Note: Descriptions are shown in the official language in which they were submitted.
CA 02499302 1996-11-07
QG/4
HackQrour~d of the Invention
Viral and bacterial diseases in fin-fish,
shellfish or other aquatic lifeforms pose a serious
problem for the aquaculture industry. Owing to the
S high density of animals in the hatchery tanks or
enclosed marine farming areas, infectious diseases may
eradicate a large proportion of the stock in, for
example, a fin-fish, shellfish, or other aquatic
lifefo~ms facility. Prevention of disease is a more
10 desired remedy to these threats to fish than
intervention once the disease is in progress.
Vaccination of fish is the only preventative method
which may offer long-term protection through immunity.
The fish immune system has many features
15 similar to the mammalian immune system, such as the
presence of B cells, T cells, lymphokines, complement,
and immunoglobulins. Fish have lymphocyte subclasses
with roles that appear similar in many respects to
those of the B and T cells of mammals. Additionally,
20 the efficiency of the immune response of fish can be
affected by outside stresses, as is true in mammals.
However, fish, unlike mammals, display a
temperature-dependent development of protective
immunity in response to antigens.
25 Most vaccines for fish have been developed
against bacteria while there have been very few fish
vaccines made for combating viral or parasitic
diseases. Fish have been immunized by antigen-based
CA 02499302 1996-11-07
- 2 -
immunization methods using live attenuated pathogens,
killed whole pathogens, or more recently, in laboratory
settings, recombinant proteins. While live attenuated
vaccines induce good humoral and cell-mediated immune
5 responses and can be administered orally or by
immersion or injection, there is the important risk of
reversion to a virulent form. Whole live attenuated
vaccines are not preferred in industrial farming due to
the risk of contaminating other fish - a live
10 attenuated vaccine which may be generally safe for the
target species of fish may be virulent in other species
of fish.
Fish vaccines using whole killed bacteria
(i.e. bacterins) or recombinant proteins from pathogens
15 expressed in cell lines (subunit vaccines) have the
disadvantage of inducing short-lived immune responses.
Injected antigen, including recombinant protein, is
processed solely in an exogenous form usually causing
induction of a humoral response (i.e., production of
20 antibodies) but often a failure to induce cell-mediated
immunity (i.e., cytotoxic T-cells).
Another disadvantage of whole killed and
subunit vaccines~'is that they almost always must be
injected and they require an adjuvant to induce an
25 effective immune response. Intramuscular injections of
these adjuvants can cause granuloma formation which
scars the flesh and lowers the market value of the
fish. Intraperitoneal injection of adjuvants may cause
adhesions between the viscera which can affect the
30 health of the fish and retard fish growth.
Recombinant protein vaccines are difficult
and expensive to make especially if the protein must be
purified. Far example, bacterially-expressed
recombinant proteins may form inclusion bodies from
r
CA 02499302 1996-11-07
- 3 -
which recovery of protein in correct configuration may
be low or nonexistent. Induction of an immune response
may require that the antigenic protein be correctly
glycosylated and folded, which may not be accomplished
5 in a cell other than an animal cell.
Some of the current methodologies for
administering vaccines are not technically or
economically practical. For example, direct injection
of recombinant and whole killed pathogen vaccines into
10 the fish is labor intensive and expensive relative to
the future market value of the fish. Furthermore,
injection needles can cross-infect fish with
contaminating pathogenic organisms, and accidental
injection of humans can cause severe or fatal
15 infections and anaphylactic reactions. Moreover,
noninjurious injection of small fish is very difficult,
especially in young fry, which are particularly
susceptible to disease.
A less expensive and easier method which has
20 been used to administer killed viral or bacterial
vaccines is an oral method wherein the vaccine is added
directly to the water or incorporated into fish food.
Oral vaccines have historically shown inconsistent and
relatively low levels of protection suggesting that
25 they may be best used as a method of revaccination.
Genes have been introduced directly into
animals by using live viral vectors containing
particular sequences from an adenovirus, an
adeno-associated virus, or a retrovirus genome. The
30 viral sequences allow the appropriate processing and
packaging of a gene into a virion, which can be
introduced to animals through invasive or non-invasive
infection. Viral vectors have several disadvantages.
Viral vectors being live pathogens, still carry the
CA 02499302 1996-11-07
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risk of inadvertent infection. Furthermore, proteins from
viral vector sequences induce undesirable inflammatory or
other immune responses which may prevent the possibility of
using the same vector fox a subsequent vaccine or boost.
Viral vectors also limit the size of the target gene that can
be expressed due to viral packaging constraints.
Naked DNA transfects relatively efficiently if
injected into skeletal muscle but poorly or not at all if
injected into other tissues (Wolff et al., Science 247;1465-
1468 (1990)). Plasmid DNA coated onto the surface of small
gold particles and introduced into the skin by a helium-
driven particle accelerator or "gene-gun" can directly
transfect cells of tree epidermis and dermis (Pecorino and Lo,
Current Biol., 2:30-32 (1992).
DNA has also been introduced into animal cells by
liposome-mediated gene transfer. DNA-liposome complexes,
usually containing a mixture of cationic and neutral lipids,
are injected into various tissues or instilled into the
respiratory passages. Nabel et al., Hum. Gene Ther., 3:649-
656 (1992) have shown that liposomes may be used to transfect
a wide variety of cell types by intravenous injection in
mammals. In addition, liposome-mediated gene transfer has
been used to transfer the cystic fibrosis transmembrane
conductance gene into the nasal epithelium of mice and humans
suffering from cystic fibrosis (Yoshimura et al., Nucleic
Acids ReQ., 12:3233-3240 (1992)) and Caplan et al., Nature
Med. 1:39-46 (1995), respectively.
Substances may also be administered using
CA 02499302 1996-11-07
- 5 -
biodegradable microspheres composed of polymers such as
polyester poly(lactide~-co-glycolide) (Marx et al., Science,
260:1323-1328 (1993)). It is notable that these particles
can survive the upper digestive system and arrive intact in
cells of gut-associated lymphoid tissue (Eldridge et al.,
Adv. Ex~. Med. Biol., 251:191-202 (1989). Biodegradable
microspheres have been used to deliver recombinant antigens,
toxoids or attenuated virus into mammals by systemic and oral
routes (O'Hagan et al., Immunology 73:239-242 (1991); O'Hagan
et al., Vaccine 11:149-154 (1993); Eldridge et al., Mol.
Immunol. 228:287-293 (1991)). They may also be useful to
deliver recombinant p7_asmid DNA to gut-associated lymphoid
tissue for the purposE~ of immunization.
While most work has been carried out on mammals,
plasmid DNA encoding reporter genes have been successfully
introduced into fish by intramuscular injection (Hansen, et
al., FEBS Lett. 290:7:3-76 (1991). Thus, cells in fish can
express proteins from a foreign gene with the same types of
vector constructs (i.e., backbones, promoter and enhancer
elements) that are used in mammals.
The induction of an immune response to a protein
expressed from an introduced gene was first suggested by
Acsadi et al., New Biologist 3:71-81 (1991), who found that
after plasmid DNA transfer into rat cardiac muscle, reporter
gene expression was transient but could be prolonged by
treatment with an immunosuppressant. Subsequently, it was
shown that antibodies were induced in rodents against human
growth hormone (Tang et al., Nature, 356:152-154 (1992);
CA 02499302 1996-11-07
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Eisenbraun et al., DNA Cell. Biol., 12:791-797 (1993)), or
human a-antitrypsin (Tang et al., Nature, 356:152-154
(1992)), when the proteins were expressed from DNA coated
onto gold particles and introduced into cells of the skin by
bombardment.
DNA-based immunization refers to the induction of
an immune response to an antigen expressed in vivo from a
gene introduced into the animal. This method offers two
major advantages over classical vaccination in which some
form of the antigen itself is administered. First, the
synthesis of antigen in a self-cell mimics in certain
respects an infection and thus induces a complete immune
response but carries absolutely no risk of infection.
Second, foreign gene expression may continue for a sufficient
length of time to induce strong and sustained immune
responses without boost.
Several mammalian animal models of DNA-based
immunization against specific viral, bacterial or parasitic
diseases have been reported. These include influenza [(Fynan
et al., Proc. Nat'1 Acad. Sci. USA, 90:11478-11482 (1993);
Montgomery et al., DNA Cell. Biol., 12:777-783 (1993);
Robinson et al., Vaccine, 11:957-960 (1993); Ulmer et al.,
Science, 259:1745-1749 (1993)], HIV [Wang et al. (1993)],
hepatitis B [Davis et al., Hum. Molec. Genet., 2:1847-1851
(1993)], malaria [Sed.agah et al., Proc. Nat'1 Acad. Sci.,
USA, 91:9866-9870 (1994)], bovine herpes [Cox et al.,
J.Virol, 67:5664-5667 (1993)], herpes simplex [Rousse et al.,
J. Virol., 68:5685-5689 (1994); Manicken et al. J. Immunol.,
CA 02499302 1996-11-07
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155:259-265 (1995)], rabies [Xiang et al., Virolocry, 199:132-
140 (1994)]; lymphocytic choriomeningitis [Yokoyama et al.,
J. Virol., 6964:2684-2688 (1995)] and tuberculosis [Lowrie et
al., Vaccine, 12:1537-1540 (1994)]. In most of these studies
a full-range of immune responses including antibodies,
cytotoxic T lymphocytes (CTL), T-cell help and (where
evaluation was possible) protection against challenge was
obtained. In these studies naked DNA was introduced by
intramuscular or intradermal injection with a needle and
syringe or by instillation in the nasal passages, or the
naked DNA was coated onto gold particles which were
introduced by a particle accelerator into the skin.
There is a need for novel systems to vaccinate fin-
fish, shellfish, and other aquatic animals against disease.
These systems should :be inexpensive to produce and
administer, avoid the use of live, attenuated organisms, and
induce strong and long-lasting immunity preferably without
boost and with induction of both antibodies and cell-mediated
immunity. More preferably, the system should be applicable
to small fish, be less stressful to fish during
administration, and have the capacity of simultaneously
immunizing many animals for reduced labor-related costs.
Surmnarv of the Invention
The present invention relates to the immunization
of cultured fin-fish, shellfish, or other aquatic animals
("aquaculture species") by DNA expression systems to overcome
many disadvantages associated with antigen-based vaccines.
The present invention. relates to introduction of DNA plasmids
CA 02499302 1996-11-07
(alone or in a formulation) containing sequences encoding
antigenic components of viral, bacterial or parasitic
diseases by transfection into aquaculture species. The
methods and compositions of this invention are useful for
immunization (i.e., far prophylactic vaccination or
therapeutic immunization) of fin-fish, shellfish or other
aquatic animals against infectious diseases. The DNA
sequences according to this invention are preferably present
in vectors capable of inducing protein expression of these
sequences (i.e., expreasion vectors) and may be administered
alone or in combination with other DNA sequences in the same
or other expression vectors or as oligonucleotides. These
additional DNA sequences may encode cytokines, costimulatory
molecules, or may include immunostimulatory sequences (e. g.,
CpG motifs). The DNA sequences may also be given with other
adjuvants, such as alum.
The present invention also relates to methods of
administration of DNA expression vectors to aquaculture
species, which may or may not encode polypeptides from
pathogens. DNA vectors of this invention may be administered
to aquaculture species by oral route, injection, spray, or
immersion. In a preferred embodiment, the DNA expression
vectors of this invention are administered by immersion
techniques or automated injection devices.
More specifically, the present invention provides a
composition for inducing a protective immune response in
aquaculture species, comprising:
an expression vector having an expression control
CA 02499302 1996-11-07
8a
sequence capable of directing expression in said aquaculture
species of at least one immunogenic polypeptide and a
polypeptide-encoding DNA sequence encoding at least one
immunogenic polypeptide from the genome of a pathogen,
together with a pharmaceutically acceptable carrier.
The present invention also provides a method of
immunizing an aquaculture species against infection from
pathogens, comprising administering to the aquaculture
species a composition for inducing a protective immune
response for aquaculture species comprising an expression
vector having an expression control sequence capable of
directing expression :in said aquaculture species of an
immunogenic polypeptide and a polypeptide-encoding DNA
sequence encoding an :immunogenic polypeptide from the genome
of a pathogen.
The present invention also provides a method for
expressing a polypeptide in an aquaculture species
comprising administering to an aquatic animal a DNA
expression vector comprising an expression control sequence
capable of directing expression in said aquaculture species
of a polypeptide and a polypeptide-encoding DNA sequence
encoding at least one polypeptide.
In another aspect, the invention provides use, for
inducing a protective immune response in cultured fin-fish
by injection, of a composition comprising: an expression
vector comprising an expression control sequence capable of
directing expression in said aquaculture species of at least
one immunogenic polypeptide and a polypeptide-encoding DNA
sequence encoding at least one immunogenic polypeptide
selected from the group consisting of the VP3 protein of
infectious pancreatic necrosis virus (IPNV) and glycoprotein
CA 02499302 1996-11-07
51440-9D
- 8b -
(G) of viral haemorrhagic septicemia virus (VHSV), together
with a pharmaceutically acceptable carrier.
In another aspect, the invention provides use, in
the manufacture of a medicament for inducing a protective
immune response in cultured fin-fish by injection, of an
expression vector comprising an expression control sequence
capable of directing expression in said aquaculture species
of at least one immunogenic polypeptide and a polypeptide-
encoding DNA sequenca_ encoding at least one immunogenic
polypeptide selected from the group consisting of the vP3
protein of infectious pancreatic necrosis virus (IPNV) and
glycoprotein (G) of ,;riral haemorrhagic septicemia virus
(VHSV).
In another aspect, the invention provides a
composition as described above, for use for inducing a
protective immune response in cultured fin-fish by
injection.
In another aspect, the invention provides a kit
comprising a composition as described above, and
instructions for use of the composition for inducing a
protective immune response in cultured fin-fish by
injection.
In another aspect, the invention provides a method
for expressing a pol:ypeptide in a fin-fish comprising
immersing the fin-fish in a DNA plasmid solution encoding
the polypeptide.
In another aspect, the invention provides a
composition for use in expressing a polypeptide in a fin-
fish by immersion of the fin-fish in the composition,
wherein the composition comprises a DNA plasmid solution
encoding the polypeptide.
CA 02499302 1996-11-07
51440-9D
- 8c -
In another aspect, the invention provides use of
plasmid DNA encoding an antigenic polypeptide for the
preparation of a med_Lcament for immunizing by immersing fin-
fish.
Description of the Invention
The present, invention provides for methods and
compositions for immunizing cultured'fin-fish.
CA 02499302 1996-11-07
g _
shellfish, and other aquatic animals against infection
by viral, bacterial or parasitic pathogens. In basic
outline, DNA encoding a polypeptide component of a
pathogen is introduced into an animal, and the
5 polypeptide is expressed in cells of the animal, thus
inducing an immune response that confers protection
against natural infection by the pathogen or helps
overcome an cngcing and possibly chronic infection.
In a preferred embodiment, the present
10 invention provides a method for immunizing cultured
fin-fish, shellfish, or other aquatic animals against
disease, comprising immersion of the animals in an
aqueous solution containing formulated plasmid DNA
encoding one or more antigenic determinants of an
15 infectious agent (regardless of codon usage), whereby
the DNA enters cells of the animal where it is
expressed leading to induction of immune responses.
The immunization procedure may be prophylactic to
prevent infecaion from occurring or may be therapeutic
20 to treat pre-existing infections.
Few anti-viral vaccines have been marketed
for fish. This is largely due to the difficulty of
growing virus in culture for the production of whole
killed viral vaccines or safe attenuated strains of
25 virus. Antigen-based vaccines using purified
recombinant proteins are difficult and Expensive to
produce in large scale and may have poor immunogenicity
in fish.
DNA-based immunization has several
30 advantages. The antigenic protein is synthesized in
vivo giving rise to both humoral and cell-mediated
(cytotoxic T lymphocytes) immune responses. However,
unlike live attenuated pathogens, which also synthesize
protein in v:ivo, DNA vaccines carry no risk of
CA 02499302 1996-11-07
- 10 -
inadvertent infection. Unlike antigen-based
immunization, DNA-based vaccination does not require
the use of traditional adjuvants to generate an
effective immune response. Furthermore, DNA used in
the methods of this invention is inexpensive and easy
to manufacture and purify.
DNA-based immunization also allows the host
animal to produce foreign antigens within its own
tissue thereby resulting in several advantages. One
10 advantage is the efficient presentation of the foreign
antigen to the immune system due to the expression of a
protein within a self-cell, which could be an
antigen-presenting cell. Another advantage is the
correct folding, protein modification, and disulfide
15 bonding of a protein expressed in a host cell,
especially fo:r viral proteins, which are normally
produced in cells of hosts. Recombinant viral proteins
synthesized in bacterial or yeast cells may be
incorrectly post-translationally modified and are often
20 massed in inclusion bodies, which make the proteins
difficult to purify or ineffective if administered in
unpurified form.
Immune responses in fish are temperature
dependent. Antigen-based vaccines may give rise to
25 sub-optimal immune responses if such vaccines are given
at the wrong temperature. DNA-based immunization is
advantageous because expression of the antigenic
protein could continue over a long period until such
time as to stimulate an immune response when the
30 temperature is optimal.
Another advantage of prolonged synthesis of
antigen is the induction of immune responses as soon as
the immune system is mature. Fish may be unable to
induce sufficient immune responses at a young age. For
CA 02499302 1996-11-07
- 11 -
example, trout and halibut may not produce lymphoid
cells until as late as ten and thirty days after
hatching, respectively, and T-dependent immune
responses do not appear until months after hatching.
5 Using the methods of this invention, expression of
foreign protein in fish can continue at least four
months after transfection indicating that DNA-based
immunization may be preferred for vaccination of young
fish.
10 The term "vaccine" herein refers to a
material capable of producing an immune response. A
vaccine according to this invention would produce
immunity against disease in cultured fin-fish,
shellfish and other aquatic species. One of skill in
15 the art would readily appreciate that activation of CTL
activity resulting from in vivo synthesis of antigen
would produce immunity against disease not only
prophylactically but also therapeutically (after
development of disease in culture).
20 Aquaculture species treated by methods of
this invention will include a diversity of species of
cultured fin-fish, shellfish, and other aquatic
animals. Fin-fish include all vertebrate fish, which
may be bony or cartilaginous fish. A preferred
25 embodiment of this invention is the immunization of
fin-fish. These fin-fish include but are not limited
to salmonids, carp, catfish, yellowtail, seabream, and
seabass. Salmonids are a family of fin-fish which
include trout (including rainbow trout), salmon, and
30 Arctic char. Examples of shellfish include, but are
not~limited to, clams, lobster, shrimp, crab, and
oysters. Other cultured aquatic animals include, but
are not limited to eels, squid, and octopi.
CA 02499302 1996-11-07
- 12 -
Purification of DNA on a large scale may be
accomplished by anion exchange chromatography (for
example, resins manufactured by Qiagen, U.S. FDA Drug
Master File (DMF-6224)).
S DNA which is introduced to aquaculture
species will encode foreign polypeptides te.g., those
derived from viral, bacterial or parasitic pathogens).
Polypeptides of this invention refer to complete
proteins or fragments thereof, including peptides which
10 are epitopes ~e.g., a CTL epitope) associated with an
infectious virus, bacterium or parasite.
DNA sequences encoding a complete or large
parts of an antigenic protein are preferred where
humoral immunity is desired rather than DNA sequences
15 encoding smaller parts, such as only CTL epitopes, as
are preferred where cell-mediated immunity is desired
and humoral immunity may be deleterious. In preferred
embodiments, the DNA sequences encoding polypeptides of
viral pathogens may be selected from the group
20 consisting of glycoprotein (G) or nucleoprotein (N) of
viral hemorrhagic septicemia virus (VHSV); G or N
proteins of infectious hematopoietic necrosis virus
(IHNV) : VPI, VP2, VP3 or N structural proteins of
infectious pancreatic necrosis virus (IPNV); ~ protein
25 of spring viremia of carp (SVC); and a
membrane-associated protein, tegumin or capsid protein
or glycoprotein of channel catfish virus (CCV).
In other preferred embodiments, the DNA
sequences encoding polypeptides of bacterial pathogens
30 may be selected from the group consisting of an
iron-regulated outer membrane protein, (IROMP), an
outer membrane protein (OMP), and an A-protein of
Aeromonis salmonicida which causes furunculosis, p5~
CA 02499302 1996-11-07
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protein of Renibacterium salmoninarum which causes
bacterial kidney disease (8KD), major surface
associated antigen (msa), a surface expressed cytotoxin
(mpr), a surface expressed hemolysin (ish), and a
5 flagellar antigen of Yersiniosis: an extracellular
protein (ECP), an iron-regulated outer membrane protein
(IROMP), and a structural protein of Pasteurellosis: an
OMP and a flagellar protein of Vibrosis anguillarum and
V. ordalii; a flagellar protein, an OMP protein, aroA,
10 and purA of Edwardsiellosis ictaluri and E. tarda~ and
surface antigen of Ichthyophthirius: and a structural
and regulatory protein of Cytophaga columnari; and a
structural and regulatory protein of Rickettsia.
In yet another preferred embodiment, the DNA
15 sequences encoding polypeptides of a parasitic pathogen
may be selected from one of the surface antigens of
Ich thyoph thiri us .
The methods of this invention could also be
used to introduce plasmid vectors encoding polypeptides
20 endogenous to the animal, but which might be normally
present in low concentrations (e. g., growth hormones).
In this case the expression proteins would serve a
. physiological role (i.e. enhanced growth) rather than
induce an immune response.
25 Vectors useful in the making of expression
plasmids include, but are not limited to, vectors
containing constitutive promoters, inducible promoters,
tissue-specific promoters, or promoters from the gene
of the antigen being expressed. Constitutive promoters
30 may include strong viral promoters, for example,
promoter sequences from cytomegalovirus (CMV), Rous
sarcoma virus (RSV), simian virus-40 (SV40), ~or herpes
simplex virus (HSV). Tissue-specific promote-rs may
CA 02499302 1996-11-07
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include the muscle beta-actin promoter or the thymidine
kinase promoter. An inducible or regulatable promoter,
for example, may include a growth hormone regulatable
promoter, a promoter under the control of 1ac operon
5 sequences or an antibiotic inducible promoter or a
Zinc-inducible metallothionein promoter.
The vector should include an expression
control sequence comprising a promoter (e. g., inducible
or constitutive promoters described above) DNA
10 sequence, and rnay include, but is not limited to, an
enhancer element, an RNA processing sequence such as an
intronic sequence for splicing of a transcript or a
polyadenylation signal (e.g., from simian virus-40
(SV40) or bovine growth hormone (BGH)), a signal
15 sequence for secretion of the expressed protein, or one
or more copies of immunostimulatory DNA sequences known
as CpG motifs. The vector should also include one or
more of the following DNA sequences: bacterial origin
of replication sequences, a selectable marker, which
20 may be for antibiotic resistance (e.g., kanamycin) or
for non-antibiotic resistance (e. g., ~i-galactosidase
gene ) .
Oligonucleotides having unmethylated CpG
dinucleotides have been shown to activate the immune
25 system (A. Krieg, et al., "CpG motifs in Bacterial DNA
Trigger Directed B Cell Activation" nature ~:54fi-549
(1995)). Depending on the flanking sequences, certain
CpG motifs may be more immunostimulatory for B cell or
T cell responses, and preferentially stimulate certain
30 species. Copies of CpG motifs in DNA expression
vectors act as adjuvants facilitating the induction of
an immune response against an expressed protein. A CpG
motif, a stretch of DNA containing CpG dinucl.eotides
CA 02499302 1996-11-07
- 15 -
within a specified sequence, may be as short as 5-40
base pairs in length. Multiple CpG motifs may be
inserted into the non-coding region of the expression
vector. When a humoral response is desired, preferred
5 CpG motifs will be those that preferentially stimulate
a B cell response. When cell-mediated immunity is
desired, preferred CpG motifs will be those that
stimulate secretion of cytokines known to facilitate a
CD8+ T cell response.
10 Other CpG motifs have be found to inhibit
immune responses. In a preferred embodiment of the
application, these immunoinhibitory CpG motifs would be
removed or mutated in a DNA expression vector used by
the methods of this invention, without disrupting the
15 expression of polypeptides therefrom.
An additional preferred embodiment of this
invention relates to the administration of a vector
containing one or more different DNA sequences, one
sequence encoding an antigen and the others encoding
20 polypeptides which may or may not be antigenic. For
example, the vector may encode two antigens from the
same pathogen. Alternatively, the different antigens)
may induce an immune response against a different
pathogen and thus serve as a multivalent vaccine.
25 Alternatively, the other polypeptides may serve to
enhance an immune response against a targeted pathogen
(e. g., helper epitopes, cytokines, carrier
polypeptides, cholera toxin subunits, or other
immunostimulants).
30 When two or more polypeptide-encoding DNA
sequences are present in one vector, the transcription
of each antigen-encoding DNA sequence may be directed
from its own promoter. Alternatively, one promoter may
drive the expression of two or more antigen-encoding
CA 02499302 1996-11-07
- 16 -
DNA sequences joined in frame to each other to express
a fusion protein. For example. VP2 and VP3 proteins of
infectious pancreatic necrosis virus (IPNV) may be
fused. In another embodiment, DNA sequences encoding
5 two or more antigens from different diseases may be
joined to form a multivalent vaccine when expressed.
Alternatively, a DNA sequence encoding an
antigenic polypeptide may be fused to a DNA sequence
encoding a carrier polypeptide. In a preferred
10 embodiment, the carrier polypeptide may contain one or
more envelope proteins of the hepatitis B virus,
preferably from the human hepatitis B virus. In a more
preferred embodiment, the envelope proteins of
hepatitis B virus will be the small and major protein
15 (also referred to as surface antigen).
In another embodiment, each
polypeptide-encoding DNA sequence in the vector may be
under the control of its own promoter for expression of
two or more non-fused polypeptides.
20 Alternatively, the DNA sequences encoding
additional antigens may be administered by using a
second vector containing such sequences. Such
sequences may encode antigens from the same pathogen or
different pathogens, or cytokines, cholera toxin
25 subunits, or ather immunostimulants. Such a vector may
be administered concurrently or sequentially with the
first expression vector. A preferred embodiment of
this invention is the concurrent administration of
expression vectors. One vector may be induced to
30 express protein simultaneously with or after expression
of protein from the other vector.
In vet another embodiment of this invention,
antigen-expressing vectors may be administered
concurrently with an antigen-based vaccine such as a
CA 02499302 1996-11-07
- 1 I -
recombinant protein or whole-killed vaccine. In a
preferred embodiment, the antigen-expressing vector is
administered simultaneously with a protein antigen
(i.e. recombinant protein or whole killed pathogen).
S Prother preferred embodiment would be to first
ad.'ninister a DNA vaccine to prime the immune response
followed by administration of the protein antigen ~wo
to eight weeks later, preferably orally o._- by
i.~uners ion, to boos t the iaunune response .
10 The DNA used in the met~~.od of t'~is invention
is preferably purified plasmid DNA(s) simply dissolved
in an acueous solution or in a formulation. One o.
skill in the art would readily appreciate how to
formulate DNA used in the methods of this invention
15 with known transfection reagents such as cationic
liposomes, fluorocarbon emulsions, cochleates, tubules,
gold particles, biodegradable microspheres, or catior_ic
polymers.
Li~osomes useful for transfection of DNA of
20 this invention include commercially available liposomes
and liposomes containing either cationic lipids or
cationic polymers. In a preferred embodiment of this
invention, liposomes would include a mixture of a
neutral lipid such as dioleoylphosphatid~_rlethanolamize
25 (DOPE) or cholesterol and a cationic lipid.
In a more preferred aspect of the invention,
liposomes would include a mixture of cat_onic polymers
and neutral lipids such as DOPE or cholesterol. Suez
liposomes may be prepared as described herein, unlike
30 cationic lipids, cationic polymers do
CA 02499302 1996-11-07
- 16 -
not have ester-linkages and have greater stability in
vivo as a result. Cationic polymers (also referred to
as dendrimers) may be dimeric, cyclic, oligomeric, or
polymeric in structure.
5 Cationic polymers in an aqueous solution
without neutral lipids are also preferred transfection
reagents according to the preferred embodiments of this
invention. Cationic polymers have been shown to work
well for transfecting fish cells in vitro with plasmids
expressing fish pathogen antigens (see Table 1, Example
1) .
Cochleates, which are stable
phospholipid-calcium precipitates composed of
phosphati~dylserine, cholesterol and calcium are
15 desirable non-toxic and non-inflammatory transfection
reagents that can survive the digestive system.
Biodegradable microspheres composed of polymers such as
polyester poly(lactide-co-glycolide) have been used to
microencapsulate DNA for transfection.
20 Tubules have been previously described in the
literature as lipid-based microcylinders consisting of
helically wrapped bilayers of lipid, the edges of which
are packed together. DNA may be placed in the hollow
center for delivery and controlled release in animals.
25 With immersion, DNA may enter cells of the
epithelium of the skin, the gills or the gut wall.
With injection, DNA may enter muscle cells or other
cells in muscle tissue (e. g. fibroblasts. immune cells)
or cells of viscera within the intraperit~oneal cavity.
30 DNA may then be expressed in these transfected cells
leading to induction of appropriate immune responses in
regional or systemic lymphoid tissue.
The invention provides for pharmaceutical
compositions comprising DNA vaccines in an amount
CA 02499302 1996-11-07
- Z9 _
effective for the treatment and prevention of diseases
caused by pathogens of aquaculture species. According
to another embodiment, the pharmaceutical compositions
of this invention further comprise a second DNA
5 vaccine, an adjuvant, a recombinant protein, a
transfection reagent, or some combination thereof.
Methods of this invention may be useful in
the immunization of aquaculture species against many
pathogens. much pathogens include but are not limited
10 to hemmorrhagic septicemia virus, infectious
hematopoietic necrosis virus, infectious pancreatic
necrosis virus, virus causing spring viremia of carp,
channel catfish virus (Herpesvirus ictaluri), grass
carp hemorrhagic virus, nodaviridae such as nervous
15 necrosis virus or striped jack nervous necrosis virus,
infectious salmon anaemia virus, Aeromonis salmonicida,
Renibacterium salmoninarum, Yersinia, Pasteurella
(including piscicida), Vibrosis (including anguillarum
and ordalii), Edwardsiella (including ictaluri and
20 tarda), Streptococci, and Ichthyophthirius.
In one embodiment of this invention,
recombinant plasmid DNA is introduced into animals
orally. DNA for oral use may be formulated with
biodegradable microspheres, fluorocarbon emulsions,
25 cochleates, or tubules. This is a non-stressful method
of immunizing aquaculture species by which DNA may be
coated onto or milled into feed in the form of a paste
or liquid suspension or incorporated into gelatin
capsules and introduced into the environment of the
30 aquaculture species. Preparations of DNA for oral use
may include lactose and corn starch. The DNA can be
used with or without products to enhance entry into
CA 02499302 1996-11-07
- 20 -
cells of the gut epithelium or more deeply situated
cells.
In another embodiment, pure recombinant
plasmid DNA is introduced into animals by injection
5 with a needle or a jet-injection system, which does not
have a needle. Injection areas of the fin-fish include
but are not limited to intraperitoneal, intramuscular,
and subcutaneous areas of the fish. In a preferred
embodiment, large fin-fish are immunized by injection
10 methods of this invention. Typically, fish are
injected with 0.1-0.5 ml of a solution containing DNA.
DNA may be injected in a pure form or may be formulated
with liposomes, cationic polymers, fluorocarbon
emulsions, cochleates, or tubules.
15 In yet another embodiment of this invention,
pure DNA is introduced into a fin-fish by particle
bombardment. This method introduces DNA-coated gold
particles into the epidermis of a fin-fish using a
"gene-gun", which uses compressed helium to shoot the
20 gold particles at high speed into the skin. This
method has been shown to be particularly efficient for
induction of cell-mediated immune responses with small
quantities of DNA in mice.
In another embodiment of this invention,
25 plasmid DNA is introduced to fish by spray. Typically,
fish are exposed to spray for at least 2 seconds.
Fish may pass through a mist of DNA solution by forcing
the vaccine through high-pressure paint-sprayer-type
nozzles. Typically, any pressure up to 90 psi is
30 satisfactory. Due to the number of pounds of fish per
unit volume that can be vaccinated by spray, it may be
more economical to immunize larger fish by this method
than by immersion. The DNA can be used with or without
CA 02499302 1996-11-07
- 21 -
products to enhance entry into cells of the skin. For
example, the DNA may be associated with liposomes or
cationic polymers.
In a more preferred embodiment of this
5 invention, a large number of animals can be immunized
simultaneously by immersion in a solution containing
DNA. In one embodiment, fish are dip-netted into
suspensions containing DNA formulations (e.g., DNA
formulated with cationic polymers or liposomes) for at
10 least several seconds. The fish are then returned to
the holding tanks in which they develop immunity. In
another embodiment, fin-fish, shellfish, or other
aquatic animals are placed into tanks containing a
relatively small volume of water. Concentrated DNA
15 formulations (e. g., DNA formulat;:d with cationic
polymers or liposomes) is added to the tank, and
animals are left for a period of time up to several
hours before the tank is refilled with water to restore
the normal aquatic environment. This method of
20 immersion is preferred for the immunization of small
fry, which cannot be immunized by direct injection.
The amount of the expression plasmid DNA that
may be combined with a carrier material to produce a
single dosage form will vary depending upon the host
25 treated, and the particular mode of administration. It
should be understood, however, that a specific dosage
and treatment regimen for any particular fish will
depend upon a variety of factors, including the
expression of the particular plasmid DNA employed, the
30 stability and activity of the particular protein or
peptide expressed. age, body weight, general health,
species of fish, the progress of the disease being
treated, and nature of the disease being immunized
against or dreaded. The amount of expression plasmid
CA 02499302 1996-11-07
- 22 -
DNA may also depend upon whether other therapeutic or
prophylactic agents including additional expression
plasmid DNAs and adjuvants, if any, are co-administered
with the expression plasmid.
5 Without being bound by the values listed
below, dose ranges for the administration of DNA used
in the methods of this invention may be generalized as
follows. For immunization of fish via oral routes, 0.1
to 50 ug DNA per fish administered over several
10 consecutive days may be used. For DNA-based
immunization by intramuscular or intraperitoneal
injection, O.:L to 10 ug of DNA may be used. For spray
immunization, a volume of 1 ml per fish of 0.1 to 10
mg/ml DNA solution may be useful. Fish immunized by
15 immersion methods of this invention may be incubated in
a 1 to 100 ug/ml DNA solution at a volume sufficient
for fish to survive for a time period necessary for
uptake of DNA to produce an immune response by the
fish. An effective dosage range for immunization of
20 fish via gene-gun route may be 10 ng to 1 ug.
Adjuvants for immunization are well known in
the art and suitable adjuvants can be combined with the
DNA sequences described herein by a person skilled in
the art to form a pharmaceutical composition. Oil
25 adjuvants are least desirable for the methods of this
invention because they create undesirable side-effects
such as visceral adhesions (which can restrict growth)
and melanized granuloma formations (which can lower the
grade of the fish at market) and because they cannot
30 form a homogeneous mixture with DNA preparations.
DNA-based immunization does not require oil adjuvants
and thus avoids these undesirable effects.
Adjuvants used in immunization with DNA
expression plasmids of this invention may include alum
CA 02499302 1996-11-07
- 23 -
or a DNA molecule having unmethylated CpG dinucleotides
therein (also referred to as CpG adjuvant).
Oligonucleotides having unmethylated CpG dinucleotides
have been shown to activate the immune system (A.
5 Krieg, et al., "CpG motifs in Bacterial DNA Trigger
Directed H Cell Activation" Nature ~7 ,:546-549
(1995)). CpG motifs may be inserted into a plasmid DNA
vaccine vector, and replicated in bacteria thereby
allowing the CpG motifs to retain their unmethylated
10 form. As such, administration of a CpG adjuvant cloned
into plasmid vectors would be simultaneous with the
administration of a plasmid DNA vaccine.
Alternatively, a CpG adjuvant in the form of free
oligonucleotides may be administered before, during or
15 after the administration of a plasmid DNA vaccine.
Oligonucleotides having CpG motifs may be
optionally modified at their phosphodiester linkages
for stability purposes. Such modifications are well
known by those of skill in the art. For example,
20 phosphodiester bonds in an oligonucleotide may be
replaced by phosphorothioate linkages.
The present invention also includes
pharmaceutical products for all of the uses
contemplated in the methods described herein. For
25 example, a pharmaceutical product comprising pure
plasmid DNA vector or formulations thereof, operatively
coding for an immunogenic polypeptide or peptide, may
be prepared in physiologically acceptable administrable
form (e.g., saline). The pharmaceutical product may be
30 placed in a container, with a notice associated with
the container in the form prescribed by a governmental
agency regulating the manufacture, use or sale of
pharmaceuticals, which notice is reflective of approval
by the agency of the form of the DNA for veterinary
CA 02499302 1996-11-07
- 24 -
administration. Such notice, for example, may be
labeling approved by the Biologics Division of
Agriculture and Agri-Food Canada or the United States
Department of Agriculture (USDA) or the approved
5 product insert.
In order that this invention may be more
fully understood, the following examples are set forth.
These examples are for the purpose of illustration only
and are not ~o be construed as limiting the scope of
10 the invention in anyway.
xam8lo 1 - Toning of DNA encoding Antigenic Proteins
Tntn Plasmid DNA Vectors
DNA encoding proteins of fish pathogens is
15 useful in developing DNA fish vaccines. Table 1 below
recites fish pathogen protein expression plasmids.
Table 1 describes nucleotide sequences encoding
proteins from pathogens cloned into vectors having the
cytomegalovirus promoter (CMV), i.e., pcDNA3 (from
20 Invitrogen) or a vector containing the CMV promoter and
intron A of CMV to promote better expression of protein
(pCMVA vector). For example, genetic sequences coding
for the major glycaprotein (G) or nucleoprotein (N) of
the viral hemorrhagic septicemia virus (VHSV) were
25 cloned into the EcoRI site of either the pcDNA3 or pCMVa
vector. Nucleotide sequences encoding the VP2 and VP3
structural proteins of the infectious pancreatic
necrosis virus (IPNV) were cloned into same vectors.
The gene encoding the ferric siderophore receptor
30 (fstA) of Aeromonas salmonicida has also been cloned
into expression vectors. The fstA protein is one of
CA 02499302 1996-11-07
- 25 -
several possible iron-regulated outer membrane proteins
that could be expressed as an antigen from a DNA
vaccine.
5 Plasmid Vector Antigen Pathogen
pCNIV-G pcDNA3 G glycoproteinviral hemorrhagic
(EcoIZI site) (#1-1565)* septicemia virus
pC'~IV,; pC~IV.4 vector G glycoproteinviral hemorrhagic
G
(EcoRI site) (# 1-1565)* septicemia virus
pCMV-N pcDNA3 N nucleoproteinviral hemorrhagic
(EcoRI site) (#92-1306)* septicemia virus
pCMV~ N pCMV,, vector N nucleoproteinviral hemorrhage
(EcoRI site) (#92-1306)* septicemia virus
10 pCMV-VP2 pcDNA3 VP2 infectious pancreatic
(I-~ndIIt/XbaI (#117-1760)* necrosis virus
site)
pCMVA VP2 pCMVA vector VP2 infectious pancreatic
(Sall/Xbal) (#117-1760)* necrosis virus
pCMV-VP3 pcDNA3 VP3 infectious pancreatic
(EcoRI/XbaI site)(#2325-3011)*necrosis virus
pCMVA VP3 pCMVA vector VP3 infectious pancreatic
(EcoRUXbaI site) (#2325-3011)*necrosis virus
pCMV-fstA pcDNA3 IROMP fstA Aeromonis salmonicida
(EcoRI/XbaI site)(#76-2630)*
15 pCMV~ fstA pCMVA vector IROMP fstA Aeromonis salmonicida
(EcoRIlXbaI site)(#76-2630)*
* # indicates the nucleotlae sequences wm~n one
genome of the pathogen which have been cloned to code
for the antigen
Examg~p 2 - Expression of F r~e~gn pro ein In Fish
20 ~n,~A~~-Ad with Pure Plasmid DNA Vector
The pCMV-luc plasmid used in the following
experiments contains the luciferase reporter gene (luc)
under the control of the cytomegalovirus promoter.
CA 02499302 1996-11-07
- 26 -
Purified plasmid DNA was prepared by using commercially
available Qiagen DNA purification columns. The
purified plasmid DNA was then dissolved in
endotoxin-free Dulbecco's phosphate buffered saline
5 (DPBS) without calcium chloride or magnesium chloride,
or in 0.15 M NaCl dissolved in deionized distilled
water for a final concentration of 0.001 mg/ml to
S mg/ml DNA. Fish were anaesthetized with 0.168 mg/ml
tricaine (3-amino benzoic acid ethylester) in water or
10 by placing the fish on ice for 30-60 seconds before
injection. Trout and zebra fish were injected
intramuscularly between the dorsal fin and the lateral
line with 10 ~1 of the DNA solution.
Luciferase activity was measured in the
15 muscle and gills 2.5 days after injection. Rainbow
trout were euthanized by an overdose of tricaine (0.1~
w/v). Zebra fish were killed by immersion in ice. The
muscle or gills of the fish were removed on ice,
homogenized, centrifuged to pellet cellular debris, and
20 the supernatants containing soluble proteins were
assayed for luciferase activity. Luciferase assays
were carried out using a kit commercially available
from Promega Corporation. Light emission in relative
light units (RLU) was quantitated by a luminometer
25 (Analytical Luminescence Laboratory) over a ten second
interval and background values from control samples
were subtracted. The concentration of protein in the
supernatants was determined and luciferase activity was
expressed as RLU/sec/mg protein.
30 The results summarized in Table 2 (below)
indicate that purified plasmid DNA can efficiently
transfect fish cells after intramuscular injection.
Nanogram amounts of plasmid DNA were able to induce
detectable protein expression in both the injected
CA 02499302 1996-11-07
- 27 -
muscle as well as more distant cells (e. g., in gills),
showing that different types of cells, possibly
including antigen presenting cells (APC), are
transfectable by plasmid DNA and are capable of
5 synthesis of foreign protein. Cells distant to the
site of injection (i.e. gills) expressed lower amounts
of protein than the injected muscle cells. Zebra fish
and trout are not closely related species of fish.
Therefore, the results in Table 2 indicate that most
10 species of fish could take up and express foreign
proteins from injected plasmids.
Luciferase
Acti= U/sec/mg
of protein)
Dose
of DNA Trout Zebra Fish
15 (pg)
Muscle Gills Muscle Gills
0 3,449 4 502 18
01
. (t1548) (t3) 0307) 08)
0 22,768 36 11,665 94
1
. 012,708) 014) 02,989) 066)
1 78,408 618 826,486 228
051,523) (t567) 0368,790) 0115)
10 280,051 982 199,285 833
0172,749) 0743) 097,134) 0621)
0 50 417,226 980 145,891 5,519
2 0165,164) 0393) 085,645) 04791)
*mean ~ standard error of mean (n=lU tlsh per group)
F~,~1 A 3 -- Kinetics And LoncrevitY of Forei gn Gene
F,~,~ression in Fish
One microgram of pCMV-luc plasmid in 10 ul of
25 saline was injected intramuscularly into adult zebra
fish and 3-4 month old rainbow trout as previously
CA 02499302 1996-11-07
- 28 -
described in Example 2. Luciferase activity in the
muscle and gi:Lls of the injected fish was determined at
various times between 2.5 days and 8 weeks using the
methods described in Example 2. In Table 3 (below),
5 the plasmid DNA directs protein expression within days
of injection and protein expression in post-mitotic
muscle remains stable for at least eight weeks.
Luciferase expression in the gills falls off over time,
possibly due to cell turnover.
10 Table 3
Luciferase
Activity
(RLU/sec/mg
of Protein)*
Time
Trout Zebra Fish
(days)
Muscle Gills Muscle Gills
0 0 0 0 0
78,408 618 2,107,048 8,705
2.5 051,523) 0567) 01,281,284) 06853)
54,004 211 4,160,080 6,965
15 14 (t19,411) 0133) 02,553,955) 03,672)
90,686 39 5,236,613 6,056
28 (t46,044) (t20) 04,536,744) 04048)
18,219 30 6,395,781 3,246
56 011,785) 023) 01,764,195) 01,040)
*mean ~ standard error or mean W =LV L~sn per ~LVU~r
Kinetics and Longevity of Foreign Gene
20 FxgrP~s~on in Zebra Fish
Zebra fish were injected intramuscularly with
0.1 ug of purified plasmid pCMV-luc DNA in 10 ul of
saline as previously described in Example 2. The
results from Table 4 (below) indicate that even
25 ten-fold less DNA than used in Example 3 is capable of
CA 02499302 1996-11-07
- 29 -
producing detectable levels of protein for at least
sixteen weeks. The results also indicate that protein
expression begins within hours after injection.
5 Time Luciferase Activity
(RLU/sec/mg of Protein)*
r
(days) Muscle Gills
0.16 64 035) 1 01)
0.33 1,620 01418) 3 (t2)
0.5 2,739 01359) 30 019)
10 1 2,629 01,129) 15 02)
2.5 11,665 02,989) 94 (~66)
112 82,424 (t49,208) 103 (t50)
*mean ~ standard error of mean (n=10 fish per group)
15
Zebra fish were injected intraperitoneally
(IP) (i.e.~ in the abdomen) with 0.1 ug of pCMV-luc
alone or associated with 0.5 ug of a cationic lipid,
6304 (obtained from Gibco BRL, New York. USA) in 10 ul.
20 Luciferase activity in muscle. gills, and
viscera (liver spleen, intestine, stomach, swim
bladder, pylaric caeca. and ovary or testis) was
measured 2.5 days after DNA injection. The fish
tissues were prepared as described previously (Example
25 2) .
Table S (below) shows that protein is
expressed fram DNA that is injected intraperitoneally
into fish. Injection of plasmid DNA formulated with
cationic lipid resulted in higher foreign protein
CA 02499302 1996-11-07
- 30 -
expression in the viscera than injection of DNA alone.
Muscle tissue, on the other hand, expressed greater
levels of luciferase enzyme when the plasmid pCMV-luc
DNA was injected without the cationic lipid.
5 Therefore, a cationic lipid may increase transfection
efficiency depending the target tissue.
Table
Zebra Fish Luciferase Activity
(RLU/sec/mg of
protein)*
Tissue DNA alone DNA + lipid
Muscle 151 (t 127) 32 (t 10)
Gills 15 (t15) 8 (t8)
Viscera 3 (t2) 85 (t40)
*mean ~ standard error of mean (n=to fish per group)
Fxa~~e 6 - Comparison of Foreign Gene Expression
aft-e_r Infection of DNA in Fish and in Mice
Fish and mice were injected intramuscularly
with a range of 0.1 ug to 50.0 ug of pCMV-luc plasmid
DNA in 50 ul. Total luciferase activity for the whole
muscle of mouse or fish was assayed 2.5 days after
20 injection. The fish and mouse tissues were prepared as
described previously (Example 2).
For each dose of DNA, injected trout
demonstrated higher levels of luciferase activity than
injected mice (Table 6 below?. In general, for a given
25 dose of DNA, luciferase activity was approximately 100
times higher in fish than in mouse. Therefore, the
knowledge that mice can be immunized against numerous
diseases using doses of DNA within the range tested
here. and the finding that fish muscle is more easily
30 transfected and/or that fish muscle expresses
CA 02499302 1996-11-07
- 31 -
transgenes more efficient) y, indicate that fis:~ should
be gocd candidates for DNr-based immunization.
Dose of DNA Luciferase Activity
(RLU/sec total)*
5 (~~) mouse trout
0.01 N/A 65,898
( '-=3 0, 7 7 4)
0.1 1, 649 3 27, 724
(1542 ) (=177, 83 )
1.0 5,466 1,100,347
(=1536) (=569,634)
10 43,082 3,3?5,068
05,419) (~ l, 869,474)
I ~ 0 70, 713 4, ~ 20, 741
0
(=15,921) (=1,609,457)
~:nean ~ standard error of mean (n = 5 zor tout except
for the 50ug dose group for which n = 12; n=10 .or
mouse groups)
~~n, A ~ _- Ex~ ~~ss; on o= c~ asm~ d DNP 3fia~ T~m~rJ; or
15 ~r Dish in DNA-containing Solutions
Cationic lipid, 6304, was obtained ==om Gibco
BRL, New York, USA. Cat=onic polymer liposomes
designated Q203, Q205, Q206, Q208, Q250, and QX we=
obtained from Qiagen Gmb~~~, Hilden, Germany. Cationic
20 polymer lipasomes are composed of a mixture of cationic
polymers and neutral lipids.
CA 02499302 1996-11-07
- 32 -
For example, a cationic polymer (either Q203,
Q205, Q206, c~208, Q250 or QX, described below in Table
7) and a neutral lipid.
dioleoyloxiphosphatidylethanolamine (DOPE) were mixed
5 together for a final concentration of 2 mM in
chloroform, which was then evaporated off in a rotary
evaporator at 60°C. The mixture was dried for 10
minutes under a reduced pressure of 10 to 15 mbar.
Under sterile conditions, endotoxin free deionized
10 water was added to the mixture, which was then heated
while stirring at 60°C.
Next. Q203, Q205, Q250, and QX were sonicated
once for 300 seconds at 60°C. In the case of Q250,
trans (3 carotene was added to a final concentration of
15 0.37 mM before sonication. Q206 and Q208 were not
sonicated but were stirred at 60°C until the solutions
became transparent or slightly opalescent. The total
concentration of DOPE+cationic polymer for all
liposomes was 2 mM. The concentration of DOPE in each
20 liposome can be calculated by multiplying the X(DOPE)
value in Table 7 by 2 mM so that. for example,
Q203-containing liposomes are 1.7 mM DOPE and 0.3 mM
Q203. Table 7 (below) summarizes the cationic polymer
liposomes used in the methods of this invention.
CA 02499302 1996-11-07
- 33 -
Table 7
Cationic
Polymer Cationic Polymers X(DOPE) Method of
Liposome Preparation
5 Rea ent
butandiyl-1,4-bis(octadecyl with sonication.
Q203 0.85
d~ethylammonium bromide)
butandiyl-1,4-bis(octadecyl with sonication.
Q205 0.82
dimethylammonium bromide)
Q206 butandiyl-1,4-bis(octadecyl0.78 without sonication.
dimethylammonium bromide)
Q208 butandiyl-1,4-bis(octadecyl0,75 without sonication.
dimethylammonium bromide)
with sonication.
didodecyldimethyl 0 Add trans ~3 carotene
ammonium 571
10 Q250 bromide . to final concentration
of D.37 mM.
QX didodecyldimethyl 0.375 with sonication.
ammonium
bromide
DNA:liposome complexes were prepared by
independently diluting DNA and liposome solutions in
0.15 M NaCl, then mixing the two solutions and
15 vortexing, and then incubating the mixture at room
temperature for 30-45 minutes. The solutions were
diluted further with water and incubated for an
additional 10-15 minutes at room temperature prior to
use with fish.
20 Each fish was immersed in the solution of
liposome formulated DNA (2.5 ml or 5 ml per fish) for
90 minutes and then returned to its normal holding
tank. After 2.5 days, the fish were homogenized or
gills and muscle were homogenized separately and
25 assayed for luciferase activity.
w CA 02499302 1996-11-07
- 34 -
Table 8 (below) shows luciferase activity
above background in individual zebra fish after
immersion. Thus, the results of Table 8 indicate that
the majority of fish were successfully transfected and
able to express foreign protein after immersion in
DNA:liposome solutions. No fish expressed luciferase
activity after immersion in pCMV-luc DNA without
liposomes. Therefore, lipid-containing transfection
reagents appear to significantly contribute to the
transfection efficiency of DNA into fish with the
immersion technique.
TransfectionTotal Luciferase Activity
Reagent ~RLU/ second)
6304 330, 65, 0, 1643, 1581, 143, 5, 165, 0, 0,
257
Q203 268, 82, 106, 264
Q205 188, 268, 166, 136
Q206 208, 286, 170, 108, 174
2 Q208 668, 204, 1060, 0, 0, 180, 842, 242, 90,
0 36
Q250 :358, 398, 60, 10, 134, 1742, 54, 136, 84,
136
QX 166, 80, 302, 74, 432, 630, 28, 28, 260,
260
~'xa le 8 -- Induction of an Immune Response against a
Protein Derived from a Fish Pathogen in Fish and Mice
~~ Injection of Antigen-Encoding Plasmid DNA
Purified pCMVA-VP3 DNA (encoding the VP3
protein of infectious pancreatic necrosis virus) was
prepared as described previously for pCM~T-luc DNA and
injected intramuscularly in mice or trout. Each of
three adult female BALH/C mice received 100 ug in
tibialis anterior muscle while a single one-year old
CA 02499302 1996-11-07
- 35 -
female trout received 200 ug in the tail muscle. Two
weeks later, the humoral immune response against the
expressed antigen was determined by ELISA assay of
plasma taken from the mice and fish to detect anti-VP3
5 antibody.
The ELISA assay was performed using standard
techniques. In particular, 96-well plates were coated
with infectious pancreatic necrosis virus (IPNV)
particles, blocked with a non-specific protein, and
10 then washed. Ten-fold serial dilutions of trout or
mice plasma and control plasma (obtained from non-
injected mice and fish or from animals injected with
control DNA not encoding VP3) were put in the
appropriate wells (100 ,ul/well) were incubated for 2
15 hours. After washing, bound anti-VP3 antibodies in
trout and mouse plasma were detected by addition of
horse-radish peroxidase-labeled (HRP) mouse anti-trout
or goat anti-mouse IgG monoclonal antibodies,
respectively. Amounts of bound antibody were
20 quantitated by reaction with O-phenylenediamine
dihydrochloride, which is cleaved by HRP producing a
color measurable by a spectrophotometer at OD45o-
ELISA titer values in Table 9 (below)
indicate the dilution factor which gave an ODQSO value
25 twice that of background. Table 9 shows that DNA-based
immunization of fish or mice by intramuscular injection
of plasmid DNA can induce an immune response against an
antigenic protein of a fish pathogen such as the VP3
protein of IPNV.
30
CA 02499302 1996-11-07
-36-
Species anti-VP3 ELISA titers
Mouse 527.2
Trout I4.1
5 In another example, pCMV-G plasmid (encoding
the G glycoprotein of viral hemorrhagic septicemia
virus) was prepared as described previously and 100 ug
was injected intramuscularly into an adult trout. Two
and four weeks later, the humoral immune response
10 against the expressed antigen was determined by ELISA
assay of plasma taken from the fish to detect anti-G
antibody. The ELISA assay was performed as described
above except the 96-well plates were coated with
purified recombinant G protein from VHSV.
15 ELISA titer values in Table IO (below)
indicate the mean dilution factor which gave an OD4so
value twice that of background. Each value is the mean
of two (14d) or three (28d) separate ELISA assays.
Table 10 shows that DNA-based immunization of fish by
20 intramuscular injection of another plasmid DNA, pCMV-G
can induce an immune response against an antigenic
protein of a second fish pathogen namely VHSV. The
humoral response was detected as late as 28 days after
injection.
Time (days) Anti-G ELISA titers
0 0
14 32 (1 )
28 34 (4)
CA 02499302 1996-11-07
-37-
The collective results of the examples show
the expression of foreign proteins in cells of fish
after administration of pure plasmid DNA, either by
intramuscular or intraperitoneal injection of pure or
formulated plasmid DNA, or by injection of or immersion
in DNA formulated with cationic liposomes.
Furthermore, the collective results show that an immune
response can be induced if the protein is antigenic,
for example a protein of a fish pathogen. This should
lead to protection against natural infection by
virulent pathogen.
While we have hereinbefore presented a number
of embodiments of this invention, it is apparent that
our basic construction can be altered to provide other
embodiments which utilize the methods of this
15 invention. Therefore, it will be appreciated that the
scope of this invention is to be defined by the claims
appended hereto rather than the specific embodiments
which have been presented hereinbefore by way of
example.
