EP0714399A1 - Gene hsv-2 ul26, proteines capsides, dosages immunologiques et inhibiteurs de protease - Google Patents

Gene hsv-2 ul26, proteines capsides, dosages immunologiques et inhibiteurs de protease

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Publication number
EP0714399A1
EP0714399A1 EP94925924A EP94925924A EP0714399A1 EP 0714399 A1 EP0714399 A1 EP 0714399A1 EP 94925924 A EP94925924 A EP 94925924A EP 94925924 A EP94925924 A EP 94925924A EP 0714399 A1 EP0714399 A1 EP 0714399A1
Authority
EP
European Patent Office
Prior art keywords
hsv
ala
protein
pro
protease
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP94925924A
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German (de)
English (en)
Other versions
EP0714399A4 (fr
Inventor
Anthony G. Dilella
Christine Marie Debouck
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SmithKline Beecham Corp
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SmithKline Beecham Corp
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Publication of EP0714399A1 publication Critical patent/EP0714399A1/fr
Publication of EP0714399A4 publication Critical patent/EP0714399A4/fr
Withdrawn legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/503—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/705—Specific hybridization probes for herpetoviridae, e.g. herpes simplex, varicella zoster
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56994—Herpetoviridae, e.g. cytomegalovirus, Epstein-Barr virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/01—DNA viruses
    • G01N2333/03—Herpetoviridae, e.g. pseudorabies virus
    • G01N2333/035—Herpes simplex virus I or II

Definitions

  • HSV-2 UL26 GENE CAPSID PROTEINS, IMMUNOASSAYS AND PROTEASE INHIBITORS
  • the present invention relates to HSV-2 UL26 and HSV-2 UL26.5 genes; to essentially pure HSN-2 UL26 and HSN-2 UL26.5 gene products; to compositions and methods of producing and using HSN-2 UL26 and HS V-2 UL26.5 D ⁇ A sequences and gene products.
  • the herpes viruses consist of large icosahedral enveloped virions containing a linear double stranded genome.
  • six human herpes viruses have been isolated and are known to be responsible for a variety of disease states from sub-clinical infections to fatal disease states in the immunocompromised.
  • One human herpes virus, herpes simplex virus type 2, designated HSV-2 is usually acquired through sexual contact and gives rise to genital herpes.
  • the frequency of recurrence of secondary genital herpes ranges between one and six times per year. It is estimated that genital HSV-2 infections occur in ten to sixty million individuals in the USA.
  • HS V-2 presents a major public health problem. Individuals continue to become infected by the virus and no completely satisfactory anti-viral agents or vaccines are available. There is a need for a method of identifying anti-HSV-2 agents. There is a need for reagents useful in such methods. There is a need for a method of identifying compounds which modulate the activity of HSV-2 proteins and affect the ability of the virus to replicate and produce multiple infectious virions in an infected cell. There is a need for methods of and kits for distinguishing HS V-2 infections from other herpesvirus infections.
  • the present invention relates to essentially pure HSV-2 UL26 gene products and fragments thereof including HS V-2 protease precursor protein, mature HSV-2 protease and active fragments thereof, HS V capsid precursor protein and mature HSV-2 capsid protein.
  • the present invention relates to essentially pure HSV-2 UL26.5 gene products and fragments thereof including HSV-2 capsid precursor protein and mature HSV-2 capsid protein.
  • the present invention relates to isolated nucleic acid molecules comprising the HSV-2 UL26 gene or portions thereof including isolated nucleic acid molecules that encode mature HS V-2 protease and active fragments thereof and nucleic acid molesules that encode precursor or mature HSV-2 capsid protein, regulatory, e.g., promoter regions, or functional fragments thereof.
  • the present invention relates to expression vectors comprising the HSV-2 UL26 gene or portions thereof including nucleotide sequences that encode mature HS V-2 protease and active fragments thereof and nucleotide sequences that encode precursor or mature HSV-2 capsid protein or functional fragments thereof.
  • the present invention relates to host cells that contain expression vectors comprising the HS V-2 UL26 gene or portions thereof including nucleotide sequences that encode mature HS V-2 protease and active fragments thereof and nucleotide sequences that encode precursor or mature HS V-2 capsid protein or functional fragments thereof.
  • the present invention relates to isolated nucleic acid molecules comprising the HS V-2 UL26.5 gene or portions thereof including isolated nucleic acid molecules that encode mature HSV-2 capsid protein, regulatory, e.g., Promoter regions or fragments thereof and nucleotide sequences that encode precursor or mature HSV-2 capsid protein or functional fragments thereof.
  • the present invention relates to expression vectors comprising the HSV-2 UL26.5 gene or portions thereof including nucleotide sequences that encode mature HS V-2 capsid protein or fragments thereof and nucleotide sequences that encode precursor or mature HSV-2 capsid protein or functional fragments thereof.
  • the present invention relates to host cells that contain expression vectors comprising the HS V-2 UL26.5 gene or portions thereof including nucleotide sequences that encode mature HSV-2 capsid protein or fragments thereof and nucleotide sequences that encode precursor or mature HS V-2 capsid protein or functional fragments thereof.
  • the present invention relates to methods of identifying compounds that inhibit HSV-2 protease activity comprising contacting HSV-2 protease or active fragments thereof with an HS V-2 protease substrate in the presence of a test compound, detecting the level of proteolytic cleavage of the substrate and comparing that level to the level that occurs in the absence of the test compound.
  • the present invention relates to methods of identifying compounds that inhibit HS V-2 virion assembly by contacting HS V-2 capsid proteins in the presence of a test compound, detecting the level of capsid-capsid association and comparing that level to the level that occurs in the absence of the test compound.
  • the present invention relates to HSV-2 protease substrates produced by means of chemical synthesis or recombinantly produced and predicated on fragments or all of the UL26 gene product.
  • the present invention relates to antibodies that selectively bind to HSV-2 protease processed substrates but not unprocessed substrates or that selectively bind to unprocessed substrates but not to processed substrates.
  • the present invention relates to methods of distinguishing between HSV-1 DNA and HS V-2 DNA comprising PCR amplification of DNA using primers which will amplify HSV-1 DNA but not HSV-2 DNA and/or PCR amplification of DNA using primers which will amplify HSV-2 DNA but not HSV-1 DNA.
  • the present invention relates to PCR primers which will amplify HSV-1
  • kits for distinguishing between HSV-1 DNA and HS V-2 DNA comprising a container comprising PCR primers which will amplify HSV-1 DNA but not HSV-2 DNA and a positive control and size marker to determine if HSV-1 DNA has been amplified by the primers and/or a container comprising PCR primers which will amplify HSV-2 DNA but not HSV-1 DNA and a positive control and size marker to determine if HS V-2 DNA has been amplified by the primers.
  • the present invention relates to methods of distinguishing between HS V- 1 protein and HSV-2 protein comprising an immunoassay using antibodies that selectively bind to HSV-1 protein but not HSV-2 protein and/or an immunoassay using antibodies that selectively bind to HSV-2 protein but not HSV-1 protein.
  • the present invention relates to antibodies which selectively bind to HS V- 1 protein but not HSV-2 protein or antibodies which selectively bind to HSV-2 protein but not HSV-1 protein.
  • the present invention relates to kits for distinguishing between HSV-1 protein and HS V-2 protein. Said kit comprising a carrier being compartmented to receive a series of containers in close confinement which comprises a first container comprising antibodies which selectively bind to HSV-1 protein but not HSV-2 protein and a means to detect whether the antibodies are bound to HS V- 1 protein and/or a second container comprising antibodies which selectively bind to HSV-2 protein but not HSV-1 protein and a means to detect whether the antibodies are bound to HS V-2 protein.
  • the present invention relateds to the HSV-2 protease promoter and/or enhancer elements and their uses.
  • the present invention relates to the HS V-2 capsid protein promoter and/or enhancer elements and their uses.
  • FIGURES Figure 1 illustrates the HSV-2 UL26 gene.
  • the symbol ⁇ > denotes the limits of the
  • HSV-2 UL26 gene product A putative termination codon is underlined.
  • the symbol [ ] denotes the limits of two major proteolytic sites.
  • the cissile bond is indicated by the *.
  • TATA box is underlined.
  • Figure 2 illustrates the expression of chloramphenicol acetyltransferase (CAT) when regulated in the HS V-2 UL26.5 promoter.
  • CAT chloramphenicol acetyltransferase
  • the term UL26 gene refers to a DNA molecule comprising a nucleotide sequence that encodes the HS V-2 protease and a form of the HS V-2 capsid protein.
  • the UL26 gene is disclosed in SEQ ID NO:l.
  • the coding region of the UL26 gene consists of nucleotides 534-2447 of SEQ ID NO: 1.
  • the UL26 gene encodes a 638 amino acid active protease precursor disclosed in SEQ ID NO: l and SEQ ID NO:2.
  • active protease precursor refers to the unprocessed UL26 translation product.
  • the active protease precursor is an active HSV-2 protease.
  • the active protease precursor autocleaves at an internal protease cleavage site between amino acid residues 247 and 248.
  • the amino terminal 247 amino acid portion retains protease activity.
  • mature protease refers to the amino terminal 247 amino acid protein that is produced by autocleavage of the active protease precursor.
  • the amino acid sequence of the mature protease is disclosed as amino acids 1-247 of SEQ ID NO:l and SEQ ID NO:2.
  • the term “HS V-2 protease” is meant to refer to, interchangeably, active protease precursor, mature protease or active fragments thereof.
  • the term "UL26.5" gene refers to a DNA molecule comprising a nucleotide sequence that encodes the HSV-2 capsid protein.
  • the UL26.5 gene is an internal sequence within the UL26 gene which is separately transcribed.
  • the UL26.5 gene is disclosed in SEQ ID NO:l and includes the coding region from nucleotide 1461-2447.
  • the UL26.5 gene encodes a 329 amino acid capsid precursor disclosed in SEQ ID NO:l and SEQ ID NO:2 as amino acids 310-638.
  • capsid precursor refers to the unprocessed UL26.5 translation product.
  • the capsid precursor is cleaved by the HS V-2 protease at an internal protease cleavage site between amino acid residues 613 and 614 of SEQ ID NO.l and SEQ ID NO:2.
  • the 304 amino acid portion is the capsid protein used in viral assembly and viral DNA packaging. It is the C-terminal processing of UL26.5 that enables packaging of viral DNA into mature capsids. Inhibition of this processing event results in the inability to package DNA into mature capsids.
  • mature capsid protein refers to the 304 amino acid protein that is produced by cleavage of the capsid precursor by the HSV-2 protease.
  • the amino acid sequence of the mature capsid protein is disclosed as amino acids 310-613 of SEQ ID NO:l and SEQ ID NO:2.
  • HSV-2 capsid protein is meant to refer to, interchangeably, capsid precursor and mature capsid protein.
  • the term "functional fragments" when used to modify a specific gene or gene product means a less than full length portion of the gene or gene product which retains substantially all of the biological function associat-ed with the full length gene or gene product to which it relates. To determine whether a fragment of a particular gene or gene product is a functional fragment one merely generates the fragments by well-known nucleolytic or proteolytic techniques and tests the thus generated fragments for the described biological function.
  • the present invention relates to essentially pure HSV-2 protease, to compositions and methods for producing and using HSV-2 protease, to nucleic acid molecules that encode HSV-2 protease and to methods for producing and using nucleic acid molecules that encode HSV-2 protease.
  • the present invention relates to essentially pure HSV-2 capsid protein, to compositions and methods for producing and using HSV-2 capsid protein, to nucleic acid molecules that encode HS V-2 capsid protein, to methods for producing and using nucleic acid molecules that encode HS V-2 capsid protein.
  • the present invention relates to substrates which are cleaved by HS V-2 protease, to methods of identifying compounds that inhibit HSV-2 protease activity, to methods of identifying compounds which inhibit HSV-2 capsid assembly, to methods of distinguishing between samples containing HSV-1 DNA and samples containing HSV-2 DNA, to methods of distinguishing between samples containing HSV-1 protein and samples containing HSV-2 protein, and to reagents, including oligonucleotides and antibodies, for performing such methods.
  • Some embodiments of the present invention provide methods for identifying compounds which inhibit or otherwise modulate the activity of HS V-2 protease.
  • the present invention provides methods for identifying compounds useful as anti-HSV-2 agents since the activity of the HSV-2 protease is essential for the viral life cycle.
  • HSV-2 protease is contacted with an HS V-2 protease substrate (substrate) in the presence of a test compound to determine whether or not the test compound affects proteolytic activity.
  • the effect of the test compound on the HS V-2 protease may be determined by comparing the proteolytic activity in the presence of the test compound to the proteolytic activity that would be observed in the absence of the compound.
  • proteolytic activity refers to the ability of the HS V-2 protease to enzymatically process the substrate into products, i.e. cleave a single substrate peptide molecule into two or more peptide molecules (proteolytic products).
  • protease precursor is processed into mature protease and capsid precursor is processed into mature capsid by such proteolytic cleavage. This conversion is necessary for virion assembly and viral DNA packaging.
  • the level of proteolytic activity may be determined by a variety of means well known by those having ordinary skill in the art. Essentially, a means is provided to distinguish unprocessed substrate from proteolytic product.
  • the present invention provides essentially pure HS V-2 protease which is useful in an assay to identify compounds which modulate HSV-2 protease activity.
  • the present invention provides methods of producing essentially pure HS V-2 protease.
  • the amino acid sequence of HSV-2 protease is disclosed in SEQ ID NO:l and SEQ ID NO:2.
  • the 638 amino acid active protease precursor is disclosed in SEQ ID NO:l and SEQ ID NO:2.
  • the active protease precursor is an active HS V-2 protease which is processed by autocleavage at an internal protease cleavage site between amino acid residues 247 and 248 to produce a 247 amino acid protein referred to as mature protease.
  • Purified active protease precursor, mature protease and active fragments thereof may be produced by routine peptide synthesis methods or by using recombinant DNA technology using the information provided in SEQ ID NO:l. Using standard procedures and readily available starting materials, one having ordinary skill in the art can produce HSV-2 protease.
  • one having ordinary skill in the art can determine whether a fragment and/or derivative of the active protease precursor or mature protease is an active fragment. Assays for determining whether or not a protein or peptide is capable of cleaving a specific substrate is disclosed herein. To determine if an HS V-2 protease fragment has proteolytic activity, one having ordinary skill in the art can perform protease activity assays as described herein without test compounds and using the fragment or derivative of the protease instead of the protease identical to SEQ ID NO:2. If the fragment or derivative cleaves the substrate, it is active, i.e. the fragment or derivative possesses proteolytic activity. Thus, one having ordinary skill in the art can routinely determine if a fragment or derivative of the protease is an active fragment or derivative.
  • the present invention relates to nucleotide sequences that encode HSV-2 protease and to nucleotide sequences that encode HSV-2 capsid protein.
  • the UL26 gene including a nucleotide sequence which encodes HSV-2 protease and a precursor form of HSV-2 capsid protein is disclosed in SEQ ID NO:l.
  • the UL26.5 gene including a nucleotide sequence which encodes HSV-2 capsid protein is also disclosed in SEQ ID NO:l.
  • One having ordinary skill in the art can, using standard techniques and readily available starting materials, use the information disclosed herein including SEQ ID NO: 1 to obtain or synthesize a nucleic acid molecule that encodes HSV-2 protease or a nucleic acid molecule that encodes HSV-2 capsid protein. Further, using standard techniques, readily available starting materials and the information disclosed herein including SEQ ID NO:l, one having ordinary skill in the art can produce essentially pure HSV-2 protease including, active precursor protease, mature protease or active HS V-2 protease fragments.
  • HSV-2 capsid protein including capsid precursor, mature capsid, or HS V-2 capsid fragments capable of assembly functional fragments.
  • One having ordinary skill in the art can, using standard techniques and readily available starting materials, use the information disclosed herein including SEQ ID NO: 1 to obtain or synthesize a nucleic acid molecule that encodes HSV-2 protease or HSV-2 capsid protein using codons which provide optimum protein production in a given host cell used in an expression system.
  • Nucleic acid molecules encoding HSV-2 protease or HSV-2 capsid protein may be generated by those having ordinary skill in the art without undue experimentation using a variety of techniques. Using, for example, Polymerase Chain Reaction (PCR) methodology, primers may be designed and used to produce multiple copies of the nucleotide sequences that encode the HS V-2 protease or HSV-2 capsid protein. The entire nucleotide sequence encoding active protease precursor may be obtained routinely by amplifying the viral DNA. Similarly, the nucleotide sequence encoding mature protease may be obtained routinely by amplifying the viral DNA.
  • PCR Polymerase Chain Reaction
  • nucleotide sequence encoding an active HS V-2 protease fragment may be obtained routinely by amplifying the viral DNA.
  • the entire nucleotide sequence encoding capsid precursor, mature capsid or functional fragments thereof may be obtained routinely by amplifying the viral DNA.
  • DNA encoding HS V-2 protease, including the active protease precursor, the mature protease, or active fragments thereof or HSV-2 capsid protein including capsid precursor, mature capsid or functional fragments thereof may be obtained from viral DNA cloned into vectors and identified by hybridization using probes designed from the disclosed nucleotide sequence.
  • nucleic acid molecules that encode the HSV-2 protease or the HS V-2 capsid protein may also be synthesized using techniques well known to those having ordinary skill in the art. Codons which encode HSV-2 protease or HSV-2 capsid protein may be selected to optimize protein production in a host cell selected for recombinant production of the HS V-2 protease or HS V-2 capsid protein.
  • the HSV-2 genome is highly rich in G+C nucleotides. This is particularly true for the UL26 gene which encodes HSV-2 protease. Such high G+C character poses a problem in overexpressing genes in E. coli because of codon usage and an increased chance of frame-shift mutations.
  • the UL26 gene and fragments thereof were changed to provide codons preferred in E. coli yet maintaining the authentic amino acid sequence of the protease.
  • the reference for preferred codon usage is: Wada et al, (1992) "Codon Usage Tabulated from the GenBank Genetic Sequence Data", Nucleic Acid Research, Vol. 20 Supplement, pages 2111-2118, which is incorporated herein by reference. Optimization of codon usage is well known and can be employed to design nucleic acid molecules according to the present invention which can be expressed at an improved level of efficiency in a selected host.
  • One having ordinary skill in the art can, using well known techniques, insert such DNA molecules into vectors such as commercially available expression vectors for use in well known expression systems.
  • commercially available plasmids such as pS ⁇ 420 (Invitrogen, San Diego, CA) or pET- 16(b) (Novagen, Madison W.I.) may be used for production of HS V-2 protease in E. coli.
  • the commercially available plasmid pYES2 (Invitrogen, San Diego, CA) may, for example, be used for production in S. cerevisiae strains of yeast.
  • the commercially available MAXBACTM complete baculovirus expression system (Invitrogen, San Diego, CA) may, for example, be used for production in insect cells.
  • the commercially available plasmid pcDNA I may, for example, be used for production in mammalian cells such as Chinese Hamster Ovary cells.
  • mammalian cells such as Chinese Hamster Ovary cells.
  • One having ordinary skill in the art can use these commercial expression vectors and systems or others to produce the HS V-2 protease or HS V-2 capsid protein using routine techniques and readily available starting materials. (See e.g., Sambrook et al., Molecular Cloning a Laboratory Manual, Second Ed. Cold Spring Harbor Press (1989) which is incorporated herein by reference.)
  • the desired proteins can be prepared in both prokaryotic and eukaryotic systems, resulting in a spectrum of processed forms of the protein.
  • the DNA encoding the polypeptide is suitably ligated into the expression vector of choice.
  • the DNA is operably linked to all regulatory elements which are necessary for expression of the DNA in the selected host.
  • One having ordinary skill in the art can, using well known techniques, prepare expression vectors for recombinant production of the polypeptide.
  • the expression vector including the DNA that encodes the HSV-2 protease or HS V-2 capsid protein is used to transform or transfect the compatible host which is then cultured and maintained under conditions wherein expression of the foreign DNA takes place.
  • the protein of the present invention thus produced is recovered from the culture, either by lysing the cells or from the culture medium as appropriate and known to those in the art.
  • One having ordinary skill in the art can, using well known techniques, isolate the protein that is produced using such expression systems.
  • protein may be produced and purified as follows.
  • a DNA molecule that comprises a nucleotide sequence that encodes the HSV-2 protease or the HSV-2 capsid protein is produced which includes a nucleotide sequence that encodes multiple histidine residues at a terminal portion of the protein.
  • This DNA molecule is incorporated into an expression vector which is introduced into suitable host cells.
  • the DNA is expressed and the protein, including the terminal histidine residues, which are referred to herein as the histidine tag or His-tag, is produced.
  • the cells are collected and maintained on ice in phosphate buffered saline at pH 8.5. The cells are then lysed by sonication.
  • the sonicated cellular material is centrifuged at 30,000 x g.
  • the supernatant is then filtered through a .2 micron filter.
  • the filtered supernatant is incubated with a metal chelating resin (e.g., a nitrilo triacetic acid nickel resin is one of many such resins useful for such a purpose) for 2 hours at room temperature, after which time the resin is separated from unbound material by centrifugation.
  • the resin is then packed into a column and washed with 50 mM imidazole to eliminate non specifically bound proteins.
  • the His-tagged protease is then eluted from the Ni column with 150 mM imidazole buffer.
  • the eluate from the column is further purified by column chromatography using Pharmacia Superdex 75 sizing column in phosphate buffered saline.
  • the DNA molecule may be engineered to include a specific cleavage site between the histidine tag and authentic HSV-2 protease to enable removal of the histidine tag from the expressed protein. Removal of the histidine tag may be accomplished as follows:
  • the (asparte)4 lysine sequence can be engineered to follow the histidine tag and precede the HSV-2 sequence when the histine tag is placed at the amino-terminus of the HS V-2 protease. Enterokinase specifically cleaves after the (aspartate)4lysine sequence thereby generating authentic HSV-2 protease.
  • automated peptide synthesizers may also be employed to produce the HSV-2 protease or the HS V-2 capsid protein. Such techniques are well known to those having ordinary skill in the art.
  • the present invention provides essentially pure substrates for HS V-2 protease cleavage activity including synthetic substrates.
  • An HSV-2 protease substrate is a peptide which can be cleaved at least into two separate peptides by HSV-2 protease mediated proteolysis.
  • the size differential between cleaved and uncleaved substrates may be used to detect whether or not the protease is active.
  • the substrates of the present invention are labelled so that they may be detected.
  • the substrates are fixed to a solid phase.
  • either the substrate or a proteolytic product has a biologically or chemical activity not present in the other which can be used to distinguish one from the other. Examples of biological activities include enzyme activity and the ability to bind with specific antibodies.
  • a substrate according to the present invention have either the formula R, - SEQ ID NO:3 - R 2 or the formula wherein R x and R 2 are, independently, hydrogen or one or more amino acids.
  • the substrate is the UL26 gene product which contains two protease cleavage sites: one comprising SEQ ID NO:3 and one comprising SEQ ID NO:4.
  • the substrate is the UL26.5 gene product which contains a protease cleavage sites comprising SEQ ID NO:4.
  • Rj is preferably 1-20 amino acids, more preferably 1-10, and most preferably 3, 4, 5, 6, 7, 8 or 9 amino acids.
  • R 2 is preferably 1-20 amino acids, more preferably 1-10, and most preferably 3, 4, 5, 6, 7, 8 or 9 amino acids.
  • VNA*S ALVNA*SS AAHVDVD SEQ ID NO: 15
  • the asterisk (*) indicates the scissile bond where cleavage by HSV-2 protease occurs.
  • the substrates may be obtained from proteolytic cleavage of the UL26 or
  • UL26.5 protein product They may be produced recombinantly by expression of UL26 or UL26.5 gene or fragment thereof containing the cleavage site or may be made by means of synthetic organic chemical means using standard peptide synthetic procedures well known in the art such as Merrifield synthesis.
  • One having ordinary skill in the art can readily design assays using the HSV-
  • test assay refers to assays that include a mixture of HSV-2 protease, substrate and test compound
  • control assay refers to assays that include a mixture of HSV-2 protease and substrate without test compound.
  • the level of HSV-2 protease activity in a test assay may be compared to the level of HSV-2 protease activity in a control assay.
  • the size differential between cleaved and uncleaved substrate is used to determine whether or not substrates are cleaved when contacted with HSV-2 protease in the presence of a test compound.
  • an HPLC assay is performed. Sample containing protease is incubated with a substrate, for example HTYLQASEKFKMWGAE (SEQ ID NO: 14), for 4 hrs at 37° C in phosphate buffered saline after which the reaction is terminated with trifluoroacetic acid. The reaction is then run on an HPLC column, showing activity manifested by the peptide cleavage products.
  • immunoassays are used to detect whether or not substrates are cleaved when contacted with HS V-2 protease in the presence of a test compound.
  • antibodies are provided which specifically bind to uncleaved substrate but not HS V-2 protease cleavage products. Such antibodies are referred to herein as “substrate- specific antibodies”.
  • antibodies are provided which specifically bind to HS V-2 protease cleavage products but not uncleaved substrate. Such antibodies are referred to herein as "product-specific antibodies”.
  • Antibodies which react to either a product or a substrate but not both are referred to herein as "non-crossreactive antibodies”.
  • antibodies are fixed to a solid phase.
  • antibodies are labelled.
  • a mixture containing HS V-2 protease, substrate and test compound is maintained under appropriate conditions and for a sufficient amount of time to allow the proteolytic reaction to occur unless the test compound affects the reaction.
  • the mixture can be added to a container which has non-crossreactive antibodies attached to the inner surface. If the non-crossreactive are substrate- specific antibodies, any uncleaved substrate remaining in the mixture will bind to the antibodies. If the substrate is labelled, the contained may be rinsed and the amount of label present may be detected. The level of HSV-2 protease activity is determined accordingly. If the non-crossreactive are product- specific antibodies, any HSV-2 protease products in the mixture will bind to the antibodies.
  • the substrate is labelled at a portion which is liberated as the product, the contained may be rinsed and the amount of label present may be detected.
  • the level of HS V-2 protease activity is determined accordingly.
  • ICP35 antibodies Catalog No.: 13-118-100; Rivers Park, 9108 Gulford Rd.
  • Such antibodies are product specific and only bind to capsid protein after it has been proteolytically processed by the HSV-2 protease.
  • the exemplified immunoassays may be modified as sandwich assays in which antibodies specific for the bound antigen complex are detected. Such antibodies are referred to herein as complex-specific antibodies.
  • the container is again rinsed and sufficient time is allowed for the binding of the complex specific antibody to any complex present.
  • the level of complex specific antibody is detected and indicative of the level of HS V-2 protease activity.
  • unlabelled substrate is used in the reaction mixture.
  • reaction mixture After the reaction mixture is added to a container comprising a non-crossreactive antibody and maintained for a sufficient time for the non- crossreactive antibody to bind to either substrate br product, either labelled substrate or labelled product, respectively, is added and will bind to any non-crossreactive antibody not bound with substrate or product from the reaction mixture. Detecting the amount of labelled substrate or labelled product indicates the level of proteolytic cleavage.
  • the substrate is labeled and the label is released when the substrate is converted to proteolytic products. Detecting the release of the label, which indicates the HSV-2 protease activity, may be accomplished by a variety of well known means.
  • labelled substrate is fixed to a solid phase. Upon cleavage by HSV-2 protease, the label attached to the portion of the substrate that becomes an unattached product, is released. Comparing the level of label present before and after the reaction mixture indicates how much label is released and thus the level of HSV-2 protease activity. Alternatively, detecting the amount of label freed from the solid phase indicates the level of HSV-2 protease activity.
  • methods of detecting HSV-2 protease activity include fluorescence liberation assays in which substrate contains fluorescent label adjacent to the scissile bond. At such a location, the label is not detectable in uncleaved substrate. However, when the substrate is cleaved by HSV-2 protease at the cleavage site, the fluorescent group becomes exposed and the fluorescence becomes detectable. Thus, the level of proteolytic activity may be measured by measuring detectable fluorescence after contacting the substrate with HSV-2 protease in the presence of a test compound.
  • methods of detecting HSV-2 protease activity include scintillation proximity assays in which radiolabelled substrate is conjugated to solid beads which, when in close proximity to the radiolabel, are excited and become detectable by scintillation. When the substrate is cleaved, the radiolabel is no longer in close proximity to the beads and the beads are not excited and not detectable by scintillation. Thus, the level of proteolytic activity may be measured by measuring the excitation of the beads by scintillation after contacting the conjugated substrate with HSV-2 protease in the presence of a test compound.
  • kits for identifying compounds that modulate HSV-2 protease activity include separate containers which comprise HSV-2 protease, substrate, and optionally, antibodies or other reagents for detecting HSV-2 protease activity or distinguishing between uncleaved substrate and products.
  • the substrate or antibodies may be fixed to the inner surface of a container.
  • the substrate or antibodies may be labelled.
  • Some embodiments of the present invention also provide methods of identifying compounds which inhibit or otherwise modulate HSV-2 capsid assembly using a multimerization assay.
  • the present invention provides methods of identifying compounds useful as anti-HSV-2 agents since capsid assembly is essential for viral replication and infectivity.
  • chimeric genes are provided which comprise either a sequence including the HSV-2 UL26.5 gene or a portion thereof which encodes an HSV-2 capsid protein linked to a sequence encoding the yeast GALA DNA-binding protein or a sequence including the HSV-2 UL26.5 gene or a portion thereof which encodes an HS V-2 capsid protein linked to a sequence encoding the yeast GALA activation protein.
  • the portion of the chimeric gene that encodes the HSV-2 capsid protein encodes the mature capsid
  • the capsid precursor protein may also be usefully employed.
  • Chimeric genes are inserted into Saccharomyces cerevisiae plasmids and the plasmids are introduced in S. cerevisiae which contains an integrated GALA- responsive lacZ indicator gene.
  • S. cerevisiae which contains an integrated GALA- responsive lacZ indicator gene.
  • fusion proteins are produced.
  • the portions of the fusion proteins comprising the HSV-2 capsid protein will, under selected condition bind to each other and thereby bring together the DNA-binding domain and activation domain of GALA.
  • the indicator gene When the two GALA domains which are in close proximity interact with the GAL4-responsive lacZ indicator gene, the indicator gene is expressed and, under the proper conditions a detectable blue color is observed. If the fusion proteins are prevented from binding, the two GALA domains will not be present in proximity to each other and the indicator gene will not be activated. Thus, no blue color will be present to observe.
  • this yeast system provides a rapid and specific assay for the interaction of HSV-2 capsid proteins that occur ' during virion assembly. In the presence of compounds which interrupt or inhibit HSV-2 capsid protein interaction, the GALA domains in the fusion proteins produced by expression of the chimeric genes will not associate and thereby will not activate the lacZ gene in the yeast system.
  • compounds may be identified by the absence of activation of the lacZ gene in transformed yeast which inhibit HS V-2 capsid assembly and therefore possess anti-viral properties.
  • Some embodiments of the present invention provides methods of distinguishing between samples containing HSV-1 DNA and samples containing HSV-2 DNA or samples containing HSV-1 proteins and samples containing HSV-2 proteins. Accordingly, the present invention provides a method of diagnosing whether an individual is infected with HSV-1 and/or HSV-2. Methods are disclosed for identifying whether an individual is infected with HSV-1 and/or HSV-2 wherein HSV-1 infection can be distinguished from HSV-2 infection.
  • PCR technology is used to distinguish between samples containing HSV-1 DNA and samples containing HSV- 2 DNA.
  • Such methods provide a means for distinguishing between HSV-1 and HS V-2 infections and allow for the diagnosis of the type of HS V infection an individual has.
  • Specific primers are designed that will provide for amplification of HSV-1 DNA but not HSV-2 DNA and/or HSV-2 DNA but not HSV-1 DNA.
  • the nucleotide sequence of the UL26 gene including the nucleotide sequence which encodes the HSV-2 protease and the HSV-2 capsid protein is disclosed in SEQ ID NO: 1.
  • the nucleotide sequence encoding HS V- 1 protease and HSV-1 capsid protein are disclosed in SEQ ID NO: 16.
  • a set of PCR primers were designed which amplify HSV-2 sequences but not HSV-1 sequences. Thus, detection of amplified DNA indicates that HSV-2 is present.
  • a set of PCR primers were designed which amplify HSV-1 sequences but not HSV-2 sequences. Thus, detection of amplified DNA indicates that HSV-1 is present.
  • both sets of primers are provided and used in separate amplification protocols with material from the same sample in order to provide an additional control.
  • Other optional controls include positive controls which contain DNA sequences that will be amplified and/or negative controls that cannot be amplified by the primers.
  • Amplified DNA may be detected by running the material on an electrophoresis gel after the amplification protocol is complete. DNA molecules of the expected length of an amplification product may be provided as size markers.
  • Present invention also relates to kits for distinguishing whether a sample contains DNA from HSV-1 or HSV-2. The kits of the present invention are useful to diagnose whether an individual is infected with HSV- 1 and/or HS V-2.
  • kits contain containers which comprise primers that will amplify HSV-1 DNA but not HSV-2 DNA or containers that will amplify HSV-2 DNA but not HSV-1 DNA. Kits may optionally contain both sets of primers in separate containers for running separate amplification procedures using different portions of the same sample. Kits may optionally contain positive and/or negative controls in separate containers. Kits may optionally contain DNA molecules in a separate container which can serve as a size marker. The DNA molecule may be of the expected length of a DNA molecule amplified using the primers.
  • immunoassays are used to distinguish between samples containing HSV-1 protein and samples containing HSV-2 protein.
  • the immunoassays are used to distinguish between HSV-1 and HS V-2 infections and diagnose the type of HSV infection an individual has.
  • Such immunoassays are based upon differences between UL26 gene products of HSV- 1 and HSV-2 or between UL26.5 gene products of HSV- 1 and HSV-2.
  • Immunoassays may be based upon differences in proteases and/or capsid proteins of HSV-1 and HS V-2.
  • Specific antibodies are provided which selectively bind to epitopes on HSV-1 antigens not present on HSV-2 antigens or which selectively bind to epitopes on HSV-2 antigens not present on HSV-1 antigens.
  • specific antibodies are provided which selectively bind to HSV-1 protease but. not HSV-2 protease or which selectively bind to HSV-2 protease but not HSV-1 protease.
  • specific antibodies are provided which selectively bind to HSV- 1 capsid but not HSV-2 capsid or which selectively bind to HSV-2 capsid but not HSV-1 capsid.
  • HSV- 1 -specific antibodies or the HSV-2-specific antibodies bind to proteins in the sample and therefore whether the individual from which the sample was taken is infected with HSV-1 and/or HSV-2.
  • the amino acid sequence of HSV- 2 active protease precursor spans amino acids 1-638 in SEQ ID NO:l and SEQ ID NO:2.
  • the amino acid sequence of HSV-2 mature protease spans amino acids 1-247 of SEQ ID NO:l and SEQ ID NO:2.
  • the amino acid sequence of HSV-2 capsid precursor spans amino acids 310-638 in SEQ ID NO:l and SEQ ID NO:2.
  • the amino acid sequence of HSV-2 mature capsid spans amino acids 310-613 of SEQ ID NO:l and SEQ ID NO:2.
  • the amino acid sequence of HSV-1 protease and capsid are disclosed in SEQ ID NO:17.
  • the amino acid sequence of HSV-1 active protease precursor spans amino acids 1-635 in SEQ ID NO: 17.
  • the amino acid sequence of HSV-1 mature protease spans amino acids 1-247 of SEQ ID NO:17.
  • the amino acid sequence of HSV- 1 capsid precursor spans amino acids 307-635 in SEQ ID NO: 17.
  • the amino acid sequence of HSV- 1 mature capsid spans amino acids 307-610 of SEQ ID NO: 17.
  • Antibodies which specifically bind to HSV-2 protease but not HSV-1 protease may be produced by those having ordinary skill in the art using routine methods and widely available starting materials. Likewise, antibodies which specifically bind to HSV-2 capsid but not HSV-1 capsid may be produced by those having ordinary skill in the art using routine methods and widely available starting materials. Either of these HSV-2 specific antibodies are used to detect HSV-2 in an immunoassay which can distinguish HSV-1 from HSV-2. Similarly, antibodies which specifically bind to HSV-1 protease but not HSV-2 protease may be produced by those having ordinary skill in the art using routine methods and widely available starting materials.
  • HSV-1 capsid antibodies which specifically bind to HSV-1 capsid but not HSV-2 capsid may be produced by those having ordinary skill in the art using routine methods and widely available starting materials.
  • HSV-1 specific antibodies are used to detect HSV-1 in an immunoassay which can distinguish HSV-1 from HSV-2. It is preferred that both immunoassays be performed using material from the same sample in order to provide an additional control.
  • Other optional controls include positive controls which include peptides which will bind to the antibody used in the immunoassay and/or negative controls which include peptides which will not bind to the antibody used in the immunoassay.
  • Antibodies may be labelled.
  • an antibody that specifically binds to the HSV specific antibodies may be used.
  • One having ordinary skill in the art can readily produce immunoassays including all necessary reagents using the information provided herein.
  • HSV-1 protease antibody produced by Serotech as Antibody 45KD and commercially available from Bioproducts for Science Inc. as catalog number MCA406 (P.O. Box 29176, Indianapolis, IN) can be used in immunoassays to distinguish HSV-2 from HSV-1.
  • the Serotech antibody binds to HSV-1 precursor or mature capsid protein but not HS V-2 precursor or mature capsid protein. Accordingly, an immunoassay using the Serotech antibodies may be performed to determine if a sample contains HSV-1 or HSV-2 and thus if the individual from whom the sample was taken is infected with HSV- 1 or HS V-2.
  • kits for diagnosing whether an individual is infected with HSV-1 or HSV-2 may comprise a container comprising antibodies which bind to HSV-1 protease but not HSV-2 protease and/or a container comprising antibodies which bind to HS V-2 protease but not HSV-1 protease. It is preferred that the kit comprises both types of antibodies in separate containers. Antibodies used in the kits may be labelled. The kits contain all other reagents and materials for performing an immunoassay with the antibodies. Kits may optionally contain positive and/or negative controls in separate containers. Kits may optionally contain means to detect the antibody including, for example a second antibody which specifically binds to the anti-HSV protease antibody.
  • kits of the present invention may comprise a container comprising antibodies which bind to HSV-1 capsid but not HSV-2 capsid and/or a container comprising antibodies which bind to HSV-2 capsid but not HSV-1 capsid. It is preferred that the kit comprises both types of antibodies in separate containers. Antibodies used in the kits may be labelled. The kits contain all other reagents and materials for performing an immunoassay with the antibodies. Kits may optionally contain positive and/or negative controls in separate containers. Kits may optionally contain means to detect the antibody including, for example a second antibody which specifically binds to the anti-HSV capsid antibody. Kits may comprise the Serotech antibody.
  • the HSV-2 protease promoter may be synthesized or isolated and linked to coding sequences which encode proteins other than HSV-2 protease. Accordingly, the present invention relates to recombinant DNA molecules which comprise at least a portion of the nucleotide sequence between nucleotides 1-534 of SEQ ID NO:l operably linked to a nucleotide sequence that encodes a protein other than HS V-2 protease.
  • the present invention relates to cells which comprise DNA molecules which comprise at least a portion of the nucleotide sequence between 1 and 534 of SEQ ID NO:l operably linked to a nucleotide sequence that encodes a protein other than HS V-2 protease.
  • Another aspect of the invention applies to bacteriophage lambda clones which harbor HSV-2 UL26 gene (SEQ. I.D. No.:l) and sequences upstream and downstream of the gene. Accordingly, the linked sequences can be used to screen for UL26 promoter regulatory and/or enhancer regions.
  • the HSV-2 capsid protein promoter is located upstream of nucleotide 1461 of SEQ ID NO:l. It may be synthesized or isolated and linked to coding sequences which encode proteins other than HSV-2 capsid protein. Accordingly, the present invention relates to recombinant DNA molecules which comprise at least a portion of the nucleotide sequence upstream of nucleotide 1461 of SEQ ID NO: 1 operably linked to a nucleotide sequence that encodes a protein other than HSV-2 capsid protein.
  • the present invention relates to cells which comprise DNA molecules which comprise at least a portion of the nucleotide sequence upstream of nucleotide 1461 of SEQ ID NO:l operably linked to a nucleotide sequence that encodes a protein other than HS V-2 capsid protein.
  • Nucleotides 1191 to 1461 (SEQ ID NO: 1 ), for example, were linked to the chloramphenicol acetyl transferase gene and shown to possess significant promoter activity when transfected into VERO cells
  • the HSV-2 protease can be employed using molecular and biochemical technology in in vitro assays identify inhibitors of this activity by rational design and screening and to test these inhibitors for antiviral activity in infected cells.
  • the HSV-2 UL26 gene was cloned as an Ncol-EcoRI fragment (1938 base pairs) which contained the start codon, the entire open reading frame, the stop codon, and 22 base pairs of 3'-unrranslated sequence.
  • Full-length HSV-2 UL26 was expressed in E. coli using the pOTS vector system in which the gene is inserted downstream of the strong and tightly regulated P L promoter from bacteriophage lambda of the pOTS-207 vector. Tight regulation of the promoter is essential when expressing genes that are likely to be toxic to the cells, such as proteases.
  • the 27 KD protease domain corresponding to one of the autoproteolytic products derived from the HSV-2 UL26 primary translation product was produced in E. coli using the tightly regulated expression vector p ⁇ T-16(b) (Novagen, Madison W.I.) which contains the T7 promoter.
  • Each construct was designed to include six histidine codons and the (aspartate)4lysine codons preceding the HS V-2 UL26 start codon so that the expressed protein will contain a cleavable histidine tag at the N-terminus for purification of the protein on Nickel columns. Other chelating columns may be used.
  • the His-tagged protein is eluted from the column by addition of imidazole Alternatively, it can be eluted by other means such as pH change. Columns and technical protocols useful to purify protein may be obtained from commercially available sources such as Qiagen.
  • the recombinant constructs are then introduced into E. coli AR120 (nalidixic acid inducible strain) and E.
  • coli AR58 heat inducible strain
  • T7 promoter vectors the recombinant constructs are introduced into E. coli BL21, an IPTG inducible strain.
  • the proteins can be readily purified by chromatography on nickel chelate column.
  • the p27 protease fragment is active as shown by its ability to remove the last 25 amino acids from a construct comprising most of the UL26.5 coding region.
  • the p27 protease gene was synthesized to contain codons characteristic of highly expressed E. coli genes, yet maintaining the amino acid sequence of p27 protease.
  • the synthesized gene was placed downstream of the tightly regulated T7 promoter in the expression vector pET-16(b). Following IPTG (ImM) induction the 27 k Da protein domain was highly expressed in E. coli.
  • HSV-2 UL26 gene (N ⁇ ?l-Ec ⁇ RI fragment) and the p27 protease is cloned into the insect cell expression vector pVL1392.
  • the recombinant construct is then introduced into insect cells derived from Spodopterafrugiperda. High titer viral stocks are then prepared for protease activity analysis and subsequent scale up for protein production.
  • UL26 gene and HSV-2 UL26 gene that shared the least amount of identity to ensure the specificity of the assay.
  • Such a region can easily be viewed by computer analysis comparing the two DNA sequences disclosed in SEQ ID NO:l and SEQ ID NO: 16, respectively.
  • the region of least identity between the two homologs lies within the UL26.5 domain, i.e. the portion of the gene that encodes the capsid.
  • the following provides the sequences of the primers used and the locations of the primers are given based on the nucleotide numbers given in the nucleotide sequence comparison provided in the enclosed computer analysis. As shown below it is helpful to design a system to generate HSV-1 and HSV-2 specific products of different sizes to improve the analysis.
  • SEQ ID NO:20 HSV-1 S'- ⁇ CCGGCTCCCCCACCTGA-S' (#1560-1542)
  • SEQ ID NO:21 HSV-2 S'-ATTCGGATCCTGGAGGCGA-S' (#2470-2452)
  • Separate PCR amplification protocols are performed on samples suspected of containing either HSV-1 or HSV-2 DNA using SEQ ID NO: 18 and SEQ ID NO:20 in the HSV-1 assay or SEQ ID NO: 19 and SEQ ID NO:21 in the HSV-2 assay. If a DNA fragment of 696 base pairs is generated in the HSV-1 assay, the presence of HSV-1 DNA in the sample is indicated. To detect the presence of a 696 base pair fragment, the amplification product is migrated through an electrophoresis matrix. A size marker of DNA of about 696 base pairs is run through the same matrix simultaneously. If a DNA fragment of 1073 base pairs is generated in the HSV-2 assay, the presence of HSV-2 DNA in the sample is indicated. To detect d e presence of a 1073 base pair fragment, the amplification product is migrated through an electrophoresis matrix. A size marker of DNA of about 1073 base pairs is run through the same matrix simultaneously.
  • a kit which comprises a container comprising SEQ ID NO: 18 and SEQ ID NO:20 in the HSV-1 assay.
  • a kit which comprises a container comprising SEQ ID NO: 19 and SEQ ID NO:21 in the HSV-2 assay.
  • a kit which comprises both a container comprising SEQ ID NO: 18 and SEQ ID NO:20 in the HSV-1 assay and a container comprising SEQ ID NO: 19 and SEQ ID NO:21 in the HSV-2 assay.
  • Size marker DNA may optionally be provided.
  • a size marker of 696 base pairs is provided.
  • a size marker of 1073 base pairs is provided.
  • a size marker of 696 base pairs and a size marker of 1073 base pairs are provided.
  • the DNA fragment was cloned by the polymerase chain reaction using the sense strand primer (5'-AACATGAGCTGCGTGACC-3') spanning nucleotide # 1191 to # 1209 of SEQ ID NO: 1 and the antisense strand primer (5'-AAAGAAGAAGAAGAAGAC-3') spanning nucleotides #1447 to # 1429 of SEQ ID NO: 1
  • Promoter activity is tested by cloning the 256 base pair PCR fragment upstream of the chloramphenicol acetyltransferase (CAT) reporter gene in the commercially available vector pCAT Basic (Promega).
  • the resulting construct can then be introduced into a suitable mammalian cell line, e.g., Vero cells, to test for promoter activity by analyzing the levels of CAT activity.
  • a suitable mammalian cell line e.g., Vero cells
  • the cell line is devoid of endogenous CAT activity; hence, after introducing the promoter construct into such a cell line, the levels of CAT activity is a direct measure of HSV-2 UL26.5 promoter activity.
  • Vero cells were grown in DMEM+10% FCS containing Gentamicine
  • HSV-2 UL26.5/pCAT construct 15 micograms of the HSV-2 UL26.5/pCAT construct was electroporated into 5 million Vero cells using standard protocols. 48 hrs after electroporation cells were harvested in 100 microliters of 0.25 M Tris buffer pH 8.0. Cells were lysed by repeated freeze-thaw, spun down at 15,000 rpm and the supernatants were transferred to fresh tubes. Total protein concentration was determined using Bio- Rad Protein Assay Dye Reagent Kit (Cat. # 500-0006).
  • control vector pCAT control (contains the S V40 promoter and enhancer) was used as a comparison of HSV-2 UL26.5 promoter strength.
  • Figure 2 summarizes the results of four experiments.
  • Column 1 is a negative control and represents CAT expression in the absense of promoter and enhancer transcriptional control elements.
  • Column 2 a positive control, employs SV40 promoter and SV40 enhancer elements to drive CAT gene expression.
  • Column 3 represents CAT gene expression driven by UL26.5 promoter alone and
  • Column 4 represents CAT gene expression when the UL26.5 promoter is used in combination with the S V40 enhancer element.
  • the promoter described here are useful for regulating the expression of heterologous genes when operably linked thereto.
  • MOLECULE TYPE protein
  • MOLECULE TYPE peptide
  • SEQUENCE DESCRIPTION SEQ ID NO : 6 :
  • ATC GTC ACC TAC GAC ACC GGT CTC GAC GCC GCC ATC GCG CCC TTT CGC 528 He Val Thr Tyr Asp Thr Gly Leu Asp Ala Ala He Ala Pro Phe Arg 165 170 175

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Abstract

L'invention porte sur des produits géniques pratiquement purs dits HSV-2 UL26 et ses fragments parmi lesquels la protéase mature HSV-2 et ses fragments actifs, sur des produits géniques pratiquement purs dits HSV-2 UL26.5 et ses fragments parmi lesquels la protéine capside HSV-2 et ses fragments fonctionnels, sur des molécules d'acide nucléique comprenant tout ou partie des gènes HSV-2 UL26 et/ou HSV-2 UL26.5, sur des vecteurs d'expression et des cellules hôtes comprenant les acides nucléiques et les molécules susdits, sur des méthodes d'identification des composés inhibant l'activité de la protéase HSV-2 et sur des méthodes d'identification des composés inhibant les ensembles de virions du HSV-2, sur des substrats de synthèse du HSV-2, sur des anticorps se fixant sélectivement aux substrats traités par la protéase du HSV-2 mais pas aux substrats non traités ou alors à des substrats non traités et pas à des substrats traités , g) sur des méthodes et trousses permettant de distinguer l'ADN ou les protéines HSV-1, de l'ADN et les protéines HSV-2 ainsi que les réactifs associés.
EP94925924A 1993-08-20 1994-08-19 Gene hsv-2 ul26, proteines capsides, dosages immunologiques et inhibiteurs de protease Withdrawn EP0714399A4 (fr)

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US8460674B2 (en) 2009-02-07 2013-06-11 University Of Washington HSV-1 epitopes and methods for using same
WO2010115172A2 (fr) 2009-04-03 2010-10-07 University Of Washington Peptide antigénique de hsv-2 et procédés d'utilisation de celui-ci
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CN101974613A (zh) * 2010-10-15 2011-02-16 成都医学院 一种筛选抗hsv-1药物的试剂盒和方法
CA2856697A1 (fr) 2010-11-24 2012-06-07 Genocea Biosciences, Inc. Vaccins contre le virus de type 2 de l'herpes simplex: compositions et procedes pour la stimulation d'une reponse immunitaire
JP6199878B2 (ja) 2011-11-23 2017-09-20 ジェノセア バイオサイエンシーズ， インコーポレイテッド 単純ヘルペスウイルス２型に対する核酸ワクチン：免疫応答を誘発する組成物及び方法
CN107305213A (zh) * 2016-04-25 2017-10-31 赵芳 一种检测有机磷类和氨基甲酸酯类农药的方法及试剂盒
CN107305212B (zh) * 2016-04-25 2019-08-30 赵芳 一种有机磷类和氨基甲酸酯类农药的免疫学检测方法及试剂盒
JP2019537555A (ja) 2016-09-28 2019-12-26 ジェノセア バイオサイエンシーズ， インコーポレイテッド ヘルペスを処置するための方法および組成物

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EP0714399A4 (fr) 1999-01-27
CA2169748A1 (fr) 1995-03-02
JPH09503385A (ja) 1997-04-08
WO1995006055A1 (fr) 1995-03-02
MXPA94006367A (es) 2004-09-09
AU7568294A (en) 1995-03-21

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