Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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METHODS AND COMPOSITIONS FOR ENGINEERING OF
ATTENUATED VACCINES
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
Not Applicable.
CROSS-REFERENCE TO RELATED APPLICATIONS
Not Applicable.
BACKGROUND OF THE INVENTION
Field of the Invention
This invention pertains to the field of vaccine development. Methods are
provided for obtaining attenuated vaccines that exhibit improvements compared
to
previously available attenuated vaccines.
Background
Edward Jenner demonstrated in 1796 that inoculation of a person with the
cowpox virus would confer protection against its deadly relative, smallpox.
Jenner's
discovery was followed by Louis Pasteur's development in 1879-1881 of
attenuated
vaccines for chicken cholera, anthrax, and rabies. Since these early
discoveries, attenuated
vaccines have provided a significant addition to medicine's arsenal of weapons
against a
wide variety of infectious and other diseases.
Attenuated vaccines are otherwise pathogenic organisms that lack certain
characteristics that are necessary to produce disease. Both bacteria and
viruses are suitable
for use as attenuated vaccines. Attenuated virus vaccines include, for
example, vaccinia virus
and other attenuated poxvirus vectors. One such poxvirus attenuated vaccine,
the NYVAC
vaccine, was obtained by attenuating the Copenhagen strain of vaccinia by
complete deletion
of the reading frames of 18 genes involved in virulence, tissue tropism and
host range
(Tartaglia et al., Yirolo~ 188: 217-32 (1992)). The resultant vector, which is
highly
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attenuated but nevertheless has the immunogenic potential of the original
Copenhagen strain,
is used in clinical trials as a human vaccine vector (Lanar et al., Infect.
Immure. 64: 1666-71
(1996); Limbach and Paoletti, Epidemiol. Infect. 116: 241-56 (1996); Paoletti
et al., Dev.
Biol. Stand. 84:19-63 (1995)). Another poxvirus vector, the ALVAC vector, is a
highly
attenuated strain derived from canarypox and is licensed as a veterinary
vaccine for canaries.
It is a safe and efficacious vector in several mammalian species, including
man. The
TROVAC vector is based on a highly attenuated fowlpox vaccine strain and is
used to
immunize day-old chicks against fowlpox disease. TROVAC can also be used as a
vector for
vaccination of chicks against avian influenza virus and Newcastle disease
virus (Paoletti et
al., supra.). Other attenuated viral vaccines include those that are useful
against polio,
measles, mumps, rubella, yellow fever and varicella. Examples of attenuated
bacteria that are
useful as vaccines include, for example, BCG (Bacillus Calmette-Guerin),
Salmonella and E.
coli.
Attenuated viruses and bacteria have been produced by the various methods,
including UV irradiation (Hristov and Karadjov, Yet. Med. Nauki, 13: 8
(1975)), chemical
attenuation by formalin or ethanol treatment (Zuschek et al., J. Am. Vet. Med
Assoc. 139:
236 (1961); Haralambiev, Acta Vet. Acad. Sci. Hung. 26: 215 (1976)), and
passage under
conditions of stress in vitro or in vivo. An example of in vitro passage under
stress was the
development of the first attenuated virus licensed as a smallpox vaccine,
which was obtained
by conducting 36 passages of the original Lister strain (LO) of vaccinia in
primary rabbit
kidney cells at 30°C, followed by another 6 passages. Selection then
yielded LC16m0 as a
temperature sensitive and medium pock-forming virus. LC-16m8 was cloned from
LC 16m0
as a small-pock forming variant (see, e.g., Sugimoto, Vaccine 12: 675-681
(1994); Morita et
al., Vaccine 5: 65-70 (1987)). Another example of the development of a vaccine
by serial
passage in vitro is the passage of vaccinia virus through >570 passages in
chick embryo
fibroblasts to generate the modified vaccinia virus (MVA) that is host
restricted, unable to
replicate in human and other mammalian cells. MVA is avirulent in normal and
immunosuppressed animals and produced no side effects, unlike the conventional
smallpox
vaccine strains. MVA had at least 30,000 by of DNA deleted with loss of at
least two host
range genes.
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Another approach to development of attenuated vaccines involves the specific
deletion of genes known to confer virulence. For example, in the case of
vaccinia virus the
following genes have been deleted to alter the viral phenotype: TK, growth
factor,
hemagglutinin, 13.8 kb secreted protein, ribonucleotide reductase, envelope
proteins, steroid
dehydrogenase, complement control protein, host range genes (Moss, Dev. Biol.
Stand. 82:
55-63 (1994); Blanchard et al., J. Gen. Virol. 79: 1159-1167 (1998); Carroll
and Moss,
Virology 238: 198-211 (1997)). The example of development of the NYVAC vector,
discussed above, is notable in that the entire genome was sequenced and
relevant genes were
precisely deleted. Thus, this is an example of the construction of an
attenuated vaccine
wherein the exact alterations are known and distance from the wild type
understood. Such
precise delineations of the genetic alterations of an attenuated strain are
obviously more
accessible technically-with a virus than with the larger genomes of bacteria.
Similarly, HSV
lacking an essential glycoprotein (gH gene) can undergo a single round of
replication in
normal cells, but the virus particles derived from this infection are
noninfectious. They are
called DISC viruses (disabled infectious single cycle) and in the case of an
HSV-2 lacking
gH sequences have been shown in a guinea pig model of genital HSV-2 infection
to protect
against infection and against primary and recurrent disease (Boursnell et al.,
J. Infect. Dis.
175 : 16-25 ( 1997)).
The insertion of specific genes into the viral genome provides another
approach to developing attenuated vaccines. For example, lymphokine genes have
been
inserted into the genome of vaccinia in order to decrease virulence without
affecting
immunogenicity. Murine or human IL-2 or interferon gamma have been inserted
into the
genome of vaccinia to produce virus of much lower pathogenicity yet unaltered
immunogenicity (Moss, Dev. Biol. Stand., 82: 55-63 (1994)). A similar approach
involved
the insertion of the B5R gene of the LO strain of vaccinia virus into LC16m8
infected RK13
cells with derivation of the LOTC virus strains (LOTC-1 through 5). The BR5
gene is
responsible for plaque and pock size and host range and corresponds to-the
pslhr gene
(Sugimoto, Vaccine 12: 675-678 (1994)).
In some of the cases described above, defined genetic alterations have been
performed with an already attenuated virus while in others, particularly with
bacterial
vaccines, there is little or no knowledge of the genetic lesions that confer
attenuation.
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Diverse types of genetic alterations are presumed to have been generated in
the course of
attenuation, including point mutations, DNA deletion and rearrangement. These
methods of
attenuation do not, in themselves, dictate the precise nature of the genetic
alterations that
confer the attenuated phenotype. Even though mutations may be chemically or UV
irradiation induced via characterized chemical mechanisms, the positions of
mutation are not
controllable by current technologies. Nor are the number of sites in the
genome that have
been altered controlled or characterized. Although there may be a dose
response relationship
between concentration of mutagen, for example, and degree of phenotypic
alteration,
whether there are 10 or 100 genes whose function has been disrupted in the
process of
attenuation is unknown. Consequently, these mutagenesis methods do not control
which
genes or control elements have been modified. In the absence of sequencing the
entire
genome of the bacterium or virus (until recently not a practical or technical
feasibility), the
positions of mutations/deletions/rearrangements in the genome are unknown. An
attenuated
phenotype could therefore reflect modification in one virulence gene and many
other genes
not relevant to virulence, or modification of 10 virulence genes as ~.vell as
many other genes
unrelated to virulence. In such cases, the degree of difference between the
attenuated
genotypes is very different genetically but may appear little different
phenotypically.
Other problems that are associated with current methods of attenuation
include, for example, the reversion of attenuated organisms to wild type
phenotype in
vaccinees, with consequent disease pathology including severe morbidity and
death. Vaccine
strains of infectious bronchitis virus (IBV) are one example of attenuated
vaccines that easily
revert to more virulent strains in vivo (Hopkins and Yoder;rvian Dis., 30: 221-
3 (1986)). A
related problem is the inability to monitor the stability of the genetic
alterations in the
attenuated vaccine organism during the process of developing, manufacturing,
and
distributing the vaccine over many years or decades. If one has not defined
what is altered, it
is impossible to identify whether the genotype is in flux and whether the
organism is
reverting closer to wild type phenotype. Vaccine manufacturers monitor the
phenotype of the
attenuated organism by standard protocols to ensure that the organism appears
stable under
standard conditions, but such methods are imprecise and do not allow any
understanding of
whether the number of mutations that separate the attenuated organism from
wild type
phenotype may have decreased to such a number that a very small number of
additional
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reversions in genotype might confer wild type phenotype). An additional
problem with
previously available methods for developing attenuated vaccines is a loss of
immunogenicity
due alteration of genes, by loss or altered sequence, that are important for
elicitation of
desired immune responses in vaccinees.
Another problem, the retention of replication competence in an attenuated
vaccine or vaccine vector raises safety concerns, particularly in
immunocompromised
persons or animals in whom even a substantially attenuated virus or bacterium
may cause
disease. The retention of replication competence, on the other hand, can be an
advantage for
the stimulation of broad based and long lasting immunity. One approach to
solving this
dilemma is to engineer the virus to undergo only one round of replication such
that its
capacity to cause disease is eliminated yet capacity to immunize effectively
enhanced
compared to replication incompetent virus or inactivated virus. An example is
the DISC
virus vaccine approach, as exemplified with HSV-1 or HSV-2 viruses in which
the essential
gH gene is deleted. Reactivation of latent Herpes zoster virus elicited by
immunization with
a live attenuated varicella virus vaccine is another drawback to the use of
attenuated vaccines
(Garnett and Grenfell, Epidemiol. Infect. 108: 513-528 (1992)).
Although the potential problems associated with attenuated vaccines are
significant, the attenuated vaccines have shown promise against infectious
diseases for
which other vaccines are not yet available. For example, an International AIDS
Vaccine
Initiative report indicates that some people infected with a weak strain of
HIV have remained
healthy for more than a dozen years, and at least one person with a weakened
HIV strain
may have successfully warded off multiple exposures to other HIV strains. This
finding is
supported by some primate studies. Accordingly, given the great need for an
AIDS vaccine,
attenuated vaccines are currently receiving a great deal of attention.
However, a recent study
which used attenuated versions of the HIV analog SIV, which infects monkeys,
found that
some monkeys may have acquired AIDS from the attenuated vaccine. Therefore, a
need
exists for improved attenuated vaccines, and for methods of developing such
vaccines. The
present invention fulfills these and other needs.
SUMMARY OF THE INVENTION
The present invention provides methods for obtaining attenuated vaccines.
The vaccines are useful for therapeutic and prophylactic purposes, and are
effective against
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pathogenic agents such as viruses, bacteria, parasites, and others. In some
embodiments, the
methods involve recombining a first set of one or more nucleic acid segments
that comprise
a complete or partial genomic library of a virus or a cell with at least a
second set of one or
more nucleic acid segments. Viruses or cells that contain members of the
resulting library of
recombinant nucleic acid fragments are then screened to identify those that
are attenuated
under physiological conditions that exist in a host organism. For example, the
viruses or
cells can be ''attenuated" in that they are less able to propagate andlor
cause disease in the
host organism than a naturally occurring isolate of the viruses or cells.
In presently preferred embodiments, the attenuated viruses or cells are
screened to identify those that can induce an immune response against a
pathogenic agent
that displays an immunogenic determinant that is also displayed by the
attenuated viruses or
cells. Attenuated viruses or cells that can induce the immune response are
useful as
attenuated vaccines against the pathogenic agent.
In some embodiments, the methods involve performing recursive
recombination and screening/selection. This involves recombining
polynucleotides from the
attenuated cells or viruses obtained in a first round of recombination and
screening/selection
with a further set of one or more forms of a nucleic acid, to form a further
library of
recombinant nucleic acids. Viruses or cells that contain members of the
further library of
recombinant nucleic acid fragments are screened to identify those viruses or
cells that are
further attenuated under physiological conditions that exist in a host
organism. The
recombination and selection/screening can be repeated until the resulting
attenuated viruses
or cells have lost the ability to replicate or cause disease under
physiological conditions that
exist in the host organism.
The nucleic acid segments of at least one of the sets are, in some
embodiments, obtained from a non-pathogenic strain of a virus or cell. In such
cases, at least
one of the other sets of nucleic acid segments are typically derived from a
pathogenic agent,
which can be of the same species as the nonpathogenic strain, or of a
different species. One
or more of the sets of nucleic acid segments can comprise a complete or
substantially
complete genomic library of the cell or virus from which the segments are
derived, or can be
a partial genomic library of the cell or virus. The nucleic acid segments can
also include
those that encode all or part of an immunogenic polypeptide that is displayed
on a
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pathogenic agent, or that encode a polypeptide, such as an immunomodulatory
molecule or
therapeutic protein, that has a desirable effect on an immune response induced
by the
vaccine.
In presently preferred embodiments of the invention, the attenuated viruses or
cells are backcrossed to remove superfluous mutations. Backcrossing, according
to the
invention, involves recombining nucleic acids from the attenuated viruses or
cells with a
library of nucleic acids from a wild-type or naturally occurring strain of the
virus or cell to
form a further library of recombinant nucleic acids. Viruses or cells that
contain members of
the further library of recombinant nucleic acids are then screened to identify
backcrossed
viruses or cells that remain attenuated under physiological conditions present
in an
inoculated host organism. The library of nucleic acids from the wild-type
strain is, in some
embodiments, a partial or complete genomic library of the naturally occurring
strain. The
backcrossed attenuated viruses or cells can also be screened to identify those
that can induce
an immune response against a pathogenic agent that displays an immunogenic
determinant
that is also displayed by the attenuated viruses or cells. The backcrossing
can be repeated
one or more times, as desired.
The methods can also involve screening the attenuated viruses or cells to
identify those that propagate under permissive conditions used for production
of the
attenuated viruses or cells, but do not propagate significantly in an
inoculated host organism.
The permissive conditions used for production can differ from the
physiological conditions
in the host in, for example, temperature, pH, sugar content, a compromised
immune system,
absence of complement or complement components, and presence or absence of
serum
proteins.
In some embodiments, the permissive condition used for the production of the
attenuated vaccine is the presence of a suppressor tRNA molecule that can
suppress
termination of translation at non-naturally occurring stop codons that are
introduced into the
genome of the attenuated virus or cell. The nucleic acid segments subjected to
recombination
include one or more polynucleotides that encode all or part of a polypeptide
that is involved
in replication or pathogenicity of the virus or cells. The polypeptide-
encoding
polynucleotides can be included within a complete or partial genomic library
of a virus or
cell. Also included in the recombination is a population of oligonucleotides
that have one or
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more stop codons interspersed within the coding sequences for the polypeptide.
The
oligonucleotides undergo recombination with the polypeptide-encoding
polynucleotides to
form a library of recombinant nucleic acids in which at least one nonnaturally
occurring stop
codon is interspersed within the coding sequence of the replication
polypeptide. The
attenuated viruses or cells are obtained by contacting the library of
recombinant nucleic acid
fragments with suppressor tRNA molecules that suppress the termination of
translation at the
nonnaturally occurring stop codons and collecting progeny viruses or cells
that propagate in
the presence of the suppressor tRNA molecules but not in the absence of the
suppressor
tRNA molecules.
The invention also provides methods of obtaining an attenuated vaccine by
introducing a library of nucleic acid fragments into a plurality of cells,
whereby at least one
of the fragments undergoes recombination with a segment in the genome or an
episome of
the cells to produce modified cells. The modified cells are screened to
identify conditionally
defective cells that have evolved toward loss of the ability to proliferate
under physiological
conditions as found in a host organism. The conditionally defective cells are,
in turn,
screened to identify those modified cells that have maintained the ability to
replicate under
permissive conditions used for production of the attenuated vaccine. The
conditionally
defective cells that replicate under permissive conditions but not in a host
mammal are
suitable for use as an attenuated vaccine organism.
In other embodiments, the invention provides methods of obtaining a
chimeric attenuated vaccine. These methods generally involve recombining a
first set of one
or more nucleic acid segments from a virus or cell with at least a second set
of one or more
nucleic acid segments. The nucleic acid segments of the second set generally
confer upon
viruses or cells that contain the nucleic acid segments a property that is
desirable for
vaccination. A library of recombinant DNA fragments is .thus formed.
Attenuated viruses or
cells are then identified by screening viruses or cells that contain members
of the library of
recombinant DNA fragments to identify those viruses or cells that are
attenuated under
physiological conditions present in a host organism inoculated with the
viruses or cells. The
attenuated viruses or cells are then screened to identify those that exhibit
an improvement in
the property that is desirable for vaccination. The screening can be conducted
in any order.
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In some embodiments, at least one of the sets of nucleic acid segments is a
partial or substantially complete genomic li'orary of a virus or cell. For
example, one set can
be from a pathogenic virus or cell, while another set is from a non-pathogenic
isolate of virus
or cell. The pathogenic and non-pathogenic isolates can be of the same or
different species.
The recombination is performed, in some embodiments, by introducing the
second set of nucleic acid segments into a plurality of nonpathogenic cells.
At least one
member of the second set of nucleic acid segments undergoes recombination with
a segment
in the genome or an episome of the nonpathogenic cells to produce modified
cells. The
modified cells are then screened to identify attenuated chimeric cells that
are nonpathogenic
and exhibit an improvement in the property that is desirable for vaccination.
In some embodiments, the methods of obtaining chimeric vaccines involve
recursive recombination and screening. For example, one can recombine nucleic
acids from
the attenuated chimeric cells or viruses with a further set of nucleic acid
segments to form a
further library of recombinant nucleic acids. Attenuated chimeric viruses or
cells that exhibit
further improvement in attenuation or in the property that is desirable for
vaccination are
identified by screening viruses or cells that contain members of the further
library of
recombinant DNA fragments to identify those that exhibit further improved
attenuation or
desirable property. The recombination and screening can be repeated one or
more times as
desired until the attenuated chimeric viruses or cells have achieved a desired
level of
pathogenicity loss or improvement in the property that is desirable for
vaccination.
The invention also provides attenuated and chimeric viruses and cells that are
produced using the methods described herein. Also provided are vaccine
compositions and
methods of vaccinating using the vaccine compositions of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows a schematic representation of recombinatorial shuffling of a
collection of families of viral genomes having a variety of mutations or
distinct genome
portions; distinct genome segments (e.g., obtained from the genomes of
different virus
isolates) are indicated by shaded boxes.
Figure 2 shows a schematic of a protocol for screening chimeric viral
vaccines that are obtained using the recombination methods of the invention.
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Figure 3 shows a summary of a strategy for using the methods of the
invention to evolve a multivalent viral vaccine. HPV is used as an
illustrative example. T'ne
arrows in the right lane of the figure point to each subsequent task after
successful
accomplishment of the previous. The broken arrows in the left lane outline
steps of
alternative methods if the previous task fails to lead to the next step.
Figure 4 shows a phylogenetic tree of papillomavirus (source: Human
Papillomavirus comp. 1997).
Figure 5 shows a high throughput (HTP) in vitro screening assay for
identifying recombinant nucleic acids that encode an improved antigen.
Figure 6 shows a schematic of a protocol for an antigen library immunization
and cross-neutralization assay.
DETAILED DESCRIPTION
Definitions
A "pathogenic agent" refers to an organism or virus that is capable of
infecting a host cell. Pathogenic agents are typically capable of causing a
disease or other
adverse effect on an infected cell or organism. Pathogenic agents include, for
example,
viruses, bacteria, fungi, parasites, and the like. The term "virus" includes
not only complete
virus particles, but also virus-like particles (VLPs) that include one or more
viral
polypeptides.
The term "attenuated," when used with respect to a virus or cell, means that
the virus or cell has lost some or all of its ability to proliferate and/or
cause disease or other
adverse effect when the virus or cell infects an organism. For example, an
"attenuated" virus
or cell can be unable to replicate at all, or be limited to one or a few
rounds of replication,
when present in an organism in which a wild-type or other pathogenic version
of the
attenuated virus or cell can replicate. Alternatively or additionally, an
"attenuated" virus or
cell might have one or more mutations in a gene or genes that are involved in
pathogenicity
of the viruses or cells.
A "host organism" is an animal that is a target of vaccination with the
attenuated and chimeric vaccines of the invention. Such host organisms have an
immune
system that is responsive to inoculation with an immunogen. Suitable host
organisms
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include, for example, humans, livestock, birds, and other animals in which it
is desirable to
vaccinate for either therapeutic or prophylactic purposes.
A "vaccine," as used herein, refers to an immunogen that, upon inoculation
into a host organim, can induce complete or partial immunity to pathogenic
agents, or can
reduce the effects of diseases associated with pathogenic agents. Vaccines are
also useful to
alleviate immune system disorders other than those associated with pathogenic
agents, such
as autoimmune conditions.
The term "screening" describes, in general, a process that identifies vaccines
that have optimal properties, such as attenuation. Selection is a form of
screening in which
identification and physical separation of attenuated vaccines are achieved
simultaneously.
For example, expression of a selection marker, which, in some genetic
circumstances, allows
cells expressing the marker to survive while other cells die (or vice versa)
can be used as a
selection method. Screening markers include, for example, genes that express
luciferase, (3-
galactosidase and green fluorescent protein, or other gene products that are
readily detected
upon expression. Selection markers include, for example, drug and toxin
resistance genes.
Because of limitations in studying primary immune responses in vitro, in vivo
studies are
particularly useful screening methods. In these studies, the putative vaccines
are introduced
into test animals, and the immune responses are subsequently studied by
analyzing
protective immune responses or by studying the quality or strength of the
induced immune
response using, for example, lymphoid cells derived from the immunized animal.
Although
spontaneous selection can and does occur in the course of natural evolution,
in the present
methods selection is performed by man.
A "exogenous DNA segment", "heterologous sequence" or a "heterologous
nucleic acid", as used herein, is one that originates from a source foreign to
the particular
host cell, or, if from the same source, is modified from its original form.
Thus, a
heterologous gene in a host cell includes a gene that is endogenous to the
particular host cell,
but has been modified. Modification of a heterologous sequence in the
applications
described herein typically occurs through the use of DNA shuffling. Thus, the
terms refer to
a DNA segment which is foreign or heterologous to the cell, or homologous to
the cell but in
a position within the host cell nucleic acid in which the element is not
ordinarily found.
Exogenous DNA segments are expressed to yield exogenous polypeptides.
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The term "gene" is used broadly to refer to any segment of DNA associated
with a biological function. Thus, genes include coding sequences and/or the
regulatory
sequences required for their expression. Genes also include nonexpressed DNA
segments
that, for example, form recognition sequences for other proteins. Genes can be
obtained from
a variety of sources, including cloning from a source of interest or
synthesizing from known
or predicted sequence information, and may include sequences designed to have
desired
parameters.
The term "isolated", when applied to a nucleic acid or protein, denotes that
the nucleic acid or protein is essentially free of other cellular components
with which it is
associated in the natural state. It is preferably in a homogeneous state
although it can be in
either a dry or aqueous solution. Purity and homogeneity are typically
determined using
analytical chemistry techniques such as polyacrylamide gel electrophoresis or
high
performance liquid chromatography. A protein which is the predominant species
present in a
preparation is substantially purified. In particular, an isolated gene is
separated from open
reading frames which flank the gene and encode a protein other than the gene
of interest.
The term "purified" denotes that a nucleic acid or protein gives rise to
essentially one band
in an electrophoretic gel. Particularly, it means that the nucleic acid or
protein is at least
about 50% pure, more preferably at least about 85% pure, and most preferably
at least about
99% pure.
The term "naturally-occurring" is used to describe an object that can be found
in nature as distinct from being artificially produced by man. For example, an
organism, or a
polypeptide or polynucleotide sequence that is present in an organism
(including viruses,
bacteria, protozoa, insects, plants or mammalian tissue) that can be isolated
from a source in
nature and which has not been intentionally modified by man in the laboratory
is naturally-
occurring.
The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and
polymers thereof in either single- or double-stranded form. Unless
specifically limited, the
term encompasses nucleic acids containing known analogues of natural
nucleotides which
have similar binding properties as the reference nucleic acid and are
metabolized in a manner
similar to naturally occurring nucleotides. Unless otherwise indicated, a
particular nucleic
acid sequence also implicitly encompasses conservatively modified variants
thereof (e.g.
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degenerate codon substitutions) and complementary sequences and as well as the
sequence
explicitly indicated. Specifically, degenerate codon substitutions may be
achieved by
generating sequences in which the third position of one or more selected (or
all) codons is
substituted with mixed-base and/or deoxyinosine residues (Batzer et al. (1991)
Nucleic Acid
Res. 19: 5081; Ohtsuka et al. (1985) J. Biol. Chem. 260: 2605-2608; Cassol et
al. (1992) ;
Rossolini et al. (1994) l~Iol. Cell. Probes 8: 91-98). The term nucleic acid
is used
interchangeably with gene, cDNA, and mRNA encoded by a gene.
"Nucleic acid derived from a gene" refers to a nucleic acid for whose
synthesis the gene, or a subsequence thereof, has ultimately served as a
template. Thus, an
mRNA, a cDNA reverse transcribed from an mRNA, an RNA transcribed from that
cDNA, a
DNA amplified from the cDNA, an RNA transcribed from the amplified DNA, etc.,
are all
derived from the gene. and detection of such derived products is indicative of
the presence
and/or abundance of the original gene and/or gene transcript in a sample.
A nucleic acid is "operably linked" when it is placed into a functional
relationship with another nucleic acid sequence. For instance, a promoter or
enhancer is
operably linked to a coding sequence if it increases the transcription of the
coding sequence.
Operably linked means that the DNA sequences being linked are typically
contiguous and,
where necessary to join two protein coding regions, contiguous and in reading
frame.
However, since enhancers generally function when separated from the promoter
by several
kilobases and intronic sequences may be of variable lengths, some
polynucleotide elements
may be operably linked but not contiguous.
A specific binding affinity between two molecules, for example, a ligand and
a receptor, means a preferential binding of one molecule for another in a
mixture of
molecules. The binding of the molecules can be considered specific if the
binding affinity is
about 1 x 104 M -1 to about 1 x 106 M ~~ or greater.
The term "recombinant" when used with reference to a cell or virus indicates
that the cell or a cell infected by the virus, replicates a heterologous
nucleic acid, or
expresses a peptide or protein encoded by a heterologous nucleic acid.
Recombinant cells
and viruses can contain genes that are not found within the native (non-
recombinant) form of
the cell or virus. Recombinant cells and viruses can also contain genes found
in the native
form of the cell or virus wherein the genes are modified and re-introduced
into the cell by
13
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artificial means. The term also encompasses cells and viruses that contain a
nucleic acid
endogenous to the cell that has been modified without removing the nucleic
acid from the
cell; such modifications include those obtained by gene replacement, site-
specific mutation,
and related techniques.
A "recombinant expression cassette" or simply an "expression cassette" is a
nucleic acid construct, generated recombinantly or synthetically, with nucleic
acid elements
that are capable of effecting expression of a structural gene in hosts
compatible with such
sequences. Expression cassettes include at least promoters and optionally,
transcription
termination signals. Typically, the recombinant expression cassette includes a
nucleic acid
to be transcribed (e.g., a nucleic acid encoding a desired polypeptide), and a
promoter.
Additional factors necessary or helpful in effecting expression may also be
used as described
herein. For example, an expression cassette can also include nucleotide
sequences that
encode a signal sequence that directs secretion of an expressed protein from
the host cell.
Transcription termination signals, enhancers, and other nucleic acid sequences
that influence
gene expression, can also be included in an expression cassette.
The terms "identical" or percent "identity," in the context of two or more
nucleic acid or polypeptide sequences, refer to two or more sequences or
subsequences that
are the same or have a specified percentage of amino acid residues or
nucleotides that are the
same, when compared and aligned for maximum correspondence, as measured using
one of
the following sequence comparison algorithms or by visual inspection.
The phrase "substantially identical," in the context of two nucleic acids or
polypeptides, refers to two or more sequences or subsequences that have at
least 60%,
preferably 80%, most preferably 90-95% nucleotide or amino acid residue
identity, when
compared and aligned for maximum correspondence, as measured using one of the
following
sequence comparison algorithms or by visual inspection. Preferably, the
substantial identity
exists over a region of the sequences that is at least about ~0 residues in
length, more
preferably over a region of at least about 100 residues, and most preferably
the sequences are
substantially identical over at least about 150 residues. In a most preferred
embodiment, the
sequences are substantially identical over the entire length of the coding
regions.
For sequence comparison, typically one sequence acts as a reference sequence
to which test sequences are compared. When using a sequence comparison
algorithm, test
14
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and reference sequences are input into a computer, subsequence coordinates are
designated,
if necessary, and sequence algorithm program parameters are designated. The
sequence
comparison algorithm then calculates the percent sequence identity for the
test sequences)
relative to the reference sequence, based on the designated program
parameters.
Optimal alignment of sequences for comparison can be conducted, e.g., by
the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482
(1981), by the
homology alignment algorithm ofNeedleman & Wunsch, J. ~~Iol. Biol. 48:443
(1970), by the
search for similarity method of Pearson & Lipman, Proc. Nat'1. Acad. Sci. USA
85:2444
(1988), by computerized implementations of these algorithms (GAP, BESTFIT,
FASTA,
and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer
Group, 57~
Science Dr., Madison, WI), or by visual inspection (see generally Ausubel et
al., infra).
One example of an algorithm that is suitable for determining percent
sequence identity and sequence similarity is the BLAST algorithm, which is
described in
Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing
BLAST analyses
is publicly available through the National Center for Biotechnology
Information
(http://www.ncbi.nlm.nih.gov~. This algorithm involves first identifying high
scoring
sequence pairs (HSPs) by identifying short words of length W in the query
sequence, which
either match or satisfy some positive-valued threshold score T when aligned
with a word of
the same length in a database sequence. T is referred to as the neighborhood
word score
threshold (Altschul et al., supra). These initial neighborhood word hits act
as seeds for
initiating searches to find longer HSPs containing them. The word hits are
then extended in
both directions along each sequence for as far as the cumulative alignment
score can be
increased. Cumulative scores are calculated using, for nucleotide sequences,
the parameters
M (reward score for a pair of matching residues; always > 0) and N (penalty
score for
mismatching residues; always < 0). For amino acid sequences, a scoring matrix
is used to
calculate the cumulative score. Extension of the word hits in each direction
are halted when:
the cumulative alignment score falls off by the quantity X from its maximum
achieved
value; the cumulative score goes to zero or below, due to the accumulation of
one or more
negative-scoring residue alignments; or the end of either sequence is reached.
The BLAST
algorithm parameters W, T, and X determine the sensitivity and speed of the
alignment. The
BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of
11, an
CA 02377084 2001-12-21
WO 01/00234 PCT/US00/16984
expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both
strands. For
amino acid sequences, the BLASTP program uses as defaults a wordlength (~ of
3, an
expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff &
Henikoff (1989)
Proc. Natl. Acad. Sci. USA 89:10915).
In addition to calculating percent sequence identity, the BLAST algorithm
also performs a statistical analysis of the similarity between two sequences
(see, e.g., Karlin
& Altschul (1993) Proc. Nat'1. Acad. Sci. US4 90:5873-5787). One measure of
similarity
provided by the BLAST algorithm is the smallest sum probability (P(I~), which
provides an
indication of the probability by which a match between two nucleotide or amino
acid
sequences would occur by chance. For example, a nucleic acid is considered
similar to a
reference sequence if the smallest sum probability in a comparison of the test
nucleic acid to
the reference nucleic acid is less than about 0.1, mere preferably less than
about 0.01, and
most preferably less than about 0.001.
Another indication that two nucleic acid sequences are substantially identical
is that the two molecules hybridize to each other under stringent conditions.
The phrase
"hybridizing specifically to", refers to the binding, duplexing, or
hybridizing of a molecule
only to a particular nucleotide sequence under stringent conditions when that
sequence is
present in a complex mixture (e.g., total cellular) DNA or RNA. "Bind(s)
substantially"
refers to complementary hybridization between a probe nucleic acid and a
target nucleic acid
and embraces minor mismatches that can be accommodated by reducing the
stringency of
the hybridization media to achieve the desired detection of the target
polynucleotide
sequence.
"Stringent hybridization conditions" and "stringent hybridization wash
conditions" in the context of nucleic acid hybridization experiments such as
Southern and
northern hybridizations are sequence dependent, and are different under
different
environmental parameters. Longer sequences hybridize specifically at higher
temperatures.
An extensive guide to the hybridization of nucleic acids is found in Tijssen
(1993)
Laboratory Techniques in Biochemistry and Molecular Biology--Hybridization
with Nucleic
Acid Probes part I chapter 2 "Overview of principles of hybridization and the
strategy of
nucleic acid probe assays", Elsevier, New York. Generally, highly stringent
hybridization
and wash conditions are selected to be about 5° C lower than the
thermal melting point (Tm)
16
CA 02377084 2001-12-21
WO 01/00234 PCT/US00/16984
for the specific sequence at a defined ionic strength and pH. Typically, under
"stringent
conditions" a probe will hybridize to its target subsequence, but to no other
sequences.
The Tm is the temperature (under defined ionic strength and pH) at which
50% of the target sequence hybridizes to a perfectly matched probe. Very
stringent
conditions are selected to be equal to the Tm for a particular probe. An
example of stringent
hybridization conditions for hybridization of complementary nucleic acids
which have more
than 100 complementary residues on a filter in a Southern or northern blot is
50%
formamide with 1 mg of heparin at 42°C, with the hybridization being
carried out overnight.
An example of highly stringent wash conditions is O.15M NaCI at 72°C
for about 15
minutes. An example of stringent wash conditions is a 0.2x SSC wash at
65°C for 15
minutes (see, Sambrook, infra., for a description of SSC buffer). Often, a
high stringency
wash is preceded by a_low stringency wash to remove background probe signal.
An example
medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is lx
SSC at 45°C
for 1 ~ minutes. An example low stringency wash for a duplex of, e.g., more
than 100
nucleotides, is 4-6x SSC at 40°C for 15 minutes. For short probes
(e.g., about 10 to 50
nucleotides), stringent conditions typically involve salt concentrations of
less than about 1.0
M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts)
at pH 7.0 to
8.3, and the temperature is typically at least about 30°C. Stringent
conditions can also be
achieved with the addition of destabilizing agents such as formamide. In
general, a signal to
noise ratio of 2x (or higher) than that observed for an unrelated probe in the
particular
hybridization assay indicates detection of a specific hybridization. Nucleic
acids which do
not hybridize to each other under stringent conditions are still substantially
identical if the
polypeptides which they encode are substantially identical. This occurs, e.g.,
when a copy of
a nucleic acid is created using the maximum codon degeneracy permitted by the
genetic
code.
A further indication that two nucleic acid sequences or polypeptides are
substantially identical is that the polypeptide encoded by the first nucleic
acid is
immunologically cross reactive with, or specifically binds to, the polypeptide
encoded by the
second nucleic acid. Thus, a polypeptide is typically substantially identical
to a second
polypeptide, for example, where the two peptides differ only by conservative
substitutions.
17
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WO 01/00234 PCT/US00/16984
The phrase "specifically (or selectively) binds to an antibody" or
"specifically
(or selectively) immunoreactive with", when referring to a protein or peptide,
refers to a
binding reaction which is determinative of the presence of the protein in the
presence of a
heterogeneous population of proteins and other biologics. Thus, under
designated
immunoassay conditions, the specified antibodies bind to a particular protein
and do not bind
in a significant amount to other proteins present in the sample. Specific
binding to an
antibody under such conditions may require an antibody that is selected for
its specificity for
a particular protein. For example, antibodies raised to the protein with the
amino acid
sequence encoded by any of the polynucleotides of the invention can be
selected to obtain
antibodies specifically immunoreactive with that protein and not with other
proteins except
for polymorphic variants. A variety of immunoassay formats may be used to
select
antibodies specifically immunoreactive with a particular protein. For example,
solid-phase
ELISA immunoassays, Western blots, or immunohistochemistry are routinely used
to select
monoclonal antibodies specifically immunoreactive with a protein. See Harlow
and Lane
(1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New
York
"Harlow and Lane"), for a description of immunoassay formats and conditions
that can be
used to determine specific immunoreactivity. Typically a specific or selective
reaction will
be at least twice background signal or noise and more typically more than 10
to 100 times
background.
"Conservatively modified variations" of a particular polynucleotide sequence
refers to those polynucleotides that encode identical or essentially identical
amino acid
sequences, or where the polynucleotide does not encode aw amino acid sequence,
to
essentially identical sequences. Because of the degeneracy of the genetic
code, a large
number of functionally identical nucleic acids encode any given polypeptide.
For instance,
2~ the codons CGU, CGC, CGA, CGG, AGA, and AGG all encode the amino acid
arginine.
Thus, at every position where an arginine is specified by a codon, the codon
can be altered to
any of the corresponding codons described without altering the encoded
polypeptide. Such
nucleic acid variations are "silent variations," which are one species of
"conservatively
modified variations." Every polynucleotide sequence described herein which
encodes a
polypeptide also describes every possible silent variation, except where
otherwise noted.
One of skill will recognize that each codon in a nucleic acid (except AUG,
which is
18
CA 02377084 2001-12-21
WO 01/00234 PCT/LTS00/16984
ordinarily the only codon for methionine) can be modified to yield a
functionally identical
molecule by standard techniques. Accordingly, each "silent variation" of a
nucleic acid
which encodes a polypeptide is implicit in each described sequence.
Furthermore, one of skill will recognize that individual substitutions,
deletions or additions which alter, add or delete a single amino acid or a
small percentage of
amino acids (typically less than 5%, more typically less than 1 %) in an
encoded sequence are
"conservatively modified variations" where the alterations result in the
substitution of an
amino acid with a chemically similar amino acid. Conservative substitution
tables providing
functionally similar amino acids are well known in the art. See, e.g.,
Creighton (1984)
Proteins, W.H. Freeman and Company. In addition, individual substitutions,
deletions or
additions which alter, add or delete a single amino acid or a small percentage
of amino acids
in an encoded sequence are also "conservatively modified variations".
A "subsequence" refers to a sequence of nucleic acids or amino acids that
comprise a part of a longer sequence of nucleic acids or amino acids (e.g.,
polypeptide)
respectively.
Description of the Preferred Embodiments
The present invention provides a new approach to the development of
attenuated vaccines. This approach is evolution-based, using methods such as
DNA shuffling
in particular, and is optimal for developing attenuated vaccines. DNA
shuffling is a process
for recursive recombination and mutation and is performed by random
fragmentation of
related DNA sequences followed by reassembly of the fragments by primerless
PCR. As in
natural evolution, the technique takes advantage of deletions, insertions,
inversions and point
mutations in the DNA sequences to generate large pools of recombinant
sequences, from
which the best for a particular purpose are identified by screening or
selection that is based
on the improved function sought. If further improvement is desired, the
optimized nucleic
acids can be subjected to new rounds of shuffling and selection. DNA shuffling
is the most
efficient known method to generate large libraries of homologous DNA sequences
and, of
particular relevance to the development of attenuated vaccines, it can also be
applied to
whole organisms, such as viruses, bacteria and other pathogens.
The use of such recombination/screening methods to generate attenuated
vaccines provides significant advantages over previously available methods.
Generally, little
19
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information is available on particular mutations that might reduce
pathogenicity while not
hampering the replication of the vaccine viruses or other organisms in
manufacturing cells or
media or the capacity of the vaccine organisms to induce protective immune
responses in
host cells. The methods of the invention overcome this obstacle by eliminating
any reliance
on a priori assumptions regarding attenuation or the mechanisms that regulate
replication
and pathogenicity. One can simply carry out the recombination and
screening/selection
methods of the invention and obtain an attenuated vaccine that has the desired
properties.
Moreover, attenuated vaccines obtained using previously available methods,
e.g., those that involve mutagenesis, generally have many diverse types of
mutation both in
genes that are involved in virulence and in other genes. For example, the use
of chemical or
ultraviolet irradiation-induced mutagenesis to develop attenuated vaccines
does not provide
any information as to how many genes that are not involved in pathogenesis are
mutated in
order to obtain a mutation in a pathogenicity gene. This is particularly true
in the case of
bacterial vaccines. Because the efficacy of an attenuated vaccine depends on
the presence of
1 ~ particular antigens that remain immunogenic in the vaccinated host, it is
desirable to
maintain as much of the wild-type amino acid sequence as possible. Thus, the
introduction
of mutations into genes that encode these antigens that can result from
previously available
attenuation methods can decrease the immunogenicity of the vaccine.
This problem is avoided by the present invention, which provides attenuated
vaccines that generally have only one or a few mutations that result in the
attenuation. In
presently preferred embodiments, the recombinant vaccines that are obtained
using the
methods of the invention are subjected to molecular backcrossing. This allows
one to move a
mutant gene or genes that are responsible for conferring attenuation back to a
parental or
wildtype genome, thus retaining those few mutations that are critical to the
desired evolved
attenuation phenotype while eliminating at least some of the mutations that
are not involved
in attenuation. Thus, backcrossing can be used to retain the sequences from
the wild-type
organism that are critical for induction of protective immune responses, while
also retaining
those mutant genes that are responsible for the attenuated phenotype of the
vaccine
organism. Backcrossing can also be used to identify the mutations that are
critical to the
desired phenotype.
CA 02377084 2001-12-21
WO 01/00234 PCT/US00/16984
Other problems that are associated with current methods of attenuation are
also avoided by the methods and attenuated vaccines of the invention. For
example, the
methods of the invention can greatly reduce or eliminate the reversion of
attenuated
organisms to the wild type, disease-causing phenotype in vaccinees that often
occurs with
attenuated vaccines prepared using previously available methods. Moreover,
because the
attenuated vaccines obtained using the methods of the invention can have well-
characterized
mutations, one can monitor the stability of the genetic alterations in the
attenuated vaccine
organism during the process of developing, manufacturing, and distributing the
vaccine over
many years or decades. Thus, the invention provides a means by which one can
obtain an
attenuated vaccine that has improved ability to induce an immune response to a
pathogenic
agent without causing the potential problems associated with previously
available attenuated
vaccines.
1. General Approach to Attenuated Vaccine Evolution
Attenuated vaccines of the invention are created by first creating a library
of
recombinant nucleic acids. The library is created by recombining two or more
variant forms
of a nucleic acid are recombined to produce a library of recombinant nucleic
acids. The
library is then screened to identify those recombinant nucleic acids that
include mutations
that result in attenuation of a potential vaccine virus or other organism. For
example, the
recombinant nucleic acid can include one or more mutations that render a
normally
pathogenic organism non-pathogenic. Importantly, complete or partial genomes
of viruses,
bacteria, fungi, parasites or other pathogens can be fragmented and subjected
to the
recombination and screening methods of the invention. Single genes or other
nucleic acid
fragments can also be subjected to the recombination and screening methods,
either alone or
in combination with a complete or partial genome of a virus or organism.
Recombination
and selection of single pathogen gene is useful in cases when the critical
genes that regulate
pathogenicity are known. For example, single gene shuffling is useful when the
protein
responsible for binding to the natural host cell is known. Whole genome
shuffling is
particularly useful when, for example, the genome is relatively small and
little is known of
the critical sequences affecting attenuation.
A number of different formats are available by which one can create a library
of recombinant nucleic acids for screening or selection. In some embodiments,
the methods
21
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WO 01/00234 PCT/US00/16984
of the invention entail performing recombination ("shuffling" or "sequence
recombination")
and screening or selection to "evolve" individual genes, whole plasmids or
viruses,
multigene clusters, or even whole genomes (Stemmer (1995) BiolTechnolo~ 13:549-
553;
PCT US98/00852; US Patent Appl. No. 09/116,188, filed July 15, 1998).
Reiterative cycles
of recombination and screening/selection can be performed to further evolve
the nucleic
acids of interest. Such techniques do not require the extensive analysis and
computation
required by conventional methods for polypeptide engineering. Shuffling allows
the
recombination of large numbers of mutations in a minimum number of selection
cycles, in
contrast to traditional, pairwise recombination events (e.g., as occur during
sexual
replication). Thus, the sequence recombination techniques described herein
provide
particular advantages in that they provide recombination between any or all of
the mutations,
thereby providing a very fast way of exploring the manner in which different
combinations
of mutations can affect a desired result. In some instances, however,
structural and/or
functional information is available which, although not required for sequence
recombination,
provides opportunities for modification of the technique.
Sequence recombination can be achieved in many different formats and
permutations of formats, as described in further detail below. These formats
share some
common principles. A group of nucleic acid molecules that have some sequence
identity to
each other, but differ in the presence of mutations, is typically used as a
substrate for
recombination. In any given cycle, recombination can occur in vivo or in
vitro, intracellularly
or extracellularly. Furthermore, diversity rese~ing from recombination can be
augmented in
any cycle by applying other methods of mutagenesis (e.g., error-prone PCR or
cassette
mutagenesis) to either the substrates or products of recombination. In some
instances, a new
or improved property or characteristic can be achieved after only a single
cycle of in vivo or
in vitro recombination, as when using different, variant forms of the
sequence, as homologs
from different individuals or strains of an organism, or related sequences
from the same
organism, as allelic variations. However, recursive sequence recombination,
which entails
successive cycles of recombination and selection/screening, can generate
further
improvement.
The DNA shuffling methods can involve one or more of at least four different
approaches to improve attenuation of an otherwise pathogenic vaccine
candidate, as well as
22
CA 02377084 2001-12-21
WO 01/00234 PCT/US00/16984
improve other properties that are of interest for a vaccine (e.g., increased
immunogenicity).
First, DNA-shuffling can be performed on a single gene. Secondly, several
highly
homologous genes can be identified by sequence comparison with known
homologous
genes. These genes can be synthesized and shuffled as a family of homologs, to
select
recombinants with the desired activity. This "family shuffling" procedure is
shown
schematically in Figure 1. The shuffled genes can be introduced into
appropriate host cells,
which can include E. coli, yeast, plants, fungi, animal cells, and the like,
and those having
the desired properties can be identified by the methods described herein.
Third, whole
genome shuffling can be performed to shuffle genes that are involved in
pathogenicity
(along with other genomic nucleic acids), thus obtaining mutated pathogeniciry
genes that
reduce or eliminate pathogenicity of the organism. For whole genome shuffling
approaches,
it is not even necessary to identify which genes are being shuffled. Instead,
e.g., bacterial cell
or viral genomes are combined and shuffled to acquire recombinant nucleic
acids that, either
itself or through encoding a polypeptide, have enhanced ability to induce an
immune
response, as measured in any of the assays described herein. Fourth,
polypeptide-encoding
genes can be codon modified to access mutational diversity not present in any
naturally
occumng gene.
Exemplary formats and examples for sequence recombination, sometimes
referred to as DNA shuffling, evolution, or molecular breeding, have been
described by the
present inventors and co-workers in co-pending applications U.S. Patent
Application Serial
No. 08/198,431, filed February 17, 1994; Serial No. PCT/LJS95/02126, filed
February 17,
1995; Serial No. 08/425,684, filed April 18, 1995; Serial No, 08/537,874,
filed October 30,
1995; Serial No. 08/564,955, filed November 30, 1995; Serial No. 08/621,859,
filed March
25, 1996; Serial No. 08/621,430, filed March 25, 1996; Serial No.
PCT/LJS96/05480, filed
April 18, 1996; Serial No. 08/650,400, filed May 20, 1996; Serial No.
08/675,502, filed July
3, 1996; Serial No. 08/721, 824, filed September 27, 1996; Serial No.
PCT/US97/17300,
filed September 26, 1997; and Serial No. PCT/US97/24239, filed December 17,
1997. See
also, Stemmer, Science 270: 1510 (1995); Stemmer et al., Gene 164: 49-53
(1995);
Stemmer, Bioll'echnology 13: 549-553 (1995); Stemmer, Proc. Natl. Acad. Sci.
U.S.A.
91:10747-10751 (1994); Stemmer, Nature 370: 389-391 (1994); Crameri et al.,
Nature
23
CA 02377084 2001-12-21
WO 01/00234 PCT/US00/16984
~Lledicine 2(1):1-3 (1996); Crameri et al., Nature Biotechnology 14: 315-319
(1996). Each of
these references is incorporated herein by reference in its entirety for all
purposes.
Other methods for obtaining libraries of recombinant polynucleotides and/or
for obtaining diversity in nucleic acids used as the substrates for shuffling
include, for
example, homologous recombination (PCT/LTS98/05223; Publ. No. W098/42727);
oligonucleotide-directed mutagenesis (for review see, Smith, Ann. Rev. Genet.
19: 423-462
(1985); Botstein and Shortle, Science 229: 1193-1201 (1985); Carter, Biochem.
J. 237: 1-7
(1986); Kunkel, "The efficiency of oligonuc~otide directed mutagenesis" in
Nucleic acids &
Molecular Biology, Eckstein and Lilley, eds., Springer Verlag, Berlin (1987)).
Included
among these methods are oligonucleotide-directed mutagenesis (Zoller and
Smith, Nucl.
Acids Res. 10: 6487-6500 (1982), Methods in Enzymol. 100: 468-500 (1983), and
Methods in
Enrymol. 154: 329-350 (1987)) phosphothioate-modified DNA mutagenesis (Taylor
et al.,
Nucl. Acids Res. 13: 8749-8764 (1985); Taylor et al., Nucl. Acids Res. 13:
8765-8787
(1985); Nakamaye and Eckstein, Nucl. Acids Res. 14: 9679-9698 (1986); Sayers
et al., Nucl.
Acids Res. 16: 791-802 (1988); Sayers et al., Nucl. Acids Res. 16: 803-814
(1988)),
mutagenesis using uracil-containing templates (Kunkel, Proc. Nat'1. Acad. Sci.
USA 82: 488-
492 (1985) and Kunkel et al., Methods in Enrymol. 154: 367-382)); mutagenesis
using
gapped duplex DNA (Kramer et al., Nucl. Acids Res. 12: 9441-9456 (1984);
Kramer and
Fritz, Methods in Enrymol. 154: 350-367 (1987); Kramer et al., ~Vucl. Acids
Res. 16: 7207
(1988)); and Fritz et al., Nucl. Acids Res. 16: 6987-6999 (1988)). Additional
suitable
methods include point mismatch repair (Kramer et al., Cell 38: 879-887
(1984)),
mutagenesis using repair-deficient host strains (Carter et al.,.Nucl. Acids
Res. 13: 4431-4443
(1985); Carter, Methods in Enrymol. 154: 382-403 (1987)), deletion mutagenesis
(Eghtedarzadeh and Henikoff, Nucl. Acids Res. 14: 5115 (1986)), restriction-
selection and
restriction-purification (Wells et al., Phil. Traps. R. Soc. Lond. A 317: 415-
423 (1986)),
mutagenesis by total gene synthesis (Na.mbiar et al., Science 223: 1299-1301
(1984);
Sakamar and Khorana, Nucl. Acids Res. 14: 6361-6372 (1988); Wells et al., Gene
34: 315-
323 (1985); and Grundstrom et al., Nucl. Acids Res. 13: 3305-3316 (1985). Kits
for
mutagenesis are commercially available (e.g., Bio-Rad, Amersham International,
Anglian
Biotechnology).
24
CA 02377084 2001-12-21 pCT/US00/16984
WO 01/00234
The recombination procedure starts with at least two nucleic acid substrates
that generally show substantial sequence identity to each other (i.e., at
least about 30%, 50%,
70%, 80% or 90% sequence identity), but differ from each other at certain
positions. The
difference can be any type of mutation, for example, substitutions, insertions
and deletions.
Often, different segments differ from each other in about 5-20 positions. For
recombination
to generate increased diversity relative to the starting materials, the
starting materials must
differ from each other in at least two nucleotide positions. That is, if there
are only two
substrates, there should be at least two divergent positions. If there are
three substrates, for
example, one substrate can differ from the second at a single position, and
the second can
differ from the third at a different single position. The 5tartli-~g DNA
segments can be natural
variants of each other, for example, allelic or species variants. The segments
can also be
from nonallelic genes showing some degree of structural and usually functional
relatedness
(e.g., different genes within a superfamily, such as the family of human
papillomavirus L1
and L2-encoding genes, for example). The starting DNA segments can also be
induced
variants of each other. For example, one DNA segment can be produced by error-
prone PCR
replication of the other, the nucleic acid can be treated with a chemical or
other mutagen, or
by substitution of a mutagenic cassette. Induced mutants can also be prepared
by
propagating one (or both) of the segments in a mutagenic strain, or by
inducing an error-
prone repair system in the cells. In these situations, strictly speaking, the
second DNA
segment is not a single segment but a large family of related segments. The
different
segments forming the starting materials are often the same length or
substantially the same
length. However, this need not be the case; for example; one segment can be a
subsequence
of another. The segments can be present as part of larger molecules, such as
vectors, or can
be in isolated form.
The starting DNA segments are recombined by any of the sequence
recombination formats provided herein to generate a diverse library of
recombinant DNA
segments. Such a library can vary widely in size from having fewer than 10 to
more than
105, 109, 1012 or more members. In some embodiments, the starting segments and
the
recombinant libraries generated will include full-length coding sequences and
any essential
regulatory sequences required for expression, such as a promoter and
polyadenylation
sequence. In other embodiments, the recombinant DNA segments in the library
can be
CA 02377084 2001-12-21
WO 01/00234 PCT/US00/16984
inserted into a common vector providing sequences necessary for expression
before
performing screening/selection.
A further technique for recombining mutations in a nucleic acid sequence
utilizes "reassembly PCR." This method can be used to assemble multiple
segments that
have been separately evolved into a full length nucleic acid template such as
a gene. This
technique is performed when a pool of advantageous mutants is known from
previous work
or has been identified by screening mutants that may have been created by any
mutagenesis
technique known in the art, such as PCR mutagenesis, cassette mutagenesis,
doped oligo
mutagenesis, chemical mutagenesis, or propagation of the DNA template in vivo
in mutator
strains. Boundaries defining segments of a nucleic acid sequence of interest
preferably lie in
intergenic regions, introns, or areas of a gene not likely to have mutations
of interest.
Preferably, oligonucleotide primers (oligos) are synthesized for PCR
amplification of
segments of the nucleic acid sequence of interest, such that the sequences of
the
oligonucleotides overlap the junctions of two segments. The overlap region is
typically about
10 to 100 nucleotides in length. Each of the segments is amplified with a set
of such
primers. The PCR products are then "reassembled" according to assembly
protocols such as
those discussed herein to assemble randomly fragmented genes. In brief, in an
assembly
protocol the PCR products are first purified away from the primers, by, for
example, gel
electrophoresis or size exclusion chromatography. Purified products are mixed
together and
subjected to about 1-10 cycles of denaturing, reannealing, and extension in
the presence of
polymerase and deoxynucleoside triphosphates (dNTP's) and appropriate buffer
salts in the
absence of additional primers ("self priming"). Subsequent PCR with primers
flanking the
gene are used to amplify the yield of the fully reassembled and shuffled
genes.
In a further embodiment, PCR primers for amplification of segments of the
nucleic acid sequence of interest are used to introduce variation into the
gene of interest as
follows. Mutations at sites of interest in a nucleic acid sequence are
identified by screening
or selection, by sequencing homologues of the nucleic acid sequence, and so
on.
Oligonucleotide PCR primers are then synthesized which encode wild type or
mutant
information at sites of interest. These primers are then used in PCR
mutagenesis to generate
libraries of full length genes encoding permutations of wild type and mutant
information at
the designated positions. This technique is typically advantageous in cases
where the
26
WO 01/00234 CA 02377084 2001-12-21
PCT/US00/16984
screening or selection process is expensive, cumbersome, or impractical
relative to the cost
of sequencing the genes of mutants of interest and synthesizing mutagenic
oligonucleotides.
In a presently preferred embodiment, DNA shuffling is used to obtain the
library of recombinant nucleic acids. DNA shuffling can result in attenuation
of a pathogen
even in the absence of a detailed understanding of the mechanism by which the
pathogenicity is mediated. Examples of candidate substrates for acquisition of
a property or
improvement in a property include bacterial, viral and nonviral vectors used
in genetic and
classical types of vaccination, as well as nucleic acids that are involved in
mediating a
particular aspect of an immune response (e.g., a nucleic acid that encodes an
antigen). The
methods require at least two variant forms of a starting substrate. The
variant forms of
candidate components can have substantial sequence or secondary structural
similarity with
each other, but they should also differ in at least two positions. The initial
diversity between
forms can be the result of natural variation, e.g., the different variant
forms (hoinologs) are
obtained from different individuals or strains of an organism (including
geographic variants;
termed "family shuffling" (Figure 1 )) or constitute related sequences from
the same
organism (e.g., allelic variations). Alternatively, the initial diversity can
be induced, e.g., the
second variant form can be generated by error-prone transcription, such as an
error-prone
PCR or use of a polymerise which lacks proof reading activity (see, Liao
(1990) Gene
88:107-111), of the first variant form, or, by replication of the first form
in a mutator strain.
1. Attenuated viral vaccines
In some embodiments, the invention provides attenuated viral vaccines and
methods for obtaining the attenuated viral vaccines. By using the methods of
the invention,
one can generate novel variant viruses having genotypes and phenotypes that do
not
naturally occur or would not otherwise be anticipated to occur at a
substantial frequency. A
preferred aspect of the method employs recursive nucleotide sequence
recombination,
termed "DNA shuffling," which enables the rapid generation of a collection of
broadly
diverse viral phenotypes that can be selectively bred for a broader range of
novel phenotypes
or more extreme phenotypes than would otherwise occur by natural evolution in
the same
time period. The method typically involves: (1) shuffling of a plurality of
viral genomes, and
(2) selection of the resultant shuffled viral genomes to isolate or enrich a
plurality of shuffled
viral genomes having a desired phenotypes) (e.g., attenuation), and optionally
(3) repeating
27
CA 02377084 2001-12-21
WO 01/00234 PCT/US00/16984
steps ( 1 ) and (2) on the plurality of shuffled viral genomes conferring on a
virus tlhe desired
phenotypes) until one or more variant viral genomes conferring a sufficiently
optimized
desired phenotype is obtained. In this general manner, the method facilitates
the "forced
evolution" of a viral genome to encode an attenuated virus which natural
selection and
evolution has heretofore not generated. Figure 2 shows a block diagram of a
basic method
for viral genome shuffling and selection for a desired phenotype; the
recursion option is
generally selected each cycle until one or more viral genomes conferring a
satisfactory
optimization for the desired phenotypes) are obtained.
Typically, a plurality of viral genomes of the sa.'ne taxonomic classification
are shuffled and selected by the present method. It is believed that a common
use of the
method will be to shuffle mutant variants of a clinical isolates) or of a
laboratory strain of a
virus to obtain a variant of the clinical isolate or laboratory strain that
possesses a novel
desired phenotype (e.g., attenuation). However, the method can be used with a
plurality of
strains (or Glades) of a virus, or even with a plurality of related viruses
(e.g., lentiviruses,
herpesviruses, adenoviruses, etc.), and in some instances with unrelated
viruses or portions
thereof which have recombinogenic portions (either naturally or generated via
genetic
engineering). The method can be used to shuffle xenogeneic viral sequences
into a viral
genome (e.g., incorporating and evolving a gene of a first virus in the genome
of a second
virus so as to confer a desired phenotype to the evolved genome of the second
virus).
Furthermore, the method can be used to evolve a heterologous nucleic acid
(e.g., a non-
naturally occurring mutant viral gene) to optimize its phenotypic expression
(e.g.,
immunogenicity) in a viral genome, and/or in a particular host cell or
expression system
(e.g., an expression cassette or expression replicon). Figure 1 shows a
schematic
representation of recombinatorial shuffling of a collection of families of
viral genomes
having a variety of mutations or distinct genome portions; distinct genome
segments (e.g.,
obtained from the genomes of different virus isolates) are indicated by shaded
boxes.
Availability of infectious cDNA clones of RNA viruses_is useful, but not
necessary, for the development of improved strains of attenuated viral
vaccines. Infectious
cDNA clones have been established, for example, from porcine reproductive and
respiratory
syndrome virus (Meulenberg et al., Adv. Exp. Med. Biol. (1998) 440:199-206),
hepatitis C
virus (Yanagi et al., Proc. Nat'1. Acad. Sci. USA (1999) 96:2291-5), tick-
borne encephalitis
28
CA 02377084 2001-12-21
WO 01/00234 PCT/US00/16984
virus (Gritsun et al., J. Virol. Nlethods (1998) 76:109-20; Mandl et al., J.
Gen. Virol. (1997)
78:1049-57), plum pox potyvirus (Guo et al., Virus Res. (1998) X7:183-95),
respiratory
syncytial virus (Jin et al., Virology (1998) 251:2D6-14), paramyxovirus (He et
al., Virology
(1998) 20:30-40), bovine viral diarrhea virus (Zhong et al., J. Virol. (1998)
72:9365-9),
feline calicivirus (Sosnovtsev et al., J. Virol. (1998) 72:3051-9), infectious
bursal disease
virus (Yao et al., J. Virol. (1998) 72:2647-54), dengue virus type 2 (Gualano
et al., J. Gen.
Virol. (1998) 79:437-46), swine fever virus (Mittelholzer et al., Virus. Res.
,(1997) 51:125-
37), coxsackievirus B3 (Lee et al., Vzrus. Res. (1997) 50:22-35), Hoffman and
Banerjee, J.
Virol. (1997) 71:4272-7), equine arteritis virus (van Dinten et al., Proc.
Nat'1. Acad. Sci.
USA (1997) 94: 991-6), yellow fever virus (Galley et al., Braz. J. Med. Biol.
Res. (1997)
30:157-68), human astrovirus serotype 1 (Geigenmuller et al., J. Virol. (1997)
71: 1713-7).
The fact that infectious cDNA clones of these viruses have been established
indicates that
same approaches can be used to generate infectious cDNA clones of other
viruses belonging
to the same families and their shuffled variants. Therefore, family shuffling
of these and
related viruses provides an excellent starting point for development of
attenuated vaccine
strains.
The methods of the invention are applicable to generation of attenuated
versions of many different viruses. Examples of viruses that are of particular
interest include,
but are not limited to, rotavirus, parvovirus B 19, herpes simplex-1 and -2,
CMV, RSV,
varicella zoster virus, influenza viruses, HPV, HIV, EBV, hepatitis A, B, C, D
& E virus.
Also of particular interest are viruses of the picornavirus family, which
includes the
following genera: Rhinoviruses, which are responsible for approximately 50%
cases of the
common cold, and thus are of interest for medical applications; Enteroviruses,
including
polioviruses and coxsackieviruses; echoviruses and human enteroviruses such as
hepatitis A
virus; and the Apthoviruses, which are the foot and mouth disease viruses, and
thus of
interest particularly for veterinary uses (target antigens include VPI, VP2,
VP3, VP4 and
VPG. The Calcivirus family, which includes the Norwalk group of viruses which
are an
important causative agent of epidemic gastroenteritis, are also of particular
interest for use as
attenuated vaccines. Other viruses for which the methods of the invention are
useful for
generating attenuated vaccines include, for example , bovine viral diarrhea
virus, Marek's
Disease Virus (MDV), bovine herpes virus type-1 (BHV-1), infectious bronchitis
virus,
29
WO 01/00234 cA o23~~oa4 2ooi-i2-2i PCT/US00/16984
infectious bursal disease virus (IBDV), porcine reproductive and respiratory
syndrome
virus, canine cistemper virus (CDV).
Also of interest for use as attenuated vaccines are the Togavirus family,
including the following genera: Alphaviruses, which are of interest for both
medical and
S veterinary use and include, for example, Senilis viruses, the RossRiver
virus and Eastern &
Western equine virus, as well as the Reovirus family, which includes Rubella
virus. The
Flariviridue family of viruses are also of particular interest for the
development of attenuated
vaccines using the methods of the invention. Examples include: for example,
dengue, yellow
fever, Japanese encephalitis, St. Louis encephalitis and tick borne
encephalitis viruses. The
hepatitis C virus, when attenuated using the methods of the invention, is also
of particular
interest for medical use.
Attenuated viral vaccines of interest also include those of the Coronavirus
family, which find use for both medical and veterinary applications. Examples
of
coronaviruses that are useful for veterinary applications include, but are not
limited to,
infectious bronchitis virus (poultry), porcine transmissible gastroenteric
virus (pig), porcine
hemagglutinatiny encephalomyelitis virus (pig), feline infectious peritonitis
virus (cats),
feline enteric coronavirus (cat) and canine coronavirus (dog). For medical
use, coronavirus
family members of particular interest include, for example, the human
respiratory
coronaviruses, which cause about 40 percent of cases of the common cold (see,
e.g., Winther
et al., Am. J. Rhinol. 12: 17-20 (1998)). Coronaviruses may also cause non-A,
B or C
hepatitis. Target antigens include, for example, El (also called M or matrix
protein), E2
(also called S or Spike protein), E3 (also called HE or hemagglutinelterose
glycoprotein (not
present in all coronaviruses), and N-nucleocapsid.
The Rhabdovirus family is another family of viruses that, when attenuated
using the methods of the invention, are useful as vaccines. Genera within this
family that are
of particular use include, for example, vesiliovirus, Lyssavirus (rabies;
finds both medical
and veterinary use. Target antigens for this family of viruses include, for
example, G protein
and N protein. Also of interest are viruses of the Filoviridue famlily, which
includes the
hemorrhagic fever viruses such as Marburg and Ebola viruses.
Viruses of the Paramyxovirus family, when attenuated using the methods of
the invention, also provide vaccines that find both medical and veterinary
use. Examples of
CA 02377084 2001-12-21
WO 01/00234 PCT/US00/16984
genera of interest include, for example, paramyxovirus (both medical and
veterinary use),
the mumps virus, New Castle disease virus (important pathogen in chickens),
morbillivirus:
(medical and veterinary use), measles, canine distemper, pneumonia viruses,
and respiratory
syncytial virus. The Orthomyxovirus family of viruses are also of interest for
medical use
when attenuated using the methods of the invention. These include, for
example, the
influenza virus.
The Bungavirus family is also of interest, including the following genera:
bungavirus (including California encephalitis virus, LA Crosse virus),
Phlebovirus
(including Rift Valley Fever virus), Hantavirus (Puumala is a hemorrhagic
fever virus),
Nairovirus (causes Nairobi sheep disease, and thus vaccines find use in
veterinary
applications). Many unassigned bungaviruses are known and are also useful as
vaccines
when attenuated using the methods of the invention.
Also of interest for use as attenuated vaccines are viruses of the arenavina
family, which includes the LCM and Lassa fever virus. The Reovirus family,
when
attenuated, also provides a vaccine of interest. Genera of particular interest
include, for
example, reovirus (a possible human pathogen), rotavirus (causes acute
gastroenteritis in
children), orbiviruses: (which find both medical and veterinary use and
include Colorado
Tick fever, Lebombo (humans), equine encephalosis, and blue tongue.
The Retrovirus family includes many viral pathogens that cause significant
diseases that are recalcitrant to existing treatment methods. Attenuated
vaccines derived
from these viruses find both veterinary and medical use. Sub-families of the
retrovirus
family include, for example, the oncorivirinal retroviruses (e:g., feline
leukemia virus,
HTLVI and HTLVII), the lentivirinal retroviruses (e.g., HIV, feline
immunodeficiency virus,
equine infections and anemia virus), and the spumavirinal retrovirus family.
Also of interest for use as vaccines are attenuated viruses of the papovavirus
family. This family includes the sub-families: polyomaviruses (including BKU
and JCU
viruses), papillomavirus (which includes many viral types associated with
cancers or
malignant progression of papilloma), adenovirus (useful for medical
applications, including
AD7, ARD., O.B; some adenoviruses cause respiratory disease, while others
(e.g., 275) can
cause enteritis).
31
CA 02377084 2001-12-21
WO 01/00234 PCT/US00/16984
Attenuated viruses of the parvovirus family find use for veterinary
applications in particular. For example, attenuated virus vaccines can be
obtained using the
methods of the invention for feline parvovirus (causes feline enteritis),
feline
panleucopeniavirus, canine parvovirus and porcine parvovirus.
Viruses of the herpesvirus family can also be subjected to the attenuation
methods of the invention to obtain attenuated vaccines. Herpesvirus sub-
families of
particular interest include the alphaherpesviridue subfamily (ilzcluding the
simplexvirus
genus (e.g., HSVI, HSVII, both of which are suitable for medical use;
Varicellovirus (useful
for both medical and veterinary use), and pseudorabies (varicella zoster)),
the
betaherpesviridue (which includes the cytomegalovirus (e.g., HC~IV) and
muromegalovirus
genera), and the gammaherpesvirdiue sub-family (including the genera
lymphocryptovirus,
EBV (Burkitts lymphoma), and rhadinovirus.
The poxvirus family is also of particular interest for the development of
attenuated vaccines. The poxvirus family includes the Chordopoxviridue
subfamily (includes
viruses that, when attenuated using the methods of the invention, are useful
for both medical
and veterinary applications; genera include variola (smallpox), vaccinia
(cowpox),
parapoxvirus (veterinary), auipoxvirus (veterinary), capripoxvirus,
leporipoxvirus and
suipoxvirus) and the entemopoxviridue subfamily.
Another viral family of interest is the hepadnavirus family, which includes,
for example, hepatitis B virus. Unclassified viruses of interest for
development of attenuated
vaccines include, for example, hepatitis delta virus. A list of viruses of
interest is presented
in Table 1.
32
CA 02377084 2001-12-21
WO 01/00234 PCT/US00/16984
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CA 02377084 2001-12-21
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40
CA 02377084 2001-12-21
WO 01/00234 PCT/US00/16984
2. Attenuated bacterial, fungal and parasite vaccines
The invention also provides attenuated vaccines against bacterial, fungal and
other pathogens, such as parasites. Methods for obtaining these attenuated
vaccines are also
provided. The methods of the invention provides a means by which one can
generate novel
variant bacteria or parasites that have genotypes and phenotypes that do not
naturally occur
or would not otherwise be anticipated to occur at a substantial frequency. A
preferred aspect
of tl:e method employs recursive nucleotide sequence recombination, termed
"sequence
shuffling" or ''DNA shuffling," which enables the rapid generation of a
collection of broadly
diverse bacterial or parasite phenotypes that can be selectively bred for a
broader range of
novel phenotypes or more extreme phenotypes than would otherwise occur by
natural
evolution in the same time period.
Similarly to the case for viral vaccines described above, the presently
preferred methods involve (1) sequence shuffling of a plurality of whole or
partial bacterial
or parasite genomes, and (2) selection of the resultant shuffled bacterial or
parasite genomes
to isolate or enrich a plurality of shuffled genomes that result in an
organism that has a
desired phenotypes) (e.g., attenuation). In preferred embodiments, the method
includes (3)
repeating steps (1) and (2) on the plurality of shuffled genomes that confer
the desired
phenotypes) until one or more variant genomes that confer a sufficiently
optimized desired
phenotype is obtained. In this general manner, the method facilitates the
"forced evolution"
of a bacterial or other pathogen genome to encode an attenuated organism which
natural
selection and evolution has heretofore not generated. The recursion option is
generally
selected each cycle until one or more genomes that confer a satisfactory
optimization for the
desired phenotypes) are obtained.
Typically, a plurality of bacterial, parasite, or other genomes of the same
taxonomic classification are shuffled and selected by the present method. A
common use of
the method will be to shuffle mutant variants of a clinical isolates) or of a
laboratory strain
of an organism to obtain a variant of the clinical isolate or laboratory
strain that possesses a
novel desired phenotype (e.g., attenuation). However, the method can be used
with a
plurality of strains (or Glades) of a pathogenic organism, or even with a
plurality of related
organisms (e.g., Mycobacterium tuberculosis, Mycobacterium vaccae and
Mycobacterium
bovis (BCG)), and in some instances with unrelated pathogens or portions
thereof which
41
CA 02377084 2001-12-21
WO 01/00234 PCT/US00/16984
have recombinogenic portions (either naturally or gene:ated via genetic
engineering). The
method can be used to shuffle xenogeneic sequences into a pathogen's genome
(e.g.,
incorporating and evolving a gene of a first pathogenic organism in the genome
of a second
organism so as to confer a desired phenotype (such as immunogenicity) to the
evolved
genome of the second organism). Furthermore, the method can be used to evolve
a
heterologous nucleic acid (e.g., a non-naturally occurring mutant gene) to
optimize its
phenotypic expression (e.g., immunogenicity) when present in a bacterial or
parasite
genome, and/or in a particular host cell or expression system (e.g., an
expression cassette or
expression replicon).
In some embodiments, the methods of the invention are used to create
chimeras of pathogenic and non-pathogenic bacteria, fungi or parasites. In
these applications,
whole genome or partial genome shuffling is preferred for generating the
libraries of
recombinant nucleic acids. For example, a specific and broad-spectrum
bacterial vaccine
against nosocomical infections can be obtained by whole genome shuffling of
pathogenic
bacteria with, for example, Lactococcus lactis. Protocols for whole genome
shuffling are
described in, for example, PCT patent application No. US98/00852 (Publ. No. WO
98/31837).
The methods of the invention, including family shuffling of single genes and
whole genomes, can also be used to generate attenuated strains of bacteria
that are useful as
vaccines or vaccine antigen delivery vehicles. In addition, these methods can
be used to
improve expression levels of vaccine antigens in bacterial strains used as
vaccines. For
example, Mycobacterium bovis bacillus Calmette-Guerin (BCG) has been widely
used as
human tuberculosis vaccine, and it has several features that make it a
particularly attractive
live recombinant vaccine vehicle. BCG, like other mycobacteria, are potent
adjuvants, and
the immune response to mycobacteria has been studied extensively (Orme, Int.
J. Tuberc.
Lung Dis. (1997) 1: 95-100). More than two billion immunizations with BCG have
been
performed with a long record of safe use in man. It is one of the few vaccines
that can be
given at birth, and it provides long- lived immune responses after a single
dose. Foreign
genes have been successfully introduced into BCG enabling the generation of
BCG-based
vaccines against non-mycobacterial diseases, including HIV (Aldovini and
Young, Nature
(1991) 351: 479-82). Another useful bacterial strain for evolution by DNA
shuffling is
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Mycobacterium vaccae (M vaccae), which has previously been implicated in the
treatment
of psoriasis (Lehrer et al., FEMS Immunol. Med. Microbiol. (1998) 21: 71-7).
The methods of the invention enable the generation of chimeric bacteria or
other pathogenic organisms that have antigenic determinants from other
bacteria or
pathogens. For example, whole genome shuffling can be used to generate BCG-
like strains
that have multiple antigenic determinants derived from Mycobacterium
tuberculosis (Mt).
More specifically, BCG and Mt can be crossed by whole genome shuffling and the
optimal
vaccine strains selected by Mt-specific antibodies. Alternatively, chimeras of
M. vaccae and
Mt can be generated using similar type of approach. The attenuated phenotype
of the new
shuffled strains can be confirmed in animals models, which will simultaneously
allow the
analysis of the immunogenicity of such strains. In fact, the optimal vaccine
strains can be
selected by using in vivo immunizations. The strains that induce potent Mt-
specific antibody
responses in vivo, while retaining their attenuated phenotype, can also be
selected for new
rounds of whole genome shuffling and selection. Challenge of the immunized
animals with
live Mt will enable the analysis of the quality of the protective immune
response.
Further examples of useful targets include, but are not limited to, whole
genome shuffling of Bacillus subtilis and Bacillus anthracis to generate
nonpathogenic
bacillus strains that have antigenic determinants from Bacillus anthracis,
which can provide
protective immune responses against anthrax. Moreover, shuffling attenuated
and pathogenic
strains of Salmonella species can be used to generate strains that have
attenuated phenotype,
while expressing immunogenic determinants from pathogenic Salmonellae,
providing
protective immune responses against Salmonella infection.
DNA shuffling can also be used to improve expression levels of antigens to
be expressed in the attenuated bacteria. Because pathogens infecting mammalian
cells have
generally not coevolved with bacteria, expression of viral antigens in
bacteria is problematic
often resulting in poor expression levels. Expression, solubility and folding
of pathogens
antigens, viral antigens in particular, are also often impaired in BCG,
reducing the efficacy
of immunizations. DNA shuffling can be used to improve solubility of proteins
in bacteria.
For example, one can generate libraries of pathogen antigens and select the
most efficiently
expressed variants in bacteria. For example, HIV antigen gp120 can be fused to
GFP, and
the fusion proteins expressed in BCG or M. vaccae. Expression of GFP is an
indication of
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gp120 expression, and the brightest cells can be selected for example by flow
cytometry
based cell sorting.
These approaches using Mycobacteria and DNA shuffling also provide
opportunities to improve orally or intranasally delivered vaccines. BCG has
also been
shown to provide protective immune responses via aerogenic vaccination
(Lagranderie et al.,
Tubercle and Lung Disease (1993) 74: 38-46). Improved expression of foreign
antigens in
BCG by DNA shuffling can substantially improve the e~cacy of BCG as an oral or
inhaled
vaccine delivery strain. Further improvements can be obtained by fusing the
antigen of
interest to adjuvant enterotoxins, such as cholera toxin (CT) or heat-labile
enterotoxin of E
coli (LT), which can then be secreted from the cells. In the most desired
approach, a library
of enterotoxins are generated by DNA shuffling (for example by shuffling CT
and LT), and
these shuffled enterotoxins are fused to shuffled vaccine antigens of
interest. These libraries
can be screened as expressed purified proteins or they can be expressed in BCG
or M.
vaccae, and these strains will subsequently be screened in animals for
immunogenicity.
1 ~ Because several enterotoxins, such as CT and LT, have been shown to act as
adjuvants,
particularly in the skin and mucosal membranes, this approach is expected to
further
improve the efficacy of oral, intranasal, transdermal and inhaled vaccines.
Screening for ability of the attenuated vaccines to induce an immune response
can be performed using methods known to those of skill in the art. In a
presently preferred
embodiment, an in vitro screen is employed to test for attenuation. The in
vitro screen will
be followed up by further testing in vivo. Moreover, the ability of the
modified cells to
induce protective immunity upon inoculation into a mammal will be studied.
The methods of the invention are useful for producing attenuated vaccines
against a wide range of bacterial and other pathogenic cells. For example, one
can obtain
vaccines against pathogenic gram-positive cocci, including pneumococcal,
staphylococcal
and streptococcal bacteria. Pathogenic gram-negative cocci are also suitable
targets. Of
particular interest are the meningococcal and gonococcal bacteria.
Also of interest are vaccines against the pathogenic enteric gram-negative
bacilli. Examples include, but are not limited to, enterobacteriaceae
(pseudomonas,
acinetobacteria and eikenella), melioidosis, salmonella, shigellosis,
hemophilus, chancroid,
brucellosis, tularemia, yersinia (pasteurella), streptobacillus moniliformis
and spirillum,
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listeria monocytogenes, erysipeloL'~.rix rhusiopathiae, diphtheria, cholera,
anthrax,
donovanosis (granuloma inguinale), and bartonellosis.
The pathogenic anaerobic bacteria are also suitable targets for the
development of attenuated vaccines. Those of particular interest include, for
example,
tetar_us, botulism, other clostridia, tuberculosis, leprosy, and other
mycobacteria. Pathogenic
spirochetal diseases include syphilis, treponematoses (yaws, pima and endemic
syphilis), and
leptospirosis.
Vaccine targets also include other infections caused by higher pathogen
bacteria and pathogenic fungi, including, for example, actinomycosis,
nocardiosis,
cryptococcosis, blastomycosis, histoplasmosis and coccidioidomycosis;
candidiasis,
aspergillosis, and mucormycosis; sporotrichosis, paracoccidiodomycosis,
petriellidiosis,
torulopsosis, mycetoma and chromomycosis; and dermatophytosis. The methods of
the
invention are also useful for obtaining attenuated vaccines against
rickettsial infections (e.g.,
rickettsial and rickettsioses) and mycoplasma and chlamydial infections (e.g.,
mycoplasma
pneumoniae, lymphogranuloma venereum, psittacosis and perinatal chlamydial
infections).
Other targets include parasites, including but not limited to, amebiasis,
malaria, leishmaniasis, trypanosomiasis, toxoplasmosis, pneumocystis carinii,
babesiosis,
giardiasis, trichinosis, filariasis, schistosomiasis, nematodes, trematodes or
flukes, and
cestode (tapeworm) infections.
II. Methods to Screen for Attenuated Organisms
A recombination cycle is usually followed by at least one cycle of screening
or selection for recombinant nucleic acid molecules having a desired property
or
characteristic that is of interest for vaccination. The nature of screening or
selection depends
on what property or characteristic is to be acquired or the property or
characteristic for which
improvement is sought, and many examples are discussed below. It is not
usually necessary
to understand the molecular basis by which particular products of
recombination
(recombinant segments) have acquired new or improved properties or
characteristics relative
to the starting substrates. For example, an attenuated vaccine of the
invention can have many
component sequences each having a different intended role (e.g., coding
sequence,
regulatory sequences, targeting sequences, stability-conferring sequences,
CA 02377084 2001-12-21
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immunomodulatory sequences and sequences affecting antigen presentation). Each
of these
component sequences can be varied and recombined simultaneously.
Screening/selection can
then be performed to identify recombinant segments that have, for example,
increased
attenuation and/or immunogenicity without the need to attribute such
improvement to any of
the individual component sequences of the vector.
If a recombination cycle is performed in vitro, the products of recombination,
i. e., recombinant segments, are sometimes introduced into cells before tl~.e
screening step.
Recombinant segments can also be linked to an appropriate vector or other
regulatory
sequences before screening. The products of in vitro recombination are
sometimes packaged
as viruses before screening, or as part of the screening. For example, an
attenuated viral
vaccine can be identified by the inability of the recombinant nucleic acids to
direct synthesis
of viruses upon introduction into a non-permissive host cell (e.g., a cell
from the species that
is to be vaccinated). If recombination is performed in vivo, recombination
products can
sometimes be screened in the cells in which recombination occurred. In other
applications,
recombinant segments are extracted from the cells, and optionally packaged as
viruses;
before screening.
The introduction of the recombinant nucleic acids into cells for screening can
introduce multiple copies of the recombinant nucleic acids. For some
applications, it is
desirable to insert only a single copy of the modified gene into each cell.
Another preferred
variation of this assay involves reducing the amount of variability in
transcription of a
recombinant nucleic acid that can result from differences in chromosomal
location of
integration sites. This requires a means for defined, site-specific
integration of the
recombinant nucleic acids. These methods can also be used to evolve an
episomal vector
(which can replicate inside the cell) that can site-specifically integrate
into a chromosome.
One way to obtain single copy integrations of recombinant nucleic acids is to
use
retroviruses as a shuttle vector. Retroviruses integrate as a single copy.
However, this
insertion is not site-specific, i.e., the retrovirus inserts in a random
location in the
chromosome. Adenoviruses and ars-plasmids are also used to shuttle modified
transgenes,
however, they integrate as multiple copies. While wild type AAV integrates as
a single copy
in chromosome q19, commonly used modified versions of AAV do not. Homologous
recombination is also used to insert a modified recombinant segment into a
chromosome, but
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this method can be inefficient and may result in the integration of two copies
in the pair of
chromosomes.
To solve these problems, some embodiments of the invention utilize
site-specific integration systems to target the transgene to a specific,
constant location in the
genome. A preferred embodiment uses the Cre/LoxP or the related FLP/FRT site-
specific
integration system. The Cre/LoxP system uses a Cre recombinase enzyme to
mediate site-
specific insertion and excision of viral or phage vectors into a specific
palindromic 34 base
pair sequence called a "LoxP site." LoxP sites can be inserted to a mammalian
genome of
choice, to create, for example, a transgenic animal containing the LoxP site,
by homologous
recombination (see Rohlmann (1996) Nature Biotech. 14:1562-1565). If a cell's
genome is
engineered to contain a LoxP site in a desired location, infection of such
cells with
recombinant nucleic acids that are flanked by LoxP sites, in the presence of
Cre recombinase
(e.g., expressed by a vector that expresses a gene for the Cre recombinase)
results in the
efficient, site-specific integration of the recombinant nucleic acids into the
LoxP site. This
1 ~ approach is reproducible from cycle to cycle and provides a single copy of
the recombinant
sequence at a constant, defined location. Thus, a recombinant nucleotide
obtained using the
methods of the invention in vitro can be reinserted into the cell for in
vivolin situ selection
for the new or improved property in the optimal way with minimal noise. This
technique can
also be used in vivo. See, for example, Agah (1997) J. Clin. Invest. 100:169-
179; Akagi
(1997) Nucleic Acids Res. 25:1766-1773; Xiao (1997) Nucleic Acids Res 25:2985-
2991;
Jiang (1997) Curr Biol 7:321-8323, Rohlmann (1996) Nature Biotech. 14:1562-
1565; Siegal
(1996) Genetics 144: 715-726; Wild (1996) Gene 179:181-188. The evolution of
Cre is
discussed in further detail in PCT patent application US97/17300 (Publ. No.
W098/13487),
filed September 26, 1997.
In presently preferred embodiments, the attenuated vaccines of the invention
are screened in mammalian cells or organisms. Once a group of attenuated
strains have been
identified, these vaccine strains are subsequently analyzed for their
immunogenicity in vivo.
Useful animal species for such studies include, but are not limited to, mice,
rats, guinea pigs,
cats, dogs, cows, pigs, horses, chicken. These experiments are useful in
identifying improved
veterinary vaccines, and they also provide information about their safety and
efficacy for use
as human vaccines. The attenuated vaccines that are intended for use in humans
are often
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subjected to further testing in humans. In some instances, cells used for
screening can be
obtained from a patient to be treated with a view, for example, to minimizing
problems of
immunogenicity in this patient. Use of an attenuated vaccine in treatment can
itself be used
as a round of screening. That is, attenuated vaccines that are successively
taken up and/or
expressed by the intended target cells in one patient are recovered from those
target cells and
used to treat another patient. The attenuated vaccines that are recovered from
the intended
target cells in one patient are enriched for vectors that have evolved, t. e.,
have been modified
by recursive recombination, toward improved or new properties or
characteristics for
attenuation, specific uptake, immunogenicity, stability, and the like.
The screening or selection step identifies a subpopulation of recombinant
segments (e.g., viral or bacterial whole or partial genomes, or other nucleic
acid segments)
that have evolved toward acquisition of improved attenuation, and/or other new
or improved
desired properties useful in vaccination. Depending on the screen, the
recombinant segments
can be screened as components of cells, components of viruses or other
vectors, or in free
form. More than one round of screening or selection can be performed after
each round of
recombination.
If further improvement in a property is desired, at least one and usually a
collection of recombinant segments surviving a first round of
screening/selection are subject
to a further round of recombination. These recombinant segments can be
recombined with
each other or with exogenous segments representing the original substrates or
further
variants thereof. Again, recombination can proceed in vitro or in vivo. If the
previous
screening step identifies desired recombinant segments as components of cells,
the
components can be subjected to further recombination in vivo, or can be
subjected to further
recombination in vitro, or can be isolated before performing a round of in
vitro
recombination. Conversely, if the previous screening step identifies desired
recombinant
segments in naked form or as components of viruses or other vectors, these
segments can be
introduced into cells to perform a round of in vivo recombination. The second
round of
recombination, irrespective how performed, generates further recombinant
segments which
encompass additional diversity compared to recombinant segments resulting from
previous
rounds.
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The second round of recombination can be followed by a further round of
screening/selection according to the principles discussed above for the first
round. The
stringency of screening/selection can be increased between rounds. Also, the
nature of the
screen and the property being screened for can vary between rounds if
improvement in more
than one property is desired or if acquiring more than one new property is
desired.
Additional rounds of recombination and screening can then be performed until
the
recombinant segments have su~ciently evolved to acquire the desired new or
improved
property or function.
After a desired phenotype is acquired toga satisfactory extent by a selected
shuffled viral or other pathogen genome or portion thereof, it is often
desirable to remove
mutations which are not essential or substantially important to retention of
the desired
phenotype ("superfluous mutations"). Superfluous mutations can be removed by
backcrossing, which involves shuffling the selected shuffled genome(s) with
one or more
parental genome and/or naturally-occurring genome(s) (or portions thereof) and
selecting or
screening the resultant collection of shufflants to identify those that retain
the desired
phenotype. By employing this method, typically in one or more recursive cycles
of shuffling
against parental or naturally-occurring genome(s) (or portions thereof) and
selection or
screening for retention of the desired phenotype, it is possible to generate
and isolate
selected shufflants that incorporate substantially only those mutations
necessary to confer the
desired phenotype (e.g., attenuation), while having the remainder of the
genome (or portion
thereof) consist of sequence which is substantially identical to the parental
(or wild-type)
sequence(s). As one example of backcrossing, a viral genome can be shuffled
and selected
for attenuation in target host cells; the resultant selected shufflants can be
backcrossed with
one or more genomes of clinical isolates of the virus and selected for
retention of the
attenuation. After several cycles of such backcrossing, the backcrossing will
yield viral
genome(s) that contain the mutations necessary for attenuation, and will
otherwise have a
genomic sequence substantially identical to the genome(s) of the clinical
isolates) of the
virus.
Examples of the types of approaches that are useful for obtaining attenuated
vaccines, and screening/selection techniques that are suitable for identifying
vaccines having
the desired properties, are described in the following section.
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III. Illustrative Examples o_fAttenuated Vaccines
The invention provides attenuated vaccines, and methods for obtaining
attenuated vaccines, that have a wide variety of properties. To obtain
attenuated vaccines
that have these and other desired properties, a suitable screening and/or
selection method is
used which is specific for the particular properties desired. The screening
and/or selection
methods can be used in combination to obtain attenuated vaccines that have
more than one
desired improvement. The following are illustrative examples of types of
attenuated viral,
and bacterial vaccines, and methods for obtaining such vaccines. Different
selection/
screening methods can also be used as is appropriate to identify attenuated
vaccines that
have other desirable properties. Analogous methods are useful for developing
attenuated
fungal and parasite vaccines.
1. Non-pathogenic chimeric vaccines
In some embodiments, the invention provides chimeric viruses, bacteria or
other organisms into which are introduced nucleic acids that encode one or
more
immunogenic polypeptides from a pathogenic virus or other organism. In
presently preferred
embodiments, the chimeric vaccine is non-pathogenic. Both the pathogenic and
non-
pathogenic virus or organism can be of the same species (e.g., a coding region
for an
immunogenic polypeptide from a pathogenic strain is introduced into a non-
pathogenic
strain). These methods are useful, for example, in the development of
polyvalent vaccines
that express immunogenic polypeptides from multiple strains of an organism or
virus. As
one example, vaccines derived from human papillomavirus strains are typically
not cross-
protective against other HPV strains. By using the methods of the invention,
one can obtain
polyvalent, cross-protective HPV strains. Alternatively, the pathogenic and
non-pathogenic
virus or organism can be of different species. As one example, a vaccinia
virus can function
as a non-pathogenic carrier for a nucleic acid that encodes an immunogenic
polypeptide such
as, for example, gp120 of HIV.
These methods typically involve recombining a first set of one or more
nucleic acid segments from a virus or cell with a second set of one or more
nucleic acid
segments. The nucleic acid segments of the second set typically encode one or
more
polypeptides, or portions thereof, that confer upon a viruses or cells that
include the
polypeptide a property that is desirable for vaccination. For example, the
second set of
CA 02377084 2001-12-21
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nucleic acid segments can encode an immunogenic polypeptide from a pathogenic
strain of a
virus or cell, an adjuvant or immunomodulatory molecule, and the like.
The resulting library of recombinant DNA fragments is then screened to
identify those that confer upon a virus or cell an improvement in the desired
property. In
presently preferred embodiments, the viruses or cells that contain the
recombinant fragments
are screened to identify those viruses or cells that have become, or remain,
attenuated (i. e.,
nonpathogenic) under physiological conditions present in a host organism
inoculated with
the virus or cell. The screening for attenuation can be conducted before or
after, or
simultaneously with, the screening for the improvement in the other desired
property.
In some embodiments, the recombination is performed in vivo. For example,
one can introduce a library of DNA fragments that comprises at least a partial
genomic
library of a pathogenic cell into a plurality of nonpathogenic cells. At least
one of the
fragments from the pathogenic cell undergoes recombination with a segment in
the genome
or an episome of the non-pathogenic cells to produce modified cells. The
modified cells are
screened to identify those that are nonpathogenic but have evolved towards an
ability to
induce an immune response against the pathogenic cells. The resulting
nonpathogenic cells
that have evolved towards an ability to induce an immune response against the
pathogenic
cells are suitable for use as an attenuated vaccine.
If desired, further improvement can obtained by subjecting the DNA from the
modified cells that are nonpathogenic and have evolved an ability to induce an
immune
response against the pathogenic cells to recombination with a further library
of DNA
fragments from a pathogenic organism. At least one of the fragments from the
pathogenic
organism undergoes recombination with a segment in the genome or the episome
of the
modified cells to produce further modified cells. Alternatively, one can
recombine DNA
from the modified cells that are nonpathogenic and have evolved an ability to
induce an
immune response with DNA from the pathogenic cells to produce further modified
cells.
The further modified cells are then screened to identify further modified
cells that are
nonpathogenic and have evolved a further ability to induce an immune response
against the
pathogenic cells. The recombination and selection/screening steps can be
repeated as
required until the further modified cells are nonpathogenic and have acquired
the ability to
induce an immune response against the pathogenic cells.
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The recombination and selection/screening methods of the invention provide
a means not only for obtaining attenuated viruses or cells for use as the
carrier, but can be
used to obtain chimeric viruses or organisms that exhibit improvements in
properties such as
enhanced expression of the antigen and improved immunogenicity of the antigen.
The genes
that encode the antigen can be subjected to recombination separately from the
non-
pathogenic virus or other vaccine organism; alternatively, one can perform the
recombination on whole or partial viral, bacterial or parasite genomes.
Methods for
improving antigen expression and immunogenicity are described in co-pending,
commonly
assigned US patent application Ser. No. 09/247,890 (filed February 10, 1999).
A
polynucleotide that encodes a recombinant antigenic polypeptide can be placed
under the
control of a promoter, e. g., a high activity or tissue-specific promoter. The
promoter used to
express the antigenic polypeptide can itself be optimized using recombination
and selection
methods analogous to those described herein (see, e.g., US Ser. No.
09/247,888, filed
February 10, 1999).
In some embodiments, the methods of the invention are used to obtain virus-
like particles (VLP'n) that have desired characteristics. VLPs lack the viral
components that
are required for virus replication and, therefore, represent a highly
attenuated form of a virus.
The VLPs can display antigens from multiple viral strains, and thus are useful
as a
polyvalent vaccine. Viral proteins from several viruses are known to form
VLPs, including
human papillomavirus, HIV (Kang et al., Biol. Chem. 380: 353-64 (1999)),
Semliki-Forest
virus (Notka et al., Biol. Chem. 380: 341-52 (1999)), human.polyomavirus
(Goldmann et al.,
J. Virol. 73: 4465-9 (1999)), rotavirus (Jiang et al., Vaccine 17: 1005-13
(1999)), parvovirus
(Canal, Biotechnology and Applied Biochemistry, Vol 29, Part 2, pp 141-150
(1999)), canine
parvovirus (Hurtado et al., J. Yirol. 70: 5422-9 (1996)), and hepatitis E
virus (Li et al., J.
Virol. 71: 7207-13 (1997)).
Screening for ability to induce an immune response against the pathogenic
cells can be performed using methods known to those of skill in the art. In a
presently
preferred embodiment, the screening is performed by testing for ability of the
modified cells
to induce protective immunity upon inoculation into a mammal.
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Z. Replication-deficient viral vaccines that are evolved for high
efficiency infectivity and protectivity
In other embodiments, the invention provides replication-deficient viruses
that are evolved to infect target cells with high efficiency, but with only
one round of
replication or no replication once the cells are infected. Replication-
deficient viruses can be
obtained either by rational design (e.g., targeted disruption of a gene that
is involved in viral
replication) or by the recombination and screening/selection methods of the
invention. The
nucleic acids of the replication deficient viruses thus obtained are then
subjected to
recombination and selection for those viruses that exhibit improved entry into
host cells. The
screening can be accomplished by, for example, fluorescence-activated cell
sorting of cells
that contain the virus (based on expression of a gene such as Mx). Viruses can
be recovered
from these cells, re-infected into host cells, re-sorted. After one or more
repetitions of the
screening/selection, individual colonies analyzed for their capacity to
replicate in cell
culture. Those that do not replicate in host cells are suitable for further
testing as vaccines,
e.g., for ability to induce an "antiviral state" in infected cells. Again,
backcrossing can be
used to obtain viral vaccines that include the mutations that prevent
replication but lack other
mutations that reduce immunogenicity.
3. Vaccines attenuated by insertions of stop codons in polypeptide
coding regions
In another embodiment, the invention provides attenuated vaccines, and
methods of obtaining attenuated vaccines, in which numerous stop codons are
introduced
into the nucleic acids of the vaccine virus or other organism. These
attenuated vaccines thus
require the presence of a suppressor tRNA in a host cell in order to replicate
(Drabkin et al.,
Mol. Cell. Biol. 16, 907-13 (1996); Park and RajBhandary, Mol. Cell. Biol. 18,
4418-25
(1998)). Accordingly, a production cell is used that contains the appropriate
suppressor
tRNA (e.g., amber, ochre, frameshift or other suppressor) that corresponds to
the stop
codons that are introduced into the genome of the vaccine virus or other
organism.
Preferably, the stop codons are of a type that is not frequently used in the
respective
naturally occurring virus or organism.
The stop codon-containing attenuated vaccines are obtained by recombining a
nucleic acid segment, or mixture of fragments, from a virus or other vaccine
organism with a
population of oligonucleotides that include one or more stop codons
interspersed within one
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or more polynucleotide sequences that code for at least a portion of a
polypeptide necessary
for replication of the virus or organism. For example, one can use a library
of stop codon-
containing oligonucleotides, wherein the sequences of the oligos are
determined based on
known sequence information for the viral proteins. The mixture of fragments
can be a full or
partial fragmented genome of the organism. A library of recombinant nucleic
acid segments
is produced by subjecting the oligonucleotides to recombination together with
nucleic acids
of the virus or other organism. This results in the non-naturally occu.-ring
stop codons
becoming incorporated into the coding regions of the cell or virus, thus
causing premature
translational termination in the absence of a corresponding suppressor tRNA.
The library of recombinant nucleic acid segments are then screened by
contacting introduced into production cells, for example, that contain
suppressor tRNA
which suppresses the translational termination that would otherwise occur at
the introduced
stop codons (see, e.g., (Drabkin et al., lt~Iol. Cell. Biol. 16, 907-13
(1996); Park and
RajBhandary, Mol. Cell. Biol. 18, 4418-25 (1998)). Those viruses or other
organisms that
reproduce in the production cells, and resulting progeny, are then collected.
In presently
preferred embodiments, those that replicate are then tested for ability to
replicate in non-
suppressor cells. Preferably, cells of the mammal that is to be inoculated are
tested. Progeny
viruses or other organisms that do not replicate in the non-suppressor cells
are suitable for
use as attenuated vaccine organisms.
4. Selection of conditionally replicating mutant strains
In some embodiments, the invention provides attenuated vaccines that are
unable to proliferate under physiological conditions in an inoculated host
mammal, but are
capable of replication under permissive conditions such as those used for
production of the
vaccine. Permissive conditions can include a property of a production cell or
growth
conditions, that differs from the corresponding physiological condition in the
cells of an
inoculated host mammal. For example, a permissive condition can be a
temperature, pH,
sugar content, the presence or absence of complement components and/or serum
proteins,
and the like, that differs from the physiological condition found in the
inoculated mammal.
A permissive condition can also be the presence of an essential nutrient that
is absent in the
inoculated host mammal.
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Methods of obtaining such vaccines are also provided by the invention. For
bacterial, parasite, and other whole-cell vaccines, these methods typically
involve
introducing a library of recombinant DNA fragments into population of the
bacterial cells.
The recombinant fragments can be a whole or partial bacterial genome, or a
recombinant
gene or genes, that can become incorporated into the genome or an episome of
the cells. The
modified cells are then screened to identify conditionally defective cells
that have evolved
toward loss of the ability to proliferate under physiological conditions as
found in a host
organism. The conditionally defective cells are then screened to identify
those modified cells
that have evolved toward ability to replicate under the permissive conditions.
Modified cells
that replicate under permissive conditions but not in a host mammal are
suitable for testing
as an attenuated vaccine organism.
If further improvement in attenuation is desired, additional rounds of
recombination and screening can be performed. DNA from the modified cells that
have
evolved toward inability to replicate ~.mder physiological conditions and
ability to replicate
under permissive conditions is recombined with a further library of DNA
fragments,
genomes, or partial genomes. The recombined DNA is introduced into the
modified cells to
produce further modified cells. Alternatively, one can recombine DNA among the
modified
cells that have evolved toward the desired function to produce the further
modified cells. The
further modified cells are then screened to identify those cells that have
further evolved
toward loss of ability to replicate under physiological conditions and toward
ability to
replicate under permissive conditions. These steps can be repeated as required
until the
further modified cells have lost the ability to replicate under.physiological
conditions in a
host mammal and have acquired the ability to replicate under permissive
conditions.
To obtain conditional-sensitive attenuated viral vaccines, the recombinant
libraries of viral genomes can be introduced into suitable test cells which
are similar to those
of the target mammal in terms of the conditions that can affect viral
replication. The virus-
containing cells are cultured in increasing or decreasing temperatures, pHs,
sugar content, or
other condition, and the surviving viruses are chosen for new rounds of
recombination and
selection. The viruses that grow in altered temperatures or other condition
are further
analyzed to identify those that do not replicate at the temperature or other
condition found in
the mammal to be inoculated. In addition, it is generally desirable to analyze
the conditional-
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sensitive viruses for their capacity to induce pathology and protective immune
response in
the natural host. Backcrossing can be employed as discussed herein to obtain
an attenuated
vaccine in which the mutated gene or genes that are responsible for the
conditional
sensitivity are found in a virus that is otherwise unmodified in terms of its
immunogenicity
or other properties related to effectiveness as a vaccine.
In some embodiments, viral vaccines are screened by introducing the
recombinant nucleic acids into allantoid cavities of embryonal eggs.
Alternatively, in vitro
tissue culture can be used. This selection scheme can employ as the culture
cells a mixture of
cells from various species that have different requirements (e.g., for
temperature or other
condition) in tissue culture. This can overcome potential loss of survival and
growth of the
host cells that could occur when cultured under changing conditions in vitro.
5. Altered host cell specificity
Also provided by the invention are attenuated vaccines that are evolved to
exhibit altered host specificity. One aim is to evolve viral, bacterial, or
parasite strains that
can specifically grow in cell types and/or organisms that allow efficient
production of the
vaccine strain, but cannot grow in the natural host cells or organisms in
which they could
cause a disease.
As one example, one can gradually change the selection pressure by using
cell lines from different species. One can start by adapting the virus or
other organism to
simultaneously grow in the natural host and in phylogenetically related
species. When
generating attenuated human viruses, for example, one can start by adapting
the virus to
grow in both monkey cells and human cells. Thereafter, one would start
selecting mutants
that also grow in bovine cell lines, and the human cells are removed from the
culture system.
After recursive rounds of shuffling and selection, one is likely to be able to
find a mutant
strain that specifically grows in nonnatural host cell. In presently preferred
embodiments, the
mutant strain will not replicate in human cells. These screening methods can
be done using
pooled whole libraries of shuffled viruses, significantly reducing the numbers
of samples
that are handled.
In addition, the attenuated strains can be selected by screening for virus
growth only in selected host cells rather than in many cell types (i. e.
restriction of host cell
specificity). This selection system allows generation of viruses that can
replicate in certain
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cells of the body, sufficient to elicit an immune response, but the restricted
cell specificity
will reduce the pathogenicity of the virus, thus preventing clinical symptoms.
Because the genes that regulate replication in host cells and the important
antigenic determinants are likely to be encoded by different genes,
backcrossing provides a
means to retain the maximal number of the epitopes that are important for
induction of
protective immune responses.
6. Rapid growth in manufacturing cells but reduced proliferation in
host cells
The invention also provides attenuated vaccines that exhibit rapid growth in
manufacturing cells, but reduced proliferation in cells of the inoculated
host. For example,
one can first select for growth in manufacturing cell lines or culture
conditions, and then
generate a library of recombinant viruses and test those individually for
growth in natural
host cells. The clones with the slowest growth rate in the natural host cells
are selected and
subjected to new rounds of shuffling and selection. The selection for high
growth in
manufacturing cells and slow growth in host cells can be repeated. An
advantage of this
method is that simultaneous selection and screening for attenuation and high
yield
manufacturing is performed. Alternatively, one can first select individual
clones that exhibit
slow growth in host cells, and then select for growth in manufacturing cells.
Again, selected
mutants/chimeras are selected for new rounds of shuffling and selection.
7. Screening based on adherence to target cells or target cell receptors
The invention also provides methods for screening to identify viral vaccines
that exhibit reduced adherence to target host cells. A library of
mutant/chimeric viruses is
incubated in the presence of cells of the type in which virus entry and
replication is not
desired. The viruses that do not bind or enter the cells are harvested from
the supernatant.
Kinetic studies can be performed to identify the optimal incubation time to
most e~ciently
remove viruses which demonstrate specific binding to the cells. Also, several
rounds of cell
panning may be required to achieve optimal removal of the mutants/chimeras
that have
retained their capacity to bind to their specific cell surface receptors.
In addition to the intact cells, one can also use purified virus receptors in
the
panning in cases when the cell surface receptors for the given virus have been
identified. In
this system the viruses are mixed with purified (e.g., recombinant) receptor
in solution or
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crosslinked to a plate. The viruses that bind to the receptors are captured by
using, for
example, monoclonal antibodies that are specific for the receptor or simply by
allowing them
to bind to the receptor crosslinked to the plate. The viruses are subsequently
selected for
growth in manufacturing cells, and the shuffling and selection is repeated as
desired for
further optimization. The selection is oscillated between binding to the
specific receptors
and growth in manufacturing cells.
8. Selection based on sensitivity to complement
Attenuated viruses can also be selected based on their sensitivity to
complement or complement components. The library of recombinant viruses is
generated by
a method such as DNA shuffling, preferably family shuffling. Individuals
clones of virus are
e~cpanded in manufacturing cells, and subsequently cultured in the presence of
complement
or complement components. Clones that demonstrate decreased virus titer upon
exposure to
complement are selected for new rounds of shuffling and selection. Viruses
that are
susceptible to killing by complement are likely to have strongly reduced
capacity to induce
pathology in vivo, yet they are likely to elicit immune responses that protect
from future
infections.
In addition, one can select virus mutants/chimeras that bind purified
complement components (e.g., C3 or components thereof). Binding of complement
components directly to the virus may induce the cascade of complement mediated
killing,
and it may also cause opsonization of the virus rendering them more
susceptible for killing
by phagocytic cells, such as monocytes. The selection of mutants/chimeras that
bind
complement components can be done for example by panning or amity column
chromatography.
9. Selection for growth in immunocompromised animals only
Additional selection systems for attenuated vaccine strains include screening
for growth in immunocompromised hosts only. Genetically immunocompromised
strains of
certain species are available, such immunodeficient mouse strains. Such
strains include, but
are not limited to, SCID mice, nude mice and mice rendered deficient in their
genes
encoding RAGI or RAG2 genes. Importantly, however, practically any host
species can be
rendered transiently immunodeficient by drug treatment or irradiation.
Immunosuppressive
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drugs include, but are not limited to cyclosporin A, FK~06, IL-10, soluble IL-
2 receptor,
steroids and anti-proliferative cancer drugs, such as methotrexate.
Individual virus, bacterial, or parasite clones are tested for their capacity
to
propagate in either genetically immunodeficient or transiently
immunocompromised hosts.
The individual clones that grow well in immunocompromised hosts are then
tested for
growth in normal hosts. Clones that grow in imm~unocompromised hosts but
demonstrate
slow growth in immunocompetent hosts are selected for new rounds of shuffling
and
selection.
In addition, viruses or other pathogens may grow, in immunocompromised
hosts, in tissues where they normally do not grow. This allows selection of
virus, bacterial,
or parasite mutants/chimeras that have altered tissue specificity in
immunocompromised
hosts. In this selection system, one can infect the host with a library of
viruses or other
organisms and harvest viruses, bacteria, or parasites from novel target
tissues. Individual
clones from these tissues can then be tested for growth in immunocompetent
hosts and
manufacturing cells. Like in other shuffling formats, the selected
mutants/chimeras can be
subjected to additional rounds of shuffling and selection.
10. Selection of pathogen variants that are killed by Ab response alone or
by components of normal serum rendering them less virulent
Immune defense against viral and other infections generally includes both cell
mediated and humoral immune responses. Attenuated virus or cell strains can be
selected by
screening mutants/chimeras that are killed by either one of these mechanisms
alone. For
example, virus or cell mutants can be selected that are killed by specific
antibodies alone, in
the absence of T cell mediated immune responses. First, antisera are generated
against the
wild-type virus or cell by immunization. Thereafter, the shuffled library of
virus or cell
mutants/chimeras is mixed with the antisera and viruses or cells that are
recognized by the
antisera are selected for further analysis. These clones are then tested
individually whether
the antiserum neutralizes the function of the virus or cell. These studies can
be done either in
vitro or in vivo, or both. For example, one can first analyze whether the
antisera, or pools of
antisera, neutralize the virus or cell variants in vitro and then test these
individual clones for
their capacity to induce pathology and immunity in vivo.
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In a va~~iant of this selection system, one can also use sera from non-
immunized hosts in order to identify virus or cell variants that are
neutralized by serum
components normally present in the host. As described above, the serum
components that
limit virus growth can include complement and complement components. However,
the use
of whole serum can allow evolution for killing by additional agents present in
normal serum.
Such mutants/chimeras are also likely to have an attenuated phenotype in vivo.
As in other
recombination and screening/selection formats, the selected mutants/chimeras
can be
subjected to new rounds of shuffling and selection if further optimization is
desired.
11. Combinations of selection systems to obtain optimal attenuation
The optimal attenuation may often be obtained by combining several different
selection mechanisms. For example, optimal attenuation can be achieved by
simultaneously
selecting mutants that grow at altered temperatures in a nonconventional host
cell, e.g., a cell
line from a species other than the normal host for the virus or other
pathogen. To achieve
such a large change in the function of the virus or other organism, gradual
selection pressure
can be important. The temperature is readily increased or decreased gradually,
and mixtures
of cells derived from different species can be used to allow gradual
adaptation to grow in
nonhost tissues.
Furthermore, in vitro and in vivo selection systems can be used in
combination. One can first select mutants that do not bind cells that normally
are target cells
for the given virus in vitro. In this format the natural host cells and the
virus library are
incubated as a mixture, and the mutants that do dot bind are hen harvested,
and the process
is repeated until minimal or no viruses are removed from the pool by the host
cells.
Thereafter, the remaining population is analyzed in vivo either as pools or as
individual
recombinant viruses. Both induction of pathology and immune responses are
measured. For
example, one can inject the mutant viruses into animals, observe pathology,
omit the ones
that become sick, and then analyze the remaining animals by challenging them
by the wild-
type virus. Animals that have developed protective immunity after inoculation
of the
attenuated mutant strain will survive the challenge with the wild-type
infectious virus.
These can be selected for new rounds of shuffling and selection. Again,
backerossing with
the wild-type virus can be used to retain maximal number of immunologically
important
residues.
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12. Evolution of vaccine strains that can escape recognition by maternal
antibodies
The invention is also useful for generating vaccines that are not recognized
by
maternal antibodies. Vaccines that exhibit reduced maternal antibody binding
are expected to
have improved efficacy. To obtain such vaccines, nucleic acids that encode
either a whole or
partial genome of a potential vaccine organism, or that encode a particular
immunogenic
polypeptide, are subjected to recombination as described herein. The resulting
recombinant
nucleic acids are then introduced into cells or viruses, which are then
negatively selected so
that the vaccines recognized by maternal antibodies are removed from the
libraries. Typical
methods to negatively select vaccines that are not recognized by maternal
aaibodies include,
but are not limited to, affinity selection using flasks or columns coated with
the maternal
antibodies.
The family shuffling approach has an advantage in that one will
simultaneously generate chimeras of the different strains. These chimeras can
then be
screened for optimal immunogenicity and crossprotection in vivo. These
multivalent strains
are likely to be more potent in inducing crossprotection against all different
existing and
emerging variants of the parent virus or cell. In some embodiments, libraries
of the virus or
cells that contain the recombinant nucleic acids are generated, and these
libraries are initially
screened for lack of binding to antibodies derived from animals that were
previously
immunized against the respective cell or virus. The immunogenicity of the
selected vaccines
can be verified by immunizing animals, and subsequently challenging the
vaccinated
animals with different strains of live virus or cells.
13. Evolution of more stable attenuated vaccine strains
The inherent lability of live organisms presents a challenge in terms of
stabilizing and preserving viability of attenuated vaccine strains during
manufacturing,
storage, and administration (Burke et al., Crit. Rev. Ther. Drug. Carrier
Syst. (1999) 16: 1-
83). Due to instability of several attenuated vaccine strains, shelf life of
the vaccines is often
limited, reducing the practicality of the vaccines for veterinary application
or limiting their
usage in undeveloped or tropical areas. Such examples include, but are not
limited to,
rinderpest vaccine for cattle (House and Manner, Dev. Biol. Stand. (1996) 87:
235-44) and
Vanzcella-Zoster virus (VZV) vaccines (Fanget and Francon, Dev. Biol. Stand.
(1996) 87:
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167-71). The stability of these vaccine strains can be improved by the methods
of the
invention.
Libraries of vaccine strains are generated by the recombination methods
described herein, e.g., DNA shuffling, and the resulting libraries are
analyzed for stability.
Different temperatures, formulations and time periods can be used to generate
a selection
pressure that only allows propagation of viruses or cells that have the
desired properties. In
addition, the virus and cell libraries can be freeze dried, reconstituted, and
the most stable
viruses or cells selected varying periods after reconstitution. The viruses or
cells that
demonstrate improved stability can be subjected to new rounds of shuffling and
selection.
Subsequent immunizations and challenge studies can be used to further evaluate
the degree
of attenuation, immunogenicity and stability.
1Y. Use of the Attenuated Vaccines
The attenuated vaccines of the invention are useful for treating and/or
preventing the various diseases and conditions that are caused by viral or
cellular pathogens.
The attenuated vaccines obtained using the methods of the invention can be
further modified
to enhance their effectiveness in vaccination. For example, one can
incorporate into the
attenuated vaccines immunostimulatory sequences such as are described in
copending,
commonly assigned US Patent Application Serial No. 09/248,716, filed February
10, 1999.
The vaccine vector can be modified to direct a particular type of immune
response, e.g., a
THl or a TH2 response, as described in US Patent Application Serial No.
09/247,888, filed
February 10, 1999. It is sometimes advantageous to employ a vaccine that is
targeted for a
particular target cell type (e.g., an antigen presenting cell or an antigen
processing cell);
suitable targeting methods are described in copending, commonly assigned US
patent
application Serial No. 09/247,886, filed February 10, 1999.
The attenuated vaccines obtained using the methods of the invention find use
not only for inducing a prophylactic or therapeutic immune response against
the vaccine
itself, but the backbone of the vaccines can be used to carry other
pharmaceutically useful
proteins into a cell. Such molecules include, for example, vaccine antigens,
immunomodulatory molecules, therapeutic proteins, and the like.
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Suitable formulations and dosage regimes for vaccine delivery are well
known to those of skill in the art. The vaccines of the invention can be
delivered to a
mammal (including humans) to induce a therapeutic or prophylactic immune
response.
Vaccine delivery vehicles can be delivered in vivo by administration to an
individual patient,
typically by systemic administration (e.g., intravenous, intraperitoneal,
intramuscular,
subdermal, intracranial, anal, vaginal, oral, buccal route or they can be
inhaled) or they can
be administered by topical application. Alternatively, vaccines can be
delivered to cells ex
vivo, such as cells explanted from an individual patient (e.g., lymphocytes,
bone marrow
aspirates, tissue biopsy) or universal donor hematopoietic stem cells,
followed by
reimplantation of the cells into a patient, usually after selection for cells
which have
incorporated the vector.
A large number of delivery methods are well known to those of skill in the
art. St~~ methods include, for example liposome-based gene delivery (Debs and
Zhu ( 1993)
WO 93/24640; Mannino and Gould-Fogerite (1988) BioTechniques 6(7): 682-691;
Rose
U.S. Pat No. 5,279,833; Brigham (1991) WO 91/06309; and Felgner et al. (1987)
Proc. Natl.
Acad. Sci. USA 84: 7413-7414), as well as use of viral vectors (e.g.,
adenoviral (see, e.g.,
Berns et al. (1995) Ann. NYAcad. Sci. 772: 95-104; Ali et al. (1994) Gene
Ther. 1: 367-384;
and Haddada et al. (1995) Curr. Top. Microbiol. Immunol. 199 ( Pt 3): 297-306
for review),
papillomaviral, retroviral (see, e.g., Buchscher et al. (1992) J. Virol. 66(5)
2731-2739;
Johann et al. (1992) J. Virol. 66 (5):1635-1640 (1992); Sommerfelt et al.,
(1990) Virol.
176:58-59; Wilson et al. (1989) J. Virol. 63:2374-2378; Miller et al., J.
Virol. 65:2220-2224
(1991); Wong-Staal et al., PCT/US94/05700, and Rosenburg and Fauci (1993) in
Fundamental Immunology, Third Edition Paul (ed) Raven Press, Ltd., New York
and the
references therein, and Yu et al., Gene Therapy (1994) supra.), and adeno-
associated viral
vectors (see, West et al. (1987) Virology 160:38-47; Carter et al. (1989) U.S.
Patent No.
4,797,368; Carter et al. WO 93/24641 (1993); Kotin (1994) human Gene Therapy
5:793-
801; Muzyczka (1994) J. Clin. Invst. 94:1351 and Samulski (supra) for an
overview of AAV
vectors; see also, Lebkowski, U.S. Pat. No. 5,173,414; Tratschin et al. (1985)
Mol. Cell.
Biol. 5(11):3251-3260; Tratschin, et al. (1984) Mol. Cell. Biol., 4:2072-2081;
Hermonat and
Muzyczka (1984) Proc. Natl. Acad. Sci. USA, 81:6466-6470; McLaughlin et al.
(1988) and
Samulski et al. (1989) J. Virol., 63:03822-3828), and the like.
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"Naked" DNA and/or RNA that comprises a genome of an attenuated vaccine
cag-be introduced directly into a tissue, such as muscle. See, e.g., USPN
5,580,859. Other
methods such as "biolistic" or particle-mediated transformation (see, e.g.,
Sanford et al.,
USPN 4,945,050; USPN 5,036,006) are also suitable for introduction of vaccines
into cells
of a mammal according to the invention. These methods are useful not only for
in vivo
introduction of DNA into a mammal, but also for ex vivo modification of cells
for
reintroduction into a mammal. As is the case for other methods of delivering
vaccines,
vaccine adminsstration is repeated, if necessary, in order to maintain the
desired level of
immunomodulation.
Attenuated vaccines can be administered directly to the mammal. The
vaccines obtained using the methods of the invention can be formulated as
pharmaceutical
compositions for administration in any suitable manner, including parenteral
(e.g.,
subcutaneous, intramuscular, intradermal, or intravenous), topical, oral,
rectal, intrathecal,
buccal (e.g., sublingual), or local administration, such as by aerosol or
transdermally, for
prophylactic and/or therapeutic treatment. Pretreatment of skin, for example,
by use of hair-
removing agents, may be useful in transdermal delivery. Although more than one
route can
be used to administer a particular composition, a particular route can often
provide a more
immediate and more effective reaction than another route.
Pharmaceutically acceptable carriers are determined in part by the particular
composition being administered, as well as by the particular method used to
administer the
composition. Accordingly, there is a wide variety of suitable formulations of
pharmaceutical
compositions of the present invention. See, e.g., Lieberman, Pharmaceutical
Dosage Forms"
Marcel Dekker, Vols. 1-3 (1998); Remington's Pharmaceutical Science, 15th ed.,
Mack
Publishing Company, Euston, Pennsylvania (1980) and similar publications. A
variety of
aqueous carriers can be used, e.g., buffered saline and the like. These
solutions are sterile
and generally free of undesirable matter. These compositions can be sterilized
by
conventional, well known sterilization techniques. The compositions can
contain
pharmaceutically acceptable auxiliary substances as required to approximate
physiological
conditions such as pH adjusting and buffering agents, toxicity adjusting
agents and the like,
for example, sodium acetate, sodium chloride, potassium chloride, calcium
chloride, sodium
lactate and the like. The concentration of attenuated vaccine in these
formulations can vary
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widely, and will be selected primarily based on fluid volumes, viscosities,
body weight and
the like in accordance with the particular mode of administration selected and
the patient's
needs.
Formulations suitable for oral administration can consist of (a) liquid
solutions, such as an effective amount of the packaged nucleic acid suspended
in diluents,
such as water, saline or PEG 400; (b) capsules, sachets or tablets, each
containing a
predetermined amount of the active ingredient, as liquids, solids, granules or
gelatin; (c)
suspensions in an appropriate liquid; and (d) suitable emulsions. Tablet forms
can include
one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn
starch, potato
starch, tragacanth, microcrystalline cellulose, acacia, gelatin, colloidal
silicon dioxide,
croscarmellose sodium, talc, magnesium stearate, stearic acid, and other
excipients,
colorants, fillers, binders, diluents, buffering agents, moistening agents,
preservatives,
flavoring agents, dyes, disintegrating agents, and pharmaceutically compatible
carriers.
Lozenge forms can comprise the active ingredient in a flavor, usually sucrose
and acacia or
tragacanth, as well as pastilles comprising the active ingredient in an inert
base, such as
gelatin and glycerin or sucrose and acacia emulsions, gels, and the like
containing, in
addition to the active ingredient, carriers known in the art. It is recognized
that the attenuated
vaccines, when administered orally, must be protected from digestion. This is
typically
accomplished either by complexing the vaccines with a composition to render it
resistant to
acidic and enzymatic hydrolysis or by packaging the vaccines in an
appropriately resistant
carrier such as a liposome. Means of protecting vectors from digestion are
well known in the
art. The pharmaceutical compositions can be encapsulated, e.g., in liposomes,
or in a
formulation that provides for slow release of the active ingredient.
The attenuated vaccines, alone or in combination with other suitable
components, can be made into aerosol formulations (e.g., they can be
"nebulized") to be
administered via inhalation. Aerosol formulations can be placed into
pressurized acceptable
propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.
Suitable formulations for rectal administration include, for example,
suppositories, which consist of the packaged nucleic acid with a suppository
base. Suitable
suppository bases include natural or synthetic triglycerides or paraffin
hydrocarbons. In
addition, it is also possible to use gelatin rectal capsules which consist of
a combination of
CA 02377084 2001-12-21
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the packaged nucleic acid with a base, including, for example, liquid
triglycerides,
polyethylene glycols, and paraffin hydrocarbons.
Formulations suitable for parenteral administration, such as, for example, by
intraarticular (in the joints), intravenous, intramuscular, intradermal,
intraperitoneal, and
subcutaneous routes, include aqueous and non-aqueous, isotonic sterile
injection solutions,
which can contain antioxidants, buffers, bacteriostats, and solutes that
render the formulation
isotonic with the blood of the intended recipient, and aqueous and non-aqueous
sterile
suspensions that can include suspending agents, solubilizers, thickening
agents, stabilizers,
and preservatives. In the practice of this invention, compositions can be
administered, for
example, by intravenous infusion, orally, topically, intraperitoneally,
intravesically or
intrathecally. Parenteral administration and intravenous administration are
the preferred
methods of administration. The formulations of attenuated vaccines can be
presented in
unit-dose or mufti-dose sealed containers, such as ampoules and vials.
The dose administered to a patient, in the context of the present invention
1 ~ should be sufficient to effect a beneficial therapeutic and/or
prophylactic response in the
patient over time. The dose will be determined by the efficacy of the
particular attenuated
vaccine employed and the condition of the patient, as well as the body weight
or vascular
surface area of the patient to be treated. The size of the dose also will be
determined by the
existence, nature, and extent of any adverse side-effects that accompany the
administration
of a particular vaccine in a particular patient.
In determining the effective amount of the vaccine to be administered in the
treatment or prophylaxis of an infection or other condition, the physician
evaluates vaccine
toxicities, progression of the disease, and the production of anti-vaccine
vector antibodies, if
any. In general, the dose equivalent of a naked nucleic acid from a vector is
from about 1 p.g
2~ to 1 mg for a typical 70 kilogram patient, and doses of vectors used to
deliver the nucleic
acid are calculated to yield an equivalent amount of therapeutic nucleic acid.
Administration
can be accomplished via single or divided doses.
In therapeutic applications, compositions are administered to a patient
suffering from a disease (e.g., an infectious disease or autoimmune disorder)
in an amount
sufficient to cure or at least partially arrest the disease and its
complications. An amount
adequate to accomplish this is defined as a "therapeutically effective dose."
Amounts
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effective for this use will depend upon the severity of the disease and the
general state of the
patient's health. Single or multiple administrations of the compositions may
be administered
depending on the dosage and frequency as required and tolerated by the
patient. In any
event, the composition should provide a sufficient quantity of the proteins of
this invention
to effectively treat the patient.
In prophylactic applications, compositions are administered to a human or
other mammal to induce an immune response that can help protect against the
establishment
of an infectious disease or other condition. Subsequent challenge by the
corresponding
pathogen will trigger the immune response that has been primed by pre-exposure
to the
vaccine.
The toxicity and therapeutic efficacy of the attenuated vaccines provided by
the invention are determined using standard pharmaceutical procedures in cell
cultures or
experimental animals. One can determine the LD;o (the dose lethal to 50% of
the population)
and the EDSO (the dose therapeutically effective in 50% of the population)
using procedures
presented herein and those otherwise known to those of skill in the art.
The attenuated vaccines of the invention can be packaged in packs, dispenser
devices, and kits for administering genetic vaccines to a mammal. For example,
packs or
dispenser devices that contain one or more unit dosage forms are provided.
Typically,
instructions for administration of the compounds will be provided with the
packaging, along
with a suitable indication on the label that the compound is suitable for
treatment of an
indicated condition. For example, the label may state that the active compound
within the
packaging is useful for treating a particular infectious disease, autoimmune
disorder, tumor,
or for preventing or treating other diseases or conditions that are mediated
by, or potentially
susceptible to, a mammalian immune response.
EXAMPLES
The following examples are offered to illustrate, but not to limit the present
invention.
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Example 1
Specific examples of using DNA shuffling to generate attenuated viruses to be
used as
vaccines or vaccine vectors
This Example describes several illustrative methods for using the methods of
the invention to generate attenuated viral vaccines or vaccine vectors.
A. Bovine viral diarrhea virus
Bovine viral diarrhea virus (BVDV) is a togavirus that is the most insidious
and devastating viral pathogen of cattle in the United States (Vassilev et
al., J. Virol. (1997)
71:471-8). The virus causes immunosuppression, diarrhea, respiratory distress,
abortion and
persistent infection in calves. There are at least two serotypes of BVDV, as
well as two
biotypes (cytopathic and non-cytopathic). Because of the existing natural
diversity in the
BVDV strains, the virus offers an excellent starting point for evolution by
family shuffling.
The approach is particularly feasible, because stable full-len'th cDNA copies
of BVDV have
been established (Mendez et al., J. Yirol. 1998;72: 4737-45; Vassilev et al.,
J. Yirol. (1997)
71:471-8). As assayed by transfection of MDBK cells, uncapped RNAs transcribed
from
these cDNA clones were highly infectious (>105 PFU/~,g). The recovered virus
was similar
in plaque morphology, growth properties, polyprotein processing, and
cytopathogenicity to
the parental BVDV strain (Mendez et al., J. Virol. (1998) 72: 4737-45).
In addition, the principle of generating chimeras of infectious BVDV and
antigenic determinants from other viruses has been demonstrated using rational
design. More
specifically, the coding region for the major envelope glycoprotein E2/gp53 in
the molecular
genomic clone of BVDV was substituted with that of the Singer strain, giving
rise to a
chimeric virus (Vassilev et al., J. Yirol. (1997) 71:471-8). However, such
approaches to
design of chimeric viruses suffer a significant drawback in that
immunogenicity against the
original virus is lost when the immunogenic envelope proteins are replaced by
those derived
from other pathogens.
To obtain chimeric viruses that have maintained the immunogenicity of the
parental virus, DNA is used shuffling to generate chimeras between different
viruses or their
immunogenic fragments. Nucleic acids of different serotypes of BVDV are
shuffled using
family shuffling approach, for example. Either the entire infectious cDNA
clones or the
nucleic acids that encode the envelope proteins are shuffled, and a library of
chimeric viruses
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is generated. Shuffling the entire viral genome has the advantage that one is
likely to
simultaneously find solutions to attenuation and immunogenicity. MDBK cells,
for example,
are suitable for use in screening for attenuation in vitro, because the wild-
type BVDV is
highly infectious in these cells, and some strains cause a cytopathic effect
(Mendez et al., J.
Virol. (1998) 72: 4737-45). Other ways by which one can screen for attenuation
include, for
example, analysis of temperature sensitivity, altered host-cell specificity,
selection based
upon sensitivity to complement, and selection for growth in immunocompromised
animals
only. Alternatively, one can also choose to shuffle only one virus strain to
attenuate the virus
first, and thereafter use family DNA shuffling of the immunogenic regions to
generate
chimeras that provide potent crossprotective immunity.
B. Marek's Disease Virus (MDV); improved manufacturine of viral vaccines by
DNA shuffling
Marek's disease (MD) is a lymphoproliferative disease of chicken, which is
characterized by malignant T cell-lymphoma formation (Morimura et al., J. Vet.
Med. Sci.
1998; 60:1-8). Relatively efficient vaccines are available to prevent the
disease, but methods
for manufacturing of the vaccine in particular need major improvements. The
previously
available attenuated MDV-vaccine is propagated in primary chick embryo
fibroblasts, and
the vaccine is the frozen, virus-infected cell preparation. Cell-free vaccines
have also been
tested, but their immunogenicity is inferior as compared to the cell-
associated vaccines. In
addition, MDV has been significantly evolving over the past 40 years to gain
greater
oncogenicity, and some of these viruses are not adequately controlled by the
vaccines that
are currently available (Biggs, Philos. Traps. R. Soc. Lond. B. Biol Sci.
(1997) 352:1951-62).
The development of vaccines against MDV is also hampered by the existence of
multiple
serotypes. Mixtures of different serotypes have been used in the vaccine
preparations; such
as vaccines based on the attenuated strains of serotype 1, 2 and 3, but
failures resulting in
disease progress have been reported indicating a need for production of new,
more effective
vaccines (Zelnik, Acta Virol. (1995) 39:53-63).
Molecular evolution of MDV by DNA shuffling provides solutions to the
problems otherwise associated with the manufacturing, attenuation and
immunogenicity of
the vaccine. Generation of cell-free vaccine preparations provides a major
improvement in
the manufacturing process. Libraries of recombinant MDV nucleic acids, or
fragments
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thereof, are generated and screened for efficient propagation in cell culture
in a cell-free
manner. Cell lines from other species can be used to simultaneously achieve
proper
attenuation. The cell-free viruses that are then analyzed for their
immunogenicity in ovo or in
chicks. These cell-free preparations will provide major improvements to the
manufacturing
and storage of the vaccine, because the current vaccine preparations have to
be shipped in
liquid nitrogen containers to ensure the stability of the cells.
Because family DNA shuffling allows one to generate chimeric antigens and
viruses, the approach is useful in generating vaccine strains that provide
efficient cross-
protection against all or most different serotypes of MDV. Furthermore,
because maternal
antibodies interfere with the vaccine, thus reducing its efficacy (Sharma and
Graham, Avian
Dis. (1982) 26: 860-70; Nazerian et al., Avian Dis. (1996) 40: 368-76), and
because DNA
shuffling can generate new antigenic variants, this approach allows one to
generate vaccine
strains that are not recognized-~r~naternal antibodies of previously
vaccinarted animals. By
generating large libraries of vaccine strains, several different immunogenic
variants are
found. This enables vaccinations of different generations with antigenically
variable
vaccines, reducing the interference by maternal antibodies induced by
immunization.
Screening for reduced interference by maternal antibodies can be done in vitro
using sera
derived from vaccinated animals. For example, negative selection techniques,
such as
panning, are used to remove all strains that are recognized by antibodies from
immunized
animals. The antigenicity and immunogenicity of the remaining strains can
subsequently be
verified in in vivo studies. Protective immunity will be analyzed by
challenging the chicken
by different live MDV serotypes.
C. Bovine heroes virus type-1 (BHV-1), also known as Infectious Bovine
Rhinotracheatis virus (IBRV)
Bovine herpesvirus 1 (BHV-1 ), also called Infectious bovine rhinotracheatis
virus (IBRV), replicates in a wide range of cell types and the disease
manifestations include
respiratory tract disease, conjunctivitis, vulvovaginitis, abortion,
balanoposthitis,
meningoencephalitis, alimentary tract disease and fatal systemic infection
(Lupton and Reed,
Am. J. Vet. Res. (1980) 41: 383). Immune responses to BHV-1 have been observed
after
exposure of animals to virulent virus, conventional live or killed vaccines,
genetically
engineered live virus vaccines, subunit vaccines and, more recently, following
immunization
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with plasmids encoding immunogenic antigens (Babiuk et al., Vet. Microbiol.
(1996) 53:31-
42). Exposure to BHV-1 or its glycoproteins induced specific responses to the
virus which
are capable of neutralizing virus and killing virus infected cells. Killing of
virus infected
cells occurs after the expression of viral antigens on the cell surface of
infected cells (Babiuk
et al., Vet. Microbiol. (1996) 53:31-42). BHV-1 may spread in the infected
host by viremia,
gaining access to a broader range of tissues and organs, and it may cause a
variety of
symptoms (Engels and Ackermann, Vet. Microbiol. (1996) 53: 3-15).
Herpesviruses may
also establish latency in neuronal or lymphoid cells, and during latency few
viral antigens
are synthesized. Upon reactivation, the viruses re-establish the lytic cycle
of replication.
Although a vigorous immune response is often induced during the primary viral
infection,
these mechanisms help the herpesviruses to escape the host immune system
during latency
and to a lesser degree also during reactivation (Id. ).
Evolution of attenuated BHV-1 strains is achieved by random DNA shuffling
of the virus or targeted evolution of virus components that are critical to
the penetration and
propagation of the virus. One example of such virus component is the
glycoprotein H
(Meyer et al., J. Gen. Virol. (1998) 79: 1983-7). gH is a structural component
of the virus
and forms a complex with glycoprotein gL. Experiments with gH-deficient BHV-1
demonstrated that gH is crucial in the infectious cycle of the virus and is
involved in virus
entry and cell-to-cell spread, but not in the attachment of the virus (Id.).
Another example of
useful component of BHV-1 for molecular evolution by DNA shuffling is the
glycoprotein D
(gD), which has also been shown to be an essential component involved in virus
entry
(Hanon et al., Virology (1999) 257: 191-197). BHV-1 viruses devoid of gD (BHV-
1 gD-/-)
are able to bind to BL-3 cells, but they are no longer able to induce
apoptosis (Id.).
Furthermore, immunity against gD has been shown to confer resistance to BHV-1
replication
in cattle (Zhu and Letchworth, Vaccine (1996) 14: 61-9).
DNA shuffling is used to cause molecular evolution of gH or gD so as to
generate BHV-1 variants that have altered capacity to spread from cell to
cell, a crucial event
in pathogenesis. Taken together, family DNA shuffling of gD and or gH provides
means to
simultaneously attenuate the virus and to generate chimeric viruses that
provide protection
against multiple serotypes.
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More specifically, in this approach the virus, or fragments thereof, such as
gH
or gD, will be shuffled and a library are generated. The shuffled fragments
can be
incorporated into the virus backbone using conventional techniques known to
those skilled
in the art. The library of viruses are then selected for attenuation. A number
of different
approaches for selection can be taken, some of which have been previously
described during
attempts to attenuate the wild-type virus. Such selection techniques can also
be applied to
selection of attenuated, DNA shuffled vaccine strains. These methods include
rapid passage
in bovine cell culture (Schwartz et al., Proc. Soc. Exp. Biol. Mod. (1957) 96:
453) or by
adaptation to porcine or canine cell cultures (Schwartz et al., Proc. Soc.
Exp. Biol. Mod.
(1958) 97: 680; Zuschek et al., J. Am. Vet. Mod. Assoc. (1961) 139: 236). In
addition, the
virus can be adapted to grow in cell culture in reduced temperature
(30°C), or by selection of
temperature sensitive mutants (56 °C for 40 minutes) (Inaba, J. Jpn.
Vet. Mod. Assoc. ( 1975)
28: 410; Bartha, Dev. Biol. Stand. (1974) 26:5).
Intranasally administered BHV-1 has also been attenuated by serial passage
in rabbit cells cultured in vitro, or were modified by treatment with HNOZ
followed by
selection of temperature sensitive mutants (Todd, Can. Vet. J. (1974) 15: 257;
Zygraich et
al., Res. Vet. Sci. (1974) 16: 328). These selection techniques are also
useful in identifying
attenuated strains of shuffled viruses; the library of shuffled viruses can
contain viruses that
have additional improvements in addition to attenuation. A crucial advantage
as compared to
previously described attenuation techniques is the fact that efficient
chimerism between the
different serotypes can be achieved, which enables one to generate
crossprotective strains
that provide improved immune responses in vivo. The analysis of the efficacy
of the immune
response in animals can be done by analyzing immune parameters in the sera and
circulating
lymphocytes in immunized animals. Moreover, the protective and crossprotective
immune
responses can be studied by challenging the immunized animals with the wild-
type viruses
of different serotypes. The viruses that demonstrate the best attenuation
combined with the
most potent crossprotective immune response in vivo are selected for further
rounds of
shuffling and selection, when desired.
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D. Infectious bronchitis virus
Infectious bronchitis virus (IBV) is a member of the family Coronaviridae
and causes highly contagious respiratory and reproductive disease in chickens.
IBV has a
single-stranded, positive sense RNA genome of 27.6 kb. The construction of a
full-length
clone of IBV downstream of the bacteriophage T7 promoter has been described
(Penzes et
al., J. Virol. (1996) 70: 8660-8). Electroporation of in vitro T7-transcribed
RNA from the
two different constructs into IBV helper virus-infected cells resulted in the
replication and
packaging of the RNA (Penzes et al., J. Virol. (1996) 70:8660-8).
The three structural proteins of IBV are the spike glycoprotein (S protein),
the
membrane glycoprotein (IV protein) and the nucleocapsid protein (N protein)
(Jia et al.,
Arch. Virol. (1995) 140: 259). There are at least ten serotypes of IBV.
Massachusetts,
Connecticut, Arkansas and California serotypes are the most persistent in the
US. Mutations
and crossovers are common mechanisms for the generation of new serotypes and
provide
means for the virus to escape naturally existing immunity or that induced by
vaccinations
(Keck et al., J. Virol. (1988) 62: 1810). More immunogenic and crossprotective
vaccine
strains are needed, and molecular evolution technologies provide improved and
faster means
to generate novel chimeras that can be screened for the desired properties
using in vitro and
in vivo screenings.
Libraries of IBV strains are generated by shuffling, and the desired clones
are
selected from the library by analyzing the entire library, pools thereof, or
individual clones.
More specifically, the degree of attenuation can be studied for example by
using tracheal
organ cultures (TOC) and oviduct organ cultures (00C) (Raj and Jones, Vaccine
(1997) 15:
163-8). Ciliostasis (CD50), immunofluorescence staining (IFID50) and organ
culture
infectivity (OCID50) have been shown to associate with attenuation and are
useful methods
for screening candidate live respiratory viral vaccines for attenuation (la'.
). These methods
are also useful when selecting shuffled, attenuated strains of IBV. Subsequent
immunizations and challenge studies in animals can be used to further evaluate
the degree of
attenuation, immunogenicity and cross-protection.
E. Evolution of stable Yellow Fever vaccine strains by DNA shuffling
The stability of these vaccine strains can be improved by DNA shuffling
technology. This Example describes the evolution of yellow fever (YF) virus,
which is an
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example of a vaccine strain, the stability of which can be improved by the
methods described
here. YF is an acute mosquito-borne viral haemorrhagic fever that has
reemerged across
Africa and in South America. A total of 18,735 yellow fever cases and 4,522
deaths were
reported from 1987 to 1991. This represents the greatest reported amount of
yellow fever
activity for any 5-year period since 1948 (Robertson et al., JAMA (1996) 276:
1157-62). In
Africa, a large proportion of cases have occurred in children. There is an
efficient vaccine
against yellow fever available, but financing the vaccine has been difFcult
for the poorest in
the world (Robertson et al., JAMA (1996) 276: 1157-62).
The stability of YF vaccine is a major problem in undeveloped countries and
tropical areas. The lyophilized vaccine strain without stabilizers
deteriorates rapidly when
exposed to temperatures above -20 °C (Monath, Dev. Biol. Stand. (1996)
87: 219-25).
Additives, such as sugars, amino acids, and divalent cations have improved the
stability of
the vaccine preparations. However, despite the relatively good stability of
these vaccine
formulations stability when freeze dried, the vaccine is unstable after
reconstitution and must
be discarded after one hour (Monath, Dev. Biol. Stand. ( 1996) 87: 219-25).
Improvements in
vaccine stability after reconstitution would significantly reduce cost,
stretch supplies of the
vaccine, and would also reduce the frequency of vaccine failures due to use of
degraded
vaccine.
In this Example, infectious cDNA clones of YF are subjected to
recombination by, for example, DNA shuffling. The resulting virus libraries
are analyzed for
stability. Selection is conducted under different temperatures, formulations
and time periods,
as desired, to obtain suitable YF viruses that are stable under the conditions
of interest. Only
those viruses that can propagate under such conditions survive the selection.
In addition, the
virus libraries can be freeze dried, reconstituted, and the most stable
viruses selected varying
periods after reconstitution. The viruses that demonstrate improved stability
can be subjected
to new rounds of shuffling and selection. Subsequent immunizations and
challenge studies
will further evaluate the degree of attenuation, immunogenicity and stability.
F. Influenza A virus
Epidemic infections with influenza A continue to associate with significant
morbidity and mortality in the general population, particularly among the
elderly and other
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high risk patients (Calfee and Hayden, Drugs (1998) ~6: 537-53). Tens of th
ousands of
deaths occur each year despite the availability of relatively efricient
vaccines. E~cient
control of the disease has not been achieved through immunization programs
because of
incomplete protective efficacy and antigenic variations of the virus.
Vaccinations must be
given annually because of the antigenic changes that the virus undergoes, and
because the
antibody responses decrease significantly over time (Rimmelzwaan ~t al.,
Vaccine (1999)
17: 1355-8).
Influenza A virus belongs to family Orthomyxoviridae, which are segmented,
negative-stranded viruses. Additional members of the family are Influenza B
and Influenza
C viruses. Viral replication occurs after synthesis of the mRNAs and requires
synthesis of
the viral proteins. Complete infectious segmented negative-strand viruses have
been
successfully recovered from cloned cDNA (Bridgen and Elliott, Proc. Nat'1.
Acad. Sci. USA
(1996) 93: 15400-4). Plasmids encoding full-length cDNA copies of three
Bunyamwera
bunyavirus RNA genome segments flanked by bacteriophage T7 promoter and
hepatitis
1 ~ delta virus ribozyme sequences were capable of encoding infectious virus
with the
characteristic of the parental cDNA clones (Bridgen and Elliott, Proc. Nat'1.
Acad. Sci. USA
(1996) 93: 15400-4). Similarly, full-length, infectious vesicular stomatitis
virus (VSV), the
prototypic nonsegmented negative-strand RNA virus, has been recovered from a
cDNA
clone (Whelan et al., Proc. Nat'1. Acad. Sci. USA (1990 92: 8388-92). These
data illustrate
the feasibility of shuffling the entire genomes of negative-strand RNA
viruses. In addition,
foreign genes have been successfully introduced into Influenza A genome
(Luytjes et al.,
Cell (1989) 59: 1107-13), indicating that Influenza A virus genomes can be
successfully
engineered. Infectious Influenza A virus has been recovered after transfection
of cDNA
encoding the PB2 polymerase gene, followed by transfection of the RNA
transcripts
(Subbarao et al., J. Virol. (1993) 67, 7223-8). Therefore, as an alternative
to whole viral
genome shuffling, novel attenuated vaccine strains of Influenza A can be
generated by
shuffling individual segments of the virus, further illustrating the
feasibility of DNA
shuffling approach in evolution of Influenza A viruses.
Libraries of Influenza A viruses are generated by shuffling the entire genome,
or segments thereof (such as, for example, the PB2 polymerase gene). Because
of the large
antigenic diversity of Influenza viruses, the segments encoding the
immunogenic proteins,
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such as the nucleocapsid, matrix proteins, hemaggluti_nia, nucleoprotein or
neuramididase,
provide additional targets for molecular evolution by DNA shuffling. Because
live
attenuated influenza A virus vaccines have been widely produced by the
transfer of
attenuating genes from a donor virus to new epidemic variants of influenza A
virus
(Subbarao et al., J. Virol. (1993) 67: 7223-8), the shuffled segments can be
introduced back
to other Influenza strains of interest. The shuffled viruses can be selected
using mufti-tiered
screening process including selection for growth in manufacturing cells,
identification of
temperature sensitive mutants, screening for presence of multiple epitopes by
polyclonal
antibodies, analysis of potent crossprotective immune response in vivo, or all
of the above.
The best variants can be selected for new rounds of shuffling and screening
when desired.
G. Respiratory svncvtial virus (RSVP
Respiratory syncytial virus (RSV) is the most important cause of lower
respiratory tract infection during infancy and early childhood (Domachowske
and
Rosenberg, Clin. Microbiol. Rev. (1999) 12: 298-309). RSV infection can be
devastating in
elderly and immunosuppressed individuals (Wyde, Antiviral Res. (1998) 39: 63-
79). The
infection generally results in the development of anti-RSV neutralizing-
antibodies, but these
are often suboptimal during an infant's initial infection. Reinfection during
subsequent
exposures is common, and efficient vaccines are highly desired.
Functional, infectious RSV has been recovered from expressed, cloned
cDNAs. RSV was expressed in a functional form by coexpressing the viral
polymerase
protein, phosphoprotein, and nucleocapsid protein from cDNA clones (Yu et al.,
J. Virol.
(1995) 69: 2412-9). Such cDNA clones provide an excellent starting point for
molecular
evolution by DNA shuffling. Several different antigenic groups of RSV have
been identified
(Sanz et al., Virus Res. (1994) 33: 203-17), and very high mortality rates, up
to 78%, have
2~ been observed in immunocompromised patients (Harrington et al., J. Infect.
Dis. (1992) 165:
987-93). Therefore, efficient vaccines that provide protection against
multiple different
variants of RSV are highly desired.
Although the antigenic heterogeneity of RSV is a challenge for vaccine
development, these naturally existing variants of the pathogen provide a pool
of existing
sequences that can be used to generate a family shuffled library of RSV.
Libraries of RSV
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viruses are generated by shuffling infectious cDNA clones derived from various
RSV
isolates. The resulting RSV variants are screened for attenuation and for
their properties as
vaccines. The stability of the viruses can be selected in vitro by storing the
vaccine strains
for prolonged periods of time. Moreover, the attenuation will be evaluated in
animal models
for lack of disease or for reduced levels of symptoms. These attenuated
strains are be further
analyzed for their capacity to induce protective immune responses in vivo.
This can be
achieved by challenging tl'~e immunized animals by live wild-type pathogens
and scoring the
different strains for their level of attenuation and efficacy in inducing
protective immune
responses. The optimal strains with desired properties can be selected for new
rounds of
shuffling and screening.
H. Canine Distemper virus (CDR
Canine distemper virus (CDV) is a morbillivirus that affects the neurologic
system and causes a frequently fatal systemic disease in a wide range of
carnivore species,
including domestic dogs. Classical serology provides data of diagnostic and
prognostic
values and is also used to predict the optimal vaccination age of pups,
because maternal
antibodies can interfere with the vaccines (Appel and Harris, J. Am. Yet. Med.
Assoc. (1988)
193: 332-3). Several antigenically different strains of CDV have been
identified (Ohashi et
al., J. Vet. Med. Sci. (1998) 60: 1209-12; Carpenter et al., Vet. Immunol.
Immunopathol.
(1998) 65(2-4): 259-66), and the virus appears to frequently cross host
species among
carnivores (Id.). The antigenic heterogeneity of the different strains is a
challenge for
vaccine development, but it also provides and excellent genetic diversity that
enables further
evolution in vitro using the methods of the invention.
Infectious morbilliviruses have been reconstituted from cDNA (Cathomen et
al., EMBO J. (1998) 17: 3899-908), indicating that the use of DNA shuffling to
generate
attenuated morbilliviruses, such as attenuated vaccine strains of CDV, is
feasible. More
stable and immunogenic viruses are highly desired. In addition, because
maternal antibodies
can interfere with CDV vaccines, vaccines not recognized by such antibodies
are expected
have improved e~cacy (Appel and Harris, J. Am. Vet. Med Assoc. (1988) 193: 332-
3).
Libraries of CDV viruses are generated by DNA shuffling of infectious cDNA,
and the
resulting viruses are screened for their properties as vaccines. Studies in
vivo in dogs are
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used to identify attenuated strains (strains that cause inhibited or no
clinical disease) that
provide efficient immune response upon challenge of the immunized animals by
wild-type
viruses. In addition, the viruses can be selected for increased stability in
vitro by storing the
vaccine strains for prolonged periods of time. Strains with desired properties
can be
subjected to new rounds of shuffling and screening if further improvement is
desired.
Example 2
Evolution of attenuated alnhaviruses; VEE as a vehicle for airborne
vaccinations
This Example describes the use of DNA shuffling to evolve attenuated
alphaviruses, which are useful as a vehicle for vaccines that are suitable for
airborne
administration.
The alphaviruses are a genus of 26 enveloped viruses that cause disease in
several species, including humans and domestic animals. Mosquitoes and other
hematophagous arthropods serve as vectors (Strauss and Strauss, Microbiol.
Rev. (1994) 58:
491-562). Alphaviruses include Venezuelan Equine Encephalitis virus (VEE),
Semliki
Forest virus (SFV) and Sindbis virus (SIN), which have also been targets of
interest as
vaccine vectors, because of the broad host range and superior infectivity of
these viruses.
The generation of high-titer recombinant alphavirus stocks has enabled high-
level expression
of several nuclear, cytoplasmic, membrane-associated and secreted proteins in
a variety of
cell lines and primary cell cultures (Lundstrom, J. Recept. Signal. Transduct.
Res. (1999) 19:
673-86).
The complete sequences of the positive stranded RNA genomes of at least
eight alphaviruses have been determined, and partial sequences are known for
several others
(Strauss and Strauss, Microbiol. Rev. (1994) 58: 491-562). Importantly, full-
length cDNA
clones from which infectious RNA can be recovered have been constructed for
four
2~ alphaviruses, including VEE, SFV and SIN (Davis et al., Virology (1989)
171:189-204;
Polo et al., Proc. Nat'l. Acad. Sci. USA (1999) 96: 4598-603; Atkins et al.,
Mol. Biotechnol.
(1996) 5: 33-8). Therefore, VEE, SIN and SFV are particularly good examples of
alphaviruses that can be attenuated by DNA shuffling. In the present Example,
although
VEE is described in detail, the high degree of structural and functional
relatedness among
alphaviruses allows one to use a similar approach for other alphaviruses.
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VEE is an unusual alphavirus in that it is also highly infectious for both
humans and rodents by aerosol inhalation. Therefore, attenuated strains of VEE
provide
vehicles to deliver the vaccines in an aerosol formulation, which enables
rapid vaccinations
of large populations of humans or animals at the same time. Aerosol
vaccination with
inactivated or attenuated recombinant patihogens has been shown to be an
efficient way to
induce local protection against lung diseases, and aerosol vaccinations have
also been shown
to protect against infectious diseases (Hensel and Lubitz, Behring. Inst.
Mitt. (1997) 98: 212-
9). Because VEE can also be used as a vector to deliver antigens from other
pathogenic
organisms, aerosol mediated vaccinations with attenuated strains of VEE are
expected to
provide very efficient and rapid vaccination protocols against a variety of
diseases. VEE is
an unusual virus also because its primary target outside the central nervous
system is the
lymphoid tissue, and therefore, attenuated variants may provide means to
target vaccines or
pharmaceutically useful proteins to the immune system.
There are at least seven subtypes of VEE that can be identified genetically
and serologically. Based on epidemiological data the virus isolates fall into
two main
categories: I-AB and I-C strains, which are associated with VEE
epizootics/epidemics, and
the remaining serotypes, which are associated primarily with enzootic
vertebrate-mosquito
cycles and circulate in specific ecological zones (Johnston and Peters, In
Fields Virology,
Third Edition, eds. B.N. Fields et al., Lippincott-Raven Publishers,
Philadelphia, 1996).
Libraries of recombinant VEE viruses are created and screened to identify
those recombinant viruses that exhibit an attenuated phenotype. Libraries are
generated by
subjecting to DNA shuffling either the entire infectious cDNA clones or the
regions known
to play a role in pathogenesis and protection, such as the envelope protein.
These libraries
and individuals chimeras/mutants thereof are subsequently screened for their
capacity to
2~ induce widely crossreacting and protective antibody responses.
Alternatively or additionally,
the libraries are screened to identify those recombinant viruses that have the
capacity to
immunize mammals through aerosol delivery. The libraries are delivered in
aerosol
formulations and the optimal viruses are subsequently identified in animals
that develop
specific antibodies and survive the infection, as an indication that the
strain was sufficiently
attenuated. When the libraries are generated by family shuffling of the
different serotypes of
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VEE, one also is able to identify the most crossprotective strains by
challenging the
immunized animals with various serotypes of the live, wild-type pathogen.
Furthermore, because VEE can act as a vehicle to deliver foreign antigens,
attenuated VEE strains encoding antigens from other pathogens can be useful in
vaccinations
against a variety of diseases. Packaging cell lines can be used to enable
production of
biologically active vector particles even when the structural proteins of the
virus have been
replaced by foreign antigens (Polo et al., supra.). These packaging cells can
be engineered
to encode those structural proteins that were shown to mediate efficient
immunization, for
example through aerosol mediated immunization. As an e:~ample, the gene that
encodes
VEE envelope protein is replaced with that which encodes hantavirus
glycoproteins.
Because VEE is known to infect mice by airborne challenge, the approach
provides a
method for aerosol mediated vaccinations of wild mice against hantavirus
infections, which
should greatly reduce the risk to humans of encountering hantavirus in endemic
areas. A
significant advantage of the method is that the laboratory setting is useful
in the initial
screening, because mice can be infected with VEE by airborne challenge (Wright
and
Phillpotts, Arch. Yirol. (1998) 143: 1155-62). The best clones are subjected
to new rounds
of shuffling and selection, when desired.
Example 3
Infectious bursal disease virus (IBDV1
Infectious bursal disease (IBD) emerged in 1957, spread rapidly, and became
recognized throughout the U.S. broiler and commercial egg production areas by
1965
(Lasher and Davis, Avian Dis. (1997) 41: 11-9). Infectious bursal disease
virus (IBDV)
attacks the Bursa of Fabritius causing immunosuppression and death. The acute
stage of
IBD, the immunosuppression that follows, and the widespread distribution of
IBD virus
(IBDV), contribute to the major economic significance of the disease (Saif,
Poult. Sci.
(1998) 77:1186-9). A live attenuated vaccine was developed and demonstrated a
rather good
efficacy (Lasher and Davis, supra.). However, in the mid-1980s novel variants
emerged,
such as the Delaware variants, and variants with increased virulence were
identified in
Europe and Asia in 1989 (Id.). In addition, maternal immunity significantly
reduces the
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efficacy of the vaccines (Bayyari et al., Avian Dis. (1996) 40: 588-99), and
vaccines with
improved efficacy are desired.
IBDV is a double stranded RNA virus and belongs to the family Birnaviridae
of the genus Avibirnavirus (Nagarajan and Kibenge, Can. J. Vet. Res. (1997)
61: 81-8). The
genome consists of two segments, designated A and B. cDNA clones encoding the
segments
A and B of IBDV have been shown to generate viable virus progeny. Independent
full-length
cDNA clones were constructed that contained the entire coding and noncoding
regions of
R~'~tA segments A and B (Mundt and Vakharia, Proc. Nat'l. Acad. Sci. USA
(1996) 93:
11131-6). These cDNAs provide an excellent starting point for DNA shuffling.
In addition,
because the NS protein is dispensable for viral replication in vitro and in
vivo plays an
important role in viral pathogenesis (Yao et al., J. Virol. (1998) 72: 2647-
54), the segment
encoding the NS protein is a particularly useful target for evolution by DNA
shuffling.
Furthermore, the existence of several natural variants of IBDV (Nagarajan and
Kibenge,
supra.), provides a source of natural diversity for family DNA shuffling to
generate
attenuated strains and chimeras of the different serotypes.
Libraries of recombinant IBDV nucleic acids are generated by shuffling both
A and B segments. In addition, libraries of viruses that contain shuffled NS
genes are
generated by shuffling NS genes that are then incorporated back into the viral
genome. The
libraries of viruses is initially screened for attenuation in vitro. However,
because chicken
are accessible in large numbers, the primary screen is for attenuation and
immunogenicity in
vivo. More specifically the viruses or pools of viruses are injected in ovo,
at one day of age
or applied to the drinking water at 7-14 days of age. Attenuated viruses not
causing clinical
disease are identified, and the immunized animals are subsequently screened
for protective
immune responses by challenging them with the live wild-type viruses. The best
viruses are
subjected to new rounds of shuffling and selection, when desired.
Example 4
Canine parvovirus~ evolution of vaccine strains that can escape interference
by
maternal antibodies
Canine parvovirus is a newly emerged pathogen of dogs that was identified in
the late 1970s (Pollock and Coyne, Yet. Clin. North Am. Small. Anim. Pract.
(1993) 23:555-
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68). Although parvoviral enteritis was initially seen as epidemic disease in
all dogs, the
disease now primarily occurs in 1- to 6-month-old dogs. Interference by
maternal antibodies
accounts for the vast majority of vaccine failures (Id.; Waner et al., J. Vet.
Diagn. Invest.
(1996) 8: 427-32). Maternally derived hemagglutination inhibition (HI) titers
have also been
shown to correlate to the efficacy of attenuated CPV vaccines (Hoare et al.,
Vaccine (1997)
15: 273-5). Intranasal vaccination of pups with maternal antibodies has
demonstrated some
success in avoiding the interference, but vaccines that have improved
immunogenicity in ti'~e
presence of such antibodies are desired (Buonavoglia et al., Zentralbl.
Veterinarmed. (1994)
41: 3-8). Molecular virologic methods have also revealed continued evolution
of the virus
adding to the antigenic heterogeneity of the virus and to the challenges in
vaccine
development.
In this Example, canine parvovirus strains are_evolved and screened to
identify those that, when used as vaccines, do not interfere with maternal
antibodies. Either
the entire vaccine strain, or the immunogenic antigens of the vaccine alone,
is shuffled and
negatively selected so that the vaccines that are recognized by maternal
antibodies are
removed from the libraries. For example, amity selection using flasks or
columns coated
with the maternal antibodies are used. To identify strains that have epitopes
derived form
multiple different strains of CPV, monoclonal antibodies that discriminate
among different
strains of CPV (Sagazio et al., J. Virol. Methods (1998) 73: 197-200) can be
used, provided
these antibody specificities are not a major component of the maternal
antibodies. These
multivalent strains are likely to be more potent in inducing crossprotection
against all
different existing and emerging variants of CPV.
Molecular infectious clones of parvoviruses have been generated (Bloom et
al., J. Virol. (1993) 67: 5976-88). Such clones provide a suitable substrate
for family DNA
shuffling. The family shuffling approach has the advantage that one will
simultaneously
generate chimeras of the different strains. These chimeras can then be
screened for optimal
immunogenicity and crossprotection in vivo. In one approach, libraries of
canine
parvoviruses are generated, and these libraries are initially screened for
lack of binding to
antibodies derived from dogs previously immunized against CPV. The
immunogenicity of
the selected vaccines can be verified by immunizing dogs, and subsequently
challenging the
vaccinated animals with different strains of live CPV. Vaccines that have
reduced binding to
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maternal antibodies are expected have improved efficacy, and chimeric vaccines
are
expected to provide crossprotective immune responses.
Example 5
Flaviviruses; evolution of chimeric attenuated vaccine strains of Dengue
viruses
Dengue viruses are transmitted through mosquito bites and 50-100 million
people each year. The virus causes serious clinical manifestations, such as
dengue
hemorrhagic fever (DHF). There are four major serotypes of Dengue virus,
namely Dengue
1, 2, 3 and 4. The spread of the four dengue virus serotypes had led to
increased incidence of
DHF and estimated 2.~ billion people at risk of the infection (Cardosa, Br.
Med. Bull. (1998)
54: 395-40~). No efficient vaccine against dengue infections are currently
available.
The envelope protein of Dengue virus has been shown to provide an immune
response that protects from a future challenge with the same strain of virus.
However, the
levels of neutralizing antibodies produced in response to subunit vaccines are
relatively low
and protection from live virus challenge is not always observed. For example,
mice injected
with genetic vaccines encoding envelope protein of Dengue-2 virus develop
neutralizing
antibodies when analyzed by zn vitro neutralization assays, but the mice did
not survive the
challenge with live Dengue-2 virus (Kochel et al., Vaccine (1997) ~15: 547-
552). However,
protective immune responses were observed in mice immunized with recombinant
vaccinia
virus expressing Dengue 4 virus structural proteins (Bray et al., (1989) J.
Yirol. 63: 2853.
Furthermore, live attenuated Dengue-2 vaccines protected monkeys against
homologous
challenge (Velzing et al., Vaccine (1999) 17: 1312-20). These data suggest
that live
attenuated Dengue vaccines will be an efficient approach to Dengue vaccine
development.
However, a tetravalent vaccine that induces neutralizing antibodies against
all four strains of
Dengue is required to avoid antibody-mediated enhancement of the disease when
the
individual encounters Dengue virus of different serotype. Family DNA shuffling
provides a
technology that simultaneously enables attenuation and generation of chimeric
viruses that
protect against all four serotypes.
Infectious cDNA clones of Dengue viruses have been generated and the
immunogenic envelope genes sequenced, providing a good starting point for
shuffling (Lai
et al., Proc. Nat'l. Acad. Sci. USA (1991) 88: 5139-43; Lanciotti et al., J.
Gen. Virol. (1994)
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75: 65-75; Kinrey et al., Virology (1997) 230: 300-8; Puri et al., Virus Genes
(1998) 17: 85-
8).
In this Example, the entire cDNAs encoding dengue viruses are shuffled to
obtain proper attenuation. Alternatively, selected regions of the virus are be
shuffled to
generate attenuated strains. Such viral genes that are useful targets for
shuffling include the
prM, E, NS1 and NS3 genes (Pryor et al., J. Gen. Virol. (1998) 79: 2631-9;
Valle and
Falgout, J. Virol. (1998) 72: 624-32). Additionally, the envelope genes of all
four serotypes
can be shuffled separately to generate crossprotective antigens, which can
subsequently be
incorporated back into the attenuated clones. Efficient chimerism is expected
to be achieved
also by family shuffling the entire infectious cDNA clones. The E proteins of
the different
dengue viruses share 62% to 77% of their amino acids. Dengue l and Dengue 3
are most
closely related (77% identical), followed by Dengue 2 (69%) and Dengue 4
(62%). These
identities are well in the range that allows efficient family shuffling.
Initially, screening for proper attenuation can be performed in vitro. For
example, passaging in primary dog kidney cells can be used (Puri et al., J.
Gen. Virol.
(1997) 78: 2287-91). One can also select temperature sensitive mutants. The
immunogenicity and crossprotection provided by the.attenuated vaccine strains
can be
further studied in mice challenged with the various live wild-type pathogens.
If desired,
further studies are performed in large non-human primates to verify the level
of attenuation.
This enables simultaneous studies of the immune responses. Infectivity in
monkeys has been
shown to correlate with that in humans, indicating that the monkey model is
useful in
selecting the properly attenuated strains (Marchette et al., Am. J. Trop. Med.
Hyg. (1990) 43:
212-8). Eventually, the attenuated, shuffled vaccines are tested in humans for
their capacity
to protect against infections with the different dengue serotypes.
Example 6
Flaviviruses: Hepatitis C virus (HCVI
Hepatitis C virus (HCV) infection is a major health problem that leads to
cirrhosis and hepatocellular carcinoma in a substantial number of infected
individuals,
estimated to be 100-200 million worldwide (Martin et al., Biochemistry (1998)
37: 11459-
68). Vaccines or effective treatments for HCV infection are not available.
Antigenic
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heterogeneity of different strains of hepatitis C virus (HCV) is a major
problem in
development of efficient vaccines against HCV. Antibodies or CTLs specific for
one strain
of HCV typically do not protects against other strains. Multivalent vaccine
antigens that
simultaneously protect against several strains of HCV would be of major
importance when
developing efficient vaccines against HCV.
HCV is a particularly suitable target for attenuation by DNA shuffling
because full-length cDNAs have been constructed (Yanigi et al., Proc. Nat'l.
Acid Sci.
USA (1997) 94: 8738-43) and infectious HCV cDNA can be obtained from
infectious blood
samples (Aizaki et al., Hepatolo~ (1998) 27:621-7). Thus, the naturally
existing diversity of
HCV can be directly isolated from the blood of infected individuals providing
an excellent
starting point for family DNA shuffling.
Several approaches to attenuation of HCV by DNA shuffling are available.
First, the entire cDNA clones may shuffled and the libraries screened using
the methods
described below. Secondly, the polymerise gene may be replaced by that derived
from other
related viruses, such as other flaviviruses. Polymerise genes from
flaviviruses, such as
Dengue virus, may allow replication of the HCV in tissue culture cells, which
is not possible
with the wild-type HCV. The NSSB protein of HCV is an RhIA-dependent RNA
polymerise
that is required for virus replication (Behrens et al., EMBO J. (1996) 15: 12-
22). Because
other flaviviruses readily replicate in vitro, RNA-polymerise genes derived
from other
flavivirus family members may allow HCV to replicate in vitro. Importantly,
family DNA
shuffling of flavivirus derived polymerise genes can be used to optimize the
genes to enable
the most potent replication in vitro. Generation of a library of HCV viruses
with shuffled
polymerise genes improves the chance to identify the viruses that efficiently
grow in vitro.
Another useful target for DNA shuffling is the internal ribosome entry site of
HCV, which is
required for viral translation (Honda et al., J. Virol. (1999) 73: 1165-74).
The region has
been shown to be relatively conserved in the different flaviviruses,
indicating that family
shuffling is a particularly attractive approach (Id.).
The libraries of shuffled HCV, generated by shuffling the entire genomes, or
fragments thereof, are generally screened for growth in vitro and in vivo.
Family DNA
shuffling allows the simultaneous generation of chimeric viruses and viral
proteins. This is
expected to improve the crossprotection against the different strains of HCV.
One important
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approach to attenuation is the selection of variants that grow in tissue
culture cells as
described above. A particularly significant selection is that which occurs in
vivo. Virus
clones isolated during the acute phase from patients and chimpanzees had
identical
sequences in the hypervariable region, and the infectious clones derived from
patients were
infectious in chimpanzees (Aizaki et al., Hepatology (1998) 27:621-7).
Therefore, the
chimpanzee model will be useful when analyzing the level of attenuation and
immune
response in animals. The degree of attenuation of the selected HCV variants
can thus be
readily analyzed in chimpanzees. The animals are subsequently challenged with
various
wild-type HCV isolates to detect the most protective and crossprotective
vaccine strains.
The best variants can be selected for new rounds of shuffling and screening,
when desired.
Example 7
Porcine reproductive and respiratory syndrome virus
Porcine reproductive and respiratory syndrome virus (PRRSV) is a recently
identified virus that continues to challenge swine producers, veterinary
practitioners, and
animal health researchers. The prevalence of infection is high, approximately
60% to 80% of
herds, and the clinical effects of infection vary widely among the farms
(Zimmerman et al.,
Vet. Microbiol. (1997) 55: 187-96). In many herds, infection is subclinical
and productivity
seemingly unaffected. However, some infected herds report occasional
respiratory disease
outbreaks in young pigs, periodic outbreaks of reproductive disease, or
severe, chronic
disease problems, particularly in young pigs. In these herds, secondary
infections with viral
or bacterial pathogens, such as Salmonella choleraesuis, Streptococcus suis,
or Haemophilus
parasuis typically occur concurrently with PRRSV infections (Zimmerman et al.,
Yet.
Microbiol. (1997) 55: 187-96).
There is evidence that existing candidate vaccine strains may persist and
mutate to a less attenuated form in vivo (Mengeling et al., Am. J. Vet. Res.
(1999) 60: 334-4).
Moreover, different isolates of PRRSV have been shown to differ substantially
in their
antigenic properties increasing the challenges in vaccine development
(Pirzadeh et al., Can.
,l. Yet. Res. (1998) 62: 170-7). Protection against heterologous strains is
poor (van Woensel
et al., Vet. Rec. (1998) 142: 510-2), and need for improved vaccines is
evident.
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DNA shuffling is used to generate improved, attenuated vaccine strains of
PRRSV. Infectious transcripts from cloned genome-length cDNA of PRRSV have
been
generated (Meulenberg et al., .I. Virol. (1998) 72: 380-7). Thus, family DNA
shuffling of
such infectious cDNA clones derived from various isolates provides a means to
generate
chimeras that simultaneously protect against the different serotypes.
Furthermore, the
genetic heterogeneity of the different isolates provides an excellent starting
pool to generate
optimally attenuated strains that provide efficient protection without
clinical disease. As an
example, porcine alveolar lung macrophages or CL2621 cells can be used to grow
the
viruses to address the level of attenuation (Meulenberg et al., J. Virol.
(1998) 72: 380-7).
Furthermore, BHK-21 cells are useful in the initial screening, because they
are readily
transfectable by the cDNA clones (Meulenberg et al., J. Virol. (1998) 72: 380-
7). The
subsequent analysis of the attenuated strains can be done in pigs or other
suitable test animal.
For example, pigs are immunized with the attenuated strains, and the animals
that do not
become ill are challenged with the wild-type viruses to assess the efficacy of
the vaccines.
The attenuated viruses that provided the most efficient protection can be
subjected to new
rounds of shuffling and selection, when desired.
Example 8
Human immunodeficiency virus (Hf~
The development of a safe and effective vaccine for the prevention of HIV
infections has proven to be extremely difficult, at least in part because of
the complexity
associated with HIV-1 and its pathogenesis (Hulskotte et al., Vaccine (1998)
16: 904-15).
Previous studies suggest that that HIV infections in human may be abrogated by
the host
immune system supporting the conclusion that it is possible to generate a
weakened virus
that induces a protective immune response, but does not cause a disease. In
addition, some
individuals survive the infection for more than 15 years, further suggesting
that the immune
response can control HIV-1 infection at least in some individuals (Id.). Live,
attenuated
viruses have been the most successful vaccines in monkey models of HIV-1
infection
(Berkhout et al., J. Virol. (1999) 73: 1138-45).
A number of infectious molecular clones from various HIV isolates have been
constructed (Srinivasan et al., Gene (1987); 52: 71-82; Sauermann et al., AIDS
Res. Hum.
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Retroviruses (1990) 6: 813-23; Collman et al., J. Virol. (1992) 66: 717-21),
thus providing
suitable substrates for using family DNA shuffling to achieve evolution of
attenuated
vaccine strains of HIV. Libraries of HIV viruses are generated by family
shuffling the entire
molecular clones or fragments thereof. Useful HIV regions for evolution by DNA
shuffling
are the genes encoding the Gag and Env structural proteins MA (matrix), CA
(capsid), NC
(nucleocapsid), p6, SU (surface), and TM (transmembrane); the Pol enzymes PR
(protease),
RT (reverse transcriptase), and IN (integrase); the gene regulatory proteins
Tat and Rev; and
the accessory proteins Nef, Vif, Vpr, and Vpu (Frankel and Young, Annu. Rev.
Biochem.
(1998) 67: 1-25; Turner and Summers, J. Mol. Biol. (1999) 285: 1-32). Nucleic
acids that
encode any of these proteins can be targeted by DNA shuffling. In addition,
combination
libraries can be generated by combining shuffled libraries of different viral
genes.
The shuffled libraries are screened for attenuation using in vitro and in vivo
methods. In vitro methods include, but are not limited to, selection of
strains with altered
host cell specificity, selection of temperature sensitive mutants and
selection of variants that
can enter the human host cells but do not replicate. In vivo methods include
selection of
variants that grow in animals that cannot be infected with the wild-type
virus. The
chimpanzee model provides an excellent in vivo system to address the degree of
attenuation
of the selected variants (Murthy et al., AIDS Res. Hum. Retroviruses (1998)
Suppl 3:S271-
6). Because chimpanzees can also be challenged with the live viruses resulting
in AIDS, the
model simultaneously provides an opportunity to address the efficacy of the
vaccines. The
chimpanzees that are immunized with the attenuated, shuffled vaccine strains
of HIV can be
challenged with various wild-type strains to analyze the level of protection
and
crossprotection. The clones demonstrating optimal level of attenuation with
efficient
protection against subsequent challenge can be chosen for additional rounds of
shuffling and
selection, if so desired.
Example 9
Evolution of Multivalent HPV Vaccines
Background
Mucosal/genital HPV is one of the most common sexually transmitted
diseases worldwide, with approximately 5.5 million new cases diagnosed per
year in the US
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alone. Estimates of up to 40 million infected individuals have been reported
in the United
States alone (Cancer Weekly Plus, June 29, 1998). Approximately 20 different
HPV types
infect the mucosal area, the majority of which are classified as "high risk"
HPV types
because of their association with over 90 % of cervical carcinomas (Bosch et
al., J. Natl.
Cancer Inst. 87:796-802 (1995)) and other ano-genital and oral malignancies.
Human
papillomavirus infections are the primary cause of cervical cancers (Bosch et
al, J. Natl.
Cancer Inst. 87:796-802, 1995) and have been also linked to anal, vaginal,
vulvar, oral and
cutaneous cancers. In the United States alone, 1 x,000 women a year are
diagnosed with
cervical cancer, resulting in 5,000 annual deaths (U.S. CDC). A summary of the
association
of different HPV types with human diseases is outlined is Table 2 (source: M.
Stanley,
Antiviral Research 24:1-1~, 1994).. It has been estimated that up to 50% of
sexually active
women are infected with these so-called 'high risk' HPVs. There are no
curative treatments
to date.
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Table 2
Skin
Common warts, hands and 1. 2. 4. (26), (27), 29.
feet etc. 57
Plane warts 3. 10, 28, (49)
Butchers' warts 7
Epidcrrnodysplasia verruciformis, 5, 8, 9, 12, 14, 15, 17,
bcnign 19, 20,
21, 22, 23, 24,25,36,4b,47,50
Epidermodysplasia verruciformis, 5, 8, 14, 17, 20
SCC
Keiatocanthoma 37
Malignant melanoma 38
Actinic kcratosis 5,8
SCC - 41 , (48)
Epidcrmoid cyst 60
Genitalia and mucous membranes
Normal cervix 16,53
Gcnital warts 6,11,44,54
Busclike Lowenstein tumours 6,11
Cervical intracphlielial 6,11, 16, 18, 30, 31, 33,
ncoplasia 34, 35, 39, 40, 42, (43),
(44), 45, 51, 52, 56, 57,
58, 66*
Cervical carcinoma 16, 18, 31, 33, 35, 39, 45,
51, 52, 56,.66*
Vulvar intracpithelial 16,18,43,59
neoplasia
Penile intracpithelial 16,18,39,40
ncoplasia
Bowen's disease 34
Bowenoid papulosis 16,39,55
Laryngeal papillomas 6,11
Laryngeal carcinoma 30
Focal epithelia] hyperplasia 13,32
(Ileck's)
Oral paoillomis 32
Papillomavirus are small, non-enveloped DNA virus with a circular genome
of 7.8 kb in size. Expression of the viral early and late proteins as well as
episomal
replication are regulated by a complex interaction of viral and host
transcription and
replication factors (Bernard and Apt, Arch. Dermatol. 130:210 (1994)). The
viral early
proteins E6 and E7 of "high risk" HPVs are powerful oncogenes and are
constitutively
expressed in cancer cells. They can thus serve as tumor antigens for
therapeutic vaccine
development. Vaccination of mice with recombinant E6 and E7 proteins has been
shown to
elicit a CTL mediated protective immune response against challenge with E6/E7
expressing
tumors (Hariharan et al, Int. J. Oncol., 12:1229-35 (1998)).
Two structural proteins, L1 and L2, form papillomavirus viruses, which are
expressed in the late viral life cycle. A virus is composed of 72 capsomers,
each of which is
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formed by 5 L 1 molecules. The ratio of the maj or capsid protein L 1 to the
minor capsid
protein L2 is estimated as 30:1. The inability of HPVs to productively grow in
cell culture
has severely hampered attempts to generate sufficient amounts of viruses for
experimental
vaccination. The discovery that papillomavirus Ll proteins have the intrinsic
capacity to
self assemble into viral like-particles (VLPs) when expressed in the absence
of other viral
gene products and epithelial differentiation (Kirnbauer et al., Proc. Nat'1.
Acad. Sci. USA 89:
12180-84 (1992)) was the technological breakthrough that has driven the recent
flurry of
prophylactic vaccine development. Papillomavirus VLPs were used in animal
models as
effective virus surrogates to induce protective immunity. They provide
essential
conformational epitopes without being infectious. There is no experimental
system for
testing prophylactic anti-HPV vaccines due to the species specificity of all
papillomaviruses.
However, experimental animal vaccinations have resulted in antibody-mediated
protection of
domestic rabbits, cows and dogs following infections with cotton-tail rabbit
papillomavirus,
bovine papillomavirus and canine oral papillomavirus, respectively (Lowy and
Schiller,
Biochim. Biophys. Acta, 1423: M1-8, 1998, and references herein). Post-
attachment
neutralization of PVs by antibodies could also be demonstrated.
Effective immune surveillance of human papillomavirus may be difficult to
achieve. Natural HPV infections at the genital mucosal surface are poorly
immunogenic,
presumably reflecting the non-lytic viral life cycle and the co-evolution of
the viruses with
their natural hosts. Individuals infected with HPVs are usually seropositive
both for the late
viral proteins and for the viral early oncogenes, but antibody titers are low.
For
epitheliotropic genital HPVs, which have no blood-borne phase in the viral
cycle, the
relevance of circulating antibodies will be restricted to their availability
on the mucosal
surface. The demonstration that systemic immunization with viral-like
particles (VLPs) in
African green monkeys induced neutralizing IgG in both sera and cervical-
vaginal secretions
makes it likely that VLP immunization will give similar results in humans
(Lowe et al, J.
Infect. Dis. 176: 1141-1145 (1997)). However, any enhancement of the elicited
immune
response will result in greater clinical benefit.
HPV pseudotype assays have shown that VLP based prophylactic vaccines
are type specific and do not mediate cross-protection (Roden et al., J.
Virol., 70: 587-3383
(1996)). Efficient prophylactic vaccines should therefore include antigenic
determinants
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from several HPV types. Recent experimental studies point to the minor capsid
protein L2 as
an alternative target for vaccine development. Immunization of mice with L2
proteins led to
the induction of neutralizing antibodies with some degree of cross-
neutralization. The
antibody titers were, however, very low compared to the titers induced by VLP
immunization.
Immunotrerapy may offer a novel and more effective means for both
prevention and treatment of HPV infection and associated diseases. The
majority of current
efforts in vaccine development are directed against two of the more prevalent
"high risk"
types, HPV-16 and HPV-18, which are most commonly found in malignant cancers.
However, in vztro neutralization assays have demonstrated that the immune
surveillance of
HPV is type specific (Roden et al, J. Virol. 70:5875-5883 (1996)). Eradication
of the rivo
major HPV types, HPV-16 and HPV-18 could, therefore, drive the evolution of
the large
number of serological distinct genotypes. Additionally, different HPV-type
specific variants,
co-evolved with human races, need to be considered in effective prophylactic
vaccine
1 ~ development. There is epidemiological evidence that minor variations
within HPV-types
may be more strongly associated with the risk of developing cancer (Xi et al,
J. Nat'1.
Cancer Inst. 89: 796-802 (1997)). Broadly protective HPV vaccines therefore
must be
multivalent.
Rational design of cross protective, multivalent VLP vaccines is extremely
difficult given the lack of knowledge concerning the structure, localization
and sequences
involved in the antigenic epitopes exposed on the virus surfaces. Furthermore,
direct
approaches to study HPV immune surveillance are not possible due to the lack
ef animal
models. Papillomavirus evolved with their hosts and are strictly species-
specific.
Evolution of Polwalent HPV Vaccines
In this Example, the challenges that have hampered development of HPV
vaccines are addressed by the use of molecular breeding by recursive rounds of
DNA family
shuffling and screening. Since shuffling does not require an understanding of
the mode or
mechanism of infection, but simply relies on a functional screen for desired
improvements, it
is the tool most likely to quickly yield a product of clinical and commercial
relevance.
Recombination of antigenic sequences from related "high risk" HPVs are used to
generate
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large pools of functionally diverse chimeric sequences from which the best are
selected
based on improved immunogenic and cross-reactive properties. For example, one
can
generate potent multivalent VLP vaccines by shuffling nucleic acids that
encode antigen
epitopes from different L 1 proteins and/or L2 genes to improve cross-
neutralizing epitopes
and antibody titers.
Nucleic acids that encode the antigens are used to generate complex, high-
quality antigen libraries that are screened with high throughput (HTP)
screening assays in
vitro and in vivo for the selection of superior cross protective antigens
against the major
"high risk" HPV types. An example of a suitable strategy is summarized in
Figure 3. The
naturally existing diversity of HPV virus antigens is combined to generate
complex antigen
libraries by DNA shuffling. HTP in vitro assay systems are then used for the
production of
VLPs and subsequent screening to enrich the libraries for antigenic epitope
display. An in
vivo antigen library screen with subsequent neutralization assays then allows
one to select
for broad-spectrum VLP vaccines.
A. Generation of chimeric LI anPigen libraries
First, different L1 genes are isolated from "high risk" HPVs and associated
variants to generate complex libraries. Papillomaviruses are a large family of
related viruses
with specific tropism for different epithelia. Based on sequence alignment of
the viral
genomes, papillomaviruses can be divided into several distinct groups. A
phylogenetic tree,
computed for 108 different papillomavirus L 1 genes, contains three
supergroups
(mucosal/genital, cutaneous/EV, and certain animal PVs) and 24 subgroups
(Figure 4). The
phylogenetic relationship is reflected by a similar tissue tropism (cutaneous,
mucosal/oral,
ano-genital) of the virus and the pathogenic lesions they induce (benign or
malignant
tumors). Eight "high risk" HPV types (HPV-18, 39, 45 and HPV-16, 31, 33, 35,
52) are
found in the majority of malignant cancers and cluster in two distinct
subgroups of the
phylogenetic tree (A7 and A9, Figure 4).
The phylogenetic distance between the two subgroups is, however, greater
than desired for successful DNA family shuffling. Therefore, two different
libraries are
generated, one for each subgroup. HPV-16 and HPV-18, which are associated with
80 % of
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the HPV-related cancers, are used as major templates and different amounts of
sequences
from related types are added to the shuffling reactions.
HPV-16 L1 genes are pooled with the closely related variants HPV-31, 33,
3~, 52 as well as different HPV-16 variants, and HPV-18 genes are pooled with
HPV-45 and
HPV-39. Pools of related Ll genes are subjected to random fragmentation and
subsequent
reassembly in a primerless PCR reaction according to established DNA family
shuffling
protocols as described herein (see also, Crameri et al., Nature 391: 288
(1998)). Additional
sequence heterogeneity can be added by spiking homologous sequences from more
distant
"high risk" HPV types (e.g., HPV-51, 56 and 66, subgroup A~ and A6, Figure 4)
into the
assembly reaction, in the form of short oligonucleotides with homologous ends.
Reassembled L 1 chimeras are amplified by PCR with primers flanking the L 1
genes and
subcloned into shuttle vectors, which allow for high throughput DNA
amplification in E. coli
and protein expression in mammalian cells. The complexity of the libraries is
estimated by
restriction analyses and sequencing of randomly selected clones. The
quantitative goal is to
gain large libraries (>105), with 90 - 100 % chimeric sequences. The same
experimental
strategy can be applied to the L2 genes.
B. Development of high throughput in vitro screening assays
The next step is to establish HTP assay systems for selection of L1 and L2
chimeras displaying antigenic epitopes. The ultimate goal is to select the
best L1 and L2
chimeras for their ability to induce broadly reactive antibodies in vivo. DNA
family
shuffling, however, can generate "wrongly" assembled genes, which give rise to
abortive
protein expression. Therefore, pre-screening the libraries in vitro for L 1
and L2 protein
expression and for ability to display immunogenic epitopes will help to avoid
unnecessary
animal studies during subsequent in vivo screening.
A general strategy of a high throughput in vitro assay is outlined in Figure
5.
Chimeric L 1 and L2 genes are transfected into mammalian cells for L T, L2
protein
expression and VLP assembly. Cell lysates are prepared from random clones to
check the
library quality by immunoblotting or simple plate ELISA assays. If the
libraries show a
higher than desired "knock out" rate, one can apply milder shuffling
conditions by using
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lower percentages of L 1 genes which are most distant from the main types, HPV
-16 and
HPV-18.
The in vitro screening assays generally involve the following components:
(i) For expression in mammalian cells and amplification in E. coli, the
shuffled L 1 and L2 genes are linked to a strong eukaryotic promoter and
cloned into plasmid
vectors containing a bacterial origin of replication and a drug selection
marker. For Ll, it is
particularly preferred to use a strong promoter, e.g., a CMV promoter or a
promoter that has
been improved using DNA shuffling. High expression of Ll is desirable to
obtain expression
levels that are sufficiently high for efficient VLP assembly.
(ii) Different mammalian cell lines are tested with different transfection
agents to optimize transfection efficiencies. An efficiency of 80-90 % is
desirable. Human
293 cells are often suitable. Transfection efficiency levels and promoter
strength can be
tested using a control plasmid (e.g., one that expresses GFP), which allows
rapid
fluorescence read outs. Random clones of the library are transfected into the
selected cell
line. ELISA or Western blotting is used to examine the cell lysates for L1 and
L2 protein
expression.
(iii) Preferred screening assays are based on immune recognition, for which
specific antibodies are needed. For some embodiments, it is sufficient for the
in vitro
screening assays to use antibodies against HPV-16 and HPV-18 Ll and L2
proteins, since
they are most prevalent in malignant cancers and selected chimeras should have
the property
to induce high levels of antibodies against these two types. Cross protection
of the antigens
against the related HPV types is tested in the final in vivo screening assays.
For the generation of polyclonal antisera against L1/L2 proteins and
conformational L1 VLP epitopes, the L1 and L2 genes of HPV-16 and HPV-18 are
cloned
into bacterial expression vectors (e.g., pET-3a) from which proteins can be
expressed in
quantitative amounts in appropriate bacterial strains (BL21/DE3, HMS174/DE3).
It has been
shown that HPV-L 1 proteins can reassemble into VLPs during the subsequent
protein
purification steps (Gripe et al., J. Virol. 71: 2988-2995 (1997)). Purified Ll
and L2 proteins
and sucrose gradient purified L1 VLPs can be used to inject rabbits or mice
for induction
antibodies against conformational antigen epitopes.
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C. HTP in vitro screening assay
To select and enrich for chimeric clones that give rise to L1/L2 proteins that
expose conformational antigenic epitopes, the libraries are subjected to
subsequent rounds of
screening in vitro. A schematic overview of the in vitro screening assay is
illustrated in
Figure 5. The libraries are transfected into mammalian cells for protein
expression and VLP
formation. For subsequent screening, Ll/L2 proteins and VLPs are preferably
purified from
the crude cell lysates. To achieve high throughput purification for the L 1
VLPs, one can use
expression vectors LI'~at direct the expression of the chimeric L1/L2 proteins
as fusions with a
heterologous antigenic epitope (e.g., a hexahistidine tag). The presence of
such heterologous
amino acid sequences does not hamper the self assembly of L 1 proteins into
VLPs and the
display of conformational antigenic epitopes (Peng et al., Virology 240:1800-
1805 (1998)).
Fusion of a hexahistidine tag to the C-terminus of the protein chimeras
provides an efficient
and fast way of protein purification in HTP plate assays. The hexahistidine
tag is uncharged
at physiological pH and rarely interferes with protein structure and function.
The His tag can
be linked to the C-terminal part of the shuffled chimeras by simply adding a
short sequence
coding for 6 histidine residues to the 3'-PCR primers used for the final
amplification of the
shuffled products.
Following amplification of the shuffled libraries in E. coli, plasmid DNA
from individual clones can be robotically prepared in a high throughput 96
well format.
Robotic plasmid purification protocols that allow purification of 600-800
plasmids per day
or more are feasible. The quantity and purity of the DNA can also be analyzed
on the plates.
The libraries are transfected into mammalian cells seeded in 96 well formats,
allowing for up
to 1000 individual transfections at a time. Crude lysates are prepared after
culturing the cells
for two days. The lysates are transferred to new 96 well plates coated with
nickel-
nitrilotriacetic acid (Ni-NTA HisSorb plates, Qiagen), to effciently
immobilize the 6xHis
tagged L 1 / L2 proteins and VLPs on the plates. The plates are incubated with
the anti-
HPV-16 and HPV-18 L1/L2 antibodies and the detection conjugate, and analyzed
by
automated plate read out. The HPV-16 and 18 wild type genes will serve as
positive
controls for the assay. HPV-16 and HPV-18 do not display cross-reactive
epitopes and can
be used as background control. The quantitative goal of the in vitro assay is
to select 1000
or more chimeras from each library for subsequent immune stimulation in mice.
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D. Alternative assays for YAP production and screening:
An alternative HTP assay can be set up by making use of the ability of VLPs
to package plasmid DNA of up to 10 kb in size (Stauffer et al., J. Mol. Biol.
263: 529-536
(1998)). A marker gene expressing a photon emitting protein (e.g., GFP, LacZ,
luciferase) is
cloned into the Ll expression plasmids, which are packaged during the VLP
assembly.
After VLP production in one cell line, all cells from a 96 well plate are
pooled, and VLPs
can be purified in a single reaction. Subsequent incubation of the VLPs with
mammalian
cells leads to marker transfer for L1 chimeras, which have the capacity to
assemble into
infectious VLPs. Cells expressing the marker gene can be easily monitored by
fluorescence
microscopy and plate ELISA, and selected by FACS sorting. The plasmid DNA can
be
purified by Hirt preparation, followed by amplification in E. coli. This
direct screening
assay is more stringent, but selection is only for VLPs which are able to
package DNA, so
variants that have lost the ability to package DNA but express strong
immunogenic epitopes
might be missed.
Other alternative transfection protocols and low-throughput (LTP)
chromatographic VLP purification steps (affinity chromatography, capillary
electrophoresis
or sucrose centrifugation) can also be used.
E. Analyses of the shuffled library in vivo
Pre-selected chimeric library clones from in vitro assays are used to
immunize mice. Two different routes of application can be envisioned: (1)
Injection of L2
proteins and purified L 1 VLPs, which has been successfully used in other
experimental
studies, and (2) naked DNA delivery, which offers the advantage of easy
commercial scale
vaccine manufacturing and non-invasive dermal application, specifically for
future clinical
applications. Naked DNA vaccinations have resulted in sufficient
conformational L 1 epitope
delivery and immune protection in an experimental rabbit model (Sundaram et
al., Vaccine
15: 664-671 (1997)).
One can initially conduct the in vivo screening experiments using dermal
naked DNA application. If this assay is not sufficiently sensitive, one can
express and purify
VLPs. L 1 proteins can be quantitatively expressed in, for example, E. coli
(or yeast),
assembled into VLPs in vitro and purified by sucrose/CsCI gradients for
injection. Using the
naked DNA delivery approach in the experimental system has the additional
advantage of
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enabling one to select concomitantly for L1 chimeras with the highest ability
to assemble
into VLPs in vivo.
Pooling plasmids or VLPs and deconvoluting in subsequent screening rounds
can reduce the number of small animals required to identify potent immunogens.
The lowest
concentration of plasmids or VLPs, which leads to induction of neutralizing
antibodies in
mice, are evaluated with the HPV-16 wild-type L1 and L2 plasmids or proteins.
Pools of 10
or 20 clones of the library can be used in a small number of experiments to
examine whether
the strategy of pooling and deconvolution in subsequent screening rounds is
feasible. If no
significant differences can be detected between different pools and the wild
type control,
single clones can be used for library immunization.
F. Neutralization assay and analyses of cross protective immunity
Sera from immunized mice is collected and tested for cross-neutralization
efficiency of the wild type VLPs in 96 well plate assays. Papillomavirus
Ll/VLPs retain the
ability of natural viruses to agglutinate mouse erythrocytes in culture and
antibodies raised
against VLPs can inhibit agglutination (Roden et al., J. Virol. 70: 3298-3201
(1996)).
Hemagglutination inhibition assays (HIA) provide therefore reliable and
sensitive surrogate
neutralization assays. VLPs from all wild-type high risk HPVs can be prepared
by
expression in E. coli and seeded together with the mouse erythrocytes in 96
well plates.
Collected sera will be added in serial dilutions to evaluate the
neutralization titers and cross-
neutralization ability. L1 and L2 chimeras from pooling experiments, which
induced
antibodies with improved neutralization titers compared to anti-wild type
antigens and are
cross reactive with other related wild type VLPs, can then be deconvoluted in
the next round
of in vivo screening. The improved characteristics of selected clones can be
confirmed in
direct neutralization assays using marker gene transfer as described above.
To further improve the quality of the novel antigens, a second round of
shuffling and screening is preferably applied to obtain the best variants.
Improved variants--
defmed as those inducing potent cross-reactive immunity against a broad range
of related
HPV--are reshuffled and screened. Shuffled chimeras can be backcrossed with
the wild type
genes to further improve the antibody titers. Backcrossing is performed by
shuffling the
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improved sequence with a large molar excess of the parental sequence and
provides a means
to breed the shuffled chimeras/mutants back to a parental or wild-type
sequence, while
retaining the mutations that are critical to the phenotype that provides cross-
protective
antibody responses. In addition to removing the neutral mutations, molecular
backcrossing
can also be used to characterize which of the many mutations in an improved
variant
contribute most to the phenotype. This cannot be accomplished in an efficient
library
fashion by any other method.
It is understood that the examples and embodiments described herein are for
illustrative purposes only and that various modificatior_s or changes in light
thereof will be
suggested to persons skilled in the art and are to be included within the
spirit and purview of
this application and scope of the appended claims. All publications, patents,
and patent
applications cited herein are hereby incorporated by reference for all
purposes.
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