Patent Grant
7994295
The present invention relates to a recombinant Rhabdovirus and methods of generating same comprising a deletion in a nucleic acid sequence encoding a Rhabdoviral G protein with an insertion of a nucleic acid encoding a Rhabdoviral G stem polypeptide within a region of the G protein. Furthermore, the recombinant Rhabdovirus of the present invention is capable of expressing foreign nucleic acids within their genome.
Rhabdoviridae are membrane-enveloped viruses that are widely distributed in nature where they infect vertebrates, invertebrates, and plants. The Rhabdoviridae are divided into 6 genera, wherein Vesicular stomatitis virus (VSV) serves as a prototype for the vesiculovirus genus. Rhabdoviridae possess non-segmented, negative-strand RNA genomes of 11-12,000 nucleotides. The virus particles contain a helical, nucleocapsid core composed of the genomic RNA and protein. Generally, three proteins, termed N (nucleocapsid, which encases the genome tightly), P (formerly termed NS, originally indicating nonstructural), and L (large) are found to be associated with the nucleocapsid. An additional matrix (M) protein lies within the membrane envelope, perhaps interacting both with the membrane and the nucleocapsid core. A single glycoprotein (G) species spans the membrane and forms the spikes on the surface of the virus particle. The G protein is responsible for binding to host cells and facilitating membrane fusion. Because the Rhabdoviral genome is negative sense [i.e., complementary to host cell RNA sequences (positive sense) that comprise host mRNA], for transcription and translation of Rhabdoviral genes to occur, the virus must encode and package an RNA-dependent RNA polymerase in its virion. The RNA-dependant RNA polymerase is composed of the P and L proteins, which transcribes genomic RNA to subgenomic mRNA's encoding the 5-6 Rhabdoviral proteins and also replicates full-length positive and negative sense RNAs. Rhabdoviral genes are transcribed sequentially, starting at the 3′ end of the genome.
Due to its broad host range, simple genetic organization and rapid growth in cell culture, VSV has been used widely to study various aspects of Rhabdoviral entry, assembly and release. The virus enters cells via receptor-mediated endocytosis. Approximately 1200 VSV glycoprotein (G) molecules, organized as homotrimeric spikes anchored in the viral envelope, are responsible for virus attachment as well as for mediating fusion of the viral envelope with the endosomal membrane of the host cell following endocytosis. Low endosomal pH causes a conformational change in the G protein facilitating fusion of the viral envelope with the endosomal membrane (9, 2, 26). Fusion of the two membranes results in the release of the viral nucleocapsid into the host cell cytoplasm where viral replication then occurs.
VSV G protein differs from the prototypic viral fusion protein, influenza hemagglutinin (HA), in that G protein does not require proteolytic processing to become fusion-competent (33, 17). Also unlike influenza HA, the N-terminus of G, apart from the signal sequence, is not particularly hydrophobic and there is no obvious region in the amino acid sequence that can be defined as a “classical” fusion peptide (51). It was postulated that the VSV G fusion peptide is internal and that the region between amino acids (aa) 118-139 could be the putative fusion domain (33). Mutational analysis has provided evidence that the region between aa 118-136 corresponds to the G protein fusion peptide (14, 25, 53, 55).
Other regions of G protein have also been shown to be important for its fusion activity. Insertion of a 3 aa linker in the membrane-proximal domain at positions 410 and 415 abolished membrane fusion activity, indicating that this region may be important for fusion (25). Substitution of amino acids in the region between 395 and 418 also affected the fusogenic activity of G protein (47). When mutations in the fusion peptide were combined with point mutations in the membrane-proximal region between amino acids 395-418 fusion activity was inhibited additively. However, one double mutant G131A-G404A was more fusogenic than the two individual mutations alone, suggesting that these regions may interact during fusion (46).
The membrane proximal region of the VSV G protein is highly conserved among vesiculovirus members (41). Structure predictions for the region between aa 385 and 444 of the VSV GIND serotype glycoprotein have indicated that this region has a propensity to form α-helices suggestive of its direct interaction with cellular membranes (16). Recently the Applicants have demonstrated that the membrane proximal cctodomain of the “stem” region of the VSV G protein (G-stem or GS) is responsible for efficient VSV budding (41), a finding which may perhaps be technically exploited for the development of the next generation of recombinant VSV yectors. There is, therefore, an advantage in developing recombinant VSV vectors containing the membrane proximal “stem” region of the VSV G protein ectodomain as these may provide accelerated fusion activity, a result providing a multiple of biologically relevant applications.
In one embodiment, the present invention relates to a recombinant Rhabdovirus and methods of generating same. The recombinant Rhabdovirus comprises a deletion in a nucleic acid sequence encoding a Rhabdoviral G protein with an insertion of a nucleic acid encoding for a fragment of a Rhabdoviral G stem polypeptide within a region of the G protein. Specifically, the present invention relates to recombinant Rhabdoviruses expressing G stem polypeptides lacking significant portions of the G protein, potentiating their fusion with target cell membranes. Fusion is potentiated by the incorporation of a heterologous fusion facilitating polypeptide, and further still with the incorporation of an anti-receptor, which aids in specific targeting of the recombinant Rhabdovirus to a given cell type. The invention relates to isolated nucleic acids comprising the components as delineated above, including nucleic acid vectors comprising the isolated nucleic acids, and cells comprising the sequences and/or vectors.
In another embodiment of the present invention, there is provided a recombinant Rhabdovirus comprising a nucleic acid sequence of a Rhabdoviral genome, wherein the Rhabdoviral genome comprises a deletion in the nucleic acid sequence encoding a Rhabdoviral G protein, and an insertion in a nucleic acid sequence encoding a Rhabdoviral G stem polypeptide, or fragment thereof.
In another embodiment, the recombinant Rhabdovirus further comprises a nucleic acid, which encodes for a fusion-facilitating polypeptide, or fragment thereof, which may be expressed from a separate transcriptional unit. The fusion facilitating polypeptide may be derived from a simian virus 5 F protein, or an HIV envelope protein.
In another embodiment, the recombinant Rhabdovirus lacks amino acids 1 to 404 of the mature G protein.
In another embodiment, the recombinant Rhabdovirus encodes for a G stem polypeptide, or fragment thereof that comprises between at least 10 and 42 amino acid residues of a membrane-proximal region of a Rhabdoviral G protein. The G stem polypeptide may also comprise a transmembrane anchor and cytoplasmic tail region of a Rhabdoviral G protein. The encoded G stem polypeptide or fragment thereof may be further engineered to express an N-terminal epitope tag, of which c-myc, FLAG®, human influenza hemagglutinin (HA) or polyhistidine (including His6 or His 10) tags may be utilized.
In another embodiment, the nucleic acid sequence encoding a Rhabdoviral G stem polypeptide, or fragment thereof corresponds to or is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more, including 95%-100% homologous to SEQ ID No: 1. The encoded Rhabdoviral G stem polypeptide or fragment thereof, in turn corresponds to or is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more, including 95%-100% homologous to SEQ ID Nos: 2, 4, 5, 6, 7, 8 or 9.
In yet another embodiment, the recombinant Rhabdovirus may further comprise an insertion of a heterologous nucleic acid sequence encoding for a second polypeptide. The second polypeptide may function as a receptor that specifically binds a cognate ligand on a target cell membrane functioning in targeting the recombinant Rhabdovirus to a given host cell. In another embodiment, the stated polypeptide may comprise a ligand for binding to specific target cell receptors. Such ligands are also referred to as “anti-receptor” proteins and may comprise polypeptide fragments thereof, uncompromised in their targeting ability. The anti-receptor proteins may comprise antibodies or antibody fragments that recognize a target antigen, such as for example CD4, or erbB proteins expressed on target cell membranes. Other cell surface proteins used for targeting the recombinant Rhabdoviruses of the invention may include chemokine receptors, adhesion molecule receptors, MHC molecules, and others, all of which may be similarly applied.
In another embodiment, there is provided a recombinant virus comprising a nucleic acid sequence encoding for a Rhabdoviral G stem polypeptide, or fragment thereof. The Rhabdoviral G stem polypeptide may comprise between at least 10 and 42 amino acid residues of a membrane-proximal region, a transmembrane anchor and cytoplasmic tail region of a Rhabdoviral G protein, and/or be further engineered to express an N-terminal epitope tag, The Rhabdoviral G stem polypeptide will correspond to, or exhibit similar homology to sequences listed hereinabove.
The recombinant virus, may, according to another aspect of the invention, further comprise a nucleic acid sequence encoding for a fusion facilitating polypeptide. In other embodiments, the recombinant virus further comprises an insertion of a non-Rhabdoviral nucleic acid sequence, which may function as a receptor that specifically binds a cognate ligand on a target cell membrane to facilitate targeting the recombinant virus to a given host cell. In another embodiment, the nucleic acid sequence may comprise a ligand for binding to specific target cell receptors. Such ligands are also referred to as “anti-receptor” proteins and may comprise polypeptide fragments thereof, uncompromised in their targeting ability.
In another embodiment of the present invention, vectors comprising the recombinant Rhabdoviruses or recombinant viruses, and cells comprising these vectors are included. Viral particles and/or liposomes comprising the recombinant Rhabdoviruses or recombinant viruses are additional applications of the present invention.
The present invention provides for a method of fusing a cell with the recombinant Rhabdoviruses or recombinant viruses or cells expressing the viral products. The to method comprises contacting a cell with the recombinant Rhabdovirus, or recombinant virus, under conditions facilitating fusion of the viral envelope or outer cell membrane with a lipid bilayer of a target cell. In another embodiment, a method of fusing viral particles comprising recombinant Rhabdoviruses or recombinant viruses with a target cell is provided, the method as described herein.
The present invention further provides for a method of potentiating cell fusion, comprising the step of contacting a cell with a recombinant Rhabdovirus or recombinant virus, wherein the recombinant Rhabdovirus or recombinant virus expresses a heterologous fusion facilitating peptide, and a G stem polypeptide, under conditions facilitating fusion of the Rhabdoviral envelope with the lipid bilayer membrane of a target cell, resulting in potentiated fusion. In another embodiment, the method of potentiating cell fusion if via contacting a cell with a cell expressing the recombinant Rhabdovirus, or recombinant virus as described herein.
The present invention further provides a method of producing a recombinant Rhabdovirus comprising a deletion in the nucleic acid sequence encoding the Rhabdoviral G protein and an insertion of a nucleic acid sequence encoding a Rhabdoviral G stem polypeptide fragment and a nucleic acid sequence encoding a heterologous fusion facilitating polypeptide, the method comprising the steps of: (A) infecting a suitable cell with a minivirus comprising the cis-acting elements necessary for Rhabdoviral replication; (B) inserting into said cell a polynucleotide sequence encoding Rhabdoviral proteins, a nucleic acid sequence encoding a Rhabdoviral G stem polypeptide or a fragment thereof and a nucleic acid sequence encoding a heterologous fusion facilitating polypeptide; (C) culturing said cell under conditions that permit expression of the nucleic acid sequence to produce the recombinant Rhabdovirus; and (D) isolating said recombinant Rhabdovirus.
In another embodiment, there is provided an isolated nucleic acid molecule comprising a nucleic acid sequence encoding for a Rhabdoviral G stem polypeptide, or a fragment thereof, corresponding to or at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more, including 95%-100% homologous to SEQ ID Nos: 2, 4, 5, 6, 7, 8 or 9. The G stem polypeptide, or fragment thereof comprises between at least 10 and 42 amino acid residues of a membrane-proximal region of a Rhabdoviral G protein, and comprises a transmembrane anchor and cytoplasmic tail region of a Rhabdoviral G protein. The Rhabdoviral G stem polypeptide or fragment thereof may further comprise an N-terminal epitope tag, of which the FLAG synthetic or human influenza hemagglutining-derived polypeptide tags may be utilized.
In another embodiment, the isolated nucleic acid further comprises a nucleic acid sequence encoding for a fusion facilitating peptide. The isolated nucleic acid, may according to another embodiment, comprise a nucleic acid encoding for an anti-receptor protein or a polypeptide fragment thereof, as described herein, and may additionally comprise other non-Rhabdoviral nucleic acid sequences.
The invention provides, in another embodiment, a method of targeted cell lysis. The method comprises contacting a cell with an effective amount of a recombinant Rhabdovirus comprising a deletion in the nucleic acid sequence encoding a Rhabdoviral G protein, an insertion of a nucleic acid sequence encoding a Rhabdoviral G stem polypeptide, an insertion of a nucleic acid sequence encoding a heterologous fusion facilitating polypeptide, and an insertion of a nucleic acid sequence encoding an anti-receptor protein. In one embodiment of this aspect of the invention, following binding of the expressed anti-receptor to its cognate ligand or receptor on a target cell, the recombinant Rhabdovirus fuses with the target cell surface, facilitating viral entry, and subsequent target cell lysis.
—WT-VSV, ▬—GSHA virus,
—ΔG virus)
The present invention relates to a recombinant Rhabdovirus and methods of generating same. The recombinant Rhabdovirus is engineered to express a Rhabdoviral G stem polypeptide, or a fragment thereof and optionally a heterologous fusion facilitating polypeptide, in strains deleted for the Rhabdoviral glycoprotein (G).
As used herein, the term “recombinant Rhabdovirus” refers to a virus genetically engineered to express proteins not natively expressed in Rhabdoviridae. Engineering of the virus in this manner therefore creates a “pseudotype” or “chimeric” virus that can subsequently be isolated.
In one embodiment, the recombinant Rhabdovirus utilized for this invention is derived from Vesicular Stomatitis Virus (VSV), though the invention provides for the utilization of any virus of the Vesiculovirus and Lysavirus genus. The Vesiculovirus genus includes: Vesicular Stomatitis Virus (VSV) of the New Jersey serotype (VSVNJ), the Indiana serotype (VSVInd), the VSV-Alagoas strain, Cocal virus, Jurona virus, Carajas virus, Maraba virus, Piry virus, Calchaquivirus, Yug Bogdanovac virus, Isfahan virus, Chandipura virus, Perinet virus, and Porton-Svirus (Rose and Whitt. IN B. N. FIELDS' VIROLOGY 4th ED. VOL. 1 (1996)). The Lyssavirus genus includes: Rabies virus (RV), Lagos bat virus, Mokola virus, Duvenhagevirus, Obodhiang virus, and Kotonkan virus (ID.)
Methods for generating recombinant Rhabdoviruses may entail utilizing cDNAs and a Minivirus or a Helper Cell Line. In this case, both “miniviruses” and “helper cells” (also known as “helper cell lines”) provide a source of Rhabdoviral proteins for Rhabdovirus virion assembly, which are not produced from the transfected DNA encoding genes for Rhabdoviral proteins.
The generation of recombinant Rhabdovirus can be accomplished using: (1) cDNA's alone; (2) cDNAs transfected into a helper cell in combinations; or (3) cDNA transfection into a cell, which is further infected with a minivirus providing in trans the remaining components or activities needed to produce either an infectious or non-infectious recombinant Rhabdovirus. Using any of these methods (e.g., minivirus, helper cell line, or cDNA transfection only), the minimum components required are an to RNA molecule containing the cis-acting signals for (1) encapsidation of the genomic (or antigenomic) RNA by the Rhabdovirus N protein, and (2) replication of a genomic or antigenomic (replicative intermediate) RNA equivalent.
The term “minivirus” is meant to include incomplete viral particles containing a polycistronic nucleic acid molecule encoding N-P-M-L, N-P-L, N-P-G-L, M-G, G only, M only or any combination of four or fewer Rhabdoviral genes. This incomplete virus particle is incapable of viral replication, a process of the Rhabdoviral lifecycle involving a complete copying of its genome.
Copying of the Rhabdoviral genome, referred to as “Rhabdoviral replication” requires, the presence of a replicating element or replicon, which, herein signifies a strand of RNA minimally containing at the 5′ and 3′ ends the leader sequence and the trailer sequence of a Rhabdovirus. In the genomic sense, the leader is at the 3′ end and the trailer is at the 5′ end. Any RNA placed between these two replication signals will in turn be replicated. The leader and trailer regions further must contain the minimal cis-acting elements for purposes of encapsidation by the N protein and for polymerase binding, which are necessary for initiating Rhabdoviral transcription and replication.
In order to produce the recombinant Rhabdovirus of the present invention the cDNA's encoding the modified Rhabdoviral genome listed above must be contacted with a cell under conditions facilitating expression of the vectors employed, permitting production of the recombinant Rhabdovirus. It is to be understood that any cell permitting assembly of the recombinant Rhabdovirus for any one of the three methods disclosed above are included as part of the present invention.
Culturing of Cells to Produce Virus: Transfected cells are usually incubated for at least 24 hours at the desired temperature, usually about 37° C. For generation of infectious virus particles, the supernatant, which contains recombinant virus is harvested and transferred to fresh cells. The fresh cells expressing the G protein (either via transient or stable transfection) are incubated for approximately 48 hours, and the supernatant is collected.
Purification of the Recombinant Rhabdovirus: The terms “isolation” or “isolating” a Rhabdovirus signifies the process of culturing and purifying virus particles such that very little cellular debris remains. One example would be to collect the is virion-containing supernatant and filter (0.2μ pore size) (e.g., Millex-GS, Millipore) the supernatant thus removing Vaccinia virus and cellular debris. Alternatively, virions can be purified using a gradient, such as a sucrose gradient. Recombinant Rhabdovirus particles can then be pelleted and resuspended in whatever excipient or carrier is desired. Viral titers can be determined by serial dilution of supernatant used to infect cells, whereupon following expression o viral proteins, infected cells are quantified via indirect immunofluorescence using for example, anti-M (23H12) or anti-N (10G4) protein specific antibodies (L. Lefrancois et al., (1982) Virology 121: 157-67). It is therefore to be understood that in recombinant Rhabdoviral particles are considered as part of the invention, as well.
For purposes of infecting cells (such as, for example, tissue culture cells or cells from a tissue sample, such as a biopsy), the isolated recombinant Rhabdovirus is incubated with the cells using techniques known in the art. Detection of infection by the recombinant Rhabdovirus could proceed by determining the presence of a reporter gene, such as a green fluorescent protein (GFP), or via assessment of viral protein expression, as determined by indirect immunofluorescence, as discussed above.
To prepare infectious virus particles, an appropriate cell line (e.g., BHK cells) is first infected with vaccinia virus vTF7-3 (T. R. Fuerst et al., (1986) Proc. Natl. Acad. Sci. USA 3. 8122-26) or equivalent which encodes a T7 RNA polymerase or other suitable bacteriophage polymerase such as the T3 or SP6 polymerases (see Usdin et al., (1993) BioTechniques 14:222-224 or Rodriguez et al. (1990) J. Virol. 64:4851-4857). Alternatively, a vaccinia-free system may be utilized which provides an RNA polymerase. The cells are then transfected with individual cDNA containing the genes encoding the N, P, G and L Rhabdoviral proteins. These cDNAs will provide the proteins for building the recombinant Rhabdovirus particle. Cells can be transfected by to any method known in the art (e.g., liposomes, electroporation, etc.).
Thus recombinant Rhabdoviruses and/or particles can be prepared, assembled and isolated. The present invention discloses a recombinant Rhabdovirus comprising a deletion in a nucleic acid sequence encoding a Rhabdoviral G protein, an insertion of a nucleic acid sequence encoding a Rhabdoviral G stem polypeptide or a fragment thereof.
The full nucleic acid sequence encoding for a Rhabdoviral G stem polypeptide is as set forth in SEQ ID No: 1, and is a sequence encoding for a Rhabdoviral G stem polypeptide of the Indiana strain. The full amino acid sequence of a Rhabdoviral G stem polypeptide of the Indiana strain is as set forth in SEQ ID No: 2. Truncations resulting in Rhabdoviral G stem polypeptide fragments are listed in SEQ ID Nos: 3-9, and their functional efficacy is disclosed in the examples section below.
The recombinant Rhabdoviruses of the present invention further comprise a nucleic acid sequence encoding a heterologous fusion facilitating polypeptide. In another embodiment of the present invention, the nucleic acid sequence encoding for a fusion facilitating polypeptide is expressed from a separate transcriptional unit.
The present invention also provides for nucleic acid vectors comprising the recombinant Rhabdoviruses described above. The present invention additionally provides for nucleic acid vectors containing isolated nucleic acids described herein.
The isolated nucleic acid sequences of the present invention comprise Rhabdoviral genomes containing a deletion in the nucleic acid sequence encoding/a Rhabdoviral G protein, and insertion of nucleic acid sequences encoding Rhabdoviral G stem polypeptides or fragments thereof, with or without the inclusion of sequences encoding a heterologous fusion facilitating polypeptide.
In another embodiment, the isolated nucleic acid described may be with or without the inclusion of sequences encoding an anti-receptor for cell targeting.
Another aspect of the present invention provides for an isolated nucleic acid sequence encoding for a Rhabdoviral G stem polypeptide alone. The isolated nucleic acid may contain additional sequences encoding non-Rhabdoviral proteins, such as fusion-facilitating polypeptides and/or anti-receptors for targeting. It is to be understood that these isolated nucleic acid sequences may comprise part of a nucleic acid vector.
In another embodiment, there is provided a recombinant virus comprising a nucleic acid sequence encoding for a Rhabdoviral G stem polypeptide alone. The recombinant virus may comprise additional nucleic acid sequences encoding for a fusion-facilitating peptide and/or an anti-receptor, facilitating direction of the recombinant virus to a target cell. It is to be understood that any virus that may be engineered to include the sequences listed herein are to be considered as part of the invention.
In one embodiment, nucleic acid sequences encoding Rhabdoviral G stem polypeptides, or fragments thereof, correspond to, or are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more, including 95%-100%, homologous to the sequence delineated in SEQ ID No: 1. The nucleic acid sequences delineated herein encode for a G stem polypeptide, or a fragment thereof, corresponding to, or being at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more, including 95%-100%, homologous to SEQ ID No: 2, 4, 5, 6, 7, 8 or 9.
According to another embodiment, nucleic acid vectors comprising the isolated nucleic acid sequences delineated herein include a promoter for regulating expression of the isolated nucleic acid. Such promoters are known to be cis-acting sequence elements required for transcription as they serve to bind DNA dependent RNA polymerase, which transcribes sequences present downstream thereof.
A vector according to the present invention preferably further includes an appropriate selectable marker. The vector may further include an origin of replication, and may be a shuttle vector, which can propagate both in bacteria, such as, for example, E. coli (wherein the vector comprises an appropriate selectable marker and origin of replication) and be compatible for propagation in vertebrate cells, or integration in the genome of an organism of choice. The vector according to this aspect of the present invention can be, for example, a plasmid, a bacmid, a phagemid, a cosmid, a phage, a virus or an artificial chromosome.
Consequently, according to yet another aspect of the present invention there is provided a host cell containing the recombinant Rhabdoviruses and/or nucleic acid vectors as described herein. The cell may be a prokaryotic or a eukaryotic cell.
As used herein, the term “Rhabdoviral G protein” is meant to include the glycoprotein (G) encoded by Rhabdoviridae, whose presence contributes to the overall efficiency of the viral budding process (Whitt M. A., (1998) The Journal of Microbiology 36: 1-8). The phrase “G stem polypeptide” or “fragment of a G stem polypeptide” refers to segments of a Rhabdoviral G protein, or polypeptides with at least at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more, including 95%-100% homology to at least some, and in one embodiment, at least 10 of the 42 membrane-proximal amino acid residues of the G protein ectodomain. In another embodiment, the G stem polypeptide or fragment thereof also includes the transmembrane anchor and cytoplasmic tail regions of the mature G protein.
As is evidenced in the Examples section herein, the G stem protein or polypeptide fragment thereof functions to potentiate membrane fusion. Consequently, utilization of G stem polypeptides may enhance recombinant virus production, viral titers and infectivity via their potentiation of membrane fusion. The addition of a G stem polypeptide can increase infectivity of a Rhabdovirus vector by 10 to 100 fold.
In one embodiment, the G stem polypeptide utilized is comprised of a Rhabdoviral G protein lacking amino acids 1 to 403 of the mature G protein. The G stem polypeptide may comprise between 10 and 42 amino acid residues of the membrane-proximal region of the G protein. The Rhabdoviral G stem polypeptide may be engineered to include an N-terminal epitope tag, of which c-myc, FLAGS®, human influenza hemagglutinin (HA) or polyhistidine (including His6 or His 10) tags may be utilized.
One example of a VSV G stem polypeptide is that derived from the VSV G stem polypeptide of the VSV-Indiana serotype (VSVInd) (GenBank Accession No. 61834). G stem polypeptides derived from VSVInd, beginning with amino acid residue 336 (N) are contemplated herein. More preferred G stem polypeptides are derived from VSVInd G stem polypeptides beginning with residue 392 (T). Most preferred G stem polypeptides include residues spanning 404 (G)-511 (K). Another preferred VSVInd G stem includes the polypeptide comprising 427 (Q)-511 (K), or 440 (F)-511 (K). Other contemplated VSVInd G stems include G stems beginning with residue 404 (G) of the G protein. Less preferred are those VSVInd G stems derived from residues downstream of 449 (N), because although G stems comprising these residues possess a high assembly phenotype, they demonstrate poor potentiation of membrane fusion, as compared to G stem polypeptides derived from residues upstream of 449 (N). The preferred VSVInd G stem polypeptides comprise the entire cytoplasmic tail domain and all of the transmembrane domain of the G protein, as well as portions of the carboxy terminus of the membrane proximal ectodomain (e.g., at least 14 to roughly 127 amino acids of the carboxy terminus of the G protein ectodomain).
Additional G stem polypeptides comprising preferred embodiments of the present invention include the incorporation of similar regions of G proteins derived from all VSV serotypes and all strains within those serotypes, as well as analogous G stem polypeptides derived from alternate Rhabdoviral G proteins. It is to be understood that other polypeptides exhibiting at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more, including 95%-100%, homology or identity to SEQ ID Nos: 2, 4, 5, 6, 7, 8 or 9 are to be included as part of this invention, as they display sequence homology/identity with Rhabdoviral G stem polypeptides. The invention is also meant to include polypeptides with a lesser degree of homology, yet exhibiting a functional equivalence, in terms of potentiation of membrane fusion are to be considered included as part of this invention.
The recombinant Rhabdovirus of the present invention may be comprised of a nucleic acid sequence encoding for a G stem polypeptide or fragment thereof that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more, including 95%-100% homology or identity to SEQ ID NO: 1, as determined by, for example, the Smith-Waterman algorithm, utilized in analyzing sequence alignment protocols, as in for example, the GAP, BESTFIT, FASTA and TFASTA programs in the Wiconsin Genetics Software Package release 7.0, Genetcis Computer Group, 575 Science Dr., Madison, Wis.).
Alternatively or additionally, polynucleotide homology may be determined by hybridization to SEQ ID NO: 1, which may be effected by stringent or moderate hybridization conditions. An example of stringent hybridization is the use of a hybridization solution containing 10% dextran sulfate, 1 M NaCl, 1% SDS and 5×106 cpm 32P labeled probe, at 65° C., with a final wash solution of 0.2×SSC and 0.1% SDS and final wash at 65° C.; whereas an example of moderate hybridization would be the use of a hybridization solution containing 10% dextran sulfate, 1 M NaCl, 1% SDS and 5×106 cpm 32P labeled probe, at 65° C., with a final wash solution of 1×SSC and 0.1% SDS and final wash at 50° C.
The recombinant Rhabdovirus of the invention preferably additionally comprises a fusion facilitating polypeptide participating in G stem mediated potentiation of membrane fusion. As used herein, the term “fusion-facilitating polypeptide” refers to any protein (or fusion-facilitating polypeptide fragment thereof) that following expression on the surface of a vesicular membrane precipitates fusion of the vesicular membrane with a lipid-bilayer encasing a target vesicle or cell. In another embodiment of the present invention, the fusion-facilitating polypeptide is: (1) is derived from a virus characterized as having a lipid envelope; and (2) when expressed as a heterologous protein in a genetically engineered virus, facilitates the fusion of the viral envelope with a cell membrane, resulting in a complete bilayer fusion between participating membranes. It is thus envisioned that a fusion-facilitating polypeptide according to the present invention can function in a non-specific fashion in facilitating the association of an attachment protein on the viral envelope other than the native viral attachment protein. One example of a fusion-facilitating polypeptide as contemplated herein is the viral envelope fusion protein known in the literature as the “F protein” of the SV5 strain of Paramyxoviruses, which specifically is referred to herein as the “F protein” rather than the more generic “F Protein” or “Fusion Protein”.
In addition to simian virus 5 (SV5)-derived F proteins, fusion facilitating polypeptides may be selected from HIV envelope proteins, as well as VSV GNJ (New Jersy serotype) or VSV GIND (Indiana serotype) proteins. Also included are polypeptides exhibiting at least 70% amino acid sequence homology to the above mentioned fusion polypeptides, as well as polypeptides exhibiting significant functional homology in terms of stimulating target cell fusion with the recombinant Rhabdoviruses and expressed nucleic acid sequences of the present invention. It is to be understood that utilization of any protein stimulating membrane fusion, or a fragment thereof is to considered within the scope of the invention, as are homologues of such proteins and their fragments, and that these proteins may be of prokaryotic or eukaryotic origin. Proteins and polypeptides derived by protein evolution techniques well known to those skilled in the art are included as well.
The recombinant Rhabdoviruses of the invention, as described above enable potentiation of viral fusion, in the absence of the Rhabdoviral G protein, as a function of expression of fusion facilitating polypeptides, where the additional presence of the Rhabdoviral G stem polypeptide provides for an enhanced rate/magnitude of fusion.
Similarly, prokaryotic and/or eukaryotic cells expressing fusion facilitating polypeptides and Rhabdoviral G stem polypeptides on their cell surfaces may provide a fusion potentiation effect, as a result of surface expression of said polypeptides.
In another embodiment of the invention, there is provided a recombinant virus, comprising a nucleic acid sequence encoding a Rhabdoviral G stem polypeptide or a fragment thereof. The Rhabdoviral G stem polypeptide or fragment thereof, as above, will be at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more, including 95%-100%, homologous to SEQ ID No:1. The Rhabdoviral G stem polypeptide will comprise between at least 10 and 42 amino acid residues of a membrane-proximal region of the G protein, corresponding to, or at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more, including 95%-100%, homologous to SEQ ID Nos: 2, 4, 5, 6, 7, 8 or 9. The Rhabdoviral G stem polypeptide or fragment thereof may also comprise a transmembrane anchor and cytoplasmic tail region of a Rhabdoviral G protein, and may additionally contain an N-terminal epitope tag, as described hereinabove.
In another embodiment, the recombinant virus further comprises a nucleic acid sequence encoding a fusion facilitating polypeptide. Other embodiments provide for the inclusion of nucleic acid sequences encoding anti-receptor proteins, or polypeptide fragments thereof, and the inclusion of additional non-Rhabdoviral sequences. The invention provides for a vector, a cell, a liposome or a viral particle comprising the recombinant virus herein disclosed, as well as compositions comprising some or all of these elements, as will be discussed further hereinbelow.
The present invention provides for a method of potentiating target cell fusion, comprising the steps of contacting the cell with an effective amount of a recombinant Rhabdoviral particle as described herein, where the recombinant Rhabdoviral particle envelope fuses with a membrane of a target cell at a rate or quantity greater than fusion of wild-type Rhabdoviral particles alone, thereby potentiating target cell fusion.
In another embodiment of the present invention, the method of potentiating target cell fusion, comprising contacting the target cell with an effective amount of cells expressing the recombinant Rhabdoviruses or isolated nucleic acid molecules/vectors described herein, where the membrane of the cell expressing the recombinant Rhabdoviruses or isolated nucleic acid molecules/vectors fuses with a membrane of a is target cell at a rate or quantity greater than fusion of wild-type cells alone, thereby potentiating target cell fusion.
It is to be understood that any vector, or cell expressing such a vector, encoding Rhabdoviral G stem polypeptides or fragments thereof, alone, or in combination with heterologous fusion facilitating polypeptides, are to be considered part of the present invention.
In keeping with this, in another embodiment of the invention, target cell fusion is potentiated via contacting a cell with an effective amount of recombinant virus, recombinant viral particles, or cells expressing the recombinant virus, as disclosed herein.
As a result of the disclosure herein of a fusion-potentiation effect mediated by a G stem polypeptide or fragment thereof, encoded by a negative-stranded RNA virus, including Rhabdoviridae, the present invention additionally relates to a method of interfering with, inhibiting or abrogating cell fusion. The method comprises the steps of contacting target cells infected with a Rhabdovirus, with an effective amount of an inhibitor of a Rhabdoviral G stem polypeptide or a fragment thereof, wherein the administration of the inhibitor serves to prevent expression or function of the Rhabdoviral G stem polypeptide, thereby interfering with, inhibiting or abrogating cell fusion.
In one embodiment, the target cells are infected with a negative stranded RNA virus, and the inhibitor is directed against a nucleic acid or a polypeptide that is at least 70% homologous to nucleic acid sequences encoding, or polypeptide sequences comprising Rhabdoviral G stem polypeptides, as disclosed herein. Rhabdoviral G stem polypeptide inhibitors may comprise isolated nucleic acid or recombinant viruses comprising an antisense molecule, siRNA or ribozyme directed against a nucleic acid molecule encoding for the Rhabdoviral G stem polypeptide. Construction of these inhibitors are by methods well known to one skilled in the art (See for example US Patent Application No. 20020086356A1 or Welch et al., Clin Diagn Virol. (1998) 10: 163-71).
In another embodiment, the Rhabdoviral G stem polypeptide inhibitor comprises a chemical inhibitor or the polypeptide preventing protein function. The inhibitor may comprise an antibody or antibody fragment, the design of which is via methodology well known to those skilled in the art.
The present invention further provides a means of directing recombinant Rhabdoviruses, and cells expressing translated products of the recombinant sequences delineated herein, to a particular cell. Direction may be a function of cell type per se, or differentiation state, or other factors, such as specific direction to infected cells within a given tissue.
The term “specific cell targeting” or “targeted cell” is therefore meant to refer to modifications introduced into the recombinant Rhabdoviruses, recombinant viruses, or cells expressing the translated products of the recombinant sequences delineated herein, facilitating direction of the virus/cell to interact with a particular cell. For example, as will be described, it will be possible to direct recombinant Rhabdoviruses to tumor cells expressing the surface marker erbB. Such erbB+ cells, in turn, would be referred to herein as “target cells” as these cells are the population with which the recombinant virus/cell expressing nucleic acids comprising sequences corresponding to the recombinant Rhabdovirus will ultimately fuse. Target cells often express a surface marker (referred to herein as “target antigen”) that may be utilized for directing the recombinant Rhabdovirus to the cell, as opposed to neighboring cells, that are not tumor to cells in origin and hence do not express erbB. Similarly, appropriately manipulated prokaryotic or eukaryotic cells engineered to incorporate nucleic acid sequences corresponding to those found in recombinant Rhabdoviruses, may be utilized, with these cells expressing targeting moieties, as above, on their cell surfaces.
The target antigen may be a receptor, therefore an “antireceptor,” also referred to as “attachment protein,” signifies a protein displayed on a recombinant Rhabdoviral envelope, or cell surface as described above, responsible for attachment of the viral particle/modified cell to its corresponding “receptor” on the target cell membrane. For example, the native antireceptor of the pararmyxovirus SV5 is the viral HN protein, which binds sialic acid on host cell membranes. Fusion thus accomplished is mediated via the binding of an attachment protein (or “antireceptor”) on the viral envelope to a cognate receptor on the cell membrane.
As used herein, the term “attachment” refers to the act of antireceptor (expressed on viral particle lipid envelopes or engineered cell sufaces) recognition and binding to a target cell surface “receptor” during infection. The skilled artisan will recognize that attachment occurs prior to fusion of the attaching membrane with the target cell plasma membrane.
The anti-receptor proteins or polypeptide fragments thereof may be derived from members of the following viral families: Arenaviridae, Bunyaviridae, Coronaviridae, Filoviridae, Flaviviridae, Herpesviridae, Hepadnaviridae, Orthomyxoviridae, Paramyxoviridae, Poxyiridae, Retroviridae, and Rhabdoviridae. Additional viral targeting agents may be derived from the following: African Swine Fever Virus, Boma Disease Virus, Hepatitis X, HIV-1, Human T Lymphocyte virus type-I (HTLV-1), HTLV-2, 1 5 lentiviruses, Epstein-Barr Virus, papilloma viruses, herpes simplex viruses, hepatitis B and hepatitis C.
Bacterial proteins expressed during intracellular infection are also potential targets contemplated for therapeutic intervention by recombinant Rhabdovirus of the present invention. The intracellular bacteria may include, amongst others: Shigella, Salmonella, Legionella, Streptococci, Mycobacteria, Francisella and Chlamydiae (See G. L. Mandell, “Introduction to Bacterial Disease” IN CECIL TEXTBOOK OF MEDICINE, (W.B. Saunders Co., 1996) 1556-7). These bacteria would be expected to express a bacteria-related protein on the surface of the infected cell to which the recombinant Rhabdovirus would attach.
Proteins of parasitic agents, which reside intracellularly, also are targets contemplated for infection by the recombinant Rhabdovirus. The intracellular parasites contemplated include for example, Protozoa. Protozoa, which infect cells, include: parasites of the genus Plasmodium (e.g., Plasmodium falciparum, P. Vivax, P. ovale and P. malariae), Trypanosoma, Toxoplasma, Leishmania, and Cryptosporidium.
Diseased and/or abnormal cells may be targeted using the recombinant Rhabdoviruses and modified cells by the methods described above. The diseased or abnormal cells contemplated include: infected cells, neoplastic cells, pre-neoplastic cells, inflammatory foci, benign tumors or polyps, cafe au lait spots, leukoplakia, and other skin moles.
The recombinant Rhabdoviruses will be targeted using an anti-receptor that will recognize and bind to its cognate receptor or ligand expressed on the diseased or abnormal cell. Fusion of the recombinant Rhabdoviral virion is dependant upon incorporation of a fusion facilitating protein as previously described.
Similarly, cells may be engineered to express Rhabdoviral genome components, by methods well known in the art. Nucleic acid vectors comprising the Rhabdoviral G stem polypeptide may be expressed in conjunction with a fusion facilitating polypeptide, on the expressing cell surface. Additional incorporation of “anti-receptor” proteins, or polypeptide fragments thereof, facilitates specific targeting of the expressing cell to the target cell, and provides for fusion of the two cell types. Possible anti-receptors used in the recombinant Rhabdovirus will include the natural anti-receptor for any particular cell receptor.
Alternatively, recombinant Rhabdoviruses and modified cells can be further engineered to express an antibody or polypeptide fragment thereof, a bi-functional antibody, Fab, Fc, Fv, or single chain Fv (scFv) as their attachment protein. Such antibody fragments may be constructed to identify and bind to a specific receptor. These antibodies can be humanized, human, or chimeric antibodies (for discussion and additional references see S. L. Morrison “Antibody Molecules, Genetic Engineering of,” in MOLECULAR BIOLOGY AND BIOTECHNOLOGY: A COMPREHENSIVE DESK REFERENCE 1995; S. D. Gillies et al., (1990) Hum. Antibod. Hybridomas 1(1): 47-54; E. HARLOW AND D. LANE, ANTIBODIES: A LABORATORY MANUAL (1988) Cold Spring Harbor Press, NY). Expression of functional single chain antibodies on the surface of viruses has been reported using Vaccinia virus (M. C. Galmiche et al., (I 997) J. Gen. Virol. 78: 3019-3027). Similar methods would be utilized in creating a recombinant Rhabdovirus expressing a fusion facilitating protein and an antibody or antibody fragment. The genes encoding monoclonal antibodies that target, for example, tumor associated antigens (TAAs) expressed on a cell surface (e.g., prostate specific antigen (PSA)), can be isolated and used to produce the desired recombinant Rhabdovirus, or subcloned into an appropriate expression vector and expressed on a cell surface, as described above, through methodology well known to an individual skilled in the art.
For example, the genes encoding antibodies recognizing TAAs can be fused to the Rhabdoviral G stem for anchoring the antibody protein or polypeptide fragment thereof within the viral membrane. Examples of antibodies include those antibodies, which react with malignant prostatic epithelium but not with benign prostate tissue (e.g., ATCC No. HB-9119; ATCC HB-9120; and ATCC No. HB-1 1430) or react with malignant breast cancer cells but not with normal breast tissue (e.g., ATCC No. HB-8691; ATCC No. HB-10807; and 21HB-108011). Other antibodies or fragments thereof, which react with diseased tissue and not with normal tissue, would be apparent to the skilled artisan.
For purposes of targeting cells in vivo, the isolated recombinant Rhabdovirus or cells expressing Rhabdoviral G stem polypeptides and additional components described above, could be administered by intravascular (i.v.), intramuscular (i.m.), intranasal (i.n.), subcutaneous (s.c.), oral, rectal, intravaginal, or by any means in which the recombinant virus can be delivered to tissue (e.g., needle or catheter). Alternatively, topical administration may be desired for insertion into epithelial cells. Another method of administration is via aspiration or aerosol formulation.
The routes of administration utilized for recombinant Rhabdoviruses facilitate viral circulation, attachment and infection, thereby enabling viral expression of encoded proteins, which may be assayed via the incorporation of reporter proteins within the recombinant Rhabdovirus. It is expected that following Rhabdoviral administration viral protein expression, (as determined, for example, by reporter protein detection) should occur within 24 hours and certainly within 36 hours.
As disclosed herein, the present invention further provides a method for targeted cell or tissue lysis, the method comprising contacting a cell or tissue with an effective amount of a recombinant Rhabdovirus. The recombinant Rhabdovirus comprises a genome containing a deletion in the nucleic acid sequence encoding a G protein, an insertion of a nucleic acid sequence encoding a Rhabdoviral G stem polypeptide, a nucleic acid sequence encoding a heterologous fusion facilitating polypeptide and a nucleic acid sequence encoding an anti-receptor protein or a polypeptide fragment thereof, wherein the anti-receptor protein or a polypeptide fragment thereof binds to a cognate receptor or ligand that is associated with the target cell and that is expressed on the surface of the target cell or tissue, facilitating Rhabdoviral entry and thereby specific lysis of the target cells or tissue. When the term “effective amount” is utilized in this context, it therefore refers to the amount of Rhabdovirus necessary to produce lysis in targeted cells or tissue. The terms “cognate receptor” and “ligand” refer to surface exposed moieties imparting specificity to their interaction with molecules expressed on the target cell or vesicle.
In another embodiment of this aspect of the invention, the targeted cell or tissue is diseased, and the anti-receptor binds a cell surface marker whose expression is a function of the diseased state of the cell or tissue. Targeted lysis of diseased cells or tissue is therefore therapeutic, resulting in destruction of diseased cells alone, and not neighboring, healthy cells that differ in terms of cell surface expression of the target antigen, thereby facilitating control of the disease. For example, recombinant Rhabdoviral targeting may result in specific destruction of tumor cells, without damaging neighboring healthy cells, selectively inhibiting tumor growth, and facilitating disease control. Additionally, targeted lysis of diseased cells may impart further activation to a developing local immune response via a “by-stander” effect, or, via direct activation, such as, for example, via stimulation of expression of immune system costimulatory molecules. Hence the vectors, cells and compositions comprising the recombinant Rhabdovirus and isolated nucleic acids may function as a vaccine for a specific disease or condition. It is to be understood that the recombinant Rhabdovirus and isolated nucleic acids alone may be similarly utilized as vaccines for preventative or prophylactic application.
Some diseases contemplated for use with the targeted recombinant Rhabdovirus and nucleic acid therapeutic lysis protocols discussed above are cancer, cell proliferative disorders, inflammatory cell disorders and infection.
Other aspects of the present invention include the provision of compositions comprising a Rhabdoviral vector for administration to a cell or to a multi cellular organism. The Rhabdoviral vectors of the invention may be employed in combination with a non-sterile or sterile carrier or carriers for administration to cells, tissues or organisms, such as a pharmaceutical carrier suitable for administration to an individual. Such compositions comprise, for instance, a media additive or a therapeutically effective amount of a recombinant virus of the invention and a pharmaceutically acceptable carrier or excipient. Such carriers may include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, and combinations thereof. The formulation should suit the mode of administration.
The invention further relates to diagnostic and pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention.
The recombinant vectors of the invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.
The pharmaceutical compositions may be administered in any effective, convenient manner including, for instance, administration by intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes among others. In therapy or as a prophylactic, the active agent may be administered to an individual as an injectable composition, for example as a sterile aqueous dispersion, preferably isotonic. The pharmaceutical compositions of the invention are preferably administered by injection to achieve a systematic effect against relevant viral pathogens.
For administration to mammals, and particularly humans, it is expected that the physician will determine the actual dosage and duration of treatment, which will be most suitable for an individual and can vary with the age, weight and response of the particular individual.
The following examples are presented in order to more fully illustrate the preferred embodiments of the invention. They should, in no way be construed, however, as limiting the broad scope of the invention.
The construct pCAGGS-GSHA was obtained by subcloning an MluI and NheI fragment encoding GSHA from pVSV-ΔG-GSHA (41) into a modified form of the eukaryotic expression vector pCAGGS (32). The constructs pCAGGS-G124-E, A133-K, P127-D, D137-L and Qn-1 were obtained by subcloning MluI and NheI fragments from the appropriate GMMG mini-genomes (13).
The constructs pCAGGS-CD4-G and CD4-GS (CD4 ectodomain fused to GS at F421) were prepared by subcloning the KpnI to SphI restriction digest fragments encoding CD4-G and CD4-GS from VSV-ΔG-CD4-G/GS (41) into the pCAGGS vector previously digested with the same enzymes. To produce ΔG-F the cDNA encoding the fusion protein (F) from the paramyxovirus simian virus 5 (SV5) (36) was subcloned into pVSV-ΔG-PL from pGEM-F (kindly provided by R. A. Lamb, Northwestern University) using the XhoI site, and clones were screened for proper orientation. The SV5-F cDNA was then moved from ΔG-F into pCAGGS by using the KpnI-NheI sites to produce pC-SV5-F. Plasmids containing the measles virus F and H proteins (pCG-F and pCG-H5, respectively) and the corresponding antibodies were generously provided by Dr. Roberto Cattaneo, Mayo Clinic (5b). The cDNA for the Moloney murine leukemia virus envelope protein was provided by Dr. Lorraine Albritton (University of Tennessee Health Science Center).
Consecutive seven amino acid (aa) truncations of GS, beginning with an amino acid containing an N-terminal FLAG or HA epitope tag (
The construct gp160w3IGS was made by a PCR-based mutagenesis approach. This glycoprotein is composed of the gp120-gp41 ectodomain from HIV-1 clone BH10 (39) through residue 671 which is connected by a linker encoding Ile-Ser-Gly to the C-terminal portion of VSV-GIND at residue 454. The gp160w3IGS cDNA was constructed by amplifying the sequence encoding the transmembrane-ectodomain junction of HIV-1 gp160 from plasmid pBH10 using a sense-strand oligonucleotide (5′-TGGATGGAGTGGGACAG-3′) (SEQ ID NO: 14) (BH10-CR1+), and the non-coding primer (5′-GTTATACCCGAGATATTCCACAAACTTGCCCATTTATC-3′) (SEQ ID NO: 15) (BH10-W3IAva), which introduced an AvaI site (underlined), and substituted the codon for tryptophan at position 672 of gp160 with an isoleucine codon (bold faced) (44) The amplicon was digested with HindIII and AvaI and then ligated together with an EcoRI-HindIII fragment from wt-BH10 into the EcoRI-AvaI digested vector pGEM-CD4-V454 (41). The resulting construct was subcloned as an EcoRI-SphI fragment into pCAGGS-MCS, resulting in pC-gp160w3IGS (a.k.a. pC-W3IGS). This chimera expresses the HIV Env protein at high levels on the cell surface and retains the same chemokine receptor tropism as WT BH10 Env.
Approximately 7×105 baby hamster kidney (BHK-21) cells were transfected with a DNA: liposome suspension containing 2 μg of the appropriate plasmid and 10 μl of lipofectamine (GIBCO-BRL) according to the manufacturer's instructions. Three hours post-transfection, the media was replaced with Dulbecco's modified Eagles medium (DMEM) containing 10% fetal bovine serum (FBS) and the cells were incubated for 36 hours at 37° C. Cells were then fixed with 3% paraformaldehyde and processed for indirect immunofluorescence microscopy. Syncytia formation with HIV-env expressing cells was done as follows. Approximately 7×105 BHK-21 cells were transfected with pCAGGS plasmids encoding GSHA, CD4, CD4-G or CD4-GS alone or co-transfected with plasmids expressing CD4-G and GSHA. After 24 hours, the cells were removed from the dish using trypsin-EDTA and then replated either alone or mixed with cells expressing gp160W3IGS. The cultures were incubated at 37° C. for an additional 24 hours and then cells were fixed with 3% paraformaldehyde and probed with a monoclonal antibody (Sim.2) specific for the CD4 molecule or with a monoclonal antibody specific for the HA epitope.
The following antibodies were used for staining the cells in the syncytia assays. The F1a monoclonal antibody (mAb), which is specific for the SV5 F protein, was a kind gift of Dr. R. A. Lamb, Northwestern University and Dr. Rick Randall, St. Andrews University (38). The Sim.2 mAb (28, 34), which binds to the CD4 ectodomain and the goat-anti-gp160 sera HT3 (27) was obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institute, of Health. The other antibodies that were used include mAb VIII, which is specific for the VSV GNJ glycoprotein (4), mAbs 12CA5 MAb and HA.11 (Covance, USA) which are specific for the HA epitope, and M2 (Sigma-Aldrich, Switzerland) which is specific for the FLAG epitope.
Virus recovery was conducted as previously described (49) with the following modifications. Confluent monolayers of BHK-21 cells in 35 mm plates were infected with a recombinant Vaccinia virus encoding the T7 RNA polymerase (vTF7-3) (15) at a multiplicity of infection (MOI) of 5 for 1 hour at 31° C. The cells were then transfected with a DNA: liposome suspension consisting of 5 μg of pVSV-ΔG-GSHA/FLAG, 3 μg, 5 μg, 8 μg and 1 μg, respectively of plasmids containing the N, P, G and L genes from VSVIND and 90 μl of TransfectACE (43, 52). After 3 hours, the transfection mix was replaced with DMEM containing 10% FBS and cells were incubated at 37° C. The supernatants were collected after 48 hours and filtered through a 0.2μ filter (Millipore, Millex-GS) to remove vaccinia virus. The filtrates were then applied to BHK-21 cells, which had been transfected with 2 μg of pCAGGS-GIND 24 hours earlier. Recovery of the virus was assessed by examining the cells for cytopathic effects that are typical of a VSV infection after 24-36 hours. The recovered viruses were plaque purified and then passaged on BHK-21 cells expressing G protein to make high titer G-complemented virus stocks. Expression of the G-stem proteins in virus infected cells was confirmed by immunofluorescence microscopy using antibodies specific for the HA or FLAG epitopes.
To produce GS virus stocks that have no full-length G protein in their envelopes (non-G complemented viruses) BHK-21 cells were infected with the G-complemented stock viruses at an MOI of 10. One hour post-infection the inoculum was removed and the cells were washed twice with serum free DMEM (SF-DMEM) and once with medium containing G-protein specific monoclonal antibody (I1) (24) to remove any residual G-complemented virus inoculum that may be present in the supernatants. At 16 hours post-infection, the supernatants were collected and the virus particles were pelleted through a 20% sucrose cushion by ultracentrifugation. The viral pellet was resuspended in sterile Dulbecco's phosphate buffered saline (dPBS) and stored at −80° C. until further use.
Viral binding was conducted as previously described (12). Approximately, 80,000 cpm of [35S]-methionine labeled WT-VSV, non-G complemented GSHA or ΔG virus was added to 500 μl of binding media (2 mM Na2HPO4, 2 mM NaH2PO4, 30 mM NaCl, 2 mM 2-[N-morpholino]-ethanesulfonic acid (IVIES), and 2 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), 0.1% BSA, DMEM, titrated to pH 7.0 or pH 5.9 with HCl) and incubated at room temperature for 30 minutes. Virus suspensions were cooled on ice for 10 minutes and then added to confluent monolayers of BHK-21 cells that had been pre-washed with ice-cold binding media at the appropriate pH. Virus adsorption was done for 3 hours on ice. The is inoculum was removed and the amount of unbound virus in the supernatants was determined via scintillation counting. Cells were washed 3 times with ice-cold binding media and the amount of virus eluted in the wash was determined as above. Cells were then solubilized with PBS containing 1% triton X-100 (TX-100) and the amount of bound virus was determined.
The assay was based on the redistribution of fluorescent probes between donor cells and target cells upon fusion. The fluorescent probes DiI-DS, calcein-AM and CMAC were obtained from Molecular Probes (Eugene, Oreg.). The assay was conducted as described previously (30) with the following modifications.
a) Preparation of donor cells: BHK-21 cells (7×106) were infected with WT-VSV, GSHA or ΔG virus at an MOI=10. At 2 hours post-infection, the cells were washed twice with SF-DMEM and once with I1 MAb to remove any unbound G-complemented virus. At 8 hours post-infection, the cells were incubated with 10 μM calcein-AM in D-PBS for 30 minutes at 37° C., washed twice with SF-DMEM to remove excess dye and then incubated in fresh DMEM+5% FBS. After 2 hours, the cells were labeled with DiI by incubating the cells in D-PBS containing 5 μM of DiI-DS for 20 minutes at 37° C., according to the manufacturer's instructions. Cells were washed three times with SF-DMEM to remove the unincorporated label. Labeled cells were then removed from the plates using PBS containing 50 mM EDTA, washed two times with D-PBS (changing tubes each time) and resuspended in 1 ml of SF-DMEM.
b) Preparation of target cells: Three hours prior to the assay, 4×106 BHK-21 cells in 6 cm dishes were labeled with the blue fluorescent dye CMAC by incubating them for 30 minutes at 37° C. with D-PBS containing 20 μM of CMAC. Cells were washed two times with SF-DMEM and then incubated with fresh DMEM+5% FBS at 37° C. for 3 hours.
The donor cells were overlayed on the CMAC-labeled target cells and the culture was incubated at 4° C. for one hour to allow the donor cells to settle and make contact with the target cells. Unbound cells were then removed by washing twice with pre-warmed SF-DMEM and the attached cells were incubated at 37° C. for 15 minutes in 2 ml of SF-DMEM. Fusion was triggered by incubating cells for 1 minute at room temperature with fusion medium (10 mm Na2HPO4, 10 mM NaH2PO4, 150 mM NaCl, 10 mM 2-[N-morpholino]ethanesulfonic acid (IVIES), 10 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES)) buffered to pH 7.0 or pH 5.9. The fusion medium was immediately replaced with SF-DMEM and fusion was allowed to proceed for 30 minutes at 37° C. The cells were then incubated on ice to arrest fusion. Phase-contrast and fluorescence micrographs were acquired using a 40× water immersion lens on a Zeiss Axiophot microscope equipped with a Zeiss Axiocam digital camera and accompanying AxioVision software. Images were adjusted for brightness and contrast using Adobe Photoshop 6.0 after conversion to tiff files.
Stocks of VSV-GFP virus (19), non-G complemented GSHA-GFP (41) and non-G complemented ΔG-GFP virus (41) were produced in BHK cells. Virions were concentrated by ultra-centrifugation, and an aliquot of the viral pellet was loaded on a 10% SDS-PAGE gel and stained with Coomassie blue (GelCode Blue, Pierce Chemical Company). Virus quantitation was based on L-protein concentration using ImageQuant software (Molecular Dynamics).
The assay was conducted as previously described (54) with the following modifications. An equivalent amount of each virus, based on viral protein content, was added to 500 μl of binding buffer (pH 7.0) and the virus suspension was incubated at room temperature for 30 minutes. The virus suspension was cooled on ice for 10 minutes and then added to pre-chilled BHK-21 cells on ice. Virus adsorption was conducted on ice for 3 hours. The inoculum was removed, cells were rinsed with pre-warmed DMEM containing 10% FBS and incubated at 37° C. for 10 minutes. Cells were then treated with DMEM+10% FBS containing CPZ (0.4 mM) for 1 minutes, washed twice, returned to drug-free medium immediately and incubated at 37° C. for 2 hours to allow the cells to recover from the effect of the drug. Cells were then overlayered with DMEM containing 5% FBS and 0.5% methylcellulose. Twelve hours later the cells were examined using a fluorescence microscope (Zeiss Axiophot, Germany) and the number of GFP positive cells were counted.
GSHA (G-stem or GS containing a N-terminal hemagglutinin [HA] epitope tag) is a truncation mutant of the Rhabdoviral G protein in which amino acids 1 to 404 of the mature protein have been deleted.
While conducting studies on GS-mediated viral assembly, the Applicants noticed that cells co-expressing GS and another membrane fusion protein produced extensive and large syncytia, but when GS was expressed alone no syncytia were observed. This phenomenon is illustrated in
Although it is well-established that VSV G protein requires a low pH trigger to obtain the fusion competent conformation (11, 3, 35), the Applicants have found that when G protein is overexpressed at very high levels in BHK-21 cells the G proteins of both the Indiana and New Jersey serotypes can induce some cell-cell fusion at neutral pH (42). The ability of VSV G to cause cell-cell fusion without prior exposure to low pH and the possible mechanism by which this occurs in the polarized endometrial cell line HEC-1A has been described previously (40). Therefore, it is more likely that the GS-mediated fusion potentiation observed following co-expression of GS with GNJ or GIND is not due to activation of G protein, per se, but rather to an enhancement of the low level fusion activity normally present in cells expressing VSV G protein.
To determine if GS could potentiate the membrane fusion activity of other viral glycoproteins, the Applicants coexpressed GSAH with the simian virus 5 (SV5) F protein. SV5 (strain W3A) F protein can directly induce membrane fusion in the absence of its cognate HN protein (18). When SV5 F protein was expressed alone in cells a few small syncytia containing 4-5 nuclei were observed (
To determine the extent to which GS could enhance the membrane fusion activity of other viral glycoproteins, the Applicants next asked if GS had an effect on HIV-1 env mediated fusion. For these studies the Applicants used a cell fusion assay in which non-human (BHK-21) cells expressing an X4-specific HIV envelope protein derived from the clone BH10 were mixed with cells expressing either human CD4 alone, CD4 and GS, or a CD4-GS chimera previously described (41). As expected, no syncytia were seen in the control cells expressing i) HIV-1 env alone, ii) CD4 and GSHA together, iii) a CD4-GS chimera alone, or iv) GSHA alone (
In contrast to the results described above, when GS was co-expressed with measles virus (MV) F protein, no discernable cell-cell fusion was observed. In contrast to the SV5 F protein, MV F protein required coexpression of viral H protein for membrane fusion activity, as evidenced by enhanced syncytium formation following MV F and H proteins coexpression with GS. A summary of the results that were obtained by coexpressing GSHA with various viral glycoproteins is shown in Table 1.
The results demonstrate that GS can potentiate the fusion activity of some but not all viral glycoproteins and that the fusion potentiating activity can occur with glycoproteins that require a pH trigger as well as those that are pH independent. This suggests that GS functions at a point common to both fusion pathways.
The Applicants next asked if GS could relieve the fusion defects of some G protein mutants that have been described previously. The mutants that were used for these assays were A133-K (14) and QN-1 (53), which have no detectable fusion activity, and D137-L, G124-E and P127-D (14, 13), which have a reduced pH threshold for fusion and can fuse as well as wild type G protein at a pH below 5.7. All the mutants have wild type (wt) G protein cell binding activity. When the Applicants co-expressed these constructs with GS in BHK-21 cells there was no enhancement of membrane fusion, indicating that GS did not rescue the fusion defective phenotypes of these mutants when cells were maintained at neutral pH or at pH 5.9 (data not shown). These results together with the results from the MV F experiment (Table 1) indicated that fusion potentiation by GS requires a fully functional viral glycoprotein.
To understand the basis for the membrane fusion enhancing activity of GS, the Applicants performed a series of experiments designed to examine various steps in the fusion process. The first step of glycoprotein-mediated membrane fusion requires binding of the glycoprotein to the target membrane. To determine whether GS exhibits cell-binding activity, radiolabeled GSHA virus was incubated with BHK-21 cells in ice-cold media buffered to pH 7.0 or pH 5.9 for 3 hours. Wild type VSV (WT) served as the positive control while “bald” viruses lacking G protein (ΔG virus) served as the negative control for binding. Virus binding was conducted on ice to prevent endocytosis of the virus as well as to inhibit fusion of the viral envelope with the host cell membranes following exposure to acidic pH. As shown in
The Applicants next examined whether GS could mediate the next step in the fusion pathway, namely the mixing of the outer leaflets of the two membranes. For this analysis the Applicants used a 3-color fusion assay similar to that described previously for other viral fusion proteins (30). In this assay BHK-21 cells were infected with WT virus, GSHA virus, or ΔG virus, functioning as the donor cells. By six hours post-infection the virus infected cells expressed high levels of either G protein or GSHA on their plasma membranes (data not shown). Virus-infected donor cells were then labeled with a red lipidic dye (DiI-DS) and a green cytoplasmic dye (calcein-AM). The target cells were labeled with a blue cytoplasmic dye (CMAC). The donor cells were then coincubated for 1 hour with the target cells via overlay, on ice, to facilitate cell attachment. After warming the culture to 37° C. for 15 minutes, fusion was triggered by incubating the cells in fusion medium buffered to pH 5.9 or 7.0 for 1 minute. This assay is based on the redistribution of the dyes between the donor cell and target cell during the fusion process. When membrane fusion occurs the donor cells redistribute the lipidic dye DiI first and then upon opening of the fusion pores calcein-AM is transferred to the cytoplasm of the target cell. The dye CMAC forms a glutathione adduct with a molecular wt of ˜600 Da within the cytoplasm of the target cells that cannot pass through the small fusion pores and thus helps in distinguishing the target cells from donor cells (31). If there is redistribution of DiI only to the target cells, this indicates that outer leaflet mixing has occurred, but that the fusion reaction did not proceed to completion. Mixing of the outer leaflet only is known as the hemi-fusion stage. If both DiI and calcein-AM are transferred to the target cells then complete fusion has occurred. As shown in
To determine if GS induced lipid mixing, as measured by DiI transfer, was a biologically relevant lipid destabilization event, the Applicants examined whether the membrane curvature agent CPZ could rescue GSHA virus infection. CPZ is an aliphatic phenothiazinea that can destabilize hemifusion diaphragms and drive the formation of a fusion pore (6). CPZ has been used previously to rescue hemifusion intermediates of influenza heamagglutinin (29), and MoMuLV glycoprotein (54). For this assay, GSHA-GFP virus bound to cells at 4° C. enabled the formation of GS-dependent hemifusion diaphragms, when cells were shifted to 37° C. The cells were then treated with 0.4 mM CPZ for 1 minute and incubated overnight in drug free media. The number of GFP positive cells in the presence or absence of CPZ was then quantified. GFP expression can occur only if the virus successfully entered the cells and initiated replication. As shown in
To determine how much of the G protein ectodomain is required for GS fusion potentiation, a series of G protein deletion mutants were generated. Constructs were generated containing nucleotide coordinates 1-273 of SEQ ID No: 1 (corresponding to nucleotide coordinates 1289-1562 and amino acid coordinates 421-497 of the VSV G protein), and truncations thereof to generate G stem polypeptides (
This application is a Continuation-in-part Application of U.S. application Ser. No. 09/217,967, filed Dec. 22, 1998 now U.S. Pat. No. 6,497,873, which claims priority of Provisional Application No. 60/068,472, filed Dec. 22, 1997, which are incorporated herein by reference.
| Number | Name | Date | Kind |
|---|---|---|---|
| 6168943 | Rose | Jan 2001 | B1 |
| 20020086356 | Tuschl et al. | Jul 2002 | A1 |
| Number | Date | Country |
|---|---|---|
| WO 03092582 | Nov 2003 | WO |
| Number | Date | Country | |
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| 20100167377 A1 | Jul 2010 | US |
| Number | Date | Country | |
|---|---|---|---|
| 60068472 | Dec 1997 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 09217967 | Dec 1998 | US |
| Child | 10327673 | US |
