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WO1992007940A2 - Genes de virus syncytiaux respiratores chez les bovins - Google Patents

Genes de virus syncytiaux respiratores chez les bovins Download PDF

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WO1992007940A2
WO1992007940A2 PCT/US1991/008177 US9108177W WO9207940A2 WO 1992007940 A2 WO1992007940 A2 WO 1992007940A2 US 9108177 W US9108177 W US 9108177W WO 9207940 A2 WO9207940 A2 WO 9207940A2
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brsv
sequence
protein
leu
ser
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PCT/US1991/008177
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WO1992007940A3 (fr
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Siba Kumar Samal
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Siba Kumar Samal
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18511—Pneumovirus, e.g. human respiratory syncytial virus
    • C12N2760/18522—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • Bovine Respiratory Syncytial Virus is an RNA virus which is recognized as an important cause of lower respiratory disease of cattle in Asia, Europe and the United States. In cattle, BRSV infection is more significantly associated with respiratory disease than any other virus. Furthermore, the highest incidence of severe BRSV disease in cattle is between 2 and 4.5 months of age. About 70 percent of cattle have been infected by BRSV by 9 months of age.
  • BRSV infection The characteristics of BRSV infection include extensive damage to the mucuous membranes, leaving the respiratory tract susceptible to dust, debris and secondary infectious agents. BRSV infection also causes a condition which resembles systemic anaphylaxis or hypersensitivity. Outbreaks of BRSV infection usually last about 10 to 14 days, and are characterized by high mortality.
  • BRSV is not easily grown, is closely associated with the cell membrane and is inherently unstable (Stott and Taylor, Arch. Virolo ⁇ v 84. 1 (1985)). Therefore, purification of the virus by biophysical techniques is extremely difficult.
  • BRSV human respiratory syncytial virus
  • HRSV human respiratory syncytial virus
  • pneumonia virus of mice comprise the genus Pneumovirus which is within the family Paramyxoviridae.
  • Viruses of this family have enveloped pleomorphic virions which contain helical, elongated nucleocapsids.
  • the genome is linear, single-stranded RNA which replicates in the cytoplasm.
  • the BRSV virion appears as round or pleomorphic forms which measure about 80 to 500 mm across, or as filamentous forms up to several um in length.
  • the outer membrane of the virion is studded with projections about 12 mm long, each of which is about 10 mm apart.
  • Purified virions contain a unique species of single-stranded RNA of which at least 93 percent is negative sense. Discrepancies regarding the size and number of polypeptides in the virion are due, in part, to differences between virus strains (Stott and Taylor, supra) .
  • the smaller polypeptide has a molecular weight between about 10,000 and 13,000 daltons, and the. larger polypeptide has a molecular weight between about 19,000 and 25,000 daltons.
  • the large polypeptide is a non-glycosylated protein. Neither of the small polypeptides has any known functions (Stott and Taylor, supra) .
  • M protein A larger protein, the M protein (about 27,000 to 28,000 daltons) is believed to be the membrane protein.
  • the M protein has 256 amino acids, and is relatively basic with two hydrophobic regions in the C-terminal third of the protein.
  • the P protein is a phosphorylated protein with a molecular weight of about 32,000 to 38,000 daltons. This protein is associated with the nucleocapsid.
  • nucleocapsids isolated from purified BRSV contain primarily NP protein in association with RNA.
  • the NP protein has a molecular weight of between about 40,000 and 44,000 daltons, and has 467 amino acids, most of which are basic amino acids.
  • F protein Two glycoproteins which are believed to be located on the surface of the virion are the F protein and G protein.
  • the F protein has a molecular weight of between about 66,000 and 68,000 daltons
  • the G protein has a molecular weight of between about 79,000 and 90,000 daltons. These two proteins have a rod-shaped morphology, suggesting that they may be the studded projections of the virion.
  • the F protein is comprised of two smaller glycoproteins linked by disulfide bonds.
  • One of the smaller glycoprotein has a molecular weight between about 43,000 and 56,000 daltons and the other smaller glycoprotein has a molecular weight between about 19,000 and 22,000 daltons.
  • the F protein has been shown to be the fusion protein by the inhibition of cell fusion by a monoclonal antibody to the F protein.
  • the G protein is believed to be the attachment protein of the virion.
  • Monoclonal antibodies to either the F protein or the G protein neutralize infectivity of the virus.
  • L protein There is little known about the largest polypeptide, the L protein. This protein has a molecular weight of between about 160,000 daltons and 200,000 daltons and is believed to be the RNA polymerase of the virion.
  • BRSV and HRSV differ in plaque reduction tests using bovine sera, cattle have been reported to be equally protected from BRSV infection by either BRSV or HRSV antibodies. Any differences detected by neutralization are believed to reflect changes in the epitopes on either the F protein or G protein (Stott and Taylor, supra) . Furthermore, two monoclonal antibodies to the BRSV fusion protein appear to react complement fixation and immunofluoresence tests, all strains of respiratory syncytial virus cross-react (Stott and Taylor, supra) .
  • the three vaccines which have been tested are: 1) an inactivated antigen combined with adjuvant given intramuscularly; 2) live attenuated viruses given intranasally; and 3) live modified virus given intramuscularl .
  • BRSV and HRSV have traditionally been classified in the same genus, Pneumovirus. as stated above, at least one group of researchers have suggested that these two viruses be classified in separate groups of the genus (Lerch et al., J. Virol. 64, 5559 (1990)).
  • a comparison of the amino acid sequences of the G protein of BRSV with the G protein of either subgroup A or B of HRSV showed only a 29 to 30% amino acid identity.
  • antisera to the BRSV G protein made by using a recombinant vector to immunize animals, recognized the BRSV G protein but not the HRSV G protein, and vice versa (Lerch et al. , supra) .
  • the present invention relates to genes derived from BRSV, vectors produced with the genes and expression systems for the genes.
  • the genes comprise the nucleotide sequences set forth as in the Sequence Listing as SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6; SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14; SEQ ID NO:16, and SEQ ID NO:18.
  • the invention further relates to fragments of these genes.
  • the invention also encompasses diagnostic probes comprising these genes or fragments thereof. All of the genes or their fragments are useful as diagnostic probes.
  • the invention further encompasses purified BRSV proteins or fragments thereof. These purified proteins and fragments can be used to detect BRSV antibodies, and can be used as vaccines.
  • the purified proteins which are useful for the detection of BRSV antibodies and as vaccines are proteins set forth in the Sequence Listing as SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:ll, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, and SEQ ID NO:19.
  • the protein of SEQ ID NO:3 is particularly useful for diagnostic testing for the presence of BRSV antibodies.
  • a further aspect of the invention are antibodies to the gene products or fragments thereof.
  • Fig. 1 shows the genetic map of BRSV strain A2
  • Fig. 2 shows an SDS-PAGE gel in which [ 3 H] glucosamine labeled antigens were immunoprecipitated with BRSV (strain A51908) antiserum; and
  • Fig. 3 shows an autoradiograph of the hybridization of 3 P-labeled BRSV-N gene probe to the RNA of a variety of bovine respiratory viruses.
  • Adjacent A position in a nucleotide sequence immediately 5' or 3' to a defined sequence.
  • Cell Culture A proliferating mass of cells which may be in an undifferentiated or differentiated state.
  • “cell”, “cell line”, and “cell culture” are used interchangeably and all such designations include progeny.
  • progeny includes the primary subject cell and cultures derived therefrom without regard for the number of tranfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny which have the same functionality as sreened for in the originally transformed cell, are included. Where distinct designations are intended, it will be clear from the context.
  • Coding Sequence A deoxyribonucleotide sequence which when transcribed and translated results in the formation of a cellular protein, or a ribonucleotide sequence which when translated results in the formation of a cellular protein.
  • Control Sequences refers to DNA sequences necessary for the expression of an operablylinked coding sequence in a particular host organism.
  • the control sequences which are suitable for procaryotes include a promoter, optionally an operator sequence, a ribosome binding site, and possibly, other as yet poorly understood, sequences.
  • Eucaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
  • Expression System Refers to DNA sequences containing a desired coding sequence and control sequences in operable linkage, so that hosts transformed with these sequences are capable of producing the encoded proteins.
  • the expression system may be included on a vector; however, the relevant DNA may then also be integrated into the host chromosome.
  • Gene A discrete nucleic acid region which is responsible for a discrete cellular product.
  • Operably Linked refers to juxtaposition such that the normal function of the components can be performed.
  • a coding sequence "operably linked" to control equences refers to a configuratin wherein the coding sequence cn be expressed under the control of these sequences.
  • Promoter The 5'- flanking, non-coding sequence adjacent a coding sequence which is involved in the initiation of transcription of the coding sequence.
  • Substantial Sequence Homology Donates nucleotide sequences that are substantially functionally equivalent to one another. Nucleotide differences between such sequences having substantial sequence homology will be de minimus in affecting the function of the gene products or an RNA coded for such a sequence.
  • the present invention relates to the novel genes and gene fragments derived from BRSV.
  • the entire genome for BRSV strain A 51908 is presented in SEQ ID NO:l.
  • the genes and gene fragments thereof are:
  • nucleocapsid protein gene (the N gene) (SEQ ID NO:2) ;
  • the matrix protein gene (the M gene) (SEQ ID NO:4) ;
  • the phosphoprotein gene (the P gene) (SEQ ID NO:6) ;
  • the fusion protein gene (the F gene) (SEQ ID NO:10) ;
  • glycoprotein gene (the G gene) (SEQ ID NO:12) ;
  • genes were derived from two strains of BRSV.
  • the genes designated by SEQ ID NOS. 2, 4, 6, 8, 10, 12 and 14 above were derived from BRSV strain A 51908, which is available from the American Type * Culture Collection (ATCC #VR-794) .
  • the other strain is strain FS-l, which is available from the National Veterinary Services Laboratory (NVSL) U.S.D.A., Ames, Iowa.
  • NVSL National Veterinary Services Laboratory
  • the P gene of BRSV strain FS-l is set forth in the Sequence Listing as SEQ ID No:16, and the F gene of this strain is set forth as SEQ ID NO:18.
  • the present inventors have compared the polypeptides of four strains of BRSV. Based on the size of the F 2 fragment and P protein the BRSV strains examined could be classified into groups.
  • Fig. 2 shows the different migration patterns of the F 2 protein of different strains of BRSV.
  • lanes 3-6 show four different strains of BRSV.
  • the BRSV strains in lanes 3 and 5 (FS-l and VC-464, respectively) have a heavier F 2 protein than the BRSV strains in lanes 4 and 6 (A51908 and Md-x, respectively) .
  • BRSV strains A51908 and Md-x have an F 2 protein of the same size as that of HRSV (lane 2)
  • the F 2 protein of BRSV strains FS-l and VC-464 is smaller than that of HRSV.
  • the difference in the size of the F 2 fragment is significant because the F 2 fragment contains most of the immunogenic and neutralizing epitopes of the most important envelope protein. Therefore, like HRSV, an effective vaccine against BRSV will require incorporation of genes from viruses of both structural groups.
  • BRSV strain 391-2 which was used by Lerch et al.. supra, was isolated from an outbreak of respiratory disease in calves in North Carolina. The immunogenic and pathogenic potential, based on the characterization of the F 2 fragment, of this strain has never been reported.
  • strain A 51908 and strain FS-l of BRSV used by the present inventors, are the reference strains in the United States. Strain A 51908 has been found to cause respiratory disease and induction of neutralizing and protective antibodies in experimental infections (Mohanty et al.. J. Inf. Pis. 134. 4095 (1976)). Strain FS-l is the first isolate of BRSV in the United States, and also has been found to cause respiratory disease in calves (Smith et al.. Arch. Viral. 47. 237 (1975)).
  • BRSV Molecular cloning of BRSV genes from any strain is very difficult due to a number of attributes characteristic of BRSV replication. Some of these attributes are that: (1) BRSV has a very narrow host range; (2) BRSV yield is very low; (3) BRSV fails to depress host cell protein synthesis; and (4) BRSV is very labile.
  • the fresh virus stocks which were used were BRSV strain A 51908 (ATCC # VR-794) and BRSV strain FS-l from NVSL, U.S.D.A., Ames, Iowa. Furthermore, the maintenance medium used was Eagle's minimum essential medium with Earle's salts (MEM) .
  • MEM Eagle's minimum essential medium with Earle's salts
  • RNAs isolated from the BRSV infected cells were used to construct cDNA libraries.
  • identification of BRSV-specific clones is very difficult for the following reasons: (1) a very small proportion of the cDNA clones are virus-specific; (2) cDNA clones of HRSV do not hybridize well with mRNAs of BRSV of Northern blot hybridization; and (3) due to the pleomorphic nature of BRSV, it is difficult to purify this virus and use its genomic RNA as a probe to identify virus-specific clones.
  • BRSV-specifc clones used a number of methods to identify BRSV-specifc clones. Some of these methods are: (1) hybrid arrest and in vitro translation; (2) Northern blot hybridization; and (3) use of cDNA clones of HRSV in different hybridization conditions.
  • viral mRNA was isolated from the BRSV-infected MDBK cells by the guanidine thiocyanate-CsCl procedure (Chirgwin et al. , Biochemistry 18. 5294 (1979)). Poly(A) + -RNA was then purified using oligo (dT) cellulose (Aviv and Leder, Proc. Natl. Acad. Sci. USA 6£, 1408 (1972)), and double-stranded cDNA was synthesized by the RNase H method of Gubler and Hoffman, Gene 25, 264 (1983) .
  • the double-stranded cDNA molecules were then ligated into the EcoRI site of plasmid Bluescript (Stratagene) , and the resulting hybrid plasmids were used to transform E.coli JM109 cells using the method described by Cohen et al. , Proc. Natl. Acad. Sci. USA 69. 2110 (1972) .
  • Bacterial clones containing recombinant DNA were selected on the basis of their color and resistance to ampicillin. N-specific clones were identified by in vitro translation of mRNA obtained by hybrid-selection of randomly selected cDNA clones (Ricciardi et al.. Proc. Natl. Acad. Sci. USA 76. 4927 (1979)).
  • A60 One clone, designated A60, was selected for further analysis as it contained the largest insert among the N-specific clones. The viral specificity of this clone was further confirmed by Northern blot analysis. The cDNA clone hybridized to poly(A) + -RNA from infected cells, but not to poly(a) + -RNA from uninfected cells.
  • the nucleotide sequence of the mRNA of the N protein was obtained by sequencing the clone A60 by the dideoxynucleotide chain termination method Sanger et al.. Proc. Natl. Acad. Sci. USA 74, 5463 (1977) .
  • Clone A60 (1196 bp exclusive of poly(dA)) contained the entire N mRNA sequence lacking only 10 nucleotides of the 5' end of the mRNA.
  • the nucleotide sequence was confirmed using clone A1032 (1099 bp exclusive of poly(dA)), which expanded from nucleotide 97 to the poly(A) tail of the N mRNA.
  • nucleotide sequence of the N mRNA SEQ ID NO:2
  • deduced amino acid sequence of the N protein SEQ ID NO:3
  • the nucleotide sequence of the 5'-end untranslated region was obtained by direct sequencing of the N mRNA using a primer complementary to nucleotides 41 to 63.
  • the BRSV N mRNA contains 1196 nucleotides, excluding the poly(A) tail.
  • the mRNA has a single long open reading frame, extending from nucleotides 16 to 1188.
  • the BRSV N mRNA encodes a polypeptide of 391 amino acids with a calculated molecular weight of 42.6 kD. This value is consistent with the apparent molecular weight of 43 kD determined earlier in SDS-PAGE (Cash et al.. Virology 82. 369 (1977) and Lerch et al.. J. Virol. 63. 833 (1989)).
  • the untranslated region at the 3'-end of the BRSV N gene also shares high homology with the conserved gene end sequence present in all HRSV genes.
  • the consensus HRSV gene start sequence and gene end sequence have also been observed in other BRSV mRNAs. Thus, the presence of these consensus sequences at the start and end of each gene is believed to be a general feature of the respiratory syncytial viruses.
  • the homology at the 5'-untranslated region (exclusive of the consensus sequence) between the bovine and the human strains is nearly as high as homology between the two strains in the coding region.
  • the homology of the 5'-noncoding region is significantly higher than the homology of the coding regions.
  • the predicted amino acid sequence of the BRSV N protein was compared to that of the N protein of HRSV.
  • the N protein of BRSV is identical to that of A2 and 19537 strains of HRSV at 93% of amino acid positions. Most of the amino acid changes correspond to amino acid substitutions.
  • the apparent structure of the N protein does not seem to be affected by the amino acid changes observed. Thus, it appears that the pneumovirus N proteins are highly conserved.
  • the BRSV N gene (SEQ ID NO:2) is the preferred gene for use as a diagnostic gene probe for the detection of BRSV infection.
  • the N gene is transcribed in the largest quantity.
  • the N gene probe is the most sensitive.
  • the probe made from the N gene does not hybridize with cognate genes of other bovine respiratory viruses, but did hybridize well with RNAs extracted from different strains of BRSV.
  • Fig. 3 shows the specificity of the BRSV N gene probe for BRSV strains as opposed to other bovine respiratory viruses. There is clear hybridization of the 32 P-labeled BRSV N gene probe to the RNA of RSV strains A51908, AMES (i.e. FS-l), VC-464 and GRSV, whereas there is no hybridization with the RNA of three other bovine respiratory viruses.
  • the M gene and the P gene are also particularly useful as probes for BRSV RNA. These two genes also exhibit significant homology in various strains of BRSV. The use of nucleotide sequences as probes is fully explained in Keller and Manak, DNA Probes. Stockton Press (1989) .
  • the product of the BRSV N gene (the BRSV N protein) is also very useful as an antigen for the detection of antibodies against BRSV in serum samples.
  • BRSV antibodies are preferably detected by N gene product antigens, because the N gene product (the nucleocapsid protein) is the first protein to appear after BRSV infection (Westenbrink et al. , J. Gen. Viral 70. 591 (1989)) and is produced in the largest quantity of all the BRSV proteins produced by virus-infected cells.
  • the BRSV N protein, as well as the other BRSV proteins can be produced through recombinant means or by polymerase chain reaction (PCR) , as explained further below.
  • the BRSV proteins can also be used to produce BRSV protein antibodies.
  • a rabbit is immunized with either a naturally occurring BRSV protein or a recombinant BRSV protein.
  • a monoclonal anti-BRSV antibody is produced using conventional techniques (eth. Enzymol. , Vol. 121, Langone, J.J. and Van Vinakis, H. , Ed., Academic Press, Orlando (1986) and Roitt, I., in Essential Immunology. 5th Ed. Blackwell Scientific Publications, Boston, pp. 145-175 (1984)).
  • the anti-BRSV antibody generated is then labeled, e.g., radioactively, fluorescently or with an enzyme such as alkaline phosphatase.
  • the BRSV N gene is also useful for vaccine production because: 1) the N protein is the most abundant protein in BRSV-infected cells; 2) calves naturally infected with BRSV have high titer antibodies to N protein; and 3) N protein is necessary for cytotoxic T cell activity.
  • the production of a vaccine from the BRSV N gene or any of the other BRSV genes requires the use of the genes or fragments thereof to manufacture recombinant proteins.
  • the gene (or gene fragment) for the desired protein is operably linked to control sequences to form an expression vector. The expression vector is then used to transform a suitable host, and the transformed host, under suitable conditions, produces a recombinant form of the desired protein.
  • Recombinant forms of any of the identified BRSV proteins or fragments thereof may be produced in this manner.
  • recombinant forms of the BRSV strain A51908 nucleocapsid protein (SEQ ID NO:3), matrix protein (SEQ ID NO:5), phosphoprotein (SEQ ID NO:7), small hydrophobic protein (SEQ ID NO:9), fusion protein (SEQ ID NO:11), glycoprotein (SEQ ID NO:13) and M2 protein (SEQ ID NO:15) are produced in this manner.
  • recombinant forms of the BRSV proteins or fragments thereof of strain FS-l can also be produced in the manner set forth above.
  • the proteins or fragments thereof of strain FS-l which have been identified are the P protein (SEQ ID No:17) and the F protein (SEQ ID NO:19).
  • each of the steps for production of a recombinant protein can be done in a variety of ways.
  • the desired coding sequences can be obtained by preparing suitable cDNA from cellular messenger and manipulating the cDNA to obtain the complete sequence.
  • genomic fragments may be obtained and used directly in appropriate hosts.
  • the constructions for expression vectors operable in a variety of hosts are made using appropriate replicons and control sequences, as set forth below. Suitable restriction sites can, if not normally available, be added to the ends of the coding sequence so as to provide an excisable gene to insert into these vectors.
  • control sequences, expression vectors, and transformation methods are dependent on the type of host cell used to express the gene.
  • procaryotic, yeast, or mammalian cells are presently useful as hosts.
  • Procaryotic hosts are in general the most efficient and convenient for the production of recombinant proteins.
  • eucaryotic cells, and, in particular, mammalian cells are sometimes used for their processing capacity.
  • the preferred vectors for the BRSV genes are ba ⁇ ulovirus and IBR herpes virus.
  • suitable host cells are insect cells such as Drosophila cells, Trichoplusia ni cells (cell line TN-368) and SF-9 cells, grown maintained under conventional conditions.
  • the preferred host cells are the SF-9 cells. The culturing, maintenance and growth of insect cell lines by Agathos et al. , in Annals of the NY Acad of Sciences 589. 372 (1990) .
  • IBR herpes virus is used as the vector, the host for producing recombinant BRSV protein is a calf.
  • the procaryotes most frequently used are represented by various strains of E. coli.
  • other microbial strains may also be used, such as bacilli, for example Bacillus subtilis, various species of Pseudomonas, or other bacerial strains.
  • plasmid or bacteriophage vectors which contain replication sites and control sequences derived from a species compatible with the host are used.
  • a wide variety of vectors for many procaryotes are known (Sa brook et al. , (1989) , Molecular Cloning: A Laboratory Manual. 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) .
  • procaryotic control sequences which are defined herein to include promoters for transcription initiation, optionally with an operator, along with ribosome binding site sequences, include such commonly used promoters as the beta-lactamase (penicillinase) and lactose (lac) promoter systems, the tryptophan (trp) promoter system and the lambda derived PL promoter and N-gene ribosome binding site, which has been made useful as a portable control cassette (U.S. Patent No. 4,711,845) .
  • any available promoter system compatible with procaryotes can be used (Sambrook et al. , supra) .
  • yeast In addition to bacteria, eucaryotic microbes, such yeast, may also be used as hosts. Laboratory strains of Saccharomyces cerevisiae. Baker's yeast, are most used, although a number of other strains are commonly available. Vectors employing the 2 micron origin of replication and, other plasmid vectors suitable for yeast expression are known (Sambrook et al. , supra) . Control sequences for yeast vectors include promoters for the synthesis of glycolytic enzymes.
  • promoters known in the art include the promoter for 3-phosphoglycerate kinase, and those for other glycolytic enzymes, such as glyceraldehyde-3-phosphase dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, hosphoglycose isomerase, and glucokinase.
  • glycolytic enzymes such as glyceraldehyde-3-phosphase dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate
  • promoters which have the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and enzymes responsible for maltose and galactose utilization. (See Sambrook et al. , supra) .
  • terminator sequences are desirable at the 3' end of coding sequences. Such terminators are found in the 3' untranslated region following the coding sequences in yeast-derived genes. Many of the vectors illustrated contain control sequences derived from the enolase gene containing plasmid peno46 or the LEU2 gene obtained from YEpl3, however, any vector containing a yeast compatible promoter, origin or replication and other control sequences is suitable (Sambrook et al. , supra) .
  • Expression vectors for such cells ordinarily include promoters and control sequences compatible with mammalian cells such as, for example, the commonly used early and later promoters from Simian Virus 40 (SV 40) , or other viral promoters such as those derived from polyoma, adenovirus 2, bovine papilloma virus, or avian sarcoma viruses, or immunoglobulin promoters and heat shock promoters (Sambrook est gl . , supra, . General aspects of mammalian cell host system transformations have been described by Axel (U.S. Patent No. 4,399,216).
  • Enhanccer regions are important in optimizing expressin; these are, generally, sequences found upstream of the promoter region. Origins of replication may be obtained, if needed, from viral sources. However, integration into the chromosome is a common mechanism for DNA replication in eucaryotes. Plant cells are also now available as hosts, and control sequences compatible with plant cells such as the nopaline synthase promoter and polyadenylation signal sequences are available fMeth. Enzy ol.. Vol. 118, Academic Press, Orlando (1979)) .
  • transformation is done using standard techniques appropriate to such cells.
  • Such techniques include, but are not .limited to, calcium treatment employing calcium chloride for procaryotes or other cells which contain substantial cell wall barriers; infection with Agrobacterium tumefaciens for certain plant cells; calcium phosphate precipitation method for mammalian cells without cell walls; and, microprojectile bombardment for many cells including plant cells.
  • vaccines may be made by using the protein directly or by attaching a carrier to the recombinant BRSV proteins at the appropriate terminal amino acid.
  • a carrier include tetanus toxoid linked to the appropriate amino acid through tyrosine, Lipid A , hepatitis B antigen and/or a microorganism to which the protein may be linked.
  • the preferred carrier is tetanus toxoid linked to the appropriate terminal amino acid through tyrosine.
  • a conventional initial dose of such a vaccine would be 50 ⁇ g of vaccine in 0.1 ml of Freund's complete adjuvant. Conventionally, two boosts of 50 ⁇ g of vaccine in 0.1 ml of Freund's incomplete adjuvant are given at two week intervals after the initial dose.
  • the envelope proteins are also protective antigens, and the genes of these envelope proteins are also necessary for a recombinant vaccine.
  • These envelope proteins are the F protein and the G protein.
  • the M protein and the SH protein have properties which suggest that they may also be necessary for an effective vaccine.
  • the SH protein was previously thought to be a non-structural protein, the present inventors have shown that the SH protein is a third glycosylated envelope protein, in addition to the F and G proteins.
  • the nucleotide sequence of the SH gene of BRSV ID SEQ. NO:8 is significantly different from the nucleotide sequence of the SH gene of HRSV .
  • cDNA clones were constructed from intracellular poly (A + )-RNA isolated from BRSV A51908-infected cells. Recombinant DNA clones containing the M and SH genes were identified by in vitro translation of mRNA obtained by hybrid selection of randomly selected cDNA clones (Ricciardi et al. , supra) . The nucleotide sequence of the polytranscript mRNA coding for the M and SH proteins was derived from two independent clones, A564 and A22, by the dideoxynucleotide chain termination method (Sanger et al. , supra) .
  • the nucleotide sequence coding for the small hydrophobic (SH) protein is 466 nucleotides long (nucleotides 3045 to 3511) (SEQ ID NO:8).
  • the homology of the coding region, at the nucleotide level, is 45-50% depending on the HRSV strain (Table 1) .
  • the gene-start signal was identified as nucleotides 2902 to 2910 by comparison with the HRSV SH mRNA (Collins and Wertz, Virology 141. 283 (1985) and Collins et al. , J. Virol. 71 1571 (1990)).
  • the 5' untranslated region excluding the gene-start signal, is the same length as its counterpart in HRSV and shares 56% sequence identity with HRSV A2 strain but only 41% with HRSV 18537 strain.
  • the 3' untranslated region is 132 nucleotides (compared to 99 in HRSV) with a sequence homology of 50-58%.
  • the predicted SH proteins from BRSV (A51908 strain) (SEQ ID NO:9) and HRSV (A2 and 18537 strains) can be compared.
  • the predicted BRSV SH protein is 155 amino acids long. It contains a 8-amino acid extension at the carboxyl-end, relative to the HRSV SH protein.
  • BRSV SH protein has a central hydrophobic core (amino acids 14 to 41) flanked by two lysine residues (at positions 13 and 43) , which are conserved in the HRSV SH proteins (A2 and 18537 strains) .
  • This hydrophobic core contains a potential membrane-spanning region (amino acids 20 to 40) similar to the one predicted for the HRSV SH proteins (strains Al and 18537) .
  • the overall homology between the BRSV and HRSV SH proteins is surprisingly low (less than 60%) (Table 1) .
  • the amino acid identities are located mainly in the amino-end region (amino acids 1 to 23) (>65%) .
  • the central hydrophobic core (amino acids 24 to 41) has no more than 34% homology
  • the carboxyl-terminal region (amino acids 42 to 65) is highly divergent (>30% homology) (Table 1) .
  • the deduced M protein contains 316 amino acids and has a molecular weight of 28,713 daltons.
  • the BRSV M protein (SEQ ID NO:5) shares an 89% homology with the HRSV M protein , with most of the differences being due to amino acid substitutions.
  • the M protein of BRSV is moderately basic. Computer analysis predicts a single hydrophobic region (residues 188 to 204) that could act as a transmembrane domain in the BRSV M protein. The same region was predicted for the HRSV M protein (Satake and Venkateson, J. Virol. 50. 92(1984)). Comparison of HRSV M gene and BRSV M gene (SEQ ID NO:4) reveals a homology of 80% between coding regions.
  • the gene-start signal (Collins et al. , Proc- Natl. Acad. Sci. USA 82 . 4594(1988)) for the BRSV M gene (nucleotides 1 to 10) contains a single nucleotide difference at position 5 compared with the HRSV M gene-start signal, excluding the 5'-terminal nucleotide.
  • the gene-end consensus signal was identified as the sequence from nucleotide .876 to nucleotide 888.
  • the untranslated region at the 3' end is 8 nucleotides shorter in the BRSV mRNA and, as seen in other RSV genes, has a lower homology (51%) with the HRSV counterpart region (Table 1) than the coding region. As in HRSV, there is no untranslated region, other the gene-start sequence, at the 5'-end of the M mRNA of BRSV. TABLE 1
  • Intergenic region 24 % The intergenic region between M and SH genes of BRSV is 25 nucleotides long (vs 9 nucleotides in HRSV) and shares only 24% homology with the corresponding region in HRSV, suggesting that this region acts as a mere bridge between genes.
  • the polycistronic mRNA studied contains two additional open reading frames.
  • One open reading frame from nucleotides 111 to 266 encodes a protein of 52 amino acids and overlaps with the M gene.
  • a second open reading frame has also been reported for HRSV, encoding a protein of 75 amino acids, overlapping with the M gene (Satake and Venkateson, supra) .
  • HRSV high-density polycistronic mRNA was found from nucleotides 1271 to 1423 which encodes a protein of 51 amino acids. No similar protein has been described for HRSV. Since these second open reading frames are not conserved, either at the sequence level or at the relative position in the genome, they probably do not play any role in the virus replication.
  • BRSV Other strains of BRSV can be grown and their genomes cloned in accordance with the description herein.
  • the DNA sequences disclosed herein can be used as probes or to prepare degenerative primers for PCR to isolate the specific individual genes which can be cloned and utilized as described above for these additional strains of BRSV.
  • Madin-Darby bovine kidney (MDBK) cells were grow in Eagle's minimum essential medium with Earle's salts (MEM) containing 6% bovine fetal serum (BFS) .
  • MDBK Madin-Darby bovine kidney
  • BFS bovine fetal serum
  • the BRSV strain A51908 was then prepared in MDBK cell cultures propagated in MEM containing 3% BFS.
  • the viral mRNA was isolated from the BRSV-infected MDBK cells by the guanidinium thiocyanate-CsCl procedure (Chirgwin et al. , supra) , followed by two cycles of oligo (DT)-cellulose column chromotography.
  • Double-stranded cDNA was synthesized by the RNase H method of Gubler and Hoffman, supra.
  • the double-stranded cDNA molecules were ligated into the EcoRI site of plasmid Bluescript (Stratagene) .
  • the resulting hybrid plasmids were used to transform E. coli JM109 cells using the method described by Cohen et al. , supra.
  • Bacterial . clones containing recombinant DNA were selected on the basis of their color and resistance to ampicillin.
  • a cDNA clone was identified to contain sequences of the major nucleocapsid (N) gene of BRSV. This clone was used to identify other N gene clones by dot blot hydridization. One clone, designated A60, was selected for further analysis as it contained the largest insert among the N-specific clones.
  • the nucleotide sequence of the mRNA of the N protein was obtained by sequencing the clone A60 by the dideoxynucleotide chain termination method.
  • Clone A60 (1196 bp exclusive of poly(dA)) contained the entire N mRNA sequence lacking only 10 nucleotides of the 5' end of the mRNA. The nucleotide sequence was confirmed using clone A 1032 (1099 bp exclusive of poly (dA) ) , which expanded from nucleotide 97 to the poly (A) tail of the N mRNA.
  • the other BRSV genes of strain A51908 (the matrix protein gene, the phosphoprotein gene, the small hydrophobic protein gene, the fusion protein gene, the glycoprotein gene and the M2 protein gene) were isolated and sequenced using the same techniques. Furthermore, the phosphoprotein gene and the fusion protein gene of BRSV strain FS-l were also isolated and sequenced in the same manner. The sequences of the BRSV genes are set forth in the Sequence Listing as follows:
  • SEQ ID NO:2 nucleocapsid protein (N) gene
  • PCR polymerase chain reaction
  • each primer 10 copies of the BRSV genome, 200 ⁇ M of each dNTP, 2 mM MgCl 2 , 10 mM Tris-HCl (pH 8.3), and 50 mM KC1 are placed in a Perkin-Elmer Cetus Instruments Thermal Cycler. Thirty cycles of 96°C for 15 seconds, 50°C for 30 seconds and 75°C for 30 seconds are run. The BRSV N gene is then recovered using conventional techniques.
  • the other BRSV genes can also be produced using the above procedure.
  • the BRSV N gene isolated in Example 1 or produced in Example 2 is inserted into baculovirus (Autoqrapha California Nuclear Polyhedrosis Vrisu (AcNPV) (Voil et al. , J. Invertebrate Pathol. 22., 231 (1971)) using conventional techniques to form an expression vector.
  • the expression vector is then used to infect Spodoptera frugiperda (SF-9) cells.
  • the SF-9 cells are maintained in a serum-free/protein-free medium developed by Maiorella et al. , Bio/Technology .6, 1406 (1988).
  • the serum-free/ protein-free medium is composed of IPL/41 medium (JR Scientific, Woodland, CA) supplemented with tryptose phosphate broth (Oxoid USA, Columbia, MD) , fetal bovine serum (Gibco, Grand Island, NY) , and pluronic polyol F68 (BASF Wyandotte, Porsippamy, NJ) .
  • the SF-9 cells After infection with the baculovirus expression vector, the SF-9 cells are grown in a medium composed of IPL/41 medium supplemented with cod liver oil polyunsaturated fatty acid methyl esters, cholesterol, alpha-tocopherol acetate, Tween 80 and diluted Yestolate (Difco) .
  • the cells are grown in spinner flasks, stirred at 75-100 rpm, at 27°C with an air atmosphere.
  • BRSV N protein titre peaks as cell lysis begins. SDS-PAGE analysis is then used to identify and isolate the recombinant BRSV N protein.
  • the other BRSV genes from Example 1 may be used in the same procedure described above to isolate and identify their respective recombinant proteins.
  • ADDRESSEE Venable, Baetjer, Howard & Civiletti
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI- SENSE NO
  • ORGANISM Bovine respiratory syncytial virus
  • CAACTGTTAT CAACCAGCAA ATATACTATT CAACGTAGTA CAGGTGACAA CATTGATATA 120
  • CTGCAAATCA AGTTCATATC AGAAAGCCTT TGGTAAGCTT CAAAGAAGAA CTGCCATCAA 1500
  • GCATCCAATC AAGCACAGCA CACACCGGAC ACTCCTTGAA TCCACCAGCT GGTTGAACTT 3120
  • GCAACCCCCC CGAAAACCAC CAAGACCACA ACAACTCCCA AACACTCCCT CATGTGCCCT 4080
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • ORGANISM Bovine respiratory syncytial virus
  • GGC AAT ATA GAA ATA GAG TCA AGG AAG TCT TAC AAA AAG ATG CTA AAA 435 Gly Asn He Glu He Glu Ser Arg Lys Ser Tyr Lys Lys Met Leu Lys 125 130 135 140
  • GGT ATG ATA GTG CTA TGT GTT GCT GCT TTG GTT ATA ACA AAA TTA GCA 531 Gly Met He Val Leu Cys Val Ala Ala Leu Val He Thr Lys Leu Ala 160 165 170
  • GTA CTA AGG AAT GAA ATG AAA CAA TAC AAA GGA CTT ATC CCG AAA GAT 627 Val Leu Arg Asn Glu Met Lys Gin Tyr Lys Gly Leu He Pro Lys Asp 190 195 200
  • GCT GCC AAA GCA TAT GCG GAA CAA TTA AAA GAG AAT GGG GTC ATC AAT 1107 Ala Ala Lys Ala Tyr Ala Glu Gin Leu Lys Glu Asn Gly Val He Asn 350 355 360
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • ORGANISM Bovine respiratory syncytial virus
  • GTA ACT GAC AAT AAA GGG GCA TTC AAG TAC ATT AAA CCA CAA AGT CAA 672 Val Thr Asp Asn Lys Gly Ala Phe Lys Tyr He Lys Pro Gin Ser Gin 210 215 220
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • ORGANISM Bovine respiratory syncytial virus
  • MOLECULE TYPE protein
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • ORGANISM Bovine resoiratory syncytial virus
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • ORGANISM Bovine respiratory syncytial virus
  • GTA CTA CAC TTG GAG GGA GAG GTG AAC AAA ATT AAA AAT GCA CTG CTA 529 Val Leu His Leu Glu Gly Glu Val Asn Lys He Lys Asn Ala Leu Leu 160 165 170
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • ORGANISM Bovine respiratory syncytial virus
  • CAC AGA ACC AGC CCT GAA GCC AAA CTG CAA ACC AAA AAA AAC ACG GCA 723 His Arg Thr Ser Pro Glu Ala Lys Leu Gin Thr Lys Lys Asn Thr Ala 225 230 235
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • ORGANISM Bovine respiratory syncytial virus
  • AAA AAG ACC ATC AAG AAC ACA ATA GAT ATT CAC AAC GAA ATA AAT GGT 528 Lys Lys Thr He Lys Asn Thr He Asp He His Asn Glu He Asn Gly 160 165 170
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • ORGANISM Bovine respiratory syncytial virus
  • ORGANISM Bovine respiratory syncytial virus
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • ORGANISM Bovine respiratory syncytial virus
  • GAGCTTCTAC CTAAAGTTAA CAATCATGAT TGTAGGATAT CCAACATAGG AACTGTGATA 660
  • GAATTCCAAC AAAAAAACAA TAGATTGTTA GAAATTGCTA GGGAATTTAG TGTAAATGCT 720
  • GGTATTACCA CACCCCTCAG TACATACATG TTGACCAATA GTGAATTACT ATCACTAATT 780
  • ORGANISM Bovine respiratory syncytial virus
  • STRAIN FS-l

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Abstract

L'invention se rapporte à des gènes dérivés du virus syncytial respiratoire des bovins (BRSV). L'invention se rapporte également à des vecteurs produits avec les gènes, à des systèmes d'expression des gènes, ainsi qu'à des sondes de diagnostic comportant les gènes, à des protéines recombinées et à des vaccins. La protéine recombinée de nucléo-encapsidation est particulièrement efficace dans des contrôles diagnostiques de détection précoce d'une infection par BRSV.
PCT/US1991/008177 1990-11-05 1991-11-04 Genes de virus syncytiaux respiratores chez les bovins WO1992007940A2 (fr)

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WO1996040945A3 (fr) * 1995-06-07 1997-01-23 Connaught Lab Vaccins a acides nucleiques du virus respiratoire syncytial
WO1997012032A1 (fr) 1995-09-27 1997-04-03 The Government Of The United States Of America, As Represented By The Department Of Health And Human Services Production de virus syncytial respiratoire infectieux a partir de sequences de nucleotides clones
US5716821A (en) * 1994-09-30 1998-02-10 Uab Research Foundation Prevention and treatment of respiratory tract disease
WO1998004710A3 (fr) * 1996-07-26 1998-04-09 Akzo Nobel Nv Vaccin recombine vivant contre l'herpes virus bovin/le virus respiratoire syncytial bovin
US5789229A (en) * 1994-09-30 1998-08-04 Uab Research Foundation Stranded RNA virus particles
WO1999024564A1 (fr) * 1997-11-10 1999-05-20 University Of Maryland PRODUCTION DE NOUVEAUX VIRUS RESPIRATOIRES SYNCYTIAUX BOVINS A PARTIR DE L'ADNc
US6017897A (en) * 1995-06-07 2000-01-25 Pasteur Merieux Connaught Canada Nucleic acid respiratory syncytial virus vaccines
US6060280A (en) * 1990-07-24 2000-05-09 The Uab Research Foundation Nucleotide sequences encoding bovine respiratory syncytial virus immunogenic proteins
US6083925A (en) * 1995-06-07 2000-07-04 Connaught Laboratories Limited Nucleic acid respiratory syncytial virus vaccines
WO2000068392A1 (fr) * 1999-05-11 2000-11-16 The Board Of Trustees Of The University Of Illinois Antigenes d'origine vegetale contre le virus respiratoire syncytial
US6730305B1 (en) 2000-05-08 2004-05-04 The Uab Research Foundation Nucleotide sequences encoding bovine respiratory syncytial virus immunogenic proteins
WO2004073737A1 (fr) * 2003-02-19 2004-09-02 Merial Limited Vaccination ou immunisation utilisant un schema posologique de double administration d'amorçage et de rappel contre le vrs, le virus-herpes bovin 1, le virus bvd, le virus de parainfluenza bovin de type 3
US6908618B2 (en) 1997-11-10 2005-06-21 University Of Maryland Production of novel bovine respiratory syncytial viruses from cDNAs

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Dissertation Abstract Internatonal B, volume 51, no. 7, January 1991, University North Carolina, US; R.A. Lerch et al.: "Molecular analysis of the genes and gene products of bovine respiratory syncytial virus", page 3254, order no. DA 9034789, see the whole abstract *
J. Gen. Virol., volume 70, no. 3, March 1989, Soc. Gen. Microbiol. (GB) F. Westenbrink et al.: "Analysis of the antibody response to bovine respiratory syncytial virus proteins in calves", pages 591-601, see page 593, line 8 - page 598, line 17 (cited in the application) *
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Journal of Virology, volume 64, no. 11, November 1990, Am. Soc. Microbiol., (Baltimore, US) R.A. Lerch et al.: "Nucleotide sequence analysis and expression from recombinant vectors demonstrate that the attachment protein of bovine respiratory syncytial virus is distinct from that of human respiratory syncytial virus", pages 5559-5569, see page 5560, left-hand column, line 58 (L documents: cited to support the non-unity) *
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US6060280A (en) * 1990-07-24 2000-05-09 The Uab Research Foundation Nucleotide sequences encoding bovine respiratory syncytial virus immunogenic proteins
WO1995003070A1 (fr) * 1993-07-22 1995-02-02 Syntro Corporation Suipoxvirus recombine
US5789229A (en) * 1994-09-30 1998-08-04 Uab Research Foundation Stranded RNA virus particles
US5716821A (en) * 1994-09-30 1998-02-10 Uab Research Foundation Prevention and treatment of respiratory tract disease
US6083925A (en) * 1995-06-07 2000-07-04 Connaught Laboratories Limited Nucleic acid respiratory syncytial virus vaccines
US6486135B1 (en) 1995-06-07 2002-11-26 Aventis Pasteur Limited Nucleic acid respiratory syncytial virus vaccines
US5843913A (en) * 1995-06-07 1998-12-01 Connaught Laboratories Limited Nucleic acid respiratory syncytial virus vaccines
US5880104A (en) * 1995-06-07 1999-03-09 Connaught Laboratories Limited Nucleic acid respiratory syncytial virus vaccines
US6677127B1 (en) 1995-06-07 2004-01-13 Aventis Pasteur Limited Nucleic acid respiratory syncytial virus vaccines
US6017897A (en) * 1995-06-07 2000-01-25 Pasteur Merieux Connaught Canada Nucleic acid respiratory syncytial virus vaccines
WO1996040945A3 (fr) * 1995-06-07 1997-01-23 Connaught Lab Vaccins a acides nucleiques du virus respiratoire syncytial
WO1997012032A1 (fr) 1995-09-27 1997-04-03 The Government Of The United States Of America, As Represented By The Department Of Health And Human Services Production de virus syncytial respiratoire infectieux a partir de sequences de nucleotides clones
US6264957B1 (en) 1995-09-27 2001-07-24 The United States Of America As Represented By The Department Of Health And Human Services Product of infectious respiratory syncytial virus from cloned nucleotide sequences
US6790449B2 (en) 1995-09-27 2004-09-14 The United States Of America As Represented By The Department Of Health And Human Services Methods for producing self-replicating infectious RSV particles comprising recombinant RSV genomes or antigenomes and the N, P, L, and M2 proteins
KR100905760B1 (ko) * 1995-09-27 2009-10-01 더 가번먼트 오브 더 유나이티드 스테이츠 오브 아메리카, 에즈 레프리젠티드 바이 더 디파트먼트 오브 헬쓰 앤드 휴먼 서비시즈 클론된 뉴클레오타이드 서열로부터 감염성 호흡기세포 융합 바이러스의 생산방법
WO1998004710A3 (fr) * 1996-07-26 1998-04-09 Akzo Nobel Nv Vaccin recombine vivant contre l'herpes virus bovin/le virus respiratoire syncytial bovin
WO1999024564A1 (fr) * 1997-11-10 1999-05-20 University Of Maryland PRODUCTION DE NOUVEAUX VIRUS RESPIRATOIRES SYNCYTIAUX BOVINS A PARTIR DE L'ADNc
US6908618B2 (en) 1997-11-10 2005-06-21 University Of Maryland Production of novel bovine respiratory syncytial viruses from cDNAs
WO2000068392A1 (fr) * 1999-05-11 2000-11-16 The Board Of Trustees Of The University Of Illinois Antigenes d'origine vegetale contre le virus respiratoire syncytial
US8591915B2 (en) 1999-05-11 2013-11-26 Dennis E. Buetow Plant-derived vaccines against respiratory syncytial virus
US6730305B1 (en) 2000-05-08 2004-05-04 The Uab Research Foundation Nucleotide sequences encoding bovine respiratory syncytial virus immunogenic proteins
WO2004073737A1 (fr) * 2003-02-19 2004-09-02 Merial Limited Vaccination ou immunisation utilisant un schema posologique de double administration d'amorçage et de rappel contre le vrs, le virus-herpes bovin 1, le virus bvd, le virus de parainfluenza bovin de type 3

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