WO2000061794A1 - Methods and compositions involving posttranslational modification of herpesvirus protein us1.5 - Google Patents

Methods and compositions involving posttranslational modification of herpesvirus protein us1.5 Download PDF

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WO2000061794A1
WO2000061794A1 PCT/US2000/009215 US0009215W WO0061794A1 WO 2000061794 A1 WO2000061794 A1 WO 2000061794A1 US 0009215 W US0009215 W US 0009215W WO 0061794 A1 WO0061794 A1 WO 0061794A1
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seq
polypeptide
peptide
amino acids
noj
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PCT/US2000/009215
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French (fr)
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Bernard Roizman
William O. Ogle
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Arch Development Corporation
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    • 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
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
    • C07K16/085—Herpetoviridae, e.g. pseudorabies virus, Epstein-Barr virus
    • C07K16/087—Herpes simplex virus
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16622—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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

  • the present invention relates generally to the fields of molecular biology and virology. More particularly, it concerns the use of polypeptides, peptides, and polynucleotides derived from U s 1.5 of he ⁇ es simplex virus to inhibit infection by a he ⁇ esvirus and to identify candidate substances that inhibit infection by the virus.
  • He ⁇ esviridae is comprised of members (he ⁇ esviruses) generally possessing (1) linear, double-stranded DNA, (2) an icosadeltahedral capsid, (3) tegument protein, and (4) an envelope.
  • He ⁇ esviruses have been isolated from humans, and these include the he ⁇ es simplex viruses (HSV), Epstein-Barr virus, human cytomegalovirus, varicella-zoster virus, human he ⁇ esvirus 6, and human he ⁇ esvirus 8 (Fields et al, 1991).
  • He ⁇ es simplex viruses designated with subtypes 1 and 2 are among the most common infectious agents encountered by humans, infecting millions worldwide. These viruses cause a broad spectrum of disease, ranging from relatively benign to severe and life-threatening.
  • Human he ⁇ esviruses can cause productive lytic infections, where infectious virus is produced and cells are lysed, or nonproductive lytic infection, where viral DNA remains intact but cells survive because full replication does not occur. They also have the properties of latency and reactivation; following an acute lytic infection, these viruses frequently persist in a latent state until reactivation causes a recurrent lytic infection.
  • HSV-1 and HSV-2 The only known natural known host for HSV is a human. Infection by either subtypes of he ⁇ es simplex viruses, HSV-1 and HSV-2, can be identified by their characteristics, such as antigenicity, clinical and epidemiologic patterns, DNA base composition, biologic properties, and sensitivity to physical and chemical stress. (Whiteley et al. 1998). Primary infection by HSV-1 occurs mostly during childhood, while it occurs by HSV-2 mainly in sexually active adolescents and young adults.
  • the primary mode of transmission is direct contact with infected secretions.
  • HSV-1 infection can be evidenced by certain clinical syndromes including oral-labial he ⁇ es, ocular he ⁇ es, and encephalitis. Infection by HSV-2 is associated with genital he ⁇ es, perianal and anal he ⁇ es, and he ⁇ etic whitlows.
  • the invention encompasses methods for identifying substances that inhibit infection by a he ⁇ esvirus as well as preventative and therapeutic compounds that permit treatment or prevention of he ⁇ esvirus infections using one or more regions of
  • the present invention includes, in some embodiments, a method for screening candidate substance for inhibition of he ⁇ esvirus infections. This method can be accomplished by obtaining a polypeptide or peptide that contains a U s 1.5 phosphorylation site, adding the candidate inhibitor to a mixture containing the U s 1.5 polypeptide or peptide, and further incubating a kinase with the mixture under conditions appropriate to allow phosphorylation of the UJ .5 polypeptide or peptide.
  • the method also includes determining the phosphorylation state of the U s 1.5 polypeptide or peptide and comparing its phosphorylation state to a polypeptide or peptide similar to the U s 1.5 moiety initially used, such as an aliquot of the same U s 1.5 polypeptide initially used and incubated with a kinase but that was not exposed to the candidate inhibitor.
  • Contacting refers to the coming together or touching of compounds. It is understood that a "similar polypeptide” or “similar polypeptide” is a U s l .5-related polypeptide or peptide that contains a U s l .5 phosphorylation site.
  • the method employs a kinase, such as U L 13 from HSV, that is incubated with a U s 1.5 phosphorylation site to do a comparative analysis of phosphorylation levels of a U s 1.5 peptide or polypeptide that has been exposed to a candidate inhibitor and one that has not.
  • a kinase such as U L 13 from HSV
  • the kinase U s 3 could be used to practice this method.
  • the U s 1.5 polypeptide that contains a phosphorylation site also includes a detectable label, which will aid in determining the phosphorylation state of the polypeptide.
  • the invention's methods further include the use of polypeptide regions including at least about 10 consecutive amino acids, at least about 15 consecutive amino acids, and at least about 20 consecutive amino acids, and at least about 25 consecutive amino acids from one or more of the following sets of sequences: SEQ ID NOJ, SEQ ID NO:2, SEQ ID NOJ. SEQ ID NOJ, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8. Therefore, it is contemplated that a polypeptide can contain amino acids from more than one set of sequences.
  • the present invention also discloses the use of p60, a cellular protein, to determine the phosphorylation state of the U s 1.5 polypeptide based on p60's ability to bind the fast-migrating, unde ⁇ rocessed isoform of wild-type ICP22, a protein that is colinear with U s 1.5.
  • the present invention also describes peptide and polypeptide compositions that can inhibit posttranslational modification of U s 1.5 produced by a he ⁇ esvirus. Because posttranslational modification of ICP22 and U s 1.5 is required for infection to progress, these compositions effectively act to inhibit or prevent he ⁇ esvirus infections, and thus, constitute a treatment of he ⁇ esvirus infections. An excess of these compositions can inhibit a he ⁇ esvirus infection by competing with protein products produced by he ⁇ esvirus as a cellular target for posttranslational modification, thereby preventing the modification of the he ⁇ esvirus protein.
  • Such a peptide includes at least about 10 consecutive amino acids, at least about 15 consecutive amino acids, at least about 20 consecutive amino acids, and at least about 25 consecutive amino acids from one or more of the following sets of sequences: SEQ ID NOJ , SEQ ID NO:2, SEQ ID NOJ, SEQ ID NOJ, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8.
  • a polypeptide can contain amino acids from more than one set of sequences.
  • the peptide includes a U s l .5 phosphorylation site or at least two U s 1.5 phosphorylation sites.
  • UJ..5 produced by he ⁇ esvirus are composed of about 20 consecutive amino acids to about 100 consecutive amino acids; the peptides contain a first region of at least about
  • peptides in some examples of the present invention, contain at least one UJ .5 phosphorylation site; while in others, the peptide contains at least two U s 1.5 phosphorylation sites.
  • first region and the second region are linked by one or more amino acids that are not the native U s 1.5 sequence (U s 1.5 amino acids 172-379).
  • Non-native sequence linkers include the use of synthetic linkers such as a non-amino acid linking moiety.
  • the invention also includes a polypeptide that binds to a phosphorylation site in U s 1.5, which would include an antibody, a single-chain antibody, or an anti- idiotypic antibody that binds immunologically to the antigen binding region of an anti-U, 13 antibody.
  • he ⁇ esvirus infection by giving a cell an inhibitor of UJ .5 phosphorylation.
  • This method contemplates that the cell is infected with a he ⁇ esvirus either prior to or after it being given an inhibitor of UJ .5 phosphorylation.
  • the he ⁇ esvirus is a he ⁇ es simplex virus, such as HSV-1 or HSV-2.
  • a polypeptide that contains a site similar to a site for U s l .5 phosphorylation but that is incapable of being modified is utilized.
  • Another method of the present invention is inhibiting he ⁇ esvirus infection by contacting a cell with a polypeptide that binds to a UJ .5 phosphorylation site.
  • this polypeptide prevents phosphorylation of a U 1.5 phosphorylation site by masking the site, and thereby effects inhibition of infection.
  • Other examples of a polypeptide that binds to a U s 1.5 phosphorylation site include an antibody.
  • FIG. 1 Schematic representation of the construction of recombinant viruses.
  • FIG 1A Line 1, sequence arrangement of the HSV-1 genome showing the unique long (U ), unique short sequences, and the location of the ⁇ genes 0, 4, and 22. Lines 2- 6, domains of the HSV-1 cosmid set used for the construction of recombinants.
  • FIG IB construction of the ⁇ 22/ ⁇ U s 1.5 virus. Line 2, expansion of the S component of HSV-1 DNA. Lines 3, 4 and 5, domains of cosmids pBClOl ⁇ and pBC1004 and plasmid pRB5213. Plasmid pBR5213 was constructed to bridge the gap in the nonoverlapping cosmids pBClOl ⁇ and pBC1004. In this plasmid the ⁇ 22/U s l .5 open reading frame was replaced with a unique Eagl restriction site.
  • FIG. 2 Schematic representation of the recombinants with an amino-terminal deletion in the ⁇ 22 gene.
  • Line 1 Bam HI N fragment of HSV- 1 (F) showing the open reading frames of ICP22 (open rectangles) and U s 1.5 along with their mRNA transcripts (thin lines).
  • the open reading frames of ICP22 and UJ .5 were replaced with a unique Eag I restriction site.
  • Line 3 R7804 (R7802 repair).
  • the Bam HI N fragment (pRB5214) was restored.
  • Line 4 schematic diagram of the Bam HI N in R7805 ( ⁇ 22/U s 1.5) recombinant virus.
  • Line 5 representation of Bam HI N fragment of R7806 (R7805 repair).
  • the Bam HI N fragment (pRB5210) was restored.
  • Line 6, representation of the Bam HI N fragment in R7808 ( ⁇ 22/U s 1.5).
  • the open reading frame of U s 1.5 was cloned into the unique Eagl site of pBR5212 to yield pRB5215.
  • BamHI N fragment (pRB5210) from wild type virus HSV- 1(F) was restored.
  • FIG. 3 Autoradiographic images of electrophoretically-separated Bam HI fragments of recombinant viral DNA hybridized with 32 P-labeled pRB5210. The fragments were electrophoretically separated in a 0.8% agarose gel, transferred to Zeta-probe membrane, and hybridized with 32 P-labeled pRB5210 carrying the HSV-1 (F) BamHI N fragment.
  • Lane 1 the 4.9-kb Bam HI N fragment from HSV-1 (F); lane 2, the 4.9-kb Bam HI N fragment from the R7905 (HSV- 1(F)) cosmid virus; lane 3, the 3.6-kb Bam HI N fragment form R7802 recombinant virus; lane 4, the 3.4-kb BamHI N fragment from the R7805 recombinant virus.
  • FIG. 4 Schematic representation of terminal sequences of the recombinants carrying 3' terminal deletions in the ⁇ 22/U s 1.5 genes of HSV-1 (F).
  • FIG1 A Bam HI N sequence arrangements in recombinants. The rectangles represent the open reading frames. The filled boxes represent the carboxyl-terminal 40 codons. The number in parentheses indicates the number of codons deleted from the terminus of the open reading frame.
  • FIG. 5 Photograph of an immunoblot of electrophoretically separated lysates of cells mock-infected or infected with HSV-1 (F) or R7356 ( ⁇ U L 13) and reacted with antibody to ICP22/ J .5.
  • Vero cells (VC) and rabbit skin cells (RSC) harvested at various times (4 - 24 h) after infection were solubilized, subjected to electrophoresis in a denaturing 10% polyacrylamide gel, transferred to a nitrocellulose sheet, and reacted with the rabbit polyclonal antibody (W2) made against the carboxyl-terminal 138 amino acids of ICP22/U S 1.5.
  • FIG. 6 Photograph of an immunoblot of electrophoretically-separated lysates of cells mock-infected or infected with HSV- 1(F), R7802, R7804, R7805,
  • Vero cells harvested at 18 h after infection were solubilized, subjected to electrophoresis in a denaturing 8% polyacrylamide gel, transferred to a nitrocellulose sheet, and reacted with the polyclonal antibody W2.
  • FIG. 7 Schematic representations of the ICP22 open reading frame of wild type and mutant HSV-1 showing the location of methionine codons.
  • Line 1 position of the four methionine codons in the first 200 amino acids of HSV-1 ;
  • line 2 position of the four methionine codons in the first 200 amino acids of HSV-1 ;
  • R7815 ( ⁇ 22 N1 /U s 1.5)[ ⁇ 47a.a.] lacking 47 amino acids deleted from the amino-terminal.
  • the first methionine available for translational initiation is at codon 90; line 3, R7805 ( ⁇ 22 T /U s 1.5)[ ⁇ 138a.a.], lacking the amino-terminal 138 amino acids.
  • the first methionine available for translational initiation is at codon 147; line 4, R7808 ( ⁇ 22 Nr /U s 1.5)[ ⁇ 171a.a.] lacking amino-terminal 170 amino acids.
  • the first methionine available for translational initiation is at codon 171.
  • Vero cells harvested at 18 h after infection were solubilized, subjected to electrophoresis in a denaturing 10% polyacrylamide gel, transferred to a nitrocellulose sheet, and reacted with the polyclonal antibody prepared against the carboxyl-terminal amino acids of ICP22/U S 1.5.
  • Lane 1 R7815 ( ⁇ 22 /U s 1.5)[ ⁇ 47a.a.]; lane 2,- R7805( ⁇ 22/UJ .5); lane 3, R7808 ( ⁇ 22 U s 1.5).
  • the numbers next to the arrows indicate the initiator methionine for the unprocessed protein product in each lane.
  • FIG. 9 Photograph of an immunoblot of electrophoretically-separated lysates of cells mock-infected or infected with HSV- 1(F), R7802, R7804, R7805, R7806, R7808 or R7828 and reacted with antibodies to U L 38 and U s l l .
  • Replicate cultures of Vero cells (VC) or rabbit skin cells (RSC) harvested at 18 h after infection were solubilized, subjected to electrophoresis in a denaturing polyacrylamide gel, transferred to a nitrocellulose sheet, then sequentially reacted with the monoclonal antibody to U s l l, and the polyclonal antibody to U L 38.
  • VC Vero cells
  • RSC rabbit skin cells
  • Lanes 1 and 2 HSV-l(F); lanes 3 and 4, R7802 ( ⁇ 22/ ⁇ U s 1.5); lanes 5 and 6, R7804 (R7802 repair); lanes 7 and 8, R7805 ( ⁇ 22 T /U s 1.5); lanes 9 and 10, R7806 (R7805 Repair); lanes 1 1 and
  • R7808 ( ⁇ 22 NT /U s 1.5 ⁇ 22p); lanes 13 and 14, R7828 (R7808 repair).
  • FIG. 10 Photograph of an immunoblot of electrophoretically-separated nuclear and cytoplasmic fractions of HEp-2 cells infected with HSV-l(F), R7805, R7808, or R7810. Infected Hep-2 cells were harvested and lysed by the addition of
  • Nonidet P-40 Nuclear and cytoplasmic fractions prepared as described in Materials and Methods were solubilized, subjected to electrophoresis on an SDS-10% polyacrylamide gel, transferred to nitrocellulose, and reacted with the polyclonal antibody to ⁇ 22/UJ .5 protein. ICP22 and U s 1.5 protein are indicated on the right, and molecular weights (in thousands) are shown on the left. Lanes 1 and 2,
  • N nuclear fraction
  • C cytoplasmic fraction.
  • FIG. 11 Photograph of an immunoblot of electrophoretically-separated lysates of cells infected with HSV-l(F), R7819, R7822. R7823, R7820, R7810 or
  • Vero cells harvested at 18 h after infection were solubilized and subjected to electrophoresis in denaturing 10% polyacrylamide gel, transferred to a nitrocellulose sheet, and reacted with the polyclonal antibody R77 against ICP22.
  • Lane 1 HSV- 1(F); lane 2, R7819 ( ⁇ 22/UJ 5 ⁇ c ⁇ l ⁇ a a ); lane 3, R7822 ( ⁇ 22/U s 1.5 ⁇ r ⁇ 5a a ); lane 4 R7823 ( ⁇ 22/U s 1.5 ⁇ c ⁇ l8a a ); lane 5, R7820 ( ⁇ 22/US1.5ACT22a.a.); lane 6, R7810 ( ⁇ 22/U s 1.5 ⁇ CT40a a .), lane 7, R7821 (R7810 repair).
  • FIG. 12 Schematic representation of the functional domains of the ⁇ 22 gene and its products, ICP22 and U s 1.5 protein.
  • FIGJ2A Functional maps. The zones are assigned on the basis of amino acid composition. Zones 1, 3, 5 and 8 are basic whereas zones 2, 4 and 7 are acidic.
  • FIGJ2B The sequence of the internal homologous repeats. The sequence alignments are Line 1, HSV-1 amino-terminal vs HSV-1 carboxyl-terminal; Line 2, HSV-2 amino-terminal vs. HSV-2 carboxyl terminal; Line 3, HSV-1 amino-terminal vs. HSV-2 amino-terminal; Line 4, HSV-1 carboxyl-terminal vs.
  • the present invention provides methods of screening for substances that may prevent or inhibit he ⁇ esvirus infections, as well as compositions containing peptides or polypeptides derived from U s 1.5 that prevent, inhibit, or treat infection by a he ⁇ esvirus.
  • the he ⁇ es simplex virus genome encodes greater than 80 genes whose expression is coordinately regulated and sequentially ordered during productive infection (Cassady et al., 1998; Ejercito et al, 1968; Ogle et al. 1997).
  • the first set of genes expressed immediately after productive infection is the genes followed by ⁇ and ⁇ genes.
  • ⁇ genes initially described four have regulatory functions, and of these three, the ⁇ genes 0, 4 and 27 have attracted considerable attention from the scientific community because they are essential for viral replication under all conditions tested.
  • ⁇ O encodes the infected cell protein (ICP) 0, a promiscuous transactivator important in early stages of infection; ICP4, the product of the ⁇ 4 gene, regulates gene expression both positively and negatively, whereas ICP27, the product of the ⁇ 27 gene, regulates posttranslational processing and transport of RNA (Pearson and Lipman, 1988).
  • ICP22 the product of the ⁇ 22 gene, attracted less attention, possibly because its functions were less apparent, obscured by the observation that it was dispensable for viral replication in cells in culture (Leopardi et al, 1997 ). Although the functions of the ⁇ 22 gene are the least well understood, five lines of evidence suggest that it plays an important role in viral replication:
  • the domain of the ⁇ .22 gene yields two mRNAs each expressed by its own promoter.
  • the ⁇ 22 mRNA initiates upstream from the open reading frame and is spliced; the first exon is in its 5' non-coding domain (Hardy and Sandri-Goldin, 1994; Ogle and Roizman, unpublished data; Rice et al, 1995).
  • Its product, ICP22 is a protein of 420 amino acids with alternating acidic and basic domains.
  • the second mRNA initiates in the coding domain of the ⁇ 22 gene and is driven by an independent promoter (B ni and Roizman, 1996).
  • ICP22 is extensively posttranslationally processed (Ackermann et al, 1984) as evidenced by the detection of phosphorylation and changes in electrophoretic mobility. ICP22 was shown to be phosphorylated largely by the protein kinase encoded by U, 13 and to a lesser extent by protein kinase encoded by U s 3 (Meignier et al, 1988; Mitchell et al, 1994). ICP22 is also nucleotidylylated by casein kinase II (Honess and Roizman, 1975; Kawaguchi et al, 1997).
  • the deletion mutant R325 generated by Post and Roizman (Leopardi et al, 1997) lacked the carboxyl-terminal 220 amino acids.
  • This mutant was highly attenuated in experimental animal systems (Honess and Roizman, 1974; Puives et al, 1987). It replicated to wild-type vims levels in Vero and HEp-2 cells but its ability to replicate in rodent or rabbit cells or in primary human fibroblasts was diminished. In these restricted cell lines, a subset of ⁇ 2 proteins exemplified by the product of s l 1 gene was significantly reduced (Mitchell et al, 1994).
  • ICP22 localizes in both the nucleus and cytoplasm (Everett et al.,
  • Nuclear ICP22 localizes early in infection in small dense nuclear stmctures. On onset of DNA synthesis, ICP22 colocalizes with ICP4, nascent DNA, RNA polymerase II and other cellular proteins (Hardwicke and Sandri-Goldin, 1 94). The displacement of ICP22 from the small nuclear dense structures requires the expression of the protein kinase encoded by U, 13. These results suggest that the products of the ⁇ 22 gene are involved in transcription of late genes - a conclusion consistent with the report that ⁇ 22 mediates an intermediate level of phosphorylation of the RNA polymerase II (Ng et al, 1997: Ogle and Roizman, 1998).
  • p78 was recently discovered to be identical in sequence to a protein reported to localize in nucleoli and designated MSP58 (Mitchell et al, 1997). Studies in this laboratory showed that p78 is made early in the S phase, has a short half-life and it binds the amino terminal domain of ICP22. In synchronized cells, during the expression of p78, the ICP22 exhibits novel posttranslationally processed forms.
  • the second protein designated p60, bound fast-migrating, unde ⁇ rocessed wild-type ICP22 and ICP22 lacking the carboxyl-terminal 24 amino acids but not
  • ICPO was independent of that of ICP22.
  • p60 was distributed throughout the cell.
  • wild-type vims infected HEp-2 cells p60 was translocated into the nucleus and formed dense bodies that colocalized with ICPO.
  • the posttranslational processing of p60 present in HEp-2 cells infected with wild-type or ICP22 mutant vimses could not be differentiated from that of uninfected cells whereas the p60 accumulating in rabbit skin cells infected with wild type vims differed in electrophoretic mobility from that made in uninfected cells.
  • p60 appears to be a linker protein capable of binding to and mediating the interaction of
  • the screening methods and therapeutic and preventative compounds related to he ⁇ esvirus infection disclosed herein take advantage of several sets of sequences encoded by ⁇ 22 that are required to be posttranslationally modified for viral replication in permissive cells to occur. More specifically, the methods and compositions of the present invention further take advantage of the observation that UJ .5 must be posttranslationally modified in a certain region or in certain regions by the U L 13 protein kinase to enable expression of a subset of late genes exemplified by U L 38 and U s l l .
  • Posttranslational processing is determined by several sets of sequences of U s 1.5: one set spans amino acids 38-66 (HSV-1 ) or amino acids 37-62 (HSV-2); another set spans from metl47 to metl 71 (HSV-1 ) or amino acids 143-167 (HSV-2), and this region is essential for posttranslational processing of U s l .5.
  • a third set contains amino acids 300-328 (HSV-1 ) or amino acids 295-320 (HSV-2), while another set includes lys402, met403, and arg404 and thus, comprises amino acids 380-
  • HSV-1 Due to the extensive homology between HSV-1 and HSV-2 in this region, the information disclosed herein is understood to extend to both HSV-1 and HSV-2. See FIG. 12B. For convenience, HSV-1 numbering is used, unless otherwise specified.
  • domains corresponding to sequences within the ⁇ 22 gene have been identified, it is contemplated that these domains — as either a polypeptide or peptide, or their cognate polynucleotides that contain or encode a UJ .5 phosphorylation site
  • U s 1.5 can be used to inhibit infection by a he ⁇ esvirus or to treat infection by a he ⁇ esvirus, whereby a subject infected by he ⁇ esvirus is conferred a therapeutic benefit. It is more specifically envisioned that domains of U s 1.5 containing sites for posttranslational modifications, particularly posttranslational modifications shown to be necessary for infection to progress, can be used to inhibit or treat a he ⁇ esvirus infection. It is understood that a region containing a U s 1.5 phosphorylation site could contain ICP22 sequences, including the entire ICP22 sequence.
  • polypeptide will be understood to mean an amino acid segment that is greater than about 100 contiguous amino acids in length, and the term “peptide” will be understood to mean an amino acid segment that is between about 6 and about 100 contiguous amino acids in length.
  • ICP22 and U s 1.5 segments of varying overall length that retain catalytic, regulatory or stmctural properties and functions are provided herein.
  • the use of the term “protein” in the context of ICP22 or U s 1.5 is understood according to the present invention to be synonymous with the term "polypeptide.”
  • a polypeptide is used as a target for posttranslational modification.
  • the ⁇ 22 gene of he ⁇ es simplex vims encodes two overlapping polypeptides, ICP22 and U s 1.5.
  • ICP22 contains unique sequences from ⁇ .22, while the entire sequence of U s 1.5 is colinear with the last 274 amino acids of ICP22. Since post-translation modification of U s 1.5 is required for viral infection to progress, a polypeptide product from the ⁇ 22 gene that contains U s 1.5 or fragments and peptides thereof, can be used in the screening method if the polypeptide, fragment, or peptide contains a site for post-translation processing. Because of their colinearity, a sequence that contains sequences from
  • U s 1.5 necessarily contains sequences from ICP22.
  • a U s 1.5 protein, polypeptide, or peptide comprises a contiguous amino acid sequence of at least between about 10 amino acids and about 50 amino acids, and between about 15 and about 30 amino acids, and between about 20 and about 25 amino from SEQ ID NOJ or SEQ ID NO:2, or biologically functional equivalents thereof.
  • an isolated 22 gene that encodes an U s 1.5 protein, polypeptide, or peptide comprises a contiguous amino acid sequence of at least about 10 amino acids from SEQ ID NOJ or SEQ ID NO:2, or biologically functional equivalents thereof.
  • consecutive amino acids will be understood to include a contiguous amino acid sequence of at least about 4, about, 5, about 6, about
  • a peptide or polypeptide includes amino acids encoded by regions of the ⁇ 22 gene of he ⁇ es simplex vims, such as SEQ ID NOJ, SEQ ID NO:2, SEQ ID NOJ, SEQ ID NOJ, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8.
  • peptides and polypeptides of the present invention are understood to include an amino acid sequence from at least one of the regions defined as SEQ ID NOJ, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NOJ, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO: 8, and in some cases, to include amino acid sequences from more than one region defined as SEQ ID NOJ, SEQ ID NO:2, SEQ ID NOJ, SEQ ID NOJ, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8.
  • the present invention is understood to contemplate a peptide or polypeptide that includes amino acid sequences of a given length from both SEQ ID NOJ and SEQ ID NOJ, such as at least 10 consecutive amino acids from SEQ ID NO:3 and at least 10 consecutive amino acids from SEQ ID NO:4.
  • This application may refer to a UJ .5 peptide or polypeptide, and this will be understood to refer to sequences derived from UJ .5 of he ⁇ esvirus, including a UJ .5 of HSV-1 and a UJ .5 of HSV-2.
  • the amino acid sequence of SEQ ID NOJ corresponds to amino acids 147-171 of HSV-1 ; and, the amino acid sequence of SEQ ID NO:2 corresponds to amino acids 380-405 of HSV-1.
  • the amino acid sequence of SEQ ID NO:3 corresponds to amino acids 38-66 of HSV-1; and the amino acid sequence of SEQ ID NOJ corresponds to amino acids 300-328 of HSV-1.
  • the amino acid sequence of SEQ ID NO:5 corresponds to amino acids 143-167 of HSV-2; and, the amino acid sequence of SEQ ID NO:6 corresponds to amino acids
  • amino acids 37-62 of HSV-2 corresponds to amino acids 37-62 of HSV-2; and, the amino acid sequence of SEQ ID NO:8 corresponds to amino acids 295-320 of HSV-2.
  • a protein can be produced by transforming a host cell with a vector containing a promoter operatively linked to a gene encoding the protein, , and by purifying the desired protein.
  • Some methods are provided in other portions of this disclosure, such as in the section describing methods of identifying compounds that inhibit a he ⁇ esvirus infection. Such methods are not intended to be limited to use with respect to that aspect of the invention only and, instead, apply, to any aspect of the invention disclosed herein.
  • Amino acid sequence variants of a U s 1.5 polypeptide or peptide can be substitutional, insertional or deletion variants.
  • Deletion variants lack one or more residues of the native protein that are not essential for function or immunogenic activity and that are exemplified by the variants lacking a transmembrane sequence described above.
  • Another common type of deletion variant is one lacking secretory signal sequences or signal sequences directing a protein to bind to a particular part of a cell.
  • Insertional mutants typically involve the addition of material at a non-terminal point in the polypeptide. This may include the insertion of an immunoreactiveepitope or simply a single residue. Terminal additions, called fusion proteins, are discussed below.
  • Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the polypeptide, and they may be designed to modulate one or more properties of the polypeptide, such as stability against proteolytic cleavage, without the loss of other functions or properties. Substitutions of this kind preferably are conservative, that is. one amino acid is replaced with one of similar shape and charge.
  • Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine.
  • biologically functional equivalent is well understood in the art and is further defined in detail herein. Accordingly, sequences that have between about 70% and about 80%; or more preferably, between about 81% and about 90%; or even more preferably, between about 91 % and about 99%; of amino acids that are identical or functionally equivalent to the amino acids of a U s 1.5 polypeptide provided the biological activity of the protein is maintained. Therefore, reference to one region in a particular HSV subtype can be understood to refer also to a corresponding region in another subtype, unless otherwise specified.
  • amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids or 5' or 3' sequences, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned.
  • the addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5' or 3' portions of the coding region or may include various internal sequences, i.e., introns, which are known to occur within genes. The following is a discussion based upon changing of the amino acids of a protein to create an equivalent, or even an improved, second-generation molecule.
  • amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with stmctures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid substitutions can be made in a protein sequence, and in its underlying DNA coding sequence, and nevertheless produce a protein with like properties. It is thus contemplated by the inventors that various changes may be made in the DNA sequences of genes without appreciable loss of their biological utility or activity, as discussed below. Table 1 shows the codons that encode particular amino acids.
  • the hydropathic index of amino acids may be considered.
  • the importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte & Doolittle, 1982). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary stmcture of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.
  • Patent 4,554,101 the following hydrophihcity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ⁇ 1); glutamate (+3.0 ⁇ 1 ); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ⁇ 1 ); alanine (-0.5); histidine *-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5): leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).
  • an amino acid can be substituted for another having a similar hydrophihcity value and still produce a biologically equivalent and immunologically equivalent protein.
  • substitution of amino acids whose hydrophihcity values are within ⁇ 2 is preferred, those that are within ⁇ 1 are particularly preferred, and those within ⁇ 0.5 are even more particularly preferred.
  • amino acid substitutions generally are based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophihcity, charge, size, and the like.
  • Exemplary substitutions that take into consideration the various foregoing characteristics are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
  • Mimetics are peptide-containing molecules that mimic elements of protein secondary stmcture. See, e.g., Johnson (1993).
  • the underlying rationale behind the use of peptide mimetics is that the peptide backbone of proteins exists chiefly to orient amino acid side chains in such a way as to facilitate molecular interactions, such as those of antibody and antigen.
  • a peptide mimetic is expected to permit molecular interactions similar to the natural molecule.
  • a specialized kind of insertional variant is the fusion protein.
  • This molecule generally has all or a portion of a native molecule, such as UJ .5, linked at the N- or C- terminus, to all or a portion of a second polypeptide.
  • fusions typically employ leader sequences from other species to permit the recombinant expression of a protein in a heterologous host.
  • Another useful fusion includes the addition of a ⁇ mmunolog ⁇ callyact ⁇ e domain, such as an antibody epitope.
  • cleavage site at or near the fusion junction will facilitate removal of the extraneous polypeptide after purification
  • Other useful fusions include linking of functional domains, such as act ⁇ e sites from enzymes such as a hydrolase, glycosylation domains, cellular targeting signals, or transmembrane regions
  • the present invention also desc ⁇ bes U s l 5-related peptides for use in various embodiments of the present invention
  • the peptides of the invention can also be synthesized in solution or on a solid support in accordance with conventional techniques
  • Va ⁇ ous automatic synthesizers are commercially available and can be used in accordance with known protocols See, for example, Stewart and Young, ( 1984), Tarn et al , (1983), Mer ⁇ field, (1986), and Barany and Mer ⁇ field (1980), each mco ⁇ orated herein by reference
  • Short peptide sequences, or libraries of overlapping peptides usually from about 6 up to about 35 to
  • 50 ammo acids which correspond to the selected regions desc ⁇ bed herein, can be readily synthesized and then screened screening assays designed to identify reactive peptides
  • recombinant DNA technology may be employed wherein a nucleotide sequence which encodes a peptide of the invention is inserted into an expression vector, transformed or transfected into an approp ⁇ ate host cell and cultivated under conditions suitable for expression
  • the present invention also contemplates the use of nonnatural amino acid substitutes that cannot be processed as natural amino acids can Since U s l 5 is required to be posttranslationally modified for viral infection to progress, the use of a
  • U s l 5 peptide that is not capable of being modified because, for example, certain synthetic ammo acids have been inco ⁇ orated into the U s l 5 peptide sequence can also inhibit infection 4.
  • Protein purification techniques are well known to those of skill in the art. These techniques involve, at one level, the cmde fractionation of the milieu from cells infected by a he ⁇ esvirus to polypeptide and non-polypeptide fractions. Once the polypeptide is separated from other proteins, the polypeptide of interest may be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suited to the preparation of a pure peptide are ion-exchange chromatography, exclusion chromatography; polyacrylamide gel electrophoresis; isoelectric focusing. A particularly efficient method of purifying peptides is fast protein liquid chromatography or even HPLC.
  • Certain aspects of the present invention concern the purification, and in particular embodiments, the substantial purification, of an encoded polypeptide or peptide.
  • the term "purified polypeptide or peptide” or “purified protein” as used herein, is intended to refer to a composition, isolatable from other components, wherein the polypeptide, protein, or peptide is purified to any degree relative to its naturally-obtainable state.
  • a purified protein, polypeptide, or peptide therefore also refers to a protein, polypeptide, or peptide, free from the environment in which it may naturally occur.
  • purified will refer to a protein, polypeptide, or peptide composition that has been subjected to fractionation to remove various other components, and which composition substantially retains its expressed biological activity. Where the term “substantially purified” is used, this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the proteins in the composition.
  • Various methods for quantifying the degree of purification of the protein, polypeptide, or peptide will be known to those of skill in the art in light of the present disclosure.
  • a preferred method for assessing the purity of a fraction is to calculate the specific activity of the fraction, to compare it to the specific activity of the initial extract, and to thus calculate the degree of purity, herein assessed by a "-fold purification number.”
  • the actual units used to represent the amount of activity will, of course, be dependent upon the particular assay technique chosen to follow the purification and whether or not the expressed protein or peptide exhibits a detectable activity.
  • Partial purification may be accomplished by using fewer purification steps in combination, or by utilizing different forms of the same general purification scheme. For example, it is appreciated that a cation-exchange column chromatography performed utilizing an HPLC apparatus will generally result in a greater "-fold" purification than the same technique utilizing a low pressure chromatography system. Methods exhibiting a lower degree of relative purification may have advantages in total recovery of protein product, or in maintaining the activity of an expressed protein.
  • High Performance Liquid Chromatography is characterized by a very rapid separation with extraordinary resolution of peaks. This is achieved by the use of very fine particles and high pressure to maintain an adequate flow rate. Separation can be accomplished in a matter of minutes, or at most an hour. Moreover, only a very small volume of the sample is needed because the particles are so small and close-packed that the void volume is a very small fraction of the bed volume. Also, the concentration of the sample need not be very great because the bands are so narrow that there is very little dilution of the sample.
  • Gel chromatography is a special type of partition chromatography that is based on molecular size.
  • the theory behind gel chromatography is that the column, which is prepared with tiny particles of an inert substance that contain small pores, separates larger molecules from smaller molecules as they pass through or around the pores, depending on their size.
  • the sole factor determining rate of flow is the size.
  • molecules are eluted from the column in decreasing size, so long as the shape is relatively constant.
  • Gel chromatography is unsu ⁇ assed for separating molecules of different size because separation is independent of all other factors such as pH, ionic strength, temperature, etc.
  • Affinity Chromatography is a chromatographic procedure that relies on the specific affinity between a substance to be isolated and a molecule that it can specifically bind to. This is a receptor-ligand type interaction.
  • the column material is synthesized by covalently coupling one of the binding partners to an insoluble matrix. The column material is then able to specifically adsorb the substance from the solution. Elution occurs by changing the conditions to those in which binding will not occur (e.g.. alter pH, ionic strength, and temperature).
  • a particular type of affinity chromatography useful in the purification of carbohydrate containing compounds is lectin affinity chromatography.
  • Lectins are a class of substances that bind to a variety of polysaccharides and glycoproteins.
  • Lectins are usually coupled to agarose by cyanogen bromide.
  • Conconavalin A coupled to Sepharose was the first material of this sort to be used and has been widely used in the isolation of polysaccharides and glycoproteins other lectins that have been include lentil lectin, wheat germ agglutinin which has been useful in the purification of N-acetyl glucosaminyl residues and Helix pomatia lectin.
  • Lectins themselves are purified using affinity chromatography with carbohydrate ligands.
  • Lactose has been used to purify lectins from castor bean and peanuts; maltose has been useful in extracting lectins from lentils and jack bean; N-acetyl-D galactosamine is used for purifying lectins from soybean; N-acetyl glucosaminyl binds to lectins from wheat germ; D-galactosamine has been used in obtaining lectins from clams and L-fucose will bind to lectins from lotus.
  • the matrix should be a substance that itself does not adsorb molecules to any significant extent and that has a broad range of chemical, physical and thermal stability.
  • the ligand should be coupled in such a way as to not affect its binding properties.
  • the ligand also should provide relatively tight binding. And it should be possible to elute the substance without destroying the sample or the ligand.
  • affinity chromatography One of the most common forms of affinity chromatography is immunoaffinity chromatography. The generation of antibodies that would be suitable for use in accord with the present invention is discussed below.
  • U s l -5 polypeptide, or peptide a natural or recombinant composition
  • at least some U s 1.5 proteins, polypeptides, or peptides will be subjected to fractionation to remove various non-U s 1.5 components from the composition.
  • fractionation to remove various non-U s 1.5 components from the composition.
  • various other techniques suitable for use in protein purification will be well known to those of skill in the art.
  • U s 1.5 protein Another example is the purification of U s 1.5 protein using a specific binding partner, such as p60.
  • a specific binding partner such as p60.
  • any fusion protein purification method can now be practiced. This is exemplified by the generation of an U s 1.5 S- transferase fusion protein, expression in E. coli, and isolation to homogeneity using affinity chromatography on glutathione-agarose or the generation of a polyhistidine tag on the N- or C-terminus of the protein, and subsequent purification using Ni- affinity chromatography.
  • U s 1.5 protein, polypeptide, or peptide there is no general requirement that the U s 1.5 protein, polypeptide, or peptide always be provided in their most purified state. Indeed, it is contemplated that less substantially purified U s l .5 protein, polypeptide or peptide, which are nonetheless enriched in U s l .5 protein compositions, relative to the natural state, will have utility in certain embodiments. These include, for example, antibody generation where subsequent screening assays using purified U s 1.5 proteins are conducted. Methods exhibiting a lower degree of relative purification may have advantages in total recovery of protein product, or in maintaining the activity of an expressed protein. Inactive products also have utility in certain embodiments, such as, e.g. , in antibody generation.
  • the present invention describes the use of polypeptides and peptides containing sequences from U s 1.5. In some embodiments, noncontiguous regions of
  • U 1.5 can be used to inhibit an infection by he ⁇ esvirus. It is contemplated that a single region of U s 1.5, such as from the region encoding amino acids 147-171 or amino acids 380-405 can be joined to nonnative U s 1.5 sequence. This compound can be created by joining regions via a biologically-releasable bond, such as a selectively- cleavable linker or non-native U s 1.5 amino acid sequence.
  • Non-native U s 1.5 sequence refers to any sequence not identical with the amino acid sequence of U s 1.5 between amino acid 172 and amino acid 379.
  • peptide linkers that include a cleavage site for an enzyme preferentially located or active within a tumor environment are contemplated.
  • Exemplary forms of such peptide linkers are those that are cleaved by urokinase, plasmin, thrombin, Factor IXa, Factor Xa, or a metallaproteinase, such as collagenase, gelatinase, or stromelysin.
  • any of the above components, to a U 1.5 region will generally employ the same technology as developed for the preparation of immunotoxins. It can be considered as a general guideline that any biochemical cross-linker that is appropriate for use in an immunotoxin will also be of use in the present context, and additional linkers may also be considered.
  • Cross-linking reagents are used to form molecular bridges that tie together functional groups of two different molecules, e.g., a stablizing and coagulating agent.
  • a stablizing and coagulating agent e.g., a stablizing and coagulating agent.
  • hetero-bifunctional cross-linkers can be used that eliminate unwanted homopolymer formation. Examples of such cross-linkers can be found in Table 2 below.
  • An exemplary hetero-bifunctional cross-linker contains two reactive groups: one reacting with primary amine group (e.g.. N-hydroxy succinimide) and the other reacting with a thiol group (e.g., pyridyl disulfide, maleimides, and halogens).
  • primary amine group e.g.. N-hydroxy succinimide
  • a thiol group e.g., pyridyl disulfide, maleimides, and halogens.
  • the cross-linker may react with the lysine residue(s) of one protein (e.g., the selected antibody or fragment) and through the thiol reactive group, the cross-linker, already tied up to the first protein, reacts with the cysteine residue (free sulfhydryl group) of the other protein (e.g., the selective agent).
  • a U s l .5 peptide can have, or be derivatized to have, a functional group available for cross-linking pu ⁇ oses.
  • This requirement is not considered to be limiting in that a wide variety of groups can be used in this manner.
  • primary or secondary amine groups, hydrazide or hydrazine groups, carboxyl alcohol, phosphate, or alkylating groups may be used for binding or cross- linking.
  • Ghose & Blair (1987) For a general overview of linking technology, one may wish to refer to Ghose & Blair (1987).
  • the spacer arm between the two reactive groups of a cross-linkers may have various length and chemical compositions.
  • a longer spacer arm allows a better flexibility of the conjugate components while some particular components in the bridge (e.g., benzene group) may lend extra stability to the reactive group or an increased resistance of the chemical link to the action of various aspects (e.g., disulfide bond resistant to reducing agents).
  • the use of peptide spacers, such as L-Leu-L-Ala-L-Leu-L-Ala, is also contemplated.
  • cross-linker having reasonable stability in blood will be employed.
  • Numerous types of disulfide-bond containing linkers are known that can be successfully employed to conjugate targeting and therapeutic/preventative agents. Linkers that contain a disulfide bond that is sterically hindered may prove to give greater stability in vivo, preventing release of the targeting peptide prior to reaching the site of action. These linkers are thus one group of linking agents.
  • Another cross-linking reagents for use in immunotoxins is SMPT, which is a bifunctional cross-linker containing a disulfide bond that is "sterically hindered" by an adjacent benzene ring and methyl groups.
  • stearic hindrance of the disulfide bond serves a function of protecting the bond from attack by thiolate anions such as glutathione which can be present in tissues and blood, and thereby help in preventing decoupling of the conjugate prior to the delivery of the attached agent to the tumor site.
  • thiolate anions such as glutathione which can be present in tissues and blood, and thereby help in preventing decoupling of the conjugate prior to the delivery of the attached agent to the tumor site.
  • the SMPT agent may also be used in connection with the bispecific coagulating ligands of this invention.
  • the SMPT cross-linking reagent lends the ability to cross-link functional groups such as the SH of cysteine or primary amines (e.g., the epsilon amino group of lysine).
  • Another possible type of cross-linker includes the hetero-bifunctional photoreactive phenylazides containing a cleavable disulfide bond such as sulfosuccinimidyl-2-(p-azido salicylamido) ethyl- l,3'-dithiopropionate.
  • the N-hydroxy-succinimidyl group reacts with primary amino groups and the phenylazide (upon photolysis) reacts non-selectively with any amino acid residue.
  • non-hindered linkers also can be employed in accordance herewith.
  • Other useful cross-linkers not considered to contain or generate a protected disulfide. include SATA, SPDP and 2-iminothiolane
  • the U s 1.5 peptide generally will be purified to separate the conjugate from unconjugated targeting agents or coagulants and from other contaminants.
  • a large a number of purification techniques are available for use in providing conjugates of a sufficient degree of purity to render them clinically useful. Purification methods based upon size separation, such as gel filtration, gel permeation or high performance liquid chromatography, will generally be of most use. Other chromatographic techniques, such as Blue-Sepharose separation, may also be used.
  • a purified UJ .5 polypeptide or peptide may be modified at the protein level.
  • U s 1.5 polypeptide fragments or other derivatives or analogs that are differentially modified during or after translation for example by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, and proteolytic cleavage. Any number of chemical modifications may be carried out by known techniques, including but not limited to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH ; acetylation, formylation, farnesylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin.
  • U s 1.5 Because posttranslational modification of U s 1.5 is required for progression of a he ⁇ esvirus infection, inhibition of modification can be used to inhibit viral infection by HSV.
  • the present invention discloses particular regions that are necessary to mediate posttranslational processing of the UJ .5. Therefore, it is contemplated that these regions can be administered as peptides or polypeptides, or fragments thereof, to a subject to inhibit he ⁇ esvirus infection by, for example, competing with U s 1.5 produced by the vims as a target for phosphorylation by kinases such as U L 13.
  • a therapy approach is the provision, to a subject, of U s 1.5 polypeptides, active fragments, synthetic peptides, mimetics or other analogs thereof.
  • the protein may be produced by recombinant expression means or, if small enough, generated by an automated peptide synthesizer.
  • Formulations would be selected based on the route of administration and pu ⁇ ose including, but not limited to, liposomal formulations and classic pharmaceutical preparations.
  • Aqueous compositions of the present invention comprise an effective amount of the U s 1.5 protein, polypeptide, peptide, epitopic core region, inhibitor, or such like. dissolved or dispersed in a pharmaceutically acceptable earner or aqueous medium
  • Aqueous compositions of gene therapy vectors expressing any of the foregoing are also contemplated
  • pharmaceutically or pharmacologically acceptable refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, or a human, as approp ⁇ ate
  • pharmaceutically acceptable earner includes any and all solvents, dispersion media, coatings, antibacte ⁇ al and antifungal agents, lsotonic and abso ⁇ tion delaying agents and the like.
  • dispersion media includes any and all solvents, dispersion media, coatings, antibacte ⁇ al and antifungal agents, lsotonic and abso ⁇ tion delaying agents and the like.
  • the use of such media and agents for pharmaceutical active substances is well known in the art Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated Supplementary active ingredients can also be inco ⁇ orated into the compositions For human administration, preparations should meet ste ⁇ hty, pyrogenicity, general safety and purity standards as required bv FDA Office of Biologies standards
  • the biological matenal should be extensively dialyzed to remove undesired small molecular weight molecules and/or lyophilized for more ready formulation into a desired vehicle, where approp ⁇ ate
  • the active compounds will then generally be formulated for parenteral administration, e g , formulated for injection via the intravenous, intramuscular, sub-cutaneous, mtralesional, or even mtrape ⁇ toneal routes
  • parenteral administration e g
  • aqueous composition that contains a U l 5 agent as an active component or ingredient will be known to those of skill m the art in light of the present disclosure
  • such compositions can be prepared as injectables, either as liquid solutions or suspensions, solid forms suitable for using to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared, and the preparations can also be emulsified
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions, formulations including sesame oil, peanut oil or aqueous propylene glycol, and sterile powders for the extemporaneous preparation of ste ⁇ le injectable solutions or dispersions.
  • the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • a U s 1.5 protein, polypeptide, peptide, agonist or antagonist of the present invention can be formulated into a composition in a neutral or salt form.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • the carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged abso ⁇ tion of the injectable compositions can be brought about by the use in the compositions of agents delaying abso ⁇ tion, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by inco ⁇ orating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by inco ⁇ orating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.
  • the preparation of more, or highly, concentrated solutions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small area.
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but dmg release capsules and the like can also be employed.
  • aqueous solutions For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • sterile aqueous media which can be employed, will be known to those of skill in the art in light of the present disclosure.
  • one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-
  • the active U s 1.5 protein-derived peptides or agents may be formulated within a therapeutic mixture to comprise about 0.0001 to 1.0 milligrams, or about 0.001 to 0J milligrams, or about 0J to 1.0 or even about 10 milligrams per dose or so. Multiple doses can also be administered.
  • other pharmaceutically acceptable forms include, e.g., tablets or other solids for oral administration; liposomal formulations; time release capsules; and any other form currently used, including cremes.
  • Nasal solutions are usually aqueous solutions designed to be administered to the nasal passages in drops or sprays. Nasal solutions are prepared so that they are similar in many respects to nasal secretions, so that normal ciliary action is maintained. Thus, the aqueous nasal solutions usually are isotonic and slightly buffered to maintain a pH of 5.5 to 6.5.
  • antimicrobial preservatives similar to those used in ophthalmic preparations, and appropriate dmg stabilizers, if required, may be included in the formulation.
  • Various commercial nasal preparations are known and include, for example, antibiotics and antihistaminesand are used for asthma prophylaxis.
  • vaginal suppositories are solid dosage forms of various weights and shapes, usually medicated, for insertion into the rectum, vagina or the urethra. After insertion, suppositories soften, melt or dissolve in the cavity fluids.
  • traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably l%-2%.
  • Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders.
  • oral pharmaceutical compositions will comprise an inert diluent or assimilable edible carrier, or they may be enclosed in hard or soft shell gelatin capsule, or they may be compressed into tablets, or they may be inco ⁇ orated directly with the food of the diet.
  • the active compounds may be inco ⁇ orated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • Such compositions and preparations should contain at least 0.1% of active compound.
  • the percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 75% of the weight of the unit, or preferably between 25-60%.
  • the amount of active compounds in such therapeutically useful compositions is such that a suitable dosage will be obtained.
  • the tablets, troches, pills, capsules and the like may also contain the following: a binder, as gum tragacanth, acacia, cornstarch, or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as com starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin may be added or a flavoring agent, such as peppermint, oil of wintergreen, or cherry flavoring.
  • a binder as gum tragacanth, acacia, cornstarch, or gelatin
  • excipients such as dicalcium phosphate
  • a disintegrating agent such as com starch, potato starch, alginic acid and the like
  • a lubricant such as magnesium stearate
  • a sweetening agent such as sucrose, lactose or saccharin may be added or
  • Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit.
  • tablets, pills, or capsules may be coated with shellac, sugar or both.
  • a symp of elixir may contain the active compounds sucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor.
  • liposomes and/or nanoparticles are contemplated for the introduction of U s 1.5 protein, polypeptides, peptides or agents, or gene therapy vectors, including both wild-type and antisense vectors, into host cells.
  • the formation and use of liposomes is generally known to those of skill in the art, and is also described below.
  • Nanocapsules can generally entrap compounds in a stable and reproducible way. To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 ⁇ m) should be designed using polymers able to be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present invention, and such particles may be are easily made.
  • Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs).
  • MLVs generally have diameters of from 25 nm to 4 ⁇ m. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 A. containing an aqueous solution in the core.
  • SUVs small unilamellar vesicles
  • Phospholipids can form a variety of stmctures other than liposomes when dispersed in water, depending on the molar ratio of lipid to water. At low ratios the liposome is the preferred stmcture.
  • the physical characteristics of liposomes depend on pH, ionic strength and the presence of divalent cations. Liposomes can show low permeability to ionic and polar substances, but at elevated temperatures undergo a phase transition which markedly alters their permeability.
  • phase transition involves a change from a closely packed, ordered stmcture, known as the gel state, to a loosely packed, less-ordered stmcture, known as the fluid state. This occurs at a characteristic phase-transition temperature and results in an increase in permeability to ions, sugars and dmgs.
  • Liposomes interact with cells via four different mechanisms: Endocytosis by phagocytic cells of the reticuloendothelial system such as macrophages and neutrophils; adso ⁇ tion to the cell surface, either by nonspecific weak hydrophobic or electrostatic forces, or by specific interactions with cell-surface components; fusion with the plasma cell membrane by insertion of the lipid bilayer of the liposome into the plasma membrane, with simultaneous release of liposomal contents into the cytoplasm; and by transfer of liposomal lipids to cellular or subcellular membranes, or vice versa, without any association of the liposome contents. Varying the liposome formulation can alter which mechanism is operative, although more than one may operate at the same time.
  • the present invention concerns polynucleotides, isolatable from HSV-infected cells, which are free from total genomic DNA and that are capable of expressing a protein, polypeptide, or peptide that is derived from the ⁇ .22 gene product.
  • DNA segment refers to a DNA molecule that has been isolated free of total genomic DNA of a particular species. Therefore, a DNA segment encoding a U s 1.5 polypeptide refers to a DNA segment that contains U s 1.5 polypeptide-coding sequences yet is isolated away from, or purified free from, total mammalian or human genomic DNA. Included within the term “DNA segment” are a polypeptide or polypeptides. DNA segments smaller than a polypeptide, and recombinant vectors, including, for example, plasmids, cosmids, phage, vimses, and the like.
  • polynucleotide refers to a nucleic acid molecule that has been isolated free of total genomic nucleic acid. Therefore, a "polynucleotide encoding an U s 1.5 polypeptide” refers to a DNA segment that contains U s 1.5 polypeptide-coding sequences yet is isolated away from, or purified free from, total mammalian or viral genomic DNA; similarly, a “polynucleotide encoding ICP22” refers to a DNA segment that contains ICP22 polypeptide coding sequences yet is isolated away from, or purified free from, total mammalian or human genomic DNA.
  • U s 1.5 or to "U s 1.5 polypeptide” that is encoded by ⁇ 22, it is meant that the polynucleotide encodes a molecule that has the ability to promote the infectivity of a he ⁇ es simplex vims in restrictive cells.
  • cDNA is intended to refer to DNA prepared using messenger RNA (mRNA) as template.
  • mRNA messenger RNA
  • a given ⁇ 22 gene from a given viral strain may be represented by natural variants that have slightly different nucleic acid sequences but, nonetheless, encode the same protein (see Table 1 above). Consequently, the present invention also encompasses derivatives of an U s 1.5 polypeptide that have minimal amino acid changes, but that possess the vims-promoting activities of the known sequence.
  • the invention concerns isolated DNA segments and recombinant vectors inco ⁇ orating DNA sequences that encode the UJ .5 polypeptide or peptide that includes within its amino acid sequence a contiguous amino acid sequence in accordance with, or essentially as set forth in, SEQ ID NOJ , corresponding to amino acids 147-171 of the polypeptide encoded by the ⁇ 22 gene of he ⁇ es simplex virus.
  • the invention concerns isolated DNA segments and recombinant vectors inco ⁇ orating DNA sequences that encode a polypeptide or peptide that includes within its amino acid sequence a contiguous amino acid sequence in accordance with, or essentially as set forth in, SEQ ID NO:2, corresponding to amino acids 380-405 of the polypeptide encoded by the ⁇ 22 gene of
  • the invention concerns isolated DNA segments and recombinant vectors inco ⁇ orating DNA sequences that encode a polypeptide or peptide that includes within its amino acid sequence a contiguous amino acid sequence in accordance with, or essentially as set forth in, SEQ ID NOJ, corresponding to amino acids 38-66 of the polypeptide encoded by the ⁇ 22 gene of
  • the invention concerns isolated DNA segments and recombinant vectors inco ⁇ orating DNA sequences that encode a polypeptide or peptide that includes within its amino acid sequence a contiguous amino acid sequence in accordance with, or essentially as set forth in, SEQ ID NOJ, corresponding to amino acids 300-328 of the polypeptide encoded by the ⁇ 22 gene of
  • the invention concerns isolated DNA segments and recombinant vectors inco ⁇ orating DNA sequences that encode a polypeptide or peptide that includes within its amino acid sequence a contiguous amino acid sequence in accordance with, or essentially as set forth in, SEQ ID NO:5, corresponding to amino acids 143-167 of the polypeptide encoded by the ⁇ 22 gene of
  • the invention concerns isolated DNA segments and recombinant vectors inco ⁇ orating DNA sequences that encode a polypeptide or peptide that includes within its amino acid sequence a contiguous amino acid sequence in accordance with, or essentially as set forth in, SEQ ID NO:6, co ⁇ esponding to amino acids 373-598 of the polypeptide encoded by the ⁇ 22 gene of HSV-2.
  • the invention concerns isolated DNA segments and recombinant vectors inco ⁇ orating DNA sequences that encode a polypeptide or peptide that includes within its amino acid sequence a contiguous amino acid sequence in accordance with, or essentially as set forth in, SEQ ID NO:7.
  • the invention concerns isolated DNA segments and recombinant vectors inco ⁇ orating DNA sequences that encode a polypeptide or peptide that includes within its amino acid sequence a contiguous amino acid sequence in accordance with, or essentially as set forth in, SEQ ID NO:8, corresponding to amino acids 295-320 of the polypeptide encoded by the ⁇ .22 gene of HSV-2 type 1.
  • gene is used for simplicity to refer to a functional protein-, polypeptide-, or peptide-encoding unit.
  • this functional term includes viral DNA sequences, cDNA sequences, and smaller engineered gene segments that express, or may be adapted to express, proteins, polypeptides, domains, peptides, fusion proteins, and mutants.
  • isolated substantially away from other coding sequences means that the gene of interest, in this case the ⁇ 22 gene, forms the significant part of the coding region of the DNA segment, and that the DNA segment does not contain large portions of naturally-occurring coding DNA, such as large viral DNA genomic fragments or other functional genes or cDNA coding regions. Of course, this refers to the DNA segment as originally isolated, and does not exclude genes or coding regions later added to the segment by the hand of man.
  • sequence essentially as set forth in SEQ ID NOJ means that the sequence substantially corresponds to a portion of SEQ ID NOJ and has relatively few amino acids that are not identical to, or a biologically functional equivalent of, the amino acids of SEQ ID NOJ .
  • sequence essentially as set forth in SEQ ID NO: can be used with any identified sequence to connote the corresponding definition as applied to SEQ ID NOJ by example.
  • the DNA segments used in the present invention encompass biologically functional equivalent U s 1.5 protein and peptides. Such sequences may arise as a consequence of codon redundancy and functional equivalency that are known to occur naturally within nucleic acid sequences and the proteins thus encoded.
  • proteins or peptides may be created via the application of recombinant DNA technology, in which changes in the protein stmcture may be engineered, based on considerations of the properties of the amino acids being exchanged. Changes designed by humans may be introduced through the application of site-directed mutagenesis techniques, e.g., to introduce improvements to the antigenicity of the protein or to test mutants in order to examine DNA binding activity at the molecular level. Accordingly, sequences that have between about 70% and about 80%; or more preferably, between about 81% and about 90%>; or even more preferably, between about 91% and about 99%; of amino acids that are identical or functionally equivalent to the amino acids of SEQ ID NOJ, SEQ ID NOJ, SEQ ID
  • SEQ ID NO:3 SEQ ID NOJ, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and/or SEQ ID NO:8 will be sequences that are "essentially as set forth in SEQ ID NOJ,” “essentially as set forth in SEQ ID NO:2,” “essentially as set forth in SEQ ID NOJ,” “essentially as set forth in SEQ ID NO:4,” “essentially as set forth in SEQ ID NO:5,” “essentially as set forth in SEQ ID NO:6,” “essentially as set forth in SEQ ID NO: 7,” and/or “essentially as set forth in SEQ ID NO: 8,” provided the biological activity of the polypeptide is maintained.
  • the term "consecutive nucleic acid segment” will be understood to include a contiguous nucleic acid sequence of at least about 8, about, 9, about 10, about 1 1 , about 12, about 13, about 14, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 60, about 70, about 80, about 90, about 100, about 1 10, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 300, about 310, about 320, about 330, about 340, about 350, about 360, about 370, about 380, about 390, about 400, about 410, about 420.
  • nucleic acids or so about 500, about 600, about 700, about 800, about 900, or about 1000, about 1100, about 1200, about 1300, about 1400, about 1500, about 1600, about 1700, about 1800, about 1900. about 2000, about 3000, about 4000, or about 5000 nucleic acids or so.
  • DNA segments encoding relatively small peptides such as, for example, peptides of from about 8, about 9, about 10, about 1 1. about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31 , about 32, about 33, about 34, about 35, about 35, about 40, about 45, to about 50 amino acids in length, and additionally, between about 10 amino acids and about 50 amino acids, between about 15 and about 30 amino acids in length, and between about 20 amino acids and about 25 amino acids, as set forth in SEQ ID NOJ and SEQ ID NO:2 and also larger polypeptides up to the full-length amino acid sequences of U s 1.5.
  • peptides of from about 8, about 9, about 10, about 1 1. about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31 , about 32, about 33, about 34, about 35, about 35, about
  • the invention concerns isolated DNA segments and recombinant vectors inco ⁇ orating DNA sequences that encode a U s 1.5 polypeptide or peptide that includes within its amino acid sequence a contiguous amino acid sequence in accordance with, or essentially co ⁇ esponding to U s 1.5 polypeptides.
  • the invention concerns isolated DNA segments and recombinant vectors inco ⁇ orating DNA sequences that encode a U s l .5 polypeptide or peptide that includes within its amino acid sequence a contiguous amino acid sequence in accordance with, or essentially corresponding to the U s l .5 polypeptide.
  • Site-specific mutagenesis is a technique useful in the preparation of individual peptides, or biologically functional equivalent proteins or peptides, through specific mutagenesis of the underlying DNA.
  • the technique further provides a ready ability to prepare and test sequence variants, inco ⁇ orating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA.
  • Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed.
  • a primer of about 17 to 25 nucleotides in length is preferred, with about 5 to 10 residues on both sides of the junction of the sequence being altered.
  • the technique of site-specific mutagenesis is well known in the art.
  • the technique typically employs a bacteriophage vector that exists in both a single stranded and double stranded form.
  • Typical vectors useful in site-directed mutagenesis include vectors such as the Ml 3 phage. These phage vectors are commercially available and their use is generally well known to those skilled in the art.
  • Double stranded plasmids are also routinely employed in site directed mutagenesis, which eliminates the step of transferring the gene of interest from a phage to a plasmid.
  • site-directed mutagenesis is performed by first obtaining a single- stranded vector, or melting of two strands of a double stranded vector which includes within its sequence a polynucleotide sequence encoding the desired protein.
  • An oligonucleotide primer bearing the desired mutated sequence is synthetically prepared. This primer is then annealed with the single-stranded DNA preparation, and subjected to DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand.
  • DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment
  • This heteroduplex vector is then used to transform appropriate cells, such as E. coli cells, and clones are selected that include recombinant vectors bearing the mutated sequence arrangement.
  • appropriate cells such as E. coli cells
  • clones are selected that include recombinant vectors bearing the mutated sequence arrangement.
  • sequence variants of the selected gene using site-directed mutagenesis is provided as a means of producing potentially useful species and is not meant to be limiting, as there are other ways in which sequence variants of genes may be obtained.
  • recombinant vectors encoding the desired gene may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants.
  • nucleic acid segments used in the present invention may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol.
  • the present invention involving a polypeptide or peptide encoded by a polynucleotide can be combined with other nucleic acid segments, including segments from an expression vector.
  • expression vectors are employed to express the U s 1.5 polypeptide or peptide that encompasses a posttranslational modification site.
  • the expression vectors can be used to deliver a polynucleotidesequence encoding a U s l .5 polypeptide or peptide to a cell either in vitro or in vivo.
  • the vector can be used to deliver such a sequence, so that its amino acid product which can then be purified and, for example, be used to vaccinate animals or humans to generate antisera or monoclonal antibody.
  • the expression vectors are used in gene therapy. Expression requires that appropriate signals be provided in the vectors, and which include various regulatory elements, such as enhancers/promoters from both viral and mammalian sources that drive expression of the genes of interest in host cells. Elements designed to optimize messenger RNA stability and translatability in host cells also are defined. The conditions for the use of a number of dominant dmg selection markers for establishing permanent, stable cell clones expressing the products also are provided, as is an element that links expression of the dmg selection markers to expression of the polypeptide.
  • various regulatory elements such as enhancers/promoters from both viral and mammalian sources that drive expression of the genes of interest in host cells.
  • Elements designed to optimize messenger RNA stability and translatability in host cells also are defined.
  • the conditions for the use of a number of dominant dmg selection markers for establishing permanent, stable cell clones expressing the products also are provided, as is an element that links expression of the dmg selection markers to expression of the polypeptid
  • expression vector is meant to include any type of genetic constmct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed.
  • the transcript may be translated into a protein, but it need not be.
  • expression includes both transcription of a gene and translation of mRNA into a gene product. In other embodiments, expression only includes transcription of the nucleic acid encoding a gene of interest.
  • the nucleic acid encoding a gene product is under transcriptional control of a promoter.
  • a “promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene.
  • under transcriptional control means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene.
  • promoter will be used here to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase II.
  • Much of the thinking about how promoters are organized derives from analyses of several viral promoters, including those for the HSV thymidine kinase (tk) and SV40 early transcription units. These studies, augmented by more recent work, have shown that promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins. At least one module in each promoter functions to position the start site for
  • RNA synthesis The best known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation.
  • promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-1 10 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well.
  • the spacing between promoter elements frequently is flexible, so that promoter function is preserv ed when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription.
  • the particular promoter employed to control the expression of a nucleic acid sequence of interest is not believed to be important, so long as it is capable of direction the expression of the nucleic acid in the targeted cell.
  • a human cell it is preferable to position the nucleic acid coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell.
  • a promoter might include either a human or viral promoter.
  • the human cytomegalovims (CMV) immediate early gene promoter can be used to obtain high-level expression of the coding sequence of interest.
  • CMV cytomegalovims
  • the use of other viral or mammalian cellular or bacterial phage promoters which are well-known in the art to achieve expression of a coding sequence of interest is contemplated as well, provided that the levels of expression are sufficient for a given pii ⁇ ose. Selection of a promoter that is regulated in response to specific physiologic or synthetic signals can permit inducible expression of the gene product.
  • transgenes that may be toxic to the producer cell line are pro-apoptotic and cytokine genes.
  • inducible promoter systems are available for production of viral vectors where the transgene product may be toxic.
  • An inducible promoter may be used to control the expression of UJ .5.
  • the ecdysone system (Invitrogen, Carlsbad, CA) is one such inducible system.
  • This system is designed to allow regulated expression of a gene of interest in mammalian cells. It consists of a tightly regulated expression mechanism that allows virtually no basal level expression of the transgene, but over 200-fold inducibility.
  • the system is based on the heterodimeric ecdysone receptor of Drosophila, and when ecdysone or an analog such as muristerone A binds to the receptor, the receptor activates a promoter to turn on expression of the downstream transgene such that high levels of mRNA transcripts are attained.
  • both monomers of the heterodimeric receptor are constitutively expressed from one vector, whereas the ecdysone-responsive promoter which drives expression of the gene of interest is on another plasmid.
  • Tet-OffTM or Tet-OnTM system (Clontech, Palo Alto, CA) originally developed by Gossen and Bujard (Gossen and Bujard, 1992; Gossen et al, 1995).
  • This system also allows high levels of gene expression to be regulated in response to tetracycline or tetracycline derivatives such as doxycycline.
  • Tet-OnTM system gene expression is turned on in the presence of doxycycline, whereas in the Tet-Of M system, gene expression is turned on in the absence of doxycycline.
  • the Tet-Of M system would be preferable so that the producer cells could be grown in the presence of tetracycline or doxycycline and prevent expression of a potentially toxic transgene, but when the vector is introduced to the patient, the gene expression would be constitutively on.
  • a transgene in a gene therapy vector.
  • different viral promoters with varying strengths of activity may be utilized depending on the level of expression desired.
  • the CMV immediate early promoter if often used to provide strong transcriptional activation. Modified versions of the CMV promoter that are less potent have also been used when reduced levels of expression of the transgene are desired.
  • retroviral promoters such as the LTRs from MLV or MMTV are often used.
  • Other viral promoters that may be used depending on the desired effect include SV40,
  • RSV LTR HIV-1 and HIV-2 LTR
  • adenovims promoters such as from the El A, E2A, or MLP region
  • AAV LTR cauliflower mosaic vims
  • HSV-TK avian sarcoma vims.
  • Enhancers are genetic elements that increase transcription from a promoter located at a distant position on the same molecule of DNA. Enhancers are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins. The basic distinction between enhancers and promoters is operational. An enhancer region as a whole must be able to stimulate transcription at a distance; this need not be tme of a promoter region or its component elements.
  • Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression constmct.
  • NCAM Neural Cell Adhesion Molecule
  • SAA Human Semm Amyloid A
  • a cDNA insert where a cDNA insert is employed, one will typically desire to include a polyadenylation signal to effect proper polyadenylation of the gene transcript.
  • the nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any such sequence may be employed such as human growth hormone and SV40 polyadenylation signals.
  • a terminator Also contemplated as an element of the expression cassette is a terminator. These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.
  • the cells contain nucleic acid constmct of the present invention
  • a cell may be identified in vitro or in vivo by including a marker in the expression vector.
  • markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression vector.
  • a dmg selection marker aids in cloning and in the selection of transformants, for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selectable markers.
  • enzymes such as he ⁇ es simplex vims thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be employed.
  • lmmunologic markers also can be employed.
  • the selectable marker employed is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product. Further examples of selectable markers are well known to one of skill in the art.
  • IRES elements are used to create multigene, or polycistronic, messages.
  • IRES elements are able to bypass the ribosome scanning model of 5' methylated Cap dependent translation and begin translation at internal sites (Pelletier and Sonenberg, 1988).
  • IRES elements from two members of the picanovims family polio and encephalomyocarditis have been described (Pelletier and Sonenberg, 1988), as well an IRES from a mammalian message (Macejak and Samow, 1991 ).
  • IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages. By virtue of the IRES element, each open reading frame is accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter/enhancer to transcribe a single message.
  • Any heterologous open reading frame can be linked to IRES elements. This includes genes for secreted proteins, multi-subunit proteins, encoded by independent genes, intracellular or membrane-bound proteins and selectable markers. In this way, expression of several proteins can be simultaneously engineered into a cell with a single vector and a single selectable marker.
  • the expression vector comprises a vi s or engineered vector derived from a viral genome.
  • the development of viral vectors is an improvement to the field of gene transfer, as demonstrated by U.S. Patent Application No. 60/078205, hereby incorporated by reference.
  • the ability of certain vimses to enter cells via receptor-mediated endocytosis, to integrate into host cell genome and express viral genes stably and efficiently have made them attractive candidates for the transfer of foreign genes into mammalian cells (Ridgeway, 1988; Nicolas and Rubenstein, 1988; Baichwal and Sugden, 1986; Temin, 1986).
  • He ⁇ esviruses as described herein can be used or altered for use as a delivery system.
  • HSV is neurotropic, it has generated considerable interest in treating nervous system disorders. Moreover, the ability of HSV to establish latent infections in non- dividing neuronal cells without integrating into the host cell chromosome or otherwise altering the host cell's metabolism, along with the existence of a promoter that is active during latency. Although much attention has focused on the neurotropic applications of HSV, this vector can be exploited for other tissues.
  • adenovims expression vector is meant to include those vectors containing adenovirus sequences sufficient to (a) support packaging of the vector and (b) to express a polynucleotide that has been cloned therein. In this context, expression may require that the gene product be synthesized.
  • Adenovims is particularly suitable for use as a gene transfer vector because of its mid-sized genome, ease of manipulation, high titer, wide target cell range and high infectivity. Both ends of the viral genome contain 100-200 base pair inverted repeats (ITRs), which are cis elements necessary for viral DNA replication and packaging.
  • ITRs inverted repeats
  • the early (E) and late (L) regions of the genome contain different transcription units that are divided by the onset of viral DNA replication.
  • the El region (El A and E1B) encodes proteins responsible for the regulation of transcription of the viral genome and a few cellular genes.
  • the expression of the E2 region results in the synthesis of the proteins for viral DNA replication.
  • MLP major late promoter
  • TPL 5'-tripartite leader
  • Adenovims vectors have been used in eukaryotic gene expression (Levrero et al, 1991 ; Gomez-Foix et al, 1992) and vaccine development (Gmnhaus and Horwitz, 1992; Graham and Prevec, 1992). Recently, animal studies suggested that recombinant adenovims could be used for gene therapy (Stratford-Perricaudet and Perricaudet, 1991 ; Stratford-Perricaudet et al, 1990; Rich et al, 1993).
  • RNA vimses are a group of single-stranded RNA vimses characterized by an ability to convert their RNA to double-stranded DNA in infected cells by a process of reverse-transcription (Coffin. 1990). The resulting DNA then stably integrates into cellular chromosomes as a provims and directs synthesis of viral proteins.
  • the integration results in the retention of the viral gene sequences in the recipient cell and its descendants.
  • the retroviral genome contains three genes, gag, pol, and env that code for capsid proteins, polymerase enzyme, and envelope components, respectively.
  • a sequence found upstream from the gag gene contains a signal for packaging of the genome into virions.
  • Two long terminal repeat (LTR) sequences are present at the 5' and 3' ends of the viral genome. These contain strong promoter and enhancer sequences and also are required for integration in the host cell genome (Coffin, 1990).
  • a nucleic acid encoding a gene of interest is inserted into the viral genome in the place of certain viral sequences to produce a vims that is replication-defective.
  • a packaging cell line containing the gag, pol, and env genes but without the LTR and packaging components is constmcted (Mann et al, 1983).
  • Retroviral vectors are able to infect a broad variety of cell types. However, integration and stable expression require the division of host cells (Paskind et al,
  • viral vectors may be employed as expression constmcts in the present invention.
  • Vectors derived from vimses such as vaccinia vims (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al. 1988) adeno-associated vims (AAV)
  • the expression vector In order to effect expression of sense or antisense gene constmcts, the expression vector must be delivered into a cell. This delivery may be accomplished in vitro, as in laboratory procedures for transforming cells lines, or in vivo or ex vivo, as in the treatment of certain disease states. One mechanism for delivery is via viral infection where the expression vector is encapsidated in an infectious viral particle. These methods are described above.
  • Non-viral methods for the transfer of expression vectors into cultured mammalian cells include calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al, 1990) DEAE-dextran (Gopal, 1985), electroporation (Tur-Kaspa et al, 1986; Potter et al, 1984), direct microinjection (Harland and Weintraub, 1985), DNA-loaded liposomes (Nicolau and Sene, 1982; Fraley et al, 1979) and lipofectamine-DNA complexes, cell sonication (Fechheimer et al, 1987), gene bombardment using high velocity microprojectiles (Yang et al, 1990), and receptor- mediated transfection (Wu and Wu, 1987; Wu and Wu, 1988). Some of these techniques may be successfully adapted for in vivo or ex vivo use.
  • the nucleic acid encoding the gene of interest may be positioned and expressed at different sites.
  • the nucleic acid encoding the gene may be stably integrated into the genome of the cell. This integration may be in the cognate location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non-specific location (gene augmentation).
  • the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or "episomes" encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. How the expression vector is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression vector employed.
  • the expression vector may simply consist of naked recombinant DNA or plasmids. Transfer of the vector may be performed by any of the methods mentioned above which physically or chemically permeabihze the cell membrane. This is particularly applicable for transfer in vitro but it may be applied to in vivo use as well.
  • Dubensky et al. (1984) successfully injected polyomavirus DNA in the form of calcium phosphate precipitates into liver and spleen of adult and newborn mice demonstrating active viral replication and acute infection. Benvenisty and Neshif (1986) also demonstrated that direct intraperitoneal injection of calcium phosphate-precipitated plasmids results in expression of the transfected genes.
  • DNA encoding a gene of interest also may be transferred in a similar manner in vivo and express the gene product.
  • a naked DNA expression vector into cells may involve particle bombardment. This method depends on the ability to accelerate DNA-coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them (Klein et al, 1987).
  • Several devices for accelerating small particles have been developed. One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force (Yang et al, 1990).
  • the microprojectiles used have consisted of biologically inert substances such as tungsten or gold beads.
  • Selected organs including the liver, skin, and muscle tissue of rats and mice have been bombarded in vivo (Yang et al. 1990; Zelenin et al. 1991). This may require surgical exposure of the tissue or cells, to eliminate any intervening tissue between the gun and the target organ, i.e., ex vivo treatment.
  • DNA encoding a particular gene such as ⁇ 22, or fragments thereof may be delivered via this method and still be inco ⁇ orated by the present invention.
  • the expression vector may be entrapped in a liposome.
  • Liposomes are vesicular stmctures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed stmctures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991). Also contemplated are lipofectamine-DNA complexes.
  • Liposome-mediated nucleic acid delivery and expression of foreign DNA in vitro has been very successful. Wong et al, (1980) demonstrated the feasibility of liposome-mediated delivery and expression of foreign DNA in cultured chick embryo, HeLa and hepatoma cells. Nicolau et al. (1987) accomplished successful liposome- mediated gene transfer in rats after intravenous injection.
  • the liposome may be complexed with a hemagglutinating vims (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA (Kaneda et al, 1989).
  • the liposome may be complexed or employed in conjunction with nuclear non-histone chromosomal proteins (HMG-1) (Kato et al,
  • the liposome may be complexed or employed in conjunction with both HVJ and HMG-1.
  • expression vectors have been successfully employed in transfer and expression of nucleic acid in vitro and in vivo, then they are applicable for the present invention.
  • a bacterial promoter is employed in the DNA vector, it also will be desirable to include within the liposome an appropriate bacterial polymerase.
  • receptor-mediated delivery vehicles that can be employed to deliver a nucleic acid encoding a particular gene into cells. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis in almost all eukaryotic cells. Because of the cell type-specific distribution of various receptors, the delivery can be highly specific (Wu and Wu, 1993).
  • Receptor-mediated gene targeting vehicles generally consist of two components: a cell receptor-specific ligand and a DNA-binding agent.
  • ligands have been used for receptor-mediated gene transfer. The most extensively characterized ligands are asialoorosomucoid (ASOR) (Wu and Wu, 1987) and transferrin (Wagner et al, 1990).
  • ASOR asialoorosomucoid
  • transferrin Wang and Wu, 1990
  • a synthetic neoglycoprotein which recognizes the same receptor as ASOR, has been used as a gene delivery vehicle (Ferkol et al, 1993;
  • EGF epidermal growth factor
  • the delivery vehicle may comprise a ligand and a liposome.
  • a ligand and a liposome For example, Nicolau et al, (1987) employed lactosyl-ceramide, a galactose-terminal asialganglioside, inco ⁇ orated into liposomes and observed an increase in the uptake of the insulin gene by hepatocytes.
  • a nucleic acid encoding a particular gene also may be specifically delivered into a cell type such as lung, epithelial or tumor cells, by any number of receptor-ligand systems with or without liposomes.
  • epidermal growth factor may be used as the receptor for mediated delivery of a nucleic acid encoding a gene in many tumor cells that exhibit upregulation of EGF receptor.
  • Mannose can be used to target the mannose receptor on liver cells.
  • antibodies to CD5 (CLL), CD22 (lymphoma), CD25 (T-cell leukemia) and MAA (melanoma) can similarly be used as targeting moieties.
  • gene transfer may more easily be performed under ex vivo conditions.
  • Ex vivo gene therapy refers to the isolation of cells from an animal, the delivery of a nucleic acid into the cells in vitro, and then the return of the modified cells back into an animal. This may involve the surgical removal of tissue/organs from an animal or the primary culture of cells and tissues.
  • Continuous perfusion of an expression vector or a viral vector also is contemplated.
  • the amount of vector or peptide delivered in continuous perfusion can be determined by the amount of uptake that is desirable.
  • Primary mammalian cell cultures may be prepared in various ways. In order for the cells to be kept viable while in vitro and in contact with the expression vector, it is necessary to ensure that the cells maintain contact with the correct ratio of oxygen and carbon dioxide and nutrients but are protected from microbial contamination. Cell culture techniques are well documented and are disclosed herein by reference (Freshner,
  • the expression constmct may then deliver and express a nucleic acid encoding a U s 1.5 polypeptide or peptide into the cells. Finally, the cells may be reintroduced into the original animal, or administered into a distinct animal, in a pharmaceutically acceptable form by any of the means described below.
  • providing an ex vivo method of treating a mammal with a pathologic condition is within the scope of the invention.
  • One of the therapeutic embodiments contemplated by the present inventors is the intervention, at the molecular level, in the events involved in he ⁇ esvirus infection or reactivation.
  • the present inventors intend to provide to an infectable or an infected cell, an expression constmct capable of providing a U s 1.5 polypeptide or peptide to that cell to inhibit a he ⁇ esvirus infection through the use of a U s 1.5 polypeptide or peptide that contains a site required to be posttranslationally modified for viral progression.
  • U s 1.5 polypeptide or peptide can be provided exogenously to inhibit infection, for example, by competing with U s 1.5 produced by the vims as a target for phosphorylation.
  • the competitor U s 1.5 peptide or polypeptide may effectively prevent the posttranslational modification of U s 1.5, thereby rendering native U s 1.5 unable to assist in downstream events of infection.
  • expression vectors such as he ⁇ esvirus, adenovims, adeno-associated vims, vaccinia vims and retrovims.
  • viral vectors such as he ⁇ esvirus, adenovims, adeno-associated vims, vaccinia vims and retrovims.
  • liposomally-encapsulated expression vector is also contemplated.
  • Those of skill in the art are well aware of how to apply gene delivery to in vivo and ex vivo situations.
  • viral vectors one generally will prepare a viral vector stock. Depending on the kind of vims and the titer attainable, one will deliver 1 to 100, 10 to 50, 100-1000, or up to 1 x 10 4 , 1 x 10 5 , 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , 1 x 10 9 , 1 x 10 10 , 1 x 10", or 1 x 10 12 infectious particles to the patient. Similar figures may be extrapolated for liposomal or other non-viral formulations by comparing relative uptake efficiencies. Fo ⁇ nulation as a pharmaceutically acceptable composition is discussed below.
  • a subject is exposed to a viral vector and the subject is then monitored for expression constmct-based toxicity, where such toxicity may include, among other things, causing a condition that is injurious to the subject.
  • the detection of U s 1.5 polynucleotides or polypeptides is desirable, for example, to determine the level of expression from an expression constmct that contains a U s 1.5 polynucleotide encoding a U s 1.5 polypeptide.
  • Methods of detecting polynucleotides, such as the mRNA transcripts encoded by the ⁇ 22 polynucleotide of the expression constmct include Northern detection methods and nucleic acid amplification methods, such as the polymerase chain reaction (PCR) described in detail in U.S. Patent Nos.
  • Immunoassays encompassed by the present invention include, but are not limited to, those described in U.S. Patent No. 4,367,1 10 (double monoclonal antibody sandwich assay) and U.S. Patent No. 4,452,901
  • Immunoassays generally are binding assays. Certain preferred immunoassays are the various types of enzyme linked immunosorbent assays (ELISAs) and radioimmunoassays (RIA) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. The basic ELISA technique and its variations are known to those of skill in the art. Assays for the presence of expression of U s 1.5 may be performed directly on tissue samples. Methods for in vitro situ analysis are well known and involve assessing binding of antigen-specific antibodies to tissues, cells, or cell extracts. These are conventional techniques well within the grasp of those skilled in the art. C. Formulations and Routes for Administration to Patients of
  • compositions expression vectors, vims stocks, polypeptides, peptides, antibodies, and drugs— in a form appropriate for the intended application.
  • compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals.
  • compositions of the present invention comprise an effective amount of the vector to cells, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such compositions also are refened to as inocula.
  • pharmaceutically or pharmacologically acceptable refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and abso ⁇ tion delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art.
  • Supplementary active ingredients also can be inco ⁇ orated into the compositions.
  • compositions of the present invention may include classic pharmaceutical preparations. Administration of these compositions according to the present invention will be via any common route so long as the target tissue is available via that route. This includes oral, nasal, buccal, rectal, vaginal or topical. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such compositions would normally be administered as pharmaceutically acceptable compositions, described supra.
  • the expression vectors and delivery vehicles also may be administered parenterally or intraperitoneally.
  • Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
  • Dispersions also can be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • compositions of the present invention are advantageously administered in the form of injectable compositions either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. These preparations also may be emulsified.
  • a typical composition for such pu ⁇ ose comprises a pharmaceutically acceptable carrier.
  • the composition may contain 10 mg, 25 mg, 50 mg or up to about 100 mg of human semm albumin per milliliter of phosphate buffered saline.
  • Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like.
  • non-aqueous solvents examples include propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyloleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc.
  • Intravenous vehicles include fluid and nutrient replenishers.
  • Preservatives include antimicrobial agents, anti-oxidants, chelating agents and inert gases. The pH and exact concentration of the various components the pharmaceutical composition are adjusted according to well known parameters.
  • Oral formulations include such typical excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like.
  • the compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders.
  • the route is topical, the form may be a cream, ointment, salve or spray.
  • an effective amount of the therapeutic agent is determined based on the intended goal, for example (i) inhibition of tumor cell proliferation or (ii) eliminafion of tumor cells.
  • unit dose refers to physically discrete units suitable for use in a subject, each unit containing a predetermined-quantity of the therapeutic composition calculated to produce the desired responses, discussed above, in association with its administration, i.e., the appropriate route and treatment regimen.
  • the quantity to be administered both according to number of treatments and unit dose, depends on the subject to be treated, the state of the subject and the protection desired. Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual.
  • the engineered vimses of the present invention may be administered directly into animals, or alternatively, administered to cells that are subsequently administered to animals.
  • the vimses can be combined with the various ⁇ -interferon inhibiting formulations to produce transducing formulations with greater transduction efficiencies. A discussion of suitable vimses is presented above.
  • in vitro administration refers to manipulations performed on cells removed from an animal, including, but not limited to, cells in culture.
  • ex vivo administration refers to cells which have been manipulated in vitro, and are subsequently administered to a living animal.
  • in vivo administration includes all manipulations performed on cells within an animal.
  • compositions may be administered either in vitro, ex vivo, or in vivo.
  • autologous B-lymphocyte cell lines are incubated with a vaccinia vims vector of the instant invention for 24 to 48 hours or with synthetic HIV peptides for two hours.
  • the transduced cells can then be used for in vitro analysis, or alternatively for in vivo administration.
  • compositions of the present invention In vivo administration of the compositions of the present invention also are contemplated. Examples include, but are not limited to, transduction of bladder epithelium by administration of the transducing compositions of the present invention through intravesicle catheterization into the bladder (Bass et al, 1995), and transduction of liver cells by infusion of appropriate transducing compositions through the portal vein via a catheter (Bao et al, 1996). Additional examples include direct injection of tumors with the instant transducing compositions, and either intranasal or intratracheal (Dong et al, 1996) instillation of transducing compositions to effect transduction of lung cells.
  • One goal of current viral research is to find ways to improve the efficacy of anti-viral therapy.
  • One way is by combining such traditional therapies with gene therapy.
  • therapy could be used similarly in conjunction with anti-viral agents, such as acyclovir, valacyclovir, and famciclovir.
  • a target cell To inhibit he ⁇ esvirus infection or reactivation, one would generally contact a target cell with U s 1.5-derived expression vectors, vims stocks, polypeptides, or peptides and at least one other agent. These compositions would be provided in a combined amount effective to inhibit infection or reactivation by a he ⁇ esvirus. This process may involve contacting the cells with a expression or viral constmct encoding
  • U s 1.5 or with a U s 1.5 polypeptide or peptide, and the agent(s) or factor(s) at the same time.
  • This may be achieved by contacting the cell with a single composition or pharmacological formulation that includes both agents, or by contacting the cell with two distinct compositions or formulations, at the same time, wherein one composition includes a U s l .5-derived composition and the other includes the agent.
  • the gene therapy treatment may precede or follow the other agent treatment by intervals ranging from minutes to weeks.
  • the other agent and expression constmct are applied separately to the cell, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agent and expression constmct would still be able to exert an advantageously combined effect on the cell.
  • both agents are delivered to a cell in a combined amount effective to prevent viral progression.
  • agents or factors suitable for use in a combined therapy are any chemical compound or treatment method with antiviral activity; therefore, the term "antiviral agent” that is used throughout this application refers to an agent with antiviral activity.
  • U s 1.5 Two regions of U s 1.5 have been identified as containing sites requiring post- translation modification to allow the infection cycle to progress. Because U s 1.5 must be posttranslationally processed for viral progression, this modification can be monitored to identify substances capable of inhibiting a he ⁇ esvims infection.
  • posttranslational modification refers to a covalent alteration of a polypeptide or peptide amino acid sequence. Such modifications include phosphorylation, ubiquitination, nucleotidylylation, and sentrinization.
  • a U s 1.5 polypeptide containing a post-translation modification site for phosphorylation is envisioned.
  • a kinase will be used in the screening method of the present invention.
  • a kinase is a type of protein that is capable of phosphorylating another amino acid segment; there are many protein kinases, and they consist of both cellular and viral kinases (for a review, see Taylor et al, 1990;
  • kinases encoded by the viral genome include thymidine kinase, U L 13, and U s 3, and it has been previously shown that U L 13 phosphorylates ICP22 (Purves and Roizman, 1992; Purves et al, 1993).
  • the screening method of the present invention includes a step in which a polypeptide that includes a U s 1.5 phosphorylation site is exposed to a kinase under conditions that will allow the kinase to phosphorylate the polypeptide.
  • the method can be employed using the U L 13 kinase to phosphorylate U s 1.5.
  • U L 13 has been characterized as mediating the posttranslational processing of ICP22.
  • ICP22 was identified as a substrate for the UL13 protein kinase by the alteration of the phosphoprofile of ICP22 in cells infected with the UL13 deletion vims.
  • ICP22 is localized in discrete small nuclear stmctures.
  • U L 13 is a protein that is required to displace ICP22 from those stmctures so that it may colocalize with ICP4 and RNA polymerase during the onset of DNA synthesis.
  • the UL13 protein kinase is conserved among the alpha-, beta- and gammahe ⁇ esvimses and was identified by using sequence motifs diagnostic of conserved domains within protein kinases (Chee et. al. 1989, Smith and Smith, 1989).
  • Candidate substances can be evaluated for their ability to inhibit he ⁇ esvims infection by comparing the phosphorylation state of U s 1.5 in the presence of the candidate substance to its state in the absence of the candidate substance.
  • phosphorylation state refers to the extent of phosphorylation of a given polypeptide.
  • Determining the phosphorylation state of U s 1.5 can be accomplished by a number of assays, some of which are described herein and many of which are well known to those of skill in the art. These assays include alteration of polypeptide weight according to electrophoretic mobility in conjunction with Western blot analyses using an antibody that recognizes ICP22 or U s 1.5. Alternatively, proteins recognizing only a subset of ICP22 or U s 1.5 isoforms can be employed; such a protein would be capable of binding, for example, only the phosphorylated form of ICP22 or U s 1.5, and thereby, measuring binding activity is a way of determining the phosphorylation state ofU s 1.5.
  • p60 an example of a protein that selectively binds only the fast-migrating and unde ⁇ rocessed isoforms of ICP22 or U s 1.5 is p60. While p60 will bind ICP22 lacking the carboxyl-terminal 24 amino acids, it will not bind ICP22 lacking the carboxyl-terminal 40 amino acids. p60 is a cellular protein characterized in a patent application filed on April 9, 1999 by Rennet Bmni, Beatrice Fineschi,
  • proteins that will distinguish between phosphorylated forms of U s 1.5 include antibodies.
  • Means for preparing and characterizing antibodies are well known in the art (See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; inco ⁇ orated herein by reference).
  • the methods for generating monoclonal antibodies (MAbs) generally begin along the same lines as those for preparing polyclonal antibodies. Briefly, a polyclonal antibody is prepared by immunizing an animal with an immunogenic composition in accordance with the present invention (either with or without prior immunotolerizing, depending on the antigen composition and protocol being employed) and collecting antisera from that immunized animal. A wide range of animal species can be used for the production of antisera.
  • the animal used for production of anti-antisera is a rabbit, a mouse, a rat, a hamster, a guinea pig or a goat. Because of the relatively large blood volume of rabbits, a rabbit is a prefe ⁇ ed choice for production of polyclonal antibodies.
  • a given composition may vary in its immunogenicity. It is often necessary therefore to boost the host immune system, as may be achieved by coupling a peptide or polypeptide immunogen to a carrier.
  • exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine semm albumin (BSA). Other albumins such as ovalbumin, mouse semm albumin or rabbit semm albumin can also be used as carriers.
  • KLH keyhole limpet hemocyanin
  • BSA bovine semm albumin
  • Other albumins such as ovalbumin, mouse semm albumin or rabbit semm albumin can also be used as carriers.
  • Means for conjugating a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, ⁇ -maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimyde and bis-biazotized benzidine.
  • the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants.
  • Suitable adjuvants include all acceptable immunostimulatory compounds, such as cytokines, toxins or synthetic compositions.
  • Adjuvants that may be used include IL-1, IL-2, IL-4, IL-7, IL-12, ⁇ -interferon, GMCSP, BCG, aluminum hydroxide, MDP compounds, such as thur-MDP and nor- MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A (MPL).
  • MDP compounds such as thur-MDP and nor- MDP
  • CGP CGP
  • MPL monophosphoryl lipid A
  • MPL monophosphoryl lipid A
  • RIBI which contains three components extracted from bacteria, MPL, trehalose dimycolate (TDM) and cell wall skeleton (CWS) in a 2% squalene/Tween 80 emulsion.
  • MHC antigens may even be used.
  • Exemplary, often prefe ⁇ ed adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants and aluminum hydroxide adjuvant.
  • complete Freund's adjuvant a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis
  • incomplete Freund's adjuvants and aluminum hydroxide adjuvant.
  • BRM biologic response modifiers
  • BRMs include, but are not limited to, Cimetidine (CIM; 1200 mg/d) (Smith/Kline, PA); or low-dose Cyclophosphamide (CYP; 300 mg/m 2 ) (Johnson/Mead, NJ) and cytokines such as ⁇ -interferon, IL-2, or
  • IL-12 or genes encoding proteins involved in immune helper functions, such as CD80 (B7-l ) and CD86 (B7-2).
  • MAbs may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Patent 4,196,265, inco ⁇ orated herein by reference.
  • this technique involves immunizing a suitable animal with a selected immunogen composition, e.g., a purified or partially purified U s 1.5 protein, polypeptide or peptide (or any U s 1.5 composition, if used after tolerization to common antigens).
  • a selected immunogen composition e.g., a purified or partially purified U s 1.5 protein, polypeptide or peptide (or any U s 1.5 composition, if used after tolerization to common antigens).
  • the immunizing composition is administered in a manner effective to stimulate antibody producing cells.
  • MAbs will be chimeric MAbs, including "humanized” MAbs.
  • the chimeric MAb is engineered by cloning recombinant DNA containing the promoter, leader, and variable-region sequences from a mouse anti-U s l .5 producing cell and the constant-region exons from a human antibody gene.
  • CDRs mouse complementary determining regions
  • Single chain antibodies that bind to U s 1.5 phosphorylation sites or bind to U s 1.5 and inhibit its phosphorylation are also contemplated by the present invention.
  • a single chain antibody is a variation of an antibody that contains a variable region from a light chain and a variable region from a heavy chain, joined by a linker, such as a peptide linker.
  • a single chain antibody (also refened to as a "single chain antibody fragment" or scFv) has one antigen binding site.
  • multivalent single chain antibody can be created by linking single chain antibody fragments to one another. Methods of producing single chain antibodies are known to those of skill in the art and are described, for example in U.S. Patent 5,877,291 , which is herein inco ⁇ orated by reference.
  • Peptides corresponding to one or more antigenic determinants, or "epitopic core regions,” of the U s 1.5 polypeptide of the present invention can also be prepared.
  • Such peptides should generally be at least five or six amino acid residues in length, will preferably be about 10, 15, 20, 25 or about 30 amino acid residues in length, and may contain up to about 35-50 residues or so.
  • Synthetic peptides will generally be about 35 residues long, which is the approximate upper length limit of automated peptide synthesis machines, such as those available from Applied Biosystems (Foster City, CA). Longer peptides may also be prepared, e.g., by recombinant means.
  • major antigenic determinants of a polypeptide may be identified by an empirical approach in which portions of the gene encoding the polypeptide are expressed in a recombinant host, and the resulting proteins tested for their ability to elicit an immune response.
  • PCRTM can be used to prepare a range of peptides lacking successively longer fragments of the C-terminus of the protein. The immunoactivity of each of these peptides is determined to identify those fragments or domains of the polypeptide that are immunodominant. Further studies in which only a small number of amino acids are removed at each iteration then allows the location of the antigenic determinants of the polypeptide to be more precisely determined.
  • Another method for determining the major antigenic determinants of a polypeptide is the SPOTsTM system (Genosys Biotechnologies, Inc., The Woodlands, TX).
  • SPOTsTM system Geneosys Biotechnologies, Inc., The Woodlands, TX.
  • overlapping peptides are synthesized on a cellulose membrane, which following synthesis and deprotection, is screened using a polyclonal or monoclonal antibody.
  • the antigenic determinants of the peptides which are initially identified can be further localized by performing subsequent syntheses of smaller peptides with larger overlaps, and by eventually replacing individual amino acids at each position along the immunoreactive peptide.
  • polypeptides are prepared that contain at least the essential features of one or more antigenic determinants.
  • the peptides are then employed in the generation of antisera against the polypeptide.
  • Minigenes or gene fusions encoding these determinants can also be constmcted and inserted into expression vectors by standard methods, for example, using PCRTM cloning methodology.
  • peptides for antibody generation or vaccination typically requires conjugation of the peptide to an immunogenic carrier protein, such as hepatitis B surface antigen, keyhole limpet hemocyanin or bovine semm albumin. Methods for performing this conjugation are well known in the art.
  • an immunogenic carrier protein such as hepatitis B surface antigen, keyhole limpet hemocyanin or bovine semm albumin.
  • the screening method employs a detectable label that can be used to identify a polypeptide that is posttranslationally modified or to distinguish a polypeptide that is posttranslationally modified from one that is not so modified.
  • Detectable labels are compounds or elements that can be detected due to their specific functional properties, or chemical characteristics, the use of which allows the antibody to which they are attached to be detected, and further quantified if desired.
  • Examples of a detectable label include the use of radioactive compounds such as [ 32 P], [ 33 S], or [ 12 T], as well as the use of a fluorescent, light-sensitive, or photo-activatable compound such as fluorescein or fluorescein isothiocyanate, rhodamine, lissamine, indocyanine, luciferase, and Green Fluorescent Protein (GFP).
  • a detectable label can be affixed to a polypeptide, and this can occur, for example, through the formation of a covalent bond or a coupling mechanism.
  • the detectable label can be attached to the polypeptide before, during, or after it contacts a posttranslational modifier under conditions appropriate to effect modification.
  • the modification can be a detectable label.
  • radioactive [ 32 P] can be used to phosphorylate the polypeptide that includes a U s 1.5 phosphorylation site, such that the labelled polypeptide can be identified when it is exposed to film, such as XAR or XRP autoradiographic film.
  • identifying the phosphorylation state of U s 1.5 involves comparing the characteristics of U s 1.5 in the presence and absence of a candidate inhibitor. To accomplish this, one would simply conduct parallel or otherwise comparatively controlled assays to identify a compound that inhibits phosphorylation or some other posttranslational modification of U s 1.5.
  • the candidate screening assay is quite simple to set up and perform. After obtaining a relatively purified preparation of U s 1.5, either from native or recombinant sources, one will simply admix a candidate substance with the polypeptide under conditions appropriate to effect modification of the polypeptide.
  • the present invention concerns immunodetection methods for binding or quantifying or otherwise generally detecting biological components such as U s 1.5 protein components.
  • the U s 1.5 antibodies prepared in accordance with the present invention may be employed to detect a particular isoform of a U s 1.5 proteins, polypeptides or peptides, such as an isoform that is not phosphorylated, or one that is phosphorylated.
  • U s l .5-specific antibodies is contemplated.
  • the immunobinding methods include obtaining a sample containing a U s 1.5 protein, polypeptide or peptide, and contacting the sample with a first anti- U s 1.5 antibody in accordance with the present invention, as the case may be, under conditions effective to allow the formation of immunocomplexes.
  • the antibody recognizes a subset of isoforms and other isoforms are not recognized.
  • the antibody will preferably be linked to a solid support, such as in the form of a column matrix, and the sample containing the U s 1.5 protein antigenic component will be applied to the immobilized antibody. The unwanted components will be washed from the column, leaving the antigen immunocomplexed to the immobilized antibody, w hich U b l 5 protein antigen is then collected by removing the U s l 5 protein or peptide from the column
  • the lmmunobinding methods also include methods for detectmg or quantifying the amount of a particular isoform of U s l 5 protein reactive component in a sample, which methods require the detection or quantification of any immune complexes formed du ⁇ ng the binding process
  • detectmg or quantifying the amount of a particular isoform of U s l 5 protein reactive component in a sample which methods require the detection or quantification of any immune complexes formed du ⁇ ng the binding process
  • the first antibody that becomes bound within the primary immune complexes may be detected by means of a second binding ligand that has binding affinity for the antibody.
  • the second binding ligand may be linked to a detectable label.
  • the second binding ligand is itself often an antibody, which may thus be termed a "secondary" antibody.
  • the primary immune complexes are contacted with the labeled, secondary binding ligand, or antibody, under conditions effective and for a period of time sufficient to allow the formation of secondary immune complexes.
  • the secondary immune complexes are then generally washed to remove any non-specifically bound labeled secondary antibodies or ligands, and the remaining label in the secondary immune complexes is then detected.
  • Further methods include the detection of primary immune complexes by a two step approach.
  • a second binding ligand such as an antibody, that has binding affinity for the antibody is used to form secondary immune complexes, as described above.
  • the secondary immune complexes are contacted with a third binding ligand or antibody that has binding affinity for the second antibody, again under conditions effective and for a period of time sufficient to allow the formation of immune complexes (tertiary immune complexes).
  • the third ligand or antibody is linked to a detectable label, allowing detection of the tertiary immune complexes thus formed. This system may provide for signal amplification if this is desired.
  • immunoassays in their most simple and direct sense, are binding assays.
  • Certain preferred immunoassays are the various types of enzyme linked immunosorbent assays (ELISAs) and radioimmunoassays (RIA) known in the art.
  • ELISAs enzyme linked immunosorbent assays
  • RIA radioimmunoassays
  • Immunohistochemical detection using tissue sections is also particularly useful. However, it will be readily appreciated that detection is not limited to such techniques, and western blotting, dot blotting, FACS analyses, and the like may also be used.
  • the U s 1.5 antibodies of the invention are immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a test composition containing the U s 1.5 protein antigen, such as a clinical sample, is added to the wells. After binding and washing to remove non-specifically bound immune complexes, the bound U s 1.5 protein antigen may be detected. Detection is generally achieved by the addition of another anti- U s 1.5 antibody that is linked to a detectable label. This type of ELISA is a simple "sandwich ELISA". Detection may also be achieved by the addition of a second anti- U s 1.5 antibody, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label.
  • the samples containing the U s 1.5 protein antigen are immobilized onto the well surface and then contacted with the anti- U s 1.5 antibodies of the invention. After binding and washing to remove non-specifically bound immune complexes, the bound anti- U s 1.5 antibodies are detected. Where the initial anti- U s 1.5 antibodies are linked to a detectable label, the immune complexes may be detected directly. Again, the immune complexes may be detected using a second antibody that has binding affinity for the first anti-U s 1.5 antibody, with the second antibody being linked to a detectable label.
  • U s 1.5 proteins, polypeptides or peptides are immobilized, involves the use of antibody competition in the detection.
  • labeled antibodies against U s 1.5 protein are added to the wells, allowed to bind, and detected by means of their label.
  • the amount of U s 1.5 protein antigen in an unknown sample is then determined by mixing the sample with the labeled antibodies against U s 1.5 before or during incubation with coated wells.
  • the presence of U s 1.5 protein in the sample acts to reduce the amount of antibody against U s 1.5 protein available for binding to the well and thus reduces the ultimate signal.
  • ELISAs have certain features in common, such as coating, incubating or binding, washing to remove non-specifically bound species, and detecting the bound immune complexes. These are described below.
  • a plate with either antigen or antibody In coating a plate with either antigen or antibody, one will generally incubate the wells of the plate with a solution of the antigen or antibody, either overnight or for a specified period of h. The wells of the plate will then be washed to remove incompletely adsorbed material. Any remaining available surfaces of the wells are then "coated" with a nonspecific protein that is antigenically neutral with regard to the test antisera. These include bovine semm albumin (BSA), casein and solutions of milk powder.
  • BSA bovine semm albumin
  • the coating allows for blocking of nonspecific adso ⁇ tion sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.
  • a secondary or tertiary detection means rather than a direct procedure.
  • the immobilizing surface is contacted with the biological sample to be tested under conditions effective to allow immune complex (antigen/antibody) formation. Detection of the immune complex then requires a labeled secondary binding ligand or antibody, or a secondary binding ligand or antibody in conjunction with a labeled tertiary antibody or third binding ligand.
  • Under conditions effective to allow immune complex (antigen/antibody) formation means that the conditions preferably include diluting the antigens and antibodies with solutions such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS)/Tween. These added agents also tend to assist in the reduction of nonspecific background.
  • the "suitable” conditions also mean that the incubation is at a temperature and for a period of time sufficient to allow effective binding. Incubation steps are typically from about 1 to 2 to 4 h or so, at temperatures preferably on the order of 25°C to 27°C, or may be overnight at about 4°C or so.
  • the contacted surface is washed so as to remove non-complexed material.
  • a preferred washing procedure includes washing with a solution such as PBS/Tween, or borate buffer. Following the formation of specific immune complexes between the test sample and the originally bound material, and subsequent washing, the occurrence of even minute amounts of immune complexes may be determined.
  • the second or third antibody will have an associated label to allow detection.
  • this will be an enzyme that will generate color development upon incubating with an appropriate chromogenic substrate.
  • a urease glucose oxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibody for a period of time and under conditions that favor the development of further immune complex formation (e.g., incubation for
  • the amount of label is quantified, e.g., by incubation with a chromogenic substrate such as urea and bromocresol pu ⁇ le or 2,2'-azino-di-(3-ethyl- benzthiazoline-6-sulfonic acid (ABTS) and H 2 O 2 , in the case of peroxidase as the enzyme label. Quantification is then achieved by measuring the degree of color generation, e.g., using a visible spectra spectrophotometer. 2.
  • the antibodies of the present invention may also be used in conjunction with both fresh-frozen and formalin-fixed, paraffin-embedded tissue blocks prepared for study by immunohistochemistry (IHC).
  • IHC immunohistochemistry
  • the method of preparing tissue blocks from these particulate specimens has been successfully used in previous IHC studies of various prognostic factors, and is well known to those of skill in the art (Brown et al, 1990; Abbondanzo et al, 1990; Alfred et al, 1990).
  • frozen-sections may be prepared by rehydrating 50 ng of frozen "pulverized” tissue at room temperature in phosphate buffered saline (PBS) in small plastic capsules; pelleting the particles by centrifugation; resuspending them in a viscous embedding medium (OCT); inverting the capsule and pelleting again by centrifugation; snap-freezing in -70°C isopentane; cutting the plastic capsule and removing the frozen cylinder of tissue; securing the tissue cylinder on a cryostat microtome chuck; and cutting 25-50 serial sections.
  • PBS phosphate buffered saline
  • OCT viscous embedding medium
  • Permanent-sections may be prepared by a similar method involving rehydration of the 50 mg sample in a plastic microfuge tube; pelleting; resuspending in 10% formalin for 4 h fixation; washing/pelleting; resuspending in warm 2.5%> agar; pelleting; cooling in ice water to harden the agar; removing the tissue/agar block from the tube; infiltrating and embedding the block in paraffin; and cutting up to 50 serial permanent sections.
  • Kits of the present invention are kits that can be used either to identify a compound capable of inhibiting infection by he ⁇ esvims or to effect a treatment method comprising U s 1.5 protein, polypeptide, peptide, inhibitor, gene, vector or other UJ .5 effector.
  • kits will generally contain, in suitable container means, a pharmaceutically acceptable formulation of a U s 1.5 protein, polypeptide, peptide, domain, inhibitor, or a gene or vector expressing any of the foregoing in a pharmaceutically acceptable formulation.
  • other components such as a kinase and reagents used in a kinase reaction may also be provided.
  • the kit may have a single container means, or it may have distinct container means for each compound.
  • the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly prefe ⁇ ed.
  • the U s 1.5 compositions may also be formulated into a syringeable composition, particularly if the kit is for therapeutic pu ⁇ oses.
  • the container means may itself be a syringe, pipette, or other such like apparatus, from which the formulation may be applied to an infected area of the body, injected into a subject, or even applied to and mixed with the other components of the kit.
  • the components of the kit may be provided as dried powder(s).
  • the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means.
  • the container means will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which the U s 1.5 protein, gene or inhibitory formulation are placed, preferably, suitably allocated.
  • the kits may also comprise a second container means for containing a sterile, pharmaceutically acceptable buffer or other diluent.
  • kits of the present invention will also typically include a means for containing the vials in close confinement for commercial sale, such as, e.g., injection or blow-molded plastic containers into which the desired vials are retained.
  • a means for containing the vials in close confinement for commercial sale such as, e.g., injection or blow-molded plastic containers into which the desired vials are retained.
  • the kits of the invention may also comprise, or be packaged with, an instmment for assisting with the injection/administration or placement of the ultimate U s 1.5 protein or gene composition within the body of a subject.
  • an instmment may be a syringe, pipette, forceps, or any such medically approved delivery vehicle.
  • HSV-l(F) is the prototype HSV-1 strain used in this laboratory (Ejercito et al, 1968).
  • PI included in the final product the EcoNI site 100 bp upstream of the ICP22 open reading frame whereas P2 replaced the initiator methionine codon of ICP22 with an Eagl site.
  • the PCRTM product was then digested with EcoNI and Eagl and subcloned into pRB5210, which has also been digested with
  • a 1.2 kb PCRTM product was generated from pRB5210 with the Pfu polymerase (Stratagene, La Jolla, CA) and the primers (P3) GAC GTT TTC TGG CGG CCG ATG GCC GAC and (P4) GAC GCT GGG ACA AAC GCT TTG ATT TTG GTC.
  • P3 inserted an Eagl site adjacent to the initiator methionine codon of ICP22 and the primer P4 represents a sequence located 50 bp downstream from the carboxyl-terminal Eagl site of the carboxyl-terminal stop codon of the ICP22 open reading frame.
  • the PCRTM product was then digested with Eagl and subcloned into the Eagl site of pRB5212.
  • the resulting plasmid, pRB5214, contained ICP22 and U s 1.5 with an Eagl site just preceding the initiation methionine.
  • a 870 bp PCRTM product was generated from pRB5212 with Pfu polymerase (Stratagene, La Jolla, CA) and the primer (P5) ACG CAG CCC CGG GCC CCC CGG CCG TCG GCC and P4.
  • P5 created an Eagl site 25 bp upstream of the U s 1.5 initiation methionine (171) and the primer P4.
  • the PCRTM product was then digested with Eagl and subcloned into pRB5212.
  • the subsequent plasmid pRB5215 contained a U s l .5 open reading frame driven by the ⁇ 22 promoter.
  • To constmct pRB458 with EcoRI site of plasmid pucl9 was destroyed with the T4 polymerase (Stratagene, La Jolla, CA).
  • Hindlll M (126,526-133,466 bp) fragment of HSV-l(F) was subcloned from pRB201 and cloned into pRB458.
  • PCRTM product was generated from pRB5210 with the Pfu polymerase and the primers P5 and (P7) GGC CCG GGC CGTTCC ACG GAG CTG GTA TC.
  • P7 inserted an Eagl site 120 bp upstream from the stop codon of ⁇ 22/U s J5.
  • the PCRTM product was digested with Eagl and Drain and subcloned into pRB5210 digested with Dralll and Eagl.
  • both ICP22 and U s 1.5 open reading frames were tmncated by 40 3' codons.
  • PCRTM product was generated from pRB5210 with the Pfu polymerase and the primers P3 and (P8) ATA GGG CGG CCG GGTGGA GAA GCG CAT TTT.
  • P8 inserted an Eagl site 30 bp upstream from the carboxyl-terminal stop codon of ICP22 and U S J5.
  • the PCRTM product was digested with Eagl and subcloned into pRB5212 digested with Eagl.
  • both ICP22 and U s 1.5 open reading frames were tmncated by 10 3' codons.
  • a 1.2 kb PCRTM product was generated from pRB5210 with the Pfu polymerase and the primer P3 and (PI 2) GGTGGA CGGCCG CAT TTTCCG GCA GCC GTC.
  • P12 inserted an Eagl site 45 bp upstream from the stop codon of ICP22 and U s .15.
  • the PCRTM product was digested with Eagl and subeloned into Eagl digested pRB5212.
  • ICP22 and U s 1.5 open reading frames are tmncated by 15 3' codons.
  • a 1 J kb PCRTM product was generated from pRB5210 with the Pfu polymerase and the primer P3 and (PI 4) TCT GAG CGG CCG TCC GAT ACA GCC TTG GAG TCT.
  • PI 4 inserted an Eagl site 1 15 bp downstream from the initiation methionine (1) of IPC22 and U S J5.
  • the PCRTM product was digested with Eagl and subcloned into Eagl digested pRB5212.
  • the ICP22 open reading frame is tmncated by 47 5' codons, the first available codon for translational initiation is methionine (90).
  • the mutagenized plasmid pRB5273 was digested with the restriction enzyme Eagl to release a 1.2 kb product and subcloned into the Eagl site of pRB5212.
  • the resulting plasmid, pRB5274 contains the complete open reading frames of ICP22/US1.5.
  • constmct R7827 (Threonine 300 to Alanine and Serine 301 to Glycine) the plasmid pRB5274 was cotransfected with R7802 recombinant viral DNA into rabbit skin cells. Several plaques were isolated, and the stmcture of the recombinant vims R7827 was verified by hybridization of electrophoretically separated BamHI and Bgl II digested viral DNA with nick-translated pRB5210 (BamHI N fragment).
  • Cosmids The set of cosmids used in this study were derived from HSV-l(F) DNA as described elsewhere (Chang et al, 1997: Kawaguchi et al. 1997) and is illustrated in FIG. 1A, lines 2 thm 6. The sequences contained in the cosmid set were as follows: pBC1004, nucleotides 133,052 to 17,029; pBC1006, the nucleotides 2,945 to 45,035; pBC1007 nucleotides 77,933 to 1 16,016; pBC1008, nucleotides 106,750 to 142,759; and pBC1014, nucleotides 40,617 to 80,454.
  • Cosmid pBClOl ⁇ containing the nucleotides 1 10,095 to 131,534 was constmcted by digesting pBC1008 with EcoRI, followed by gel purification. The desired DNA fragment was then ligated into the multicloning site of the SuperCosl cosmid vector (Strategene, La Jolla, CA) and packaged into lambda phage with the Gigapack XLII (Stratagene, La Jolla CA) packaging extract according to the manufacturer's instmctions.
  • SuperCosl cosmid vector Strategene, La Jolla, CA
  • Gigapack XLII Stratagene, La Jolla CA
  • the constmction of recombinant virus R7802 ( ⁇ 22/ ⁇ U s 1.5) involved the following steps: (i) The cosmid pBC1008 was digested with the restriction enzyme
  • This plasmid has an 2.5 kb overlap with pBClOl ⁇ and a 1 kb overlap with the cosmid pBC1004 (FIG. IB, lines 3, 4, and 5).
  • a second plasmid was constmcted (pBR5219) that contained the open reading frame for ⁇ 22/U s 1.5 driven by the recombinant he ⁇ es vims ⁇ promoter but lacking sequences overlapping within the cosmid set.
  • the modified cosmid set in amounts of 1 ⁇ g each (pBC1004, pBC1006, pBC1007, pBC1014, and pBClOl ⁇ ), the linearized bridging plasmid pBR5213 in amounts ranging from 0 to 0.8 ⁇ g lacking the ⁇ 22/U s 1.5 gene, and the ⁇ 22/U s 1.5 ⁇ expression plasmid (pBR5219, 0.1 ⁇ g) were transfected into Vero cells with Lipofectamine (Gibco BRL, Gaithersburg MD) according to the manufacturer's instmctions. The progeny vims were designated R7802 ( ⁇ 22/ ⁇ U s l .5).
  • the cells were transfected with the cosmid set (pBC1006, pBC1014, pBC1007, pBC1004, and pBClOl ⁇ ) without the bridging plasmid (pBR5213) to yield R7805 ( ⁇ 22 NT /U s 5). (FIG. 2, line 4).
  • viral DNAs were isolated and digested with BamHI, electrophoretically separated in agarose gels, transferred to a nylon membrane, and hybridized to 32 P-labeled BamHI N (pBR5210) as described above.
  • the probe pBR5210 (BamHI N) hybridized to the 4.9-kb BamHI N fragment of wild type HSV- 1(F) and R7905 (HSV- 1(F)) (FIG. 3, lanes 1 and 2).
  • the probe hybridized to a 3.6-kb BamHI fragment corresponding to the predicted size of BamHI N in R7802 ( ⁇ 22/ ⁇ U s 1.5) (FIG. 3, lane 3).
  • the probe also hybridized to a 3.4-kb fragment corresponding to the predicted size of BamHI N in R7805 ( ⁇ 22 NT / ⁇ U s 1.5) (FIG. 3, lane 4).
  • the probe hybridizes to the BamHI Z fragment since this fragment contains a potion of the inverted repeat common with BamHI N.
  • R7808 ( ⁇ 22/ ⁇ U s 1.5) the predicted open reading frame of U s 1.5, that is, the sequence encoding codons 171 to 420 of ICP22, was PCRTM-amplified and cloned into plasmid pRB5212.
  • the new plasmid, pRB5215 contained an additional Eagl restriction site at the beginning of the U s 1.5 open reading frame (FIG. 2, line 6). This plasmid was cotransfected with the R7802 ( ⁇ 22/ ⁇ U s 1.5) recombinant viral DNA into rabbit skin cells.
  • This amplified product extended from the initiation methionine codon to codon 410 of ⁇ 22 and contained a diagnostic Eagl restriction endonuclease site at the amino terminus of the ⁇ 22/U s 1.5 genes.
  • the PCRTM product was cloned into plasmid pRB5212 to create pRB5217.
  • This plasmid (pRB5217) was cotransfected with R7802 ( ⁇ 22/ ⁇ U s 1.5) viral DNA into rabbit skin cells.
  • R7802 ⁇ 22/ ⁇ U s 1.5
  • R7804 (FIG. 2, line 3).
  • the open reading frame of 22/U s 1.5 was PCRTM amplified and cloned into plasmid (pRB5212) to create pRB5214 which was identical to BamHI N except for the presence of an additional Eagl restriction site at the beginning of the a22/U s 1.5 open reading frame (FIG. 2, compare lines 2 and 3).
  • This plasmid (pRB5214) was cotransfected with viral DNA of R7802 ( ⁇ 22/ ⁇ U s 1.5) into rabbit skin cells. The selection for the recombinant vims took advantage of the observation that R7802 viral DNA did not form plaques in transfected rabbit skin cells.
  • plaques on this cell line would signal the presence of a recombinant vims.
  • Several plaques were isolated, and the stmcture of the recombinant R7804 vims was verified by hybridization of electrophoretically separated BamHI digested viral DNA with nick-translated pRB5210 (BamHI N fragment).
  • R7828 ( ⁇ 22/U s 1.5) respectively (FIG. 2 lines 6 and 7).
  • R7810 ( ⁇ 22 c ⁇ /U s 1.5 c ⁇ )[ ⁇ 40a.a.] was repaired with plasmid pRB5212 to yield R7821
  • U s l 1 , ICPO HI 083 mouse monoclonal antibodies
  • the rabbit polyclonal antibody R77 amino-terminal ICP22, W2 against the carboxyl-terminal of ICP22 and the rabbit polyclonal Wl against U L 38 were described previously (Ackermann et al, 1985; Kemp and Latchman, 1988; Roller and Roizman, 1992; Ward et al, 1996).
  • Goat anti-rabbit or anti-mouse alkaline phosphatase-conjugated secondary antibody was purchased from Bio-Rad.
  • PFU plaque forming units
  • the cells were maintained in medium 199V consisting of a mixture of 199 supplemented with 1% calf semm.
  • PBS-A ice-cold phosphate-buffered saline lacking Ca2+ and Mg2+
  • the cells were rinsed and scraped into 1 ml of ice-cold phosphate-buffered saline lacking Ca2+ and Mg2+ (PBS-A), centrifuged for 5 min in a microcentrifuge at 4°C, and resuspended in 350 ⁇ l of PBS-A*[PBS-A with OJ mM TPCK (tosylsulfonyl phenlylalanyl chlromethyl ketone), OJ mM TLCK (tosyl-L-phenylalanine chloromethyl ketone), OJ mM PMSF (phenylmethylsulfonyl fluoride), 1.0% (v/v)
  • Vero cell lysate equivalent to one half of a 25-cm2 tissue culture flask were harvested in lysis buffer, chilled on wet ice for 30 min, and cleared by centrifugation in a microfuge for 20 min. at 4oC.
  • the supernatant fluid was then precleared by mixing with 30 ⁇ l of a 50%) slurry of protein A-conjugated Sepharose beads (Sigma) for 1 hr. with mixing and reacted with a 1 :100 dilution of UL13 (W3) or ICPO (HI 083) antisemm overnight at 4°C (or any other appropriate substrate Ab).
  • the immune complex was harvested with protein A-Sepharose beads, repeatedly rinsed with lysis buffer and either eluted from the Sepharose beads by boiling for five minutes in dismption buffer and electrophoretically separated on denaturing polyacrylamide gels or used for in vitro kinase assays as described below.
  • Cytoplasmic DNAs from infected cells were harvested by resuspending 2 roller bottle cultures (approximately 4x10 s cells) in 20 ml of 150 mM NaCl , 10 mM Tris-HCL pH 1.4, 1.5 mM MgCl, with NP-40 added to a final concentration of 0J%> (v/v).
  • the nuclei were pelleted by centrifugation at 2,500 ⁇ m for 5 min in a
  • RNase A was added to a final concentration of 20 ⁇ g/ml and incubated for 15 min at 37°C, then centrifuged through a linear 5-20% potassium acetate gradient in 10 mM Tris-HCI pH 8.0, 5 mM EDTA in a SW41 rotor at 40,000 ⁇ m for 3.5 h at 20°C. The pellet was gently rinsed once with H 2 O, resuspended in 0.4 ml of H 2 O, precipitated by the addition of 2 volumes of 100% ethanol, solubilized, digested with BamHI, electrophoretically separated on an 0.8% agarose gel and transferred to a nylon membrane (Bio-Rad Laboratories, Hercules, CA). The hybridization and membrane-stripping procedures were as recommended by the manufacturer.
  • the plasmid pRB5210 was used to make ?2 P-dCTP labeled probe using a Nick Translation Kit (Promega, Madison Wl).
  • HEp-2 cells grown in 25-cm2 flask cultures were infected with 10 PFU of HSV-l(F), R7805 ( ⁇ 22 NT /U s 1.5), R7808 ( ⁇ 22 NT U s 1.5 ⁇ 22), and R7810 ( ⁇ 22 r ⁇ / ⁇ U s 1.5 c ⁇ ) per cell. At 18 h after infection, the cells were washed with 5 mis
  • PBS(A) and then scraped into 1 ml PBS(A) and resuspended into 100 ⁇ l buffer A (50mM Tris-HCI [pH 7.5], 5 mM MgCl 2 , 1 mM PMSF, 0.1 mM TLCK, 0.1 mM TPCK).
  • the cells were lysed by addition of 4 ⁇ l of 10%> Nonident P-40 and stored at 25°C for 5 min.
  • the nuclei were separated from the cytoplasm by centrifugation in a microcentrifuge. The supernatant (cytoplasmic fraction) was removed and 50 pi of dismption buffer added.
  • the nuclei were resuspended in 75 ⁇ l PBS(A) and 50 ⁇ l of dismption buffer.
  • the plasmid was then sequenced to verify the presence of these mutations and no other.
  • the resulting plasmid pRB5273 contains the open reading frame of ICP22/US1.5 in which Threonine 300 has been changed to Alanine and Serine 301 has been changed to Glycine.
  • the Fc receptor-depleted lysates were then used for immunoprecipitation with the UL13 (W3) or ICPO (HI 083) antisemm as described above.
  • the immune complexes harvested on the protein A-conjugated Sepharose beads were rinsed twice with kinase buffer [50 mM Tris-HCI (pH 8.0), 50 mM MgCl 2 , 0.1% NP40, and 1 mM DTT].
  • Protein kinase assays were done by incubating the immune complexes with 70 ⁇ l protein kinase buffer supplemented with 100 ⁇ M ATP and 65 ⁇ Ci [ 32 P]ATP at 37°C for 30 minutes.
  • the beads were then rinsed with lysis buffer, resuspended in dismption buffer and boiled for 5 minutes.
  • the released proteins in the supernatant were electrophoretically separated on denaturing polyacrylamide gels.
  • EXAMPLE 2 Construction ofthe ___22/t_U ⁇ 1.5 and UJ.5 Recombinant Viruses
  • ICP22/US 1.5 it was necessary to constmct a vims from which the entire open reading frame of ⁇ 22/U s 1.5 had been deleted.
  • the inventors used a modified cosmid system as described previously (Chang et al, 1997; Kawaguchi et al, 1997) and in the material and methods. This cosmid set is illustrated in FIG. 1A, lines 2 thm 6.
  • Constmction of the ⁇ 22/ ⁇ U s 1.5 cosmid vims involved the following steps, (i) The cosmid pBC1008 was digested with the restriction enzyme EcoRI to create a gap within the ⁇ 22/U s 1.5 region (FIG. IB, lines 3 and 4) to create the cosmid pBClOl ⁇ . (ii) A bridging plasmid (pBR5213) from which the entire ⁇ 22/U s 1.5 open reading frame had been deleted was constmcted. (FIG. IB, line 5).
  • a second plasmid was constmcted (pBR5219) which contained the open reading frame for ⁇ 22/U s 1.5 driven by the recombinant he ⁇ es vims apy promoter but without any overlapping sequences within the cosmid set.
  • the modified cosmid set (pBC1004, pBClOO ⁇ , pBC1007, pBC1014, and pBClOl ⁇ ), the bridging plasmid pBR5213 lacking the ⁇ 22/U s 1.5 genes and the ⁇ 22/U s 1.5 expression plasmid (pBR5219) were transfected into Vero cells to yield R7802 (FIG. 2, line 2).
  • the cells were transfected with the cosmid set (pBClOO ⁇ , pBC1014, pBC1007, pBC1004, and pBClOl ⁇ ) without the bridging plasmid (pBR5213) to yield R7805 (FIG. 2, line 4).
  • the subsequent plaques were isolated and the genotypes analyzed.
  • the genotypes of the wild type parent HSV- 1(F) and of the recombinant vimses R7905 (HSV- 1(F)) derived from the original cosmid set, R7802 ( ⁇ 22/ ⁇ U s 1.5), and R7805 ( ⁇ 22/U s 1.5) were analyzed as follows: Viral DNAs were isolated and digested with BamHI, electrophoretically separated in agarose gels, transfe ⁇ ed to a nylon membrane, and hybridized to 32 P-labeled BamHI N (pBR5210) as described in Materials and Methods. The probe hybridized to the 4.9-kb BamHI N fragment of wild type HSV-l(F) and R7905 (HSV-I(F)) (FIG. 3, lanes 1 and 2). The probe hybridized to a 3.6-kb BamHI fragment corresponding to the predicted size of
  • the recombinant vims R7808 ( ⁇ 22/U s 1.5) and the repair vimses of R7802 ( ⁇ 22/ ⁇ U s 1.5), R7805 ( ⁇ 22/U s 1.5) and R7808 ( ⁇ 22/U s 1.5) were constmcted by blind selection on rabbit skin cells to yield R7804 ( ⁇ 22/U s 1.5), R7806 ( ⁇ 22/U s 1.5) and R7828 ( ⁇ 22/U s 1.5) (FIG. 2, lines 3, 5, 6 and 7).
  • EXAMPLE 3 Biologic Properties ofR7802 (Aa22/AU .5) and R7805 (Aa22/UJ5)
  • R7802 and R7805 Two are of key importance. These are R7802 and R7805.
  • R7802 lacked the coding domains of both ⁇ 22 and U s 1.5 genes (FIG. 2, line 2), whereas R7805 contained the coding sequences of U s 1.5, but not the 5' sequence that code the amino terminus of ICP22 (FIG. 2, line 4).
  • R7802 could not be differentiated from R325 with respect to biologic properties.
  • R7805 was tested for its ability to cause morbidity or mortality upon intracerebral inoculation in mice.
  • the PFU/LD 50 were 5.6 ⁇ l0 4 for R7805, 3.4x10' for R7806 in which the ⁇ 22/U s 1.5 lesions were repaired as compared with l.lxl 0 2 for the wild-type parent, HSV- 1(F).
  • the antibody to the carboxyl-terminal 138 amino acids of ICP22/U S 1.5 reacted with several bands of ICP22 (M r 67,000 to 72,000) and U s 1.5 (M r 35,000 to 48,000) corresponding to the isoforms resulting from posttranslational processing of the two proteins (FIG. 5, lanes 7 and 8).
  • ICP22 and U s 1.5 proteins were not processed at 4 h after infection and exhibited a reduced number of isoforms in lysates of cells infected with the ⁇ U, 13 (R7356) vims.
  • the decrease in the number of isoforms of U s 1.5 paralleled the decrease in the number of isoforms of ICP22 (FIG. 5, compare lane 8 with 9).
  • R7808 ( ⁇ 22/U s 1.5), or R7828 (R7808 Repair) per cell.
  • the cells were harvested, solubilized, electrophoretically separated on a denaturing polyacrylamide gel. electrically transferred to a nitrocellulose sheet, and reacted with the polyclonal antibody to the carboxyl-terminal of ICP22/U S 1.5 proteins as described in Materials and Methods.
  • FIG. 6 were as follows:
  • R7828 (R7808 Repair) exhibited wild type levels of expression and posttranslational processing of ICP22/U S 1.5 proteins (FIG. 6, compare lane 1 with lanes 3, 5, and 7). ICP4 measured by its reactivity with corresponding monoclonal antibody served as a loading control.
  • the ICP22 Function That Enhances the Expression of a Subset of , Genes Is Located at the Carboxyl-Terminal Domain Shared with the UJ 5 protein and Can Be Expressed by the Latter Protein
  • F HSV-1
  • R7802 ⁇ 22/ ⁇ U s l 5
  • R7804 R7802 Repair
  • R7805 ( ⁇ 22/U s l 5), R7806 (R7805 Repair), R7808 ( ⁇ 22/U s l 5) or R7828 (R7808 Repair) vims per cell
  • the cells were harvested, solubilized, electrophoretically separated m a denatunng polyacrylamide gel, electncally transferred to a nitrocellulose sheet and reacted sequentially with the monoclonal antibody to U l l, and the polyclonal antibody (Wl ) to U L 38
  • the expression levels of U s l 1 and U t 38 were examined The results, FIG 9, were as follows
  • Vero and rabbit skm cells infected with HSV-I(F) expressed equivalent levels of U l 1 protein whereas the level of U L 38 protein in rabbit skin cells was greater than that detected in Vero cells (FIG 9, compare lanes 1 and 2)
  • Rabbit skin cells infected with R7802 ( ⁇ 22/ ⁇ U s l 5) expressed less U s l 1 and U L 38 proteins than infected Vero cells (FIG 9, compare lanes 3 and 4)
  • This phenotype is comparable to the phenotype seen with infection of these cell lines with R7356 a recombinant vims deleted in the U L 13 protein kinase and R325 a recombinant virus delete for U s l 5 (Purves et al , 1993
  • EXAMPLE 8 All Isoforms of UJ 5 Protein Are Translocated into the Nucleus The pu ⁇ ose of this senes of studies was to determine whether posttranslational processing of the isoforms of U s l 5 proteins was dependent on nuclear localization. HEp-2 cells were exposed to 10 PFU of HSV- 1(F), R7805 ( ⁇ 22/U s 1.5), R7808 ( ⁇ 22/U s 1.5), or R7810 ( ⁇ 22/ ⁇ U s 1.5 , c ⁇ ) per cell and harvested at 18 h after infection. The nuclei and cytoplasm were separated as described in Materials and Methods.
  • ICP22 was detected in both the nucleus and the cytoplasm of cells infected with HSV- 1(F).
  • the nuclear and cytoplasmic ICP22 were posttranslationally processed to the same extent, but the slowest migrating forms of ICP22 was more abundant in the nucleus than in the cytoplasm.
  • U b 1.5 protein made in cells infected with HSV- 1(F) was present in greater abundance in the nucleus.
  • the ratio of the various electrophoretically distinct isoforms of ICP22 in the cytoplasm differed from those in the nucleus. (FIG. 10, compare lanes 1 and 2).
  • U s 1.5 was present in both fractions.
  • the nuclear form of U s 1.5 has an addition major band as compared to the cytoplasmic fraction (FIG. 10, compare lanes 5 and 6).
  • ICP22 Processed forms of ICP22 were present in lysates of cell infected with all vimses except those infected with R7810, R7820 or R7823 (FIG 1 1 , lanes 4-6) The inventors noted a slightly higher accumulation of the fastest migrating forms of ICP22 in cells infected with mutants carrymg carboxyl-terminal deletions (e g R7822, R7823, FIG 1 1 , lanes 3 and 4) (ii) Only the fastest migrating forms of ICP22 accumulated in cells infected with R7823 or R7820 (FIG. 1 1 , lanes 4 and 5).
  • ICP22 encoded by mutants lacking the carboxyl-terminal 15 or less amino acids were posttranslationally processed by the U L 13 protein kinase whereas ICP22 encoded by mutants lacking 18 or more carboxyl-terminal amino acids were not processed, (ii)
  • Threonine 300 and Serine 301 were mutagenized to Alanine and Glycine, respectively.
  • [HSV- 1(F)] or R7827 vimses per cell were harvested, solubilized, electrophoretically separated on a denaturing polyacrylamide gel, electrically transferred to a nitrocellulose, sheet, and reacted with a polyclonal antibody to ICP22/US1.5.
  • ICP22/US1.5 proteins The processing of the ICP22/US1.5 proteins is reduced in cells extracts infected with R7827 relative to those of wild-type [HSV-1 (F)] infected cells. Based on this data and a homology comparison between HSV-1 and HSV-2. it can be concluded that the domain within ICP22/US1.5 consisting of amino acids 300 to at least 310 (and probably to amino acid 328) in HSV-1 and amino acids 295 to 305 in HSV-2 (and probably to amino acid 320) is the site of phosphorylation by the UL13 protein kinase ( Figure 12B, line 4).
  • compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of prefe ⁇ ed embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents, which are both chemically and physiologically related, may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
  • Temin "Retrovims vectors for gene transfer: Efficient integration into and expression of exogenous DNA in vertebrate cell genome," In: Gene Transfer, Kucherlapati, ed., Plenum Press, New York, pp. 149-188, 1986. Tur-Kaspa et al, Mol. Cell Biol, 6:716-718, 1986.

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Abstract

The invention encompasses methods for identifying substances that inhibit infection by a herpesvirus and preventative and therapeutic compounds that constitute treatment or prevention of herpesvirus infections using one or more regions of US1.5 containing a posttranslational modification site that is required to be modified to effect progression of virus infection. The screening method of the invention involves a comparative analysis of the phosphorylation state of a polypeptide containing a US1.5 phosphorylation site that has been exposed to a kinase in the presence and absence of a candidate inhibitor. The compounds of the invention include US1.5 polypeptides and peptides, and their corresponding polynucleotides, that can be used to inhibit the posttranslational modification of US1.5, thus interfering with the progression of herpesvirus infection. A method of inhibiting infection is also disclosed and is directed at providing a cell with an inhibitor of US1.5 phosphorylation. Other therapeutic and preventative compounds and methods are also directed at polypeptides that bind to a US1.5 phosphorylation site.

Description

DESCRIPTION
METHODS AND COMPOSITIONS INVOLVING POSTTRANSLAΗONAL MODIFICAΗON OF HERPESVIRUS PROTEIN Us 1.5
BACKGROUND OF THE INVENTION
This application claims priority to U.S. Provisional Application No. 60/128,500, filed on April 9, 1999, which is incorporated by reference in its entirety. The government owns rights in the present invention pursuant to grant numbers CA47451 , CA71933, and CA78766 from the National Cancer Institute.
I. Field of the Invention
The present invention relates generally to the fields of molecular biology and virology. More particularly, it concerns the use of polypeptides, peptides, and polynucleotides derived from Us1.5 of heφes simplex virus to inhibit infection by a heφesvirus and to identify candidate substances that inhibit infection by the virus.
II. Description of Related Art
The family Heφesviridae is comprised of members (heφesviruses) generally possessing (1) linear, double-stranded DNA, (2) an icosadeltahedral capsid, (3) tegument protein, and (4) an envelope. Heφesviruses have been isolated from humans, and these include the heφes simplex viruses (HSV), Epstein-Barr virus, human cytomegalovirus, varicella-zoster virus, human heφesvirus 6, and human heφesvirus 8 (Fields et al, 1991). Heφes simplex viruses, designated with subtypes 1 and 2, are among the most common infectious agents encountered by humans, infecting millions worldwide. These viruses cause a broad spectrum of disease, ranging from relatively benign to severe and life-threatening.
Human heφesviruses can cause productive lytic infections, where infectious virus is produced and cells are lysed, or nonproductive lytic infection, where viral DNA remains intact but cells survive because full replication does not occur. They also have the properties of latency and reactivation; following an acute lytic infection, these viruses frequently persist in a latent state until reactivation causes a recurrent lytic infection.
The only known natural known host for HSV is a human. Infection by either subtypes of heφes simplex viruses, HSV-1 and HSV-2, can be identified by their characteristics, such as antigenicity, clinical and epidemiologic patterns, DNA base composition, biologic properties, and sensitivity to physical and chemical stress. (Whiteley et al. 1998). Primary infection by HSV-1 occurs mostly during childhood, while it occurs by HSV-2 mainly in sexually active adolescents and young adults.
The primary mode of transmission is direct contact with infected secretions.
HSV-1 infection can be evidenced by certain clinical syndromes including oral-labial heφes, ocular heφes, and encephalitis. Infection by HSV-2 is associated with genital heφes, perianal and anal heφes, and heφetic whitlows.
While prophylactic measures can be taken to prevent direct contact with viral secretions to avoid a primary infection, such as wearing gloves or a condom, prevention of viral infection through the use of vaccines or antibodies has not been established. Furthermore, the clinical outcome of heφesvirus infection is dependent upon early diagnosis and prompt initiation of antiviral therapy. Despite some successful efforts in treating HSV infections, dermal and epidermal lesions frequently recur, and HSV infections of neonates and infections of the brain are associated with high levels of morbidity and mortality. The need for effective substances to prevent or treat infection by heφesviruses is clear. Therefore, it is the object of the present invention to provide a screening method for identifying substances that inhibit infection by a heφesvirus, preventative compounds that can inhibit infection or reactivation of heφesviruses, and therapeutic compounds effective in treating heφesvirus infections. SUMMARY OF THE INVENTION
The invention encompasses methods for identifying substances that inhibit infection by a heφesvirus as well as preventative and therapeutic compounds that permit treatment or prevention of heφesvirus infections using one or more regions of
Usl .5 that contain a phosphorylation site.
The present invention includes, in some embodiments, a method for screening candidate substance for inhibition of heφesvirus infections. This method can be accomplished by obtaining a polypeptide or peptide that contains a Us1.5 phosphorylation site, adding the candidate inhibitor to a mixture containing the Us1.5 polypeptide or peptide, and further incubating a kinase with the mixture under conditions appropriate to allow phosphorylation of the UJ .5 polypeptide or peptide. The method also includes determining the phosphorylation state of the Us1.5 polypeptide or peptide and comparing its phosphorylation state to a polypeptide or peptide similar to the Us1.5 moiety initially used, such as an aliquot of the same Us1.5 polypeptide initially used and incubated with a kinase but that was not exposed to the candidate inhibitor. "Contacting" refers to the coming together or touching of compounds. It is understood that a "similar polypeptide" or "similar polypeptide" is a Usl .5-related polypeptide or peptide that contains a Usl .5 phosphorylation site.
In some embodiments of the present invention, the method employs a kinase, such as UL13 from HSV, that is incubated with a Us1.5 phosphorylation site to do a comparative analysis of phosphorylation levels of a Us1.5 peptide or polypeptide that has been exposed to a candidate inhibitor and one that has not. Alternatively, the kinase Us3 could be used to practice this method. In other embodiments, the Us1.5 polypeptide that contains a phosphorylation site also includes a detectable label, which will aid in determining the phosphorylation state of the polypeptide. The invention's methods further include the use of polypeptide regions including at least about 10 consecutive amino acids, at least about 15 consecutive amino acids, and at least about 20 consecutive amino acids, and at least about 25 consecutive amino acids from one or more of the following sets of sequences: SEQ ID NOJ, SEQ ID NO:2, SEQ ID NOJ. SEQ ID NOJ, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8. Therefore, it is contemplated that a polypeptide can contain amino acids from more than one set of sequences.
The present invention also discloses the use of p60, a cellular protein, to determine the phosphorylation state of the Us1.5 polypeptide based on p60's ability to bind the fast-migrating, undeφrocessed isoform of wild-type ICP22, a protein that is colinear with Us 1.5.
In addition to screening methods, the present invention also describes peptide and polypeptide compositions that can inhibit posttranslational modification of Us1.5 produced by a heφesvirus. Because posttranslational modification of ICP22 and Us1.5 is required for infection to progress, these compositions effectively act to inhibit or prevent heφesvirus infections, and thus, constitute a treatment of heφesvirus infections. An excess of these compositions can inhibit a heφesvirus infection by competing with protein products produced by heφesvirus as a cellular target for posttranslational modification, thereby preventing the modification of the heφesvirus protein. Such a peptide includes at least about 10 consecutive amino acids, at least about 15 consecutive amino acids, at least about 20 consecutive amino acids, and at least about 25 consecutive amino acids from one or more of the following sets of sequences: SEQ ID NOJ , SEQ ID NO:2, SEQ ID NOJ, SEQ ID NOJ, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8. Once again, it is contemplated that a polypeptide can contain amino acids from more than one set of sequences. Moreover, in some embodiments it is contemplated that the peptide includes a Usl .5 phosphorylation site or at least two Us1.5 phosphorylation sites.
In some embodiments, peptides that inhibit posttranslational modification of
UJ..5 produced by heφesvirus are composed of about 20 consecutive amino acids to about 100 consecutive amino acids; the peptides contain a first region of at least about
10 consecutive amino acids from one or more of the following sets of sequences: SEQ ID NOJ , SEQ ID NO:2, SEQ ID NOJ, or SEQ ID NO:4; and a second region of at least about 10 consecutive amino acids from one or more of the following sets of sequences: SEQ ID NOJ, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NOJ, such that the peptide inhibits posttranslational modification of heφesvirus Us1.5. These peptides, in some examples of the present invention, contain at least one UJ .5 phosphorylation site; while in others, the peptide contains at least two Us1.5 phosphorylation sites. It is further contemplated that the first region and the second region are linked by one or more amino acids that are not the native Us1.5 sequence (Us1.5 amino acids 172-379). Non-native sequence linkers include the use of synthetic linkers such as a non-amino acid linking moiety.
The invention also includes a polypeptide that binds to a phosphorylation site in Us1.5, which would include an antibody, a single-chain antibody, or an anti- idiotypic antibody that binds immunologically to the antigen binding region of an anti-U, 13 antibody.
Other methods included in the present invention are the inhibition of heφesvirus infection by giving a cell an inhibitor of UJ .5 phosphorylation. This method contemplates that the cell is infected with a heφesvirus either prior to or after it being given an inhibitor of UJ .5 phosphorylation. Furthermore, it is considered part of the invention that the heφesvirus is a heφes simplex virus, such as HSV-1 or HSV-2. In some examples, a polypeptide that contains a site similar to a site for Usl .5 phosphorylation but that is incapable of being modified is utilized.
Another method of the present invention is inhibiting heφesvirus infection by contacting a cell with a polypeptide that binds to a UJ .5 phosphorylation site. In one embodiment of the present invention, this polypeptide prevents phosphorylation of a U 1.5 phosphorylation site by masking the site, and thereby effects inhibition of infection. Other examples of a polypeptide that binds to a Us1.5 phosphorylation site include an antibody. The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one."
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
FIG. 1. Schematic representation of the construction of recombinant viruses. FIG 1A: Line 1, sequence arrangement of the HSV-1 genome showing the unique long (U ), unique short sequences, and the location of the α genes 0, 4, and 22. Lines 2- 6, domains of the HSV-1 cosmid set used for the construction of recombinants. FIG IB: construction of the Δα22/ΔUs1.5 virus. Line 2, expansion of the S component of HSV-1 DNA. Lines 3, 4 and 5, domains of cosmids pBClOlό and pBC1004 and plasmid pRB5213. Plasmid pBR5213 was constructed to bridge the gap in the nonoverlapping cosmids pBClOlό and pBC1004. In this plasmid the α22/Usl .5 open reading frame was replaced with a unique Eagl restriction site.
FIG. 2. Schematic representation of the recombinants with an amino-terminal deletion in the α22 gene. Line 1 , Bam HI N fragment of HSV- 1 (F) showing the open reading frames of ICP22 (open rectangles) and Us1.5 along with their mRNA transcripts (thin lines). Line 2, R7802 (Δα22/ΔUs1.5) and a schematic representation of the Bam HI N fragment (pRB5212). The open reading frames of ICP22 and UJ .5 were replaced with a unique Eag I restriction site. Line 3, R7804 (R7802 repair). The Bam HI N fragment (pRB5214) was restored. Line 4, schematic diagram of the Bam HI N in R7805 (Δα22/Us1.5) recombinant virus. Line 5, representation of Bam HI N fragment of R7806 (R7805 repair). The Bam HI N fragment (pRB5210) was restored. Line 6, representation of the Bam HI N fragment in R7808 (Δα22/Us1.5). The open reading frame of Us1.5 was cloned into the unique Eagl site of pBR5212 to yield pRB5215. Line 7, representation of BamHI N fragment of R7828 (R7808 repair). BamHI N fragment (pRB5210) from wild type virus HSV- 1(F) was restored. Abbreviations: B,- BamHI; E,- EcoRI; S,- Sau3AI.
FIG. 3. Autoradiographic images of electrophoretically-separated Bam HI fragments of recombinant viral DNA hybridized with 32P-labeled pRB5210. The fragments were electrophoretically separated in a 0.8% agarose gel, transferred to Zeta-probe membrane, and hybridized with 32P-labeled pRB5210 carrying the HSV-1 (F) BamHI N fragment. Lane 1 , the 4.9-kb Bam HI N fragment from HSV-1 (F); lane 2, the 4.9-kb Bam HI N fragment from the R7905 (HSV- 1(F)) cosmid virus; lane 3, the 3.6-kb Bam HI N fragment form R7802 recombinant virus; lane 4, the 3.4-kb BamHI N fragment from the R7805 recombinant virus.
FIG. 4. Schematic representation of terminal sequences of the recombinants carrying 3' terminal deletions in the α22/Us1.5 genes of HSV-1 (F). FIG1 A: Bam HI N sequence arrangements in recombinants. The rectangles represent the open reading frames. The filled boxes represent the carboxyl-terminal 40 codons. The number in parentheses indicates the number of codons deleted from the terminus of the open reading frame. Line 1 ,- Bam HI N of HSV-l(F); line 2, R7819 (Δα22rr Us1.5rτ)[Δ10a.a.]; line 3, R7822 (Δα22rτ/UJ.5cτ)[Δ15αα]; line 4, R7823 (Δα22rτ/Us1.5cl)[Δ18a.a.]; line 5, R7820 (Δα22rr/UJ.5cτ)[Δ22αα]; line 6,
(Δα22rτ/Us1.5cJ[Δ40a.a.]; line 7, R7821 (R7810 repair). The BamHI N fragment (pRB5214) with the open reading frames of ICP22 and Us1.5 restored. Abbreviations: B,- BamHI; E,- EcoRI. Panel B: Schematic diagram of the carboxyl-terminal 43 amino acids of α22/Us1.5 protein from amino acids 377 to end of the C-22/UJ .5 protein.
FIG. 5. Photograph of an immunoblot of electrophoretically separated lysates of cells mock-infected or infected with HSV-1 (F) or R7356 (ΔUL13) and reacted with antibody to ICP22/ J .5. Vero cells (VC) and rabbit skin cells (RSC) harvested at various times (4 - 24 h) after infection were solubilized, subjected to electrophoresis in a denaturing 10% polyacrylamide gel, transferred to a nitrocellulose sheet, and reacted with the rabbit polyclonal antibody (W2) made against the carboxyl-terminal 138 amino acids of ICP22/US1.5.
FIG. 6. Photograph of an immunoblot of electrophoretically-separated lysates of cells mock-infected or infected with HSV- 1(F), R7802, R7804, R7805,
R7806, R7808 or R7828, and reacted with antibody to ICP22/US1.5. Vero cells harvested at 18 h after infection were solubilized, subjected to electrophoresis in a denaturing 8% polyacrylamide gel, transferred to a nitrocellulose sheet, and reacted with the polyclonal antibody W2. Lane 1, HSV-l(F); lane 2, R7802 (Δα22/ΔUJ .5); lane 3, R7804 (R7802 repair); lane 4, R7805 (Δα22 Us1.5); lane 5, R7806 (R7805 repair); lane 6, R7808 (Δα22 Us1.5-α22P); lane 7, R7828(R7808 repair).
FIG. 7. Schematic representations of the ICP22 open reading frame of wild type and mutant HSV-1 showing the location of methionine codons. Line 1 , position of the four methionine codons in the first 200 amino acids of HSV-1 ; line 2,
R7815 (Δα22N1/Us1.5)[Δ47a.a.] lacking 47 amino acids deleted from the amino-terminal. The first methionine available for translational initiation is at codon 90; line 3, R7805 (Δα22 T/Us1.5)[Δ138a.a.], lacking the amino-terminal 138 amino acids. The first methionine available for translational initiation is at codon 147; line 4, R7808 (Δα22Nr/Us1.5)[Δ171a.a.] lacking amino-terminal 170 amino acids. The first methionine available for translational initiation is at codon 171. FIG. 8. Photograph of an immunoblot of electrophoretically-separated lysates of cells infected with R7815, R7805, or R7808 and reacted with the polyclonal antibody W2 to ICP22 UJ .5. Vero cells harvested at 18 h after infection were solubilized, subjected to electrophoresis in a denaturing 10% polyacrylamide gel, transferred to a nitrocellulose sheet, and reacted with the polyclonal antibody prepared against the carboxyl-terminal amino acids of ICP22/US1.5. Lane 1, R7815 (Δα22 /Us1.5)[Δ47a.a.]; lane 2,- R7805(Δα22/UJ .5); lane 3, R7808 (Δα22 Us1.5). The numbers next to the arrows indicate the initiator methionine for the unprocessed protein product in each lane.
FIG. 9. Photograph of an immunoblot of electrophoretically-separated lysates of cells mock-infected or infected with HSV- 1(F), R7802, R7804, R7805, R7806, R7808 or R7828 and reacted with antibodies to UL38 and Usl l . Replicate cultures of Vero cells (VC) or rabbit skin cells (RSC) harvested at 18 h after infection were solubilized, subjected to electrophoresis in a denaturing polyacrylamide gel, transferred to a nitrocellulose sheet, then sequentially reacted with the monoclonal antibody to Usl l, and the polyclonal antibody to UL38. Lanes 1 and 2, HSV-l(F); lanes 3 and 4, R7802 (Δα22/ΔUs1.5); lanes 5 and 6, R7804 (R7802 repair); lanes 7 and 8, R7805 (Δα22 T/Us1.5); lanes 9 and 10, R7806 (R7805 Repair); lanes 1 1 and
12, R7808 (Δα22NT/Us1.5α22p); lanes 13 and 14, R7828 (R7808 repair).
FIG. 10. Photograph of an immunoblot of electrophoretically-separated nuclear and cytoplasmic fractions of HEp-2 cells infected with HSV-l(F), R7805, R7808, or R7810. Infected Hep-2 cells were harvested and lysed by the addition of
0.4%) Nonidet P-40. Nuclear and cytoplasmic fractions prepared as described in Materials and Methods were solubilized, subjected to electrophoresis on an SDS-10% polyacrylamide gel, transferred to nitrocellulose, and reacted with the polyclonal antibody to α22/UJ .5 protein. ICP22 and Us1.5 protein are indicated on the right, and molecular weights (in thousands) are shown on the left. Lanes 1 and 2,
HSV-l(F); lanes 3 and 4, R7805 (Δα22 NUs1.5); lanes 5 and 6, R7808 (Δα/2 T/Us1.5α22p); lanes 7 and 8, R7810 (Δα22cτ/ΔUJ .5CT). Abbreviations: N, nuclear fraction; C, cytoplasmic fraction.
FIG. 11. Photograph of an immunoblot of electrophoretically-separated lysates of cells infected with HSV-l(F), R7819, R7822. R7823, R7820, R7810 or
R7821 reacted with polyclonal rabbit antibody R77 to ICP22. Vero cells harvested at 18 h after infection were solubilized and subjected to electrophoresis in denaturing 10% polyacrylamide gel, transferred to a nitrocellulose sheet, and reacted with the polyclonal antibody R77 against ICP22. The cells were infected with Lane 1 : HSV- 1(F); lane 2, R7819 (Δα22/UJ 5Δcτlϋa a ); lane 3, R7822 (Δα22/Us1.5Δrτι5a a ); lane 4 R7823 (Δα22/Us1.5Δcτl8a a ); lane 5, R7820 (Δα22/US1.5ACT22a.a.); lane 6, R7810 (Δα22/Us1.5ΔCT40a a.), lane 7, R7821 (R7810 repair).
FIG. 12 Schematic representation of the functional domains of the α22 gene and its products, ICP22 and Us1.5 protein. FIGJ2A: Functional maps. The zones are assigned on the basis of amino acid composition. Zones 1, 3, 5 and 8 are basic whereas zones 2, 4 and 7 are acidic. FIGJ2B: The sequence of the internal homologous repeats. The sequence alignments are Line 1, HSV-1 amino-terminal vs HSV-1 carboxyl-terminal; Line 2, HSV-2 amino-terminal vs. HSV-2 carboxyl terminal; Line 3, HSV-1 amino-terminal vs. HSV-2 amino-terminal; Line 4, HSV-1 carboxyl-terminal vs. HSV-2 carboxyl-terminal; Line 5, HSV-1 Us1.5 amino- terminal vs. HSV-2 Us1.5 amino-terminal; Line 6, HSV-1 amino-terminal vs. HSV-2 amino-terminal. The numbers above and below refer to amino acid numbers of the corresponding ICP22.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
The present invention provides methods of screening for substances that may prevent or inhibit heφesvirus infections, as well as compositions containing peptides or polypeptides derived from Us1.5 that prevent, inhibit, or treat infection by a heφesvirus.
The heφes simplex virus genome encodes greater than 80 genes whose expression is coordinately regulated and sequentially ordered during productive infection (Cassady et al., 1998; Ejercito et al, 1968; Ogle et al. 1997). The first set of genes expressed immediately after productive infection is the genes followed by β and γ genes. Of the five α genes initially described, four have regulatory functions, and of these three, the α genes 0, 4 and 27 have attracted considerable attention from the scientific community because they are essential for viral replication under all conditions tested. Thus, αO encodes the infected cell protein (ICP) 0, a promiscuous transactivator important in early stages of infection; ICP4, the product of the α4 gene, regulates gene expression both positively and negatively, whereas ICP27, the product of the α27 gene, regulates posttranslational processing and transport of RNA (Pearson and Lipman, 1988). ICP22, the product of the α22 gene, attracted less attention, possibly because its functions were less apparent, obscured by the observation that it was dispensable for viral replication in cells in culture (Leopardi et al, 1997 ). Although the functions of the α22 gene are the least well understood, five lines of evidence suggest that it plays an important role in viral replication:
First, the domain of the α.22 gene yields two mRNAs each expressed by its own promoter. The α22 mRNA initiates upstream from the open reading frame and is spliced; the first exon is in its 5' non-coding domain (Hardy and Sandri-Goldin, 1994; Ogle and Roizman, unpublished data; Rice et al, 1995). Its product, ICP22, is a protein of 420 amino acids with alternating acidic and basic domains. The second mRNA initiates in the coding domain of the α22 gene and is driven by an independent promoter (B ni and Roizman, 1996). It directs the synthesis of a protein of 274 amino acids beginning with metl47 of ICP22 and is colinear with the remainder of the protein. This protein, designated Us1.5, is also expressed with α gene kinetics. The possibility that the sequences unique to ICP22 perform functions different from those of sequences shared by ICP22 and UJ .5 protein emerged from the observation that insertion of a 20 codon linker at codon 200 or 240 had no apparent effect on the functions associated with ICP22 and described below.
Second, ICP22 is extensively posttranslationally processed (Ackermann et al, 1984) as evidenced by the detection of phosphorylation and changes in electrophoretic mobility. ICP22 was shown to be phosphorylated largely by the protein kinase encoded by U, 13 and to a lesser extent by protein kinase encoded by Us3 (Meignier et al, 1988; Mitchell et al, 1994). ICP22 is also nucleotidylylated by casein kinase II (Honess and Roizman, 1975; Kawaguchi et al, 1997).
Third, the deletion mutant R325 generated by Post and Roizman (Leopardi et al, 1997) lacked the carboxyl-terminal 220 amino acids. This mutant was highly attenuated in experimental animal systems (Honess and Roizman, 1974; Puives et al, 1987). It replicated to wild-type vims levels in Vero and HEp-2 cells but its ability to replicate in rodent or rabbit cells or in primary human fibroblasts was diminished. In these restricted cell lines, a subset of γ2 proteins exemplified by the product of sl 1 gene was significantly reduced (Mitchell et al, 1994). In addition, the levels of mRNA and protein products of the αO gene were also reduced (Mitchell et al, 1994). More detailed analyses showed that in rabbit skin cells infected with R325, ICP0 mRNA was unstable and the alternate splice acceptor C of ICP0 intron 1 was not used. The studies by Purves et. al. cited above showed that the phenotype of R325 deletion mutant was similar to that of the mutant lacking a functional U, 13 gene (Mitchell et al, 1994).
Fourth, ICP22 localizes in both the nucleus and cytoplasm (Everett et al.,
1991 ). Nuclear ICP22 localizes early in infection in small dense nuclear stmctures. On onset of DNA synthesis, ICP22 colocalizes with ICP4, nascent DNA, RNA polymerase II and other cellular proteins (Hardwicke and Sandri-Goldin, 1 94). The displacement of ICP22 from the small nuclear dense structures requires the expression of the protein kinase encoded by U, 13. These results suggest that the products of the α22 gene are involved in transcription of late genes - a conclusion consistent with the report that α22 mediates an intermediate level of phosphorylation of the RNA polymerase II (Ng et al, 1997: Ogle and Roizman, 1998).
Finally, studies using yeast two-hybrid systems with the entire α22 gene as bait yielded evidence of the interaction of ICP22 with at least 2 host proteins. The first, designated p78, was recently discovered to be identical in sequence to a protein reported to localize in nucleoli and designated MSP58 (Mitchell et al, 1997). Studies in this laboratory showed that p78 is made early in the S phase, has a short half-life and it binds the amino terminal domain of ICP22. In synchronized cells, during the expression of p78, the ICP22 exhibits novel posttranslationally processed forms.
These forms are replaced by the standard series of ICP22 isoforms with time after infection (Ackermann et al, 1985).
The second protein, designated p60, bound fast-migrating, undeφrocessed wild-type ICP22 and ICP22 lacking the carboxyl-terminal 24 amino acids but not
ICP22 lacking the terminal 40 amino acids. p60 also bound ICPO; the binding of
ICPO was independent of that of ICP22. In uninfected HEp-2 cells, p60 was distributed throughout the cell. In wild-type vims infected HEp-2 cells, p60 was translocated into the nucleus and formed dense bodies that colocalized with ICPO. The posttranslational processing of p60 present in HEp-2 cells infected with wild-type or ICP22 mutant vimses could not be differentiated from that of uninfected cells whereas the p60 accumulating in rabbit skin cells infected with wild type vims differed in electrophoretic mobility from that made in uninfected cells. The posttranslational processing of p60 was absent in rabbit skin cells infected with the vims lacking the sequences encoding the carboxyl-terminal half of ICP22. p60 appears to be a linker protein capable of binding to and mediating the interaction of
ICPO with the undeφrocessed form of ICP22 (Altschul et al, 1990).
The screening methods and therapeutic and preventative compounds related to heφesvirus infection disclosed herein take advantage of several sets of sequences encoded by α22 that are required to be posttranslationally modified for viral replication in permissive cells to occur. More specifically, the methods and compositions of the present invention further take advantage of the observation that UJ .5 must be posttranslationally modified in a certain region or in certain regions by the UL13 protein kinase to enable expression of a subset of late genes exemplified by UL38 and Usl l . Posttranslational processing is determined by several sets of sequences of Us1.5: one set spans amino acids 38-66 (HSV-1 ) or amino acids 37-62 (HSV-2); another set spans from metl47 to metl 71 (HSV-1 ) or amino acids 143-167 (HSV-2), and this region is essential for posttranslational processing of Usl .5. A third set contains amino acids 300-328 (HSV-1 ) or amino acids 295-320 (HSV-2), while another set includes lys402, met403, and arg404 and thus, comprises amino acids 380-
405 (HSV-1) or amino acids 373-398 (HSV-2).
Due to the extensive homology between HSV-1 and HSV-2 in this region, the information disclosed herein is understood to extend to both HSV-1 and HSV-2. See FIG. 12B. For convenience, HSV-1 numbering is used, unless otherwise specified.
II. Compositions for Inhibiting Infection by Herpesvirus
Since domains corresponding to sequences within the α22 gene have been identified, it is contemplated that these domains — as either a polypeptide or peptide, or their cognate polynucleotides that contain or encode a UJ .5 phosphorylation site
— can be used to inhibit infection by a heφesvirus or to treat infection by a heφesvirus, whereby a subject infected by heφesvirus is conferred a therapeutic benefit. It is more specifically envisioned that domains of Us1.5 containing sites for posttranslational modifications, particularly posttranslational modifications shown to be necessary for infection to progress, can be used to inhibit or treat a heφesvirus infection. It is understood that a region containing a Us1.5 phosphorylation site could contain ICP22 sequences, including the entire ICP22 sequence. As used herein, in the context of various of the instant compositions and methods, the term "polypeptide" will be understood to mean an amino acid segment that is greater than about 100 contiguous amino acids in length, and the term "peptide" will be understood to mean an amino acid segment that is between about 6 and about 100 contiguous amino acids in length. Thus, ICP22 and Us1.5 segments of varying overall length that retain catalytic, regulatory or stmctural properties and functions are provided herein. The use of the term "protein" in the context of ICP22 or Us1.5 is understood according to the present invention to be synonymous with the term "polypeptide."
A. Us1.5 Polypeptides and Peptides
In the screening method of the present invention, it is contemplated that a polypeptide is used as a target for posttranslational modification. The α22 gene of heφes simplex vims encodes two overlapping polypeptides, ICP22 and Us1.5. ICP22 contains unique sequences from α.22, while the entire sequence of Us1.5 is colinear with the last 274 amino acids of ICP22. Since post-translation modification of Us1.5 is required for viral infection to progress, a polypeptide product from the α22 gene that contains Us1.5 or fragments and peptides thereof, can be used in the screening method if the polypeptide, fragment, or peptide contains a site for post-translation processing. Because of their colinearity, a sequence that contains sequences from
Us1.5 necessarily contains sequences from ICP22.
In certain embodiments of the present invention, a Us1.5 protein, polypeptide, or peptide comprises a contiguous amino acid sequence of at least between about 10 amino acids and about 50 amino acids, and between about 15 and about 30 amino acids, and between about 20 and about 25 amino from SEQ ID NOJ or SEQ ID NO:2, or biologically functional equivalents thereof. In certain other embodiments of the present invention, an isolated 22 gene that encodes an Us1.5 protein, polypeptide, or peptide comprises a contiguous amino acid sequence of at least about 10 amino acids from SEQ ID NOJ or SEQ ID NO:2, or biologically functional equivalents thereof.
As used herein in various aspects of the invention, such as in the context of a polypeptide or peptide, the term "consecutive amino acids" will be understood to include a contiguous amino acid sequence of at least about 4, about, 5, about 6, about
7, about 8, about 9, about 10, about 12. about 13. about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21 , about 22, about 23, about 24. about 25, about 30, about 35, about 40, about 45, about 50, about 60, about 70, about 80, about 90, about 100, about 1 10, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220. about 230, about 240, about 250. about 260, about 270, about 280, about 290, about 300. about 310, about 320, about 330, about 340, about 350, about 360, about 370, about 380, about 390, about 400, about 410, about 420, about 430, about 440, about 450. about 460, about 470, about 480, about 490, about 500, about 600, about 700, about 800, about 900, or about 1000 amino acids or so.
Furthermore, in certain embodiments of the present invention, a peptide or polypeptide includes amino acids encoded by regions of the α22 gene of heφes simplex vims, such as SEQ ID NOJ, SEQ ID NO:2, SEQ ID NOJ, SEQ ID NOJ, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8. In some examples, peptides and polypeptides of the present invention are understood to include an amino acid sequence from at least one of the regions defined as SEQ ID NOJ, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NOJ, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO: 8, and in some cases, to include amino acid sequences from more than one region defined as SEQ ID NOJ, SEQ ID NO:2, SEQ ID NOJ, SEQ ID NOJ, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8. Therefore, by way of example, the present invention is understood to contemplate a peptide or polypeptide that includes amino acid sequences of a given length from both SEQ ID NOJ and SEQ ID NOJ, such as at least 10 consecutive amino acids from SEQ ID NO:3 and at least 10 consecutive amino acids from SEQ ID NO:4.
This application may refer to a UJ .5 peptide or polypeptide, and this will be understood to refer to sequences derived from UJ .5 of heφesvirus, including a UJ .5 of HSV-1 and a UJ .5 of HSV-2.
Particular regions of Us1.5 have been characterized. The amino acid sequence of SEQ ID NOJ corresponds to amino acids 147-171 of HSV-1 ; and, the amino acid sequence of SEQ ID NO:2 corresponds to amino acids 380-405 of HSV-1. The amino acid sequence of SEQ ID NO:3 corresponds to amino acids 38-66 of HSV-1; and the amino acid sequence of SEQ ID NOJ corresponds to amino acids 300-328 of HSV-1. The amino acid sequence of SEQ ID NO:5 corresponds to amino acids 143-167 of HSV-2; and, the amino acid sequence of SEQ ID NO:6 corresponds to amino acids
373-398 of HSV-2. The amino acid sequence of SEQ ID NO:7 corresponds to amino acids 37-62 of HSV-2; and, the amino acid sequence of SEQ ID NO:8 corresponds to amino acids 295-320 of HSV-2.
Methods of protein synthesis, detection, and purification are well-known to those of skill in the art. For example, one of skill in the art would know that a protein can be produced by transforming a host cell with a vector containing a promoter operatively linked to a gene encoding the protein, , and by purifying the desired protein. Some methods are provided in other portions of this disclosure, such as in the section describing methods of identifying compounds that inhibit a heφesvirus infection. Such methods are not intended to be limited to use with respect to that aspect of the invention only and, instead, apply, to any aspect of the invention disclosed herein.
1. Variants of Us1.5
Amino acid sequence variants of a Us1.5 polypeptide or peptide can be substitutional, insertional or deletion variants. Deletion variants lack one or more residues of the native protein that are not essential for function or immunogenic activity and that are exemplified by the variants lacking a transmembrane sequence described above. Another common type of deletion variant is one lacking secretory signal sequences or signal sequences directing a protein to bind to a particular part of a cell. Insertional mutants typically involve the addition of material at a non-terminal point in the polypeptide. This may include the insertion of an immunoreactiveepitope or simply a single residue. Terminal additions, called fusion proteins, are discussed below. Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the polypeptide, and they may be designed to modulate one or more properties of the polypeptide, such as stability against proteolytic cleavage, without the loss of other functions or properties. Substitutions of this kind preferably are conservative, that is. one amino acid is replaced with one of similar shape and charge. Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine.
The term "biologically functional equivalent" is well understood in the art and is further defined in detail herein. Accordingly, sequences that have between about 70% and about 80%; or more preferably, between about 81% and about 90%; or even more preferably, between about 91 % and about 99%; of amino acids that are identical or functionally equivalent to the amino acids of a Us1.5 polypeptide provided the biological activity of the protein is maintained. Therefore, reference to one region in a particular HSV subtype can be understood to refer also to a corresponding region in another subtype, unless otherwise specified.
The term "functionally equivalent codon" is used herein to refer to codons that encode the same amino acid, such as the six codons for arginine or serine, and also refers to codons that encode biologically equivalent amino acids (see Table 1 , below). TABLE 1 CODON TABLE
Amino Acids Codons
Alanine Ala A GCA GCC GCG GCU
Cysteine Cys C UGC UGU
Aspartic acid Asp D GAC GAU
Glutamic acid Glu E GAA GAG
Phenylalanine Phe F UUC uuu
Glycine Gly G GGA GGC GGG GGU
Histidine His H CAC CAU
Isoleucine He I AUA AUC AUU
Lysine Lys K AAA AAG
Leucine Leu L UUA UUG CUA CUC CUG CUU
Methionine Met M AUG
Asparagine Asn N AAC AAU
Proline Pro P CCA CCC CCG CCU
Glutamine Gin Q CAA CAG
Arginine Arg R AGA AGG CGA CGC CGG CGU
Serine Ser S AGC AGU UCA UCC UCG UCU
Threonine Thr T ACA ACC ACG ACU
Valine Val V GUA GUC GUG GUU
Tryptophan Tφ w UGG
Tyrosine Tyr Y UAC UAU
It also will be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids or 5' or 3' sequences, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned. The addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5' or 3' portions of the coding region or may include various internal sequences, i.e., introns, which are known to occur within genes. The following is a discussion based upon changing of the amino acids of a protein to create an equivalent, or even an improved, second-generation molecule. For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with stmctures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid substitutions can be made in a protein sequence, and in its underlying DNA coding sequence, and nevertheless produce a protein with like properties. It is thus contemplated by the inventors that various changes may be made in the DNA sequences of genes without appreciable loss of their biological utility or activity, as discussed below. Table 1 shows the codons that encode particular amino acids.
In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte & Doolittle, 1982). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary stmcture of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.
It also is understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophihcity. U.S. Patent 4,554,101, incoφorated herein by reference, states that the greatest local average hydrophihcity of a protein, as governed by the hydrophihcity of its adjacent amino acids, correlates with a biological property of the protein. As detailed in U.S. Patent 4,554,101 , the following hydrophihcity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ± 1); glutamate (+3.0 ± 1 ); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1 ); alanine (-0.5); histidine *-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5): leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is understood that an amino acid can be substituted for another having a similar hydrophihcity value and still produce a biologically equivalent and immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophihcity values are within ±2 is preferred, those that are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.
As outlined above, amino acid substitutions generally are based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophihcity, charge, size, and the like. Exemplary substitutions that take into consideration the various foregoing characteristics are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
Another embodiment for the preparation of polypeptides according to the invention is the use of peptide mimetics. Mimetics are peptide-containing molecules that mimic elements of protein secondary stmcture. See, e.g., Johnson (1993). The underlying rationale behind the use of peptide mimetics is that the peptide backbone of proteins exists chiefly to orient amino acid side chains in such a way as to facilitate molecular interactions, such as those of antibody and antigen. A peptide mimetic is expected to permit molecular interactions similar to the natural molecule. These principles may be used, in conjunction with the principles outline above, to engineer second generation molecules having many of the natural properties of Us1.5, but with altered and even improved characteristics.
2. Fusion Proteins
A specialized kind of insertional variant is the fusion protein. This molecule generally has all or a portion of a native molecule, such as UJ .5, linked at the N- or C- terminus, to all or a portion of a second polypeptide. For example, fusions typically employ leader sequences from other species to permit the recombinant expression of a protein in a heterologous host. Another useful fusion includes the addition of a ιmmunologιcallyactι\ e domain, such as an antibody epitope. to facilitate puπfication of the fusion protein Inclusion of a cleavage site at or near the fusion junction will facilitate removal of the extraneous polypeptide after purification Other useful fusions include linking of functional domains, such as actι\e sites from enzymes such as a hydrolase, glycosylation domains, cellular targeting signals, or transmembrane regions
3. Synthetic peptides
The present invention also descπbes Usl 5-related peptides for use in various embodiments of the present invention Because of their relatively small size, the peptides of the invention can also be synthesized in solution or on a solid support in accordance with conventional techniques Vaπous automatic synthesizers are commercially available and can be used in accordance with known protocols See, for example, Stewart and Young, ( 1984), Tarn et al , (1983), Merπfield, (1986), and Barany and Merπfield (1980), each mcoφorated herein by reference Short peptide sequences, or libraries of overlapping peptides, usually from about 6 up to about 35 to
50 ammo acids, which correspond to the selected regions descπbed herein, can be readily synthesized and then screened screening assays designed to identify reactive peptides Alternatively, recombinant DNA technology may be employed wherein a nucleotide sequence which encodes a peptide of the invention is inserted into an expression vector, transformed or transfected into an appropπate host cell and cultivated under conditions suitable for expression
The present invention also contemplates the use of nonnatural amino acid substitutes that cannot be processed as natural amino acids can Since Usl 5 is required to be posttranslationally modified for viral infection to progress, the use of a
Usl 5 peptide that is not capable of being modified because, for example, certain synthetic ammo acids have been incoφorated into the Usl 5 peptide sequence can also inhibit infection 4. Peptide and Polypeptide Purification
It may be desirable to purify Us1.5, or variants thereof. Protein purification techniques are well known to those of skill in the art. These techniques involve, at one level, the cmde fractionation of the milieu from cells infected by a heφesvirus to polypeptide and non-polypeptide fractions. Once the polypeptide is separated from other proteins, the polypeptide of interest may be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suited to the preparation of a pure peptide are ion-exchange chromatography, exclusion chromatography; polyacrylamide gel electrophoresis; isoelectric focusing. A particularly efficient method of purifying peptides is fast protein liquid chromatography or even HPLC.
Certain aspects of the present invention concern the purification, and in particular embodiments, the substantial purification, of an encoded polypeptide or peptide. The term "purified polypeptide or peptide" or "purified protein" as used herein, is intended to refer to a composition, isolatable from other components, wherein the polypeptide, protein, or peptide is purified to any degree relative to its naturally-obtainable state. A purified protein, polypeptide, or peptide therefore also refers to a protein, polypeptide, or peptide, free from the environment in which it may naturally occur.
Generally, "purified" will refer to a protein, polypeptide, or peptide composition that has been subjected to fractionation to remove various other components, and which composition substantially retains its expressed biological activity. Where the term "substantially purified" is used, this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the proteins in the composition. Various methods for quantifying the degree of purification of the protein, polypeptide, or peptide will be known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific activity of an active fraction, or assessing the amount of polypeptides within a fraction by SDS/PAGE analysis. A preferred method for assessing the purity of a fraction is to calculate the specific activity of the fraction, to compare it to the specific activity of the initial extract, and to thus calculate the degree of purity, herein assessed by a "-fold purification number." The actual units used to represent the amount of activity will, of course, be dependent upon the particular assay technique chosen to follow the purification and whether or not the expressed protein or peptide exhibits a detectable activity.
Various techniques suitable for use in protein purification will be well known to those of skill in the art. These include, for example, precipitation with ammonium sulphate, PEG, antibodies and the like or by heat denaturation, followed by centrifugation; chromatography steps such as ion exchange, gel filtration, reverse phase, hydroxylapatite and affinity chromatography; isoelectric focusing; gel electrophoresis; and combinations of such and other techniques. As is generally known in the art, it is believed that the order of conducting the various purification steps may be changed, or that certain steps may be omitted, and still result in a suitable method for the preparation of a substantially purified protein or peptide.
There is no general requirement that the protein or peptide always be provided in their most purified state. Indeed, it is contemplated that less substantially purified products will have utility in certain embodiments. Partial purification may be accomplished by using fewer purification steps in combination, or by utilizing different forms of the same general purification scheme. For example, it is appreciated that a cation-exchange column chromatography performed utilizing an HPLC apparatus will generally result in a greater "-fold" purification than the same technique utilizing a low pressure chromatography system. Methods exhibiting a lower degree of relative purification may have advantages in total recovery of protein product, or in maintaining the activity of an expressed protein.
It is known that the migration of a polypeptide can vary, sometimes significantly, with different conditions of SDS/PAGE (Capaldi et al, 1977). It will therefore be appreciated that under differing electrophoresis conditions, the apparent molecular weights of purified or partially purified expression products may vary.
High Performance Liquid Chromatography (HPLC) is characterized by a very rapid separation with extraordinary resolution of peaks. This is achieved by the use of very fine particles and high pressure to maintain an adequate flow rate. Separation can be accomplished in a matter of minutes, or at most an hour. Moreover, only a very small volume of the sample is needed because the particles are so small and close-packed that the void volume is a very small fraction of the bed volume. Also, the concentration of the sample need not be very great because the bands are so narrow that there is very little dilution of the sample.
Gel chromatography, or molecular sieve chromatography, is a special type of partition chromatography that is based on molecular size. The theory behind gel chromatography is that the column, which is prepared with tiny particles of an inert substance that contain small pores, separates larger molecules from smaller molecules as they pass through or around the pores, depending on their size. As long as the material of which the particles are made does not adsorb the molecules, the sole factor determining rate of flow is the size. Hence, molecules are eluted from the column in decreasing size, so long as the shape is relatively constant. Gel chromatography is unsuφassed for separating molecules of different size because separation is independent of all other factors such as pH, ionic strength, temperature, etc. There also is virtually no adsoφtion, less zone spreading and the elution volume is related in a simple matter to molecular weight. Affinity Chromatography is a chromatographic procedure that relies on the specific affinity between a substance to be isolated and a molecule that it can specifically bind to. This is a receptor-ligand type interaction. The column material is synthesized by covalently coupling one of the binding partners to an insoluble matrix. The column material is then able to specifically adsorb the substance from the solution. Elution occurs by changing the conditions to those in which binding will not occur (e.g.. alter pH, ionic strength, and temperature).
A particular type of affinity chromatography useful in the purification of carbohydrate containing compounds is lectin affinity chromatography. Lectins are a class of substances that bind to a variety of polysaccharides and glycoproteins.
Lectins are usually coupled to agarose by cyanogen bromide. Conconavalin A coupled to Sepharose was the first material of this sort to be used and has been widely used in the isolation of polysaccharides and glycoproteins other lectins that have been include lentil lectin, wheat germ agglutinin which has been useful in the purification of N-acetyl glucosaminyl residues and Helix pomatia lectin. Lectins themselves are purified using affinity chromatography with carbohydrate ligands. Lactose has been used to purify lectins from castor bean and peanuts; maltose has been useful in extracting lectins from lentils and jack bean; N-acetyl-D galactosamine is used for purifying lectins from soybean; N-acetyl glucosaminyl binds to lectins from wheat germ; D-galactosamine has been used in obtaining lectins from clams and L-fucose will bind to lectins from lotus.
The matrix should be a substance that itself does not adsorb molecules to any significant extent and that has a broad range of chemical, physical and thermal stability. The ligand should be coupled in such a way as to not affect its binding properties. The ligand also should provide relatively tight binding. And it should be possible to elute the substance without destroying the sample or the ligand. One of the most common forms of affinity chromatography is immunoaffinity chromatography. The generation of antibodies that would be suitable for use in accord with the present invention is discussed below. To purify a Usl -5 polypeptide, or peptide a natural or recombinant composition, at least some Us1.5 proteins, polypeptides, or peptides will be subjected to fractionation to remove various non-Us1.5 components from the composition. In addition to those techniques described in detail herein below, various other techniques suitable for use in protein purification will be well known to those of skill in the art. These include, for example, precipitation with ammonium sulfate, PEG, antibodies and the like or by heat denaturation, followed by centrifugation; chromatography steps such as ion exchange, gel filtration, reverse phase, hydroxylapatite, lectin affinity and other affinity chromatography steps; isoelectric focusing; gel electrophoresis; and combinations of such and other techniques.
Another example is the purification of Us1.5 protein using a specific binding partner, such as p60. Such purification methods are routine in the art. As the present invention provides DNA sequences Us1.5 proteins, any fusion protein purification method can now be practiced. This is exemplified by the generation of an Us1.5 S- transferase fusion protein, expression in E. coli, and isolation to homogeneity using affinity chromatography on glutathione-agarose or the generation of a polyhistidine tag on the N- or C-terminus of the protein, and subsequent purification using Ni- affinity chromatography.
It is contemplated that almost any purification method now can be employed. Although preferred for use in certain embodiments, there is no general requirement that the Us1.5 protein, polypeptide, or peptide always be provided in their most purified state. Indeed, it is contemplated that less substantially purified Usl .5 protein, polypeptide or peptide, which are nonetheless enriched in Usl .5 protein compositions, relative to the natural state, will have utility in certain embodiments. These include, for example, antibody generation where subsequent screening assays using purified Us1.5 proteins are conducted. Methods exhibiting a lower degree of relative purification may have advantages in total recovery of protein product, or in maintaining the activity of an expressed protein. Inactive products also have utility in certain embodiments, such as, e.g. , in antibody generation.
5. Linkers/coupling agents
The present invention describes the use of polypeptides and peptides containing sequences from Us1.5. In some embodiments, noncontiguous regions of
U 1.5 can be used to inhibit an infection by heφesvirus. It is contemplated that a single region of Us1.5, such as from the region encoding amino acids 147-171 or amino acids 380-405 can be joined to nonnative Us1.5 sequence. This compound can be created by joining regions via a biologically-releasable bond, such as a selectively- cleavable linker or non-native Us1.5 amino acid sequence. Non-native Us1.5 sequence refers to any sequence not identical with the amino acid sequence of Us1.5 between amino acid 172 and amino acid 379. For example, peptide linkers that include a cleavage site for an enzyme preferentially located or active within a tumor environment are contemplated. Exemplary forms of such peptide linkers are those that are cleaved by urokinase, plasmin, thrombin, Factor IXa, Factor Xa, or a metallaproteinase, such as collagenase, gelatinase, or stromelysin.
a. Biochemical cross-linkers
The joining of any of the above components, to a U 1.5 region will generally employ the same technology as developed for the preparation of immunotoxins. It can be considered as a general guideline that any biochemical cross-linker that is appropriate for use in an immunotoxin will also be of use in the present context, and additional linkers may also be considered.
Cross-linking reagents are used to form molecular bridges that tie together functional groups of two different molecules, e.g., a stablizing and coagulating agent. To link two different proteins in a step-wise manner, hetero-bifunctional cross-linkers can be used that eliminate unwanted homopolymer formation. Examples of such cross-linkers can be found in Table 2 below.
TABLE 2 HETERO-BIFUNCTIONAL CROSS-LINKERS
Figure imgf000032_0001
An exemplary hetero-bifunctional cross-linker contains two reactive groups: one reacting with primary amine group (e.g.. N-hydroxy succinimide) and the other reacting with a thiol group (e.g., pyridyl disulfide, maleimides, and halogens). Through the primary amine reactive group, the cross-linker may react with the lysine residue(s) of one protein (e.g., the selected antibody or fragment) and through the thiol reactive group, the cross-linker, already tied up to the first protein, reacts with the cysteine residue (free sulfhydryl group) of the other protein (e.g., the selective agent).
In some embodiments, therefore, a Usl .5 peptide can have, or be derivatized to have, a functional group available for cross-linking puφoses. This requirement is not considered to be limiting in that a wide variety of groups can be used in this manner. For example, primary or secondary amine groups, hydrazide or hydrazine groups, carboxyl alcohol, phosphate, or alkylating groups may be used for binding or cross- linking. For a general overview of linking technology, one may wish to refer to Ghose & Blair (1987).
The spacer arm between the two reactive groups of a cross-linkers may have various length and chemical compositions. A longer spacer arm allows a better flexibility of the conjugate components while some particular components in the bridge (e.g., benzene group) may lend extra stability to the reactive group or an increased resistance of the chemical link to the action of various aspects (e.g., disulfide bond resistant to reducing agents). The use of peptide spacers, such as L-Leu-L-Ala-L-Leu-L-Ala, is also contemplated.
It is preferred that a cross-linker having reasonable stability in blood will be employed. Numerous types of disulfide-bond containing linkers are known that can be successfully employed to conjugate targeting and therapeutic/preventative agents. Linkers that contain a disulfide bond that is sterically hindered may prove to give greater stability in vivo, preventing release of the targeting peptide prior to reaching the site of action. These linkers are thus one group of linking agents. Another cross-linking reagents for use in immunotoxins is SMPT, which is a bifunctional cross-linker containing a disulfide bond that is "sterically hindered" by an adjacent benzene ring and methyl groups. It is believed that stearic hindrance of the disulfide bond serves a function of protecting the bond from attack by thiolate anions such as glutathione which can be present in tissues and blood, and thereby help in preventing decoupling of the conjugate prior to the delivery of the attached agent to the tumor site. It is contemplated that the SMPT agent may also be used in connection with the bispecific coagulating ligands of this invention.
The SMPT cross-linking reagent, as with many other known cross-linking reagents, lends the ability to cross-link functional groups such as the SH of cysteine or primary amines (e.g., the epsilon amino group of lysine). Another possible type of cross-linker includes the hetero-bifunctional photoreactive phenylazides containing a cleavable disulfide bond such as sulfosuccinimidyl-2-(p-azido salicylamido) ethyl- l,3'-dithiopropionate. The N-hydroxy-succinimidyl group reacts with primary amino groups and the phenylazide (upon photolysis) reacts non-selectively with any amino acid residue.
In addition to hindered cross-linkers, non-hindered linkers also can be employed in accordance herewith. Other useful cross-linkers, not considered to contain or generate a protected disulfide. include SATA, SPDP and 2-iminothiolane
(Wawrzynczak & Thoφe, 1987). The use of such cross-linkers is well understood in the art.
Once conjugated, the Us1.5 peptide generally will be purified to separate the conjugate from unconjugated targeting agents or coagulants and from other contaminants. A large a number of purification techniques are available for use in providing conjugates of a sufficient degree of purity to render them clinically useful. Purification methods based upon size separation, such as gel filtration, gel permeation or high performance liquid chromatography, will generally be of most use. Other chromatographic techniques, such as Blue-Sepharose separation, may also be used. In addition to chemical conjugation, a purified UJ .5 polypeptide or peptide may be modified at the protein level. Included within the scope of the invention are Us1.5 polypeptide fragments or other derivatives or analogs that are differentially modified during or after translation, for example by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, and proteolytic cleavage. Any number of chemical modifications may be carried out by known techniques, including but not limited to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH ; acetylation, formylation, farnesylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin.
6. Peptide/Polypeptide-Based Therapies
Because posttranslational modification of Us1.5 is required for progression of a heφesvirus infection, inhibition of modification can be used to inhibit viral infection by HSV. The present invention discloses particular regions that are necessary to mediate posttranslational processing of the UJ .5. Therefore, it is contemplated that these regions can be administered as peptides or polypeptides, or fragments thereof, to a subject to inhibit heφesvirus infection by, for example, competing with Us1.5 produced by the vims as a target for phosphorylation by kinases such as UL13.
Therefore, a therapy approach is the provision, to a subject, of Us1.5 polypeptides, active fragments, synthetic peptides, mimetics or other analogs thereof. The protein may be produced by recombinant expression means or, if small enough, generated by an automated peptide synthesizer. Formulations would be selected based on the route of administration and puφose including, but not limited to, liposomal formulations and classic pharmaceutical preparations.
a. Pharmaceutically-acceptable carriers Aqueous compositions of the present invention comprise an effective amount of the Us1.5 protein, polypeptide, peptide, epitopic core region, inhibitor, or such like. dissolved or dispersed in a pharmaceutically acceptable earner or aqueous medium Aqueous compositions of gene therapy vectors expressing any of the foregoing are also contemplated The phrases "pharmaceutically or pharmacologically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, or a human, as appropπate
As used herem, "pharmaceutically acceptable earner" includes any and all solvents, dispersion media, coatings, antibacteπal and antifungal agents, lsotonic and absoφtion delaying agents and the like The use of such media and agents for pharmaceutical active substances is well known in the art Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated Supplementary active ingredients can also be incoφorated into the compositions For human administration, preparations should meet steπhty, pyrogenicity, general safety and purity standards as required bv FDA Office of Biologies standards
The biological matenal should be extensively dialyzed to remove undesired small molecular weight molecules and/or lyophilized for more ready formulation into a desired vehicle, where appropπate The active compounds will then generally be formulated for parenteral administration, e g , formulated for injection via the intravenous, intramuscular, sub-cutaneous, mtralesional, or even mtrapeπtoneal routes The preparation of an aqueous composition that contains a U l 5 agent as an active component or ingredient will be known to those of skill m the art in light of the present disclosure Typically, such compositions can be prepared as injectables, either as liquid solutions or suspensions, solid forms suitable for using to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared, and the preparations can also be emulsified
The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions, formulations including sesame oil, peanut oil or aqueous propylene glycol, and sterile powders for the extemporaneous preparation of steπle injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
A Us1.5 protein, polypeptide, peptide, agonist or antagonist of the present invention can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. In terms of using peptide therapeutics as active ingredients, the technology of U.S. Patents 4,608,251 ; 4,601 ,903; 4,599,231 ; 4,599,230; 4,596,792; and 4,578,770, each incoφorated herein by reference, may be used.
The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absoφtion of the injectable compositions can be brought about by the use in the compositions of agents delaying absoφtion, for example, aluminum monostearate and gelatin.
Sterile injectable solutions are prepared by incoφorating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incoφorating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof. The preparation of more, or highly, concentrated solutions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small area.
Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but dmg release capsules and the like can also be employed.
For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media, which can be employed, will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-
1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
The active Us1.5 protein-derived peptides or agents may be formulated within a therapeutic mixture to comprise about 0.0001 to 1.0 milligrams, or about 0.001 to 0J milligrams, or about 0J to 1.0 or even about 10 milligrams per dose or so. Multiple doses can also be administered.
In addition to the compounds formulated for parenteral administration, such as intravenous or intramuscular injection, other pharmaceutically acceptable forms include, e.g., tablets or other solids for oral administration; liposomal formulations; time release capsules; and any other form currently used, including cremes.
One may also use nasal solutions or sprays, aerosols or inhalants in the present invention. Nasal solutions are usually aqueous solutions designed to be administered to the nasal passages in drops or sprays. Nasal solutions are prepared so that they are similar in many respects to nasal secretions, so that normal ciliary action is maintained. Thus, the aqueous nasal solutions usually are isotonic and slightly buffered to maintain a pH of 5.5 to 6.5. In addition, antimicrobial preservatives, similar to those used in ophthalmic preparations, and appropriate dmg stabilizers, if required, may be included in the formulation. Various commercial nasal preparations are known and include, for example, antibiotics and antihistaminesand are used for asthma prophylaxis.
Additional formulations that are suitable for other modes of administration include vaginal suppositories and pessaries. A rectal pessary or suppository may also be used. Suppositories are solid dosage forms of various weights and shapes, usually medicated, for insertion into the rectum, vagina or the urethra. After insertion, suppositories soften, melt or dissolve in the cavity fluids. In general, for suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably l%-2%.
Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders. In certain defined embodiments, oral pharmaceutical compositions will comprise an inert diluent or assimilable edible carrier, or they may be enclosed in hard or soft shell gelatin capsule, or they may be compressed into tablets, or they may be incoφorated directly with the food of the diet. For oral therapeutic administration, the active compounds may be incoφorated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 75% of the weight of the unit, or preferably between 25-60%. The amount of active compounds in such therapeutically useful compositions is such that a suitable dosage will be obtained.
The tablets, troches, pills, capsules and the like may also contain the following: a binder, as gum tragacanth, acacia, cornstarch, or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as com starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin may be added or a flavoring agent, such as peppermint, oil of wintergreen, or cherry flavoring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A symp of elixir may contain the active compounds sucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor.
b. Liposomes and nanocapsules
In certain embodiments, the use of liposomes and/or nanoparticles is contemplated for the introduction of Us1.5 protein, polypeptides, peptides or agents, or gene therapy vectors, including both wild-type and antisense vectors, into host cells. The formation and use of liposomes is generally known to those of skill in the art, and is also described below.
Nanocapsules can generally entrap compounds in a stable and reproducible way. To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 μm) should be designed using polymers able to be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present invention, and such particles may be are easily made.
Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs). MLVs generally have diameters of from 25 nm to 4 μm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 A. containing an aqueous solution in the core.
The following information may also be utilized in generating liposomal formulations. Phospholipids can form a variety of stmctures other than liposomes when dispersed in water, depending on the molar ratio of lipid to water. At low ratios the liposome is the preferred stmcture. The physical characteristics of liposomes depend on pH, ionic strength and the presence of divalent cations. Liposomes can show low permeability to ionic and polar substances, but at elevated temperatures undergo a phase transition which markedly alters their permeability. The phase transition involves a change from a closely packed, ordered stmcture, known as the gel state, to a loosely packed, less-ordered stmcture, known as the fluid state. This occurs at a characteristic phase-transition temperature and results in an increase in permeability to ions, sugars and dmgs.
Liposomes interact with cells via four different mechanisms: Endocytosis by phagocytic cells of the reticuloendothelial system such as macrophages and neutrophils; adsoφtion to the cell surface, either by nonspecific weak hydrophobic or electrostatic forces, or by specific interactions with cell-surface components; fusion with the plasma cell membrane by insertion of the lipid bilayer of the liposome into the plasma membrane, with simultaneous release of liposomal contents into the cytoplasm; and by transfer of liposomal lipids to cellular or subcellular membranes, or vice versa, without any association of the liposome contents. Varying the liposome formulation can alter which mechanism is operative, although more than one may operate at the same time.
B. Polynucleotides
1. Polynucleotides encoding polynucleotide products of α22
The present invention concerns polynucleotides, isolatable from HSV-infected cells, which are free from total genomic DNA and that are capable of expressing a protein, polypeptide, or peptide that is derived from the α.22 gene product.
As used herein, the term "DNA segment" refers to a DNA molecule that has been isolated free of total genomic DNA of a particular species. Therefore, a DNA segment encoding a Us1.5 polypeptide refers to a DNA segment that contains Us1.5 polypeptide-coding sequences yet is isolated away from, or purified free from, total mammalian or human genomic DNA. Included within the term "DNA segment" are a polypeptide or polypeptides. DNA segments smaller than a polypeptide, and recombinant vectors, including, for example, plasmids, cosmids, phage, vimses, and the like.
As used in this application, the term "polynucleotide" refers to a nucleic acid molecule that has been isolated free of total genomic nucleic acid. Therefore, a "polynucleotide encoding an Us1.5 polypeptide" refers to a DNA segment that contains Us1.5 polypeptide-coding sequences yet is isolated away from, or purified free from, total mammalian or viral genomic DNA; similarly, a "polynucleotide encoding ICP22" refers to a DNA segment that contains ICP22 polypeptide coding sequences yet is isolated away from, or purified free from, total mammalian or human genomic DNA. Therefore, when the present application refers to the function of Us1.5 or to "Us1.5 polypeptide" that is encoded by α22, it is meant that the polynucleotide encodes a molecule that has the ability to promote the infectivity of a heφes simplex vims in restrictive cells.
The term "cDNA" is intended to refer to DNA prepared using messenger RNA (mRNA) as template. The advantage of using a cDNA, as opposed to genomic DNA or DNA polymerized from a genomic, non- or partially-processed RNA template, is that the cDNA primarily contains coding sequences of the corresponding protein. There may be times when the full or partial genomic sequence is preferred, such as where the non-coding regions are required for optimal expression or where non-coding regions such as introns are to be targeted in an anti sense strategy.
It also is contemplated that a given α22 gene from a given viral strain may be represented by natural variants that have slightly different nucleic acid sequences but, nonetheless, encode the same protein (see Table 1 above). Consequently, the present invention also encompasses derivatives of an Us1.5 polypeptide that have minimal amino acid changes, but that possess the vims-promoting activities of the known sequence. In particular embodiments, the invention concerns isolated DNA segments and recombinant vectors incoφorating DNA sequences that encode the UJ .5 polypeptide or peptide that includes within its amino acid sequence a contiguous amino acid sequence in accordance with, or essentially as set forth in, SEQ ID NOJ , corresponding to amino acids 147-171 of the polypeptide encoded by the α22 gene of heφes simplex virus. In other embodiments, the invention concerns isolated DNA segments and recombinant vectors incoφorating DNA sequences that encode a polypeptide or peptide that includes within its amino acid sequence a contiguous amino acid sequence in accordance with, or essentially as set forth in, SEQ ID NO:2, corresponding to amino acids 380-405 of the polypeptide encoded by the α22 gene of
HSV-1. In other embodiments, the invention concerns isolated DNA segments and recombinant vectors incoφorating DNA sequences that encode a polypeptide or peptide that includes within its amino acid sequence a contiguous amino acid sequence in accordance with, or essentially as set forth in, SEQ ID NOJ, corresponding to amino acids 38-66 of the polypeptide encoded by the α22 gene of
HSV-1. In other embodiments, the invention concerns isolated DNA segments and recombinant vectors incoφorating DNA sequences that encode a polypeptide or peptide that includes within its amino acid sequence a contiguous amino acid sequence in accordance with, or essentially as set forth in, SEQ ID NOJ, corresponding to amino acids 300-328 of the polypeptide encoded by the α22 gene of
HSV-1. In other embodiments, the invention concerns isolated DNA segments and recombinant vectors incoφorating DNA sequences that encode a polypeptide or peptide that includes within its amino acid sequence a contiguous amino acid sequence in accordance with, or essentially as set forth in, SEQ ID NO:5, corresponding to amino acids 143-167 of the polypeptide encoded by the α22 gene of
HSV-2. In other embodiments, the invention concerns isolated DNA segments and recombinant vectors incoφorating DNA sequences that encode a polypeptide or peptide that includes within its amino acid sequence a contiguous amino acid sequence in accordance with, or essentially as set forth in, SEQ ID NO:6, coπesponding to amino acids 373-598 of the polypeptide encoded by the α22 gene of HSV-2. In other embodiments, the invention concerns isolated DNA segments and recombinant vectors incoφorating DNA sequences that encode a polypeptide or peptide that includes within its amino acid sequence a contiguous amino acid sequence in accordance with, or essentially as set forth in, SEQ ID NO:7. coπesponding to amino acids 37-62 of the polypeptide encoded by the α22 gene of HSV-2. In other embodiments, the invention concerns isolated DNA segments and recombinant vectors incoφorating DNA sequences that encode a polypeptide or peptide that includes within its amino acid sequence a contiguous amino acid sequence in accordance with, or essentially as set forth in, SEQ ID NO:8, corresponding to amino acids 295-320 of the polypeptide encoded by the α.22 gene of HSV-2 type 1.
The term "gene" is used for simplicity to refer to a functional protein-, polypeptide-, or peptide-encoding unit. As will be understood by those in the art, this functional term includes viral DNA sequences, cDNA sequences, and smaller engineered gene segments that express, or may be adapted to express, proteins, polypeptides, domains, peptides, fusion proteins, and mutants.
"Isolated substantially away from other coding sequences" means that the gene of interest, in this case the α22 gene, forms the significant part of the coding region of the DNA segment, and that the DNA segment does not contain large portions of naturally-occurring coding DNA, such as large viral DNA genomic fragments or other functional genes or cDNA coding regions. Of course, this refers to the DNA segment as originally isolated, and does not exclude genes or coding regions later added to the segment by the hand of man.
The phrase "a sequence essentially as set forth in SEQ ID NOJ" means that the sequence substantially corresponds to a portion of SEQ ID NOJ and has relatively few amino acids that are not identical to, or a biologically functional equivalent of, the amino acids of SEQ ID NOJ . The phrase "a sequence essentially as set forth in SEQ ID NO:" can be used with any identified sequence to connote the corresponding definition as applied to SEQ ID NOJ by example. The DNA segments used in the present invention encompass biologically functional equivalent Us1.5 protein and peptides. Such sequences may arise as a consequence of codon redundancy and functional equivalency that are known to occur naturally within nucleic acid sequences and the proteins thus encoded. Alternatively, functionally equivalent proteins or peptides may be created via the application of recombinant DNA technology, in which changes in the protein stmcture may be engineered, based on considerations of the properties of the amino acids being exchanged. Changes designed by humans may be introduced through the application of site-directed mutagenesis techniques, e.g., to introduce improvements to the antigenicity of the protein or to test mutants in order to examine DNA binding activity at the molecular level. Accordingly, sequences that have between about 70% and about 80%; or more preferably, between about 81% and about 90%>; or even more preferably, between about 91% and about 99%; of amino acids that are identical or functionally equivalent to the amino acids of SEQ ID NOJ, SEQ ID NOJ, SEQ ID
NO:3, SEQ ID NOJ, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and/or SEQ ID NO:8 will be sequences that are "essentially as set forth in SEQ ID NOJ," "essentially as set forth in SEQ ID NO:2," "essentially as set forth in SEQ ID NOJ," "essentially as set forth in SEQ ID NO:4," "essentially as set forth in SEQ ID NO:5," "essentially as set forth in SEQ ID NO:6," "essentially as set forth in SEQ ID NO: 7," and/or "essentially as set forth in SEQ ID NO: 8," provided the biological activity of the polypeptide is maintained.
As used herein in various aspects of the invention, the term "consecutive nucleic acid segment" will be understood to include a contiguous nucleic acid sequence of at least about 8, about, 9, about 10, about 1 1 , about 12, about 13, about 14, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 60, about 70, about 80, about 90, about 100, about 1 10, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 300, about 310, about 320, about 330, about 340, about 350, about 360, about 370, about 380, about 390, about 400, about 410, about 420. about 430, about 440, about 450, about 460, about 470, about 480, about 490, about 500, about 600, about 700, about 800, about 900, or about 1000, about 1100, about 1200, about 1300, about 1400, about 1500, about 1600, about 1700, about 1800, about 1900. about 2000, about 3000, about 4000, or about 5000 nucleic acids or so.
Also encompassed by the invention are DNA segments encoding relatively small peptides, such as, for example, peptides of from about 8, about 9, about 10, about 1 1. about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31 , about 32, about 33, about 34, about 35, about 35, about 40, about 45, to about 50 amino acids in length, and additionally, between about 10 amino acids and about 50 amino acids, between about 15 and about 30 amino acids in length, and between about 20 amino acids and about 25 amino acids, as set forth in SEQ ID NOJ and SEQ ID NO:2 and also larger polypeptides up to the full-length amino acid sequences of Us1.5.
In particular embodiments, the invention concerns isolated DNA segments and recombinant vectors incoφorating DNA sequences that encode a Us1.5 polypeptide or peptide that includes within its amino acid sequence a contiguous amino acid sequence in accordance with, or essentially coπesponding to Us1.5 polypeptides.
In other embodiments, the invention concerns isolated DNA segments and recombinant vectors incoφorating DNA sequences that encode a Usl .5 polypeptide or peptide that includes within its amino acid sequence a contiguous amino acid sequence in accordance with, or essentially corresponding to the Usl .5 polypeptide.
2. Mutagenesis
Site-specific mutagenesis is a technique useful in the preparation of individual peptides, or biologically functional equivalent proteins or peptides, through specific mutagenesis of the underlying DNA. The technique further provides a ready ability to prepare and test sequence variants, incoφorating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA. Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed. Typically, a primer of about 17 to 25 nucleotides in length is preferred, with about 5 to 10 residues on both sides of the junction of the sequence being altered.
In general, the technique of site-specific mutagenesis is well known in the art. As will be appreciated, the technique typically employs a bacteriophage vector that exists in both a single stranded and double stranded form. Typical vectors useful in site-directed mutagenesis include vectors such as the Ml 3 phage. These phage vectors are commercially available and their use is generally well known to those skilled in the art. Double stranded plasmids are also routinely employed in site directed mutagenesis, which eliminates the step of transferring the gene of interest from a phage to a plasmid.
In general, site-directed mutagenesis is performed by first obtaining a single- stranded vector, or melting of two strands of a double stranded vector which includes within its sequence a polynucleotide sequence encoding the desired protein. An oligonucleotide primer bearing the desired mutated sequence is synthetically prepared. This primer is then annealed with the single-stranded DNA preparation, and subjected to DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand. Thus, a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation. This heteroduplex vector is then used to transform appropriate cells, such as E. coli cells, and clones are selected that include recombinant vectors bearing the mutated sequence arrangement. The preparation of sequence variants of the selected gene using site-directed mutagenesis is provided as a means of producing potentially useful species and is not meant to be limiting, as there are other ways in which sequence variants of genes may be obtained. For example, recombinant vectors encoding the desired gene may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants.
The nucleic acid segments used in the present invention, regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol.
3. Vectors for cloning, gene transfer, and expression
The present invention involving a polypeptide or peptide encoded by a polynucleotide can be combined with other nucleic acid segments, including segments from an expression vector. Within certain embodiments expression vectors are employed to express the Us1.5 polypeptide or peptide that encompasses a posttranslational modification site. The expression vectors can be used to deliver a polynucleotidesequence encoding a Usl .5 polypeptide or peptide to a cell either in vitro or in vivo. Alternatively, the vector can be used to deliver such a sequence, so that its amino acid product which can then be purified and, for example, be used to vaccinate animals or humans to generate antisera or monoclonal antibody. In other embodiments, the expression vectors are used in gene therapy. Expression requires that appropriate signals be provided in the vectors, and which include various regulatory elements, such as enhancers/promoters from both viral and mammalian sources that drive expression of the genes of interest in host cells. Elements designed to optimize messenger RNA stability and translatability in host cells also are defined. The conditions for the use of a number of dominant dmg selection markers for establishing permanent, stable cell clones expressing the products also are provided, as is an element that links expression of the dmg selection markers to expression of the polypeptide.
a. Regulatory elements
Throughout this application, the term "expression vector" is meant to include any type of genetic constmct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed. The transcript may be translated into a protein, but it need not be. In certain embodiments, expression includes both transcription of a gene and translation of mRNA into a gene product. In other embodiments, expression only includes transcription of the nucleic acid encoding a gene of interest.
In some embodiments, the nucleic acid encoding a gene product is under transcriptional control of a promoter. A "promoter" refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene. The phrase "under transcriptional control" means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene.
The term promoter will be used here to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase II. Much of the thinking about how promoters are organized derives from analyses of several viral promoters, including those for the HSV thymidine kinase (tk) and SV40 early transcription units. These studies, augmented by more recent work, have shown that promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins. At least one module in each promoter functions to position the start site for
RNA synthesis. The best known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation.
Additional promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-1 10 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserv ed when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription.
The particular promoter employed to control the expression of a nucleic acid sequence of interest is not believed to be important, so long as it is capable of direction the expression of the nucleic acid in the targeted cell. Thus, where a human cell is targeted, it is preferable to position the nucleic acid coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell. Generally speaking, such a promoter might include either a human or viral promoter.
In various embodiments, the human cytomegalovims (CMV) immediate early gene promoter, the SV40 early promoter, the Rous sarcoma vims long terminal repeat, rat insulin promoter and glyceraldehyde-3-phosphate dehydrogenase can be used to obtain high-level expression of the coding sequence of interest. The use of other viral or mammalian cellular or bacterial phage promoters which are well-known in the art to achieve expression of a coding sequence of interest is contemplated as well, provided that the levels of expression are sufficient for a given piiφose. Selection of a promoter that is regulated in response to specific physiologic or synthetic signals can permit inducible expression of the gene product. For example in the case where expression of a transgene is toxic to the host cells, it may be desirable to prohibit or reduce expression of the transgene. Examples of transgenes that may be toxic to the producer cell line are pro-apoptotic and cytokine genes. Several inducible promoter systems are available for production of viral vectors where the transgene product may be toxic. An inducible promoter may be used to control the expression of UJ .5.
The ecdysone system (Invitrogen, Carlsbad, CA) is one such inducible system.
This system is designed to allow regulated expression of a gene of interest in mammalian cells. It consists of a tightly regulated expression mechanism that allows virtually no basal level expression of the transgene, but over 200-fold inducibility. The system is based on the heterodimeric ecdysone receptor of Drosophila, and when ecdysone or an analog such as muristerone A binds to the receptor, the receptor activates a promoter to turn on expression of the downstream transgene such that high levels of mRNA transcripts are attained. In this system, both monomers of the heterodimeric receptor are constitutively expressed from one vector, whereas the ecdysone-responsive promoter which drives expression of the gene of interest is on another plasmid. Using this type of system, cotransfection of plasmids containing the gene of interest and the receptor monomers in the producer cell line would allow for the production of the gene transfer vector without expression of a potentially toxic transgene. At the appropriate time, expression of the transgene could be activated with ecdysone or muristeron A.
Another inducible system that would be useful is the Tet-Off™ or Tet-On™ system (Clontech, Palo Alto, CA) originally developed by Gossen and Bujard (Gossen and Bujard, 1992; Gossen et al, 1995). This system also allows high levels of gene expression to be regulated in response to tetracycline or tetracycline derivatives such as doxycycline. In the Tet-On™ system, gene expression is turned on in the presence of doxycycline, whereas in the Tet-Of M system, gene expression is turned on in the absence of doxycycline. For gene therapy vector production, the Tet-Of M system would be preferable so that the producer cells could be grown in the presence of tetracycline or doxycycline and prevent expression of a potentially toxic transgene, but when the vector is introduced to the patient, the gene expression would be constitutively on.
In some circumstances, it may be desirable to regulate expression of a transgene in a gene therapy vector. For example, different viral promoters with varying strengths of activity may be utilized depending on the level of expression desired. In mammalian cells, the CMV immediate early promoter if often used to provide strong transcriptional activation. Modified versions of the CMV promoter that are less potent have also been used when reduced levels of expression of the transgene are desired. When expression of a transgene in hematopoetic cells is desired, retroviral promoters such as the LTRs from MLV or MMTV are often used. Other viral promoters that may be used depending on the desired effect include SV40,
RSV LTR, HIV-1 and HIV-2 LTR, adenovims promoters such as from the El A, E2A, or MLP region, AAV LTR, cauliflower mosaic vims, HSV-TK, and avian sarcoma vims.
b. Enhancers
Enhancers are genetic elements that increase transcription from a promoter located at a distant position on the same molecule of DNA. Enhancers are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins. The basic distinction between enhancers and promoters is operational. An enhancer region as a whole must be able to stimulate transcription at a distance; this need not be tme of a promoter region or its component elements.
Below is a list of cellular promoters/enhancers and inducible promoters/enhancers that could be used in combination with the nucleic acid encoding a gene of interest in an expression construct (Table 3 and Table 4). Additionally, any promoter/enhancer combination (as per the Eukaryotic Promoter Data Base EPDB) could also be used to drive expression of the gene. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression constmct.
TABLE 3
ENHANCER
Immunoglobulm Heavy Chain
Immunoglobulm Light Chain
T-Cell Receptor
HLA DQ α and DQ β β-Interferon
Interleukin-2
Interleukin-2 Receptor
MHC Class II 5
MHC Class II HLA-DR β-Actin
Muscle Creatine Kinase
Prealbumin (Transthyretin)
Elastase/
Metallothionein
Collagenase
Albumin Gene α-Fetoprotein τ-Globin β-Globin e-fos c-HA-rαs
Insulin
Neural Cell Adhesion Molecule (NCAM) TABLE 3 (Continued)
ENHANCER αl-Antitrypsin
H2B (TH2B) Histone
Mouse or Type I Collagen
Glucose-Regulated Proteins (GRP94 and GRP78)
Rat Growth Hormone
Human Semm Amyloid A (SAA)
Troponin I (TN I)
Platelet-Derived Growth Factor
Duchenne Muscular Dystrophy
SV40
Polyoma
Retro vimses
Papilloma Virus
Hepatitis B Vims
Human Immunodeficiency Vims
Cytomegalovirus
Gibbon Ape Leukemia Vims
TABLE 4
Figure imgf000057_0001
c. Polyadenylation signals
Where a cDNA insert is employed, one will typically desire to include a polyadenylation signal to effect proper polyadenylation of the gene transcript. The nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any such sequence may be employed such as human growth hormone and SV40 polyadenylation signals. Also contemplated as an element of the expression cassette is a terminator. These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.
d. Selectable markers
In certain embodiments of the invention, the cells contain nucleic acid constmct of the present invention, a cell may be identified in vitro or in vivo by including a marker in the expression vector. Such markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression vector. Usually the inclusion of a dmg selection marker aids in cloning and in the selection of transformants, for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selectable markers. Alternatively, enzymes such as heφes simplex vims thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be employed. lmmunologic markers also can be employed. The selectable marker employed is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product. Further examples of selectable markers are well known to one of skill in the art.
e. Multigene constructs and IRES
In certain embodiments of the invention, the use of internal ribosome binding sites (IRES) elements are used to create multigene, or polycistronic, messages. IRES elements are able to bypass the ribosome scanning model of 5' methylated Cap dependent translation and begin translation at internal sites (Pelletier and Sonenberg, 1988). IRES elements from two members of the picanovims family (polio and encephalomyocarditis) have been described (Pelletier and Sonenberg, 1988), as well an IRES from a mammalian message (Macejak and Samow, 1991 ). IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages. By virtue of the IRES element, each open reading frame is accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter/enhancer to transcribe a single message.
Any heterologous open reading frame can be linked to IRES elements. This includes genes for secreted proteins, multi-subunit proteins, encoded by independent genes, intracellular or membrane-bound proteins and selectable markers. In this way, expression of several proteins can be simultaneously engineered into a cell with a single vector and a single selectable marker.
f. Viral vectors
There are a number of ways in which expression vectors may be introduced into cells. In certain embodiments of the invention, the expression vector comprises a vi s or engineered vector derived from a viral genome. The development of viral vectors is an improvement to the field of gene transfer, as demonstrated by U.S. Patent Application No. 60/078205, hereby incorporated by reference. The ability of certain vimses to enter cells via receptor-mediated endocytosis, to integrate into host cell genome and express viral genes stably and efficiently have made them attractive candidates for the transfer of foreign genes into mammalian cells (Ridgeway, 1988; Nicolas and Rubenstein, 1988; Baichwal and Sugden, 1986; Temin, 1986). Heφesviruses as described herein can be used or altered for use as a delivery system.
Because HSV is neurotropic, it has generated considerable interest in treating nervous system disorders. Moreover, the ability of HSV to establish latent infections in non- dividing neuronal cells without integrating into the host cell chromosome or otherwise altering the host cell's metabolism, along with the existence of a promoter that is active during latency. Although much attention has focused on the neurotropic applications of HSV, this vector can be exploited for other tissues.
Another method for delivery involves the use of an adenovims expression vector. "Adenovims expression vector" is meant to include those vectors containing adenovirus sequences sufficient to (a) support packaging of the vector and (b) to express a polynucleotide that has been cloned therein. In this context, expression may require that the gene product be synthesized.
Adenovims is particularly suitable for use as a gene transfer vector because of its mid-sized genome, ease of manipulation, high titer, wide target cell range and high infectivity. Both ends of the viral genome contain 100-200 base pair inverted repeats (ITRs), which are cis elements necessary for viral DNA replication and packaging. The early (E) and late (L) regions of the genome contain different transcription units that are divided by the onset of viral DNA replication. The El region (El A and E1B) encodes proteins responsible for the regulation of transcription of the viral genome and a few cellular genes. The expression of the E2 region (E2A and E2B) results in the synthesis of the proteins for viral DNA replication. These proteins are involved in DNA replication, late gene expression and host cell shut-off (Renan, 1990). The products of the late genes, including the majority of the viral capsid proteins, are expressed only after significant processing of a single primary transcript issued by the major late promoter (MLP). The MLP (located at 16.8 m.u.) is particularly efficient during the late phase of infection, and all the mRNAs issued from this promoter possess a 5'-tripartite leader (TPL) sequence, which makes them preferred mRNAs for translation.
Adenovims vectors have been used in eukaryotic gene expression (Levrero et al, 1991 ; Gomez-Foix et al, 1992) and vaccine development (Gmnhaus and Horwitz, 1992; Graham and Prevec, 1992). Recently, animal studies suggested that recombinant adenovims could be used for gene therapy (Stratford-Perricaudet and Perricaudet, 1991 ; Stratford-Perricaudet et al, 1990; Rich et al, 1993). Studies in administering recombinant adenovims to different tissues include trachea instillation (Rosenfeld et al, 1991 ; Rosenfeld et al, 1992), muscle injection (Ragot et al. 1993), peripheral intravenous injections (Herz and Gerard, 1993) and stereotactic inoculation into the brain (Le Gal La Salle et al, 1993). The retrovimses are a group of single-stranded RNA vimses characterized by an ability to convert their RNA to double-stranded DNA in infected cells by a process of reverse-transcription (Coffin. 1990). The resulting DNA then stably integrates into cellular chromosomes as a provims and directs synthesis of viral proteins. The integration results in the retention of the viral gene sequences in the recipient cell and its descendants. The retroviral genome contains three genes, gag, pol, and env that code for capsid proteins, polymerase enzyme, and envelope components, respectively. A sequence found upstream from the gag gene contains a signal for packaging of the genome into virions. Two long terminal repeat (LTR) sequences are present at the 5' and 3' ends of the viral genome. These contain strong promoter and enhancer sequences and also are required for integration in the host cell genome (Coffin, 1990).
In order to constmct a retroviral vector, a nucleic acid encoding a gene of interest is inserted into the viral genome in the place of certain viral sequences to produce a vims that is replication-defective. In order to produce virions, a packaging cell line containing the gag, pol, and env genes but without the LTR and packaging components is constmcted (Mann et al, 1983). When a recombinant plasmid containing a cDNA, together with the retroviral LTR and packaging sequences is introduced into this cell line (by calcium phosphate precipitation for example), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al, 1983). The media containing the recombinant retrovimses is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors are able to infect a broad variety of cell types. However, integration and stable expression require the division of host cells (Paskind et al,
1975).
Other viral vectors may be employed as expression constmcts in the present invention. Vectors derived from vimses such as vaccinia vims (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al. 1988) adeno-associated vims (AAV)
(Ridgeway, 1988; Baichwal and Sugden, 1986; Hermonat and Muzycska, 1984) and heφesviruses may be employed. These different viral vectors offer several attractive features for various mammalian cells (Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al, 1988; Horwich et al, 1990).
With the recent recognition of defective hepatitis B viruses, new insight was gained into the stmcture- function relationship of different viral sequences. In vitro studies showed that the vims could retain the ability for helper-dependent packaging and reverse transcription despite the deletion of up to 80% of its genome (Horwich et al, 1990). This suggested that large portions of the genome could be replaced with foreign genetic material. The hepatotropism and persistence (integration) were particularly attractive properties for liver-directed gene transfer. Chang et al, recently introduced the chloramphenicol acetyltransferase (CAT) gene into duck hepatitis B vims genome in the place of the polymerase, surface, and pre-surface coding sequences. It was co-transfected with wild-type vims into an avian hepatoma cell line. Culture media containing high titers of the recombinant vims were used to infect primary duckling hepatocytes. Stable CAT gene expression was detected for at least 24 days after transfection (Chang et al, 1991).
4. Methods of gene transfer In order to effect expression of sense or antisense gene constmcts, the expression vector must be delivered into a cell. This delivery may be accomplished in vitro, as in laboratory procedures for transforming cells lines, or in vivo or ex vivo, as in the treatment of certain disease states. One mechanism for delivery is via viral infection where the expression vector is encapsidated in an infectious viral particle. These methods are described above.
Several non-viral methods for the transfer of expression vectors into cultured mammalian cells also are contemplated by the present invention. These include calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al, 1990) DEAE-dextran (Gopal, 1985), electroporation (Tur-Kaspa et al, 1986; Potter et al, 1984), direct microinjection (Harland and Weintraub, 1985), DNA-loaded liposomes (Nicolau and Sene, 1982; Fraley et al, 1979) and lipofectamine-DNA complexes, cell sonication (Fechheimer et al, 1987), gene bombardment using high velocity microprojectiles (Yang et al, 1990), and receptor- mediated transfection (Wu and Wu, 1987; Wu and Wu, 1988). Some of these techniques may be successfully adapted for in vivo or ex vivo use.
Once the expression vector has been delivered into the cell the nucleic acid encoding the gene of interest may be positioned and expressed at different sites. In certain embodiments, the nucleic acid encoding the gene may be stably integrated into the genome of the cell. This integration may be in the cognate location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non-specific location (gene augmentation). In yet further embodiments, the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or "episomes" encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. How the expression vector is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression vector employed.
In yet another embodiment of the invention, the expression vector may simply consist of naked recombinant DNA or plasmids. Transfer of the vector may be performed by any of the methods mentioned above which physically or chemically permeabihze the cell membrane. This is particularly applicable for transfer in vitro but it may be applied to in vivo use as well. Dubensky et al. (1984) successfully injected polyomavirus DNA in the form of calcium phosphate precipitates into liver and spleen of adult and newborn mice demonstrating active viral replication and acute infection. Benvenisty and Neshif (1986) also demonstrated that direct intraperitoneal injection of calcium phosphate-precipitated plasmids results in expression of the transfected genes. It is envisioned that DNA encoding a gene of interest also may be transferred in a similar manner in vivo and express the gene product. In still another embodiment of the invention for transferring a naked DNA expression vector into cells may involve particle bombardment. This method depends on the ability to accelerate DNA-coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them (Klein et al, 1987). Several devices for accelerating small particles have been developed. One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force (Yang et al, 1990). The microprojectiles used have consisted of biologically inert substances such as tungsten or gold beads.
Selected organs including the liver, skin, and muscle tissue of rats and mice have been bombarded in vivo (Yang et al. 1990; Zelenin et al. 1991). This may require surgical exposure of the tissue or cells, to eliminate any intervening tissue between the gun and the target organ, i.e., ex vivo treatment. Again, DNA encoding a particular gene such as α22, or fragments thereof, may be delivered via this method and still be incoφorated by the present invention.
In a further embodiment of the invention, the expression vector may be entrapped in a liposome. Liposomes are vesicular stmctures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed stmctures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991). Also contemplated are lipofectamine-DNA complexes.
Liposome-mediated nucleic acid delivery and expression of foreign DNA in vitro has been very successful. Wong et al, (1980) demonstrated the feasibility of liposome-mediated delivery and expression of foreign DNA in cultured chick embryo, HeLa and hepatoma cells. Nicolau et al. (1987) accomplished successful liposome- mediated gene transfer in rats after intravenous injection. In certain embodiments of the invention, the liposome may be complexed with a hemagglutinating vims (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA (Kaneda et al, 1989). In other embodiments, the liposome may be complexed or employed in conjunction with nuclear non-histone chromosomal proteins (HMG-1) (Kato et al,
1991). In yet further embodiments, the liposome may be complexed or employed in conjunction with both HVJ and HMG-1. In that such expression vectors have been successfully employed in transfer and expression of nucleic acid in vitro and in vivo, then they are applicable for the present invention. Where a bacterial promoter is employed in the DNA vector, it also will be desirable to include within the liposome an appropriate bacterial polymerase.
Other expression vectors that can be employed to deliver a nucleic acid encoding a particular gene into cells are receptor-mediated delivery vehicles. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis in almost all eukaryotic cells. Because of the cell type-specific distribution of various receptors, the delivery can be highly specific (Wu and Wu, 1993).
Receptor-mediated gene targeting vehicles generally consist of two components: a cell receptor-specific ligand and a DNA-binding agent. Several ligands have been used for receptor-mediated gene transfer. The most extensively characterized ligands are asialoorosomucoid (ASOR) (Wu and Wu, 1987) and transferrin (Wagner et al, 1990). A synthetic neoglycoprotein, which recognizes the same receptor as ASOR, has been used as a gene delivery vehicle (Ferkol et al, 1993;
Perales et al, 1994) and epidermal growth factor (EGF) also has been used to deliver genes to squamous carcinoma cells (Myers, EPO 0273085).
In other embodiments, the delivery vehicle may comprise a ligand and a liposome. For example, Nicolau et al, (1987) employed lactosyl-ceramide, a galactose-terminal asialganglioside, incoφorated into liposomes and observed an increase in the uptake of the insulin gene by hepatocytes. Thus, it is feasible that a nucleic acid encoding a particular gene also may be specifically delivered into a cell type such as lung, epithelial or tumor cells, by any number of receptor-ligand systems with or without liposomes. For example, epidermal growth factor (EGF) may be used as the receptor for mediated delivery of a nucleic acid encoding a gene in many tumor cells that exhibit upregulation of EGF receptor. Mannose can be used to target the mannose receptor on liver cells. Also, antibodies to CD5 (CLL), CD22 (lymphoma), CD25 (T-cell leukemia) and MAA (melanoma) can similarly be used as targeting moieties.
In certain embodiments, gene transfer may more easily be performed under ex vivo conditions. Ex vivo gene therapy refers to the isolation of cells from an animal, the delivery of a nucleic acid into the cells in vitro, and then the return of the modified cells back into an animal. This may involve the surgical removal of tissue/organs from an animal or the primary culture of cells and tissues.
Continuous perfusion of an expression vector or a viral vector also is contemplated. The amount of vector or peptide delivered in continuous perfusion can be determined by the amount of uptake that is desirable.
Primary mammalian cell cultures may be prepared in various ways. In order for the cells to be kept viable while in vitro and in contact with the expression vector, it is necessary to ensure that the cells maintain contact with the correct ratio of oxygen and carbon dioxide and nutrients but are protected from microbial contamination. Cell culture techniques are well documented and are disclosed herein by reference (Freshner,
1992).
During in vitro culture conditions the expression constmct may then deliver and express a nucleic acid encoding a Us1.5 polypeptide or peptide into the cells. Finally, the cells may be reintroduced into the original animal, or administered into a distinct animal, in a pharmaceutically acceptable form by any of the means described below. Thus, providing an ex vivo method of treating a mammal with a pathologic condition is within the scope of the invention.
4. Genetic-Based Therapies One of the therapeutic embodiments contemplated by the present inventors is the intervention, at the molecular level, in the events involved in heφesvirus infection or reactivation. Specifically, the present inventors intend to provide to an infectable or an infected cell, an expression constmct capable of providing a Us1.5 polypeptide or peptide to that cell to inhibit a heφesvirus infection through the use of a Us1.5 polypeptide or peptide that contains a site required to be posttranslationally modified for viral progression. It is contemplated that a Us1.5 polypeptide or peptide can be provided exogenously to inhibit infection, for example, by competing with Us1.5 produced by the vims as a target for phosphorylation. The competitor Us1.5 peptide or polypeptide may effectively prevent the posttranslational modification of Us1.5, thereby rendering native Us1.5 unable to assist in downstream events of infection.
The lengthy discussion of expression vectors and the genetic elements employed therein is incoφorated into this section by reference. This would include viral vectors such as heφesvirus, adenovims, adeno-associated vims, vaccinia vims and retrovims. Also contemplated is a liposomally-encapsulated expression vector.
Those of skill in the art are well aware of how to apply gene delivery to in vivo and ex vivo situations. For viral vectors, one generally will prepare a viral vector stock. Depending on the kind of vims and the titer attainable, one will deliver 1 to 100, 10 to 50, 100-1000, or up to 1 x 104, 1 x 105, 1 x 106, 1 x 107, 1 x 108, 1 x 109, 1 x 1010, 1 x 10", or 1 x 1012 infectious particles to the patient. Similar figures may be extrapolated for liposomal or other non-viral formulations by comparing relative uptake efficiencies. Foπnulation as a pharmaceutically acceptable composition is discussed below.
In some embodiments of the present invention, a subject is exposed to a viral vector and the subject is then monitored for expression constmct-based toxicity, where such toxicity may include, among other things, causing a condition that is injurious to the subject.
In other embodiments of the present invention, the detection of Us1.5 polynucleotides or polypeptides is desirable, for example, to determine the level of expression from an expression constmct that contains a Us1.5 polynucleotide encoding a Us1.5 polypeptide. Methods of detecting polynucleotides, such as the mRNA transcripts encoded by the α22 polynucleotide of the expression constmct, include Northern detection methods and nucleic acid amplification methods, such as the polymerase chain reaction (PCR) described in detail in U.S. Patent Nos.
4,683,195, 4,683,202 and 4,800,159. Such techniques are known to those of ordinary skill in the art. Various methods of detecting polypeptides are also within the ordinary skill of those in the art. Immunoassays encompassed by the present invention include, but are not limited to, those described in U.S. Patent No. 4,367,1 10 (double monoclonal antibody sandwich assay) and U.S. Patent No. 4,452,901
(western blot). Other assays include immunoprecipitation of labeled ligands and immunocytochemistry, both in vitro and in vivo.
Immunoassays generally are binding assays. Certain preferred immunoassays are the various types of enzyme linked immunosorbent assays (ELISAs) and radioimmunoassays (RIA) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. The basic ELISA technique and its variations are known to those of skill in the art. Assays for the presence of expression of Us1.5 may be performed directly on tissue samples. Methods for in vitro situ analysis are well known and involve assessing binding of antigen-specific antibodies to tissues, cells, or cell extracts. These are conventional techniques well within the grasp of those skilled in the art. C. Formulations and Routes for Administration to Patients of
Polypeptide, Peptide and Polynucleotide-Based Compositions
Where clinical applications are contemplated, it will be necessary to prepare pharmaceutical compositions—expression vectors, vims stocks, polypeptides, peptides, antibodies, and drugs— in a form appropriate for the intended application.
Generally, this will entail preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals.
One will generally desire to employ appropriate salts and buffers to render delivery vectors stable and allow for uptake by target cells. Buffers also will be employed when recombinant cells are introduced into a patient. Aqueous compositions of the present invention comprise an effective amount of the vector to cells, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such compositions also are refened to as inocula. The phrase "pharmaceutically or pharmacologically acceptable" refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absoφtion delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art.
Except insofar as any conventional media or agent is incompatible with the vectors or cells of the present invention, its use in therapeutic compositions is contemplated.
Supplementary active ingredients also can be incoφorated into the compositions.
The active compositions of the present invention may include classic pharmaceutical preparations. Administration of these compositions according to the present invention will be via any common route so long as the target tissue is available via that route. This includes oral, nasal, buccal, rectal, vaginal or topical. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such compositions would normally be administered as pharmaceutically acceptable compositions, described supra.
The expression vectors and delivery vehicles also may be administered parenterally or intraperitoneally. Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions also can be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
The therapeutic compositions of the present invention are advantageously administered in the form of injectable compositions either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. These preparations also may be emulsified. A typical composition for such puφose comprises a pharmaceutically acceptable carrier. For instance, the composition may contain 10 mg, 25 mg, 50 mg or up to about 100 mg of human semm albumin per milliliter of phosphate buffered saline. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyloleate. Aqueous carriers include water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc. Intravenous vehicles include fluid and nutrient replenishers. Preservatives include antimicrobial agents, anti-oxidants, chelating agents and inert gases. The pH and exact concentration of the various components the pharmaceutical composition are adjusted according to well known parameters.
Additional formulations are suitable for oral administration. Oral formulations include such typical excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. The compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders. When the route is topical, the form may be a cream, ointment, salve or spray.
An effective amount of the therapeutic agent is determined based on the intended goal, for example (i) inhibition of tumor cell proliferation or (ii) eliminafion of tumor cells. The term "unit dose" refers to physically discrete units suitable for use in a subject, each unit containing a predetermined-quantity of the therapeutic composition calculated to produce the desired responses, discussed above, in association with its administration, i.e., the appropriate route and treatment regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the subject to be treated, the state of the subject and the protection desired. Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual.
The engineered vimses of the present invention may be administered directly into animals, or alternatively, administered to cells that are subsequently administered to animals. The vimses can be combined with the various β-interferon inhibiting formulations to produce transducing formulations with greater transduction efficiencies. A discussion of suitable vimses is presented above.
1. in vitro, ex vivo, in vivo administration
As used herein, the term in vitro administration refers to manipulations performed on cells removed from an animal, including, but not limited to, cells in culture. The term ex vivo administration refers to cells which have been manipulated in vitro, and are subsequently administered to a living animal. The term in vivo administration includes all manipulations performed on cells within an animal.
In certain aspects of the present invention, the compositions may be administered either in vitro, ex vivo, or in vivo. In certain in vitro embodiments, autologous B-lymphocyte cell lines are incubated with a vaccinia vims vector of the instant invention for 24 to 48 hours or with synthetic HIV peptides for two hours. The transduced cells can then be used for in vitro analysis, or alternatively for in vivo administration.
U.S. Patents 4,690,915 and 5,199,942, both incoφorated herein by reference, disclose methods for ex vivo manipulation of blood mononuclear cells and bone marrow cells for use in therapeutic applications.
In vivo administration of the compositions of the present invention also are contemplated. Examples include, but are not limited to, transduction of bladder epithelium by administration of the transducing compositions of the present invention through intravesicle catheterization into the bladder (Bass et al, 1995), and transduction of liver cells by infusion of appropriate transducing compositions through the portal vein via a catheter (Bao et al, 1996). Additional examples include direct injection of tumors with the instant transducing compositions, and either intranasal or intratracheal (Dong et al, 1996) instillation of transducing compositions to effect transduction of lung cells.
2. Combined therapy with anti-viral agents Persistent infection and latency represent major problems in clinical virology.
One goal of current viral research is to find ways to improve the efficacy of anti-viral therapy. One way is by combining such traditional therapies with gene therapy. In the context of the present invention, it is contemplated that therapy could be used similarly in conjunction with anti-viral agents, such as acyclovir, valacyclovir, and famciclovir.
To inhibit heφesvirus infection or reactivation, one would generally contact a target cell with Us1.5-derived expression vectors, vims stocks, polypeptides, or peptides and at least one other agent. These compositions would be provided in a combined amount effective to inhibit infection or reactivation by a heφesvirus. This process may involve contacting the cells with a expression or viral constmct encoding
Us1.5, or with a Us1.5 polypeptide or peptide, and the agent(s) or factor(s) at the same time. This may be achieved by contacting the cell with a single composition or pharmacological formulation that includes both agents, or by contacting the cell with two distinct compositions or formulations, at the same time, wherein one composition includes a Usl .5-derived composition and the other includes the agent.
Alternatively, the gene therapy treatment may precede or follow the other agent treatment by intervals ranging from minutes to weeks. In embodiments where the other agent and expression constmct are applied separately to the cell, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agent and expression constmct would still be able to exert an advantageously combined effect on the cell. In such instances, it is contemplated that one would contact the cell with both modalities within about 12-24 hours of each other and, more preferably, within about 6-12 hours of each other, with a delay time of only about 12 hours being most preferred. In some situations, it may be desirable to extend the time period for treatment significantly, however, where several days (2, 3, 4, 5, 6 or
7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations. It is further understood that the expression vector discussed above can be substituted with a Usl .5 polypeptide or peptide-based composition.
It also is conceivable that more than one administration of either the expression constmct, or Us1.5 peptide, or the other agent will be desired. Various combinations may be employed, where the US 1.5-encoding constmct or U<J .5 peptide is "A" and the other agent is "B", as exemplified below:
A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B
A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A
A/A/A/B B/A/A/A A/B/A/A A/A/B/A A/B/B/B B/A/B/B B/B/A/B Other combinations are contemplated. Again, to achieve inhibition of viral infection, both agents are delivered to a cell in a combined amount effective to prevent viral progression.
Agents or factors suitable for use in a combined therapy are any chemical compound or treatment method with antiviral activity; therefore, the term "antiviral agent" that is used throughout this application refers to an agent with antiviral activity.
The skilled artisan is directed to "Remington's Pharmaceutical Sciences" 15th Edition. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.
II. Screening Methods
Two regions of Us1.5 have been identified as containing sites requiring post- translation modification to allow the infection cycle to progress. Because Us1.5 must be posttranslationally processed for viral progression, this modification can be monitored to identify substances capable of inhibiting a heφesvims infection.
A. Posttranslational modifications
The term "posttranslational modification" refers to a covalent alteration of a polypeptide or peptide amino acid sequence. Such modifications include phosphorylation, ubiquitination, nucleotidylylation, and sentrinization. In certain embodiments, a Us1.5 polypeptide containing a post-translation modification site for phosphorylation is envisioned. In those cases, a kinase will be used in the screening method of the present invention. A kinase is a type of protein that is capable of phosphorylating another amino acid segment; there are many protein kinases, and they consist of both cellular and viral kinases (for a review, see Taylor et al, 1990;
Kikkawa, et al, 1989). In heφesvims, kinases encoded by the viral genome include thymidine kinase, UL13, and Us3, and it has been previously shown that UL13 phosphorylates ICP22 (Purves and Roizman, 1992; Purves et al, 1993).
The screening method of the present invention includes a step in which a polypeptide that includes a Us1.5 phosphorylation site is exposed to a kinase under conditions that will allow the kinase to phosphorylate the polypeptide. The method can be employed using the UL13 kinase to phosphorylate Us1.5. UL13 has been characterized as mediating the posttranslational processing of ICP22. ICP22 was identified as a substrate for the UL13 protein kinase by the alteration of the phosphoprofile of ICP22 in cells infected with the UL13 deletion vims. The absence of the higher molecular weight species of ICP22 was observed along with the hypeφhosphorylated or increasing concentration of the Mr 70,000 phosphoprotein in cells infected with the UL13 deletion vims (Purves and Roizman, 1992). Early in infection, ICP22 is localized in discrete small nuclear stmctures. UL13 is a protein that is required to displace ICP22 from those stmctures so that it may colocalize with ICP4 and RNA polymerase during the onset of DNA synthesis. (Leopardi et al. 1996). The UL13 protein kinase is conserved among the alpha-, beta- and gammaheφesvimses and was identified by using sequence motifs diagnostic of conserved domains within protein kinases (Chee et. al. 1989, Smith and Smith, 1989).
Conditions that are appropriate to effect phosphorylation of a polypeptide are well known to those of skill in the art and can be found, by way of example, in Maniatis, Molecular Cloning : A Laboratory Manual. 2nd ed. (Cold Spring Harbor, N.Y., 1989). See also Dmkoku et aL, 1992; Ng et al, 1994; Ogle et al, 1997.
B. Determination of phosphorylation state
Candidate substances can be evaluated for their ability to inhibit heφesvims infection by comparing the phosphorylation state of Us1.5 in the presence of the candidate substance to its state in the absence of the candidate substance. The term "phosphorylation state" refers to the extent of phosphorylation of a given polypeptide.
Determining the phosphorylation state of Us1.5 can be accomplished by a number of assays, some of which are described herein and many of which are well known to those of skill in the art. These assays include alteration of polypeptide weight according to electrophoretic mobility in conjunction with Western blot analyses using an antibody that recognizes ICP22 or Us1.5. Alternatively, proteins recognizing only a subset of ICP22 or Us1.5 isoforms can be employed; such a protein would be capable of binding, for example, only the phosphorylated form of ICP22 or Us1.5, and thereby, measuring binding activity is a way of determining the phosphorylation state ofUs1.5.
1. Recognition by p60
In addition to antibodies, an example of a protein that selectively binds only the fast-migrating and undeφrocessed isoforms of ICP22 or Us1.5 is p60. While p60 will bind ICP22 lacking the carboxyl-terminal 24 amino acids, it will not bind ICP22 lacking the carboxyl-terminal 40 amino acids. p60 is a cellular protein characterized in a patent application filed on April 9, 1999 by Rennet Bmni, Beatrice Fineschi,
William O. Oggle, and Bernard Roizman and entitled "Cellular Protein p60 That Interacts with Heφes Vims Regulatory Proteins," which is incoφorated herein by reference.
2. Recognition by Antibodies
Other examples of proteins that will distinguish between phosphorylated forms of Us1.5 include antibodies. Means for preparing and characterizing antibodies are well known in the art (See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; incoφorated herein by reference). The methods for generating monoclonal antibodies (MAbs) generally begin along the same lines as those for preparing polyclonal antibodies. Briefly, a polyclonal antibody is prepared by immunizing an animal with an immunogenic composition in accordance with the present invention (either with or without prior immunotolerizing, depending on the antigen composition and protocol being employed) and collecting antisera from that immunized animal. A wide range of animal species can be used for the production of antisera. Typically the animal used for production of anti-antisera is a rabbit, a mouse, a rat, a hamster, a guinea pig or a goat. Because of the relatively large blood volume of rabbits, a rabbit is a prefeπed choice for production of polyclonal antibodies.
As is well known in the art, a given composition may vary in its immunogenicity. It is often necessary therefore to boost the host immune system, as may be achieved by coupling a peptide or polypeptide immunogen to a carrier. Exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine semm albumin (BSA). Other albumins such as ovalbumin, mouse semm albumin or rabbit semm albumin can also be used as carriers. Means for conjugating a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, μ-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimyde and bis-biazotized benzidine.
As is also well known in the art, the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants. Suitable adjuvants include all acceptable immunostimulatory compounds, such as cytokines, toxins or synthetic compositions.
Adjuvants that may be used include IL-1, IL-2, IL-4, IL-7, IL-12, γ-interferon, GMCSP, BCG, aluminum hydroxide, MDP compounds, such as thur-MDP and nor- MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A (MPL). RIBI, which contains three components extracted from bacteria, MPL, trehalose dimycolate (TDM) and cell wall skeleton (CWS) in a 2% squalene/Tween 80 emulsion. MHC antigens may even be used.
Exemplary, often prefeπed adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants and aluminum hydroxide adjuvant. In addition to adjuvants, it may be desirable to coadminister biologic response modifiers (BRM), which have been shown to upregulate T cell immunity or downregulate suppressor cell activity. Such BRMs include, but are not limited to, Cimetidine (CIM; 1200 mg/d) (Smith/Kline, PA); or low-dose Cyclophosphamide (CYP; 300 mg/m2) (Johnson/Mead, NJ) and cytokines such as γ-interferon, IL-2, or
IL-12 or genes encoding proteins involved in immune helper functions, such as CD80 (B7-l ) and CD86 (B7-2).
MAbs may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Patent 4,196,265, incoφorated herein by reference.
Typically, this technique involves immunizing a suitable animal with a selected immunogen composition, e.g., a purified or partially purified Us1.5 protein, polypeptide or peptide (or any Us1.5 composition, if used after tolerization to common antigens). The immunizing composition is administered in a manner effective to stimulate antibody producing cells.
In another embodiment, MAbs will be chimeric MAbs, including "humanized" MAbs. In such an approach, the chimeric MAb is engineered by cloning recombinant DNA containing the promoter, leader, and variable-region sequences from a mouse anti-Usl .5 producing cell and the constant-region exons from a human antibody gene.
That is, mouse complementary determining regions ("CDRs") are transfeπed from heavy and light V-chains of the mouse Ig into a human V-domain. This can be followed by the replacement of some human residues in the framework regions of their murine counteφarts.
Single chain antibodies that bind to Us1.5 phosphorylation sites or bind to Us1.5 and inhibit its phosphorylation are also contemplated by the present invention. A single chain antibody is a variation of an antibody that contains a variable region from a light chain and a variable region from a heavy chain, joined by a linker, such as a peptide linker. A single chain antibody (also refened to as a "single chain antibody fragment" or scFv) has one antigen binding site. However, multivalent single chain antibody can be created by linking single chain antibody fragments to one another. Methods of producing single chain antibodies are known to those of skill in the art and are described, for example in U.S. Patent 5,877,291 , which is herein incoφorated by reference.
a. Epitopic core sequences
Peptides corresponding to one or more antigenic determinants, or "epitopic core regions," of the Us1.5 polypeptide of the present invention can also be prepared.
Such peptides should generally be at least five or six amino acid residues in length, will preferably be about 10, 15, 20, 25 or about 30 amino acid residues in length, and may contain up to about 35-50 residues or so.
Synthetic peptides will generally be about 35 residues long, which is the approximate upper length limit of automated peptide synthesis machines, such as those available from Applied Biosystems (Foster City, CA). Longer peptides may also be prepared, e.g., by recombinant means.
U.S. Patent 4,554,101, (Hopp) incoφorated herein by reference, teaches the identification and preparation of epitopes from primary amino acid sequences on the basis of hydrophihcity. Through the methods disclosed in Hopp, one of skill in the art would be able to identify epitopes from within an amino acid sequence such as Us1.5 sequences disclosed herein in SEQ ID NOJ, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NOJ, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8.
Numerous scientific publications have also been devoted to the prediction of secondary stmcture, and to the identification of epitopes, from analyses of amino acid sequences (Chou and Fasman, 1974a,b; 1978a,b, 1979). Any of these may be used, if desired, to supplement the teachings of Hopp in U.S. Patent 4,554,101.
Moreover, computer programs are currently available to assist with predicting antigenic portions and epitopic core regions of proteins. Examples include those programs based upon the Jameson-Wolf analysis (Jameson and Wolf, 1988; Wolf et al, 1988), the program PepPlot® (Bmtlag et al, 1990; Weinberger et al, 1985), and other new programs for protein tertiary structure prediction (Fetrow and Bryant, 1993). Another commercially available software program capable of carrying out such analyses is MacVector (IBI, New Haven, CT).
In further embodiments, major antigenic determinants of a polypeptide may be identified by an empirical approach in which portions of the gene encoding the polypeptide are expressed in a recombinant host, and the resulting proteins tested for their ability to elicit an immune response. For example, PCR™ can be used to prepare a range of peptides lacking successively longer fragments of the C-terminus of the protein. The immunoactivity of each of these peptides is determined to identify those fragments or domains of the polypeptide that are immunodominant. Further studies in which only a small number of amino acids are removed at each iteration then allows the location of the antigenic determinants of the polypeptide to be more precisely determined.
Another method for determining the major antigenic determinants of a polypeptide is the SPOTs™ system (Genosys Biotechnologies, Inc., The Woodlands, TX). In this method, overlapping peptides are synthesized on a cellulose membrane, which following synthesis and deprotection, is screened using a polyclonal or monoclonal antibody. The antigenic determinants of the peptides which are initially identified can be further localized by performing subsequent syntheses of smaller peptides with larger overlaps, and by eventually replacing individual amino acids at each position along the immunoreactive peptide.
Once one or more such analyses are completed, polypeptides are prepared that contain at least the essential features of one or more antigenic determinants. The peptides are then employed in the generation of antisera against the polypeptide. Minigenes or gene fusions encoding these determinants can also be constmcted and inserted into expression vectors by standard methods, for example, using PCR™ cloning methodology.
The use of such small peptides for antibody generation or vaccination typically requires conjugation of the peptide to an immunogenic carrier protein, such as hepatitis B surface antigen, keyhole limpet hemocyanin or bovine semm albumin. Methods for performing this conjugation are well known in the art.
3. Detectable Labels In some examples, the screening method employs a detectable label that can be used to identify a polypeptide that is posttranslationally modified or to distinguish a polypeptide that is posttranslationally modified from one that is not so modified. "Detectable labels" are compounds or elements that can be detected due to their specific functional properties, or chemical characteristics, the use of which allows the antibody to which they are attached to be detected, and further quantified if desired.
Examples of a detectable label include the use of radioactive compounds such as [32P], [33S], or [12T], as well as the use of a fluorescent, light-sensitive, or photo-activatable compound such as fluorescein or fluorescein isothiocyanate, rhodamine, lissamine, indocyanine, luciferase, and Green Fluorescent Protein (GFP). A detectable label can be affixed to a polypeptide, and this can occur, for example, through the formation of a covalent bond or a coupling mechanism. The detectable label can be attached to the polypeptide before, during, or after it contacts a posttranslational modifier under conditions appropriate to effect modification. Therefore, it is also contemplated that the modification can be a detectable label. For example, radioactive [32P] can be used to phosphorylate the polypeptide that includes a Us1.5 phosphorylation site, such that the labelled polypeptide can be identified when it is exposed to film, such as XAR or XRP autoradiographic film.
In many of these assays, identifying the phosphorylation state of Us1.5 involves comparing the characteristics of Us1.5 in the presence and absence of a candidate inhibitor. To accomplish this, one would simply conduct parallel or otherwise comparatively controlled assays to identify a compound that inhibits phosphorylation or some other posttranslational modification of Us1.5. The candidate screening assay is quite simple to set up and perform. After obtaining a relatively purified preparation of Us1.5, either from native or recombinant sources, one will simply admix a candidate substance with the polypeptide under conditions appropriate to effect modification of the polypeptide.
C. Immunodetection Methods
In still further embodiments, the present invention concerns immunodetection methods for binding or quantifying or otherwise generally detecting biological components such as Us1.5 protein components. The Us1.5 antibodies prepared in accordance with the present invention may be employed to detect a particular isoform of a Us1.5 proteins, polypeptides or peptides, such as an isoform that is not phosphorylated, or one that is phosphorylated. As described throughout the present application, the use of Usl .5-specific antibodies is contemplated. The steps of various useful immunodetection methods have been described in the scientific literature, such as, e.g., Deshpande, In: Enzyme Immunoassays: From Concept to Product Development, Chapter 27, Chapman & Hall, New York, 1996, incoφorated herein by reference.
In general, the immunobinding methods include obtaining a sample containing a Us1.5 protein, polypeptide or peptide, and contacting the sample with a first anti- Us1.5 antibody in accordance with the present invention, as the case may be, under conditions effective to allow the formation of immunocomplexes.
These methods include methods for identifying Usl .5 proteins, polypeptides or peptides. In these instances, the antibody recognizes a subset of isoforms and other isoforms are not recognized. The antibody will preferably be linked to a solid support, such as in the form of a column matrix, and the sample containing the Us1.5 protein antigenic component will be applied to the immobilized antibody. The unwanted components will be washed from the column, leaving the antigen immunocomplexed to the immobilized antibody, w hich Ubl 5 protein antigen is then collected by removing the Usl 5 protein or peptide from the column
The lmmunobinding methods also include methods for detectmg or quantifying the amount of a particular isoform of Usl 5 protein reactive component in a sample, which methods require the detection or quantification of any immune complexes formed duπng the binding process Here, one would obtain a sample containing a Usl 5 protein or peptide, and contact the sample with an antibody against an isoform of Usl 5, and then detect or quantify the amount of immune complexes formed under the specific conditions
Contacting a mixture containing Usl 5 with the antibody under conditions effective and for a penod of time sufficient to allow the formation of immune complexes (pnmary immune complexes) is generally a matter of simply adding the antibody composition to the sample and incubating the mixture for a penod of trme lone enough for the antrbodies to form immune complexes with, i e , to bind to, any Usl 5 protein antigens present After this time, the sample-antibody composition, such as a tissue section, ELISA plate, dot blot or western blot, will generally be washed to remove any non-specifically bound antibody species, allowing only those antibodies specifically bound within the pnmary immune complexes to be detected
In general, the detection of immunocomplex formation is well known in the art and may be achieved through the application of numerous approaches These methods are generally based upon the detection of a label or marker, such as any of those radioactive, fluorescent, biological or enzymatic tags U S Patents concerning the use of such labels include 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149 and 4,366,241, each incoφorated herein by reference Of course, one may find additional advantages through the use of a secondary binding ligand such as a second antibody or a biotin avidin ligand binding arrangement, as is known in the art The Us1.5 antibody employed in the detection may itself be linked to a detectable label, wherein one would then simply detect this label, thereby allowing the amount of the primary immune complexes in the composition to be determined. Alternatively, the first antibody that becomes bound within the primary immune complexes may be detected by means of a second binding ligand that has binding affinity for the antibody. In these cases, the second binding ligand may be linked to a detectable label. The second binding ligand is itself often an antibody, which may thus be termed a "secondary" antibody. The primary immune complexes are contacted with the labeled, secondary binding ligand, or antibody, under conditions effective and for a period of time sufficient to allow the formation of secondary immune complexes. The secondary immune complexes are then generally washed to remove any non-specifically bound labeled secondary antibodies or ligands, and the remaining label in the secondary immune complexes is then detected.
Further methods include the detection of primary immune complexes by a two step approach. A second binding ligand, such as an antibody, that has binding affinity for the antibody is used to form secondary immune complexes, as described above. After washing, the secondary immune complexes are contacted with a third binding ligand or antibody that has binding affinity for the second antibody, again under conditions effective and for a period of time sufficient to allow the formation of immune complexes (tertiary immune complexes). The third ligand or antibody is linked to a detectable label, allowing detection of the tertiary immune complexes thus formed. This system may provide for signal amplification if this is desired.
Various methods of immunodetection are further described below.
1. ELISAs
As detailed above, immunoassays, in their most simple and direct sense, are binding assays. Certain preferred immunoassays are the various types of enzyme linked immunosorbent assays (ELISAs) and radioimmunoassays (RIA) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. However, it will be readily appreciated that detection is not limited to such techniques, and western blotting, dot blotting, FACS analyses, and the like may also be used.
In one exemplary ELISA, the Us1.5 antibodies of the invention are immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a test composition containing the Us1.5 protein antigen, such as a clinical sample, is added to the wells. After binding and washing to remove non-specifically bound immune complexes, the bound Us1.5 protein antigen may be detected. Detection is generally achieved by the addition of another anti- Us1.5 antibody that is linked to a detectable label. This type of ELISA is a simple "sandwich ELISA". Detection may also be achieved by the addition of a second anti- Us1.5 antibody, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label.
In another exemplary ELISA, the samples containing the Us1.5 protein antigen are immobilized onto the well surface and then contacted with the anti- Us1.5 antibodies of the invention. After binding and washing to remove non-specifically bound immune complexes, the bound anti- Us1.5 antibodies are detected. Where the initial anti- Us1.5 antibodies are linked to a detectable label, the immune complexes may be detected directly. Again, the immune complexes may be detected using a second antibody that has binding affinity for the first anti-Us1.5 antibody, with the second antibody being linked to a detectable label.
Another ELISA in which the Us1.5 proteins, polypeptides or peptides are immobilized, involves the use of antibody competition in the detection. In this ELISA, labeled antibodies against Us1.5 protein are added to the wells, allowed to bind, and detected by means of their label. The amount of Us1.5 protein antigen in an unknown sample is then determined by mixing the sample with the labeled antibodies against Us1.5 before or during incubation with coated wells. The presence of Us1.5 protein in the sample acts to reduce the amount of antibody against Us1.5 protein available for binding to the well and thus reduces the ultimate signal.
Irrespective of the format employed, ELISAs have certain features in common, such as coating, incubating or binding, washing to remove non-specifically bound species, and detecting the bound immune complexes. These are described below.
In coating a plate with either antigen or antibody, one will generally incubate the wells of the plate with a solution of the antigen or antibody, either overnight or for a specified period of h. The wells of the plate will then be washed to remove incompletely adsorbed material. Any remaining available surfaces of the wells are then "coated" with a nonspecific protein that is antigenically neutral with regard to the test antisera. These include bovine semm albumin (BSA), casein and solutions of milk powder. The coating allows for blocking of nonspecific adsoφtion sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.
In ELISAs, it is probably more customary to use a secondary or tertiary detection means rather than a direct procedure. Thus, after binding of a protein or antibody to the well, coating with a non-reactive material to reduce background, and washing to remove unbound material, the immobilizing surface is contacted with the biological sample to be tested under conditions effective to allow immune complex (antigen/antibody) formation. Detection of the immune complex then requires a labeled secondary binding ligand or antibody, or a secondary binding ligand or antibody in conjunction with a labeled tertiary antibody or third binding ligand.
"Under conditions effective to allow immune complex (antigen/antibody) formation" means that the conditions preferably include diluting the antigens and antibodies with solutions such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS)/Tween. These added agents also tend to assist in the reduction of nonspecific background. The "suitable" conditions also mean that the incubation is at a temperature and for a period of time sufficient to allow effective binding. Incubation steps are typically from about 1 to 2 to 4 h or so, at temperatures preferably on the order of 25°C to 27°C, or may be overnight at about 4°C or so.
Following all incubation steps in an ELISA, the contacted surface is washed so as to remove non-complexed material. A preferred washing procedure includes washing with a solution such as PBS/Tween, or borate buffer. Following the formation of specific immune complexes between the test sample and the originally bound material, and subsequent washing, the occurrence of even minute amounts of immune complexes may be determined.
To provide a detecting means, the second or third antibody will have an associated label to allow detection. Preferably, this will be an enzyme that will generate color development upon incubating with an appropriate chromogenic substrate. Thus, for example, one will desire to contact and incubate the first or second immune complex with a urease, glucose oxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibody for a period of time and under conditions that favor the development of further immune complex formation (e.g., incubation for
2 h at room temperature in a PBS-containing solution such as PBS-Tween).
After incubation with the labeled antibody, and subsequent to washing to remove unbound material, the amount of label is quantified, e.g., by incubation with a chromogenic substrate such as urea and bromocresol puφle or 2,2'-azino-di-(3-ethyl- benzthiazoline-6-sulfonic acid (ABTS) and H2O2, in the case of peroxidase as the enzyme label. Quantification is then achieved by measuring the degree of color generation, e.g., using a visible spectra spectrophotometer. 2. Immunohistochemistry
The antibodies of the present invention may also be used in conjunction with both fresh-frozen and formalin-fixed, paraffin-embedded tissue blocks prepared for study by immunohistochemistry (IHC). The method of preparing tissue blocks from these particulate specimens has been successfully used in previous IHC studies of various prognostic factors, and is well known to those of skill in the art (Brown et al, 1990; Abbondanzo et al, 1990; Alfred et al, 1990).
Briefly, frozen-sections may be prepared by rehydrating 50 ng of frozen "pulverized" tissue at room temperature in phosphate buffered saline (PBS) in small plastic capsules; pelleting the particles by centrifugation; resuspending them in a viscous embedding medium (OCT); inverting the capsule and pelleting again by centrifugation; snap-freezing in -70°C isopentane; cutting the plastic capsule and removing the frozen cylinder of tissue; securing the tissue cylinder on a cryostat microtome chuck; and cutting 25-50 serial sections.
Permanent-sections may be prepared by a similar method involving rehydration of the 50 mg sample in a plastic microfuge tube; pelleting; resuspending in 10% formalin for 4 h fixation; washing/pelleting; resuspending in warm 2.5%> agar; pelleting; cooling in ice water to harden the agar; removing the tissue/agar block from the tube; infiltrating and embedding the block in paraffin; and cutting up to 50 serial permanent sections.
III. Additional Uses for Methods and Composition Involving Us1.5-Derived Sequences
A. Kits
Kits of the present invention are kits that can be used either to identify a compound capable of inhibiting infection by heφesvims or to effect a treatment method comprising Us1.5 protein, polypeptide, peptide, inhibitor, gene, vector or other UJ .5 effector. Such kits will generally contain, in suitable container means, a pharmaceutically acceptable formulation of a Us1.5 protein, polypeptide, peptide, domain, inhibitor, or a gene or vector expressing any of the foregoing in a pharmaceutically acceptable formulation. In the screening kit of the present invention, other components such as a kinase and reagents used in a kinase reaction may also be provided. The kit may have a single container means, or it may have distinct container means for each compound.
When the components of the kit are provided in one or more liquid solutions, the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly prefeπed. The Us1.5 compositions may also be formulated into a syringeable composition, particularly if the kit is for therapeutic puφoses. In that case, the container means may itself be a syringe, pipette, or other such like apparatus, from which the formulation may be applied to an infected area of the body, injected into a subject, or even applied to and mixed with the other components of the kit.
However, the components of the kit may be provided as dried powder(s). When reagents or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means.
The container means will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which the Us1.5 protein, gene or inhibitory formulation are placed, preferably, suitably allocated. The kits may also comprise a second container means for containing a sterile, pharmaceutically acceptable buffer or other diluent.
The kits of the present invention will also typically include a means for containing the vials in close confinement for commercial sale, such as, e.g., injection or blow-molded plastic containers into which the desired vials are retained. Iπespective of the number or type of containers, the kits of the invention may also comprise, or be packaged with, an instmment for assisting with the injection/administration or placement of the ultimate Us1.5 protein or gene composition within the body of a subject. Such an instmment may be a syringe, pipette, forceps, or any such medically approved delivery vehicle.
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
IV. Examples
EXAMPLE I: Materials and Methods
A. Cells and Viruses
Vero and HEp-2 cells were obtained from the American Type Culture Collection (Rockville, MD). Rabbit skin cells were originally obtained from John McClaren. HSV-l(F) is the prototype HSV-1 strain used in this laboratory (Ejercito et al, 1968). The constructions of HSV-1 recombinant vimses R325, R7356
(AUL13) and R7905 was reported elsewhere (Kawaguchi et al, 1997; Post and Roizman, 1981 ; Prod'hon et al, 1996).
B. Plasmids To constmct pRB5210 the plasmid pRB138 containing BamHI N (131,399 basepairs (bp) to 136,289 bp of the HSV-1 consensus sequence) was digested with EcoRI and BamHI releasing a BamHI N fragment that was tmncated by 121 bp. This fragment was cloned into the EcoRV and BamHI sites of the pucl9 vector.
To constmct pRB5212 a 1 10 bp PCR™ product was generated from pRB5210 with the aid of the Pfu polymerase (Stratagene, La Jolla, CA) and the primers (PI )
GGA ACG TCC TCG TCG AGG CGA CCG and (P2) GCC TGG GGA AAT GTC GGC CGT CCA GAA AAC GTC. PI included in the final product the EcoNI site 100 bp upstream of the ICP22 open reading frame whereas P2 replaced the initiator methionine codon of ICP22 with an Eagl site. The PCR™ product was then digested with EcoNI and Eagl and subcloned into pRB5210, which has also been digested with
EcoNI and Eagl to remove the ICP22 open reading frame. In the resulting plasmids, pRB5212 the sequences encoding ICP22 and Us1.5 from the initiation methionine to the carboxyl-terminal stop codon had been deleted leaving only a unique Eagl restriction endonuclease cleavage site.
To constmct pRB5214, a 1.2 kb PCR™ product was generated from pRB5210 with the Pfu polymerase (Stratagene, La Jolla, CA) and the primers (P3) GAC GTT TTC TGG CGG CCG ATG GCC GAC and (P4) GAC GCT GGG ACA AAC GCT TTG ATT TTG GTC. P3 inserted an Eagl site adjacent to the initiator methionine codon of ICP22 and the primer P4 represents a sequence located 50 bp downstream from the carboxyl-terminal Eagl site of the carboxyl-terminal stop codon of the ICP22 open reading frame. The PCR™ product was then digested with Eagl and subcloned into the Eagl site of pRB5212. The resulting plasmid, pRB5214, contained ICP22 and Us1.5 with an Eagl site just preceding the initiation methionine.
To constmct pRB5215 a 870 bp PCR™ product was generated from pRB5212 with Pfu polymerase (Stratagene, La Jolla, CA) and the primer (P5) ACG CAG CCC CGG GCC CCC CGG CCG TCG GCC and P4. P5 created an Eagl site 25 bp upstream of the Us1.5 initiation methionine (171) and the primer P4. The PCR™ product was then digested with Eagl and subcloned into pRB5212. The subsequent plasmid pRB5215 contained a Usl .5 open reading frame driven by the α22 promoter. To constmct pRB458 with EcoRI site of plasmid pucl9 was destroyed with the T4 polymerase (Stratagene, La Jolla, CA).
To constmct pRB5211 the 3.9 kb Sacl(129,088-133,046 bp) fragment from
Hindlll M (126,526-133,466 bp) fragment of HSV-l(F) was subcloned from pRB201 and cloned into pRB458.
To constmct pRB5213 the 3.2 kilobases (kb) Sacl-Xbal fragment (nucleotides 133,049 to 136,289 of BamHI N) from pRB5212 was cloned into pRB521 1. The resulting plasmid pRB5213 extended BamHI N by 2 kb to nucleotide 129,088.
To constmct pRB5216 a 740 bp PCR™ product was generated from pRB5210 with the Pfu polymerase and the primers P5 and (P7) GGC CCG GGC CGTTCC ACG GAG CTG GTA TC. P7 inserted an Eagl site 120 bp upstream from the stop codon of α22/UsJ5. The PCR™ product was digested with Eagl and Drain and subcloned into pRB5210 digested with Dralll and Eagl. In the resulting plasmid, pRB5216, both ICP22 and Us1.5 open reading frames were tmncated by 40 3' codons.
To constmct pRB5217 a 1.2 kb PCR™ product was generated from pRB5210 with the Pfu polymerase and the primers P3 and (P8) ATA GGG CGG CCG GGTGGA GAA GCG CAT TTT. P8 inserted an Eagl site 30 bp upstream from the carboxyl-terminal stop codon of ICP22 and USJ5. The PCR™ product was digested with Eagl and subcloned into pRB5212 digested with Eagl. In the resulting plasmid, pRB5217, both ICP22 and Us1.5 open reading frames were tmncated by 10 3' codons.
To constmct pRB5218 a 1.2 kb PCR™ product was generated from pRB5210 with the Pfu polymerase and the primer P3 and (P9) GCA GCC CGG CCG ACA
CTT GCG GTC TTC TGC. P9 inserted an Eagl site 66 bp upstream from the stop codon of ICP22 and USJ5. The PCR™ product was digested with Eagl and subcloned into Eagl digested pRB5212. In the resulting plasmid pRB5218 both ICP22 and Us1.5 open reading frames are tmncated by 22 3' codons.
To constmct pRB5219 a 1.3 kb PCR™ product was generated from pRB5210 with the Pfu polymerase and the primer (P10) CCG GTA CCTTTTCTG GAT GGC
CGA CAT TTC CCC AGG and (PI 1 ) GCC GGTACC ACG CTG GGA CAA ACG CTTTGA TTTTGG. P10 inserted an Kpnl site adjacent to initiator methionine codon of ICP22 whereas PI 1 placed a Kpnl site downstream and adjacent to the stop codon of ICP22 and Us1.5. The PCR™ product was digested with Kpnl and subcloned into the Kpnl site of pRB4297 downstream of the βγ promoter. The αβγ promoter was constructed by cloning the -12 to -520 bp upstream promoter region of α4 in front of a polylinker. 200 bp of the 240 bp γl promoter of UL19(VPS) was cloned in at the -12 position. This created a promoter that allows the expression of an inserted gene throughout the heφes viral infection cycle. The resulting plasmid pRB5219 contained the open reading frames of the α22 gene driven by the αβγ promoter.
To constmct pRB5243 a 1.2 kb PCR™ product was generated from pRB5210 with the Pfu polymerase and the primer P3 and (PI 2) GGTGGA CGGCCG CAT TTTCCG GCA GCC GTC. P12 inserted an Eagl site 45 bp upstream from the stop codon of ICP22 and Us.15. The PCR™ product was digested with Eagl and subeloned into Eagl digested pRB5212. In the resulting plasmid pRB5243 both ICP22 and Us1.5 open reading frames are tmncated by 15 3' codons.
To constmct pRB5244 a 1.2 kb PCR™ product was generated from pRB5210 with the Pfu polymerase and the primer P3 and (PI 3) GCG CAT CGGCCG GCA
GCC GTC CAG ACA CTT GC. PI 3 inserted an Eagl site 54 bp upstream from the stop codon of ICP22 and Us.15. The PCR™ product was digested with Eagl and subcloned into Eagl digested pRB5212. In the resulting plasmid pRB5244 both ICP22 and Usl .5 open reading frames are tmncated by 18 3' codons. To constmct pRB5251 a 1 J kb PCR™ product was generated from pRB5210 with the Pfu polymerase and the primer P3 and (PI 4) TCT GAG CGG CCG TCC GAT ACA GCC TTG GAG TCT. PI 4 inserted an Eagl site 1 15 bp downstream from the initiation methionine (1) of IPC22 and USJ5. The PCR™ product was digested with Eagl and subcloned into Eagl digested pRB5212. In the resulting plasmid pRB5251 the ICP22 open reading frame is tmncated by 47 5' codons, the first available codon for translational initiation is methionine (90).
To constmct pRB5272, a 1270 bp PCR product was generated from pRB5210 with the aid of the Pfu polymerase (Stratagene, La Jolla, CA) and the primers
(WO9713.1 ) GGG GAA TTC CGG CCG ATG GCC GAC ATT TCC CCA GGC G and (WO9714.1) CCG GGA TCC CGG CCG GAG AAA CGT GTC GCT GCA. The primer WO9713.1 inserted EcoRI and Eagl sites adjacent to the initiator methionine codon of ICP22, and the primer WO9714.1 inserts a BamHI site adjacent to the existing Eagl site at the stop codon of ICP22/US1.5. The PCR product was then digested with EcoRI and BamHI and subcloned into the EcoRI and BamHI sites within the polylinker of pUC19. The resulting plasmid pRB5272 was then used as the template for mutagenesis within the ICP22/US1.5 open reading frame.
To constmct pRB5274, the mutagenized plasmid pRB5273 was digested with the restriction enzyme Eagl to release a 1.2 kb product and subcloned into the Eagl site of pRB5212. The resulting plasmid, pRB5274, contains the complete open reading frames of ICP22/US1.5.
To constmct R7827 (Threonine 300 to Alanine and Serine 301 to Glycine) the plasmid pRB5274 was cotransfected with R7802 recombinant viral DNA into rabbit skin cells. Several plaques were isolated, and the stmcture of the recombinant vims R7827 was verified by hybridization of electrophoretically separated BamHI and Bgl II digested viral DNA with nick-translated pRB5210 (BamHI N fragment).
C. Cosmids The set of cosmids used in this study were derived from HSV-l(F) DNA as described elsewhere (Chang et al, 1997: Kawaguchi et al. 1997) and is illustrated in FIG. 1A, lines 2 thm 6. The sequences contained in the cosmid set were as follows: pBC1004, nucleotides 133,052 to 17,029; pBC1006, the nucleotides 2,945 to 45,035; pBC1007 nucleotides 77,933 to 1 16,016; pBC1008, nucleotides 106,750 to 142,759; and pBC1014, nucleotides 40,617 to 80,454. Cosmid pBClOlό containing the nucleotides 1 10,095 to 131,534 was constmcted by digesting pBC1008 with EcoRI, followed by gel purification. The desired DNA fragment was then ligated into the multicloning site of the SuperCosl cosmid vector (Strategene, La Jolla, CA) and packaged into lambda phage with the Gigapack XLII (Stratagene, La Jolla CA) packaging extract according to the manufacturer's instmctions.
D. Construction of recombinant viruses
The constmction of recombinant virus R7802 (Δα22/ΔUs1.5) involved the following steps: (i) The cosmid pBC1008 was digested with the restriction enzyme
EcoRI to create a gap in the cosmid within the α22/Us1.5 region (FIG. IB, lines 3 and 4) creating cosmid pBClOlό. (ii) The cosmids (pBC1006, ρBC1014, pBC1007, pBClOl ό, and pBC1004) were digested with the restriction enzyme Pad to release the HSV-1 sequences from the cosmid vector, (iii) A bridging plasmid (pBR5213) from which the entire α22/Us1.5 open reading frame had been deleted was constmcted. This plasmid has an 2.5 kb overlap with pBClOlό and a 1 kb overlap with the cosmid pBC1004 (FIG. IB, lines 3, 4, and 5). (iv) A second plasmid was constmcted (pBR5219) that contained the open reading frame for α22/Us1.5 driven by the recombinant heφes vims αβγ promoter but lacking sequences overlapping within the cosmid set. (v) The modified cosmid set in amounts of 1 μg each (pBC1004, pBC1006, pBC1007, pBC1014, and pBClOlό), the linearized bridging plasmid pBR5213 in amounts ranging from 0 to 0.8 μg lacking the α22/Us1.5 gene, and the α22/Us1.5 α expression plasmid (pBR5219, 0.1 μg) were transfected into Vero cells with Lipofectamine (Gibco BRL, Gaithersburg MD) according to the manufacturer's instmctions. The progeny vims were designated R7802 (Δα22/ΔUsl .5). In addition, the cells were transfected with the cosmid set (pBC1006, pBC1014, pBC1007, pBC1004, and pBClOlό) without the bridging plasmid (pBR5213) to yield R7805 (Δα22NT/Us 5). (FIG. 2, line 4).
The genotypes of the wild type parent HSV-1 (F) and ofthe recombinant vimses R7905 (HSV-1 (F) derived from the original cosmid set, R7802
(Δα22/ΔUs1.5), and R7805 (Δα22/ΔUs1.5 ) were analyzed as follows: viral DNAs were isolated and digested with BamHI, electrophoretically separated in agarose gels, transferred to a nylon membrane, and hybridized to 32P-labeled BamHI N (pBR5210) as described above. The probe pBR5210 (BamHI N) hybridized to the 4.9-kb BamHI N fragment of wild type HSV- 1(F) and R7905 (HSV- 1(F)) (FIG. 3, lanes 1 and 2).
The probe hybridized to a 3.6-kb BamHI fragment corresponding to the predicted size of BamHI N in R7802 (Δα22/ΔUs1.5) (FIG. 3, lane 3). The probe also hybridized to a 3.4-kb fragment corresponding to the predicted size of BamHI N in R7805 (Δα22NT/ΔUs1.5) (FIG. 3, lane 4). The probe hybridizes to the BamHI Z fragment since this fragment contains a potion of the inverted repeat common with BamHI N.
These results are consistent with the predicted size of the BamHI N region deleted for the open reading frame of α22/Us1.5 (R7802) and the deletion of the promoter elements and amino-terminal of α22 (R7805).
To construct R7808 (Δα22/ΔUs1.5) the predicted open reading frame of Us1.5, that is, the sequence encoding codons 171 to 420 of ICP22, was PCR™-amplified and cloned into plasmid pRB5212. The new plasmid, pRB5215, contained an additional Eagl restriction site at the beginning of the Us1.5 open reading frame (FIG. 2, line 6). This plasmid was cotransfected with the R7802 (Δα22/ΔUs1.5) recombinant viral DNA into rabbit skin cells. Several plaques were isolated, and the stmcture of the recombinant vims R7808 (Δα22/ΔUs1.5αp) was verified by hybridization of electrophoretically separated BamHI digested viral DNA with nick-translated pRB5210 (BamHI N fragment).
To constmct R7815 (Δα22NTUs1.5)[Δ47a.a.] the predicted open reading frame of ICP22/US1.5, that is, the sequence encoding codons 47 to 420 of ICP22, was PCR™-amplified and cloned into plasmid pRB5251. This plasmid was cotransfected with the R7802 (Δα22/ΔUs1.5) recombinant viral DNA into rabbit skin cells. Several plaques were isolated, and the stmcture of the recombinant vims R7815 (Δα22 Us1.5)[Δ47a.a.] (FIG. 7) was verified by hybridization of electrophoretically separated BamHI digested viral DNA with nick-translated pRB5210 (BamHI N fragment).
E. Construction of ICP22/US1.5 carboxyl-terminal truncation viruses
In order to investigate the function of the carboxyl-terminal domain of ICP22/U 1.5 a series of recombinant vimses lacking the terminal 10, 15, 18, 22, or 40 codons of the genes, respectively, were constmcted. To constmct the recombinant vims R7819, which lacks the 3' terminal 10 codons of the α221Us1.5 open reading frames (FIG. 4, panel A: line 2 and panel B: line 2), the ICP221US1.5 gene was amplified using PCR™. This amplified product extended from the initiation methionine codon to codon 410 of α22 and contained a diagnostic Eagl restriction endonuclease site at the amino terminus of the α22/Us1.5 genes. The PCR™ product was cloned into plasmid pRB5212 to create pRB5217. This plasmid (pRB5217) was cotransfected with R7802 (Δα22/ΔUs1.5) viral DNA into rabbit skin cells. Several plaques were isolated, and the stmcture of the mutant vims R7819 was verified by hybridization of electrophoretically separated BamHI digested viral DNA with nick-translated pRB5210.
In a similar fashion, co-transfection of R7802 DNA with plasmid pRB5216 yielded R7810 (Δα22cτUs1.5cτ)[Δ40a.a.], with plasmid pRB5243 yielded R7822 (Δα22cτ/Us1.5cτ)[Δ15a.a.], with plasmid pRB5244 yielded R7823 (Δα22cτ/Us1.5cτ)
[Δ18a.a.], and with plasmid pRB5218 yielded R7820 (Δα22rτ/Us1.5cτ)[Δ20a.a.],
(FIG. 4).
F. Repair of sequences deleted from the viral genomes The deletion in R7802 (Δα22/ΔUs1.5) was repaired to yield the repair vims
R7804 (FIG. 2, line 3). The open reading frame of 22/Us1.5 was PCR™ amplified and cloned into plasmid (pRB5212) to create pRB5214 which was identical to BamHI N except for the presence of an additional Eagl restriction site at the beginning of the a22/Us1.5 open reading frame (FIG. 2, compare lines 2 and 3). This plasmid (pRB5214) was cotransfected with viral DNA of R7802 (Δα22/ΔUs1.5) into rabbit skin cells. The selection for the recombinant vims took advantage of the observation that R7802 viral DNA did not form plaques in transfected rabbit skin cells. Therefore, the presence of plaques on this cell line would signal the presence of a recombinant vims. Several plaques were isolated, and the stmcture of the recombinant R7804 vims was verified by hybridization of electrophoretically separated BamHI digested viral DNA with nick-translated pRB5210 (BamHI N fragment).
The recombinant vimses R7805 (Δα22 T/UsL5) and R7808 (Δ22 1ΛJS1.5) were repaired by blind selection on rabbit skin cells to yield R7806 (α22/Us1.5) and
R7828 (α22/Us1.5) respectively (FIG. 2 lines 6 and 7). In a similar fashion, R7810 (Δα22cτ/Us1.5cτ)[Δ40a.a.] was repaired with plasmid pRB5212 to yield R7821
(α22/Us1.5) (FIG. 4, line 7).
G. Antibodies
The Usl 1 , ICPO (HI 083) mouse monoclonal antibodies, the rabbit polyclonal antibody R77 amino-terminal ICP22, W2 against the carboxyl-terminal of ICP22 and the rabbit polyclonal Wl against UL38 were described previously (Ackermann et al, 1985; Kemp and Latchman, 1988; Roller and Roizman, 1992; Ward et al, 1996). Goat anti-rabbit or anti-mouse alkaline phosphatase-conjugated secondary antibody was purchased from Bio-Rad.
H. Electrophoretic separation and immunoblotting of viral proteins
Replicate cultures of Vero or rabbit skin cells in 25 cm2 flasks were exposed to
10 plaque forming units (PFU) of the appropriate vims per cell. The cells were maintained in medium 199V consisting of a mixture of 199 supplemented with 1% calf semm. At 18 h after infection the cells were rinsed and scraped into 1 ml of ice-cold phosphate-buffered saline lacking Ca2+ and Mg2+ (PBS-A), centrifuged for 5 min in a microcentrifuge at 4°C, and resuspended in 350 μl of PBS-A*[PBS-A with OJ mM TPCK (tosylsulfonyl phenlylalanyl chlromethyl ketone), OJ mM TLCK (tosyl-L-phenylalanine chloromethyl ketone), OJ mM PMSF (phenylmethylsulfonyl fluoride), 1.0% (v/v) Nonidet-P40, 40 mM B-glycerophosphate, and 1.0 % (w/v) sodium deoxycholate]. The lysates were sonicated briefly and frozen in aliquots at
-70°C. Aliquots were thawed on wet ice and 20 μl of dismption buffer (12.5 mM Tris-HCL [pH 6.8], 0.5% sodium dodecyl sulfate, 2.5% glycerol, 5% β-mercaptoethanol) were added to 40 μl of infected cell lysate and boiled for 5 min. The solubilized proteins were subjected to electrophoresis in denaturing polyacrylamide gels (60 μl per lane), transferred to a nitrocellulose membrane
(Schleicher & Schuell, Keene, NH), and reacted with the appropriate antibody. The bound antibody was visualized with antibody conjugated to alkaline phosphatase (Bio-Rad) and visualized according to the manufacturer's instmctions.
Alternatively, Vero cell lysate equivalent to one half of a 25-cm2 tissue culture flask were harvested in lysis buffer, chilled on wet ice for 30 min, and cleared by centrifugation in a microfuge for 20 min. at 4oC. The supernatant fluid was then precleared by mixing with 30 μl of a 50%) slurry of protein A-conjugated Sepharose beads (Sigma) for 1 hr. with mixing and reacted with a 1 :100 dilution of UL13 (W3) or ICPO (HI 083) antisemm overnight at 4°C (or any other appropriate substrate Ab).
The immune complex was harvested with protein A-Sepharose beads, repeatedly rinsed with lysis buffer and either eluted from the Sepharose beads by boiling for five minutes in dismption buffer and electrophoretically separated on denaturing polyacrylamide gels or used for in vitro kinase assays as described below.
I. Analyses of viral DNA by hybridization
Cytoplasmic DNAs from infected cells were harvested by resuspending 2 roller bottle cultures (approximately 4x10s cells) in 20 ml of 150 mM NaCl , 10 mM Tris-HCL pH 1.4, 1.5 mM MgCl, with NP-40 added to a final concentration of 0J%> (v/v). The nuclei were pelleted by centrifugation at 2,500 φm for 5 min in a
Beckman (Model TJ-6) table top centrifuge. The supernatant fluid containing cytoplasmic virions was collected and sodium dodecyl sulfate, EDTA, and β-mercaptoethanol were added to a final concentration of 0.2%, 5 mM, and 50 mM respectively. Phenol-chloroform extraction was performed twice followed by a chloroform extraction. Viral DNA was then precipitated with 2 volumes of 100% ethanol and centrifuged at 10,000 φm in a SS-34 rotor. Viral DNA pellet was resuspended in 1 ml of sterile H2O. RNase A was added to a final concentration of 20 μg/ml and incubated for 15 min at 37°C, then centrifuged through a linear 5-20% potassium acetate gradient in 10 mM Tris-HCI pH 8.0, 5 mM EDTA in a SW41 rotor at 40,000 φm for 3.5 h at 20°C. The pellet was gently rinsed once with H2O, resuspended in 0.4 ml of H2O, precipitated by the addition of 2 volumes of 100% ethanol, solubilized, digested with BamHI, electrophoretically separated on an 0.8% agarose gel and transferred to a nylon membrane (Bio-Rad Laboratories, Hercules, CA). The hybridization and membrane-stripping procedures were as recommended by the manufacturer. The plasmid pRB5210 was used to make ?2P-dCTP labeled probe using a Nick Translation Kit (Promega, Madison Wl).
J. Cell fractionation
HEp-2 cells grown in 25-cm2 flask cultures were infected with 10 PFU of HSV-l(F), R7805 (Δα22NT/Us1.5), R7808 (Δα22NT Us1.5α22), and R7810 (Δα22rτ/ΔUs1.5cτ) per cell. At 18 h after infection, the cells were washed with 5 mis
PBS(A), and then scraped into 1 ml PBS(A) and resuspended into 100 μl buffer A (50mM Tris-HCI [pH 7.5], 5 mM MgCl2, 1 mM PMSF, 0.1 mM TLCK, 0.1 mM TPCK). The cells were lysed by addition of 4 μl of 10%> Nonident P-40 and stored at 25°C for 5 min. The nuclei were separated from the cytoplasm by centrifugation in a microcentrifuge. The supernatant (cytoplasmic fraction) was removed and 50 pi of dismption buffer added. The nuclei were resuspended in 75 μl PBS(A) and 50 μl of dismption buffer.
K. Mutagenesis: The amino acids Threonine 300 and Serine 301 of ICP22/US1.5 were mutated to Alanine and Glycine, respectively, by performing mutagenesis using the QuickChangeTM site-directed mutagenesis kit (Stratagene, La Jolla, CA). Briefly, two complementary oligonucleotides were synthesized to yield primers (WO9843) TCT CAG CGC GGC AGG CGA TGA TGA GAT CTC and (WO9844) GAG ATC TCA TCA TCG CCT GCC GCG CTG AGA. These primers also incoφorate a diagnostic Bglll site that alters a single base causing a silent mutation. In a final volume of 50 μl, 25 ng of pRB5272, 125 ng each of WO9843 and WO9844, dNTP mix and Pfu polymerase were combined in a 0.5 ml thin-walled microcentrifuge tube. The cycling parameters were as follows: 1 cycle at 95 °C 30 seconds, 18 cycles of 95 °C for 30 seconds, 55 °C for 1 minute and 68 °C for 8 minutes. The reaction was then digested with the restriction enzyme Dpn I at 37 °C for 1 hour. E. coli XL-1 Blue electrocompetent cells were then transformed with 1 μl of the reaction mixture. Mini- preps were performed and the colonies were then screened using the diagnostic Bglll site. The plasmid was then sequenced to verify the presence of these mutations and no other. The resulting plasmid pRB5273 contains the open reading frame of ICP22/US1.5 in which Threonine 300 has been changed to Alanine and Serine 301 has been changed to Glycine.
L. Kinase Assays
Unlabeled mock-infected or infected Vero cell lysates were reacted with 10 μl of rabbit preimmune semm for 2 hrs and then with protein A-Sepharose for 1.5 hrs.
The Fc receptor-depleted lysates were then used for immunoprecipitation with the UL13 (W3) or ICPO (HI 083) antisemm as described above. The immune complexes harvested on the protein A-conjugated Sepharose beads were rinsed twice with kinase buffer [50 mM Tris-HCI (pH 8.0), 50 mM MgCl2, 0.1% NP40, and 1 mM DTT]. Protein kinase assays were done by incubating the immune complexes with 70 μl protein kinase buffer supplemented with 100 μM ATP and 65 μCi [32P]ATP at 37°C for 30 minutes. The beads were then rinsed with lysis buffer, resuspended in dismption buffer and boiled for 5 minutes. The released proteins in the supernatant were electrophoretically separated on denaturing polyacrylamide gels.
EXAMPLE 2: Construction ofthe ___22/t_Uγ1.5 and UJ.5 Recombinant Viruses In order to conduct a comprehensive analysis of ICP22/US 1.5 it was necessary to constmct a vims from which the entire open reading frame of α22/Us1.5 had been deleted. To delete the entire α22 Us1.5 domain, the inventors used a modified cosmid system as described previously (Chang et al, 1997; Kawaguchi et al, 1997) and in the material and methods. This cosmid set is illustrated in FIG. 1A, lines 2 thm 6. Constmction of the Δα22/ΔUs1.5 cosmid vims involved the following steps, (i) The cosmid pBC1008 was digested with the restriction enzyme EcoRI to create a gap within the α22/Us1.5 region (FIG. IB, lines 3 and 4) to create the cosmid pBClOlό. (ii) A bridging plasmid (pBR5213) from which the entire α22/Us1.5 open reading frame had been deleted was constmcted. (FIG. IB, line 5). (hi) A second plasmid was constmcted (pBR5219) which contained the open reading frame for α22/Us1.5 driven by the recombinant heφes vims apy promoter but without any overlapping sequences within the cosmid set. (iv) The modified cosmid set (pBC1004, pBClOOό, pBC1007, pBC1014, and pBClOlό), the bridging plasmid pBR5213 lacking the α22/Us1.5 genes and the α22/Us1.5 expression plasmid (pBR5219) were transfected into Vero cells to yield R7802 (FIG. 2, line 2). In addition, the cells were transfected with the cosmid set (pBClOOό, pBC1014, pBC1007, pBC1004, and pBClOlό) without the bridging plasmid (pBR5213) to yield R7805 (FIG. 2, line 4). The subsequent plaques were isolated and the genotypes analyzed.
The genotypes of the wild type parent HSV- 1(F) and of the recombinant vimses R7905 (HSV- 1(F)) derived from the original cosmid set, R7802 (Δα22/ΔUs1.5), and R7805 (Δα22/Us1.5) were analyzed as follows: Viral DNAs were isolated and digested with BamHI, electrophoretically separated in agarose gels, transfeπed to a nylon membrane, and hybridized to 32P-labeled BamHI N (pBR5210) as described in Materials and Methods. The probe hybridized to the 4.9-kb BamHI N fragment of wild type HSV-l(F) and R7905 (HSV-I(F)) (FIG. 3, lanes 1 and 2). The probe hybridized to a 3.6-kb BamHI fragment corresponding to the predicted size of
BamHI N in R7802 (Aa22/ΔUs1.5) (FIG. 3, lane 3) and to a 3.4-kb fragment corresponding to the predicted size of BamHI N in R7805 (Δα22 Us1.5) (FIG. 3, lane 4). These results are consistent with the predicted size ofthe BamHI N region deleted for the open reading frame of α22/Us1.5 (R7802) and the deletion of the promoter elements and amino-terminus of α22 (R7805).
The recombinant vims R7808 (Δα22/Us1.5) and the repair vimses of R7802 (Δα22/ΔUs1.5), R7805 (Δα22/Us1.5) and R7808 (Δα22/Us1.5) were constmcted by blind selection on rabbit skin cells to yield R7804 (α22/Us1.5), R7806 (α22/Us1.5) and R7828 (α22/Us1.5) (FIG. 2, lines 3, 5, 6 and 7).
EXAMPLE 3: Biologic Properties ofR7802 (Aa22/AU .5) and R7805 (Aa22/UJ5) Of the various recombinants produced in these studies, two are of key importance. These are R7802 and R7805. R7802 lacked the coding domains of both α22 and Us1.5 genes (FIG. 2, line 2), whereas R7805 contained the coding sequences of Us1.5, but not the 5' sequence that code the amino terminus of ICP22 (FIG. 2, line 4). In the studies, R7802 could not be differentiated from R325 with respect to biologic properties. Thus it replicated in primate cell lines (Vero, HEp-2) and to a lesser extent in rabbit skin cells consistent with the studies reported earlier on R325 (Purves et al, 1993; Sears et al, 1985) and the report on a homolog of R7802 described by Poffenberger et al, 1993; Poffenberger et al, 1994. One remarkable observation with significant consequences was that R7802 did not yield plaques on transfection of rabbit skin cells. Plaques were formed however, if a plasmid expressing ICP22 was co-transfected with the cosmid set, but under conditions in which ICP22 could not recombine with the cosmids to form an infectious vims. The failure of transfected R7802 DNA to yield plaques was of special significance since virtually any plasmid containing coding sequences of ICP22 or Us1.5 genes rescued the capacity to make plaques and recombinant vimses could be easily selected on the basis of that property. R7805 was tested for its ability to cause morbidity or mortality upon intracerebral inoculation in mice. The PFU/LD50 were 5.6χl04 for R7805, 3.4x10' for R7806 in which the α22/Us1.5 lesions were repaired as compared with l.lxl 02 for the wild-type parent, HSV- 1(F).
EXAMPLE 4: The Posttranslational Modification ofUs1.5 Is Determined bv the U 3 Protein
Kinase
Earlier studies (Purves and Roizman, 1992) have shown that UL13 mediates the phosphorylation and posttranslational processing of ICP22. A central question was whether the UL13 protein kinase also mediates the posttranslational processing of
Us1.5 protein. In this study, replicate cultures of Vero or rabbit skin cells were exposed to 10 PFU of wildtype (HSV-1 (F)) or ΔUL13 (R7356) vimses per cell. At 4,
8, 12, and 24 h after infection the cells were harvested, solubilized, electrophoretically separated on a denaturing polyacrylamide gel, electrically transferred to a nitrocellulose sheet, and reacted with a polyclonal antibody to α22/Us1.5 (FIG. 5).
The antibody to the carboxyl-terminal 138 amino acids of ICP22/US1.5 reacted with several bands of ICP22 (Mr 67,000 to 72,000) and Us1.5 (Mr 35,000 to 48,000) corresponding to the isoforms resulting from posttranslational processing of the two proteins (FIG. 5, lanes 7 and 8). ICP22 and Us1.5 proteins were not processed at 4 h after infection and exhibited a reduced number of isoforms in lysates of cells infected with the ΔU, 13 (R7356) vims. The decrease in the number of isoforms of Us1.5 paralleled the decrease in the number of isoforms of ICP22 (FIG. 5, compare lane 8 with 9). These results indicate that the UL13 protein kinase mediated some of the posttranslational processing of the Us1.5 protein. The results also indicate that (i) at least one domain targeted by the UL13 protein kinase is located in the amino-terminal 250 amino acids shared by both ICP22 and Us1.5 protein and (ii) the same domain is responsible for the differentiation of several isoforms of these proteins.
EXAMPLE 5: Processing of UJ.5 Protein Expressed by Mutant Viruses
The puφose of this series of studies was to verify that R7802 did not express α22/Us1.5 proteins and to examine the expression of Us1.5 in R7805 and R7808.
Replicate cultures of Vero cells were exposed to 10 PFU of HSV- 1(F), R7802 (Δα22/ΔUs1.5), R7804 (R7802 Repair), R7805 (Δα22/Us1.5), R7806 (R7805 Repair),
R7808 (Δα22/Us1.5), or R7828 (R7808 Repair)
Figure imgf000105_0001
per cell. At 18 h after infection, the cells were harvested, solubilized, electrophoretically separated on a denaturing polyacrylamide gel. electrically transferred to a nitrocellulose sheet, and reacted with the polyclonal antibody to the carboxyl-terminal of ICP22/US1.5 proteins as described in Materials and Methods. The results, FIG. 6, were as follows:
(i) Both ICP22 and Us1.5 were present in lysates of HSV-l(F) (FIG. 6, lane 1).
(ii) Both ICP22 and Us1.5 were absent in lysates of R7802 (Δα22/ΔUs1.5) infected cells lysates (FIG. 6, lane 2).
(iii) The Us1.5 protein was detected in lysates of cells infected with R7805 (Δα22/Us1.5) and R7808 (Δα22/Us1.5) (FIG. 6, lanes 4 and 6). In R7805 Us1.5 was overexpressed as compared to Us1.5 in HSV-l(F) infected cell extracts (FIG. 6, compare lanes 1 and 4). The Us1.5 protein in R7805 was posttranslationally processed and exhibited a range of proteins from Mr 33,000 to 48,000 similar to Us1.5 of HSV-I(F). In R7808 Us1.5 was also overexpressed as compared to HSV-l(F) (FIG. 6, compare lanes 1 and 6). But, in this recombinant Us1.5 was not posttranslationally processed and formed predominantly two bands, a major band with an apparent Mr 33,000 and a minor band with an apparent Mr 36,000.
(iv) The repair vimses R7804 (R7802 Repair), R7806 (R7805 Repair), and
R7828 (R7808 Repair) exhibited wild type levels of expression and posttranslational processing of ICP22/US1.5 proteins (FIG. 6, compare lane 1 with lanes 3, 5, and 7). ICP4 measured by its reactivity with corresponding monoclonal antibody served as a loading control.
The inventors conclude from these studies that the ICP22 amino acids 147 to 171 are required for posttranslational processing of a truncated product of ICP22 that corresponds to Us1.5. Forced translation initiation at methionine 171 yielded a product that did not appear to be posttranslationally processed.
EXAMPLE 6: Translational Initiation within the Domain Containing UJ.5 Preferentially
Occurs at Amino Acid 147 ofthe ICP22 Sequence The puφose of this series of studies was two-fold. The first was to attempt to produced amino-terminal tmncations of ICP22 other than those which correspond to the sequence encoding the Us1.5 protein. The second objective was to characterize further the product of the ICP22/US1.5 genes encoded by R7808/R7805. Preliminary studies designed to meet the first objective indicated that all 5' terminal tmncations of the α22 gene 5' ofthe metl47 codon yielded proteins that resembled the Us1.5 protein. The hypothesis that emerged from the studies described above was that the prefeπed translation initiation methionine within the Us1.5 transcript was at codon 147. To test this hypothesis the inventors constmcted a mutant in which the amino-terminal 47 codons of the α22 coding sequence were deleted. The tmncated α22 gene in the resulting vims, R7815, contained three possible initiator methionine codons, met90, metl47, and metl71 (FIG. 7, line 2). Vero cells were exposed to 10 PFU of R7805 (Δα22/Us1.5), R7808 (Δα22/Us1.5), or R7815 (Δα22NT/Us1.5) vims per cell. At 18 h after infection the cells were harvested, solubilized, electrophoretically separated in a denaturing polyacrylamide gel, electrically transferred to a nitrocellulose sheet and reacted with the polyclonal antibody to the carboxyl-terminal of ICP22. The expression of Usl .5 was examined. The results, FIG. 8, were as follows:
(i) In infected cell extracts infected with R7815 the predominant species of protein initiated at amino acid 147, even though the first methionine in this constmct is at ammo acid 90 (FIG 7 A, lane 1 ) Because of the vanabihty seen between strains of Heφes simplex vims 1 the presence of this methionine in (F) was verified by sequencing
In cell extracts infected with R7805 the predominant species of protein initiated at amino acid 147, with weak initiation at amino acid 171 (FIG 8, lane 2) The initiation at amino acid 171 can be seen in infected extracts with R7808 (FIG 8, lane 3)
in) In infected cell extracts infected with R7808 the predominant species of protein initiates at amino acid 171 , with no apparent initiation at ammo acid 194 (FIG 8, lane 3) The inventors conclude from these studies that in the absence of the initiator methionine, the preferred mrtrator methionine is the codon metl47
EXAMPLE 7
The ICP22 Function That Enhances the Expression of a Subset of , Genes Is Located at the Carboxyl-Terminal Domain Shared with the UJ 5 protein and Can Be Expressed by the Latter Protein In this senes of studies replicate cultures of Vero or rabbit skin cells were exposed to 10 PFU of HSV-1 (F), R7802 (Δα22/ΔUsl 5), R7804 (R7802 Repair),
R7805 (Δα22/Usl 5), R7806 (R7805 Repair), R7808 (Δα22/Usl 5) or R7828 (R7808 Repair) vims per cell At 18 h after mfection the cells were harvested, solubilized, electrophoretically separated m a denatunng polyacrylamide gel, electncally transferred to a nitrocellulose sheet and reacted sequentially with the monoclonal antibody to U l l, and the polyclonal antibody (Wl ) to UL38 The expression levels of Usl 1 and Ut38 were examined The results, FIG 9, were as follows
(I) Vero and rabbit skm cells infected with HSV-I(F) expressed equivalent levels of U l 1 protein whereas the level of UL38 protein in rabbit skin cells was greater than that detected in Vero cells (FIG 9, compare lanes 1 and 2) (n) Rabbit skin cells infected with R7802 (Δα22/ΔUsl 5) expressed less Usl 1 and UL38 proteins than infected Vero cells (FIG 9, compare lanes 3 and 4) This phenotype is comparable to the phenotype seen with infection of these cell lines with R7356 a recombinant vims deleted in the UL13 protein kinase and R325 a recombinant virus delete for Usl 5 (Purves et al , 1993
(m) Vero and rabbit skin cells infected with a Usl 5 expressing vims (R7805) expressed equivalent levels of Usl 1 and UL38 (FIG 9, compare lanes 7 and
8)
(iv) Vero and rabbit skin cells infected with R7808 did not express equivalent levels of U l 1 and UL38 consistent with the observation that Usl 5 m this constmct is not processed (FIG 9, compare lanes 1 1 and 12) The decrese of Usl l protein m cells infected with R7808 was not as great as that seen in cells infected with R7802
(v) Vero and rabbit skin cells infected with R7804 (R7802 Repair), R7806 (R7805 Repair), and R7828 (R7808 Repair) expressed equivalent levels of U&11 and UL38 proteins in each cell line (FIG 9, compare lanes 5, 6, 9, 10, and 13, 14)
The inventors conclude that the genetic information required for optrmal expression of a subset of late γ2 genes exemplified by Usl 1 and UL38 resides in the domain shared by Usl 5 and ICP22 Earlier studies have shown that optimal expression of this subset of γ2 genes requires a functional UL13 protein kinase that, coincidentally, also mediates the posttranslational processing and phosphorylatron of
ICP22 and UJ.5
EXAMPLE 8 All Isoforms of UJ 5 Protein Are Translocated into the Nucleus The puφose of this senes of studies was to determine whether posttranslational processing of the isoforms of Usl 5 proteins was dependent on nuclear localization. HEp-2 cells were exposed to 10 PFU of HSV- 1(F), R7805 (Δα22/Us1.5), R7808 (Δα22/Us1.5), or R7810 (Δα22/ΔUs1.5 ,cτ) per cell and harvested at 18 h after infection. The nuclei and cytoplasm were separated as described in Materials and Methods. Each fraction was subjected to electrophoresis in a denaturing polyacrylamide gel and then reacted with the polyclonal antibody made against the carboxyl-terminal of ICP22. The fractionation was validated by reacting the nitrocellulose sheet as second time but with antibody against ICP4. As expected, ICP4 localized in the nuclear fractions. The results FIG. 10, were as follows:
(i) ICP22 was detected in both the nucleus and the cytoplasm of cells infected with HSV- 1(F). The nuclear and cytoplasmic ICP22 were posttranslationally processed to the same extent, but the slowest migrating forms of ICP22 was more abundant in the nucleus than in the cytoplasm. Ub1.5 protein made in cells infected with HSV- 1(F) was present in greater abundance in the nucleus. Moreover, the ratio of the various electrophoretically distinct isoforms of ICP22 in the cytoplasm differed from those in the nucleus. (FIG. 10, compare lanes 1 and 2).
(ii) In cells infected with R7805 (Δα22 r/Us1.5), the Us1.5 protein was more abundant but also present in both nucleus and cytoplasm. Some of the slow migrating forms of Us1.5 were absent or present in smaller amounts in the cytoplasm
(FIG. 10, compare lanes 3 and 4).
(iii) In cells infected with R7808 (Δα22 T/Us1.5αp ), Us1.5 was present in both fractions. The nuclear form of Us1.5 has an addition major band as compared to the cytoplasmic fraction (FIG. 10, compare lanes 5 and 6).
(iv) In cells infected with R7810 (Δα22cτ/ΔUs1.5cτ) (FIG. 4, panel A, line 6), both ICP22 and the Usl .5 protein are unprocessed and distributed in both fractions. (FIG. 10, compare lanes 7 and 8). The inventors conclude the following (1) All isoforms of ICP22 and Usl 5 localized in both the nucleus and cytoplasm Implicit in this observation is that either UJ 5 contains an as yet unidentified nuclear localization signal or it is transported to the nucleus in association with another protein (n) Posttranslational processing of Usl 5 protein requires at least two domains, the amino terminal domain between amino acids 147 and 171 and the carboxyl-terminal 40 amino acids although partial processing was noted in Usl 5 protein lacking the ammo-terminal domain (in) Processing ofthe Usl 5 protein does not require the presence of an intact ICP22
EXAMPLE 9
The Amino Acids Required for Posttranslational Modification of ICP22/U 5 Maps to Three Amino Acids within the Carboxyl-terminal Domain In the preceding section, deletion of the carboxyl-terminal 40 codons yielded a tmncated protein that was not posttranslationally processed To map the sequence required for processing, the inventors constmcted the senes of carboxyl-terminal deletion mutants shown in FIG 4A and 4B Replicate cultures of Vero cells were exposed to 10 PFU of HSV-l(F), R7819 (Δ10 codons), R7822 (Δ15 codons), R7823 (Δ18 codons), R7820 (Δ22 codons), R7810 (Δ40 codons). or R7821 (repair of R7810) per cell At 18 h after infection the cells were harvested, solubilized, electrophoretically separated in a denatunng polyacrylamide gel, electncally transferred to a nitrocellulose sheet, and reacted with the R77 polyclonal antibody R77 to ICP22 The results, FIG 1 1 , were as follows
(I) Processed forms of ICP22 were present in lysates of cell infected with all vimses except those infected with R7810, R7820 or R7823 (FIG 1 1 , lanes 4-6) The inventors noted a slightly higher accumulation of the fastest migrating forms of ICP22 in cells infected with mutants carrymg carboxyl-terminal deletions (e g R7822, R7823, FIG 1 1 , lanes 3 and 4) (ii) Only the fastest migrating forms of ICP22 accumulated in cells infected with R7823 or R7820 (FIG. 1 1 , lanes 4 and 5).
The substitution of lysine for the arginine 402 had no effect on processing of ICP22 although again the fastest-migrating form accumulated in the infected cells.
The inventors conclude from these studies the following: (1) ICP22 encoded by mutants lacking the carboxyl-terminal 15 or less amino acids were posttranslationally processed by the UL13 protein kinase whereas ICP22 encoded by mutants lacking 18 or more carboxyl-terminal amino acids were not processed, (ii)
The carboxyl-terminal 3 amino acids - - lys402, met403, and arg404 appear to be required for posttranslational processing of ICP22.
EXAMPLE 10: The Posttranslational Modification ofICP22/US1.5 Is Altered in the Presence of
ULJ3 When Threonine 300 and Serine 301 Are Mutated
A site for phosphorylation by the UL13 protein kinase within ICP22 US1.5 was identified by doing mutational analysis. A recombinant vims was constmcted,
R7827, in which Threonine 300 and Serine 301 were mutagenized to Alanine and Glycine, respectively. To investigate whether ICP22/US1.5 is still post-tranlationally processed, replicate cultures of Vero cells were exposed to 10 PFU of wild-type
[HSV- 1(F)] or R7827 vimses per cell. At 24 h after infection, the cells were harvested, solubilized, electrophoretically separated on a denaturing polyacrylamide gel, electrically transferred to a nitrocellulose, sheet, and reacted with a polyclonal antibody to ICP22/US1.5.
The processing of the ICP22/US1.5 proteins is reduced in cells extracts infected with R7827 relative to those of wild-type [HSV-1 (F)] infected cells. Based on this data and a homology comparison between HSV-1 and HSV-2. it can be concluded that the domain within ICP22/US1.5 consisting of amino acids 300 to at least 310 (and probably to amino acid 328) in HSV-1 and amino acids 295 to 305 in HSV-2 (and probably to amino acid 320) is the site of phosphorylation by the UL13 protein kinase (Figure 12B, line 4).
All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of prefeπed embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents, which are both chemically and physiologically related, may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
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Claims

CLAIMS:
1. A method for screening a candidate substance for inhibition of heφesvims infections comprising:
(a) obtaining a polypeptide comprising a Usl .5 phosphorylation site;
(b) contacting said polypeptide with a candidate inhibitor;
(c) contacting said polypeptide with a kinase under conditions appropriate to effect phosphorylation of said polypeptide;
(d) determining the phosphorylation state of said polypeptide; and
(e) comparing the phosphorylation state of said polypeptide in step (d) with that of a similar polypeptide contacted with said kinase in the absence of said candidate inhibitor.
The method of claim 1 , wherein said kinase comprises UL13.
3. The method of claim 2, wherein said polypeptide comprises at least about 10 consecutive amino acids from one or more of SEQ ID NOJ, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NOJ.
4. The method of claim 3, wherein said polypeptide comprises at least about 15 consecutive amino acids from one or more of SEQ ID NOJ, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.
5. The method of claim 4, wherein said polypeptide comprises at least about 20 consecutive amino acids from one or more of SEQ ID NOJ, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NOJ.
6. The method of claim 5, wherein said polypeptide comprises at least about 25 consecutive amino acids from one or more of SEQ ID NOJ, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.
7. The method of claim 1, wherein said polypeptide comprises a detectable label.
8. The method of claim 1, wherein determining the phosphorylation state of said polypeptide comprises determining p60 binding to a heφesvims product.
9. A peptide comprising at least about 10 consecutive amino acids from one or more of SEQ ID NOJ, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4, wherein said peptide inhibits posttranslational modification of heφesvims Us1.5.
10. The peptide of claim 9, comprising at least about 15 consecutive amino acids from one or more of SEQ ID NOJ, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NOJ, wherein said peptide inhibits posttranslational modification of heφesvims Usl.5.
11. The peptide of claim 10, comprising at least about 20 consecutive amino acids from one or more of SEQ ID NOJ, SEQ ID NO:2, SEQ ID NOJ, or SEQ ID NOJ, wherein said peptide inhibits posttranslational modification of heφesvims Usl .5.
12. The peptide of claiml l, comprising at least about 25 consecutive amino acids from one or more of SEQ ID NOJ, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NOJ, wherein said peptide inhibits posttranslational modification of heφesvims Us 1.5.
13. The peptide of claim 9, comprising a Us1.5 phosphorylation site.
14. The peptide of claim 13, comprising at least two Us1.5 phosphorylation sites.
15. A peptide of about 20 consecutive amino acids to about 100 consecutive amino acids, comprising a first region of at least about 10 consecutive amino acids from one or more of SEQ ID NOJ , SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NOJ, and a second region of at least about 10 consecutive amino i acids from one or more of SEQ ID NOJ, SEQ ID NO:2, SEQ ID NO:3, or
SEQ ID NOJ, wherein said peptide inhibits posttranslational modification of heφesvims Us1.5.
16. The peptide of claim 15, comprising a Us1.5 phosphorylation site.
17. The peptide of claim 16, comprising at least two Us1.5 phosphorylation sites.
18. The peptide of claim 15, wherein said first and second regions are linked by one or more amino acids, other than the native Usl .5 sequence.
19. The peptide of claim 18, wherein said first and second regions are linked by a non-amino acid linking moiety.
20. A polypeptide that binds to a phosphorylation site in Usl .5.
21. The polypeptide of claim 20, wherein said polypeptide is an antibody.
22. The polypeptide of claim 20, wherein said polypeptide is a single chain antibody.
23. The polypeptide of claim 21, wherein said antibody is an anti-idiotypic antibody that binds immunologically to the antigen binding region of an anti- UL13 antibody.
24. A method of inhibiting heφesvims infection comprising contacting a cell with an inhibitor of Us1.5 phosphorylation.
25. The method of claim 24, wherein said cell is infected with heφesvims prior to contacting.
26. The method of claim 24, wherein said cell is infected with heφesvims after contacting.
27. The method of claim 24, wherein said heφesvims is HSV.
28. A method of inhibiting heφesvims infection comprising contacting a cell with a polypeptide that binds to a Usl .5 phosphorylation site.
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