Title: Attenuated rabies virus with nucleoprotein mutation at the phosphorylation site for vaccination against rabies and gene therapy in the CNS
United States Patent: 6,706,523
Issued: March 16, 2004
Inventors: Fu; Zhen Fang (Athens, GA)
Assignee: University of Georgia Research Foundation, Inc. (Athens, GA)
Appl. No.: 199024
Filed: July 22, 2002
A mutant virus is provided which contains a mutation at a phosphorylation site in one or more of the proteins of the virus, which mutation causes the virus to be attenuated, and therefore, an improved vaccine composition can be produced therewith. The invention also relates to vaccine compositions which contain the mutant virus, as well as to methods of inducing an immune response, and of protecting mammals from infection by rabies virus. Also included in the invention are methods of producing the mutant virus and mutant viral proteins, including producing the mutant virus in a host cell which produces or even overproduces a wild-type counterpart of the mutant viral protein, which complements the other viral proteins such that production of the mutant viral particle is optimized. The invention also includes those host cells in which viral production is optimized, as well as vaccine compositions including the viral proteins, either alone or in combination with the intact virus, and to methods of inducing an immune response or protecting a mammal from infection, using the same. Also included in the invention are vectors suitable for delivering a gene to a cell of a human or animal, as well methods of delivery thereof.
SUMMARY OF THE INVENTION
In accordance with the present invention, a mutant virus is provided which contains a mutation at a phosphorylation site in one or more of the proteins of the virus, which mutation causes the virus to be attenuated, and therefore, an improved vaccine composition can be produced therewith.
In particular, a mutant rabies virus is provided, wherein the virus contains a mutant rabies virus N protein which has an amino acid other than serine at position 389. Additionally, the mutant virus may contain one or more mutations within the N protein, or in other of the viral proteins, for example, in the G glycoprotein.
The invention also relates to vaccine compositions which contain the mutant virus, as well as to methods of inducing an immune response, and of protecting mammals from infection by rabies virus.
Also included in the invention are methods of producing the mutant virus and mutant viral proteins, including producing the mutant virus in a host cell which produces or even overproduces a wild-type counterpart of the mutant viral protein, which complements the other viral proteins such that production of the mutant viral particle is optimized. The invention also includes those host cells in which viral production is optimized.
Also included within the invention are nucleic acids which encode the mutant viral protein(s), and nucleic acids which encode a portion of, or the entire viral nucleic acid sequence. In addition, the invention includes vectors containing the nucleic acid sequences, including expression vectors, and host cells transformed with the nucleic acid sequences.
The invention also includes the viral proteins encoded by the mutant nucleic acids, vaccine compositions including the viral proteins, either alone or in combination with the intact virus, and to methods of inducing an immune response or protecting a mammal from infection, using the same.
The invention also includes antibodies to the intact mutant virus and to the mutant viral proteins, and to methods of making and using the same.
Also included in the invention are vectors suitable for delivering a gene to a cell of a human or animal, as well methods of delivery thereof.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to effective and affordable virus vaccines for humans as well as for animals, to methods of making the same, and to methods of using the same for inducing an immune response, preferably a protective immune response in animals and humans. Suitable viruses include, but are not limited to, measles, Respiratory Syncytial virus (RSV), ebola virus and influenza virus, Sendai virus, and bovine RSV.
In particular, the invention relates to avirulent live virus vaccines containing mutant virus in which the phosphorylation on the N nucleoprotein has been disrupted. Suitable viruses include, but are not limited to, measles, Respiratory Syncytial virus (RSV), ebola virus and influenza virus, which are all phosphorylated on the N protein. The phosphorylation is disrupted by any suitable means, including alteration of the phosphorylation site by insertion, deletion or preferably by substitution. In addition, the phosphorylation may be disrupted by changes in other portions of the N protein, such as a consensus sequence at another site in the N protein. Preferably, the N protein has an amino acid other than serine at position 389, preferably a neutral amino acid, and more preferably, alanine.
The N protein may be mutated so as to affect the binding of the N protein to RNA, to a phosphate moiety, or to itself. This modulation of the binding properties of the N nucleoprotein affects vital functions of the virus, such as replication.
In a preferred embodiment, the present invention is directed to avirulent live rabies virus vaccines containing mutant virus in which the phosphorylation on the N nucleoprotein has been disrupted, either by insertion, deletion, substitution, or other appropriate means. Preferably, the virus has a reduced rate of viral replication (by mutating the nucleoprotein N or by reshuffling the genes within the rabies virus genome). In a preferred embodiment a serine at position 389 of the N nucleoprotein is substituted with alanine, glycine, glutamine, glutamic acid, aspartic acid or asparagine.
In a preferred embodiment, the viruses also have a reduced ability to spread in the nervous system (by mutation of the glycoprotein G), preferably at position 333 of the G glycoprotein.
The mutant viruses may also preferably have more than one change in either or both of the N and G proteins, such that the chances of reversion to a wild-type (WT) phenotype are reduced.
Any strain of rabies virus can be used in which the phosphorylation site is conserved. The phosphorylation site on the N protein of all presently known rabies viruses is conserved.
In addition, the mutant virus of the invention may contain a G glycoprotein of another type of virus, in order to direct the tropism of the virus within the body. Thus, the viruses of the present invention are likewise useful in gene therapy, for administering therapeutic or immunogenic proteins to the human or animal in which it is administered.
In particular, the rabies virus G glycoprotein causes a tropism for CNS cells, and thus is suitable for treating diseases of the CNS such as cancer, including but not limited to neuroblastoma, and neurodegenerative diseases including, but not limited to, Alzheimer's Disease, Parkinson's Disease, Huntington's Disease. Likewise, the human immunodeficiency virus (HIV) G protein causes a tropism for T cells, and thus is suitable for treating T-cell mediated disorders by gene delivery thereto, including various cancers, and diseases affecting T-cells, including HIV.
The vesicular stomatitis virus (VSV) G glycoprotein is pantropic, and thus may be used for administration to various cell types. The RSV G glycoprotein causes a tropism for epithelia, and thus is suitable for direction to the lung and treatment of disorders thereof, including, but not limited to, cystic fibrosis.
The invention also relates to methods of using the mutant virus for inducing an immune response, and preferably, a protective immune response in a human or animal.
Also included in the invention are host cells for producing the mutant virus, as well as a method of producing the same. Preferably, the host cell is a mammalian host cell which produces a wild-type rabies virus N protein, preferably a hamster cell, more preferably a BHK cell, and most preferably, a host cell which was deposited at the American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209, USA, as deposit number ATCC PTA-3544 on Jul. 20, 2001.
Mutation on both the G and N or relocation of these genes leads to attenuation of the virus to an extent that the virus no longer causes disease in animals at any age and by any route of infection; yet, it can induce immune responses that provide protection against virulent rabies virus challenge. This is based on recent studies showing the following. 1) Mutation of the phosphorylated serine at 389 of the N to alanine reduced the rate of viral replication by more than five-fold and virus production by more than 10,000 times. 2) Mutation of the G at residue 333 reduced dramatically the virulence and pathogenicity of rabies virus. 3) Rearrangement of the genes in a related virus, vesicular stomatitis virus (VSV), resulted in attenuation and enhancement of its immune responses. Rabies viruses with mutations on both the G and N or with rearranged genes are further attenuated than currently available attenuated rabies viruses (still induce rabies in neonatal animals). Further attenuated rabies viruses which are incapable of inducing diseases in experimental animals at any age and by any route of inoculation, yet remain immunogenic, can be developed into modified live rabies vaccines for humans and animals.
Alternatively, the vaccine of the present invention may contain isolated mutant N protein, in the absence of intact virus. Because the N phosphorylation mutant aggregates to a larger extent than its wild-type counterpart, it may have increased adjuvant effects compared to compositions containing wild-type N.
The vaccine compositions of the invention may contain an adjuvant, including, but not limited to, hepatitis B surface antigen (HbsAg) or the rabies virus G protein. The vaccine may be prepared using any pharmaceutically acceptable carrier or vehicle, including Hanks basic salt solution (HBSS) or phosphate buffered saline (PBS). The vaccine compositions can be administered by any known route, including intradermal, intramuscular and subcutaneous, which are preferred, as well as oral, via skin (epidermal abrasion) or intranasal.
In accordance with the present invention there may be employed conventional molecular biology, microbiology, immunology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook et al, "Molecular Cloning: A Laboratory Manual" (3rd edition, 2001); "Current Protocols in Molecular Biology" Volumes I-III [Ausubel, R. M., ed. (1999 and updated bimonthly)]; "Cell Biology: A Laboratory Handbook" Volumes I-III [J. E. Celis, ed. (1994)]; "Current Protocols in Immunology" Volumes I-IV [Coligan, J. E., ed. (1999 and updated bimonthly)]; "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)]; "Culture of Animal Cells, 4th edition" [R. I. Freshney, ed. (2000)]; "Immobilized Cells And Enzymes" [IRL Press, (1986)]; B. Perbal, "A Practical Guide To Molecular Cloning" (1988); Using Antibodies: A Laboratory Manual: Portable Protocol No. I, Harlow, Ed and Lane, David (Cold Spring Harbor Press, 1998); Using Antibodies: A Laboratory Manual, Harlow, Ed and Lane, David (Cold Spring Harbor Press, 1999).
The present invention further contemplates therapeutic compositions useful in practicing the therapeutic methods of this invention. A subject therapeutic composition includes, in admixture, a pharmaceutically acceptable excipient (carrier) and one or more of a mutant rabies virus, a mutant rabies virus polypeptide or fragment thereof, as described herein as an active ingredient. In a preferred embodiment, the composition comprises an antigen capable of inducing an immune response, and preferably a protective immune response against rabies.
The preparation of therapeutic compositions which contain polypeptides, analogs or active fragments as active ingredients is well understood in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions, however, solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared. The preparation can also be emulsified. The active therapeutic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents which enhance the effectiveness of the active ingredient.
A virus, polypeptide, or fragment thereof can be formulated into a therapeutic and/or immunogenic composition as neutralized pharmaceutically acceptable salt forms. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide or antibody molecule) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
The therapeutic and/or immunogenic virus-, polypeptide-, or fragment-containing compositions are conventionally administered in a unit dose, for example. The term "unit dose" when used in reference to a therapeutic composition of the present invention refers to physically discrete units suitable as unitary dosage for humans, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic and/or immunogenic effect in association with the required diluent; i.e., carrier, or vehicle.
The compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically or immunogenically effective amount. The quantity to be administered depends on the subject to be treated, capacity of the subject's immune system to utilize the active ingredient, and degree of expression desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual. However, for polypeptide administration, suitable dosages may range from about 0.1 to 20, preferably about 0.5 to about 10, and more preferably one to several, milligrams of active ingredient per kilogram body weight of individual per day and depend on the route of administration. For viral administration, suitable dosages may be from 105 infectious units (i.u.) to 107 i.u. Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed by repeated doses at 7 day intervals by a subsequent injection or other administration.
The therapeutic compositions may further include one or more of the following active ingredients: an antibiotic, a steroid.
Another feature of this invention is the expression of the DNA sequences operably inserted into the viruses disclosed herein. As is well known in the art, DNA sequences may be expressed by operatively linking them to an expression control sequence in an appropriate expression vector and employing that expression vector to transform an appropriate host.
Such operative linking of a DNA sequence of this invention to an expression control sequence, of course, includes, if not already part of the DNA sequence, the provision of an initiation codon, ATG, in the correct reading frame upstream of the DNA sequence.
A wide variety of host/expression vector combinations may be employed in expressing the DNA sequences encoding viral proteins of this invention. Useful expression vectors, for example, may consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences. Suitable vectors include derivatives of SV40 and known bacterial plasmids, e.g., E. coli plasmids col E1, pCR1, pBR322, pMB9 and their derivatives, plasmids such as RP4; phage DNAS, e.g., the numerous derivatives of phage.lambda., e.g., NM989, and other phage DNA, e.g., M13 and filamentous single stranded phage DNA; yeast plasmids such as the 2.mu. plasmid or derivatives thereof; vectors useful in eukaryotic cells, such as vectors useful in insect (baculovirus) or mammalian cells; vectors derived from combinations of plasmids and phage DNAs, such as plasmids that have been modified to employ phage DNA or other expression control sequences; and the like.
Any of a wide variety of expression control sequences--sequences that control the expression of a DNA sequence operatively linked to it--may be used in these vectors to express the DNA sequences of this invention. Such useful expression control sequences include, for example, the early or late promoters of SV40, CMV, vaccinia, polyoma or adenovirus, the lac system, the trp system, the TAC system, the TRC system, the LTR system, the major operator and promoter regions of phage .lambda., the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase (e.g., Pho5), the promoters of the yeast .alpha.-mating factors, and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.
A wide variety of host cells are also useful in expressing the DNA sequences encoding viral proteins of this invention. These hosts may include well known eukaryotic and prokaryotic hosts, such as strains of E. coli, Pseudomonas, Bacillus, Streptomyces, fungi such as yeasts, and animal cells, such as CHO, R1.1, B-W and L-M cells, African Green Monkey kidney cells (e.g., COS 1, COS 7, BSC1, BSC40, and BMT10), insect cells (e.g., Sf9), BHK cells, and human cells and plant cells in tissue culture.
It will be understood that not all vectors, expression control sequences and hosts will function equally well to express the DNA sequences of this invention. Neither will all hosts function equally well with the same expression system. However, one skilled in the art will be able to select the proper vectors, expression control sequences, and hosts without undue experimentation to accomplish the desired expression without departing from the scope of this invention. For example, in selecting a vector, the host must be considered because the vector must function in it. The vector's copy number, the ability to control that copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, will also be considered.
In selecting an expression control sequence, a variety of factors will normally be considered. These include, for example, the relative strength of the system, its controllability, and its compatibility with the particular DNA sequence or gene to be expressed, particularly as regards potential secondary structures. Suitable mutant viral vectors will be selected by consideration of, e.g., their replicative capacity as well as the toxicity to the host of the product encoded by the DNA sequences to be expressed, or by the mutant virus.
Considering these and other factors a person skilled in the art will be able to construct a variety of vector/expression control sequence/host combinations that will express the DNA sequences of this invention.
It is further intended that other mutant viral proteins may be prepared from nucleotide sequences of the present invention. Analogs, such as fragments, may be produced, for example, by pepsin digestion of viral polypeptide material. Other analogs, such as muteins, can be produced by standard site-directed mutagenesis of sequences encoding viral proteins. Mutants exhibiting immunogenic or protective activity, may be identified by known in vivo and/or in vitro assays.
As mentioned above, a DNA sequence encoding the virus or viral proteins can be prepared synthetically rather than cloned. The complete sequence is assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, e.g., Edge, Nature, 292:756 (1981); Nambair et al., Science, 223:1299 (1984); Jay et al., J. Biol. Chem., 259:6311 (1984).
Synthetic DNA sequences allow convenient construction of genes which will express viral protein mutants or "muteins". Alternatively, DNA encoding muteins can be made by site-directed mutagenesis of native viral genes or cDNAs, and muteins can be made directly using conventional polypeptide synthesis.
A general method for site-specific incorporation of unnatural amino acids into proteins is described in Christopher J. Noren, Spencer J. Anthony-Cahill, Michael C. Griffith, Peter G. Schultz, Science, 244:182-188 (April 1989). This method may be used to create analogs with unnatural amino acids.
Claim 1 of 62 Claims
What is claimed is:
1. A mutant rabies virus comprising a rabies virus N protein, wherein said N protein is not phosphorylated.