The present invention is directed to genetically engineered, membrane-enveloped viruses with deletion mutations in the protein transmembrane domains. Also provided are viral vaccines based on the engineered viruses, methods of producing and using such vaccines.

Description of the Invention

SUMMARY OF THE INVENTION

Viruses which are transmitted in nature by blood sucking insects are a major source of disease in man and domestic animals. Many of these viruses have lipid membrane bilayers with associated integral membrane proteins as part of their three dimensional structure. These viruses are hybrid structures in which the proteins are provided by the genetic information of the virus and the membrane is the product of the host cell in which the virus is grown. Differences in the composition of the membranes of the mammalian and insect host are exploited to produce virus mutants containing deletions in the membrane spanning domains of the virus membrane proteins. Some of the mutants are capable of replicating and assembling normally in the insect host cell but assemble poorly in the mammalian host cell. These host range mutants produce immunity to wild type virus infection when used as a vaccine in mice, and represent a novel strategy for the production of vaccines against arthropod vectored, membrane containing viruses.

In one embodiment of the present invention, there is provided a genetically engineered membrane-enveloped virus comprising a viral transmembrane glycoprotein that is able to span or correctly integrate into the membrane of insect cells but not that of mammalian cells due to deletion of one or more amino acids in the viral transmembrane glycoprotein. The virus is capable of infecting and producing progeny virus in insect cells, and is capable of infecting but not producing progeny virus in mammalian cells. The virus can be an Arthropod vectored virus such as Togaviruses, Flaviviruses, Bunya viruses and all other enveloped viruses which can replicate naturally in both mammalian and insect cells, as well as enveloped viruses which can be made to replicate in mammalian and insect cells by genetic engineering of either the virus or the cell. Representative examples of such engineered viruses are .DELTA.K391, TM17, TM10 and TM16 viruses.

In another embodiment of the present invention, there is provided a method of producing a viral vaccine by introducing the engineered virus disclosed herein into insect cells and allowing the virus to replicate in the insect cells to produce a viral vaccine. Representative examples of the engineered viruses are .DELTA.K391 virus, TM 17 virus and TM16 virus.

In still another embodiment of the present invention, there is provided a method for vaccinating an individual in need of such treatment comprising the step of introducing the viral vaccine of the present invention into the individual to produce viral proteins for immune surveillance and stimulate immune system for antibody production.

In still yet another embodiment of the present invention, there is provided a method of producing a viral vaccine to a disease spread by a wild mosquito population to mammals, comprising the steps of engineering a deletion of one or more amino acids in a viral transmembrane protein to produce an engineered virus similar to TM16, TM17 or delta K391, wherein the transmembrane protein is able to span the membrane envelope in mosquito cells but not in mammalian cells; introducing the engineered virus, into the wild mosquito population; and allowing the engineered virus to replicate in cells of the wild mosquito population to produce a population of mosquitoes which excludes the wild type pathogenic virus and harbors the vaccine strain of the virus so that a mosquito bite delivers the vaccine to the mammal bitten. Presence of the mutated virus renders the mosquito incapable of transmitting other membrane containing viruses (Karpf et al 1997).

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Maniatis, Fritsch & Sambrook, "Molecular Cloning: A Laboratory Manual (1982); "DNA Cloning: A Practical Approach," Volumes I and II (D. N. Glover ed. 1985); "Oligonucleotide Synthesis" (M. J. Gait ed. 1984); "Nucleic Acid Hybridization" (B. D. Hames & S. J. Higgins eds. (1985)); "Transcription and Translation" (B. D. Hames & S. J. Higgins eds. (1984)); "Animal Cell Culture" (R. I. Freshney, ed. (1986)); "Immobilized Cells And Enzymes" (IRL Press, (1986)); B. Perbal, "A Practical Guide To Molecular Cloning" (1984).

The vaccines of the present invention are based on deletion mutations in the transmembrane domains of membrane glycoproteins of membrane-enveloped viruses. Many membrane-coated viruses have membrane glycoproteins on their surface which are responsible for identifying and infecting target cells (Schlesinger, S. and M. J. Schlesinger, 1990). These membrane glycoproteins have hydrophobic membrane-spanning domains which anchor the proteins in the membrane bilayer (Rice et al 1982).

The membrane-spanning domains of these transmembrane proteins must be long enough to reach from one side of the bilayer to the other in order to hold or anchor the proteins in the membrane. Experiments have shown that if the domains are shortened by the deletion of amino acids within the domain, the proteins do not appropriately associate with the membrane and fall out (Adams and Rose. 1985).

Unlike mammalian cell membranes, the membranes of insect cells contain no cholesterol (Clayton 1964, Mitsuhashi et al 1983). Because insects have no cholesterol in their membranes, the insect-generated viral membrane will be thinner in cross section than the viral membranes generated from mammals. Consequently, the membrane-spanning domains of proteins integrated into insect membranes do not need to be as long as those integrated into the membranes of mammals. It is possible, therefore, to produce deletions in engineered viruses which remove amino acids from the transmembrane domain of the viral glycoprotein. This results in a glycoprotein which can integrate normally into the membrane of a virus replicating in an insect cell, but not into the membrane of a virus replicating in a mammal. Thus, the mutated virus can replicate and be produced in insect cells as well as the parent wild-type virus. On the other hand, the mutant virus can infect mammalian cells and produce viral proteins; however, since the mutated virus glycoprotein cannot span and be anchored in the mammalian membrane, progeny virus cannot be produced in mammalian cells. An advantage to the approach of the present invention is that the mutants are engineered as deletion mutants, hence there is absolutely no chance for reversion to wild-type phenotype, a common problem with virus vaccines.

The protocol described by the present invention works for any virus which replicates in insects and mammals and has integral membrane proteins as part of its structure, namely, Togaviruses, Flaviviruses and Bunya viruses and all other enveloped viruses which can replicate naturally in both mammalian and insect cells, as well as enveloped viruses which can be made to replicate in mammalian and insect cells by genetic engineering of either the virus or the cell.

Vaccines are made against any membrane-containing virus by removing amino acids from the membrane-spanning domain of a protein in the viral envelope. This is done by removing bases from a cDNA clone of the virus as described below. RNA transcribed from the altered clone is transfected into insect cells. The viruses produced are amplified by repeated growth in insect cells until large quantities of mutant viruses are obtained. These viruses are tested for its ability to infect and produce progeny in mammalian cells. Viruses which do not produce progeny in mammalian cells are tested for ability to produce immunity in laboratory animals. Those viruses which do produce immunity are candidates for production of human and animal vaccines by procedures known in the art.

Using the prototype of the Alphaviridea, Sindbis virus, the different compositions of insect and mammalian membranes are exploited to produce mutants which assemble efficiently in insect cells but assemble poorly in mammalian cells. The envelope glycoproteins of Sindbis virus are integrated into the membranes of the endoplasmic reticulum as a multi pass protein with 6 membrane spanning domains. There are, therefore, 6 potential targets for the production of deletion mutations which will prevent the correct integration of a transmembrane domain (TMD) (See FIG. 1, see Original Patent). Some of these targets are less satisfactory for deletion mutagensis because they have functions other than simply anchoring the protein in the membrane bilayer. For example, transmembrane domain #1 (FIG. 1) is the signal sequence which is recognized by the Signal Recognition Particle and directs protein synthesis to the membranes of the endoplasmic reticulum. Truncating this domain would likely disturb targeting in both mammalian and insect cells. TMD #3 will become a cytoplasmic domain upon protein maturation and contains specific sequences that recognize and bind capsid protein. It has been shown that this interaction is very specific in nature and requires the sequence that is in the transmembrane domain (Liu et al., 1996; Lopez et al., 1994). TMD #3, therefore, like TMD #1 has a functional as well as a structural component. A significant deletion in this domain would likely eliminate budding in both cell systems. This leaves four transmembrane domains as targets for the production of deletions which will effect membrane integration (FIG. 1, TMD #2, #4, #5, and #6).

The 6k protein is not a component of mature virus and its function in virus assembly is not clear. In the poly protein the proper integration and orientation of 6k in the endoplasmic reticulum membrane is essential for the correct integration of E1. The transmembrane domains of 6k (TMD #4 and #5) are excellent targets for deletion mutation as failure to integrate one of these domains may cause the poly protein to integrate into the membrane in a wrong configuration or cause the failure to integrate E1. TMD #2 and #6 are the membrane spanning domains of E2 and E1 and are both obvious targets for deletion mutation. Multiple membrane spanning domains in this poly protein suggest that if deletion mutations in a single transmembrane domain do not totally block virus production in mammalian cells, then deletions in additional membrane spanning domains can further reduce maturation to negligible levels.

The present invention is directed to a genetically engineered membrane-enveloped virus comprising a transmembrane protein which has a deletion of one or more amino acids in the transmembrane region of the protein such that the transmembrane protein is able to span or correctly integrate into the membrane of an infected cell when the engineered virus replicates in insect cells, but is unable to span or integrate into the membrane of an infected cell when the virus replicates in mammalian cells. Preferably, the virus is an Arthropod vectored virus selected from the group consisting of Togaviruses, Flaviviruses, Bunya viruses and all other enveloped viruses which can replicate naturally in both mammalian and insect cells, as well as enveloped viruses which can be made to replicate in mammalian and insect cells by genetic engineering of either the virus or the cell. Representative examples of such engineered viruses are .DELTA.K391, TM17, TM10 and TM16 viruses. Preferably, the insect cells are mosquito cells, such as Aedes albopictus cells, and the mammalian cells are human cells.

In a preferred embodiment, the genetically engineered, membrane-enveloped virus is Sindbis virus, and the transmembrane protein is viral glycoprotein E2. However, a person having ordinary skill in this art could readily predict that similar mutations can be successfully installed in the membrane spanning domains of other virus membrane proteins such as E1.

In another preferred embodiment, the genetically engineered membrane-enveloped virus is selected from the group consisting of HSV, HIV, rabies virus, Hepatitis, and Respiratory Syncycial virus, and the transmembrane protein is selected from the group consisting of glycoprotein E1, glycoprotein E2, and G protein.

In still another preferred embodiment, the genetically engineered membrane-enveloped virus are RNA tumor viruses, and the transmembrane protein is Env.

The present invention is also drawn to a method of producing a viral vaccine from the genetically engineered membrane-enveloped virus disclosed herein for vaccination of mammals, comprising the steps of introducing the engineered virus into insect cells and allowing the virus to replicate in the insect cells to produce a viral vaccine. Representative examples of the engineered viruses are .DELTA.K391 virus, TM17 virus and TM16 virus.

In addition, the present invention provides a method of vaccinating an individual in need of such treatment, comprising the steps of introducing the viral vaccine of the present invention into the individual and allowing the vaccine to produce viral proteins for immune surveillance and stimulate immune system for antibody production in the individual.

Furthermore, the present invention provides a method of producing a viral vaccine to a disease spread by a wild mosquito population to a mammal, comprising the steps of genetically engineering a deletion of one or more amino acids in a viral transmembrane protein to produce an engineered virus, wherein the transmembrane protein is able to span the membrane envelope when the virus replicates in mosquito cells, but is unable to span the membrane envelope when the virus replicates in mammalian cells, and wherein the virus remains capable of replicating in mosquito cells; introducing the engineered virus into a wild mosquito population; and allowing the virus to replicate in cells of the wild mosquito population to produce a population of mosquitoes which excludes the wild type pathogenic virus and harbors the vaccine strain of the virus such that the mosquito bite delivers the vaccine to a mammal bitten.

It is contemplated that pharmaceutical compositions may be prepared using the novel mutated viruses of the present invention. In such a case, the pharmaceutical composition comprises the novel virus of the present invention and a pharmaceutically acceptable carrier. A person having ordinary skill in this art readily would be able to determine, without undue experimentation, the appropriate dosages and routes of administration of this viral vaccination compound. When used in vivo for therapy, the vaccine of the present invention is administered to the patient or an animal in therapeutically effective amounts, i.e., amounts that immunize the individual being treated from the disease associated with the particular virus. It will normally be administered parenterally, preferably intravenously or subcutaineusly, but other routes of administration will be used as appropriate. The amount of vaccine administered will typically be in the range of about 10.sup.3 to about 10.sup.6 pfu/kg of patient weight. The schedule will be continued to optimize effectiveness while balancing negative effects of treatment. See Remington's Pharmaceutical Science, 17th Ed. (1990) Mark Publishing Co., Easton, Pa.; and Goodman and Gilman's: The Pharmacological Basis of Therapeutics 8th Ed (1990) Pergamon Press; which are incorporated herein by reference. For parenteral administration, the vaccine will be most typically formulated in a unit dosage injectable form (solution, suspension, emulsion) in association with a pharmaceutically acceptable parenteral vehicle. Such vehicles are preferably non-toxic and non-therapeutic. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin.
 

Claim 1 of 16 Claims

1. A method of replicating a genetically engineered Arbovirus comprising the steps: a) obtaining a genetically engineered Arbovirus comprising a transmembrane glycoprotein with a deletion of one or more amino acids in a transmembrane domain wherein said engineered Arbovirus has an ability to infect mammalian cells but a reduced ability to replicate therein relative to wild type virus; b) allowing the virus to replicate in insect cells to produce the engineered Arbovirus.
 

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