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WO1999061637A1 - Sequences genetiques du virus rubeolique associees a l'attenuation - Google Patents

Sequences genetiques du virus rubeolique associees a l'attenuation Download PDF

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WO1999061637A1
WO1999061637A1 PCT/CA1999/000479 CA9900479W WO9961637A1 WO 1999061637 A1 WO1999061637 A1 WO 1999061637A1 CA 9900479 W CA9900479 W CA 9900479W WO 9961637 A1 WO9961637 A1 WO 9961637A1
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cendehill
rna
gcc
rubella
cgc
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PCT/CA1999/000479
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English (en)
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Janet Chantler
Karen Lund
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The University Of British Columbia
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Priority to AU39237/99A priority Critical patent/AU3923799A/en
Priority to CA002298070A priority patent/CA2298070A1/fr
Publication of WO1999061637A1 publication Critical patent/WO1999061637A1/fr

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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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • 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
    • A61K39/12—Viral antigens
    • A61K39/20—Rubella virus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5254—Virus avirulent or attenuated
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/36011—Togaviridae
    • C12N2770/36211—Rubivirus, e.g. rubella virus
    • C12N2770/36222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/36011—Togaviridae
    • C12N2770/36211—Rubivirus, e.g. rubella virus
    • C12N2770/36234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/36011—Togaviridae
    • C12N2770/36211—Rubivirus, e.g. rubella virus
    • C12N2770/36261—Methods of inactivation or attenuation

Definitions

  • Rubella virus is the causative agent of German measles, a viral infection associated with a mild fever and rash.
  • the most serious complications of rubella occur during pregnancy due to transplacental passage of the virus to the fetus resulting in the widespread manifestations of congenital rubella. These include fetal loss, or multisystem defects in the newborn such as cataracts, deafness, cardiac abnormalities and microcephaly.
  • Vaccination reduced the incidence of congenital rubella but was found to be associated with a number of sequelae, particularly in women over 25 years of age. Symptoms included arthritis, neurological manifestations and chronic fatigue. The most notable complication of rubella immunisation was arthritis which has also frequently been documented as a consequence of natural rubella. The joint symptoms induced can be severe in the acute stage but usually resolve without causing permanent joint damage. Occasionally, however, chronic or recurrent arthritis develops which can persist for many months or years in certain individuals (Ford et al . , 1988)
  • the Cendehill strain (Peetermans & Huygelen, 1967) was developed in Belgium and was the predominant strain used in vaccine production in Europe until 1989.
  • the Cendehill strain is reported to be associated with a decreased incidence of complications in the adult female population in a comparative study of five vaccines. Best et al . (1974) reported that acute arthritis occurred in only 3% of individuals immunised with Cendehill vaccine but in 17% of those receiving RA27/3. Moreover the symptoms with RA27/3 were also more prolonged.
  • the disadvantage of the Cendehill vaccine was that the mean titre of HAI antibody induced in vaccine recipients was lower than that obtained with the RA27/3 strain indicating that Cendehill is less immunogenic .
  • Rubella virus is a small (60-70 nm) enveloped togavirus, the sole member of the genus Rubivirus . It has a single-stranded RNA genome approximately lOkb in size. The genomic RNA is positive-stranded which means that it can act as mRNA within the infected cell. The sequence of the entire genome has been determined for two wild-type strains Therien and M33 (Dominguez et al . , 1990; Gillam et al. , 1993, Genbank No. X72393), and the RA27/3 vaccine strain (Pugachev et al . , 1997) .
  • the genome contains two large open-reading frames (ORF's) which code for the structural proteins (3' proximal 3189 nucleotides) and non-structural proteins (5' proximal 6345 nucleotides).
  • ORF's open-reading frames
  • the current understanding is that the open-reading frames for the structural and the non-structural proteins are separated by a region of about 123 nucleotides.
  • the infected cell contains two virus-induced positive-strand RNA species, the genomic RNA (40s,• lOkb) and a sub-genomic mRNA (26s; 3kb) which encodes the major ORF for the structural proteins.
  • the ORF for structural proteins is translated into a llOkd polyprotein and is subsequently cleaved by cellular signal peptidase into the three structural viral proteins, El, E2 , and C.
  • the order of structural genes was originally determined by synchronised translation as being NH 2 -C-E2-El-COOH, which was confirmed by sequence analysis of cDNA clones of the subgenomic mRNA (Clarke et al .
  • the non-structural (NS) genes are translated from the full-length genomic RNA as a >200kD polyprotein which is subsequently cleaved into two non-structural proteins, pl50 and p90. These comprise the enzymes required for viral replication in the cell. Protein pl50, nearest the 5' terminus, is 1300 amino-acids in length and encodes the putative methyltransferase function and the viral protease. Protein p90 is 905 amino-acids long and has regions of homology with global helicase and replicase domains.
  • nucleic acids comprising one or more sequences of nucleotides corresponding to all or part of the genome of the Cendehill strain of rubella virus.
  • Nucleic acids of this invention may encode an infectious virus of the Cendehill strain or one having an attenuated phenotype equivalent to Cendehill strain.
  • DNA of this invention may be in a plasmid or viral vector which enables replication and/or transcription of the Cendehill cDNA and is referred to herein as a Cendehill infectious clone.
  • the infectious clone may be used to produce a DNA vaccine for rubella virus .
  • This invention also provides a nucleic acid (DNA or RNA) comprising a sequence of nucleotides that includes a first portion corresponding to one or more of the non-translated regions, pl50, p90, C, E2 and El gene regions of Cendehill strain and a second portion that is derived from another rubella virus strain such that the product encodes a novel infectious chimeric rubella virus strain.
  • DNA of this invention may be in a plasmid or viral vector forming an infectious clone.
  • This invention also provides a chimeric Cendehill/RA27/3 clone whose genome includes a first portion corresponding to the Cendehill 5' non-translated RNA, Cendehill pl50 and p90 and wherein a second portion corresponds to the structural gene region and the 3 ' non-translated region of RA27/3 strain.
  • This clone can be used to produce a chimeric virus that expresses the structural proteins of RA27/3 but has the genetic structure at the 5'end and in the non-structural genes of Cendehill strain that determine the non-arthrotropic nature of this strain.
  • This invention also provides RNA encoding the entire genome of Cendehill or the Cendehill/RA27/3 chimera or a fragment thereof, by transcribing the aforementioned DNA.
  • This invention also provides rubella virus produced by transcribing the DNA, transfecting cells with the RNA so derived, and recovering virus from cells so transfected.
  • This invention also provides a nucleic acid encoding one or more Cendehill strain rubella virus proteins selected from the group consisting of: pl50, p90, C, El and E2, or wherein the nucleic acid corresponds to a non-translated region of the Cendehill genome.
  • the nucleic acid may be DNA or RNA and may be incorporated into a plasmid or viral vector for expression of protein.
  • This invention also provides a method of producing Cendehill viral protein comprising the steps of expressing a DNA sequence encoding a protein corresponding to Cendehill protein pl50, p90, C, E2 or El in a cell by means of a suitable expression vector and recovering the protein so expressed.
  • the protein may be a Cendehill protein having a sequence corresponding to a portion of the cDNA sequence in Appendix 1 or the protein may be altered by modification of the Cendehill cDNA, as described herein.
  • This invention also provides a method of producing a recombinant DNA encoding a mutated or chimeric rubella virus exhibiting the lack of arthrotropicity of the Cendehill strain but with additional advantageous properties that include, but are not restricted to, increased immunogenicity or stability of another rubella strain.
  • This method comprises steps whereby;
  • nucleotides in Cendehill cDNA encoding viral structural proteins are altered such that the protein so encoded increases the immunogenicity or stability of a recombinant rubella virus comprising said protein;
  • nucleotides in the non-translated regions or non- structural gene region of cDNA for rubella virus other than Cendehill are altered to decrease arthritogenicity of a recombinant rubella virus coded for by the altered cDNA.
  • cDNA from steps (a) or (b) may be incorporated into a plasmid or viral vector to produce an infectious clone, from which RNA may be transcribed and transfected into cells to provide virus that may be used as a recombinant rubella vaccine.
  • cDNA from (a) or (b) in a suitable vector may be used as a DNA vaccine.
  • This invention also provides a rubella virus whose genetic material comprises a first portion corresponding to one or more RNA sequences selected from the group consisting of: Cendehill non-translated RNA, Cendehill pl50, p90, C, El and E2 RNA; and wherein a second portion of the genome corresponds to RNA of a rubella virus other than Cendehill .
  • This invention also provides a Cendehill viral protein free of virus, selected from the group consisting of: pl50, p90, C, El and E2 , produced by expressing Cendehill cDNA encoding said protein from an expression vector.
  • This invention also provides rubella cDNA, RNA or a rubella virus having one or more of the Cendehill strain-specific nucleotides selected from a group consisting of: 37-C, 55-G, 118-T(or U) , 358-C, 2829-A, 3060-G, 3164-C3528-T (or U) , 4530-T (or U) , 6611-C, 6770- G, 6771-G, 7428-T (or U) , 8786-G, 8788-T (or U) , 8864-A, 9180-T (or U) , 9254-A, and 9741-T (or U) .
  • nucleotide numbers are in reference to nucleotides bearing the same numbers as shown in Appendix 1 for Cendehill.
  • cDNA, RNA or virus of this invention may have the strain-specific nucleotide at a different nucleotide position number as compared to Cendehill, providing the context of the strain-specific nucleotide is the same as for Cendehill. In this instance, context defines the five nucleotides on either side of the strain-specific nucleotide in Cendehill.
  • This invention also provides a Cendehill cDNA, and genomic RNA that encodes a rubella virus protein selected from the group of proteins pl50, p90, C, El and E2 and with one or more Cendehill strain-specific amino-acids defined as pl50/929/tyr, pl50/l006/gly , pl50/l04l/his , pl50/H62/val, p90/l496/ile, C/ 34/pro, C/87/gly, E2/306/val, E2/413/ile, El/759/asp, El/785/met/ , El/890/leu, and El/915/thr.
  • a rubella virus protein selected from the group of proteins pl50, p90, C, El and E2 and with one or more Cendehill strain-specific amino-acids defined as pl50/929/tyr, pl50/l006/gly , pl50/l04l/his , pl50/H62/val,
  • strain-specific amino acids are identified by protein name, amino-acid position within the Cendehill rubella polyprotein, and the identity of an amino-acid at such a position.
  • proteins of this invention include proteins having the strain-specific amino acid at a different amino acid position number in the protein as compared to Cendehill providing the context of the strain-specific amino acid is the same as for Cendehill.
  • context is defined as including the three amino acids to either side of the strain-specific amino acid in Cendehill.
  • reference to a strain-specific amino acid such as pl50/929/tyr will be used to identify the amino acid as well as a protein (eg. pl50) containing the strain-specific amino acid, in context as described herein.
  • This invention also provides a nucleic acid (eg. DNA) for the first 5' non-translated region (NTR) and first stem loop (nucleotides 1 to 65) equivalent to that found in the Cendehill strain and characterised as being a major determinant of growth restriction in joint tissue.
  • a nucleic acid eg. DNA
  • NTR non-translated region
  • first stem loop nucleotides 1 to 65
  • Specific characteristics of this stem loop in Cendehill include two nucleotide changes from the wild-type Therien strain, a U to C at nucleotide 37 in the predicted terminal loop that alters the size of the loop from 6 to 11 nucleotides, and an A to G at nucleotide 55 that increases the size of the predicted medial loop from 6-10 nucleotides.
  • nucleotides 20-28 and 52-60 are determinants of arthrotropism.
  • Other mutations between nucleotides 20-28 and 52-60 that either increase or decrease the predicted size of the medial loop are included within the scope of this invention.
  • any mutation that alters the predicted size of the terminal loop and alters the phenotypic characteristics of the virus are within the scope of this invention.
  • Factors that define the determinants of joint cell restriction include sequence-specific changes in the medial or terminal loop or changes that alter the size of either or both of the loops. These regions include nucleotides 20-28, 33-43 and 52-60.
  • Appendix 1 sets out the sequence of cDNA representing the Cendehill genome. Location of the various non-translated regions and coding regions are shown. Two polyproteins are encoded, beginning at the start codons indicated for pl50 and the C protein, respectively. The amino acid sequence of each polyprotein and the respective structural and non-structural proteins may be determined from the nucleotide sequence of Appendix 1. In this specification, the location of an amino-acid will be given by reference to a residue number of a polyprotein, which residue number may be determined directly from the series of codons shown in Appendix 1 commencing at one or the other of the start codons.
  • nucleic acid, peptide or protein as used in this specification means that when a nucleic acid, peptide or protein is described by reference to a specified nucleic acid, peptide or protein, the nucleic acid, peptide or protein so described may include a nucleotide or amino acid sequence which differs from the sequence of the specified nucleic acid, peptide or protein.
  • nucleic acids, polypeptides or proteins will include sequences of differing length or which differ by one or more substitutions, additions or deletions.
  • Nucleic acids, peptides and proteins of this invention include fragments of specified nucleic acids, peptides or proteins and may include additional amino acid or nucleotide sequences from that specified.
  • nucleic acids include complementary nucleic acids, meaning those nucleic acids capable of base pairing with a specified nucleic acid. Nucleic acids having sequences which differ from the sequence of a specified nucleic acid due to degeneracy of the genetic code are also included within the meaning of the term "corresponding" . Further, nucleic acids which encode peptides or proteins in which there are conservative substitutions, additions or deletions as compared to a specified peptide or protein are included. Any and all such nucleotide variations and resulting amino acid polymorphisms which provide the advantages of this invention as described herein are within the scope of this invention.
  • Nucleic acids within the scope of this invention may contain linkers, modified or unmodified restriction endonuclease sites and other sequences of nucleotides useful for cloning, expression, or purification. Nucleic acids within the scope of this invention may be incorporated in a larger sequence of nucleotides, including plasmids and vectors useful for manipulation or expression of nucleic acids.
  • nucleic acids, peptides or proteins with respect to this invention is relative “identity" between sequences.
  • correspondence includes a peptide having at least about 50% identity, more preferably at least about 70% identity, even more preferably at least about 90% identity, even more preferably at least about 95% and most preferably at least about 98-99% identity to a specified peptide or protein.
  • Preferred measures of identity as between nucleic acids is the same as specified above for peptides with at least about 90% or at least about 98-99% identity being more or most preferable.
  • identity refers to the measure of identity of sequence between two peptides or between two nucleic acid molecules. Identity can be determined by comparing a position in each sequence which may be a line for purposes of comparison. Two amino acid or nucleic acid sequences are considered substantially identical if they share at least about 75% sequence identity, preferably at least about 90% sequence identity, even more preferably at least 95% sequence identity and most preferably at least about 98-99% identity.
  • Sequence identity may be determined by the BLAST algorithm described in Altschul et al . (1990) J. Mol . Biol . 215:403-410, using the published default settings. When a position in the compared sequence is occupied by the same base or amino acid, the molecules are considered to have shared identity at that position. The degree of identity between sequences is a function of the number of matching positions shared by the sequences.
  • An alternate measure of identity of nucleic acid sequences is to determine whether two sequences hybridize to each other under low stringency, and preferably high stringency conditions. Such sequences are substantially identical when they will hybridize under high stringency conditions.
  • Hybridization to filter-bound sequences under low stringency conditions may, for example, be performed in 0.5 M NaHP0 4 , 7% sodium dodecyl sulfate (SDS) , 1 mM EDTA at 65°C, and washing in 0.2 x SSC/O.l SDS at 42°C (see Ausubel et al . (eds.) 1989, Current Protocols in Molecular Biology, Vol. 1, Green Publishing Associates, Inc., and John Wiley & Sons, Inc., New York, at p. 2.10.3).
  • hybridization to filter-bound sequences under high stringency conditions may for example, be performed in
  • Hybridization conditions may be modified in accordance with known methods depending on the sequence of interest (see Tijssen, 1993, Laboratory Techniques in Biochemistry and Molecular Biology -- Hybridization with Nucleic Acid Probes, Part I, Chapter 2 "Overview of Principles in Hybridization and the Strategy of Nucleic Acid Probe Assays", Elsevier, New York) .
  • stringent conditions are selected to be about 5°C lower than the thermal melting point for the specific sequence at a defined ionic strength and pH.
  • Nucleic acids of this invention will preferably exhibit substantial identity to Cendehill, with respect to the regions of the Cendehill genome described herein which relate to the arthrotropic phenotype of Cendehill. More preferably, such regions will have at least about 98% identity. Most preferably, there will be complete identity in the "context" of Cendehill strain-specific nucleotides or amino acids, as "context" is described herein.
  • Nucleic acids of this invention include a 5'NTR having a folded structure in which one or both of the terminal and medial loops is altered in size as compared to wild-type.
  • the size of the loop may be quantified according to the number of un-paired bases in the loop region.
  • such alterations result in an increase in size of the loop as compared to wild-type.
  • such altered loops will be of at least the size of the terminal and medial loops described herein for Cendehill.
  • nucleic acids of this invention comprising a 5'NTR
  • nucleic acids of this invention may include a bulge which is increased in size as compared to the wild-type bulge and preferably will have at least four un-paired bases in a bulge to one side of the stem structure.
  • the sequence of un-paired bases in such a bulge will be substantially as described herein for the Cendehill bulge. Determination of predicted folding of a 5'NTR is carried out as described herein using the MfoldTM 3.0 program.
  • Variation in the immunogenicity, yield, stability or pathogenicity of the product may readily be determined by standard techniques by comparison to known strains such as Cendehill.
  • mutation of Cendehill to increase antigenicity may be determined by measuring increased binding of a virus or viral protein to a known antibody to rubella virus and comparing this binding to that of Cendehill virus or protein at an equivalent concentration.
  • Arthrotropism for the purpose of this specification, is defined as the ability of a rubella virus strain to replicate in pieces of human joint tissue weighing approximately 0.1 gram cultured in 2 mis of medium and yield virus of titres greater than 100 plaque-forming units per ml of medium, at 24 hours post -infection that increases over the next 24 to 48 hours. Any virus less than 100 pfu/cell and that does not show an increase in titre represents residual virus from the inoculum. Following a period to allow adsorption of virus in the inoculum to the cells (4 hours) , the joint pieces are washed 4 to 5 times to reduce this residual virus and characteristically 10-100 pfu/ml of virus remains after this procedure.
  • This invention also provides a method for constructing chimeric rubella viral strains comprising part Cendehill and part of a second rubella strain including steps whereby:
  • cDNA for one or more of the Cendehill non-translated regions, non-structural proteins pl50 and p90 and structural proteins C, E2 and El is joined to cDNA of a rubella virus other than Cendehill to produce DNA corresponding to a complete RNA genome of a chimeric rubella virus. This may also be incorporated into a plasmid or viral vector to provide a chimeric infectious clone.
  • the resulting altered cDNA clone may be transcribed to produce RNA which may be used to transfect cells to produce chimeric virus, which can be cultivated as a seed stock for vaccine production.
  • This invention also provides rubella cDNA, RNA, or virus wherein cDNA or RNA encoding one or more of the viral pl50 or p90 proteins or the cDNA or RNA corresponding to a 5' non-translated region is derived from or is mutated to correspond to Cendehill, and at least part of the DNA, RNA or viral RNA, is derived from or is mutated to correspond to rubella other than Cendehill.
  • the cDNA, RNA or genome of the virus will have one or more substitutions or deletions (as compared with Therien strain) in or near the 5' non-translated region in the areas of nucleotides 17-65; substitutions in the non-structural gene coding region resulting in one or more mutations of amino acids 929, 1006, 1041, 1162 of pl50 protein or amino acid 1496 of p90 protein; or, substitutions at or near nucleotides 118 or 358 of the non-structural gene encoding region.
  • This invention also provides the use of the aforementioned cDNA, RNA, vectors (including infectious clones) and viruses (recombinant or chimeric) in the production of modified rubella cDNA, RNA or viruses, production of modified rubella protein, and in the production of rubella vaccines (DNA vaccines, live attenuated viral vaccines and subunit vaccines) .
  • This invention also provides the entire sequence of the Cendehill strain of rubella virus, including the identification of nucleotide substitutions relative to wild-type strains which are unique to the Cendehill strain and are associated with the attenuating phenotype.
  • This phenotype includes temperature sensitivity and the restriction of growth in human joint tissue.
  • substitutions can be incorporated into other rubella strains such as the current RA27/3 vaccine to produce new vaccine strains that are not arthritogenic .
  • Such substitutions may be in the region of nucleotides 17-65 (in or near the first 5' non-translated region) which forms a stem-loop structure.
  • substitutions may be at or near nucleotides 118 or 358 of the non-structural gene region, or the substitutions may involve one or more mutations of amino acids 929, 1006, 1041, 1162 of pl50 or amino acid 1496 of p90.
  • This invention also identifies mutations in Cendehill virus structural gene regions associated with reduced immunogenicity of this strain. These include two amino acid substitutions in the E2 protein at amino acids 306 and 413 (ie. at nucleotides 7428 or 7746/47), and four amino acid substitutions in El at amino acids 759, 785, 890 and 915 (ie. at nucleotides 8786/88; 8864; 9180; or 9254) . Alterations of some or all of these nucleotides to the equivalent nucleotides found in a more immunogenic strain such as RA27/3 or wild-type, enables production of a modified Cendehill strain which would be more antigenic. This may also be used as an alternative vaccine.
  • the infectious clone of Cendehill strain exemplified herein and identified as pJCND, comprises a DNA copy of the full-length Cendehill viral genome inserted into a vector from which RNA transcripts of the genome can be synthesized in vi tro and which transcripts are infectious when transfected into cells.
  • the vector is the plasmid pCL 1921, which was originally constructed by Lerner and Inouye (1990) but modified by incorporation of the pUC19 polycloning region (Yanisch-Perron et al . , 1985) and an SP6 RNA polymerase promoter .
  • This plasmid is replicated at low copy number (approximately 5 copies per cell) and contains a spectinomycin resistance gene.
  • Transcription of pJCND or other infectious clones employing Cendehill cDNA with a suitable polymerase eg. SP6 polymerase for pJCND
  • a suitable polymerase eg. SP6 polymerase for pJCND
  • Suitable expression vectors for rubella cDNA include those described herein as well as others known in the art such as the pSI or pCI mammalian expression systems (Promega) which incorporate the SV40 and
  • bacterial plasmids such as pUC19, pGEM or PBR-322 (Promega) incorporating a suitable promoter sequence such as the SP6 promoter.
  • cDNA derived from this invention may be expressed in pSI or pCI described above or the vector could be a viral vector modified to allow expression of foreign genes.
  • Such vectors derived from adenovirus, retrovirus, alphavirus, or vaccinia virus are frequently modified to make them non-pathogenic to the host.
  • Such vectors expressing cDNA derived from this invention may be used directly as a DNA vaccine .
  • a preferred method is to synthesize cDNA from a second rubella virus by preparing RNA from virus of the second strain using established techniques and then performing reverse transcription and PCR (polymerase chain reaction) on the isolated RNA using primers which flank the region of interest (for example, primers FI or 18 as described herein for synthesis of the Cendehill/RA27/3 chimera) .
  • the cDNA is then subjected to restriction enzyme digestion and resulting fragments are ligated into the Cendehill infectious clone which has been similarly digested to remove the same segment.
  • desirable portions of the Cendehill cDNA may be obtained by digestion, and the resulting fragment ligated into an infectious clone of a second rubella strain which has been similarly digested.
  • recombinant viruses were derived from pJCND and Therien/Cendehill chimeras. These strains were compared for their ability to grow in primary human joint cells, enabling the identification of two regions associated with growth restriction in these cells, in the non-structural gene region. The identification of these regions enables the production of further recombinant virus strains which combine the phenotypic property of joint growth restriction with the immunogenicity of other rubella virus strain such as RA27/3, M33 or Therien.
  • the stem- loop region which includes a 5' non-translated region and extends into the non-structural open reading frame (ORF) , contributes to joint growth restriction. This region has been shown to be important in viral viability and virulence in some a-viruses, including Sindbis virus and rubella virus (Niesters & Strauss, 1990, Pogue et al . , 1993, Pugachev & Frey, 1998) .
  • Cendehill strain contains 67 substitutions relative to the Therien strain: three in the non-translated region (NTR) upstream of the translational start site of the subgenomic RNA, two in the 3 'NTR, and the remainder in the coding region.
  • NTR non-translated region
  • Many of the substitutions in the structural genes occur as the third base of a codon and do not affect the amino-acid composition, leaving 16 substitutions in the 1062 amino-acids comprising the structural genes (nine of which are also found in the M33 strain) .
  • the substitutions include two substitutions in the capsid protein, two in the E2 glycoprotein and four in the El glycoprotein.
  • Figure 1 is a schematic showing the organization of the rubella virus genome.
  • the RNA is polyadenylated (A n ) and both the genomic and sub-genomic species are capped (CAP) .
  • Figure 2 describes the oligonucleotide primers used for reverse transcription of Rubella virus RNA and amplification of cDNA. Identification numbers for each primer appear on the left. Viral genome positions corresponding to nucleotide positions in Appendix I for seven of the primers, appear on the right.
  • Figure 3 is a schematic showing four Cendehill cDNA fragments used to construct chimeric viruses and an Cendehill infectious clone, beneath a general representation of the viral genome. Restriction sites are identified and location of sites used for construction are indicated by the dotted lines. Primers used to generate each cDNA fragment are indicated by primer identification numbers (from Figure 2) at fragment termini.
  • Figure 4 is a schematic showing the modified polycloning site of pCLPC, which is derived from pCL1921.
  • FIG. 5 is a schematic of a cloning strategy for production of Cendehill and Cendehill chimeric clones.
  • Cendehill double stranded (ds) cDNA fragments are cut using the appropriate restriction enzymes and inserted sequentially into similarly restricted regions of pROBO302.
  • Figure 6 is a schematic comparing pROBO302 to a full-length Cendehill clone (pJCND) and two Cendehill chimeras (pR0C3) and pR0C3M) . Regions without cross-hatching are Therien and cross-hatched regions are
  • Figure 7 shows predicted 5' stem loop structures of rubella RNA' s generated by the MfoldTM 3.0 program using the published default settings and for linear RNA.
  • Figures 7A, 7B and 7C are for Cendehill, wild-type and RA27/3, respectively.
  • the wild-type structure shown in Figure 7B is the same for the Therien and M33 strains and also the HPV77 vaccine.
  • Figure 8 is a schematic showing the non-structural gene region and the position of amino acid substitutions in the Cendehill strain relative to Therien. Bars indicate mutations described by single letter amino acid codes.
  • Figure 9 is a schematic showing the structural genes, glycosylation sites and the position of the amino acid substitutions in the Cendehill strain as compared to Therien, including those shared with M33 strain (unshaded bars) . Solid bars indicate mutations unique to Cendehill.
  • An infectious clone comprising a cDNA copy of all of the RNA of the Cendehill strain of rubella virus was produced as described below.
  • Viral RNA Cendehill virions were obtained by pelleting supernatant virus from the medium of Vero cells infected with Cendehill virus (Rohm Pharma) for 4 hours @ 18000 rpm in a SorvalTM centrifuge. Viral RNA was isolated by extraction with acidified phenol/guanidinium isothiocyanate using TrizolTM (Gibco/BRL) according to the manufacturer's instructions. RNA was precipitated from the aqueous phase by the addition of isopropyl alcohol (1:1) and washed with 75% ethanol diluted in DEPC-treated H 2 0 prior to drying and resuspension in DEPC-treated ddH 2 0.
  • the standard reaction mixture contained lOmM dithiothreitol and ImM dNTPs . The volume was brought to lOO ⁇ l by addition of TE buffer and heated to 90°C to inactivate the reverse transcriptase .
  • Enzyme, primers and excess nucleotides were removed by extraction of the mixture with phenol/chloroform/isoamyl alcohol (25:24:1, by volume), followed by precipitation at -20 °C in 0.3M sodium acetate and 66% ethanol.
  • double stranded cDNA was made by thermal cycling amplification with a MinicyclerTM (MJ Research) using the specific primers (described in Figure 2 according to the scheme shown in Figure 3) and repeated cycles of incubation with Deep VentTM (NEB) thermostable polymerase with 3' -5' proof-reading exonuclease activity.
  • the standard reaction mixture contained 400 ⁇ M dNTP, 2mM MgS0 4 , 0.5 ⁇ M primer and 1 unit of polymerase.
  • the products were resuspended in H 2 0 for ligation into the plasmid vector, pCLPC, a derivative of pCL1921 with the modified cloning site shown in Figure 4.
  • the constructs were screened by restriction enzyme digestion to determine that the inserts were the correct size and had the expected restriction pattern. Each clone was also screened for infectivity as follows. Small-scale plasmid preparations were carried out by standard techniques. These preparations were linearised by restriction digestion with EcoRl at the 3' terminus of the viral sequence. Positive-polarity viral RNA was generated by transcription from the SP6 promoter and the products were transfected into BHK21 cells by electroporation. After 2 days the supernatants were transferred to Vero cells and supernatant virus was removed for plaque titration 4 days later. The 3 constructs all gave titres of progeny virus of 10 5 - 10 6 /ml after three serial passages in Vero cells. The progeny viruses were designated ROC3 , ROC3M and JCND .
  • Attenuating characteristics examined included temperature sensitivity and replication in human joint cells.
  • regions of the Cendehill genome containing sequences involved in joint cell restriction include nucleotides 2803 to 5355, which are present in pR0C3M but not pROC and the 5' end of the genome, nucleotides 1 to 2803 which are specific to pJCND.
  • nucleotide substitutions involved in attenuation was determined by sequence analysis.
  • the entire cDNA sequence corresponding to the Cendehill genome was determined using an automated sequencing system at the NAPS unit at the University of British Columbia employing Amplitaq Dye Terminator CycleTM sequencing reagents (ABI) and by analysing the fluorescent products spectrophotometrically.
  • the sequence obtained is shown in Appendix 1. It was compared with the published sequences of Therien strain (Dominguez et al . , 1990, later corrected in Pugachev et al . , 1997) , a consensus M33 sequence (Clarke et al . , 1987, Zheng et al .
  • nucleotide 37 U to C nucleotide 55 : A to G
  • substitutions are in a stem- loop region that is believed to be important in controlling viral replication and translation. Alterations in this region destabilize the stem structure and may affect binding of cellular or viral factors important in viral replication.
  • the stem loop structure may be predicted by computer programs intended to generate representations of folded structures.
  • stem loop structures are determined by use of the MfoldTM 3.0 program from Dr. Michael Zuker, Washington University School of Medicine (see: M. Zuker, et al . ; Algorithms and Thermodynamics for RNA Secondary Structure Prediction: A Practical Guide in RNA Biochemistry and Biotechnology,
  • the Mfold 3.0 program may also be obtained on the Internet at : http : //mfold2.
  • the mfold program default settings are used with the imputed RNA sequence being designated as linear.
  • the alteration at nucleotide 37 is in the terminal loop of the stem.
  • the terminal loop of Cendehill is altered as compared to the predicted terminal loop of both wild-type and RA27/3 strains.
  • the substitution at nucleotide 55 increases the size of the bulge in the stem of Cendehill as compared to the bulges of wild-type or RA27/3.
  • the medial loop of Cendehill is altered as compared to the medial loop which appears in both wild-type and RA27/3.
  • Attenuation of the wild-type rubella phenotype is expected upon alterations in the nucleotide region 15-65, particularly in the regions 20-28, 33-43 and 52-60. Alterations which increase the size of the bulge such that a bulge to one side of the stem has at least four unpaired nucleotides (such as is shown in Figure 7A) is also associated with the Cendehill phenotype.
  • NSG Non- structural Gene
  • nucleotides 2800 and 4550 are found between nucleotides 2800 and 4550, including 5 mutations specific to the Cendehill strain which are present in pR0C3M but not in pROC and are therefore associated with a significant restriction in joint cell growth as described in Table I. These mutations are delineated in Table III:
  • P90 nucleotide 4530 C to U aa 1496 thr - ile Two of the NSG mutations lie within or in proximity to a region of homology with the alphavirus NSP3 domain while the other two are in the protease domain and on either side of cys 1151 at the catalytic site.
  • the p90 mutation is in the helicase domain.
  • rubella virus The structural genes of rubella virus are produced from a 3327 nucleotide subgenomic RNA as represented in
  • Figure 1 It consists of a short (78 nucleotide)
  • NTR non-translated region
  • ORF open-reading frame
  • 3 'NTR 3 ' NTR
  • Both the 3' and 5' NTRs are capable of forming stem-loop structures, can bind host cell proteins and are believed to be important in viral replication.
  • 67 nucleotide substitutions were identified in Cendehill strain when compared with the
  • Therien strain (see Appendix 1) . Two are in the 5'NTR upstream of the translational start site, two in the 3 'NTR and the remainder are in the coding region. Many of the substitutions in the structural genes occur as the third base of a codon and do not affect the amino-acid composition, leaving 16 substitutions in the 1062 amino-acids comprising the structural genes, eight of which are also found in the M33 strain. The remaining 8 amino acid substitutions are not found in the HPV77/DE5 or RA27/3 vaccine strains either.
  • the nucleotide/amino acid substitutions specific to the Cendehill strain (other than the 5'NTR substitutions) are shown in Table V(a) - (d) in which the amino acid numbering is according to the polyprotein.
  • substitution at aa34 occurs within a stretch of 28 amino-acids (28-56) believed to be important in binding of protein C to viral RNA during encapsidation.
  • a region between amino-acids 64 and 97 has been shown to react with a monoclonal antibody, indicating that this is an antigenic region although not one of the reported major antigenic sites .
  • nucleotide 7428 C to U aa 306 ala-val
  • the two changes at nucleotides 7746 and 7747 result in the loss of a Asn-X-Thr glycosylation site, one of four N- linked glycosylation sites found in Therien strain.
  • the literature is conflicting as to whether the latter substitution is present in M33.
  • the four alterations in El all occur in the region of the protein which is extruded into the lumen of the endoplasmic reticulum, and is therefore also exposed on the surface of the mature virion.
  • the first substitution at amino-acid 759 alters an asparagine to an aspartic acid residue with the resulting loss of an N- linked glycosylation site, one of three in El, all of which are believed to be utilised. None of the substitutions in El are in regions identified as dominant epitopes of the cell-mediated immune response, nor in regions identified by monoclonal antibodies as being associated with hemagglutination or neutralisation.
  • nucleotide 9731 G to C nucleotide 9740 C to U nucleotide 9741 C to U
  • 9731 and 9740 are also found in the M33 strain, they may affect attenuation as M33 is a less cytopathic strain than Therien.
  • a chimeric strain can be produced comprising (for example) the entire structural gene region of RA27/3 inserted into the Cendehill infectious clone. Either of these constructs would provide an improved attenuated rubella vaccine.
  • Altered strains can be produced by standard recombinant DNA technology as described in many current textbooks including "Molecular Cloning: A Laboratory Manual,” edited by Maniatis,T., Fritsch E.F., and
  • oligonucleotide-directed mutagenesis and gene amplification technology can be used as described by Higuchi (1989) .
  • This procedure involves synthesis of oligonucleotides specific for the region to be modified, containing the required nucleotide substitution, as well as an appropriate restriction site. This can then be used as one primer for a gene amplification reaction encompassing the region of interest. A second primer is chosen which includes a unique restriction site and which will yield a fragment of suitable size. Following amplification of the fragment which now has the requisite nucleotide substitution incorporated, the fragment is cloned into the infectious clone replacing the original sequence. In this way, mutations can be incorporated into the gene sequence either singly or sequentially until the resulting virus has the properties wanted.
  • a cDNA clone including the entire structural gene region of a rubella stain such as RA27/3 can be made in the following steps: (i) isolation of viral RNA from high-titre virus stock, (ii) first strand cDNA synthesis using a specific primer for the 3 'end, (iii) amplification of the structural gene region using primers FI and 18
  • a chimeric Cendehill/RA27/3 clone whose genome includes a first portion which is equivalent to the Cendehill 5" non- translated RNA, Cendehill pl50 and p90 and a second portion equivalent to the structural gene region and the 3 ' non-translated region of RA27/3 strain was made.
  • This clone can be used to produce a chimeric virus that expresses the structural proteins of RA27/3 but has the determinants of arthrotropism found in the genetic structure at the 5' end and in the non-structural genes of Cendehill strain.
  • This construct was produced by synthesising a cDNA/PCR fragment, using RA27/3 RNA as template, equivalent to the 18-F1 fragment shown in Figure 2. This fragment was then inserted into the Cendehill infectious clone using the restriction enzymes Bglil and EcoRl, in an identical manner to the synthesis of pROC3 described elsewhere in this specification. The new chimeric clone was sequenced through nucleotides 6611 and 6770/6771 as well as through nucleotides 8786/8788 and 8864 to ensure that replacement of the 18 -FI fragment had occurred.
  • RA27/3 The published sequence of RA27/3 indicates that the latter strain has the same nucleotides as Therien strain at these positions (Pugachev KV, Abernathy ES and Frey TK. Archives of Virology 142 1165-1180, 1997: Genomic sequence of the RA27/3 vaccine strain of rubella virus) while Cendehill is modified in these regions as disclosed herein.
  • Modified cDNA clones incorporated in the pCL1921 plasmid can be transcribed into complete infectious RNA from the SP6 promoter.
  • the RNA produced can be transfected into BHK-21 cells by a variety of techniques including electroporation or use of LipofectamineTM (Gibco/BRL) .
  • the transfected RNA is translated and replicated in the cell to yield virus with altered phenotypic properties according to the mutations introduced.
  • seed stocks of rubella strains of this invention may be produced. Phenotypic properties of rubella strains of this invention can be monitored for characteristics associated with attenuation and immunogenicity.
  • yield, temperature sensitivity and the ability to grow in human joint tissue can be determined as described previously for pR0C3 and pR0C3M.
  • the antigenicity of the strains can be assessed using standard enzyme-linked immunosorbent assays, immunoprecipitation assays and immunoblots with human rubella seropositive antisera.
  • the efficacy of a strain for eliciting a strong neutralising antibody response can be measured in rabbits and compared with the current vaccine strain, RA27/3 and also the parental Cendehill strain. In this way, novel strains can be assessed for characteristics that would make them suitable for use as improved attenuated vaccines.
  • Attenuated rubella strains may be used as a seed stock for manufacturing vaccine .
  • Virus from such a stock may be combined with a variety of stabilisers such as saline, phosphate buffer, polyethylene glycol, glycerin as currently used in vaccine preparations.
  • the vaccine may be produced in lyophilised form to aid long-term preservation. It can also be combined with other vaccines such as mumps and measles vaccines as in the current M-M-R formulation.
  • modified infectious cDNA clones may also be used to produce a DNA vaccine against rubella virus, either singly or in combination with other DNA vaccines.
  • the cDNA of the rubella virus strain is sub-cloned into an expression vector (either plasmid or viral) which contains a suitable eukaryotic promoter. Either the entire rubella virus genome, the structural genes or immunogenic regions of the structural genes can be used in this manner to directly immunise patients.
  • the DNA vaccine is taken up by cells and transcribed from the eukaryotic promoter to yield RNA which is translated into viral proteins . These in turn elicit an immune response.
  • Cendehill infectious clone and its derivatives include the production of large quantities of virus for use as antigen in enzyme-linked immunosorbent assays to assess human antibody levels against rubella.
  • antigen known to react optimally according to the vaccine strain delivered.
  • a virus strain with the structural gene region identical to the vaccine in use, but altered in the non-structural genes or NTR regions to improve viral yield for antigen production may be propagated.
  • the strain for use in immunoassays would be treated to produce a non- infectious antigen preparation.
  • the structural proteins alone could be produced from a suitable expression vector to yield an antigen preparation with the correct specificity.

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Abstract

L'invention concerne la séquence complète de la souche rubéolique de Cendehill, y compris des clones infectieux de l'ADN complémentaire qui en sont dérivés. Des parties du génome de Cendehill responsables de la diminution de l'arthritogénicité et de l'immunogénicité ont été identifiés. L'invention concerne également un ADN complémentaire, un ARN et un virus rubéoliques modifiés comprenant des séquences de la souche rubéolique de Cendehill ou d'autres souches.
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US6835550B1 (en) 1998-04-15 2004-12-28 Genencor International, Inc. Mutant proteins having lower allergenic response in humans and methods for constructing, identifying and producing such proteins
US6897049B1 (en) 1998-04-15 2005-05-24 Genencor International, Inc. Proteins producing an altered immunogenic response and methods of making and using the same

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US6835550B1 (en) 1998-04-15 2004-12-28 Genencor International, Inc. Mutant proteins having lower allergenic response in humans and methods for constructing, identifying and producing such proteins
US6838269B1 (en) 1998-04-15 2005-01-04 Genencor International, Inc. Proteins producing an altered immunogenic response and methods of making and using the same
US6897049B1 (en) 1998-04-15 2005-05-24 Genencor International, Inc. Proteins producing an altered immunogenic response and methods of making and using the same
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WO2001059130A3 (fr) * 2000-02-08 2002-03-07 Genencor Int Proteines produisant une reponse immunogene modifiee et procedes de production et d'utilisation de ces proteines

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