Attenuated rabies virus with nucleoprotein mutation at the phosphorylation
site for vaccination against rabies and gene therapy in the CNS
United States Patent: 7,544,791
Issued: June 9, 2009
Inventors: Fu; Zhen Fang
Assignee: University of
Georgia Research Foundation, Inc. (Athens, GA)
Appl. No.: 11/700,899
Filed: February 1, 2007
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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.
Description of the
SUMMARY OF THIS 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.
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.
Preferably, the mutant rabies virus is encoded by one of the sequences of
FIG. 8 (see Original Patent), the mutant rabies virus N protein is encoded
by one of the sequences of FIG. 9 (see Original Patent), and/or the mutant
rabies virus G protein is encoded by one of the sequences of FIG. 10 (see Original Patent).
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
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
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,
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
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" (3.sup.rd 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, 4.sup.th 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 10.sup.5 infectious units (i.u.) to 10.sup.7 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 El, pCR1, pBR322, pMB9 and their derivatives, plasmids such as
RP4; phage DNAS, e.g., the numerous derivatives of phage.lamda., 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 .lamda., 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
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
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
Claim 1 of 14 Claims
1. A composition comprising an isolated
nucleic acid encoding a mutant rabies virus comprising a rabies virus N
protein, wherein said N protein comprises an amino acid other than serine
at position 389.
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