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:
RELATED APPLICATION 
       [0001]    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. 
     
    
     BACKGROUND OF THE INVENTION 
       [0002]      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. 
       SUMMARY OF THE INVENTION 
       [0003]    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. 
         [0004]    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. 
         [0005]    Also provided is a recombinant vector comprising a nucleotide sequence encoding at least two full-length mannose-6-phosphate receptors. 
         [0006]    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. 
         [0007]    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. 
         [0008]    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. 
         [0009]    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. 
         [0010]    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. 
         [0011]    Additional aspects of the present invention will be apparent in view of the description which follows. 
     
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         [0012]      FIG. 1  is an illustration of a  Varicella zoster  Virus (VZV) particle; 
           [0013]      FIG. 2  illustrates that mannose-6-phosphate receptors (MPRs) sort lysosomal enzymes and target them to endosomes; 
           [0014]      FIG. 3  illustrates intracellular transport of VZV and packing of the final envelope in the trans-Golgi network (TGN); 
           [0015]      FIG. 4  illustrates that MPRs direct newly assembled VZV to late endosomes; 
           [0016]      FIG. 5  illustrates the mechanism by which VZV particles receive their final envelope in the TGN; 
           [0017]      FIG. 6  demonstrates that human embryonic lung cells (HELF) infection by cell-free VZV is inhibited by phosphorylated sugars; 
           [0018]      FIG. 7  demonstrates that expression of cation-independent MPRs (MPR ci s) in MeWo cells is downregulated by antisense cDNA;W 
           [0019]      FIG. 8  shows that downregulation of MPR ci s inhibits infection by cell-free VZV; 
           [0020]      FIG. 9  demonstrates that MPR ci -KO (knock-out) cells can be infected by cell-associated VZV; 
           [0021]      FIG. 10  illustrates that MPR ci -KO cells release infectious VZV into the culture supernatant; 
           [0022]      FIG. 11  illustrates that VZV nucleocapsids assemble in the nuclei of infected KO-MeWo cells; 
           [0023]      FIG. 12  shows that enveloped VZV enters the perinuclear cisterna of KO-MeWo cells; 
           [0024]      FIG. 13  demonstrates that, in KO-MeWo cells, transport vesicles contain individual virions; 
           [0025]      FIG. 14  demonstrates that VZV accumulates in late endosomes in parental MeWo cells; 
           [0026]      FIG. 15  illustrates that enveloped VZV is released intact in the superficial epidermis; 
           [0027]      FIG. 16  shows that lytic infection of neurons occurs when non-neuronal cells are present; 
           [0028]      FIG. 17  illustrates that neurons die within two days when infection is lytic; 
           [0029]      FIG. 18  demonstrates that expression of HSV ICP0 causes VZV to reactivate in enteric neurons; 
           [0030]      FIG. 19  shows that latent VZV reactivates in enteric neurons forced to express HSV ICP0; 
           [0031]      FIG. 20  shows that cultured ganglia contain few non-neuronal cells; 
           [0032]      FIG. 21  demonstrates that RT-PCR reveals expression of VZV DNA and RNA in isolated ganglia; 
           [0033]      FIG. 22  shows that infected ganglia contain mRNA and DNA encoding VZV proteins; 
           [0034]      FIG. 23  illustrates that ORF29 mRNA is found by in situ hybridization in subsets of neurons; 
           [0035]      FIG. 24  demonstrates that infected neurons are ORF29p− but not gE-immunoreactive; 
           [0036]      FIG. 25  shows that ORF62p, 4p, and 21p are present in the cytoplasm of infected neurons. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0037]    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. 
         [0038]    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. 
         [0039]    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. 
         [0040]    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. 
         [0041]    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. 
         [0042]    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. 
         [0043]    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 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. The Man 6-P/IGF2R-deficient MeWo cells, finally, 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. 
         [0044]    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. 
       EXAMPLES 
     Example 1 
     Production of Man 6-P/IGF2R-Deficient Cells 
       [0045]    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). 
         [0046]    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. 
       Example 2 
     Development of Animal Model of VZV Latency 
       [0047]    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). 
         [0048]    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. 
         [0049]    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. 
         [0050]    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. 
         [0051]    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.