specific antibodies wt. Moreover, the inoculation of pigs with the mutant viruses induced significantly higher levels of neutralizing antibodies against the mutants as well as the wt PRRSV, suggesting that the loss of the glycan residues in the ectodomain of the GPS increases both of the sensitivity of these viruses to an in vitro neutralization as well as the immunogenicity of the close neutralization epitope. These results should have great significance for the development of PRRSV vaccines of increased protective efficacy. It is known that neutralizing antibodies are major correlates of protection against PRRSV. It has been found that the elimination of glycosylation sites in the GP5 protein of PRRSV results in a significant increase in: (1) the ability of the modified PRRSV strain to be neutralized by a convalescent PRRSV antiserum and (2) the ability of this modified strain of PRRSV to produce unprecedented levels of PRRSV-neutralizing antibodies when used to inoculate pigs. The application of this concept to any live virus (wt or attenuated) to immunize against PRRSV infection could have a significant impact on its use to confer effective protection against PRRSV infection.
Currently, there are three main approaches to immunize against PRRSV infections: (1) live attenuated vaccines, (2) inactive vaccines (which are based on the PRRSV wt developed in vitro chemically inactivated), and (3) the use of a premeditated infection with the wt virulent PRRSV in a systematic way to all animals in the herd. There is a great discussion and controversy about which of these 3 paths is the most effective. The invention could be beneficial, regardless of the approach used for immunization. The genetic alteration of a living PRRSV to modify the level of glycosylation of its proteins can be carried out in any of the attenuated PRRSV vaccine strains, or the wt PRRSV strain used to produce an inactive vaccine, or the wt PRRSV strain used for direct inoculation of the herd by massive infection. Porcine reproductive and respiratory syndrome virus (PRRSV) belongs to the family of Arteriviridae within the order Nidovirals that also include equine arteritis virus (EAV), lactate dehydrogenase elevating virus (LDV-by its acronym in English), and simian hemorrhagic fever virus (SHFV-for its acronym in English). The viral genome is a linear, positive-strand RNA molecule of approximately 15.0 Kb in length and has an end cap structure at the 5 'end and a poly (A) end at the 3' end. Eight open reading frames (ORF) are encoded in the viral genome. The first two reading frames (ORF1 a and ORFl ab) encode
non-structural polyproteins (NS) that are involved in the processing of polyproteins and transcription and replication of the genome. Viral structural proteins, encoded in ORF2-7, are expressed from six polyadenylated and subgenomically capped mRNAs that are synthesized as a set of 3 'cotermined nested mRNAs with a common leader sequence at the 5' end. The main viral enveloping protein is glycoprotein 5 (GP5), which is encoded in the ORF5 of the viral genome. GP5 is a glycosylated transmembrane protein approximately 25 kDa in size. It has a putative N-terminal signal peptide and has three potential N-linked glycosylation sites that are located in a small ectodomain that comprises the first 40 residues of the mature protein. In the EAV and the LDV, the main envelope glycoprotein forms a heterodimer linked to bisulfide with the product of the ORF6 gene, the viral matrix (M) protein. A similar interaction has been observed between GP5 and M proteins of PRRSV but the mode of interaction has not yet been defined. It has been postulated that the formation of heterodimers of the GP5 and M proteins can exert a critical function in the assembly of infectious PRRSV. In addition to its function in viral assembly, GP5 seems to be involved in the entry of the virus into susceptible host cells. It is presumed that GP5 interacts with the host cell receptor, sialoadesin, to enter porcine alveolar macrophages (PAM), the target cells in vivo for PRRSV. The function of GP5 in the recognition of the receiver is supported by the presence of an epitope
main neutralization in the N-terminus ectodomain, thus implying a central function for the ectodomain of GP5 in the infectious process. The N-linked glycans of the GP5 ectodomain can be critical for the proper functioning of the protein. N-linked glycosylation, in general, is important for correct replication, recognition, and biological activity of proteins. In many enveloped viruses, the enveloping proteins are modified by the addition of de-sugar moieties and the N-linked glycosylation of the envelope protein exerts various functions of the viral glycoproteins such as receptor binding, membrane fusion, penetration into the cells , and virus budding. Recent studies have demonstrated the role of N-linked glycosylation of the Hantaan virus glycoprotein in the replication of proteins and intracellular trafficking as well as in the biological activity and antigenicity of the haemagglutinin (HA) protein of the influenza virus. Moreover, it has become evident that the glycosylation of viral enveloping proteins is a major mechanism for the evasion and persistence of viral imune used by numerous different enveloped viruses to escape, block or minimize the response of virus-neutralizing antibodies. Examples of this effect have been reported for SIV and HIV-1, HBV, influence and most importantly, in the case of PRRSV, the LDV arterivirus. Recently, the development of inverse genetic systems for PRRSV has been reported by numerous laboratories, including that of applicants. Obviously, mutational studies with clones
Infectious diseases have led to a better understanding of the mechanisms of transcription and replication of the viral genome of arterivirus. Then, to examine the importance of N-linked glycosylation in the biological activity of GP5 of PRRSV in the generation of infectious viruses or in obtaining neutralizing antibodies in vivo, a series of mutant GP5 proteins has been constructed in which each of the potential N-linked glycosylation sites have been mutated either individually or in various combinations. The resulting mutant proteins were examined for their glycosylation pattern, function in the recovery of infectious virus and in cross-neutralization by antibodies raised through experimental inoculations, against wt PRRSV or against mutant viruses. The data demonstrate that the three putative glycosylation sites are used for glycosylation with superior glycans of the truss type and the glycosylation of the GP5 protein at residue 44 is critical for the recovery of infectious PRRSV. Importantly, data from neutralization and antibody response studies indicate that natural infection with PRRSV may involve immune evasion based on glycan protection mechanisms as previously described for other viruses, thus helping to explain the immune response protective humoral rather ineffective that is observed in animals infected by PRRSV.
N-linked glycosylation sites and inactivation methods N-linked glycosylation in glycoproteins typically occurs in the Asn-Xaa-Ser / Thr sequences (NXS / T), where Xaa (X) is any amino acid residue except Pro. introducing a variety of mutations at the N-linked glycosylation sites to provide for their inactivation. A preferred method of inactivation comprises the substitution of the residue asparagine with a residue encoding any amino acid other than asparagine. In more preferred embodiments of this invention, the asparagine residue is replaced with an alanine or a glutamine residue. Other methods for the inactivation of the N-linked glycosylation sites, and in particular the N-linked glycosylation sites corresponding to asparagine 34 and / or 51 of the reference protein GP5 of SEQ ID NO: 1, are also contemplated in the present. Substitutions of certain amino acids such as proline, tryptophan, aspartate, glutamate or Leucine at the Xaa position can also be used to inactivate the N-linked glycosylation sites (Kasturi et al., Biochem J. 323 (2): 415-9, 1997). Alternatively, substitutions of the final position of the hydroxy-containing amino acid of the N-linked glycosylation site (ie, the serine or threonine residue of the NXS / T sequence) with any amino acid that does not contain hydroxy (ie, any amino acid) different from serine or threonine) can also be used to inactivate the N-linked glycosylation site. Examples of non-hydroxy amino acids that have been used for
inactivating N-linked glycosylation sites include cysteine (Kasturi et al., J. Biol Chemistry 270 (24), 14756-14761, 1995). In addition to amino acid substitutions, other types of mutations that inactivate N-linked glycosylation sites such as amino acid insertions or amino acid deletions are also contemplated by this invention. Those skilled in the art will appreciate that an N-linked glycosylation site can be readily activated by deletions that remove key amino acids in the sequence of NXS / T (ie, asparagine residues) will result in the inactivation of that glycosylation site. Deletions of the X residue or serine / threonine residue can similarly inactivate certain N-linked glycosylation sites where the S or T residue is not followed by another S or T residue in the naturally occurring sequence. When X is an amino acid that does not contain hydroxy (ie, it is not serine or threonine), the insertions of any amino acid residue at the carboxy terminus of the N residue can inactivate the N-linked glycosylation site. The insertions of any amino acids that do not contain hydroxy at the carboxy terminus of residue X can also inactivate the N-linked glycosylation site. In summary, it is understood that the key feature of the mutation used to practice the invention is that it inactivates N-linked glycosylation. at the sites of asparagine 34 and / or asparagine 51 in a GP5 protein. While not limited by theory, it is believed that the characteristic
key to these mutations is that they prevent glycosylation in a certain region of the protein (ie, residues corresponding to the sites of asparagine 34 and / or asparagine 51 in a GP5 reference protein of SEQ ID NO: 1). By preventing glycosylation at these sites, the sugar residues that ordinarily protect the key epitopes of the wild-type virus are removed, thereby enabling an improved immune response to be obtained. Accordingly, it is anticipated that a number of different types of mutations (i.e. substitution, insertion or deletion of amino acids) can be used to inactivate the N-linked glycosylation sites and obtain an antigen that will obtain the improved immune response.
Description of PRRSV polynucleotides and polypeptides of the invention The methods of this invention can be practiced with a variety of different polynucleotides that can be derived from a variety of different sources. The common feature of all polynucleotides is that they encode a hypoglycosylated variant of the PRRSV GP5 polypeptide where the N-linked glycosylation sites corresponding to either asparagine 34, asparagine 51, or both asparagine 34 and asparagine 54 are inactivated. in a reference GP5 protein of SEQ ID NO: 1. To identify the N-linked glycosylation sites corresponding to asparagine 34 and asparagine 54 in the reference GP5 protein of SEQ ID NO: 1, the polypeptide sequence of the GP5 of
Non-variant and normally glycosylated PRRSV can be aligned with the reference GP5 protein of SEQ ID NO: 1. Examples of such alignment are displayed in Figures 5 and 6. The particular sequences used in this alignment are described in Table 1.
TABLE 1 Description of Sequences
To identify the N-linked glycosylation sites corresponding to asparagine 34 or asparagine 51 in a reference GP5 protein of SEQ ID NO: 1 that can be activated and used in the methods of this invention, the GP5 proteins of either of an isolated strain of the
North American PRRSV desired (Figure 5) or an isolated strain of PRRSV
European (Figure 6) are aligned with the reference GP5 protein of SEQ ID NO: 1 (North American strain NVSL 97-7895). By using the reference GP5 protein of SEQ ID NO: 1 as a reference protein, one skilled in the art can easily identify the N-linked glycosylation sites of any GP5 protein and then construct the hypoglycosylated variants of the GP5 protein of this invention. It is then apparent that the term "corresponding to (asparagine 34 and / or asparagine 51) in a reference GP5 protein of SEQ ID NO: 1" serves as a descriptor of the N-linked glycosylation site in any GP5 protein.
The hypoglycosylated GP5 protein can be obtained from a strain isolated from the North American PRRSV which includes, but is not limited to, SEQ ID NO: 1-13, a strain isolated from the North American PRRSV that is at least 85% identical at one level. of amino sequence with a consensus sequence of the North American PRRSV such as SEQ ID NO: 14, or of a consensus sequence of the North American PRRSV. The hypoglycosylated GP5 protein can also be obtained from strains isolated from the European PRRSV which include, but are not limited to, SEQ ID NO: 15, a strain isolated from the European PRRSV which is at least 85% identical at a level of amino sequence for a European PRRSV consensus sequence such as SEQ ID NO: 15, or from a consensus sequence of the European PRRSV. To obtain the hypoglycosylated variant of GP5 which encodes polynucleotides, the polynucleotides of any of the sources listed above can be mutagenized by standard site-directed mutagenesis techniques so that they will modify a polypeptide of the hypoglycosylated variant GP5. Alternatively, a complete synthetic DNA sequence encoding the hypoglycosylated variant GP5 polypeptide can be constructed. This is typically effected by the use of a sequence analysis program such as "back transíate" (reverse translation) that converts a polypeptide sequence into a corresponding polynucleotide sequence (GCG Wisconsin Package ™, Accelrys, Inc., San Diego, CA ). If desired, a "routine codon" can be incorporated into the "back transient" program to provide the design of a synthetic gene
which incorporates the appropriate codons for use in the desired expression host (i.e., mammal or yeast). The N-linked glycosylation site corresponding to asparagine 51 of the reference GP5 protein of SEQ ID NO: 1 is present in all the isolated strains of the North American PRRSV shown in Figure 5 and in the European strain Lelystad PRRSV. In these non-limiting North American and European PRRSV strains, the N-linked glycosylation site in this position will comprise the "NGT" sequence. However, it is also anticipated that other PRRSV variants may comprise other structurally interchangeable N-linked glycosylation sites in this position (ie, NXS or T) that may also be inactivated through the methods taught herein. This N-linked glycosylation site can be inactivated by substituting the codons encoding other amino acid residues such as glutamine or alanine instead of those of asparagine 51 in the corresponding polynucleotide sequence. In these examples, the corresponding amino acid sequence in the hypoglycosylated variant of the GP5 protein of the North American PRRSV could comprise sequences such as "QGT", "AGT", or "XGT", where X is any amino acid other than asparagine. These and other hypoglycosylated variants of the strains isolated from the GP5 protein of the North American PRRSV where the N-linked glycosylation site corresponding to asparagine 51 is inactivated can also be combined with other hypoglycosylated variants of GP5 where other glycosylation sites are inactivated. linked to N. Other methods to inactivate
N-linked glycosylation sites include amino acid substitutions of the "X" or "S / T" residues of the NXS / T sequence, amino acid deletions or amino acid insertions and are described in the foregoing. The N-linked glycosylation site corresponding exactly to asparagine 34 of the reference GP5 protein of SEQ ID NO: 1 is present in only certain representative North American PRRSV isolates shown here (Figure 5). More specifically, the GP5 proteins of the representative isolates of the PRRSV North American IAF-BAJ (SEQ ID NO: 3), 94-3182 (SEQ ID NO: 7), and 94-287 (SEQ ID NO: 8) which contain the N-linked glycosylation site corresponding exactly to asparagine 34 in the reference GP5 protein of SEQ ID NO: 1 and comprises the N-linked NSS glycosylation site. Of course it is anticipated that other isolated strains of PRRSV GP5 not shown herein also contain N-linked glycosylation sites corresponding to asparagine 34 of the reference GP5 protein of SEQ ID NO: 1 and that the hypoglycosylated variants of these other GP5 proteins can also be obtained using the methods described herein. This glycosylation site linked to N of SEQ ID NOs: 3, 7, and 8 or other strains isolated from PRRSV containing the N-linked glycosylation site of asparagine 34 can be inactivated by substituting codons encoding other amino acid residues such as glutamine or alanine instead of those of asparagine 34 in the sequence of corresponding polynucleotides. In these examples where the glycosylation site linked to
N in asparagine 34 is "NSS", the corresponding amino acid sequence in the hypoglycosylated variant of the GP5 protein of the North American PRRSV may comprise sequences such as "QSS", "ASS", or "XSS", where X is any amino acid different from asparagine. Alternatively, the serine residue of the "NSS" sequence can be substituted with an amino acid that does not contain hydroxy (ie, non-threonine serine). In these examples, the corresponding amino acid sequence in the hypoglycosylated variant of the GP5 protein of the North American PRRSV may comprise the sequence "NXS", wherein X is any amino acid other than asparagine. An insertion of a non-hydroxy amino acid between the two serine residues of the "NSS" sequence (ie, between serines 35 and 36) can also be used to inactivate this particular glycosylation site. In other strains isolated from the North American PRRSV lacking the N-linked glycosylation site corresponding exactly to asparagine 34 of the reference GP5 protein of SEQ ID NO: 1, other N-linked glycosylation sites located in the residue can also be inactivated. 30 (Figure 5"ÑAS" in SEQ ID NOs: 2, 3, 4, 6, 7, 8, 9, 11, 13), and residue 33 (Figure 5"NNS" in SEQ ID NOs: 3, 8; NSS "in SEQ ID NOs: 6, 10," NDS "in SEQ ID NOs: 11, 13). In other words, N-linked glycosylation sites in other North American isolates located at the amino acid positions corresponding to residues 30 and 33 of the reference GP5 protein of SEQ ID NO: 1 can also be inactivated and
used in the methods of this invention. Without being limited by theory, the particular region of the GP5 protein of PRRSV located between residues 29 and 35 of the reference GP5 protein of SEQ ID NO: 1 appears to be a hypervariable region (Figure 5) that can tolerate a variety of different amino sequences (Figure 5; see also Pirzadeh et al., Can. J. Vet Res., 1998, 62: 170-177). Although certain strains isolated from PRRSV that occur naturally contain non-N-linked glycosylation sites in this region (ie, isolated North American strains of SEQ ID NOs: 5, 12, European isolated strain of SEQ ID NO: 15), others Isolated strains can contain between 1 to 3 glycosylation sites in this region. Accordingly, the inactivation of any of the glycosylation sites of a given GP5 protein in the region located between residues 29 and 35 of the reference GP5 protein of SEQ ID NO: 1 are contemplated herein as a composition or method to obtain an improved immune response to the GP5 protein of PRRSV. Moreover, the inactivation of more than one or all of the glycosylation sites of a given GP5 protein in the region located between residues 29 and 35 of the reference GP5 protein of SEQ ID NO: 1 is also contemplated herein as a composition or method for obtaining an improved immune response to the GP5 protein of PRRSV. The alignment of the North American PRRSV sequence and
European demonstrates that the N-linked glycosylation site corresponding to asparagine 51 of the reference GP5 protein of SEQ ID NO: 1 is also present in a strain isolated from the European PRRSV
representative. In this particular example, the N-linked glycosylation site comprises the sequence "NGT" and asparagine 51 of the reference sequence of SEQ ID NO: 1 corresponds to asparagine 53 of SEQ ID NO: 15. This site of N-linked glycosylation can be inactivated by substituting the codons encoding other amino acid residues such as glutamine or alanine instead of those of asparagine 53 in the corresponding European PRRSV polynucleotide sequence. In these examples, the corresponding amino acid sequence in the β-glycosylated variant of the GP5 protein of the European PRRSV may comprise the sequences such as "QGT", "AGT", or "XGT", where X is any amino acid other than asparagine. These and other hypoglycosylated variants of strains isolated from the European PRRSV where the N-linked glycosylation site corresponding to asparagine 51 is inactivated can also be combined with other hypoglycosylated variants of GP5 where other N-linked glycosylation sites are inactivated. Hypoglycosylated variants of GP5 proteins can be encoded by PRRS viruses that can be used to prepare live vaccines, killed, or attenuated to protect pigs from PRRSV infections. In preferred embodiments of the invention, the hypoglycosylated variant proteins of the GP5 of this invention are designed in infectious PRRSV clones that are capable of producing infectious PRRSV RNA. Descriptions of infectious clones of the North American PRRSV that can be designed to encode variant proteins
hypoglycosylated from GP5 are found in the U.S. Patent. No. 6,500,662, Nielsen et al., J. Virol. 77: 3702-11, 2003, and Truong et al., Virology 325: 308-19, 2004. The sequences of an infectious clone of the North American PRRSV that can be mutagenized to obtain PRRSV viruses for use in vaccines include but are not limited to to the North American strains NVSL 97-7895 (SEQ ID NO: 16) and strain VR-2332 (SEQ ID NO: 17). Descriptions of the infectious clones of the European PRRSV that can be designed to encode the hypoglycosylated variant proteins of GP5 are found in U.S. Pat. 6,268,199. In embodiments wherein the vaccine comprises a live or attenuated PRRSV, the N-linked glycosylation site corresponding to N44 in the reference GP5 protein of SEQ ID NO: 1 (ie, the sequence "NLT" in Figures 5 and 6) is not inactivated because glycosylation at this site is required for the ineffectiveness of PRRSV. In the case of the isolated strain of the European PRRSV, the N-linked glycosylation site corresponding to N44 in the reference GP5 protein of SEQ ID NO: 1 is the NLT sequence that starts in asparagine 46 of the Lelystad strain of PRRSV Representative European (SEQ ID NO: 15, Figure 6). The glycosylation site bound to N N46 of the European PRRSV strains is also required for ineffectiveness and is not inactivated in the embodiments of the invention where a live or attenuated PRRSV vaccine is used. Alternatively, hypoglycosylated variant proteins of GP5 can be introduced into pigs with DNA vaccines. Such DNA vaccines typically comprise a DNA molecule wherein a promoter
Active in mammalian cells is operably linked to said polynucleotide encoding said hypoglycosylated variant of the GP5 polypeptide of PRRSV. Promoters that can be used to drive expression of the hypoglycosylated variant protein of GP5 include, but are not limited to, the CMV (cytomegalovirus) immediate early promoter, RSV (Rous sarcoma virus) long terminal repeat promoter, and SV40 ( Simium Virus 40) promoter of the T antigen. In certain preferred embodiments, this promoter is a CMV promoter. In still other embodiments, the isolated polynucleotide expressing the hypoglycosylated variant protein of GP5 comprises a viral vector different from PRRSV. Viral vectors other than PRRSV include, but are not limited to, vaccine virus vectors, herpes simplex viral vectors, adenovirus vectors, alphavirus vectors, and TGFE vectors. Such vectors are described in various publications such as Patents of E.U.A. Nos. 7,041, 300 (for TGEV vectors) and 6,692,750 (for alphavirus vectors).
Therapeutically acceptable carriers and adjuvants In the practice of the invention, hypoglycosylated variant polypeptides or polynucleotides of GP5 which encode hypoglycosylated variant polypeptides of GP5 can be combined with therapeutically acceptable carriers or excipients. Non-limiting examples of such vehicles include physiological saline or other similar salt solutions, proteins such
as albumin whey proteins, buffers such as carbonate, phosphate, phosphonate, or Tris-based buffers, surfactants such as NP40 or Triton X100, and polyethylene glycol polymers. Any combination of said vehicles can be used in the composition and methods of this invention. A preferred vehicle for the compositions comprising the live or attenuated PRRSV virus is dl-a-tocopherol acetate at a concentration of between 50 to 100 mg / ml. "The use of adjuvants in compositions containing any of the GP5 hypoglycosylated variant polypeptides or polynucleotides encoding GP5 hypoglycosylated variant polypeptides is also contemplated, Such adjuvants are typically either aqueous or oily in nature, adjuvants that can be used. include, but not limited to, aluminum hydroxide, Quil A, a suspension of alumina gel, mineral oils, glycerides, fatty acids, fatty acid byproducts, mycobacteria, and CpG oligodeoxynucleotides, or any combination thereof. Various types of CpG adjuvants that can be used are described in US Patents. Nos. 6,977,245 and 6,406,705. Also contemplated is the use of other adjuvants that enhance cellular immune responses (ie, subpopulation enhancers of T-helper cells (Th.sub.1 and Th.sub.2)). Such adjuvants include but are not limited to interleukin 1 (IL-1), IL-2, IL4, IL-5,
IL6, IL-12, gamma interferon (g-IFN), cell necrosis factor, MDP (muramyl dipeptide), immune stimulating complex (ISCOM), and liposomes. Administration of the composition can be effected by subcutaneous injection, intravenous injection, intradermal injection, parenteral injection, intramuscular injection, needle free injection, electroporation, oral delivery, intranasal delivery, oronasal delivery, or any combination thereof. Needle free injection is typically performed with a device such as an Agro-Jet® injector (Medical International Technologies, Montreal, Canada).
EXAMPLES
EXAMPLE 1
The following example illustrates the construction of various PRRSV polynucleotides encoding various hypoglycosylated polypeptide variants of GPRA of the North American PRRSV, compositions including such polynucleotides that are used to obtain improved immune responses to a PRRSV antigen, and methods for using the polynucleotides and compositions for obtaining an improved immune response in a pig to a PRRSV antigen.
Materials and Methods Cells, medium, and antibodies. MARC-145 cells were propagated in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 100 units of penicillin, 20 units of streptomycin and 20 units of kanamycin per ml of growth medium . These cells were used for RNA electroporation, viral infection, viral growth, and plaque assays. The baby hamster kidney cells (BHK-21) were kept in a Minimum Essential Medium (MEM) with Earl's salt containing 5% FBS and the aforementioned antibiotics. BHK-21 cells were used for transient expression of GP5 followed by either immunofluorescence (IFA) or radiolabeled assays and immunoprecipitation experiments. All cells were maintained at 37 ° C and 5% C02 environment. Rabbit polyclonal antibodies for GP5 and M proteins of PRRSV were kindly provided by Cari A. Gagnon (University of Quebec (University of Quebec), Montreal, Canada). The monoclonal antibody (SDOW17) against the nucleocapsid protein (N) was purchased from the National Veterinary Services Laboratories (NVSL, Ames, IA, USA). Alexa-488 anti-mouse was obtained from Molecular Probes, Inc. (Eugene, Oregon, USA).
Genetic manipulation of plasmids encoding GP5 and PRRSV infectious clone The infectious clone of the full-length PRRSV cDNA (FL12; SEQ ID NO: 16) in pBR322 was digested with restriction enzyme EcoRV and BstZ17 I and the fragment of -4.9 kbp spanning the majority of ORF2, complete ORF3-7, and the UTR of the complete 3 'region of the PRRSV was cloned into pBR322 using the same enzyme sites. This intermediate plasmid served as the model for mutagenesis to introduce mutations in the potential glycosylation sites linked to N (N34, N44, and N51) with GP5 (Figs 2A-2B and Table A). Mutagenesis was performed using overlapping PCR extension with synthetic primers (Table 2) using standard techniques.
TABLE 2 Primers and their sequences used in this study. The underlined codon sequences indicate the mutation site
The PCR product was digested with restriction enzymes BsrGI and BstEII and replaced back into the intermediate plasmid. The clones containing the desired mutations were identified and confirmed by sequencing. The complete transcribed region of GP5 was sequenced to ensure that no additional mutations were present in the clones. The EcoRV-Pac I fragment from the intermediate plasmid containing the mutations in the transcribed region of GP5 was placed back into the full-length cDNA clone using the same enzyme restriction sites. The transcribed region of GP5 in the full-length clones was sequenced again with internal PRSSV-specific primers to confirm the presence of the mutations. The GP5 wt and the individual mutants were cloned into a bicistronic vector where GPS is the first cistron followed by the internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV) and the transcribed M sequences (Fig. 1A). The full-length GP5 was also cloned into a CMV promoter driving vector (pcDNA 3.0 ™, Clontech Laboratories, Inc., Mountain View, CA, USA) for complementary studies. For this purpose, the transcribed region of GP5 was amplified by PCR, cloned and sequenced.
In vitro transcription and electroporation The full-length plasmids were digested with Acll and the linearized DNA was used as a model to generate transcripts from
RNA topped using the mMESSAGE mMACHINE Ultra T7 ™ device in accordance with the manufacturer's recommendations (Ambion, Inc., Austin, Tx, USA) and as described above. The reaction mixture was treated with DNasal to digest the DNA model and extracted with phenol and chloroform and finally precipitated with isopropanol. The integrity of the in vitro transcripts was analyzed by glyoxal agarose gel electrophoresis followed by staining with ethidium bromide. MARC-145 cells were electroporated with approximately 5.0 μg of in vitro transcripts together with 5.0 μg of total RNA isolated from MARC-145 cells. Approximately 2x106 cells in 400 μl of DMEM containing 1.25% DMSO were pulsed once using a Bio-Rad Gene Pulser Xcell ™ (Bio-Rad, Inc., Hercules, CA, USA) at 250V, 950μF in a 4.0 cuvette mm. The cells were diluted in normal growth medium, plated on a 60 mm cell culture plate. A small portion of the electroporated cells was plated in a 24 well plate to examine the expression of the N protein at 48 hours post-electroporation, which could indicate the replication and transcription of the genome. Once the expression of the N protein was confirmed using an indirect immunofluorescence assay (IFA), the supernatant of the volume of the electroporated cells in the 60 mm dish was collected at 48 hours post-electroporation, clarified and passed on MARC-145 cells without alteration. The infected cells were observed for the cytopathic effect (CPE-for its acronym in English) along with the expression of the
N protein using IFA. Supernatants from infected cells that showed both CPE and positive fluorescence were assigned as containing the infectious virus. After confirmation, the virus stock was grown and frozen at -80 ° C in small aliquots for further studies. In all experiments, the FL12 containing the wt PRRSV genome and the FL12 pol containing the defective polymerase PRRSV genome were used as controls.
Metabolic radiolabelling and protein analysis BHK-21 cells in six-well plates were infected with a recombinant vaccinia virus (vTF7-3) at an MOI of 3.0 and subsequently transfected with bicistronic plasmid DNA encoding wt GP5 or various mutants under a T7 RNA polymerase promoter. DNA transfection was performed using Lipofectamine2000 ™ according to the manufacturer's protocol (Life Technologies, USA). At 16 h post-transfection, cells were washed twice with PBS and fasted in methionine / cysteine-free DMEM for 1 hr and radiolabeled with 0.6 ml of methionine / cysteine-free DMEM containing 100 μCi of Mix of Marking of
Expre35S 35S protein (NEN Life Sciences, Boston, MA) per ml of medium for three hrs. Following radiolabelling, cells were washed in cold PBS three times and cell extracts were prepared in 300 μl of radioimmunoprecipitation assay buffer (RIPA) (10 mM Tris-HCl pH 8.0, 150 mM
NaCl, 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate, and inhibitor of
protease 1 x). Clarified cell extracts were incubated overnight at 40 ° C with anti-GP5 protein or anti-rabbit M protein. A slurry of approximately 4.0 mg of protein A cepharose (Pharmacia, Uppsala, Sweden) was added in 100 μl of RIPA buffer and subsequently incubated for 2 hrs. The immunoprecipitated complexes were washed 3 times with 500 μl of RIPA buffer and used for further analysis. For the treatment of endoglycosidase H (Endo H), the immunoprecipitated complexes were resuspended in 20 μl of 1x denaturing buffer (0.5% SDS, 1.0% ß-mercaptoethanol) and heated to boiling for 10 min. The supernatant was collected, was adjusted to 1 x G5 buffer (0.05 M sodium citrate pH 5.5) and incubated for 16 hrs at 37 ° C with 100 units of Endo H (New England Biolabs, Beverly, MA, USA). The undigested control samples were processed similarly but Endo H was not added following digestion with Endo H, the samples were mixed with an equal volume of sample buffer 2xSDS-PAGE, heated to boiling for 5 min and resolved by SDS-12% PAGE under denaturing conditions together with a protein marker (Protein Plus Precision Standard, Bío-Rad, Inc). The gel was fixed with 10% acetic acid for 15 min, washed three times with water, treated with 0.5 M sodium salicylate for 30 min, dried and finally exposed to X-ray film at -70 ° C. For the digestion of the peptide N-Glycosidase F (PNGase F) (New England Biolabs, Inc), the immunoprecipitated complexes were suspended in 1x G7
of buffer (sodium phosphate 0.05 M pH 7.5, 1.0% of NP-40) and the digestion was developed when incubating for 16 hrs at 37 ° C with 2 units of the enzyme. To examine the synthesis of GP5 in the presence of tunicamycin (Sigma, St.Louis, MO), the transfected cells were treated with 2.0 μg of tunicamycin per ml of medium per one hr and the radiolabelling was developed in the presence of the drug for 3 hours. hrs as in the previous. To obtain radiolabeled extracellular virions or GP5 expressed in intracellular viruses, the MARC-145 cells were infected with the wt or mutant PRRSV. At 48 h post-infection, cells were fasted for 1 hr and radiolabelled with 100 μCi of Expre35S 35S Protein Labeling Mix per ml of medium containing 90% methionine / cysteine-free DMEM and 10% Regular DMEM for 24 hrs. Following labeling, the culture supernatant was fasted, clarified from defective cells and extracellular virions were pelleted at 100,000x g for 3 hrs at 4 ° C. Viral pellets were suspended in 200 μl of RIPA buffer, immunoprecipitated with anti-GP5 antibody and proteins were examined with or without Endo H treatment. For the immunoprecipitation of GP5 expressed in intracellular virus, infection was performed as previous and at 24 h post-infection, the cells were fasted for one hr, radiolabelled as above for 2 hrs before the preparation of the cell extracts.
Kinetics of viral growth and plaque assay MARC-145 cells were infected with mutant PRRSV or wt at an MOI of 3.0 PFU per cell and incubated at 370C in an incubator. At several points in the post-infection time, aliquots of the culture supernatants from infected cells were collected and virus titration in the supernatants was determined and expressed by an infectious tissue culture dose of 50 per ml (TCID50 / ml ). The kinetics of viral growth was developed three times. To examine the plaque morphology of the mutant viruses, the plaque assay was developed using MARC-145 cells. The cells were infected with 10-fold serial dilutions of individual viruses for 1 hr at 37 ° C. The infected cell monolayer was washed with PBS and coated with DMEM-5% FBS containing 0.8% marine plankton agarose (FMC Bioproducts, Rockland, ME USA). After 96 hrs, the agarose plugs were removed and the cell monolayer was incubated with staining solution (20% Formaldehyde 9.0% Ethanol, and 0.1% crystal violet) for 30 min at room temperature. The cells were washed gently with water to remove excess dye and air dried to examine and count the plates.
Complement of virus recovery by expressing wt GP5 in the trans region BHK-21 cells were transfected with pCpc-GP5. At 40 h post-transfection, the cells were fasted and
electro pora rum with in vitro capped transcripts derived from the full-length PRRSV cDNA encoding the mutant GP5. Electroporated cells were diluted with fresh medium and plated in six-well plates. The supernatant of the electroporated cells was collected at 48 hrs post-electroporation, centrifuged to remove the defective cells and used to infect MARC-145 unaltered cells. The infected MARC-145 cells were examined at 48 h post-infection for the expression of the N protein by IFA as described above. The number of positive cells was counted to assign the number of pseudo-particles produced in the supernatant. The average number of positive cells was calculated from three independent experiments and was presented as the number of pseudo-particles produced per microgram of RNA transcribed in vitro transfected within the cells.
Neutralization serum (SN) assays The titration of PRRSV neutralizing antibodies in a serum sample was determined using the fluorescence focus neutralization assay previously described. Serial dilutions of the test serum were incubated for 60 min at 37 ° C in the presence of 200 TCID50 of the test virus, which consisted of either FL12 (PRRSV wt) or any of the viruses encoding the mutant GP5, FL-N34A, FL-N51A, and FL-N34 / 51A in Dulbecco's modified Eagle's medium containing 5% serum fetal bovine The mixtures were added to microtiter plates of
96 wells containing confluent MARC-145 cells which had been planted 48 h before. After incubation for 24 hrs at 37 ° C in a humidified atmosphere containing 5% CO 2, the cells were fixed for 10 min with a 50% methanol and 50% acetone solution. After extensive washing with PBS, the expression of the PRRSV N protein was detected with the monoclonal antibody SDOW17 using a dilution of 1: 500, followed by incubation with goat anti-mouse IgG conjugated with FITC (Sigma, St.Louis , MO, USA) at a dilution of 1: 100. Neutralization titers were expressed as the reciprocal of the highest dilution that inhibited 90% of the foci present in the control wells.
Experimental inoculation of pigs with the mutant GP5 and PRRSV wt High titration stocks (obtained through 3 passages in the MARC-145 cells) of the mutant GP5 viruses (FL-N34A, FL-N51A, and FL -N34 / 51A) and FL12 (PRRSV wt) to infect young pigs. The twenty-one-day, recently weaned pigs were purchased from a specific herd free of pathogens with a certified record of absence of PRRSV infection. All animals were negative for anti-PRRSV antibodies according to the evaluation by ELISA (Iddex Labs, Portland, ME). Three pigs were infected per group with either FL12 PRRSV wt or FL-N34A, FL-N51A, and FL-N34 / 51A mutants. In all cases, the inoculation consisted of 105 TICD50 diluted in 2 ml and administered
intramuscularly in the neck. The rectal temperatures of the inoculated animals were monitored for 15 days post-inoculation (Pl). Viremia was measured by regular isolation in MARC 145 cells on days 4, 7 and 14 Pl. Serum samples were taken weekly for a total period of 49 days Pl. Serum samples were used to detect homologous cross-neutralization titers and heterologous for each of the mutant PRRSV and wt.
Results Expression and characterization of PRRSV GP5 GP5 of PRRSV strain 97-7895 has three putative glycosylation sites (N34, N44, and N51). To examine the glycosylation pattern of GP5, a bicistronic vector was first generated in which the transcribed regions of the GP5 and M proteins flanking the IRES from the EMCV were placed under the control of the T7 RNA polymerase promoter (Figure 1A). ). The rationale for the construction of the bicistronic vector is that it is known (for the LDV and the EAV) or it postulates (for the PRRSV) that the GP5 and M proteins interact with each other and that these interactions may be important for protein folding, glycosylation , intracellular transport, and / or other biological activity of GP5. Transient expression of GP5 and M by transfection of the bicistronic plasmid followed by radiolabelling and immunoprecipitation with the anti-GP5 antibody revealed two major protein species. The species
of protein that migrated with a -25.5 kDa mass is a fully glycosylated form of GP5 (Figure 1B, band 2). Because each N-linked glycosylation adds -2.5 kDa of molecular mass to a protein, this indicates that all three potential glycosylation sites are possibly used for glycosylation of GPS. The protein species of 19.0 kDa is the viral M protein because it was also immunoprecipitated with the anti-M antibody (lane 7). The results indicate that the GP5 and M proteins interact with each other in cells that express both proteins. With the treatment of Endo H, the enzyme that removes chains of superior oligosaccharides of the mannose type, the size of GP5 was reduced to -18 kDa, while the size of the M protein remained unchanged (band 3). The treatment of GP5 with PNGase F (band 4), an enzyme that removes all types of sugars from the main structure of the protein or synthesis of GP5 in the presence of tunicamycin (band 5) resulted in a protein that migrated with an electrophoretic mobility slightly greater than the protein with the Endo-H treatment. This is expected, because the treatment with tunicamycin or digestion with PNGase F could generate non-glycosylated proteins where Endo-H treatment could result in proteins that retain the N-acetylglucosamine residues in each of the N-linked glycosylation sites It should be noted that a prominent protein species of -30 kDa molecular mass was immunoprecipitated with the anti-M antibody. The identity of this protein is not known but it could be a cellular protein that interacts with the M protein.
The results of previous studies suggest that the non-glycosylated and glycosylated forms of GP5 have apparent molecular sizes of 18.0 kDa and 25.5 kDa, respectively. It seems that these three potential glycosylation sites are used to generate the fully glycosylated form of GP5. The glycan portions added to these sites are superior to the mannose type because they are sensitive to Endo H digestion. Additionally, the results indicate that both non-glycosylated and fully glycosylated forms of GP5- appear to interact with the M protein.
Analysis of N-linked glycosylation sites used for glycosylation of GP5 To more accurately determine if all or some of the potential N-linked glycosylation sites in GP5 are used for the addition of sugar portions, a series of mutants in the bicistronic plasmid where the three potential glycosylation sites N34, N44, and N51 (Figure 2A) were altered in the alanines either individually or in various combinations, as shown in Table A.
TABLE A
In cells with transfected plasmid, the proteins were radiolabelled and immunoprecipitated with anti-GP5 antibody. Immune complexes were either left untreated or treated with Endo H and examined by SDS-PAGE. As can be seen from the data presented in Figure 2B, mutant GP5 proteins carrying single mutations (N34A, N44A or N51A) migrated as protein species of approximately 23.0 kDa (bands 4, 6 and 8, arrowhead) . With the Endo H treatment, these proteins migrated as protein species of -18.0 kDa (bands 5, 7, and 9, respectively) similar to the wt GP5 after the Endo H treatment (band 3). The minor differences in the electrophoretic mobility of the proteins is more likely to reflect the fact that the wt protein can retain the three N-acetylglucosamine residues following the Endo H treatment compared to the simple mutants that could contain two such residues. The protein species produced by double mutation (N34 / 44A, N44 / 51A and N34 / 51A) that migrated close to the -20.5 kDa protein (bands 10, 12 and
14) and with the treatment of Endo H, the size of the proteins was reduced to 18.0 kDa (bands 11, 13, and 15). The triple mutant (N34 / 44 / 51A) generated a protein that migrated as an 18.0 kDa protein (band 16) and was resistant to digestion of Endo H (band 17). Then, from the previous mutational studies, it is clear that the three potential glycosylation sites are used for glycosylation to generate the fully mature PRRSV GP5. It appears that the three glycosylation sites are modified by higher glycan portions of the mannose type.
Recovery of PRRS virus with the mutant GP5 To evaluate the importance of N-linked glycosylation in the generation of infectious PRRSV, the transcribed regions of the mutant GP5 proteins were inserted into full-length cDNA clones. The in vivo capped transcripts produced from the clones were electroporated into MARC-145 cells and the generation of the infectious PRRSV was examined. The results showed that the infectious virus recovered easily from the electroporated cells with full-length transcripts containing the mutations in N34, N51, and N34 / 51. However, under similar conditions of virus recovery, repeated attempts to recover other mutant viruses were not successful. Although the growth kinetics of the recovered viruses was similar to that of the wt virus, the total yield of the FL-N34A and FL-N51A viruses containing the
Mutations in N34 and N51 were approximately one log lower than in MARC-145 cells while FL-N34 / 51A with double mutation (N34 / 51A) was almost 1.5 log lower than PRRSV wt (Fig. 3A). The amplification of RT-PCR of RNA from infected cells followed by nucleotide sequencing indicated that these viruses are stable, contained the desired mutations and no other mutations were detected in the complete GP5 region (data not shown). The viral ~ plate assay was developed in MARC-145 cells to monitor the plaque phenotype of the mutant viruses. The plates generated by the PRRSV wt were clear and distinct while the mutant viruses produced plaques that had different phenotypes. FL-N34A, FL-N51A, and FL-N34 / 51A viruses generated plaques that were less distinct and many of the cells within the plaque appeared normal (Figure 3B, open arrow). Additionally, FL-N51A, and FL-N34 / 51A produced some plaques in which viruses failed to clear the cell monolayer (Figure 3B, solid arrow). These data indicate that the recovered mutant viruses are in fact less cytopathic compared to the PRRSV wt. Because infectious PRRSV could not be recovered with mutant models FL-N44A, FL-N31 / 44A, FL-N44 / 51A, and FL-N31 / 44 / 51A, it is possible that mutations in the transcribed region of GP5 may have affected some other functions of RNA models, such as the packaging of genomic RNA into particles. To direct this, it was examined if cells expressing wt GP5 in the trans region could support the packaging of mutant RNA models that are otherwise defective in the generation of infectious PRRSV. BHK-21 cells transfected with pCpc-GP5 were electroporated with in vitro transcripts and 48 hrs post-electroporation, culture supernatants were collected and used to infect unaltered MARC-145 cells to determine the pseudo-particle production of the PRRSV. If the pseudo-particles are generated, then one would expect to observe the N-protein expression in these infected MARC-145 cells. N expression is only possible when the unaltered MARC-145 cells receive the full length encapsidated mutant RNA genome which initiates replication following the entry of the pseudo-particles into the cells. Of all the mutants that could not be recovered previously, it was able to recover the pseudo-particles that contained two genomes of full-length mutants (FL-N44A and FL-N34 / 44A) (Figure 3B). Each green fluorescent cell in the mutant culture infected with virus represents an infectious pseudo-particle. Because these particles contain only the functional wt GP5 in the envelope but contain the sequences transcribed for the non-functional mutant GP5 in the genome, they can not produce infectious particles to spread to the surrounding cells. Multiple attempts to recover the pseudo-infectious particles were not successful with the other mutant models (FL-N44 / 51A, and FL-N31 / 44 / 51A).
A quantitative estimate of the number of infectious pseudo-particles produced from these experiments suggests that approximately 1000 particles are produced per microgram of electroporated mutant RNA within the cells (Figure 3C). This is approximately 100 times less than that obtained with the wt GP5 that encodes the RNA. The production of said low levels of infectious pseudo-particles could be due to the fact that only about 5-10% of the cells expressing the wt GP5 received the full-length transcripts observed by the expression of the N protein in these cells. It is also possible that the low expression levels of the wt GP5 in the transfected cells may have contributed to the low levels of production of these pseudo-virions.
Examination of GP5 incorporated in mutant viruses and those expressed in infected cells To determine the nature of the GP5 protein incorporated in infectious virions produced from transfected cells, radiolabeled PRRSV were generated from cells infected with wt and mutant viruses. The extracellular virions in the culture supernatant were pelleted by ultracentrifugation and the GP5 present in those virions was examined by immunoprecipitation using an anti-GP5 antibody and subsequent electrophoretic analysis. The results show that the wt GP5 incorporated in the virions migrated as a broadly diffuse band of protein species
of -25-27 kDa (Figure 4A, band 1), which is partially resistant to digestion of Endo H (band 2). The mutant GP5 (N34A and N51A) incorporated into the virions were sensitive to Endo H. Based on the size of the products generated following the digestion of Endo H, it seems that only a portion of glycan in these mutants of a single site is sensitive while another is resistant. In contrast, the double-mutant GP5 (N43 / 51A) was resistant to Endo H. Moreover, digestion of Endo H from the GP5 mutant virus also produced very small amounts of the main structure of the GP5 protein, indicating that these viruses incorporate GP5 proteins that contain glycan portions resistant to Endo-H as well as sensitive to Endo-H. Because in the cells transfected with the bicistronic vector, GP wt proteins as well as mutants were completely sensitive to Endo H (Figures 1A and 1B and 2A-2B and Table A), the observation that GP5 in PRRS virions contains highly resistant forms to Endo H was surprising. To examine whether the Endo H resistant forms of the protein are also synthesized in infected cells, MARC-145 cells infected with PRRSV were radiolabeled. wt or mutant. The GP proteins were immunoprecipitated with anti-GP5 antibodies and analyzed by electrophoresis with or without Endo H digestion. The results of said experiment are shown in Figure 4B. The majority of GP5 wt contains glycans resistant to Endo H at all three sites (lanes 2 and 3), while two simple mutants contain glycans resistant to Endo H only in one place (bands 4-7). Some of the glycan moieties in the double mutants are resistant while others are sensitive to Endo H (lanes 8 and 9). Although the resistance pattern to Endo H is similar to that observed with GP5 associated with the virion, it is different from that observed in cells expressing both GP5 and M proteins (Figures 1A and 1B and 2A-2B and Table A). These results indicate that other viral proteins may play a role in a further modification of glycans in GP5.
Influence of GP5 hypozygosity on the ability of PRRSV to be neutralized by specific antibodies It is known that the level of glycosylation of viral glycoproteins that are involved in the interaction with viral receptors affects the ability of the virions to react with the virus neutralizing antibodies . To assess whether this phenomenon occurs in the case of PRRSV, the PRRSV GPS mutants were compared with altered glycosylation patterns (FL-N34A, FL-N51A and FL-N34 / 51A) with the PRRSV wt (FL12) in their ability to be neutralized by convalescent antiserum. For this, convalescent antiserum (47 days p.i.) was used from 4 animals that were infected with the PRRSV wt. Similar doses (2,000 TCID50) of infectious mutants of PRRSV GP5 (FL-N34A, FL-N51A and FL-N34 / 51A) as well as PRRSV wt (FL12) derived from the infectious clone were used as a virus. neutralization of serum following the standard trial protocol of the applicants and the set of 4 anti-PRRSV sera wt (FL12)
It was used as a reference. Table 3 shows the different terminal points obtained from the serum neutralization titration. Normally, a convalescent serum sample of PRRSV wt collected at 47-54 days p.i. contains moderate levels of wt PRRSV neutralizing activity (1: 8 to 1: 32, Tables 3 and 4), reflecting the relatively weak and delayed nature of the neutralizing antibody response that is typical of PRRSV wt infections. However, the use of PRRSV hypoglycosylated mutants (lacking one or two portions of glycan-in the GP5 ectodomain) a test virus in the SN assay appears to have a significantly increased endpoint of the reference serum, with an increase in the assessment of the terminal point varying from six to twenty-two times (Table 3). This observation clearly suggests that the removal of one, and particularly two, of the glycan moieties increases the accessibility of the neutralizing epitope to specific antibodies. These results seem to indicate the presence of significant amounts of the PRRSV-neutralizing antibodies in the convalescent serum infected with the wt PRRSV that could otherwise not be detected due to the typical use of wt PRRSV containing the fully glycosylated GP5 in the SN assays.
TABLE 3 Effect of alteration of the glycosylation pattern of GP5 of PRRSV on the ability of the infectious virion to react with neutralizing antibodies. The numbers in Table 3 correspond to the terminal point of inverse dilution that shows the neutralization (terminal point SN)
Influence of GP5 hypoglycosylation on the ability of PRRSV to induce neutralizing antibodies in vivo A remarkable effect that has been reported where the removal of carbohydrates from a viral envelope glycoprotein leads to the production of high titers of neutralizing antibodies against them. mutant viruses when this mutant is used for in vivo inoculation of the host; in some cases also inducing higher titers of antibodies to the wt virus than the wt virus itself. Groups of pigs were infected with identical doses of either FL12 PRRSV wt or each of the mutants with altered glycosylation patterns. Interestingly, the
Clinical / philological evaluations of infection by evaluation of rectal temperature and evaluation of viremia on days 4, 7 and 10 p.i. indicated a similar pattern of infection in all groups as previously described for FL12 with no evidence of attenuation or virulence exacerbation for any of the mutants (data not shown). However, sequential serum sampling of these animals over a period of 48 days indicated pronounced differences between the wt PRRSV and the mutants in their induction kinetics of a PRRSV-neutralizing antibody response (Tables 4A and 4B). The mutants developed an early homologous neutralizing antibody response more robust than that developed by the PRRSV wt, to the point where, in the case of the mutants, the response of the neutralizing antibodies of the PRRSV characteristically slow and scarce seems to have been corrected (Table 4B). The kinetics of appearance of neutralizing homologous mutant antibodies (Table 4B) indicates a seroconversion of neutralizing antibodies plus recular consistent with that described for other viral infections such as influenza virus or Pseudorabias but not for PRRSV. Of greater importance is the fact that infection with glycosylation mutants of GP5 induced a neutralizing antibody response specific to PRRSV wt significantly higher than the response with PRRSV itself wt. The mutant viruses FL-N34 and FL-N51A induced levels five times higher (p <0.05) of neutralizing antibodies against PRRSV wt that the PRRSV itself wt
whereas the FL-N34 / 51 mutant induced a six-fold higher (p <0.01) titration of the PRRSV neutralizing antibodies wt than the PRRSV wt itself (Table 4 B).
TABLE 4A Effects of the alteration of the glycosylation pattern of GP5 of PRRSV on the ability of PRRSV strains to induce neutralizing antibodies for PRRSV wt (4A) or for the homologous, infectious strain (4B) (*) The numbers in Table 4A and B correspond to the geometric mean of the terminal point of SN for the group (n = 3)
TABLE 4B
Analysis In the present study, the influence of the glycosylation of GP5 of PRRSV on the recovery of infectious virus, its function on the ability of mutant viruses to be neutralized by antibodies, and on the introduction of neutralizing antibodies in vivo was examined. . It has been found that the three potential glycosylation sites (N34, N44, and N51) in GP5 are used for the addition of glycan portions. The results reveal that the addition of glycan in the N44 site is more critical for the recovery of the infectious virus. Moreover, the results show that PRRSV containing the hypoglycosylated forms of GP5 are exquisitely sensitive to neutralization by antibodies and that mutant viruses induce significantly higher levels of neutralizing antibodies not only to homologous mutant viruses but also to PRRSV wt . Confirmation that the three potential N-linked glycosylation sites are used for the addition of glycan in GP5 was provided by the use of mutants with single or multiple site alterations (Figure 2A-2B and Table A). Biochemical studies showed that the GP5 protein of the PRRSV when coexpressed with the M protein in the transfected cells, contained superior glycans of the tricky type sensitive to Endo H. the observation that the majority of the GP5 incorporated in virions is resistant to Endo H (Figure 4A) while the GP5 expressed in the presence of the M protein in the transfected cells is completely
sensitive to Endo H, it is intriguing. It is possible that when GP5 is expressed in the presence of the M protein in transfected cells, it accumulates mostly in the ER or in the cis-Golgi region and therefore remains sensitive to Endo H. However, in cells infected by PRRSV, GP5 can interact with additional viral proteins and the transport of GP5 beyond ER or cis-golgi is facilitated through the formation of complexes with the other viral proteins. Consistent with this interpretation, it has been observed that in cells infected with the wt or mutant PRRSV, the GP5 protein is also resistant to Endo H. It is suggested that GP5, which is synthesized in the ER in infected cells, is transported to the middle and / or trans-Golgi regions where most of the molecules of GP5 acquire resistance to Endo H before being incorporated into the PRRSV virions. Numerous studies with arterivirus including PRRSV suggest that the protein GP5 and M form heterodimers, which can play a key role in viral infectivity. In the EAV and LDV, the direct interaction of the GP5 protein and M through the formation of bisulfide bridges has been demonstrated. Such interactions may occur prior to further processing of the N-linked oligosaccharide side chains, presumably before GP5 is transported out of the ER or the cis-Golgi compartment. It is interesting to note that the Endo H resistance pattern of GP5 incorporated in wt virions and mutants is different. While most of the GP5 molecules in the wt PRRSV were resistant to
Endo H, most of the GP5 molecules in the single site mutant virions (FL-N34A and FL-N51A) were sensitive to Endo H (Figure 4A). Moreover, of the two glycan portions in these mutants, only one was sensitive while the other was resistant. The virion (FL-N34 / 51A) double mutant also incorporated the GP5 that contained glycans, some of which were also sensitive to Endo H. These data are consistent with the interpretation that PRRSV virions wt as well as mutants incorporated a mixed population of GP5 molecules that contained different portions of glycan at different sites. Previous studies demonstrated the incorporation of differentially glycosylated forms of GP5 in PRRSV virions wt further reinforces the applicants' interpretation. From the sensitivity pattern to Endo H of the GP5 incorporated in the virions, it can be speculated that the N44 site can contain the glycans resistant to Endo H, although some molecules of the GP5 with glycans sensitive to Endo H at this site were incorporated. in the virions. Whether this unusual pattern of glycans at various sites in GP5 and the incorporation of various forms of GP5 into virions has any relevance to the pattern of immune response observed in animals infected with PRRSV remains to be investigated. In a recent study, it was shown that of the two glycosylation sites linked to N (N46 and N53) in GP5 of PRRSV Lelystad, the glycosylation of residue N46 was strongly required for the production of virus particles. The infectious virus yield was reduced by
approximately 100 times with the mutation in N46. The results suggest that the addition of glycan in N44 (for North American PRRSV) is absolutely essential to recover infectious PRRSV. It is then possible that strains isolated from European and North American PRRSV may somehow differ in their requirements for N-linked glycosylation for the production of the infectious virus. In this regard, it should be noted that the Lelystad virus contained only two glycosylation sites linked to ~ N ~ while the isolated North American strain that was used in this study contained three such sites. GP5 is the most important glycoprotein of PRRSV involved in the generation of PRRSV neutralizing antibodies and protective immunity. The results reveal that the absence of glycans in residues 34 and 51 in the ectodomain of GP5, while generating viable mutants of PRRSV, increased both the sensitivity of these voters to neutralization by antibodies as well as the immunogenicity of the nearby epitope. of neutralization. The immediate effect of the absence of glycans in the GP5 of the PRRSV mutants has been the increased sensitivity of the viruses to the neutralization by convalescent serum of pigs infected with PRRSV wt (Table 3). Studies with HIV-1 and SIV have shown that the acquisition or removal of glycans in the variable circuits of gp160 modify their sensitivity to neutralization. Therefore, it has been postulated that glycans exert at least two types of essential functions during the biosynthesis of viral enveloping glycoproteins. In a
In this case, the lack of glycans implies defects in the glycoprotein and then, in the total viability of the viral strain. It is postulated that the glycans in N44 of GP5 of the PRRSV serve a similar function. In the second case, the glycans potentially serve to protect the viral proteins against neutralization by the antibodies. For PRRSV GP5, the glycans in N34 and N51 may have a similar function. In the case of HIV, "the protection of glycan" is postulated as a principal mechanism to explain the evasion of the neutralizing immune response, thus ensuring the persistence of HIV in vivo. This invites us to outline some parallel comparisons with the PRRSV. The infection with PRRSV, which is known to be persistent for many months in individual animals, presents an unusual behavior in terms of inducing a virus-neutralizing immune response. It has been well established that animals infected with PRRSV usually take longer than normal to establish a detectable response of neutralizing PRRSV antibodies. Once established, this neutralizing response of PRRSV is weak, and varies significantly from animal to animal. It has been postulated that the delay in neutralizing antibody response is due to the presence of a nearby immunodominant decoy epitope (amino acid positions 27 to 30), which evokes a non-protective, early, robust immune response that masks and / or identifies the response to the neutralizing epitope (amino acid positions 37 to 45) (26, 38). While this is a plausible explanation for the atypical character of the neutralizing antibody response of the
PRRSV, remains to be evaluated. In the applicants' laboratory, it has been consistently proven that the deletion of the decoy epitope is lethal for the recovery of infectious PRRSV (Ansari et al., Unpublished data), thus making it difficult to prove this hypothesis. It is possible that an alternative or complementary mechanism to explain the peculiar nature of the neutralizing response to PRRSV could be contemplated by the proposed "glycan protection" phenomenon for HIV and SIV. The use of the PRRSV mutant that lacks one or more glycan moieties in the studies provides evidence for the first time of the presence of large amounts of PRRSV neutralizing antibodies in the serum of animals infected with PRRSV wt that were not detected otherwise. to the use of PRRSV wt in SN trials. The neutralizing antibodies of PRRSV, although they are present in the host response, they are not able to react with the infectious virions of PRRSV wt due to blocking or protection of the neutralizing epitope by the glycan portions in GP5. An important precedent for the escape of neutralization by the glycosylation of glycoproteins in the arterivirus has been described for the lactate dehydrogenase (LDV) elevating virus. LDV is highly resistant to neutralization by antibodies due to heavy glycan protection of its main glycoprotein, VP-3, however, certain strains of LDV that exist naturally are highly susceptible to neutralization, due to the loss of two sites of glycosylation in the ectodomain of VP-3.
Interestingly, this phenotype sensitive to neutralization correlates with a high degree of neurotropimy in the host acquired by these readily neutralizable strains of LDV. This increase in neuropathicity probably reflects the ease of interaction of viral glycoproteins with receptors in neural cells, possibly due to the absence of glycan protection. In the young pig model used for inoculation with PRRSV, no pathogenic differences could be detected between any mutant PRRSV and PRRSV wt, although the observation was limited to temperature and viremia measurements. It is possible that under different experimental conditions (ie, in a pregnant sow model), some alterations in the pathogenicity of these mutant PRRSVs may be observed. It is not known if the finding of hypoglycosylated strains of PRRSV that exist naturally is a common occurrence, although previous reports have suggested its presence. A notable observation in the experiments has been that GP5 mutants, when infected with pigs in vivo, can improve PRRSV wt in their ability to mount a wrestling PRRSV neutralizing response in late phases of infection (Table 4A). In a parallel scenario, it has been observed not only higher neutralization valuations against homologous PRRSV mutants but also measurable valuations against PRRSV wt (Table 4A). Additionally, the response that occurred before, with neutralizing evaluations detectable on day 14 p.i., an observation not typically
noted with a wt PRRSV infection (Table 4B). The increased neutralization of PRRSV wt by the serum of the pigs infected with the mutant PRRSV suggests that the glycans were masking the neutralizing epitope (s) that does not induce neutralizing antibodies when the glycans are present. This observation has a great significance in the design of better, more effective PRRSV vaccines, suggesting that the new vaccines, rationally designed, must carry the modifications in the glycosylation pattern of GP5 to increase the production of neutralizing antibodies. Additionally, it will be important to study the effects that this carbohydrate removal of glycoproteins prominently immunologically from PRRSV can have on the increased SN titers not only for the homologous immunizing strain but also for various unrelated PRRSV strains.
EXAMPLE 2 Identification of N-linked glycosylation sites in isolated strains of the North American and European PRRSV
To identify the N-linked glycosylation sites corresponding to asparagine 34 or asparagine 51 in a reference GP5 protein of SEQ ID NO: 1 that can be inactivated and used in the methods of this invention, the GP5 proteins of either strain isolated from the desired North American PRRSV (Figure 5) or an isolated strain of
European PRRSV (Figure 6) are aligned with the reference GP5 protein of SEQ ID NO: 1 (North American strain NVSL 97-7895). In this example, the alignments were created with the MegAlign ™ program from DNASTAR, Inc. (Madison, Wl, USA) using the Jotun-Hein alignment method (Hein, JJ In Methods in Enzymology, Vol. 183: • pp. 626 -645, 1990). The multiple sequence alignment parameters were a separation penalty of 1 1 and a penalty of separation length of 3. For pairwise comparisons, a Ktuple value of 2 was used. In Figure 5, it is clear that the site of N-linked glycosylation corresponding to asparagine 51 of the reference GP5 protein of SEQ ID NO: 1 present in all strains isolated from the North American PRRSV showed and understood the glycosylation site bound to N "NGT". This N-linked glycosylation site can be inactivated by substituting codons encoding amino acid residues such as glutamine or alanine instead of those of asparagine 51 in the corresponding polynucleotide sequence. In these examples, the corresponding amino acid sequence in the hypoglycosylated variant of the GP5 protein of the North American PRRSV may comprise sequences such as "QGT", "AGT", or "XGT", wherein X is an amino acid other than asparagine. These or other hypoglycosylated variants of the strains isolated from the North American PRRSV where the N-linked glycosylation site corresponding to asparagine 51 is inactivated can also be combined with other hypoglycosylated GP5 variants where other N-linked glycosylation sites are inactivated.
It is also clear from Figure 5 that the N-linked glycosylation site corresponding exactly to asparagine 34 of the reference GP5 protein of SEQ ID NO: 1 is present only in certain strains isolated from the North American PRRSV. More specifically, the GP5 proteins of the strains isolated from the North American PRRSV IAF-BAJ (SEQ ID NO: 3), 94-3182 (SEQ ID NO: 7), and 94-287 (SEQ ID NO: 8) contain the site of N-linked glycosylation corresponding exactly to asparagine 34 of the reference GP5 protein of SEQ ID NO: 1 and comprises the glycosylation site linked to N "NSS". This N-linked glycosylation site of SEQ ID NOs: 3, 7 and 8 can be activated by replacing the codons encoding other amino acid residues such as glutamine or alanine for asparagine 34 in the corresponding polynucleotide sequence. In these examples, the corresponding amino acid sequence in the hypoglycosylated variant of the GP5 protein of the North American PRRSV may comprise sequences such as "QSS", "ASS", or "XSS", where X is any amino acid other than asparagine. In other strains isolated from the North American PRRSV lacking the N-linked glycosylation site corresponding exactly to asparagine 34 of the reference GP5 protein of SEQ ID NO: 1, other N-linked glycosylation sites located at residue 30 (Figure 5"YES" in SEQ ID NOs: 2, 3, 4, 6, 7, 8, 9, 11, 13), and residue 33 (Figure 5"NNS" in SEQ ID NOs: 3, 8; SEQ ID NOs: 6, 10, "NDS" in SEQ ID NOs: 11, 13) can also be activated. In other words, the glycosylation sites
linked to N in other isolated North American strains located at amino acid positions corresponding to residues 30 and 33 of the reference GP5 protein of SEQ ID NO: 1 can also be inactivated and used in the methods of this invention. In Figure 6, it is clear that the N-linked glycosylation site corresponding to asparagine 51 of the reference GP5 protein of SEQ ID NO: 1 is also present in a representative European PRRSV isolated strain and comprises the glycosylation site linked to N "NGT". This N-linked glycosylation site can be activated to replace codons encoding other amino acid residues such as glutamine or alanine instead of those of asparagine 51 in the corresponding polynucleotide sequence. In these examples, the corresponding amino acid sequence in the hypoglycosylated variant of the GP5 protein of the European PRRSV may comprise sequences such as "QGT", "AGT", or "XGT", wherein X is any amino acid different from asparagine. These or other hypoglycosylated variants of strains isolated from the European PRRSV where the N-linked glycosylation site corresponding to asparagine 51 is inactivated can also be combined with other hypoglycosylated variants of GP5 where other N-linked glycosylation sites are activated. In view of the above, it will be noted that the numerous advantages of the invention are achieved and fulfilled.
The modalities were selected and described to better explain the principles of the invention and its practical application in order to enable other experts in the art to better use the invention in various modalities and with various modifications that adapt to the particular use contemplated. . As various modifications can be made to the constructions and methods described and illustrated herein without departing from the scope of the invention, it is intended that all matter contained in the above description or shown in the accompanying drawings should be construed as illustrative rather than limiting . Then, the breadth and scope of the present invention should not be limited by any of the exemplary embodiments described above, but should be defined only in accordance with the following claims appended hereto and their equivalents. A variety of patent and non-patent references are described herein, each of which is expressly incorporated herein by reference in its entirety.
Claims (66)
- NOVELTY OF THE INVENTION CLAIMS 1. - The use of a composition comprising a polynucleotide encoding a hypoglycosylated variant of the PRRSV GP5 polypeptide wherein at least one N-linked glycosylation site corresponding to asparagine 34 tasparagine 51 is inactivated in a reference protein GP5 of SEQ ID NO: 1, to prepare a vaccine useful for obtaining an improved immune response in a pig for a Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) antigen.
- 2. The use as claimed in claim 1, wherein said polynucleotide comprises an infectious molecule of PRRSV RNA.
- 3. The use as claimed in claim 1, wherein said polynucleotide comprises a DNA molecule that encodes an infectious molecule of PRRSV RNA.
- 4. The use as claimed in claim 2 or claim 3, wherein said infectious PRRSV RNA molecule is a derivative of the North American PRRSV or a derivative of the European PRRSV.
- 5. The use as claimed in claim 2, wherein said infectious PRRSV RNA molecule is a derivative of the North American PRRSV that encodes a hypoglycosylated variant protein of the PRRSV GP5 polypeptide wherein at least one site is inactivated. of N-linked glycosylation corresponding to asparagine 34 or asparagine 51 in a reference GP5 protein of SEQ ID NO: 1.
- 6. The use as claimed in claim 5, wherein the N-linked glycosylation site corresponding to asparagine 51 is inactivated in SEQ ID NO: 1.
- 7. The use as claimed in claim 5, wherein both of said N-linked glycosylation sites corresponding to asparagine 34 and asparagine 51 are inactivated in a reference GP5 protein-of-SEQ ID NO: 1.
- 8. The use as claimed in claim 7, wherein said N-linked glycosylation sites are inactivated by replacing the codons encoding said asparagine 34 and said asparagine 51 with codons encoding an amino acid other than asparagine.
- 9. The use as claimed in claim 8, wherein said codons encode another amino acid encoding any of a glutamine alanine residue.
- 10. The use as claimed in claim 9, wherein said codons encode an alanine residue.
- 11. The use as claimed in claim 1, wherein said polynucleotide comprises a DNA molecule wherein an active promoter in mammalian cells is operably linked to said polynucleotide encoding a hypoglycosylated GP5 protein. 12. - The use as claimed in claim 11, wherein said promoter is a CMV promoter. 13. The use as claimed in claim 1, wherein said N-linked glycosylation site is inactivated by replacing a codon encoding said asparagine 51 with a codon encoding an amino acid other than asparagine. 14. The use as claimed in claim 13, wherein said codon encoding another amino acid encodes a residue of alanine or one of glutamine. 15. The use as claimed in claim 1, wherein said N-linked glycosylation site corresponding to asparagine 34 is inactivated by replacing a codon encoding said asparagine 34 with a codon encoding an amino acid other than asparagine. 16. The use as claimed in claim 15, wherein said codon encoding another amino acid encodes an alanine or a glutamine residue. 17. The use as claimed in claim 1, wherein said polynucleotide encoding a hypoglycosylated variant protein of the PRRSV GP5 polypeptide wherein both of the N-linked glycosylation sites corresponding to asparagine 34 and asparagine are inactivated. in a reference GP5 protein of SEQ ID NO: 1. 18. The use as claimed in claim 17, wherein both of said N-linked glycosylation sites are inactivated when replacing the codons encoding said asparagine 34 and said asparagine 51 with codons encoding an amino acid other than asparagine. 19. The use as claimed in claim 18, wherein said codons encode another amino acid encoding any one of a glutamine alanine residue. 20. The use as claimed in claim 17, wherein one of said N-linked glycosylation sites is inactivated by replacing a codon encoding said asparagine 34 or said asparagine 51 with a codon encoding an amino acid other than asparagine. 21. The use as claimed in claim 1, wherein said vaccine is formulated to be administrable by subcutaneous injection, intravenous injection, intradermal injection, parenteral injection, intramuscular injection, needle free injection, electroporation, oral delivery, intranasal delivery , oronasal supply, or any combination thereof. 22. The use as claimed in claim 1, wherein said vaccine additionally comprises a therapeutically acceptable vehicle. 23. The use as claimed in claim 22, wherein said carrier is selected from the group consisting of a protein, a buffer, a surfactant, and a propylene glycol polymer, or any combination thereof. 24. - The use as claimed in claim 1, wherein said vaccine additionally comprises an adjuvant. 25. The use as claimed in claim 24, wherein said adjuvant is selected from the group consisting of aluminum hydroxide, Quil A, a suspension of alumina gel, mineral oils, glycerides, fatty acids, fatty acid byproducts , mycobacteria, and CpG oligodeoxynucleotides, or any combination thereof. 26. The use as claimed in claim 1, wherein said polynucleotide comprises a viral vector selected from the group consisting of a vaccine virus vector, a herpes simplex viral vector, an adenovirus vector, an alphavirus vector , and a TGEV vector. 27. The use as claimed in claim 1, wherein said improved immune response of said pig to a PRRSV antigen comprises an increased production of the PRRSV neutralizing antibodies by said pig. 28.- The use as claimed in claim 1, wherein said pig is a sow, a first sow, a boar, or a piglet. 29. The use as claimed in claim 1, wherein said polynucleotide encoding a hypoglycosylated variant of the PRRSV GP5 polypeptide is obtained by direct synthesis, mutagenesis of a nucleotide sequence of the GP5 of the isolated PRRSV strain. North American, mutagenesis of a nucleotide sequence of GP5 consented to the North American PRRSV. 30. - The use as claimed in claim 29, wherein said nucleotide sequence of the GP5 of the isolated strain of the North American PRRSV encodes a peptide selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO : 11, SEQ ID NO: 12, and SEQ ID NO: 13. 31. The use as claimed in claim 29, wherein said nucleotide sequence of the GP5 consented to the isolated strain of the North American PRRSV encodes a consensual GP5 protein that is at least 85% identical to SEQ ID NO: 14 32. The use as claimed in claim 1, wherein said polynucleotide encoding a hypoglycosylated variant of the PRRSV GP5 polypeptide is obtained by direct synthesis, mutagenesis of a nucleotide sequence of the GP5 of the isolated strain of the European PRRSV or mutagenesis of a nucleotide sequence consented to GP5 of the European PRRSV. 33. The use as claimed in claim 32, wherein said nucleotide sequence of the GP5 of the European PRRSV isolated strain encodes a GP5 protein that is at least 85% identical to SEQ ID NO: 15. 34 The use as claimed in claim 33, wherein said nucleotide sequence of the GP5 of the isolated strain of the European PRRSV is SEQ ID NO: 15. 35. - The use of a composition comprising a hypoglycosylated variant of the PRRSV GP5 polypeptide wherein inactivated for said pig, at least one N-linked glycosylation site corresponding to asparagine 34 or asparagine 51 in a reference GP5 protein of SEQ ID NO: 1, to prepare a vaccine useful for obtaining an improved immune response in a pig for a Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) antigen. 36. A composition comprising a polynucleotide encoding a hypoglycosylated variant of the North American PRRSV GP5 polypeptide wherein at least one N-linked glycosylation site corresponding to asparagine 51 is inactivated in a reference GP5 protein of SEQ ID NO: 1, and a therapeutically acceptable vehicle. 37. The composition according to claim 36, further characterized in that said polynucleotide comprises an infectious RNA molecule of the North American PRRSV. 38.- The composition according to claim 36, further characterized in that said polynucleotide comprises a DNA molecule that encodes an infectious RNA molecule of the North American PRRSV. 39.- The composition according to claim 36, further characterized in that said polynucleotide comprises a DNA molecule in which an active promoter in mammalian cells is ligated operatively to said polynucleotide encoding said hypoglycosylated variant of the GP5 polypeptide of the North American PRRSV. 40.- The composition according to claim 39, further characterized in that said promoter is a CMV promoter. 41. The composition according to claim 36, further characterized in that said N-linked glycosylation site is inactivated by replacing a codon encoding said asparagine 51 with a codon encoding an amino acid other than asparagine. 42. The composition according to claim 41, further characterized in that said codon encoding an amino acid other than asparagine encodes an alanine or a glutamine residue. 43.- The composition according to claim 36, further characterized in that said polynucleotide encodes a hypoglycosylated variant protein of the North American PRRSV GP5 polypeptide wherein both of the N-linked glycosylation sites corresponding to asparagine 34 and asparagine 51 are inactivated. in a North American GP5 protein reference of SEQ ID NO: 1. The composition according to claim 43, further characterized in that both of said N-linked glycosylation sites are inactivated by replacing the codons encoding said asparagine 34 and said asparagine 51 with codons encoding an amino acid other than asparagine. 45. - The composition according to claim 44, further characterized in that said codons encode another amino acid encoding any one of alanine residue one of glutamine. 46. The composition according to claim 43, further characterized in that one of said N-linked glycosylation sites is inactivated by replacing a codon encoding said asparagine 34 or said asparagine 51 with a codon encoding an amino acid other than asparagine. 47. The composition according to claim 36, further characterized in that said therapeutically acceptable carrier is selected from the group consisting of a protein, a buffer, a surfactant, and a propylene glycol polymer, or any combination thereof. 48. The composition according to claim 34, further characterized in that said composition additionally comprises at least one adjuvant. 49.- The composition according to claim 48, further characterized in that said adjuvant is selected from the group consisting of aluminum hydroxide, Quil A, a suspension of alumina gel, mineral oils, glycerides, fatty acids, fatty acid byproducts , mycobacteria, and CpG oligodeoxynucleotides, or any combination thereof. 50. - The composition according to claim 48, further characterized in that said composition additionally comprises a second adjuvant that is selected from the group consisting of interleukin-1 (IL-1), IL-2, IL-4, IL-5, IL- 6, IL-12, interferon gamma (g-IFN), cell necrosis factor, MDP (muramyl dipeptide), immune stimulating complex (ISCOM), and liposomes. 51.- The composition according to claim 36, further characterized in that said polynucleotide comprises ~ a viral vector selected from the group consisting of a vector of vaccine virus, a viral vector of herpes simplex, an adenovirus vector, a vector of alphaviruses, and a TGEV vector. 52. A composition comprising a hypoglycosylated variant of the North American PRRSV GP5 polypeptide wherein at least one N-linked glycosylation site corresponding to asparagine 51 is inactivated in a reference GP5 consensus protein of SEQ ID NO: 1 and a therapeutically acceptable vehicle. 53. An isolated polynucleotide encoding a hypoglycosylated variant of the North American PRRSV GP5 polypeptide wherein at least one N-linked glycosylation site corresponding to asparagine 51 is inactivated in a reference GP5 protein of the SEQ ID NO: 1. 54.- The isolated polynucleotide according to claim 53, further characterized by inactivating both sites of N-linked glycosylation corresponding to asparagine 34 and asparagine 51 in SEQ ID NO: 1. 55.- The isolated polynucleotide according to claim 53, further characterized in that said polynucleotide comprises an infectious RNA molecule of the North American PRRSV. 56. The isolated polynucleotide according to claim 53, further characterized in that said polynucleotide comprises a DNA molecule that encodes an infectious RNA molecule of the North American PRRSV. 57. The isolated polynucleotide according to claim 53, further characterized in that said polynucleotide comprises a DNA molecule in which an active promoter in mammalian cells is operatively linked to said polynucleotide encoding said hypoglycosylated variant of the GP5 polypeptide of PRRSV North American. 58.- The isolated polynucleotide according to claim 57, further characterized in that said promoter is a CMV promoter. 59. The isolated polynucleotide according to claim 53, further characterized in that said N-linked glycosylation site corresponding to asparagine 51 is inactivated by replacing a codon encoding said asparagine 51 with a codon encoding an amino acid other than asparagine. 60. - The isolated polynucleotide according to claim 59, further characterized in that said codon encoding an amino acid other than asparagine encodes an alanine or a glutamine residue. 61.- The isolated polynucleotide according to claim 53, further characterized in that an N-linked glycosylation site corresponding to asparagine 34 is inactivated in a reference GP5 protein of SEQ ID NO: 1. 62.- The polynucleotide isolated from according to claim 61, further characterized in that, by replacing a codon encoding said asparagine 34 with a codon encoding an amino acid other than asparagine. 63. The isolated polynucleotide according to claim 61, further characterized in that said codon encoding another amino acid encodes a residue of alanine or one of glutamine. 64.- The isolated polynucleotide according to claim 61, further characterized in that one of said N-linked glycosylation sites is inactivated by replacing a codon encoding said asparagine 34 or said asparagine 51 with a codon encoding an amino acid other than asparagine . 65.- The isolated polynucleotide according to claim 53, further characterized in that said polynucleotide comprises a viral vector selected from the group consisting of a vector of vaccine virus, a viral herpes simplex vector, an adenovirus vector, an alphavirus vector, and a TGEV vector. 66.- An isolated polypeptide that is a hypoglycosylated variant of the North American PRRSV GP5 polypeptide wherein at least one N-linked glycosylation site corresponding to asparagine 51 is inactivated in a reference GP5 protein of SEQ ID NO: 1.
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
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US60/712,357 | 2005-08-30 |
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MX2008003046A true MX2008003046A (en) | 2008-09-26 |
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