Note: Descriptions are shown in the official language in which they were submitted.
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USE
This invention relates to the identification of a DNA molecule encoding an
antigen and in particular a rhabdovirus glycoprotein as new and effective
adjuvant
molecules and the use of said DNA molecules as adjuvants. The present
invention
further provides vaccine compositions comprising said adjuvant and one or more
vaccine molecules, and the use of such compositions in vaccination.
Vaccination of mammals and other organisms against various types of
infection and disease is an important goal in many fields of biotechnology. In
particular in fields of food production and farming it is vital to protect the
reared
organisms from infection and disease which can lead to illness or even death
of
organisms and thereby reduce the yield and quality of the food product. Thus,
the
development of new or improved vaccines which will protect against various
types
of infection and disease is desirable. For example, in the fish farming
industry
cultured fish are prone to viral infections. Nodavirus infection is a
particular
problem and has emerged as a major constraint on the culturing of a number of
marine fish species. Thus, vaccines which will prevent or reduce the number of
farmed fish becoming affected by nodavirus infection are particularly
desirable.
Surprisingly, it has been found that DNA molecules encoding an antigen and
in particular a rhabdovirus glycoprotein can be used to enhance the immune-
stimulating properties of antigens or to enhance the immune response to
antigens.
Put another way it has been found that DNA molecules encoding an antigen and
in
particular a rhabdovirus glycoprotein can be used as a vaccine adjuvant.
Thus, the present invention provides the use of a DNA molecule encoding an
antigen which is expressed on the surface of a host cell, or a variant, or a
fragment
thereof, as a vaccine adjuvant.
Viewed alternatively, the present invention provides the use of a DNA
molecule encoding an antigen which is expressed on the surface of a host cell,
or a
variant, or a fragment thereof, to enhance the immune stimulating properties
or
vaccination properties of a vaccine molecule, or to enhance the immune
response to
a vaccine molecule.
The term "antigen" is used herein to mean any foreign substance (i.e. a
substance not normally present in a host organism) which is capable of
triggering an
immune response in an organism. Thus, molecules normally present in a
particular
host organism are excluded. For example, molecules normally involved in the
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manifestation of an immune response in the host, for example chemokines,
cytokines, ligands expressed on antigen presenting cells associated with T
cell
stimulation, co-stimulatory molecules and molecules involved in the complement
cascade are all not antigens within the meaning of the present invention.
Whilst not wishing to be bound by theory, it is believed that the expression
of antigens on the surface of cells in the host organism in question may be
important
for the observed adjuvant effect. Thus, in addition, antigens of the present
invention
are preferably antigens which are expressed on the surface of cells in the
host
organism in question. This means that molecules which encode antigens which
remain in the cytoplasm of host cells are also preferably excluded.
Examples of antigens which are included in the scope of the present
invention are antigens which can be presented (expressed) on the surface of T
cells
in the host organism. Also, in general, appropriate antigens are antigens
which, in
their native form, are found on the surfaces of cells or other biological
particles such
as viruses or bacteria, and which are then expressed on the surface of cells
in the
particular host organism. Thus, preferred antigens are bacterial or viral
proteins
found on the surface of bacterial or viral particles, e.g. envelope proteins.
In
particular, glycoproteins are preferred antigens for use in all aspects of the
present
invention.
In all aspects of the invention which involve glycoproteins, the DNA
molecule encoding an antigen which is expressed on the surface of a host cell
is
replaced by a DNA molecule encoding a glycoprotein. It is further preferred
that,
when expressed in the host organism, such glycoproteins are expressed on the
surface of a host cell, for example such glycoproteins are anchored in the
membrane
and exposed on the surface of host cells. Such glycoproteins can be derived
from
any sources. However preferred sources are bacteria and viruses and thus
bacterial
and viral glycoproteins are preferred. Also preferred is that the virus or
bacteria
from which the glycoproteins are derived is a virus or bacteria that can
replicate in
or can infect the organism to which the adjuvant is to be administered. In
more
preferred embodiments of the invention, the encoded glycoprotein is a virus
glycoprotein, most preferably a rhabdovirus glycoprotein. Preferably the DNA
molecule encoding the antigen and the vaccine molecule for which said DNA
molecule acts as an adjuvant are expressed in the same cells in the host
organism.
The term "host cell" as used herein in connection with antigens which can be
used as adjuvants or to enhance the immune stimulating properties, etc.,
refers to
cells in the particular host to which the adjuvant is administered. Thus, if
the
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adjuvant is being administered to a host which is a fish, then the antigen is
expressed
on the surface of cells found in the host fish.
The present invention further provides the use of a DNA molecule encoding
an antigen which is expressed on the surface of a host cell, or a variant, or
a
fragment thereof, in the manufacture of a medicament for use as a vaccine
adjuvant
or to enhance the immune stimulating properties or vaccination properties of a
vaccine molecule, or to enhance the immune response to a vaccine molecule.
The present invention further provides a method of vaccination of an
organism or a method of stimulating an immune response in an organism wherein
a
DNA molecule encoding an antigen which is expressed on the surface of a host
cell,
or a variant, or a fragment thereof, is used as a vaccine adjuvant or is used
to
enhance the immune stimulating properties or vaccination properties of a
vaccine
molecule, or to enhance the immune response to a vaccine molecule.
In embodiments of the invention where the antigen is a virus glycoprotein,
DNA molecules encoding glycoproteins from any virus may be used in the present
invention. Preferred viruses include rabes and/or rhabdovirus.
In all the embodiments and aspects of the invention described herein a
preferred viral glycoprotein is a rhabdovirus glycoprotein.
Any enhancement or increase as described herein, particularly in connection
with the immune stimulating properties or vaccination properties of a vaccine
molecule or the immune response to a vaccine molecule, includes any measurable
enhancement or increase when the property in question in the presence of the
DNA
molecules (adjuvants) of the invention is compared with the equivalent
parameters
in the absence of the DNA molecules (adjuvants). Preferably the enhancement or
increase is a statistically significant one.
Methods of determining the statistical significance of differences in
parameters are well known and documented in the art. For example herein a
parameter is generally regarded as significant if a statistical comparison
using an
appropriate statistical test such as a Student t-test shows a probability
value of
<0.05.
The methods and uses of the invention can be carried out by administration
of appropriate DNA molecules encoding an antigen to any appropriate organism.
Appropriate organisms may be mammalian or non-mammalian and include humans
and all farmed or reared organisms.
Thus, appropriate organisms include humans, avian species such as chickens
or birds, cows, pigs, sheep, rodents (e.g. mouse, rat, guinea pig, hamster
etc), fish,
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insects, crustaceans, etc. Farmed or reared organisms are particularly
preferred and
fish, in particular marine species of fish such as Atlantic halibut, turbot,
spotted
wolfish, groupers, salmon, trout and cod, are especially preferred in this
regard.
Examples of farmed fish which are preferred are salmonids, cadoids, groupers,
flat
fish (turbot, halibut), sea bream, trout, sea bass and striped jack. Flat fish
are
especially preferred.
Thus, further preferred antigens for use as adjuvants or to enhance the
immune stimulating properties, etc., are antigens, for example glycoproteins,
derived
from organisms or pathogens that can replicate or infect fish, e.g. are
derived from
fish pathogens. Preferably the glycoprotein is not derived from Hepatitis B,
EBV,
plant viruses (e.g. potato viruses) and/or cholera toxin
The term "adjuvant" is used herein in its standard meaning known in the art
and refers to substances which are added to antigens (or vaccine molecules) to
enhance the immune response to such antigens (or vaccine molecules). In the
present invention the adjuvants are generally used to enhance the immune
response
to antigens which are distinct from the antigen which comprises the adjuvant.
Thus,
where the adjuvant is DNA encoding a rhabdovirus glycoprotein, this is
generally
used as an adjuvant to enhance the immune response to antigens other than the
rhabdovirus glycoprotein being used as the adjuvant.
Preferred DNA molecules used in the present invention may encode a
glycoprotein from any type of rhabdovirus and the DNA encoding the rhabdovirus
glycoprotein for use in the present invention can be derived from any
appropriate
source. However, preferably the rhabdovirus glycoprotein is a fish rhabdovirus
glycoprotein, for example a glycoprotein derived from viral hemorrhagic
septicemia
virus (VHSV) or infectious hematopoietic necrosis virus (IIINV). A
particularly
preferred rhabdovirus glycoprotein is that derived from VHSV. A preferred DNA
molecule in this regard is pVHSV-G as described in the Examples.
Although DNA molecules encoding a full length antigen, preferably a
rhabdovirus glycoprotein, can be used in the present invention, use of
functionally
equivalent variants (herein termed "variants") and functionally equivalent
fragments
of such full length molecules or variants (herein termed "fragments") are also
contemplated.
"Functionally equivalent" is used herein to define DNA molecules encoding
proteins related to or derived from the native protein, where the amino acid
sequence
has been modified by single or multiple amino acid substitution, addition
and/or
deletion, but which nonetheless retain the ability to function as an adjuvant
in the
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organism in question. Functionally equivalent variants also include degenerate
sequences, i.e. nucleic acid sequences which contain base changes (i.e.
nucleotide
changes) that do not cause a change in the encoded amino acid sequence. DNA
molecules which are substantially homologous to the DNA molecule encoding the
5 native protein or which encode sequences which are substantially homologous
to the
encoded native protein are also included. "Substantially homologous" as used
herein in connection with an amino acid or a nucleic acid sequence includes
those
sequences having a sequence homology or identity of approximately 60% or more,
e.g. 70%, 80%, 90%, 95%, 98% or more with a particular sequence and also
functionally equivalent variants and related sequences modified by single or
multiple base or amino acid substitution, addition and/or deletion.
Homology may be assessed by any convenient method. However, for
determining the degree of homology between sequences, computer programs that
make multiple alignments of sequences are useful, for instance Clustal W
(Thompson, J. D., D.G. Higgins, et al. (1994). "CLUSTAL W: Improving the
sensitivity of progressive multiple sequence alignment through sequence
weighting,
position-specific gap penalties and weight matrix choice". Nucleic Acids Res
22:
4673-4680). Programs that compare and align pairs of sequences, like ALIGN (E.
Myers and W. Miller, "Optical Alignments in Linear Space", CABIOS (1988) 4: 11-
17), FASTA (W.R. Pearson and D.J. Lipman (1988), "Improved tools for
biological
sequence analysis", PNAS 85:2444-2448, and W.R. Pearson (1990) "Rapid and
sensitive sequence comparison with FASTP and FASTA" Methods in Enzymology
183:63-98) and gapped BLAST (Altschul, S.F., T.L. Madden, et al. (1997).
"Gapped BLAST and PSI-BLAST: a new generation of protein database search
programs". Nucleic Acids Res. 25: 3389-3402) are also useful for this purpose.
Furthermore, the Dali server at the European Bioinformatics institute offers
structure-based alignments of protein sequences (Holm, J. of Mol. Biology,
1993,
Vol. 233: 123-38; Holm, Trends in Biochemical Sciences, 1995, Vol 20: 478-480;
Holm, Nucleic Acid Research, 1998, Vol. 26: 316-9).
By way of providing a reference point, sequences according to the present
invention having 60%, 70%, 80%, 90%, 95% homology etc. may be detennined
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using the ALIGN program with default parameters (for instance available on
Internet at the GENESTREAM network server, IGH, Montpellier, France).
Such functionally equivalent variants mentioned above also include natural
biological variations (e.g. allelic variants or geographical variations within
a species)
and derivatives prepared using known techniques. For example, functionally
equivalent molecules may be prepared either by chemical synthesis or in
recombinant form using the known techniques of site directed mutagenesis
(including deletion), random mutagenesis, or enzymatic cleavage and/or
ligation of
nucleic acid. Functionally equivalent variants also include analogues in
different
genera or species.
Preferred fragments for use in the present invention encode at least the
transmembrane domain of an antigen, preferably the rhabdovirus glycoprotein.
Examples of DNA molecules encoding rhabdovirus glycoproteins which
may be used in the present invention are known in the art, for example DNA
encoding the glycoproteins from VHSV and IHNV are known. However, if
necessary, DNA sequences encoding appropriate glycoproteins from any
rhabdoviruses can be readily determined using standard and routine methods.
For
example, a rhabdovirus can be taken and the glycoprotein isolated from the
viral
envelope, after which a peptide sequence and DNA sequence can readily be
determined using standard methods.
The DNA molecules encoding antigens, preferably rhabdovirus
glycoproteins, for use in the present invention can take any appropriate form
and can
be produced by any appropriate method, e.g. by recombinant techniques.
Conveniently, such DNA molecules take the form of plasmids or expression
vectors
and can be produced by any appropriate method, e.g. by recombinant techniques.
which comprise the DNA sequence encoding the antigen or variant, or fragment
thereof, together with appropriate regulatory sequences to enable the
expression of
the antigen in the organism in question. Preferably, said DNA sequences
encoding
the antigen or variant, or fragment thereof, are placed downstream of an
appropriate
promoter sequence, e.g. a eukaryotic promoter sequence such as the early CMV
promoter, or another appropriate promoter which is recognised by the organism
to
which the DNA molecule is to be administered. For example, promoters which are
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native to the organism which is being vaccinated can also be used and are
sometimes
preferred, e.g. fish promoters such as the common carp 0-actin promoter (Gomez-
Chiarri et al., 1999, Genetic Analysis: Biomolecular Engineering 15:121-124),
or a
trout interferon regulatory factor lA promoter (Alonso et al., 2003, Vaccine
21:1591-1600). Non-plasmid or non-vector forms of DNA molecules can also be
used. For example, DNA molecules encoding only the antigen without any vector
components can be used.
Plasmids or vectors containing appropriate promoter elements and other
regulatory elements are readily available commercially or can readily be
designed
and made in the laboratory using standard skills and methods. For example, an
appropriate vector for use in this regard which contains the CMV early
promoter is
the pcDNA3 vector (Invitrogen). The DNA molecules encoding the antigen,
preferably the rhabdovirus glycoprotein, may optionally be modified so that
they are
more stable in vivo using methods which are well known and standard in the
art.
For example, the DNA backbone may be modified to improve stability by methods
well known and standard in the art.
The DNA molecules encoding an antigen, preferably a rhabdovirus
glycoprotein, can be used as an adjuvant for any contemplated vaccine molecule
(or
indeed as an adjuvant for any antigen) and preferably a vaccine molecule
against
which a specific long lasting immune response is desired. In this regard, the
vaccine
molecule may be any molecule wherein it is desirable to stimulate an immune
response to that molecule or part thereof in the organism in question. Such
vaccine
molecules might be able to give rise to the stimulation of an immune response
in
their own right. However, the uses and methods of the invention are
particularly
appropriate for vaccine molecules which are not able to generate a strong or
significant or effective or adequate immune response or protective response in
their
own right (e.g. when administered by themselves), in which case the adjuvant
properties of the DNA molecule encoding the antigen are particularly valuable.
Thus, in preferred embodiments the vaccine molecule does not itself act as an
adjuvant.
Thus, said vaccine molecule can be any known or new protein or DNA
vaccine, including recombinant vaccines and peptide vaccines. Many such
vaccine
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molecules are known in the art and include all manner of bacterial or viral
antigens
or indeed antigens or antigenic components of any pathogenic species.
Appropriate
vaccine molecules may comprise whole organisms (whether live, dead or
attenuated) or may be recombinant vaccine molecules, e.g. sub-unit vaccine
molecules based on particular components of organisms, e.g. proteins, peptides
or
even carbohydrates. Such vaccine molecules may comprise the relevant protein,
peptide or carbohydrate molecules or, if DNA vaccines are to be used, may
comprise nucleic acid molecules which encode said relevant proteins or
peptides.
Vaccine molecules based on antigenic molecules associated with particular
disease
states, e.g. cancer vaccines, can also be used.
Examples of DNA vaccines are known and described in the art. However, in
general such vaccines usually take the form of an expression vector (e.g. a
plasmid)
carrying an appropriate foreign DNA fragment (e.g. a fragment encoding a
pathogenic protein or peptide) regulated by an appropriate promoter which is
recognised by the organism being vaccinated (e.g. a eukaryotic promoter). The
DNA fragment of interest is expressed when the expression vector is taken up
by
cells and transported to the nucleus where the cellular transcription
apparatus
recognise the promoter. Such DNA vaccines are thought to be more safe than
protein or peptide vaccines as they have no risk of reversion to virulence.
Any
appropriate promoter can be used in the DNA vaccines of the invention. Common
promoters for use in this regard are eukaryotic promoters such as the CMV
immediate early promoter. However, promoters which are native to the organism
which is being vaccinated are also preferred, e.g. fish promoters such as the
common
carp 0-actin promoter (Gomez-Chiarri et al., supra), or a trout interferon
regulatory
factor IA promoter could be used (Alonso et al., supra). Non-plasmid or non -
vector
forms of DNA vaccine can also be used. For example, DNA molecules comprising
only the foreign DNA fragments without any vector components can be used.
Thus, in preferred aspects of the invention, said vaccine molecule is a DNA
vaccine, more preferably a DNA vaccine which is designed to protect against
viral
or bacterial infection, more preferably viral infection. Such antiviral DNA
vaccines
generally comprise DNA encoding a viral component such as a glycoprotein or a
capsid protein.
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A vast number of vaccine candidates have been proposed in the literature, for
example in the treatment of viral or bacterial diseases and infections, or in
the
treatment of other disease states such as cancer, and DNA molecules encoding
an
antigen, preferably a rhabdovirus glycoprotein, may be used as an adjuvant for
any
of these in accordance with the present invention. However, as discussed
above,
DNA molecules encoding an antigen, preferably a rhabdovirus glycoprotein, may
also be used as adjuvants in conjunction with known vaccine molecules which
have
not been shown to stimulate an adequate immune response in their own right in
order to improve or enhance said immune response, or in conjunction with other
molecules which are candidates for use as vaccines but which have not
previously
been tested.
Thus, in a preferred embodiment of the invention, the DNA molecule
encoding the antigen, preferably a rhabdovirus glycoprotein, is used as an
adjuvant
for a vaccine molecule which alone cannot stimulate an effective or
significant or
adequate immune response. Such vaccine molecules are generally those which
alone cannot induce protection against a subsequent challenge by the
appropriate
infective agent or disease. Examples of vaccine molecules which alone cannot
stimulate an effective or significant or adequate immune response are often
those
which encode cytoplasmic proteins (and are therefore not necessarily "seen" by
the
host immune system) as opposed to proteins expressed on the surface of host
cells.
As mentioned above, vaccines which can protect fish against challenge by
viruses, and in particular nodaviruses, are particularly desired. Nodaviruses
are non -
enveloped RNA viruses consisting of an icosahedral capsid 25-30 nm in
diameter,
which contains a genome of two single stranded positive-sense RNA segments,
RNA1 (3.1 kb) and RNA2 (1.4kb). RNAl encodes the putative RNA-dependent
RNA polymerase while RNA2 encodes the capsid protein. In fish, nodavirus
infections cause viral encephalopathy and retinopathy (VER) also termed viral
nervous necrosis (VNN), and has emerged as a major constraint on the culturing
of a
number of marine fish species. Common external signs of nodavirus infection
are
related to the neuro-invasiveness of the virus and include hyperreactivity,
abnormal
swimming patterns with looping and spiral movements and reduced coordination
and changes in pigmentation. Histopathological findings include vacuolation
and
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necrosis of the central nervous system, the retina and the ganglia of the
peripheral --
nervous system.
It is known in the art that a DNA molecule encoding a rhabdovirus
glycoprotein can confer protection in fish against a challenge from
rhabdoviruses
5 (La Patra et al., Vaccine, 2001, 19:4011-19, Lorenzen et al., J. Aquatic
Animal
Health, 2000, 12:167-80). It has also been observed that a DNA molecule
encoding
a rhabdovirus glycoprotein can confer short term protection in fish against a
challenge from nodavirus. However, longer term protection against a nodavirus
challenge was not observed and by 35 days post vaccination the protective
effect
10 was reduced (Sommerset et al., Vaccine, 2003, 21:4661-7). In other reports
a DNA
molecule encoding a capsid protein of nodavirus was used to try and give a
protective effect against nodavirus infection, however no short term or long
term
protective effect was observed (Sommerset et al., Abstract 113, Proceedings of
the
10th International Conference of the EAFP, Dublin, 9-14 September 2001). Long
term protection against nodavirus for the time appropriate to rear fish for
human
consumption has thus to date proved elusive.
Surprisingly however, it has now been found that administration of a DNA
molecule encoding a rhabdovirus glycoprotein and a DNA molecule encoding a
nodavirus capsid protein can give long term protection (70 days post
vaccination)
against nodavirus infection. This is indeed surprising given the fact that, as
discussed above, the DNA molecule encoding the rhabdovirus glycoprotein
molecule alone could not confer long term protection against a nodavirus
challenge
and the DNA molecule encoding the nodavirus capsid molecule alone conferred no
protection at all, even in the short term, against a nodavirus challenge.
Thus, in a preferred embodiment of the invention, the vaccine molecules are
derived from nodaviruses and, more preferably, the use of such nodavirus
derived
vaccine molecules in conjunction with the above described DNA molecules
encoding an antigen, preferably a rhabdovirus glycoprotein, in accordance with
the
invention result in long term protection (70 days or more post vaccination)
against a
nodavirus challenge. The vaccine molecules may comprise nodavirus component
proteins such as the RNA dependent RNA polymerase (referred to as nodavirus
Protein A), the accessory proteins (referred to as nodavirus Proteins B, for
example
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protein B 1 or protein B2) or the capsid protein (referred to as nodavirus
Protein a),
or peptide fragments thereof. Preferably however the vaccine molecules are DNA
molecules encoding such nodavirus component proteins or variants, or fragments
thereof (i.e. is a DNA vaccine). A particularly preferred vaccine molecule
comprises a DNA molecule encoding the capsid protein of a nodavirus or a
functionally equivalent variant, or a fragment thereof.
As described above for DNA vaccines in general, said DNA molecule can
take any appropriate form and can be produced by any appropriate method, e.g.
by
recombinant techniques. Said DNA molecule will generally be administered as
part
of an expression vector or plasmid, for example the open reading frame
encoding the
capsid protein (or other nodavirus derived protein), or variant, or fragment
thereof,
will be present downstream of an appropriate promoter sequence, e.g. CMV early
promoter or another appropriate promoter sequence. A preferred CMV containing
vector for use in this regard is the pcDNA vector (Invitrogen). Alternatively,
the
DNA could be administered in non-plasmid or non-vector form. For example, DNA
molecules encoding only the vaccine molecules without any vector components
can
be used.
The nodaviruses from which the vaccine molecules are derived may be of
any type, e.g. may be either alphanodaviruses or betanodaviruses.
Betanodaviruses
are preferred, in particular in cases where the organism which it is desired
to
vaccinate is a fish, betanodaviruses are preferred. Particularly preferred
examples of
genotypes of betanodaviruses which may be used are striped jack nervous
necrosis
virus (SJNNV), tiger puffer nervous necrosis virus (TPNNV), barfin flounder
nervous necrosis virus (BFNNV) and red spotter grouper nervous necrosis virus
(RGNNV). Some betanodaviruses do not belong to these four major groups, but
the
use of these is also included. An example of a nodavirus in this regard is
turbot
nodavirus (TNV) which is a preferred nodavirus from which the vaccine
molecules
of the invention can be derived. Most preferred nodaviruses are genotypes of
barfin
flounder nervous necrosis virus (BFNNV) and in particular AHNV (Atlantic
halibut
nodavirus). A particularly preferred nodavirus molecule to be used as a
vaccine
molecule is the capsid protein derived from AHNV, and more particularly DNA
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encoding said capsid protein, or a fragment thereof. A preferred vaccine
molecule
in this regard is pAHNV-C as described in the Examples.
Preferred organisms for use in embodiments of the invention where the
vaccine molecules are derived from nodavirus are fish, insects or crustaceans,
or any
other organisms (for example salmon) which can be infected by nodaviruses.
Fish
are preferred, in particular cadoids, groupers, flat fish (turbot, halibut),
sea bream,
seabass and striped jack. Flat fish are especially preferred.
The adjuvant component, i.e. the DNA molecule encoding the antigen,
preferably a rhabdovirus glycoprotein, and the vaccine molecule can be
administered at the same time (or substantially the same time), or
sequentially. If
administered at the same time, which is preferred, then optionally the DNA
adjuvant
component and the vaccine component may be administered as a mixture.
Preferably both the antigen encoding DNA molecule and the vaccine molecule are
administered at the same site, however it is also envisaged that different
sites may be
used. In addition, in aspects of the invention where the vaccine molecule is a
DNA
molecule, a single DNA molecule (e.g. a single plasmid or vector) encoding
both the
antigen and the vaccine molecule can be preferably administered. Where a
single
DNA molecule is used the antigen and the vaccine molecule might be expressed
under the control of the same or different promoter elements.
Thus, a yet further aspect of the invention provides a composition
comprising (i) an adjuvant component which comprises a DNA molecule encoding
an antigen which is expressed on the surface of a host cell (preferably a
rhabdovirus
glycoprotein) or a variant, or a fragment thereof, and (ii) a vaccine
molecule. Said
compositions are suitable for use in the methods and uses described herein.
Thus,
said compositions may be vaccine compositions which are used to vaccinate
organisms or stimulate an immune response against a relevant bacterial or
viral
infection or against a relevant disease. Thus, the invention can also be seen
to
provide a vaccine composition for stimulating an immune response in an
organism
comprising (i) an adjuvant component which comprises a DNA molecule encoding
an antigen and (ii) a vaccine molecule. Said adjuvant components and vaccine
molecules and preferred examples thereof are as defined elsewhere herein.
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In addition, a method of stimulating an immune response in an organism is
provided comprising administering to said organism a vaccine composition as
defined above. Preferably, said immune response is generated against a
relevant
bacterial or viral infection or against a relevant disease depending on the
nature of
the vaccine molecule which is included in the vaccine composition.
The invention further provides the use of a vaccine composition comprising
(i) an adjuvant component which comprises a DNA molecule encoding an antigen
which is expressed on the surface of a host cell (preferably a rhabdovirus
glycoprotein) or a variant, or a fragment thereof, and (ii) a vaccine molecule
in the
manufacture of a medicament for use. in therapy, for example for stimulating
an
immune response or in vaccination.
Preferably the above described compositions are pharmaceutically
acceptable and may optionally contain a pharmaceutically acceptable carrier,
excipient or diluent.
A still further aspect of the invention provides a product comprising (i) an
adjuvant component which comprises a DNA molecule encoding an antigen which
is expressed on the surface of a host cell (preferably a rhabdovirus
glycoprotein) or a
variant, or a fragment thereof, and (ii) a vaccine molecule as a combined
preparation
for simultaneous, separate or sequential use in stimulating an immune response
or in
vaccination.
A yet further aspect of the invention provides a kit for use in stimulating an
immune response or in vaccination, said kit comprising:
(i) a DNA molecule encoding an antigen which is expressed on the
surface of a host cell (preferably a rhabdovirus glycoprotein) or a
variant, or a fragment thereof; and
(ii) a vaccine molecule.
Again a pharmaceutically acceptable excipient or diluent may be present in
either or both component (i) or (ii) of said kits, or may be supplied as a
separate
component. Components (i) and (ii) may be presented in the kit as a mixture or
as
separate components. Components (i) and (ii) may be part of the same molecular
entity, e.g. a single DNA molecule (e.g. a single plasmid or vector) might
encode
both components (i) and (ii).
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In a further aspect the invention provides a DNA molecule encoding an
antigen which is expressed on the surface of a host cell (preferably a
rhabdovirus
glycoprotein) or a variant, or a fragment thereof, together with a vaccine
molecule
for use in therapy, for example for stimulating an immune response or in
vaccination.
Put another way, the present invention provides compositions of the
invention for use in therapy, for example for stimulating an immune response
or in
vaccination.
In a further aspect the invention also provides the use of a DNA molecule
encoding an antigen which is expressed on the surface of a host cell
(preferably a
rhabdovirus glycoprotein) or a variant, or a fragment thereof, together with a
vaccine
molecule in the manufacture of a medicament for use in therapy, for example
for
stimulating an immune response or in vaccination.
Put another way, the present invention provides the use of compositions of
the invention in the manufacture of a medicament for use in therapy, for
example for
stimulating an immune response or in vaccination.
Processes for preparing compositions of the invention are also provided. A
preferred process in this regard involves mixing or otherwise combining a DNA
molecule encoding an antigen (preferably a rhabdovirus glycoprotein) or a
variant,
or a fragment thereof, with a vaccine molecule.
Thus, the compositions of the invention may be fomlulated in any
convenient manner according to techniques and procedures known in the art,
e.g.
using one or more appropriate carriers, excipients or diluents. The nature of
the
compositions and carriers or excipients or diluents may be selected in a
routine
manner according to for example the choice and desired route of
administration,
nature of molecules to be administered, etc. Appropriate effective doses of
the DNA
molecule encoding an antigen and the vaccine molecule to be administered can
be
readily determined in a routine manner according to for example the choice and
desired route of administration, the nature of molecules to be administered,
the
purpose of vaccination, type and weight of organism being vaccinated, nature
of
vaccine molecule. Doses, carriers, excipients and diluents are selected such
that
when administered to the organism in question, the DNA molecule encoding the
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antigen (preferably a rhabdovirus glycoprotein) or a variant, or a fragment
thereof,
can act as an adjuvant to stimulate an enhanced or increased immune response
to the
particular vaccine molecule in the composition. Preferably the immune response
is
such that protection against a challenge by the appropriate infective agent or
disease
5 is conferred. Examples of suitable doses of adjuvants and vaccine molecules
might
be in the order of nanogram amounts or may be higher doses such as 1-50 pg or
2-20 ug or 5-20 pg.
Preferred DNA molecules (adjuvant molecules) and vaccine molecules for
use in the above described embodiments are as described elsewhere herein. It
should
10 also be mentioned that the adjuvants as described herein could be
administered in
conjunction with multiple different vaccine molecules. Thus, in all aspects of
the
invention discussed herein, one or more types of vaccine molecule may be used.
Any appropriate mode of administration may be used to administer the DNA
molecules (adjuvant molecules), vaccine molecules, or compositions in
accordance
15 with the invention, e.g. injection (e.g. intramuscular, intravenous or
intraperitoneal
injection), infusion, topical administration, gene gun (particle bombardment
of
epidermis), scarification of the skin, immersion using DNA coated beads or
liposomes, use of ultrasound etc. However, injection and in particular
intramuscular
injection is preferred.
It can thus be seen that preferred methods and uses of the invention are
methods of vaccination or uses of the above described molecules in
vaccination.
The ultimate aim of any such vaccination method or use is to provide
protection
against a challenge by the relevant infective agent or disease, which is
determined
by the vaccine molecule component of the composition used. Such a protective
effect can be conferred prophylactically, e.g. by administering the active
agents or
vaccine compositions to organisms in advance of a challenge by the pathogenic
organism or disease. Alternatively, such a protective effect can be conferred
by
administering the active agents or vaccine compositions once the pathogenic
organism or disease is present in the organism to be vaccinated.
Preferably the protection provided by the methods and uses of the invention
is long term protection (for example protection which is still effective at
least 70
days after vaccination and preferably for the life time of the organism in
question)
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16
against challenge by the relevant disease or infective agent. The provision of
long
term protection by the adjuvants of the invention when combined with vaccine
molecules is surprising. Short term or early protection (for example
protection for
less than 70 days or less than 35 days or less than 8 days after vaccination)
is
however also acceptable, particularly if such a time scale is sufficient to
cover all or
a significant part of the lifespan of the organism in question, or, in farmed
organisms, is sufficient to protect the organism until it reaches the age of
culling.
Whilst not wishing to be bound by theory, it is believed that the adjuvants of
the invention are also effective to induce short term protection (preferably
as early
as 8 days after vaccination or earlier), against disease or infection. This is
extremely
advantageous as it means that the organisms in question are protected from
disease
from a very early time period and thus are protected from infection or disease
for a
proportion of the time during which long term protection is being established.
As discussed above, vaccines have been developed in some fields in order to
protect organisms from various types of infection and disease. However, as
such
vaccines often contain or encode part of the infective organism and the
vaccines
may persist in the organism in the long term after administration, then this
gives rise
to concerns with regard to human safety, particularly in farming applications
where
it is preferable that the final food product should not contain significant
amounts of
foreign molecules such as vaccine molecules.
A further important advantage of the DNA molecules encoding an antigen,
particularly a rhabdovirus glycoprotein, contemplated for use in the present
invention is that such molecules can stimulate quicker elimination of
coadministered
molecules such as vaccine molecules. This is extremely advantageous in both
the
health industry and in particular in the farming industry, where, if any
administered
vaccines can be eliminated prior to culling, this is seen as a great
advantage.
Surprisingly; it has been shown that administration of a DNA molecule
encoding a rhabdovirus glycoprotein can stimulate elimination of a
coadministered
DNA molecule which is not normally eliminated by the organism in question, or
increase the elimination rate of the coadministered DNA molecule. In this
regard, it
has been shown that administration of a DNA molecule encoding a rhabdovirus
glycoprotein (in the form of pVHSV-G) into fish muscle can result in the rapid
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17
decline of a coadministered DNA molecule encoding the luciferase gene (in the
form of pCMV-luc), which when administered alone remains in the organism for a
long time (DNA could still be detected at the site of injection 160 days post
injection).
Thus, a yet further aspect of the invention relates to the use of a DNA
molecule encoding an antigen which is expressed on the surface of a host cell
(preferably a rhabdovirus glycoprotein) or a variant, or a fragment thereof,
to
improve or increase the elimination of other foreign DNA molecules from an
organism. Such other foreign DNA molecules, for example vaccine molecules,
will
preferably have been coadministered with the DNA molecule encoding said
antigen,
however they may also be administered before or after the administration of
the
DNA molecule encoding said antigen. Preferably both the antigen encoding DNA
molecule and the foreign DNA molecule are administered at the same site,
however
it is also envisaged that different sites may be used.
Preferred DNA molecules encoding the antigens for use in this further aspect
of the invention are as discussed above. Preferably the other foreign DNA
molecule
is a vaccine molecule as described above. Thus, where a DNA molecule encoding
an antigen is used as an adjuvant for a vaccine molecule, then a further
advantage is
that, after the protective effect has been conferred, the elimination of said
vaccine
molecule is improved or enhanced.
Preferred compositions, organisms, modes of administration, etc for this
aspect of the invention are as described above. Preferred doses are also as
described
above, although higher doses, for example in the order of milligram amounts
(e.g. 1-
50 mg or 5-30 mg) can also be used for this aspect of the invention.
The invention thus further provides the use of a DNA molecule encoding an
antigen which is expressed on the surface of a host cell (preferably a
rhabdovirus
glycoprotein) or a variant, or a fragment thereof, in the manufacture of a
composition which can improve or increase the elimination of other foreign DNA
molecules from an organism.
The invention further provides a method of improving or increasing the
elimination of foreign DNA molecules from an organism, said method comprising
the administration of a DNA molecule encoding an antigen which is expressed on
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18
the surface of a host cell (preferably a rhabdovirus glycoprotein) or a
variant, or a
fragment thereof.
In such methods and uses the DNA molecule encoding an antigen is
preferably co-administered with said foreign DNA molecules which are to be
eliminated.
Said improvement, enhancement or increase in the elimination of foreign
DNA molecules in the presence of a DNA molecule encoding an antigen refers to
an
improvement, enhancement or increase in the elimination of such molecules as
compared to the elimination observed when said foreign DNA molecules are
administered alone, i.e. in the absence of the DNA molecule encoding an
antigen.
Preferably said improvement, enhancement or increase is statistically
significant.
Methods of determining the statistical significance of differences in
parameters are well known and documented in the art. For example herein a
parameter is generally regarded as significant if a statistical comparison
using an
appropriate statistical test such as a Student t-test shows a probability
value of
<0.05. Such improvement, enhancement or increase in elimination generally
results
in a faster total elimination time for said foreign DNA molecules. This is
clearly
advantageous. Advantageously, the DNA molecules encoding the antigen also show
increased elimination from the organism in question.
The invention will now be further described by way of the following non-
limited examples with reference to the following figure in which:
Figure 1 shows cumulative mortality curves of DNA vaccinated turbots
(diamonds) and control turbots (squares) challenged by an intra-muscular
injection
of AHNV at 70 days post vaccination.
EXAMPLES
Example 1. DNA encoding a rhabdovirus glycoprotein can act as an adjuvant
for a nodavirus DNA vaccine.
Preparation of nodavirus
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The AHNV challenge strain (AH95NorA) was propagated in the SSN-1 cell line as
obtained from the European Collection of Animal Cell Cultures (ECACC),
Salisbury, UK as described by Dannevig et al., (Dis. Aquat. Org. 2000; 43:183 -
9).
The virus titer was determined based on cytopathologic observation by end-
point
dilution (10-fold serial dilution) on SSN-1 cells cultured in 96-well cell
culture
plates (Falcon Primera), and TCID50 was determined using eight parallel wells
for
each dilution.
DNA vaccine (pAHN-C) preparation
The DNA vaccine containing the capsid-encoding region of AHNV was prepared as
described in Sommerset et al., (Vaccine 2003; 21:4661-4667). Briefly, the
capsid-
encoding region and part of the 3' UTR of AHNV RNA2 (Grotmol et al., Dis.
Aquat. Org. 2000; 39:79-88) was subcloned behind the early cytomegalovirus
promoter of pcDNA3.1 (+) (Invitrogen). The recombinant pcDNA3.1-AHNV-C
plasmid was transformed into Top 10 E. coli cells (Invitrogen) and verified by
DNA
sequencing. Large-scale preparations of the vaccine plasmid (called pAHNV-C)
were purified from overnight cultures by anion exchange chromatography
(EndoFreeTM Plasmid Kit, Qiagen). The presence of supercoiled plasmid was
verified by agarose gel electrophoresis, and DNA quality and quantity were
determined spectrophotometrically (A260/A280). The pAHNV-C was resuspended
in PBS to final concentrations of 0.5 mg/ml and 2 mg/ml respectively, and
stored at
-20 C until used.
Preparation of pVHSV-G
The DNA molecule encoding the VHSV glycoprotein (G), herein called pVHSV-G,
contains the G gene inserted downstream of the CMV promoter in the pcDNA3
vector (Invitrogen) as described in detail elsewhere (Lorenzen et al., Fish
Shellfish
Immunol., 1998; 8:261-270 and Heppell et al., Fish Shellfish Immunol., 1998;
8:271-286). Large-scale preparations of the plasmid were purified from
overnight
cultures by anion exchange chromatography (EndoFreeTM Plasmid Kit, Qiagen).
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The presence of supercoiled plasmid was verified by agarose gel
electrophoresis,
and DNA quality and quantity were determined spectrophotometrically
(A260/A280). The pVHSV-G was resuspended in PBS to final concentrations of 0.5
mg/ml and 2 mg/ml respectively, and stored at -20 C until used.
5
Vaccination:
In order to show the adjvant effect of pVHSV-G, 60 mixed sex turbots were
injected
i.m. with 10 l of a DNA vaccine composition containing 2.5 g pAHNV-C and 2.5
g pVHSV-G in PBS. At the time of vaccination the mean weight of the fish was
10 2.7 g. As a contro160 fish were injected i.m. with 10 .l PBS
Challenge:
10 weeks post vaccination the fish were labelled with VIE subcutaneously at
the
base of the tail or dorsal fin, weighed, challenged by two i.m. injections
with 50 l
15 nodavirus suspension containing a total of 106 TCID50 AHNV, and divided
into 2
tanks (30 fish from each group in each tank).
Results :
The two parallel tanks gave quite similar results. The total (both tanks)
cumulative
20 mortalities of DNA vaccinated (diamonds) and control fish (squares) is
shown in
Figure 1.
It can be seen that the fish vaccinated with pVHSV-G and pAHNV-C show a
significantly lower mortality rate (4/60 fish died, i.e. 6.7%) than the
control fish
which are not vaccinated (41/60 died, i.e. 68.3%). In this regard the
vaccinated fish
display almost complete protection against nodavirus infection when challenged
at
70 days post vaccination.
In a separate experiment reported in Vaccine 2003 (supra), when pVHSV-G alone
was administered, 5/49 fish (10.2%) died when challenged at 35 days post
vaccination, when pAHNV-C alone was administered, 13/49 fish (26.5%) died when
challenged at 35 days post vaccination, and in the control sample 13/49 fish
also
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21
died. It can thus be seen that pAHNV-C had no protective effect and that pVHSV-
G
had a relatively low protective effect compared to the control fish. In
contrast, in the
above described experiment, where a combination of pAHNV-C and pVHSV-G was
administered, the vaccinated fish showed more than a 10 fold increase in
protection
over the control fish, i.e. a significantly better protective effect was
observed.
Moreover, this significantly better protective effect was observed in fish
which were
challenged at 70 days post vaccination as opposed to 35 days post vaccination,
i.e. a
long term protective effect against nodavirus infection had been conferred.
Example 2. A DNA molecule encoding a rhabdovirus glycoprotein can enhance
the elimination of a co-administered DNA molecule.
Rainbow trout between 1-2 kg were injected at 6 sites at the left side with 25
mg
pCMV-luc and 6 sites at the right side with 25 mg pCMV-luc + 25 mg pVHSV-G
(as described above) (that means 2.5 x 10"copies of each plasmid per injection
site). The former plasmid expresses luciferase, the last expresses a
glycoprotein
from VHS virus, both under the control of a cytomegalovirus promoter.
At 0, 56, 111, 160, and 209 days post injection samples were taken from the
site of
injection. DNA was isolated by means of the DNeasy kit (Qiagen) and analysed
by
PCR with two primer sets, one specific for pCMV-luc, the other specific for
pVHSV-G.
From the left side the pCMV-luc plasmid DNA could be detected 160 days post
injection. From the right side a strong signal was obtained for both plasmids
at day
0, and some weak signal for pCMV-luc at day 56 post injection.
This shows that when pCMV-luc is injected alone, it will persist for a long
time at
the site of injection. However, when pCMV-luc is injected together with pVHSV-
G,
both plasmids will disappear much faster. The explanation for this may be that
the
pVHSV-G plasmid trigger some kind of a immune response that will destroy the
muscle cell and cause the plasmid to leak out.
