A novel HIV vaccine is provided. In particular, the vaccine comprises an avirulent and non-cytolytic recombinant HIV wherein the NSS of the virus' envelope glycoprotein is replaced with a non-cytolytic signal sequence and nef gene of the virus is deleted which renders the virus avirulent.

DETAILED DESCRIPTION OF THE INVENTION

Recombinant Retrovirus

As mentioned hereinabove, the present invention relates to an essentially non-cytolytic retrovirus wherein the natural signal sequence of HIV-1 envelope glycoprotein gp120 (NSS) is modified to be essentially non-cytolytic or is replaced with an essentially non-cytolytic signal sequence. The term "essentially non- cytolytic" as used herein means that the retrovirus does not significantly damage or kill the cells it infects.

In one embodiment, the present invention provides an essentially non-cytolytic recombinant HIV-1 capable of highly efficient replication wherein the NSS of the virus' envelope glycoprotein is modified sufficiently to prevent cell damage by the virus, preferably by eliminating positively charged amino acids, even more preferably, such elimination or modification resulting in no more than one (1) and preferably zero (0) positively charged amino acids. The positively charged amino acids which may be modified or replaced include lysine and arginine.

In another embodiment, replacement of the natural signal sequence results in a more efficient replication of HIV. Accordingly the present invention provides an essentially non-cytolytic recombinant HIV-1 capable of highly efficient replication wherein the NSS of the virus' envelope glycoprotein is replaced with an essentially non-cytolytic and more efficient signal sequence. In a preferred embodiment, replacement of the NSS of the envelope glycoprotein of HIV-1 with either the mellitin or IL-3 signal sequence decreases the cytotoxicity of the retrovirus. As such, the present invention includes within its scope replacement of NSS with any signal sequence which renders the retrovirus essentially non-cytolytic. The inventors have also shown that replacement of the NSS with mellitin or IL-3 signal sequences results in a greater level of production and secretion of gp120, in addition to the reduced cytotoxicity. The inventors have also shown that replacement of the NSS results in partial deletion the vpu gene. Studies have shown the vpu gene can be completely deleted without any measurable impact on the virus' ability to replicate (James et al. AIDS Res. Human Retrovirus 10:343 350, 1994).

In another embodiment, the retrovirus is rendered avirulent. In a preferred embodiment, the virus is rendered avirulent by deleting the nef gene. Accordingly, the present invention provides an avirulent, essentially non-cytolytic retrovirus which contains a sufficient deletion of the nef gene to render the virus non-pathogenic and wherein the virus' envelope glycoprotein gp120 coding sequence is replaced with a more efficient signal sequence. As used herein, "sufficient deletion" means deletion of enough of the sequence to prevent transcription and thereby production of the nef protein product.

In a further embodiment, the retrovirus is rendered avirulent, essentially non-cytolytic, and contains a sufficient deletion of the nef gene and the vpu gene to render the virus non-pathogenic.

The recombinant retrovirus of the present invention can be any retrovirus including HIV-1, HIV-2, SIV, HTLV-1. Preferably the retrovirus is a human immunodeficiency virus selected from HIV-1 and HIV-2, more preferably, the retrovirus is HIV-1.

The recombinant retroviruses of the present invention can be prepared using techniques known in the art. In one embodiment, the retrovirus may be introduced in a host cell under conditions suitable for the replication and expression of the retrovirus in the host. Accordingly, the present invention also provides a cell transfected with a recombinant retrovirus wherein the natural signal sequence of the virus' envelope glycoprotein gp120 is modified to provide an essentially non-cytotoxic virus or is replaced with an essentially non-cytolytic signal sequence. The cell is preferably a T-lymphocyte, more preferably a T-cell that is not derived from a transformed cell line.

The essentially non-cytolytic and avirulent retrovirus of the present invention will be extremely useful for the prevention and treatment of a retroviral infection as the retrovirus may be produced in large quantities and in a form that is non-pathogenic to the host, preferably the virus of the invention will be useful for development of HIV/AIDS vaccines for the prevention and treatment of HIV infections. Accordingly, the present invention also provides a method of preventing or treating a retroviral infection comprising administering an effective amount of a killed recombinant essentially non-cytolytic avirulent retrovirus of the present invention to an animal in need thereof. The term "effective amount" as used herein means an amount effective and at dosages and for periods of time necessary to achieve the desired result. The term "animal" as used herein includes all members of the animal kingdom including mammals, preferably humans.

In a preferred embodiment, the present invention provides a method of preventing or treating a retroviral infection comprising administering an effective amount of a killed recombinant essentially non-cytolytic avirulent retrovirus to an animal in need thereof, wherein the natural signal sequence of the virus' envelope glycoprotein, preferably gp120, is modified to provide an essentially non-cytolytic signal sequence, preferably the virus is rendered avirulent by deleting the nef gene. According to a preferred embodiment the modification to provide a non-cytolytic NSS results in no more than one positively charged amino acid in the NSS sequence, more preferably zero positively charged amino acids. Most preferably, the animal is a human, preferably the retrovirus is HIV-1.

In a further preferred embodiment, the present invention provides a method of preventing or treating a retroviral infection comprising administering an effective amount of a killed recombinant essentially non-cytolytic avirulent retrovirus to an animal in need thereof, wherein the natural signal sequence of the virus' envelope glycoprotein, preferably gp120, is replaced with an essentially non-cytolytic signal sequence, preferably the virus is rendered avirulent by deleting the nef gene. Most preferably, the animal is a human, preferably the retrovirus is HIV-1.

According to a preferred embodiment of the method wherein the NSS is replaced, the non-cytolytic signal sequence is selected from the group consisting of the mellitin sequence and the IL-3 signal sequence.

Vaccines

The present invention further includes a vaccine comprising an effective amount of an avirulent and an essentially non-cytolytic retrovirus wherein the natural signal sequence of the virus' envelope glycoprotein, preferably gp120, is replaced with an essentially non-cytolytic signal sequence and the virus is rendered avirulent by deleting a sufficient portion of the nef gene. The retrovirus may also have a portion of the vpu gene deleted as a result of replacement of the NSS. Preferably the essentially non-cytolytic signal sequence is selected from the group consisting of the mellitin sequence and the IL-3 signal sequence.

According to one embodiment, modification of the natural signal sequence of a retrovirus' envelope glycoprotein results in a more efficient replication of the virus, preferably HIV. Accordingly, the present invention provides a non-cytolytic recombinant HIV-1 capable of highly efficient replication wherein the NSS of the virus' envelope glycoprotein is modified sufficiently to prevent cell damage by the virus, preferably by eliminating positively charged amino acids, even more preferably, such elimination or modification resulting in no more than one (1) positively charged amino acid, more preferably no more than zero (0) positively charged amino acids.

In another embodiment, replacement of the non-cytolytic signal sequence results in a more efficient replication of HIV. Accordingly the present invention provides a vaccine comprised of a non-cytolytic recombinant HIV-1 capable of highly efficient replication wherein the NSS of the virus' envelope glycoprotein is replaced with a non-cytolytic and more efficient signal sequence, preferably containing no more than one positive amino acids, preferably mellitin signal sequence (MSS) or IL-3 signal sequence (ILSS).

According to another embodiment, an essentially non-cytolytic retrovirus is also avirulent, preferably through deletion of the nef gene. Accordingly, the present invention provides a vaccine comprising an avirulent, essentially non-cytolytic retrovirus comprising a nucleic acid sequence addition or deletion that renders the virus avirulent and wherein the natural signal sequence of the virus' envelope glycoprotein is either modified or replaced to provide an essentially non-cytolytic signal sequence.

Alternatively, the vaccine may comprise an effective amount of an avirulent and essentially non-cytolytic retrovirus wherein the natural signal sequence of the virus' envelope glycoprotein gp120 is modified to reduce the number of positive amino acids to no more than one positively charged amino acids, preferably no more than zero positively charged amino acids and the virus is rendered avirulent by deleting a sufficient portion of the nef gene.

Accordingly, the present invention also includes a method of preventing or treating a retroviral infection comprising administering a vaccine of the present invention to an animal in need thereof. As used herein, "vaccine" includes all prophylactic and therapeutic vaccines. According to one embodiment the vaccine contains an avirulent and essentially non-cytolytic recombinant retrovirus, wherein the NSS of the virus' envelope glycoprotein is modified to provide an essentially non- cytolytic NSS or is replaced with an essentially non-cytolytic NSS and the virus is rendered avirulent by deleting a sufficient portion of the nef gene.

The vaccine compositions of the invention are suitable for administration to subjects in a biologically compatible form in vivo. The expression "biologically compatible form suitable for administration in vivo" as used herein means a form of the substance to be administered in which any toxic effects are outweighed by the therapeutic effects. The substances may be administered to any animal, preferably humans.

The vaccines of the present invention may additionally contain suitable diluents, adjuvants and/or carriers. Preferably, the vaccines contain an adjuvant which can enhance the immunogenicity of the vaccine in vivo. The adjuvant may be selected from many known adjuvants in the art including the lipid-A portion of gram negative bacteria endotoxin, trehalose dimycolate of mycobacteria, the phospholipid lysolecithin, dimethyldictadecyl ammonium bromide (DDA), certain linear polyoxypropylene-polyoxyethylene (POP-POE) block polymers, aluminum hydroxide, and liposomes. The vaccines may also include cytokines that are known to enhance the immune response including GM-CSF, IL-2, IL-12, TNF and IFN.gamma..

The dose of the vaccine may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of antibody to elicit a desired response in the individual. Dosage regime may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. The dose of the vaccine may also be varied to provide optimum preventative dose response depending upon the circumstances.

The vaccines may be administered in a convenient manner such as by injection (subcutaneous, intravenous, intramuscular, etc.), oral administration, inhalation, transdermal administration (such as topical cream or ointment, etc.), or suppository applications.

Prevention of Apoptosis

The present invention also includes a method of preventing the NSS of a retrovirus from exerting its apoptotic effects on a cell, apoptosis induced by a retroviral infection. Accordingly, the present invention provides a method of preventing or inhibiting apoptosis comprising administering a sufficient amount of antagonist to NSS to an animal in need thereof. The antagonist may be any substance that can inhibit the NSS gene or its protein product referred to herein as "NSS protein", preferably the antagonist is an antibody or antisense molecule.

In one embodiment, the antagonist is a substance that inhibits the NSS protein such as an NSS protein specific antibody. Antibodies to NSS protein may be prepared using techniques known in the art such as those described by Kohler and Milstein, Nature 256, 495 (1975) and in U.S. Pat. No. RE 32,011, U.S. Pat. Nos. 4,902,614, 4,543,439, and 4,411,993 which are incorporated herein by reference. (See also Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.), 1980, and Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988, which are also incorporated herein by reference). Within the context of the present invention, antibodies are understood to include monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab, and F(ab').sub.2) and recombinantly produced binding partners. Consequently, the present invention provides a method of inhibiting the effects of the NSS of a retrovirus comprising administering an effective amount of an antibody that inhibits the NSS protein.

In addition to antibodies, other antagonists or ligands that bind to the NSS protein and inhibit its function may also be used. NSS protein ligands may be identified by assaying a sample for peptides that bind to NSS protein. Any assay system or testing method that detects protein-protein interactions may be used including co-immunoprecipitation, crosslinking and co-purification through gradients or chromatographic columns may be used. Biological samples and commercially available libraries may be tested for NSS protein-binding peptides. For example, labelled NSS protein or soluble NSS protein may be used to probe phage display libraries. In addition, antibodies that bind to NSS protein may be used to isolate other peptides with NSS protein binding affinity. For example, labelled antibodies may be used to probe phage display libraries or biological samples. Additionally, a nucleic acid sequence encoding a NSS protein may be used to probe biological samples or libraries for nucleic acids that encode NSS protein-binding proteins or ligands.

In another embodiment, the NSS antagonist is an antisense oligonucleotide that inhibits the expression of NSS protein. Antisense oligonucleotides that are complimentary to a nucleic acid sequence from an NSS protein gene can be used in the methods of the present invention to inhibit NSS protein.

Consequently, the present invention provides a method of inhibiting the effects of the NSS of a retrovirus comprising administering an effective amount of an antisense oligonucleotide that is complimentary to a nucleic acid sequence from an NSS protein gene to an animal in need thereof. Preferably the retrovirus is HIV-1.

The term antisense oligonucleotide as used herein means a nucleotide sequence that is complimentary to its target.

The term "oligonucleotide" refers to an oligomer or polymer of nucleotide or nucleoside monomers consisting of naturally occurring bases, sugars, and intersugar (backbone) linkages. The term also includes modified or substituted oligomers comprising non-naturally occurring monomers or portions thereof, which function similarly. Such modified or substituted oligonucleotides may be preferred over naturally occurring forms because of properties such as enhanced cellular uptake, or increased stability in the presence of nucleases. The term also includes chimeric oligonucleotides which contain two or more chemically distinct regions. For example, chimeric oligonucleotides may contain at least one region of modified nucleotides that confer beneficial properties (e.g. increased nuclease resistance, increased uptake into cells), or two or more oligonucleotides of the invention may be joined to form a chimeric oligonucletide.

The antisense oligonucleotides of the present invention may be ribonucleic or deoxyribonucleic acids and may contain naturally occurring bases including adenine, guanine, cytosine, thymidine and uracil. The oligonucleotides may also contain modified bases such as xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl and other alkyl adenines, 5-halo uracil, 5-halo cytosine, 6-aza uracil, 6-aza cytosine and 6-aza thymine, pseudo uracil, 4-thiouracil, 8-halo adenine, 8-aminoadenine, 8-thiol adenine, 8-thiolalkyl adenines, 8-hydroxyl adenine and other 8-substituted adenines, 8-halo guanines, 8-amino guanine, 8-thiol guanine, 8-thiolalkyl guanines, 8-hydroxyl guanine and other 8-substituted guanines, other aza and deaza uracils, thymidines, cytosines, adenines, or guanines, 5-trifluoromethyl uracil and 5-trifluoro cytosine.

Other antisense oligonucleotides of the invention may contain modified phosphorous, oxygen heteroatoms in the phosphate backbone, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages. For example, the antisense oligonucleotides may contain phosphorothioates, phosphotriesters, methyl phosphonates, and phosphorodithioates. In an embodiment of the invention there are phosphorothioate bonds links between the four to six 3'-terminus bases. In another embodiment phosphorothioate bonds link all the nucleotides.

The antisense oligonucleotides of the invention may also comprise nucleotide analogs that may be better suited as therapeutic or experimental reagents. An example of an oligonucleotide analogue is a peptide nucleic acid (PNA) wherein the deoxyribose (or ribose) phosphate backbone in the DNA (or RNA), is replaced with a polyamide backbone which is similar to that found in peptides (P. E. Nielsen, et al Science 1991, 254, 1497). PNA analogues have been shown to be resistant to degradation by enzymes and to have extended lives in vivo and in vitro. PNAs also bind stronger to a complimentary DNA sequence due to the lack of charge repulsion between the PNA strand and the DNA strand. Other oligonucleotides may contain nucleotides containing polymer backbones, cyclic backbones, or acyclic backbones. For example, the nucleotides may have morpholino backbone structures (U.S. Pat. No. 5,034,506). Oligonucleotides may also contain groups such as reporter groups, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an antisense oligonucleotide. Antisense oligonucleotides may also have sugar mimetics.

The antisense nucleic acid molecules may be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. The antisense nucleic acid molecules of the invention or a fragment thereof, may be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed with mRNA or the native gene e.g. phosphorothioate derivatives and acridine substituted nucleotides. The antisense sequences may be produced biologically using an expression vector introduced into cells in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense sequences are produced under the control of a high efficiency regulatory region, the activity of which may be determined by the cell type into which the vector is introduced.

Furthermore, the present invention also contemplates a method for assaying for a substance that inhibits the NSS activity of a retrovirus comprising reacting a retrovirus containing an NSS with a test substance, under conditions which permit inhibition of the NSS, assaying for the ability of the retrovirus to induce apoptosis, and comparing to the ability to induce apoptosis obtained in the absence of the test substance, to determine the effect of the substance on the NSS of the retrovirus.

Apoptosis in Cancer Cells

The present invention further includes a method of killing or destroying target cells, preferably cancer cells, comprising administering to the cell or cells, an effective amount of a recombinant virus, preferably VSV or any other carrier RNA virus, specific for the target cells, containing, preferably the NSS of HIV-1. Preferably the cells are in an animal in need thereof, most preferably in human. Cells which are infected or cancerous, express cell specific markers for which a complementary recognition site may be incorporated into a suitable vector into which the NSS of HIV-1 has been incorporated. This approach has been taken with vesicular stomatitis virus (VSV) which has been engineered to incorporate the genes for CD4 and CXCR4 thereby targeting the modified VSV to infect HIV-1 infected cells (Schnell, M. J. et al. Cell 90: 849 857 (1997)). Accordingly, the present invention provides a method of killing target cells, such as cancer cells, comprising administering a recombinant virus containing NSS and a recognition site specific to the target cells, to an animal in need thereof. In an embodiment, the NSS of HIV-1 is incorporated into a modified VSV-like vector which has been targeted to a specific cancer cell type based on a particular cancer cell surface antigen thereby providing the VSV with the ability to induce apoptosis in the targeted cancer cells.

 

Claim 1 of 13 Claims

1. A recombinant human immunodeficiency virus-1 (HIV-1), wherein the natural signal sequence (NSS) of the HIV-1 envelope glycoprotein gp120 of the virus is replaced with a signal sequence selected from the group consisting of mellitin signal sequence (MSS) and interleukin 3 signal sequence (ILSS).

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