US20080279888A1 - Genetically engineered cell lines and systems for propagating varicella zoster virus and methods of use thereof - Google Patents
Genetically engineered cell lines and systems for propagating varicella zoster virus and methods of use thereof Download PDFInfo
- Publication number
- US20080279888A1 US20080279888A1 US11/903,708 US90370807A US2008279888A1 US 20080279888 A1 US20080279888 A1 US 20080279888A1 US 90370807 A US90370807 A US 90370807A US 2008279888 A1 US2008279888 A1 US 2008279888A1
- Authority
- US
- United States
- Prior art keywords
- vzv
- cell
- varicella zoster
- zoster virus
- cells
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Images
Classifications
-
- 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
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
- A61K39/245—Herpetoviridae, e.g. herpes simplex virus
- A61K39/25—Varicella-zoster virus
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
- A61K2039/5254—Virus avirulent or attenuated
-
- 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
-
- 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
- C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
- C12N2710/16722—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
- C12N2710/16734—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
- C12N2710/16761—Methods of inactivation or attenuation
Definitions
- VZV Varicella zoster virus
- varicella is the cause of chickenpox ( varicella ) and shingles ( zoster ).
- Varicella is a primary infection in which VZV infects a naive host.
- Zoster is the result of the reactivation of VZV, which has remained latent in its host, often for many years.
- a major paradox has impeded research on VZV for many years: VZV is highly infectious and spreads readily from an infected host to susceptible individuals, yet, it is extremely difficult to propagate in vitro, because it spreads only by direct cell-to-cell contact when it is grown in tissue culture.
- VZV The dissemination of VZV among a population of susceptible subjects is mediated by cell-free virions, which are thought to be airborne.
- Cell-to-cell spread of VZV in vitro does not depend on cell-free virions, which are not released in viable form by infected cells in tissue culture. Instead, cells that are infected in vitro fuse with their uninfected neighbors enabling infection to be transferred intracellularly.
- the present invention provides a genetically engineered cell line stably transformed with a nucleotide sequence encoding at least two full-length mannose-6-phosphate receptors.
- the present invention provides a genetically engineered cell line stably transformed with a nucleotide sequence encoding a full-length mannose-6-phosphate receptor and a full-length insulin-like growth factor receptor.
- Also provided is a recombinant vector comprising a nucleotide sequence encoding at least two full-length mannose-6-phosphate receptors.
- the present invention further provides a vaccine comprising an attenuated live virus produced by culturing genetically-engineered cells stably transformed with a nucleotide sequence encoding at least two full-length mannose-6-phosphate receptors and a pharmaceutically acceptable carrier.
- the present invention is also directed to a method for generating an in vitro system for Varicella zoster virus (VZV), by: (a) isolating enteric ganglia from guinea pig; and (b) contacting the enteric ganglia with cell-free VZV to generate latent expression of the VZV. Also provided is an in vitro system for VZV generated by this method.
- VZV Varicella zoster virus
- the present invention further provides an in vitro system for Varicella zoster virus (VZV), comprising an enteric ganglion that has been contacted with cell-free VZV to produce a latent VZV, wherein the VZV is subsequently reactivated to express VZV in an active form.
- VZV Varicella zoster virus
- the present invention provides a method for reactivating Varicella zoster virus (VZV), by: (a) isolating enteric ganglia from guinea pig; (b) contacting the enteric ganglia with cell-free VZV to generate latent expression of the VZV; and (c) contacting the infected ganglia with a vector containing a nucleic acid sequence encoding VZV ORF61 or a homologue thereof. Also provided is a Varicella zoster virus reactivated by this method.
- VZV Varicella zoster virus
- the present invention provides a method of screening for an agent for treating Varicella zoster virus (VZV) infection, comprising use of an in vitro system for VZV, wherein the in vitro system comprises an enteric ganglion that has been contacted with cell-free VZV to produce a latent VZV, and wherein the VZV is subsequently reactivated to express VZV in an active form.
- VZV Varicella zoster virus
- FIG. 1 is an illustration of a Varicella zoster Virus (VZV) particle
- FIG. 2 illustrates that mannose-6-phosphate receptors (MPRs) sort lysosomal enzymes and target them to endosomes;
- MPRs mannose-6-phosphate receptors
- FIG. 3 illustrates intracellular transport of VZV and packing of the final envelope in the trans-Golgi network (TGN);
- FIG. 4 illustrates that MPRs direct newly assembled VZV to late endosomes
- FIG. 5 illustrates the mechanism by which VZV particles receive their final envelope in the TGN
- FIG. 6 demonstrates that human embryonic lung cells (HELF) infection by cell-free VZV is inhibited by phosphorylated sugars;
- FIG. 7 demonstrates that expression of cation-independent MPRs (MPR ci s) in MeWo cells is downregulated by antisense cDNA;W
- FIG. 8 shows that downregulation of MPR ci s inhibits infection by cell-free VZV
- FIG. 9 demonstrates that MPR ci -KO (knock-out) cells can be infected by cell-associated VZV;
- FIG. 10 illustrates that MPR ci -KO cells release infectious VZV into the culture supernatant
- FIG. 11 illustrates that VZV nucleocapsids assemble in the nuclei of infected KO-MeWo cells
- FIG. 12 shows that enveloped VZV enters the perinuclear cisterna of KO-MeWo cells
- FIG. 13 demonstrates that, in KO-MeWo cells, transport vesicles contain individual virions
- FIG. 14 demonstrates that VZV accumulates in late endosomes in parental MeWo cells
- FIG. 15 illustrates that enveloped VZV is released intact in the superficial epidermis
- FIG. 16 shows that lytic infection of neurons occurs when non-neuronal cells are present
- FIG. 17 illustrates that neurons die within two days when infection is lytic
- FIG. 18 demonstrates that expression of HSV ICP0 causes VZV to reactivate in enteric neurons
- FIG. 19 shows that latent VZV reactivates in enteric neurons forced to express HSV ICP0;
- FIG. 20 shows that cultured ganglia contain few non-neuronal cells
- FIG. 21 demonstrates that RT-PCR reveals expression of VZV DNA and RNA in isolated ganglia
- FIG. 22 shows that infected ganglia contain mRNA and DNA encoding VZV proteins
- FIG. 23 illustrates that ORF29 mRNA is found by in situ hybridization in subsets of neurons
- FIG. 24 demonstrates that infected neurons are ORF29p ⁇ but not gE-immunoreactive
- FIG. 25 shows that ORF62p, 4p, and 21p are present in the cytoplasm of infected neurons.
- VZV trans-Golgi network
- lysosomal enzymes Diversion of lysosomal enzymes occurs because they contain mannose-6-phosphate (Man 6-P) groups that enable them to bind to mannose-6-phosphate receptors (MPRs), which are responsible for routing the lysosomal enzymes to endosomes.
- MPRs mannose-6-phosphate receptors
- Lysosomal enzymes dissociate from MPRs in endosomes and are transported within the interiors of vesicles to lysosomes, while the MPRs are transported in membranes either back to the TGN or to the plasma membrane.
- the Man 6-P/IGF2Rs in the plasma membrane are able to bind to extracellular molecules that express Man 6-P groups, such as those of lysosomal enzymes, and media
- VZV is diverted from the secretory pathway of infected cells by Man 6-P/IGF2Rs, which is thus responsible for the delivery of enveloped virions to endosomes.
- Man 6-P/IGF2Rs glycoproteins of the VZV envelope contain Man 6-P groups.
- Man 6-P interferes with the interaction between plasma membrane Man 6-P/IGF2R and the Man 6-P groups of extracellular molecules.
- Man 6-P does not prevent the cell-to-cell spread of VZV in vitro, which because it depends on the fusion of adjacent cells, which is independent of the interaction of viral Man 6-P with Man 6-P/IGF2R.
- the inventors produced a cell line that is deficient in Man 6-P/IGF2R. Because of the strong preference of VZV for human cells, the Man 6-P/IGF2R-deficient cell line was derived from MeWo cells, a human melanoma tumor cell line, which is known to be susceptible to infection by VZV and to support the growth of VZV in vitro.
- the Man 6-P/IGF2R-deficient MeWo cells were found to be far more resistant to infection by cell-free VZV than their parental control MeWo cells; however, infected cells can transfer VZV infection to the Man 6-P/IGF2R-deficient MeWo cells as readily as to the parental control MeWo cells.
- the medium in which parental control MeWo cells is growing is not infectious and cannot be used to transfer infection with VZV to susceptible target cells (human embryonic lung cells [HELF] are employed as the susceptible targets).
- HELF human embryonic lung cells
- the medium in which Man 6-P/IGF2R-deficient MeWo cells is growing does contain infectious VZV and can be used to transfer infection with VZV to target HELF cells.
- Man 6-P/IGF2R-deficient MeWo cells This is the first cell line that, when infected with VZV, releases substantial quantities of infectious VZV particles to the ambient medium.
- the medium in which the Man 6-P/IGF2R-deficient MeWo cells are growing is thus extraordinarily useful as a source of intact, non-degraded, infectious VZV.
- the resistance of the Man 6-P/IGF2R-deficient MeWo cells to infection by cell-free VZV confirms that the Man 6-P/IGF2R plays an important role in enabling VZV to enter target cells. Infection of Man 6-P/IGF2R-deficient MeWo cells must be accomplished by adding other infected cells to them. The infected cells can thus infect the Man 6-P/IGF2R-deficient MeWo cells by cell-to-cell contact, which is Man 6-P/IGF2R-independent.
- Man 6-P/IGF2R-deficient MeWo cells The successful maintenance and easy use of the Man 6-P/IGF2R-deficient MeWo cells depends on the knockdown of the Man 6-P/IGF2R being incomplete. When the Man 6-P/IGF2R expression is abolished, the resulting cells grow poorly and slowly. The current Man 6-P/IGF2R-deficient MeWo cells express small amounts of the Man 6-P/IGF2R and thus grow adequately, can be maintained without excess difficulty, yet still release infectious VZV.
- Man 6-P/IGF2R-deficient MeWo cells Uses of the Man 6-P/IGF2R-deficient MeWo cells include the development of new and reliable methods to produce the VZV vaccine.
- This vaccine is an attenuated live virus that must be produced by cultured cells. It grows in culture, like wild-type VZV, only by cell-to-cell contact. The final product has to be liberated from the small intracellular compartment that contains newly assembled virions that have not yet been transported to endosomes. Virions that have been degraded in endosomes are useless as components of the vaccine. Yields of live virus are thus very low. Propagation in the Man 6-P/IGF2R-deficient MeWo cells should produce high yields of much more readily purified viable virus.
- Man 6-P/IGF2R-deficient MeWo cells should be just as helpful in developing new strains of VZV for future vaccines.
- virus produced in Man 6-P/IGF2R-deficient MeWo cells is likely to be much more uniform and readily controlled than that produced in ordinary tissue culture cells. The non-controllable degradation of virions is avoided, yields are improved, and a step of cell lysis can be avoided in vaccine production. Contamination of the vaccine with cellular constituents and other viruses that might be contained in tissue culture cells can also be minimized.
- Man 6-P/IGF2R-deficient MeWo cells will also help in research on VZV itself. As such, Man 6-P/IGF2R-deficient MeWo cells will be of great value and sought after by research workers hoping to develop antiviral drugs and those seeking to understand the basic properties of VZV.
- the Man 6-P/IGF2R has been most strongly implicated as important, as described in the biology of VZV, the receptor may also play roles in the biology of other related herpesviruses, such as herpes simplex virus types 1 and 2 and pseudorabies virus. These virions do spread through media in vitro, but they may well spread more readily when propagated in Man 6-P/IGF2R-deficient MeWo cells.
- Man 6-P/IGF2R-deficient MeWo cells are likely to be attractive to cell biologists who are interested in the basic properties of lysosomes, endosomes, receptor-mediated endocytosis, autophagy, and the pathogenesis of lysosomal storage disease.
- Man 6-P/IGF2Rs The deficient expression of Man 6-P/IGF2Rs was induced by stably transfecting parent MeWo cells (available from the European Collection of Cell Cultures (ECACC) Salisbury, Wiltshire) with cDNA encoding the full-length Man 6-P/IGF2R in the antisense configuration.
- the cDNA construct was packaged in a retroviral vector that also contained a puromycin resistance gene.
- the success of the knockdown of the Man 6-P/IGF2R was confirmed by Western analysis and by immunocytochemistry; both techniques demonstrated reduced Man 6-P/IGF2R expression.
- Man 6-P/IGF2R-deficient MeWo cells were found to contain less cathepsin D (a lysosomal enzyme that was investigated as a marker) than control parental MeWo cells. This demonstration was again accomplished by Western analysis and by immunocytochemistry.
- the Man 6-P/IGF2R-deficient cells also secreted a greater amount of another lysosomal enzyme, acid phosphatase, than parental MeWo cells (shown by direct measurement of acid phosphatase activity in cells and in media and by Western analyses of media and cell lysates).
- FIGS. 1-15 The foregoing results are summarized in FIGS. 1-15 .
- the data confirm that the Man 6-P/IGF2R-deficient MeWo cells have a reduced ability to divert lysosomal enzymes from their secretory pathway to endosomes.
- VZV has a narrow host range. However, it is known to be neurotropic, and to become latent in sensory ganglia. Because the enteric nervous system (ENS) contains intrinsic sensory neurons, the inventors postulated that VZV may infect, and become latent in, the ENS (e.g., enteric neurons and/or enteric ganglia).
- ENS enteric nervous system
- the ENS is an independent autonomic division that is capable of regulating the behavior of the gut without input from the central nervous system (CNS).
- the ENS has independence from the CNS because it contains two main types of intrinsic primary afferent neurons (IPANS): submucosal IPANS (containing cholinergic-CGRP or substance-P) or myenteric IPANS (containing cholinergic calbindin).
- IPANS intrinsic primary afferent neurons
- the inventors found that reactivation of VZV in enteric neurons could provide a source of visceral zoster, and present a model for understanding the origin of zoster. This was established by first testing the ability of VZV to establish latency in guinea pig enteric ganglia in vitro, and then by testing the ability of the latent VZV to reactivate.
- the inventors first isolated ganglia from guinea pig small intestine.
- the LM-MP was mechanically dissected from the bowel, and dissociated with collagenase. Myenteric ganglia then were individually selected and cultured. Mitotic inhibitors were used to decrease growth of non-neuronal cells, enabling enteric neurons to reorganize and interconnect. Cell-free VZV was adsorbed for 4 h, approximately 5 days after the ganglia were plated. Cultures were maintained for 4-6 weeks thereafter.
- VZV infected guinea pig enteric ganglia in vitro Latent infection occurred with the use of cultures highly enriched in neurons. It was observed that neurons expressed only those VZV proteins that had been reported to be expressed during VZV latency in human sensory ganglia (no glycoproteins).
- ORF62p and ORF29p are cytoplasmic, not nuclear. Lytic infection required the presence of non-neuronal cells. Both neurons and non-neuronal cells expressed glycoproteins, and ORF62p and ORF29p were found in nuclei. Reactivation occurred in neurons, and likely occurred in glia.
- HSV ICP0 the HSV homologue of VZV ORF61
- Neurons expressed glycoproteins, and ORF29p was found in nuclei.
- the model of the in vitro reactivation of VZV described herein is the first in vitro model for shingles.
Abstract
The present invention provides genetically engineered cell lines, recombinant vectors, and vaccines. The present invention also provides methods for generating an in vitro system for Varicella zoster virus (VZV), and the in vitro systems generated by these methods. The present invention further provides methods for reactivating VZV, and VZV reactivated by these methods. Finally, the present invention provides a method of screening for an agent for treating VZV infection.
Description
- This application is a Divisional of prior Application U.S. Ser. No. 10/436,706 filed May 12, 2003, which claims the benefit of U.S. Provisional Application No. 60/379,819, filed May 10, 2002.
- Varicella zoster virus (VZV) is the cause of chickenpox (varicella) and shingles (zoster). Varicella is a primary infection in which VZV infects a naive host. Zoster is the result of the reactivation of VZV, which has remained latent in its host, often for many years. A major paradox has impeded research on VZV for many years: VZV is highly infectious and spreads readily from an infected host to susceptible individuals, yet, it is extremely difficult to propagate in vitro, because it spreads only by direct cell-to-cell contact when it is grown in tissue culture. The dissemination of VZV among a population of susceptible subjects is mediated by cell-free virions, which are thought to be airborne. Cell-to-cell spread of VZV in vitro does not depend on cell-free virions, which are not released in viable form by infected cells in tissue culture. Instead, cells that are infected in vitro fuse with their uninfected neighbors enabling infection to be transferred intracellularly.
- The present invention provides a genetically engineered cell line stably transformed with a nucleotide sequence encoding at least two full-length mannose-6-phosphate receptors.
- Additionally, the present invention provides a genetically engineered cell line stably transformed with a nucleotide sequence encoding a full-length mannose-6-phosphate receptor and a full-length insulin-like growth factor receptor.
- Also provided is a recombinant vector comprising a nucleotide sequence encoding at least two full-length mannose-6-phosphate receptors.
- The present invention further provides a vaccine comprising an attenuated live virus produced by culturing genetically-engineered cells stably transformed with a nucleotide sequence encoding at least two full-length mannose-6-phosphate receptors and a pharmaceutically acceptable carrier.
- The present invention is also directed to a method for generating an in vitro system for Varicella zoster virus (VZV), by: (a) isolating enteric ganglia from guinea pig; and (b) contacting the enteric ganglia with cell-free VZV to generate latent expression of the VZV. Also provided is an in vitro system for VZV generated by this method.
- The present invention further provides an in vitro system for Varicella zoster virus (VZV), comprising an enteric ganglion that has been contacted with cell-free VZV to produce a latent VZV, wherein the VZV is subsequently reactivated to express VZV in an active form.
- Additionally, the present invention provides a method for reactivating Varicella zoster virus (VZV), by: (a) isolating enteric ganglia from guinea pig; (b) contacting the enteric ganglia with cell-free VZV to generate latent expression of the VZV; and (c) contacting the infected ganglia with a vector containing a nucleic acid sequence encoding VZV ORF61 or a homologue thereof. Also provided is a Varicella zoster virus reactivated by this method.
- Finally, the present invention provides a method of screening for an agent for treating Varicella zoster virus (VZV) infection, comprising use of an in vitro system for VZV, wherein the in vitro system comprises an enteric ganglion that has been contacted with cell-free VZV to produce a latent VZV, and wherein the VZV is subsequently reactivated to express VZV in an active form.
- Additional aspects of the present invention will be apparent in view of the description which follows.
-
FIG. 1 is an illustration of a Varicella zoster Virus (VZV) particle; -
FIG. 2 illustrates that mannose-6-phosphate receptors (MPRs) sort lysosomal enzymes and target them to endosomes; -
FIG. 3 illustrates intracellular transport of VZV and packing of the final envelope in the trans-Golgi network (TGN); -
FIG. 4 illustrates that MPRs direct newly assembled VZV to late endosomes; -
FIG. 5 illustrates the mechanism by which VZV particles receive their final envelope in the TGN; -
FIG. 6 demonstrates that human embryonic lung cells (HELF) infection by cell-free VZV is inhibited by phosphorylated sugars; -
FIG. 7 demonstrates that expression of cation-independent MPRs (MPRcis) in MeWo cells is downregulated by antisense cDNA;W -
FIG. 8 shows that downregulation of MPRcis inhibits infection by cell-free VZV; -
FIG. 9 demonstrates that MPRci-KO (knock-out) cells can be infected by cell-associated VZV; -
FIG. 10 illustrates that MPRci-KO cells release infectious VZV into the culture supernatant; -
FIG. 11 illustrates that VZV nucleocapsids assemble in the nuclei of infected KO-MeWo cells; -
FIG. 12 shows that enveloped VZV enters the perinuclear cisterna of KO-MeWo cells; -
FIG. 13 demonstrates that, in KO-MeWo cells, transport vesicles contain individual virions; -
FIG. 14 demonstrates that VZV accumulates in late endosomes in parental MeWo cells; -
FIG. 15 illustrates that enveloped VZV is released intact in the superficial epidermis; -
FIG. 16 shows that lytic infection of neurons occurs when non-neuronal cells are present; -
FIG. 17 illustrates that neurons die within two days when infection is lytic; -
FIG. 18 demonstrates that expression of HSV ICP0 causes VZV to reactivate in enteric neurons; -
FIG. 19 shows that latent VZV reactivates in enteric neurons forced to express HSV ICP0; -
FIG. 20 shows that cultured ganglia contain few non-neuronal cells; -
FIG. 21 demonstrates that RT-PCR reveals expression of VZV DNA and RNA in isolated ganglia; -
FIG. 22 shows that infected ganglia contain mRNA and DNA encoding VZV proteins; -
FIG. 23 illustrates that ORF29 mRNA is found by in situ hybridization in subsets of neurons; -
FIG. 24 demonstrates that infected neurons are ORF29p− but not gE-immunoreactive; -
FIG. 25 shows that ORF62p, 4p, and 21p are present in the cytoplasm of infected neurons. - The inventors have demonstrated that viable VZV is not released to the medium by cultured cells because newly assembled virions are diverted from the secretory pathway to late endosomes where they encounter an acidic environment and lysosomal enzymes. The viral particles are then degraded in these endosomes before the virions are released to the medium by exocytosis. Only degraded VZV, which is not infectious, thus is delivered to the extracellular medium. VZV receives its final envelope in the trans-Golgi network (TGN). This cellular organelle is an intracellular sorting location and is the site where lysosomal enzymes are separated from proteins destined to be secreted. Diversion of lysosomal enzymes occurs because they contain mannose-6-phosphate (Man 6-P) groups that enable them to bind to mannose-6-phosphate receptors (MPRs), which are responsible for routing the lysosomal enzymes to endosomes. There are 2 MPRs, a large cation-independent molecule, which is also the receptor for insulin-like growth factor 2 (Man 6-P/IGF2R), and a small cation-dependent molecule. Lysosomal enzymes dissociate from MPRs in endosomes and are transported within the interiors of vesicles to lysosomes, while the MPRs are transported in membranes either back to the TGN or to the plasma membrane. The Man 6-P/IGF2Rs in the plasma membrane are able to bind to extracellular molecules that express Man 6-P groups, such as those of lysosomal enzymes, and mediate the endocytosis of these ligands.
- The inventors surprisingly found that newly assembled VZV is diverted from the secretory pathway of infected cells by Man 6-P/IGF2Rs, which is thus responsible for the delivery of enveloped virions to endosomes. This is supported by observations that glycoproteins of the VZV envelope contain Man 6-P groups. In addition, the simple addition of Man 6-P to the medium in which cells are growing prevents these cells from becoming infected by cell-free VZV. Man 6-P interferes with the interaction between plasma membrane Man 6-P/IGF2R and the Man 6-P groups of extracellular molecules. Man 6-P does not prevent the cell-to-cell spread of VZV in vitro, which because it depends on the fusion of adjacent cells, which is independent of the interaction of viral Man 6-P with Man 6-P/IGF2R. These observations suggest that the Man 6-P groups of the glycoproteins of the VZV envelope interact with cellular Man 6-P/IGF2Rs and thus play critical roles in the ability of cell-free VZV to infect target cells and in the transport within infected cells of newly assembled VZV from the TGN to endosomes.
- Accordingly, the inventors produced a cell line that is deficient in Man 6-P/IGF2R. Because of the strong preference of VZV for human cells, the Man 6-P/IGF2R-deficient cell line was derived from MeWo cells, a human melanoma tumor cell line, which is known to be susceptible to infection by VZV and to support the growth of VZV in vitro.
- The Man 6-P/IGF2R-deficient MeWo cells were found to be far more resistant to infection by cell-free VZV than their parental control MeWo cells; however, infected cells can transfer VZV infection to the Man 6-P/IGF2R-deficient MeWo cells as readily as to the parental control MeWo cells. The medium in which parental control MeWo cells is growing is not infectious and cannot be used to transfer infection with VZV to susceptible target cells (human embryonic lung cells [HELF] are employed as the susceptible targets). In contrast, the medium in which Man 6-P/IGF2R-deficient MeWo cells is growing does contain infectious VZV and can be used to transfer infection with VZV to target HELF cells. This is the first cell line that, when infected with VZV, releases substantial quantities of infectious VZV particles to the ambient medium. The medium in which the Man 6-P/IGF2R-deficient MeWo cells are growing is thus extraordinarily useful as a source of intact, non-degraded, infectious VZV. The resistance of the Man 6-P/IGF2R-deficient MeWo cells to infection by cell-free VZV confirms that the Man 6-P/IGF2R plays an important role in enabling VZV to enter target cells. Infection of Man 6-P/IGF2R-deficient MeWo cells must be accomplished by adding other infected cells to them. The infected cells can thus infect the Man 6-P/IGF2R-deficient MeWo cells by cell-to-cell contact, which is Man 6-P/IGF2R-independent.
- The successful maintenance and easy use of the Man 6-P/IGF2R-deficient MeWo cells depends on the knockdown of the Man 6-P/IGF2R being incomplete. When the Man 6-P/IGF2R expression is abolished, the resulting cells grow poorly and slowly. The current Man 6-P/IGF2R-deficient MeWo cells express small amounts of the Man 6-P/IGF2R and thus grow adequately, can be maintained without excess difficulty, yet still release infectious VZV.
- Uses of the Man 6-P/IGF2R-deficient MeWo cells include the development of new and reliable methods to produce the VZV vaccine. This vaccine is an attenuated live virus that must be produced by cultured cells. It grows in culture, like wild-type VZV, only by cell-to-cell contact. The final product has to be liberated from the small intracellular compartment that contains newly assembled virions that have not yet been transported to endosomes. Virions that have been degraded in endosomes are useless as components of the vaccine. Yields of live virus are thus very low. Propagation in the Man 6-P/IGF2R-deficient MeWo cells should produce high yields of much more readily purified viable virus. In addition to being extremely useful in vaccine production, the Man 6-P/IGF2R-deficient MeWo cells should be just as helpful in developing new strains of VZV for future vaccines. Finally, the virus produced in Man 6-P/IGF2R-deficient MeWo cells is likely to be much more uniform and readily controlled than that produced in ordinary tissue culture cells. The non-controllable degradation of virions is avoided, yields are improved, and a step of cell lysis can be avoided in vaccine production. Contamination of the vaccine with cellular constituents and other viruses that might be contained in tissue culture cells can also be minimized.
- The Man 6-P/IGF2R-deficient MeWo cells will also help in research on VZV itself. As such, Man 6-P/IGF2R-deficient MeWo cells will be of great value and sought after by research workers hoping to develop antiviral drugs and those seeking to understand the basic properties of VZV. Although the Man 6-P/IGF2R has been most strongly implicated as important, as described in the biology of VZV, the receptor may also play roles in the biology of other related herpesviruses, such as herpes
simplex virus types - The present invention is described in the following Examples, which are set forth to aid in the understanding of the invention, and should not be construed to limit in any way the scope of the invention as defined in the claims which follow thereafter.
- The deficient expression of Man 6-P/IGF2Rs was induced by stably transfecting parent MeWo cells (available from the European Collection of Cell Cultures (ECACC) Salisbury, Wiltshire) with cDNA encoding the full-length Man 6-P/IGF2R in the antisense configuration. The cDNA construct was packaged in a retroviral vector that also contained a puromycin resistance gene. The cells that were infected by the vector, and thus expressed mRNA encoding the Man 6-P/IGF2R in the antisense configuration, was selected in media containing puromycin. The success of the knockdown of the Man 6-P/IGF2R was confirmed by Western analysis and by immunocytochemistry; both techniques demonstrated reduced Man 6-P/IGF2R expression. In addition, the Man 6-P/IGF2R-deficient MeWo cells were found to contain less cathepsin D (a lysosomal enzyme that was investigated as a marker) than control parental MeWo cells. This demonstration was again accomplished by Western analysis and by immunocytochemistry. The Man 6-P/IGF2R-deficient cells also secreted a greater amount of another lysosomal enzyme, acid phosphatase, than parental MeWo cells (shown by direct measurement of acid phosphatase activity in cells and in media and by Western analyses of media and cell lysates).
- The foregoing results are summarized in
FIGS. 1-15 . The data confirm that the Man 6-P/IGF2R-deficient MeWo cells have a reduced ability to divert lysosomal enzymes from their secretory pathway to endosomes. - VZV has a narrow host range. However, it is known to be neurotropic, and to become latent in sensory ganglia. Because the enteric nervous system (ENS) contains intrinsic sensory neurons, the inventors postulated that VZV may infect, and become latent in, the ENS (e.g., enteric neurons and/or enteric ganglia).
- The ENS is an independent autonomic division that is capable of regulating the behavior of the gut without input from the central nervous system (CNS). The ENS has independence from the CNS because it contains two main types of intrinsic primary afferent neurons (IPANS): submucosal IPANS (containing cholinergic-CGRP or substance-P) or myenteric IPANS (containing cholinergic calbindin). The inventors found that reactivation of VZV in enteric neurons could provide a source of visceral zoster, and present a model for understanding the origin of zoster. This was established by first testing the ability of VZV to establish latency in guinea pig enteric ganglia in vitro, and then by testing the ability of the latent VZV to reactivate.
- The inventors first isolated ganglia from guinea pig small intestine. The LM-MP was mechanically dissected from the bowel, and dissociated with collagenase. Myenteric ganglia then were individually selected and cultured. Mitotic inhibitors were used to decrease growth of non-neuronal cells, enabling enteric neurons to reorganize and interconnect. Cell-free VZV was adsorbed for 4 h, approximately 5 days after the ganglia were plated. Cultures were maintained for 4-6 weeks thereafter.
- As shown in
FIGS. 16-25 , VZV infected guinea pig enteric ganglia in vitro. Latent infection occurred with the use of cultures highly enriched in neurons. It was observed that neurons expressed only those VZV proteins that had been reported to be expressed during VZV latency in human sensory ganglia (no glycoproteins). In this regard, it is noted that ORF62p and ORF29p are cytoplasmic, not nuclear. Lytic infection required the presence of non-neuronal cells. Both neurons and non-neuronal cells expressed glycoproteins, and ORF62p and ORF29p were found in nuclei. Reactivation occurred in neurons, and likely occurred in glia. Reactivation was induced by expression of HSV ICP0 (the HSV homologue of VZV ORF61). Neurons expressed glycoproteins, and ORF29p was found in nuclei. The model of the in vitro reactivation of VZV described herein is the first in vitro model for shingles. - While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be appreciated by one skilled in the art, from a reading of the disclosure, that various changes in form and detail can be made without departing from the true scope of the invention in the appended claims.
Claims (10)
1. A vaccine comprising an attenuated live virus produced by culturing genetically-engineered cells stably transformed with a nucleotide sequence encoding at least two full-length mannose-6-phosphate receptors and a pharmaceutically acceptable carrier.
2. A method for generating an in vitro system for Varicella zoster virus (VZV), comprising the steps of:
(a) isolating enteric ganglia from guinea pig; and
(b) contacting the enteric ganglia with cell-free VZV to generate latent expression of the VZV.
3. An in vitro system for VZV generated by the method of claim 2 .
4. The in vitro system of claim 3 , wherein the method further comprises the step of reactivating the VZV by contacting the system with a vector containing a nucleic acid sequence coding for VZV ORF61 or a homologue thereof.
5. The in vitro system of claim 4 , wherein the homologue is HSV ICP0.
6. An in vitro system for Varicella zoster virus (VZV), comprising an enteric ganglion that has been contacted with cell-free VZV to produce a latent VZV, wherein the VZV is subsequently reactivated to express VZV in an active form.
7. A method for reactivating Varicella zoster virus (VZV), comprising the steps of:
(a) isolating enteric ganglia from guinea pig;
(b) contacting the enteric ganglia with cell-free VZV to generate latent expression of the VZV; and
(c) contacting the infected ganglia with a vector containing a nucleic acid sequence encoding VZV ORF61 or a homologue thereof.
8. The method of claim 7 , wherein the homologue is HSV ICP0.
9. A Varicella zoster virus reactivated by the method of claim 10 .
10. A method of screening for an agent for treating Varicella zoster virus (VZV) infection, comprising use of an in vitro system for VZV, wherein the in vitro system comprises an enteric ganglion that has been contacted with cell-free VZV to produce a latent VZV, and wherein the VZV is subsequently reactivated to express VZV in an active form.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US11/903,708 US20080279888A1 (en) | 2002-05-10 | 2007-09-24 | Genetically engineered cell lines and systems for propagating varicella zoster virus and methods of use thereof |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US37981902P | 2002-05-10 | 2002-05-10 | |
US10/436,706 US7273616B2 (en) | 2002-05-10 | 2003-05-12 | Genetically engineered cell lines and systems for propagating Varicella zoster virus and methods of use thereof |
US11/903,708 US20080279888A1 (en) | 2002-05-10 | 2007-09-24 | Genetically engineered cell lines and systems for propagating varicella zoster virus and methods of use thereof |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US10/436,706 Division US7273616B2 (en) | 2002-05-10 | 2003-05-12 | Genetically engineered cell lines and systems for propagating Varicella zoster virus and methods of use thereof |
Publications (1)
Publication Number | Publication Date |
---|---|
US20080279888A1 true US20080279888A1 (en) | 2008-11-13 |
Family
ID=29420560
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US10/436,706 Expired - Fee Related US7273616B2 (en) | 2002-05-10 | 2003-05-12 | Genetically engineered cell lines and systems for propagating Varicella zoster virus and methods of use thereof |
US11/903,708 Abandoned US20080279888A1 (en) | 2002-05-10 | 2007-09-24 | Genetically engineered cell lines and systems for propagating varicella zoster virus and methods of use thereof |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US10/436,706 Expired - Fee Related US7273616B2 (en) | 2002-05-10 | 2003-05-12 | Genetically engineered cell lines and systems for propagating Varicella zoster virus and methods of use thereof |
Country Status (3)
Country | Link |
---|---|
US (2) | US7273616B2 (en) |
AU (1) | AU2003229016A1 (en) |
WO (1) | WO2003095623A2 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2011016830A1 (en) * | 2009-07-28 | 2011-02-10 | The Trustees Of Columbia University In The City Of New York | Herpes virus backbone for viral vaccine and vaccine based thereon |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2011109600A1 (en) * | 2010-03-05 | 2011-09-09 | Alnylam Pharmaceuticals, Inc. | Compositions and methods for modifying the glycosylation pattern of a polypeptide |
Citations (61)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4780401A (en) * | 1984-04-09 | 1988-10-25 | Ciba-Geigy Corporation | Novel monoclonal antibodies to human renin and hybridoma cells, processes for their preparation and their applications |
US4845079A (en) * | 1985-01-23 | 1989-07-04 | Luly Jay R | Peptidylaminodiols |
US4885292A (en) * | 1986-02-03 | 1989-12-05 | E. R. Squibb & Sons, Inc. | N-heterocyclic alcohol renin inhibitors |
US4894437A (en) * | 1985-11-15 | 1990-01-16 | The Upjohn Company | Novel renin inhibiting polypeptide analogs containing S-aryl-D- or L- or DL-cysteinyl, 3-(arylthio)lactic acid or 3-(arylthio)alkyl moieties |
US4980283A (en) * | 1987-10-01 | 1990-12-25 | Merck & Co., Inc. | Renin-inhibitory pepstatin phenyl derivatives |
US5034512A (en) * | 1987-10-22 | 1991-07-23 | Warner-Lambert Company | Branched backbone renin inhibitors |
US5036054A (en) * | 1988-02-11 | 1991-07-30 | Warner-Lambert Company | Renin inhibitors containing alpha-heteroatom amino acids |
US5036053A (en) * | 1988-05-27 | 1991-07-30 | Warner-Lambert Company | Diol-containing renin inhibitors |
US5055466A (en) * | 1987-11-23 | 1991-10-08 | E. R. Squibb & Sons, Inc. | N-morpholino derivatives and their use as anti-hypertensive agents |
US5063208A (en) * | 1989-07-26 | 1991-11-05 | Abbott Laboratories | Peptidyl aminodiol renin inhibitors |
US5064965A (en) * | 1990-03-08 | 1991-11-12 | American Home Products Corporation | Renin inhibitors |
US5066643A (en) * | 1985-02-19 | 1991-11-19 | Sandoz Ltd. | Fluorine and chlorine statine or statone containing peptides and method of use |
US5071837A (en) * | 1990-11-28 | 1991-12-10 | Warner-Lambert Company | Novel renin inhibiting peptides |
US5075451A (en) * | 1990-03-08 | 1991-12-24 | American Home Products Corporation | Pyrrolimidazolones useful as renin inhibitors |
US5089471A (en) * | 1987-10-01 | 1992-02-18 | G. D. Searle & Co. | Peptidyl beta-aminoacyl aminodiol carbamates as anti-hypertensive agents |
US5095119A (en) * | 1990-03-08 | 1992-03-10 | American Home Products Corporation | Renin inhibitors |
US5095006A (en) * | 1989-05-08 | 1992-03-10 | Bayer Aktiengesellschaft | Renin inhibitors having all retro-inverted peptide bonds |
US5098924A (en) * | 1989-09-15 | 1992-03-24 | E. R. Squibb & Sons, Inc. | Diol sulfonamide and sulfinyl renin inhibitors |
US5104869A (en) * | 1989-10-11 | 1992-04-14 | American Cyanamid Company | Renin inhibitors |
US5106835A (en) * | 1988-12-27 | 1992-04-21 | American Cyanamid Company | Renin inhibitors |
US5114937A (en) * | 1989-11-28 | 1992-05-19 | Warner-Lambert Company | Renin inhibiting nonpeptides |
US5116835A (en) * | 1988-12-09 | 1992-05-26 | Hoechst Aktiengesellschaft | Enzyme-inhibiting urea derivatives of dipeptides, a process for the preparation thereof, agents containing these, and the use thereof |
US5120737A (en) * | 1990-09-28 | 1992-06-09 | Kyowa Hakko Kogyo Co., Ltd. | Hexitol derivatives |
US5256697A (en) * | 1992-04-16 | 1993-10-26 | Abbott Laboratories | Method of administering pyruvate and methods of synthesizing pyruvate precursors |
US5262165A (en) * | 1992-02-04 | 1993-11-16 | Schering Corporation | Transdermal nitroglycerin patch with penetration enhancers |
US5284872A (en) * | 1989-09-12 | 1994-02-08 | Schwarz Pharma Ag | Nitrato alkanoic acid derivatives, methods for their production, pharmaceutical compositions containing the derivatives and medicinal uses thereof |
US5344991A (en) * | 1993-10-29 | 1994-09-06 | G.D. Searle & Co. | 1,2 diarylcyclopentenyl compounds for the treatment of inflammation |
US5380790A (en) * | 1993-09-09 | 1995-01-10 | Eastman Chemical Company | Process for the preparation of acrylic polymers for pharmaceutical coatings |
US5380738A (en) * | 1993-05-21 | 1995-01-10 | Monsanto Company | 2-substituted oxazoles further substituted by 4-fluorophenyl and 4-methylsulfonylphenyl as antiinflammatory agents |
US5409944A (en) * | 1993-03-12 | 1995-04-25 | Merck Frosst Canada, Inc. | Alkanesulfonamido-1-indanone derivatives as inhibitors of cyclooxygenase |
US5428061A (en) * | 1988-09-15 | 1995-06-27 | Schwarz Pharma Ag | Organic nitrates and method for their preparation |
US5434178A (en) * | 1993-11-30 | 1995-07-18 | G.D. Searle & Co. | 1,3,5 trisubstituted pyrazole compounds for treatment of inflammation |
US5436265A (en) * | 1993-11-12 | 1995-07-25 | Merck Frosst Canada, Inc. | 1-aroyl-3-indolyl alkanoic acids and derivatives thereof useful as anti-inflammatory agents |
US5466823A (en) * | 1993-11-30 | 1995-11-14 | G.D. Searle & Co. | Substituted pyrazolyl benzenesulfonamides |
US5474995A (en) * | 1993-06-24 | 1995-12-12 | Merck Frosst Canada, Inc. | Phenyl heterocycles as cox-2 inhibitors |
US5510368A (en) * | 1995-05-22 | 1996-04-23 | Merck Frosst Canada, Inc. | N-benzyl-3-indoleacetic acids as antiinflammatory drugs |
US5552422A (en) * | 1995-01-11 | 1996-09-03 | Merck Frosst Canada, Inc. | Aryl substituted 5,5 fused aromatic nitrogen compounds as anti-inflammatory agents |
US5604253A (en) * | 1995-05-22 | 1997-02-18 | Merck Frosst Canada, Inc. | N-benzylindol-3-yl propanoic acid derivatives as cyclooxygenase inhibitors |
US5604260A (en) * | 1992-12-11 | 1997-02-18 | Merck Frosst Canada Inc. | 5-methanesulfonamido-1-indanones as an inhibitor of cyclooxygenase-2 |
US5639780A (en) * | 1995-05-22 | 1997-06-17 | Merck Frosst Canada, Inc. | N-benzyl indol-3-yl butanoic acid derivatives as cyclooxygenase inhibitors |
US5650447A (en) * | 1992-08-24 | 1997-07-22 | The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services | Nitric oxide-releasing polymers to treat restenosis and related disorders |
US5661129A (en) * | 1993-06-26 | 1997-08-26 | Schwarz Pharma Ag | Organic nitrates containing a disulfide group as cardiovascular agents |
US5703073A (en) * | 1995-04-19 | 1997-12-30 | Nitromed, Inc. | Compositions and methods to prevent toxicity induced by nonsteroidal antiinflammatory drugs |
US5807847A (en) * | 1996-06-04 | 1998-09-15 | Queen's University At Kingston | Nitrate esters |
US5859053A (en) * | 1995-05-02 | 1999-01-12 | Bayer Aktiengesellschaft | Acetylsalicylic acid nitrates |
US5874437A (en) * | 1996-11-01 | 1999-02-23 | Nitromed, Inc. | Nitrosated and nitrosylated phosphodiesterase inhibitor compounds, compositions and their uses |
US5876916A (en) * | 1996-03-18 | 1999-03-02 | Case Western Reserve University | Pyruvate compounds and methods for use thereof |
US5910316A (en) * | 1992-08-24 | 1999-06-08 | The United States Of America, As Represented By The Department Of Health And Human Services | Use of nitric oxide-releasing agents to treat impotency |
US5932538A (en) * | 1996-02-02 | 1999-08-03 | Nitromed, Inc. | Nitrosated and nitrosylated α-adrenergic receptor antagonist compounds, compositions and their uses |
US5932598A (en) * | 1996-04-12 | 1999-08-03 | G. D. Searle & Co. | Prodrugs of benzenesulfonamide-containing COX-2 inhibitors |
US5948433A (en) * | 1997-08-21 | 1999-09-07 | Bertek, Inc. | Transdermal patch |
US5958926A (en) * | 1996-11-01 | 1999-09-28 | Nitromed, Inc. | Nitrosated and nitrosylated phosphodiesterase inhibitor compounds, compositions and their uses |
US5994294A (en) * | 1996-02-02 | 1999-11-30 | Nitromed, Inc. | Nitrosated and nitrosylated α-adrenergic receptor antagonist compounds, compositions and their uses |
US6010715A (en) * | 1992-04-01 | 2000-01-04 | Bertek, Inc. | Transdermal patch incorporating a polymer film incorporated with an active agent |
US6071531A (en) * | 1995-06-07 | 2000-06-06 | Ortho-Mcneil Pharmaceutical, Inc. | Transdermal patch and method for administering 17-deacetyl norgestimate alone or in combination with an estrogen |
US6232336B1 (en) * | 1997-07-03 | 2001-05-15 | The United States Of America As Represented By The Department Of Health And Human Services | Nitric oxide-releasing amidine- and enamine-derived diazeniumdiolates, compositions and uses thereof and method of making same |
US6297260B1 (en) * | 1998-10-30 | 2001-10-02 | Nitromed, Inc. | Nitrosated and nitrosylated nonsteroidal antiinflammatory compounds, compositions and methods of use |
US6302685B1 (en) * | 1997-09-16 | 2001-10-16 | University Of Medicine And Dentistry Of New Jersey | Human lysosomal protein and methods of its use |
US6331542B1 (en) * | 1995-10-30 | 2001-12-18 | Smithkline Beecham Corporation | Protease inhibitors |
US6455542B1 (en) * | 1998-01-22 | 2002-09-24 | Oxon Medica Inc. | Piperidine and pyrrolidine derivatives comprising a nitric oxide donor for treating stress |
US6633272B1 (en) * | 1996-04-05 | 2003-10-14 | Matsushita Electric Industrial Co., Ltd. | Driving method, drive IC and drive circuit for liquid crystal display |
-
2003
- 2003-05-12 WO PCT/US2003/014825 patent/WO2003095623A2/en not_active Application Discontinuation
- 2003-05-12 AU AU2003229016A patent/AU2003229016A1/en not_active Abandoned
- 2003-05-12 US US10/436,706 patent/US7273616B2/en not_active Expired - Fee Related
-
2007
- 2007-09-24 US US11/903,708 patent/US20080279888A1/en not_active Abandoned
Patent Citations (73)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4780401A (en) * | 1984-04-09 | 1988-10-25 | Ciba-Geigy Corporation | Novel monoclonal antibodies to human renin and hybridoma cells, processes for their preparation and their applications |
US4845079A (en) * | 1985-01-23 | 1989-07-04 | Luly Jay R | Peptidylaminodiols |
US5066643A (en) * | 1985-02-19 | 1991-11-19 | Sandoz Ltd. | Fluorine and chlorine statine or statone containing peptides and method of use |
US4894437A (en) * | 1985-11-15 | 1990-01-16 | The Upjohn Company | Novel renin inhibiting polypeptide analogs containing S-aryl-D- or L- or DL-cysteinyl, 3-(arylthio)lactic acid or 3-(arylthio)alkyl moieties |
US4885292A (en) * | 1986-02-03 | 1989-12-05 | E. R. Squibb & Sons, Inc. | N-heterocyclic alcohol renin inhibitors |
US4980283A (en) * | 1987-10-01 | 1990-12-25 | Merck & Co., Inc. | Renin-inhibitory pepstatin phenyl derivatives |
US5089471A (en) * | 1987-10-01 | 1992-02-18 | G. D. Searle & Co. | Peptidyl beta-aminoacyl aminodiol carbamates as anti-hypertensive agents |
US5034512A (en) * | 1987-10-22 | 1991-07-23 | Warner-Lambert Company | Branched backbone renin inhibitors |
US5055466A (en) * | 1987-11-23 | 1991-10-08 | E. R. Squibb & Sons, Inc. | N-morpholino derivatives and their use as anti-hypertensive agents |
US5036054A (en) * | 1988-02-11 | 1991-07-30 | Warner-Lambert Company | Renin inhibitors containing alpha-heteroatom amino acids |
US5036053A (en) * | 1988-05-27 | 1991-07-30 | Warner-Lambert Company | Diol-containing renin inhibitors |
US5428061A (en) * | 1988-09-15 | 1995-06-27 | Schwarz Pharma Ag | Organic nitrates and method for their preparation |
US5116835A (en) * | 1988-12-09 | 1992-05-26 | Hoechst Aktiengesellschaft | Enzyme-inhibiting urea derivatives of dipeptides, a process for the preparation thereof, agents containing these, and the use thereof |
US5106835A (en) * | 1988-12-27 | 1992-04-21 | American Cyanamid Company | Renin inhibitors |
US5095006A (en) * | 1989-05-08 | 1992-03-10 | Bayer Aktiengesellschaft | Renin inhibitors having all retro-inverted peptide bonds |
US5063208A (en) * | 1989-07-26 | 1991-11-05 | Abbott Laboratories | Peptidyl aminodiol renin inhibitors |
US5284872A (en) * | 1989-09-12 | 1994-02-08 | Schwarz Pharma Ag | Nitrato alkanoic acid derivatives, methods for their production, pharmaceutical compositions containing the derivatives and medicinal uses thereof |
US5098924A (en) * | 1989-09-15 | 1992-03-24 | E. R. Squibb & Sons, Inc. | Diol sulfonamide and sulfinyl renin inhibitors |
US5104869A (en) * | 1989-10-11 | 1992-04-14 | American Cyanamid Company | Renin inhibitors |
US5114937A (en) * | 1989-11-28 | 1992-05-19 | Warner-Lambert Company | Renin inhibiting nonpeptides |
US5075451A (en) * | 1990-03-08 | 1991-12-24 | American Home Products Corporation | Pyrrolimidazolones useful as renin inhibitors |
US5064965A (en) * | 1990-03-08 | 1991-11-12 | American Home Products Corporation | Renin inhibitors |
US5095119A (en) * | 1990-03-08 | 1992-03-10 | American Home Products Corporation | Renin inhibitors |
US5120737A (en) * | 1990-09-28 | 1992-06-09 | Kyowa Hakko Kogyo Co., Ltd. | Hexitol derivatives |
US5071837A (en) * | 1990-11-28 | 1991-12-10 | Warner-Lambert Company | Novel renin inhibiting peptides |
US5262165A (en) * | 1992-02-04 | 1993-11-16 | Schering Corporation | Transdermal nitroglycerin patch with penetration enhancers |
US6010715A (en) * | 1992-04-01 | 2000-01-04 | Bertek, Inc. | Transdermal patch incorporating a polymer film incorporated with an active agent |
US5256697A (en) * | 1992-04-16 | 1993-10-26 | Abbott Laboratories | Method of administering pyruvate and methods of synthesizing pyruvate precursors |
US5910316A (en) * | 1992-08-24 | 1999-06-08 | The United States Of America, As Represented By The Department Of Health And Human Services | Use of nitric oxide-releasing agents to treat impotency |
US5650447A (en) * | 1992-08-24 | 1997-07-22 | The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services | Nitric oxide-releasing polymers to treat restenosis and related disorders |
US5604260A (en) * | 1992-12-11 | 1997-02-18 | Merck Frosst Canada Inc. | 5-methanesulfonamido-1-indanones as an inhibitor of cyclooxygenase-2 |
US5409944A (en) * | 1993-03-12 | 1995-04-25 | Merck Frosst Canada, Inc. | Alkanesulfonamido-1-indanone derivatives as inhibitors of cyclooxygenase |
US5380738A (en) * | 1993-05-21 | 1995-01-10 | Monsanto Company | 2-substituted oxazoles further substituted by 4-fluorophenyl and 4-methylsulfonylphenyl as antiinflammatory agents |
US5550142A (en) * | 1993-06-24 | 1996-08-27 | Merck Frosst Canada Inc. | Phenyl heterocycles as cox-2 inhibitors |
US5536752A (en) * | 1993-06-24 | 1996-07-16 | Merck Frosst Canada Inc. | Phenyl heterocycles as COX-2 inhibitors |
US5474995A (en) * | 1993-06-24 | 1995-12-12 | Merck Frosst Canada, Inc. | Phenyl heterocycles as cox-2 inhibitors |
US5661129A (en) * | 1993-06-26 | 1997-08-26 | Schwarz Pharma Ag | Organic nitrates containing a disulfide group as cardiovascular agents |
US5380790A (en) * | 1993-09-09 | 1995-01-10 | Eastman Chemical Company | Process for the preparation of acrylic polymers for pharmaceutical coatings |
US5344991A (en) * | 1993-10-29 | 1994-09-06 | G.D. Searle & Co. | 1,2 diarylcyclopentenyl compounds for the treatment of inflammation |
US5436265A (en) * | 1993-11-12 | 1995-07-25 | Merck Frosst Canada, Inc. | 1-aroyl-3-indolyl alkanoic acids and derivatives thereof useful as anti-inflammatory agents |
US5466823A (en) * | 1993-11-30 | 1995-11-14 | G.D. Searle & Co. | Substituted pyrazolyl benzenesulfonamides |
US5434178A (en) * | 1993-11-30 | 1995-07-18 | G.D. Searle & Co. | 1,3,5 trisubstituted pyrazole compounds for treatment of inflammation |
US5552422A (en) * | 1995-01-11 | 1996-09-03 | Merck Frosst Canada, Inc. | Aryl substituted 5,5 fused aromatic nitrogen compounds as anti-inflammatory agents |
US6057347A (en) * | 1995-04-19 | 2000-05-02 | Nitromed, Inc. | Compositions and methods to prevent toxicity induced by nonsteroidal antiinflammatory drugs |
US5703073A (en) * | 1995-04-19 | 1997-12-30 | Nitromed, Inc. | Compositions and methods to prevent toxicity induced by nonsteroidal antiinflammatory drugs |
US5859053A (en) * | 1995-05-02 | 1999-01-12 | Bayer Aktiengesellschaft | Acetylsalicylic acid nitrates |
US5604253A (en) * | 1995-05-22 | 1997-02-18 | Merck Frosst Canada, Inc. | N-benzylindol-3-yl propanoic acid derivatives as cyclooxygenase inhibitors |
US5639780A (en) * | 1995-05-22 | 1997-06-17 | Merck Frosst Canada, Inc. | N-benzyl indol-3-yl butanoic acid derivatives as cyclooxygenase inhibitors |
US5510368A (en) * | 1995-05-22 | 1996-04-23 | Merck Frosst Canada, Inc. | N-benzyl-3-indoleacetic acids as antiinflammatory drugs |
US6071531A (en) * | 1995-06-07 | 2000-06-06 | Ortho-Mcneil Pharmaceutical, Inc. | Transdermal patch and method for administering 17-deacetyl norgestimate alone or in combination with an estrogen |
US6331542B1 (en) * | 1995-10-30 | 2001-12-18 | Smithkline Beecham Corporation | Protease inhibitors |
US5932538A (en) * | 1996-02-02 | 1999-08-03 | Nitromed, Inc. | Nitrosated and nitrosylated α-adrenergic receptor antagonist compounds, compositions and their uses |
US5994294A (en) * | 1996-02-02 | 1999-11-30 | Nitromed, Inc. | Nitrosated and nitrosylated α-adrenergic receptor antagonist compounds, compositions and their uses |
US5876916A (en) * | 1996-03-18 | 1999-03-02 | Case Western Reserve University | Pyruvate compounds and methods for use thereof |
US6633272B1 (en) * | 1996-04-05 | 2003-10-14 | Matsushita Electric Industrial Co., Ltd. | Driving method, drive IC and drive circuit for liquid crystal display |
US5932598A (en) * | 1996-04-12 | 1999-08-03 | G. D. Searle & Co. | Prodrugs of benzenesulfonamide-containing COX-2 inhibitors |
US5883122A (en) * | 1996-06-04 | 1999-03-16 | Queen's University At Kingston | Nitrate esters and their use for neurological conditions |
US5807847A (en) * | 1996-06-04 | 1998-09-15 | Queen's University At Kingston | Nitrate esters |
US6221881B1 (en) * | 1996-11-01 | 2001-04-24 | Nitromed, Inc. | Nitrosated and nitrosylated phosphodiesterase inhibitor compounds, compositions and their uses |
US6232321B1 (en) * | 1996-11-01 | 2001-05-15 | Nitromed, Inc. | Nitrosated and nitrosylated phosphodiesterase inhibitor compounds, compositions and their uses |
US6172068B1 (en) * | 1996-11-01 | 2001-01-09 | Nitromed, Inc. | Nitrosated and nitrosylated phosphodiesterase inhibitor compounds, compositions and their uses |
US6177428B1 (en) * | 1996-11-01 | 2001-01-23 | Nitromed, Inc. | Nitrosated and nitrosylated phosphodiesterase inhibitor compounds, compositions and their uses |
US6197778B1 (en) * | 1996-11-01 | 2001-03-06 | Nitromed, Inc. | Nitrosated and nitrosylated phosphodiesterase inhibitor compounds, compositions and their uses |
US6211179B1 (en) * | 1996-11-01 | 2001-04-03 | Nitromed, Inc. | Nitrosated and nitrosylated phosphodiesterase inhibitor compounds, compositions and their uses |
US5958926A (en) * | 1996-11-01 | 1999-09-28 | Nitromed, Inc. | Nitrosated and nitrosylated phosphodiesterase inhibitor compounds, compositions and their uses |
US6133272A (en) * | 1996-11-01 | 2000-10-17 | Nitromed, Inc. | Nitrosated and nitrosylated phosphodiesterase inhibitor compounds, compositions and their uses |
US5874437A (en) * | 1996-11-01 | 1999-02-23 | Nitromed, Inc. | Nitrosated and nitrosylated phosphodiesterase inhibitor compounds, compositions and their uses |
US6316457B1 (en) * | 1996-11-01 | 2001-11-13 | Nitromed, Inc. | Nitrosated and nitrosylated phosphodiesterase inhibitor compounds, compositions and their uses |
US6232336B1 (en) * | 1997-07-03 | 2001-05-15 | The United States Of America As Represented By The Department Of Health And Human Services | Nitric oxide-releasing amidine- and enamine-derived diazeniumdiolates, compositions and uses thereof and method of making same |
US5948433A (en) * | 1997-08-21 | 1999-09-07 | Bertek, Inc. | Transdermal patch |
US6302685B1 (en) * | 1997-09-16 | 2001-10-16 | University Of Medicine And Dentistry Of New Jersey | Human lysosomal protein and methods of its use |
US6455542B1 (en) * | 1998-01-22 | 2002-09-24 | Oxon Medica Inc. | Piperidine and pyrrolidine derivatives comprising a nitric oxide donor for treating stress |
US6297260B1 (en) * | 1998-10-30 | 2001-10-02 | Nitromed, Inc. | Nitrosated and nitrosylated nonsteroidal antiinflammatory compounds, compositions and methods of use |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2011016830A1 (en) * | 2009-07-28 | 2011-02-10 | The Trustees Of Columbia University In The City Of New York | Herpes virus backbone for viral vaccine and vaccine based thereon |
CN102573898A (en) * | 2009-07-28 | 2012-07-11 | 纽约哥伦比亚大学理事会 | Herpes virus backbone for viral vaccine and vaccine based thereon |
Also Published As
Publication number | Publication date |
---|---|
WO2003095623A2 (en) | 2003-11-20 |
WO2003095623A3 (en) | 2004-09-02 |
US20060121048A1 (en) | 2006-06-08 |
US7273616B2 (en) | 2007-09-25 |
AU2003229016A1 (en) | 2003-11-11 |
AU2003229016A8 (en) | 2003-11-11 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Tischer et al. | A self-excisable infectious bacterial artificial chromosome clone of varicella-zoster virus allows analysis of the essential tegument protein encoded by ORF9 | |
Dorange et al. | Characterization of Marek's disease virus serotype 1 (MDV-1) deletion mutants that lack UL46 to UL49 genes: MDV-1 UL49, encoding VP22, is indispensable for virus growth | |
Dingwell et al. | Glycoproteins E and I facilitate neuron-to-neuron spread of herpes simplex virus | |
Schumacher et al. | The protein encoded by the US3 orthologue of Marek's disease virus is required for efficient de-envelopment of perinuclear virions and involved in actin stress fiber breakdown | |
Ringertz et al. | Cell hybrids | |
Skepper et al. | Herpes simplex virus nucleocapsids mature to progeny virions by an envelopment→ deenvelopment→ reenvelopment pathway | |
Chen et al. | Mannose 6-phosphate receptor dependence of varicella zoster virus infection in vitro and in the epidermis during varicella and zoster | |
Mulvey et al. | A herpesvirus ribosome-associated, RNA-binding protein confers a growth advantage upon mutants deficient in a GADD34-related function | |
Hahn et al. | The human cytomegalovirus ribonucleotide reductase homolog UL45 is dispensable for growth in endothelial cells, as determined by a BAC-cloned clinical isolate of human cytomegalovirus with preserved wild-type characteristics | |
Adler et al. | Virus reconstituted from infectious bacterial artificial chromosome (BAC)-cloned murine gammaherpesvirus 68 acquires wild-type properties in vivo only after excision of BAC vector sequences | |
Thomas et al. | Herpes simplex virus latency-associated transcript encodes a protein which greatly enhances virus growth, can compensate for deficiencies in immediate-early gene expression, and is likely to function during reactivation from virus latency | |
Klupp et al. | Glycoproteins required for entry are not necessary for egress of pseudorabies virus | |
CA2058955A1 (en) | Isolation growth and differentiation of human muscle cells | |
Moffat et al. | Functions of the C-terminal domain of varicella-zoster virus glycoprotein E in viral replication in vitro and skin and T-cell tropism in vivo | |
Gershon et al. | VZV infection of keratinocytes: production of cell-free infectious virions in vivo | |
Weise et al. | Replacement of glycoprotein B gene sequences in herpes simplex virus type 1 strain ANG by corresponding sequences of the strain KOS causes changes of plaque morphology and neuropathogenicity | |
Morimoto et al. | Differences in the regulatory and functional effects of the Us3 protein kinase activities of herpes simplex virus 1 and 2 | |
Tomishima et al. | In vivo egress of an alphaherpesvirus from axons | |
Lawler et al. | Gammaherpesvirus colonization of the spleen requires lytic replication in B cells | |
Wu et al. | Characterization of a replication-incompetent pseudorabies virus mutant lacking the sole immediate early gene IE180 | |
Fulmer et al. | UL20 protein functions precede and are required for the UL11 functions of herpes simplex virus type 1 cytoplasmic virion envelopment | |
CN106987559A (en) | A kind of construction method of recombinant C HOK1 cell lines and its application | |
Lebrun et al. | Varicella-zoster virus ORF9p binding to cellular adaptor protein complex 1 is important for viral infectivity | |
US20080279888A1 (en) | Genetically engineered cell lines and systems for propagating varicella zoster virus and methods of use thereof | |
Chuard et al. | The tegument protein pUL47 of Marek’s Disease Virus is necessary for horizontal transmission and is important for expression of glycoprotein gC |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: THE TRUSTEES OF COLUMBIA UNIVERSITY IN THE CITY OF Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:GERSHON, MICHAEL D.;ZHU, ZHENGLUN;SILVERSTEIN, SAUL;AND OTHERS;REEL/FRAME:021240/0412;SIGNING DATES FROM 20080703 TO 20080714 |
|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |