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Novel sea lice vaccine.
The present invention relates to a 200 kD protein, a 180 kD protein, a 100/85
kD
protein and a 79 kD protein. The invention further relates to nucleic acid
sequences
encoding a 200 kD protein, a 180 kD protein, a 100/85 kD protein and a79 kD
protein,
to vaccines comprising these proteins or a nucleic acid sequences encoding the
proteins, to DNA fragments, recombinant DNA molecules, live recombinant
carriers
and to host cells comprising such nucleic acid sequences, to vaccines
comprising such
DNA fragments, recombinant DNA molecules, live recombinant carriers and to
host
cells comprising such nucleic acid sequences, to methods for the preparation
of such
vaccines and to the use of such protein or nucleic acid sequences encoding
such
protein in vaccines and for the manufacture of a vaccine for combating sea
lice
infection in salmonids.
Sea lice form a group of ectoparasites that have marine fish as host. They are
in some
cases host specific. Merely as an example; the salmon louse (Lepeophtheirus
salmonis
(Kroyer)) is host specific in that it infects salmonids only. However, most
sea lice are
to a variable degree not host specific, infecting different marine fish
including
salmonids and cods. Caligus rogercresseyi is mostly found on salmonids while
Caligus curtus is mostly found on non-salmonids. One of the least host
specific sea
lice, Caligus elongatus, has been isolated from more than 80 different species
of fish.
The salmon louse Lepeophtheirus salmonis is a marine ectoparasitic copepod
feeding
on skin, mucus and blood of salmonid hosts. The louse has ten developmental
stages
of which two stages are free living in the water, one is infectious and seven
stages are
parasitic (reviewed in Pike, A.W. and Wadsworth, S.L., Adv. Parasitol. 44: 233-
337
(1999)).
Although under natural conditions both species are usually found in low
numbers, L.
salmonis can cause significant harm to both the wild Atlantic salmon (Salmo
salar)
and the sea trout (Salmo trutta). Salmo salar is grown more and more
frequently in
aquaculture, leading to a high local density of hosts for sea lice. As a
consequence,
infection with sea lice of salmon in aqua-farming is clearly increasing.
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For Caligus infections in cod, the same will happen in the foreseeable future,
due to
the increasing importance of cod farming.
Newly started cod production amounted to 605 tons only. However, given the
fact that
only in the UK, cod consumption is 170.000 tons yearly, and given the sharp
reduction in European catch limits, it is clear that cod farming commercially
becomes
more and more attractive.
Louse infection as such is seldom the direct cause of illness and death, since
sea lice
feed primarily on mucus and skin. This however causes damage to both mucus and
skin, making the host more vulnerable to opportunistic infections. These
opportunistic
infections thus are the direct cause of increasingly significant economic
losses.
Classical treatment with organophosphate pesticides such as Nevugon and
Aquagard
is no longer an option. This is due to the fact that lice are developing an
increasing
resistance to such pesticides, and to the fact that such pesticides are no
longer
acceptable from an environmental point of view.
Therefore, vaccines against sea lice are highly desirable. Up till now,
however, no
such vaccines are on the market.
The search for suitable vaccine candidates has been going on for many years
already.
This approach has however so far not led to vaccines for combating sea lice
infection.
It is an objective of the present invention to provide a novel vaccine that is
capable of
inducing in susceptible fish such as i.a. Salmo salar, Salmo trutta and cod, a
degree of
protection against sea lice infection and to the effects of the infection.
Surprisingly it was found now, that several proteins could be isolated from
the eggs of
adult egg producing sea lice, more specifically Lepeophtheirus salmonis,
Caligus
rogercresseyi, Caligus elongatus and Caligus curtus, that are capable of
inducing
antibodies in Salmo salar, Salmo trutta and cod to the extent that fish
vaccinated with
these proteins are to an efficient degree protected against sea lice infection
and the
effects thereof.
Even more surprisingly, a strong cross-reactivity between antibodies against
the 200
kD protein, the 180 kD protein, the 100/85 kD protein and the 79 kD protein
respectively of one species and the homologous 200 kD protein, the 180 kD
protein,
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the 100/85 kD protein and the 79 kD protein in other species appered to exist.
Antibodies raised against this 200 kD protein, the 180 kD protein, the 100/85
kD
protein and the 79 kD protein from e.g. Lepeophtheirus salmonis additionally
appeared to react strongly in a Western blot with homologous proteins from
Caligus
curtus and Caligus rogercresseyi.
As will be further clarified below, a first embodiment of the present
invention relates
to this 200 kD protein.
A second embodiment of the present invention relates to this 180 kD protein.
A third embodiment of the present invention relates to this 100/85 kD protein.
A fourth embodiment of the present invention relates to this 79 kD protein.
It is well-known in the art, that many different nucleic acid sequences can
encode one
and the same protein. This phenomenon is commonly known as wobble in the
second
and especially the third base of each triplet encoding an amino acid. This
phenomenon
can result in a heterology of about 30% for two nucleic acid sequences still
encoding
the same protein. Therefore, two nucleic acid sequences having an overall
sequence
identity as low as 70 % can still encode one and the same protein.
It will also be understood that, for the particular proteins embraced herein,
natural
variations can exist between individual sea lice strains. These variations may
be
demonstrated by (an) amino acid difference(s) in the overall sequence or by
deletions,
substitutions, insertions, inversions or additions of (an) amino acid(s) in
said
sequence. Amino acid substitutions which do not essentially alter biological
and
immunological activities, have been described, e.g. by Neurath et al. in "The
Proteins"
Academic Press New York (1979). Amino acid replacements between related amino
acids or replacements which have occurred frequently in evolution are, inter
alia,
Ser/Ala, Ser/Gly, Asp/Gly, Asp/Asn, Ile/Val (see Dayhof, M.D., Atlas of
protein
sequence and structure, Nat. Biomed. Res. Found., Washington D.C., 1978, vol.
5,
suppl. 3). Other amino acid substitutions include Asp/Glu, Thr/Ser, Ala/Gly,
Ala/Thr,
Ser/Asn, Ala/Val, Thr/Phe, Ala/Pro, Lys/Arg, Leu/Ile, Leu/Val and Ala/Glu.
Based on
this information, Lipman and Pearson developed a method for rapid and
sensitive
protein comparison (Science,227, 1435-1441, 1985) and determining the
functional
similarity between identical proteins. Such amino acid substitutions of the
exemplary
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embodiments of this invention, as well as variations having deletions and/or
insertions
are within the scope of the invention as long as the resulting proteins retain
their
immune reactivity. This explains why sea lice proteins according to the
invention,
when isolated from different field isolates, may have identity levels of about
70%,
while still representing the same protein with the same immunological
characteristics.
Those variations in the amino acid sequence of a certain protein according to
the
invention that still provide a protein capable of inducing an immune response
against
infection with sea lice or at least against the clinical manifestations of the
infection are
considered as "not essentially influencing the immunological characteristics".
Therefore, the protein according to the invention comprises those proteins and
immunogenic fragments thereof that have an amino acid sequence that is at
least 70%
identical to the amino acid sequence of the 200 kD protein as depicted in SEQ
ID NO:
2, the 180 kD protein as depicted in SEQ ID NO: 4, the 100/85kD protein as
depicted
in SEQ ID NO: 6 or the 79 kD protein as depicted in SEQ ID NO: 8.
Thus, one embodiment of the present invention relates to a 200 kD protein or
an
immunogenic fragment thereof, said protein or immunogenic fragment thereof
having
an amino acid sequence that is at least 70% identical to the amino acid
sequence as
depicted in SEQ ID NO: 2.
An immunogenic fragment is a fragment that has a length of at least 50 amino
acids.
The concept of immunogenic fragments will be defined below.
Preferably, the amino acid sequence of a 200 kD protein or an immunogenic
fragment
of that protein has at least 75%, or more preferably 80% identity with the
amino acid
sequence of SEQ ID NO: 2. Even more preferred is a identity level of 85%, 90%,
92%, 94%, 95% 96%, 97%, 98%, 99% or even 100% in that order of preference.
Therefore, a preferred form of this embodiment relates to a 200 kD protein or
an
immunogenic fragment of said protein according to the invention wherein said
protein
or immunogenic fragment thereof has a sequence identity of at least 70%,
preferably
75%, more preferably 80% or even 85%, 90%, 92%, preferably 94%, more
preferably
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95%, even more preferred 96%, 97%, 98%, 99% or even 100% in that order of
preference identity with the amino acid sequence of SEQ ID NO: 2.
A most preferred form of this embodiment relates to a 200 kD protein or an
5 immunogenic fragment of said protein, according to the invention as encoded
by a
nucleic acid sequence described in SEQ ID NO: 1.
The level of protein identity can e.g. be determined with the computer program
"BLAST 2 SEQUENCES" by selecting sub-program: "BLASTP", that can be found at
www.ncbi.nlm.nih.l!ov/blast/bl2seg/b12.htm1.
A reference for this program is Tatiana A. Tatusova, Thomas L. Madden FEMS
Microbiol. Letters 174: 247-250 (1999). Matrix used: "blosum62". Parameters
used
are the default parameters:
Open gap: 11. Extension gap: 1. Gap x_dropoff: 50.
Amino acid sequences that comprise tandem arrays of the sequences according to
the
invention are also within the scope of the invention.
A second embodiment of the present invention relates to a 180 kD protein or an
immunogenic fragment thereof, said protein or immunogenic fragment thereof
having
an amino acid sequence that is at least 70% identical to the amino acid
sequence as
depicted in SEQ ID NO: 4.
Preferably, the amino acid sequence of the 180 kD protein or an immunogenic
fragment of that protein has at least 75%, or more preferably 80% identity
with the
amino acid sequence of SEQ ID NO: 4. Even more preferred is a identity level
of
85%, 90%, 92%, 94%, 95% 96%, 97%, 98%, 99% or even 100% in that order of
preference.
Therefore, a preferred form of this embodiment relates to a 180 kD protein or
an
immunogenic fragment of said protein according to the invention wherein said
protein
or immunogenic fragment thereof has a sequence identity of at least 70%,
preferably
75%, more preferably 80% or even 85%, 90%, 92%, preferably 94%, more
preferably
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95%, even more preferred 96%, 97%, 98%, 99% or even 100% in that order of
preference identity with the amino acid sequence of SEQ ID NO: 4.
A most preferred form of this embodiment relates to a 180 kD protein or an
immunogenic fragment of said protein, according to the invention as encoded by
a
nucleic acid sequence described in SEQ ID NO: 3.
A third embodiment of the present invention relates to a 100/85 kD protein or
an
immunogenic fragment thereof, said protein or immunogenic fragment thereof
having
an amino acid sequence that is at least 70% identical to the amino acid
sequence as
depicted in SEQ ID NO: 6.
Preferably, the amino acid sequence of the 100/85 kD protein or an immunogenic
fragment of that protein has at least 75%, or more preferably 80% identity
with the
amino acid sequence of SEQ ID NO: 6. Even more preferred is a identity level
of
85%, 90%, 92%, 94%, 95% 96%, 97%, 98%, 99% or even 100% in that order of
preference.
Therefore, a preferred form of this embodiment relates to a 100/85 kD protein
or an
immunogenic fragment of said protein according to the invention wherein said
protein
or immunogenic fragment thereof has a sequence identity of at least 70%,
preferably
75%, more preferably 80% or even 85%, 90%, 92%, preferably 94%, more
preferably
95%, even more preferred 96%, 97%, 98%, 99% or even 100% in that order of
preference identity with the amino acid sequence of SEQ ID NO: 6.
A most preferred form of this embodiment relates to a 100/85 kD protein or an
immunogenic fragment of said protein, according to the invention as encoded by
a
nucleic acid sequence described in SEQ ID NO: 5.
A fourth embodiment of the present invention relates to a 79 kD protein or an
immunogenic fragment thereof, said protein or immunogenic fragment thereof
having
an amino acid sequence that is at least 70% identical to the amino acid
sequence as
depicted in SEQ ID NO: 8.
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Preferably, the amino acid sequence of the 79 kD protein or an immunogenic
fragment of that protein has at least 75%, or more preferably 80% identity
with the
amino acid sequence of SEQ ID NO: 8. Even more preferred is a identity level
of
85%, 90%, 92%, 94%, 95% 96%, 97%, 98%, 99% or even 100% in that order of
preference.
Therefore, a preferred form of this embodiment relates to a 79 kD protein or
an
immunogenic fragment of said protein according to the invention wherein said
protein
or immunogenic fragment thereof has a sequence identity of at least 70%,
preferably
75%, more preferably 80% or even 85%, 90%, 92%, preferably 94%, more
preferably
95%, even more preferred 96%, 97%, 98%, 99% or even 100% in that order of
preference identity with the amino acid sequence of SEQ ID NO: 8.
A most preferred form of this embodiment relates to a 79 kD protein or an
immunogenic fragment of said protein, according to the invention as encoded by
a
nucleic acid sequence described in SEQ ID NO: 7.
As an readily be seen from the Examples, antibodies raised against e.g. the
200 kD
protein, the 180 kD protein, the 100/85 kD protein or the 79 kD protein as
isolated
from L. salmonis react strongly in a Western blot with the homologous protein
of e.g.
Caligus curtus or Caligus rogercresseyi. This already indicates that the
epitopes
against which the antibodies are directed, are well-conserved within the
copepod
ectoparasites.
Therefore, copepod ectoparasitic proteins from the eggs of adult egg producing
sea
lice that react in a Western blot with antiserum raised against a protein
having the
amino acid sequence as depicted in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6
and SEQ ID NO: 8 are also considered to fall within the scope of the
invention.
Specific examples of such ectoparasitic copepods are of course the species
Lepeophtheirus salmonis, Caligus curtus, Caligus elongatus and Caligus
rogercresseyi as mentioned above.
Since the nucleic acid sequences encoding the nove1200 kD protein, the 180 kD
protein, the 100/85 kD protein and the 79 kD protein according to the present
invention are disclosed here, it is now feasible to obtain this protein in
sufficient
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quantities. This can e.g. be done by using expression systems to express the
whole or
parts of the gene encoding the 200 kD protein, the 180 kD protein, the 100/85
kD
protein or the 79 kD protein. An essential requirement for the expression of
the
nucleic acid sequence is an adequate promoter functionally linked to the
nucleic acid
sequence, so that the nucleic acid sequence is under the control of the
promoter. It is
obvious to those skilled in the art that the choice of a promoter extends to
any
eukaryotic, prokaryotic or viral promoter capable of directing gene
transcription in
cells used as host cells for protein expression.
Functionally linked promoters are promoters that are capable of controlling
the
transcription of the nucleic acid sequences to which they are linked.
Constructs comprising the nucleic acid sequences encoding the 200 kD protein,
the
180 kD protein, the 100/85 kD protein or the 79 kD protein according to the
invention
under the control of a functionally linked promoter will be further referred
to as
recombinant DNA molecules.
Such a promoter can be the native promoter of the protein gene or another
promoter,
provided that that promoter is functional in the cell used for expression. It
can also be
a heterologous promoter. When the host cells are bacteria, useful expression
control
sequences which may be used include the Trp promoter and operator (Goeddel, et
al.,
Nucl. Acids Res., 8, 4057, 1980); the lac promoter and operator (Chang, et
al., Nature,
275, 615, 1978); the outer membrane protein promoter (Nakamura, K. and Inouge,
M., EMBO J., 1, 771-775, 1982); the bacteriophage lambda promoters and
operators
(Remaut, E. et al., Nucl. Acids Res., 11, 4677-4688, 1983); the a-amylase (B.
subtilis) promoter and operator, termination sequences and other expression
enhancement and control sequences compatible with the selected host cell.
When the host cell is yeast, useful expression control sequences include,
e.g., a-
mating factor. For insect cells the polyhedrin or p10 promoters of
baculoviruses can
be used (Smith, G.E. et al., Mol. Cell. Biol. 3, 2156-65, 1983). When the host
cell is
of vertebrate origin illustrative useful expression control sequences include
the
(human) cytomegalovirus immediate early promoter (Seed, B. et al., Nature 329,
840-
842, 1987; Fynan, E.F. et al., PNAS 90, 11478-11482,1993; Ulmer, J.B. et al.,
Science 259, 1745-1748, 1993), Rous sarcoma virus LTR (RSV, Gorman, C.M. et
al.,
PNAS 79, 6777-6781, 1982; Fynan et al., supra; Ulmer et al., supra), the MPSV
LTR
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(Stacey et al., J. Virology 50, 725-732, 1984), SV40 immediate early promoter
(Sprague J. et al., J. Virology 45, 773 ,1983), the SV-40 promoter (Berman,
P.W. et
al., Science, 222, 524-527, 1983), the metallothionein promoter (Brinster,
R.L. et al.,
Nature 296, 39-42, 1982), the heat shock promoter (Voellmy et al., Proc. Natl.
Acad.
Sci. USA, 82, 4949-53, 1985), the major late promoter of Ad2 and the (3-actin
promoter (Tang et al., Nature 356, 152-154, 1992). The regulatory sequences
may
also include terminator and poly-adenylation sequences. Amongst the sequences
that
can be used are the well known bovine growth hormone poly-adenylation
sequence,
the SV40 poly-adenylation sequence, the human cytomegalovirus (hCMV)
terminator
and poly-adenylation sequences.
Bacterial, yeast, fungal, insect and vertebrate cell expression systems are
very
frequently used systems. Such systems are well-known in the art and generally
available, e.g. commercially through Clontech Laboratories, Inc. 4030 Fabian
Way,
Palo Alto, California 94303-4607, USA. Next to these expression systems,
parasite-
based expression systems are attractive expression systems. Such systems are
e.g.
described in the French Patent Application with Publication number 2 714 074,
and in
US NTIS Publication No US 08/043109 (Hoffman, S. and Rogers, W.: Public. Date
1
December 1993).
In view of this, another embodiment of the invention relates to Live
Recombinant
Carriers (LRCs) comprising a nucleic acid sequence encoding the 200 kD
protein, the
180 kD protein, the 100/85 kD protein or the 79 kD protein or an immunogenic
part of
any of said proteins. Preferably, the LRC comprises a DNA fragment that in
turn
comprises a nucleic acid sequence encoding the 200 kD protein, a 180 kD
protein, a
100/85 kD protein or a 79 kD protein according to the invention or an
immunogenic
part thereof. More preferably, the nucleic acid sequence encoding the 200 kD
protein,
the 180 kD protein, the 100/85 kD protein or the 79 kD protein or an
immunogenic
part of any of said proteins is brought under the control of a functionally
linked
promoter. These LRCs are micro-organisms or viruses in which additional
genetic
information, in this case a nucleic acid sequence encoding the 200 kD protein,
the 180
kD protein, the 100/85 kD protein or the 79 kD protein, or an immunogenic
fragment
of any of said proteins as described above has been cloned. Fish infected with
such
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LRCs will produce an immunological response not only against the immunogens of
the carrier, but also against the immunogenic parts of the protein(s) for
which the
genetic code is additionally cloned into the LRC, e.g. the 200 kD protein, the
180 kD
protein, the 100/85 kD protein or the 79 kD protein described in the
invention.
5 As an example of bacterial LRCs, bacteria such as Vibrio anguillarum known
in the
art can attractively be used. (Singer, J.T. et al., New Developments in Marine
Biotechnology, p. 303-306, Eds. Le Gal and Halvorson, Plenum Press, New York,
1998).
10 Also, LRC viruses may be used as a way of transporting the nucleic acid
sequence
into a target cell. Viruses suitable for this task are e.g. alphavirus-
vectors. A review on
alphavirus-vectors is given by Sondra Schlesinger and Thomas W. Dubensky Jr.,
Current opinion in Biotechnology, 10:434-439 (1999).
Preferred viral LRC's are viruses from the genus Novirhabdoviruses, especially
the
species viral hemorrhagic septicemia virus, and infectious hematopoietic
necrosis
virus (IHNV). For instance IHNV is a fish pathogen, for which an attenuated
viral
expression and delivery system for use in salmonids has been described. (WO
03/097090; Biacchesi et al., 2000, J. of Virol., vol. 74, p. 11247-11253).
Deletion of
the viral NV protein attenuates the virus and creates room for insertion of a
foreign
gene. A preferred construct is a recombinant IHNV carrying a nucleic acid
construct
capable of encoding a polypeptide or protein according to the invention. Such
an LRC
is then administered to target fish for instance by immersion vaccination
The technique of in vivo homologous recombination, well-known in the art, can
be
used to introduce a recombinant nucleic acid sequence into the genome of a
bacterium, parasite or virus of choice, capable of inducing expression of the
inserted
nucleic acid sequence according to the invention in the host animal.
Finally another form of this embodiment of the invention relates to a host
cell
comprising a recombinant DNA molecule or a live recombinant carrier as
described
above.
A host cell may be a cell of bacterial origin, e.g. Escherichia coli, Bacillus
subtilis and
Lactobacillus species, in combination with bacteria-based plasmids as pBR322,
or
bacterial expression vectors as pGEX, or with bacteriophages. The host cell
may also
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be of eukaryotic origin, e.g. yeast-cells in combination with yeast-specific
vector
molecules, or higher eukaryotic cells like insect cells (Luckow et al; Bio-
technology
6: 47-55 (1988)) in combination with vectors or recombinant baculoviruses,
plant
cells in combination with e.g. Ti-plasmid based vectors or plant viral vectors
(Barton,
K.A. et al; Ce1132: 1033 (1983), mammalian cells like Hela cells, Chinese
Hamster
Ovary cells (CHO) or Crandell Feline Kidney-cells, also with appropriate
vectors or
recombinant viruses.
As is mentioned above, it is one of the merits of the present invention that
it was
found that the 200 kD protein, the 180 kD protein, the 100/85 kD protein and
the 79
kD protein each induce a degree of protection against sea lice species.
Therefore, the
200 kD protein, the 180 kD protein, the 100/85 kD protein and the 79 kD
protein each
constitute a major compound of a vaccine for the protection of fish against
fish sea
lice species. Therefore, another embodiment relates to vaccines for combating
sea lice
infection, comprising at least one or, preferably, more of these proteins.
When a protein is used for vaccination purposes or for raising antibodies, it
is not
necessary to use the whole protein. It is also possible to use a fragment of
that protein
that is capable, as such or coupled to a carrier such as e.g. KLH, of inducing
an
immune response against that protein, a so-called immunogenic fragment. An
"immunogenic fragment" is understood to be a fragment of the full-length
protein that
still has retained its capability to induce an immune response in a vertebrate
host, i.e.
comprises a B- or T-cell epitope. Shortly, an immunogenic fragment is a
fragment that
is capable of inducing antibodies that react with the full length protein,
i.e. the 200 kD
protein, the 180 kD protein, the 100/85 kD protein or the 79 D protein
according to
the invention. At this moment, a variety of techniques is available to easily
identify
DNA fragments encoding immunogenic fragments (determinants). The method
described by Geysen et al (Patent Application WO 84/03564, Patent Application
WO
86/06487, US Patent NR. 4,833,092, Proc. Natl Acad. Sci. 81: 3998-4002 (1984),
J.
Imm. Meth. 102, 259-274 (1987), the so-called PEPSCAN method is an easy to
perform, quick and well-established method for the detection of epitopes; the
immunologically important regions of the protein. The method is used world-
wide
and as such well-known to man skilled in the art. This (empirical) method is
especially suitable for the detection of B-cell epitopes. Also, given the
sequence of the
gene encoding any protein, computer algorithms are able to designate specific
protein
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12
fragments as the immunologically important epitopes on the basis of their
sequential
and/or structural agreement with epitopes that are now known. The
determination of
these regions is based on a combination of the hydrophilicity criteria
according to
Hopp and Woods (Proc. Natl. Acad. Sci. 78: 38248-3828 (1981)), and the
secondary
structure aspects according to Chou and Fasman (Advances in Enzymology 47: 45-
148 (1987) and US Patent 4,554,101). T-cell epitopes can likewise be predicted
from
the sequence by computer with the aid of Berzofsky's amphiphilicity criterion
(Science 235, 1059-1062 (1987) and US Patent application NTIS US 07/005,885).
A
condensed overview is found in: Shan Lu on common principles: Tibtech 9: 238-
242
(1991), Good et al on Malaria epitopes; Science 235: 1059-1062 (1987), Lu for
a
review; Vaccine 10: 3-7 (1992), Berzofsky for HIV-epitopes; The FASEB Journal
5:2412-2418 (1991).
Thus, one form of this embodiment of the invention relates to vaccines for
combating
sea lice infection, that comprise a 200 kD protein, a 180 kD protein, a 100/85
kD
protein or a 79 kD protein according to the invention or an immunogenic
fragment of
any of said proteins as described above, together with a pharmaceutically
acceptable
carrier.
Still another embodiment relates to the use of a 200 kD protein, a 180 kD
protein, a
100/85 kD protein or a 79 kD protein according to the invention or an
immunogenic
fragment of any of said proteins for the manufacturing of a vaccine for
combating sea
louse infections.
Vaccines based upon the 200 kD protein, the 180 kD protein, the 100/85 kD
protein or
the 79 kD protein, or an immunogenic fragment of any of said proteins can
easily be
made by admixing the protein or immunogenic fragments thereof with a
pharmaceutically acceptable carrier as described below.
Another possibility for such vaccines is a vaccine comprising a host cell as
described
above, and a pharmaceutically acceptable carrier.
Alternatively, a vaccine according to the invention can comprise live
recombinant
carriers as described above, capable of expressing the protein according to
the
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13
invention or immunogenic fragments thereof. Such vaccines, e.g. based upon a
Vibrio
carrier or a viral carrier e.g. an alphavirus vector have the advantage over
subunit
vaccines that they better mimic the natural way of infection of sea lice.
Moreover,
their self-propagation is an advantage since only low amounts of the
recombinant
carrier are necessary for immunization.
All vaccines described above contribute to active vaccination, i.e. they
trigger the
host's defense system. Alternatively, antibodies can be raised in e.g. rabbits
or can be
obtained from antibody-producing cell lines as described below. Such
antibodies can
then be administered to the fish. This method of vaccination, passive
vaccination, is
the vaccination of choice when an animal is already infected, and there is no
time to
allow the natural immune response to be triggered. It is also the preferred
method for
vaccinating animals that are prone to sudden high infection pressure. The
administered antibodies against the 200 kD protein, the 180 kD protein, the
100/85 kD
protein or the 79 kD protein according to the invention or immunogenic
fragments
thereof can in these cases bind directly to the protein of the sea lice. This
has the
advantage that it decreases the load of the sea lice infection.
Therefore, one other form of this embodiment of the invention relates to a
vaccine for
combating sea lice infection that comprises antibodies against the 200 kD
protein, the
180 kD protein, the 100/85 kD protein or the 79 kD protein, or an immunogenic
fragment of any of said proteins, and a pharmaceutically acceptable carrier.
The proteins or immunogenic fragments thereof, e.g. expressed as indicated
above can
be used to produce antibodies, which may be polyclonal, monospecific or
monoclonal
(or derivatives thereof). If polyclonal antibodies are desired, techniques for
producing
and processing polyclonal sera are well-known in the art (e.g. Mayer and
Walter, eds.
Immunochemical Methods in Cell and Molecular Biology, Academic Press, London,
1987).
Monoclonal antibodies, reactive against the protein according to the invention
or an
immunogenic fragment thereof according to the present invention, can be
prepared by
immunizing inbred mice by techniques also known in the art (Kohler and
Milstein,
Nature, 256, 495-497, 1975).
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Still another embodiment of this invention relates to antibodies against the
sea lice
protein described in the invention or against an immunogenic fragment of that
protein.
Methods for large-scale production of antibodies according to the invention
are also
known in the art. Such methods rely on the cloning of (fragments of) the
genetic
information encoding the protein according to the invention in a filamentous
phage
for phage display. Such techniques are described i.a. at the "Antibody
Engineering
Page" under "filamentous phage display" at htt ://axinitl.init.u.ni-
~'narburg.de/--rekJae ~hage.htrr~l., and in review papers by Cortese, R. et
al., (1994) in
Trends Biotechn. 12: 262-267., by Clackson, T. & Wells, J.A. (1994) in Trends
Biotechn. 12: 173-183, by Marks, J.D. et al., (1992) in J. Biol. Chem. 267:
16007-
16010, by Winter, G. et al., (1994) in Annu. Rev. Immunol. 12: 433-455, and by
Little, M. et al., (1994) Biotechn. Adv. 12: 539-555. The phages are
subsequently
used to screen camelid expression libraries expressing camelid heavy chain
antibodies. (Muyldermans, S. and Lauwereys, M., Journ. Molec. Recogn. 12: 131-
140
(1999) and Ghahroudi, M.A. et al., FEBS Letters 414: 512-526 (1997)). Cells
from
the library that express the desired antibodies can be replicated and
subsequently be
used for large scale expression of antibodies.
Still another embodiment relates to a method for the preparation of a vaccine
according to the invention that comprises the admixing of antibodies according
to the
invention and a pharmaceutically acceptable carrier.
An alternative and efficient way of vaccination is direct vaccination with DNA
encoding the relevant antigen. Direct vaccination with DNA encoding proteins
has
been successful for many different proteins. (As reviewed in e.g. Donnelly et
al., The
Immunologist 2: 20-26 (1993)). This way of vaccination is also attractive for
the
vaccination of fish against sea lice infection. Therefore, still other forms
of the
vaccine embodiment of the invention relate to vaccines comprising nucleic acid
sequences encoding a protein according to the invention or immunogenic
fragments
thereof, to vaccines comprising DNA fragments that comprise such nucleic acid
sequences and to recombinant DNA molecules comprising such nucleic acid
sequences.
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Examples of DNA fragments that are suitable for use in a DNA vaccine according
to
the invention are conventional cloning or expression plasmids for bacterial,
eukaryotic
and yeast host cells, many of said plasmids being commercially available. Well-
known examples of such plasmids are pBR322 and pcDNA3 (Invitrogen). The DNA
5 fragments or recombinant DNA molecules should be able to induce protein
expression
of the nucleic acid sequences. The DNA fragments or recombinant DNA molecules
may comprise one or more protein-encoding nucleic acid sequences. In addition,
the
DNA fragments or recombinant DNA molecules may comprise other nucleic acid
sequences such as the immune-stimulating oligonucleotides having unmethylated
10 CpG di-nucleotides, or nucleic acid sequences that code for other antigenic
proteins or
adjuvating cytokines.
Thus, both in view of their use in expression systems and their use in DNA
vaccination, another embodiment of the invention relates to nucleic acid
sequences
15 encoding an 200 kD protein or an immunogenic fragment of that protein
comprising a
nucleic acid sequence that has a identity of at least 70%, preferably 75%,
more
preferably 80% or even 85%, 90%, 92%, preferably 94%, more preferably 95%,
even
more preferred 96%, 97%, 98%, 99% or even 100% in that order of preference
identity with the the nucleic acid sequence depicted in SEQ ID NO: 1.
The percentage of identity between any nucleic acid and a nucleic acid
according to
the invention can be determined with the computer program "BLAST 2
SEQUENCES" by selecting sub-program: "BlastN" (T. Tatusova & T. Madden, 1999,
FEMS Microbiol. Letters, vol. 174, p. 247-250), that can be found at the
internet
address www.ncbi.nlm.nih.gov/blast/bl2seq/b12.htm1. Parameters that are to be
used
are the default parameters: reward for a match: +l; penalty for a mismatch: -
2; open
gap penalty: 5; extension gap penalty: 2; and gap x_dropoff: 50.
Unlike the output of the BlastP program described above, the BlastN program
does not list similarities, only identities: the percentage of nucleotides
that are
identical is indicated as "Identities".
Next to using computer algorithms for determining the level of
identity/mismatch
between any nucleic acid and a nucleic acid according to the invention,
experimental
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techniques can also be used. Especially by hybridisation under conditions of
controlled stringency.
The definition of stringent hybridisation conditions, as a function of the
identity
between two nucleotide sequences, follows from the formula for the melting
temperature Tm of Meinkoth and Wahl (1984, Anal. Biochem., vol. 138, p. 267-
284):
Tm =[81.5 C + 16.6(log M) + 0.41(%GC) - 0.61(%formamide) - 500/L] - 1 C/1%
mismatch
In this formula: M is the molarity of monovalent cations; %GC is the
percentage of
guanosine and cytosine nucleotides in the DNA; L is the length of the hybrid
in base
pairs; and "mismatch" is the lack of an identical match.
Washing conditions subsequent to the hybridization can also be made more or
less stringent, thereby selecting for higher or lower percentages of identity
respectively.
In general, higher stringency is obtained by reducing the salt concentration,
and increasing the incubation temperature. It is well within the capacity of
the skilled
person to select hybridisation conditions that match a certain percentage-
level of
identity as determined by computer analysis.
Merely as an example, and of course depending upon the G/C content and the
length
of the fragment: "stringent" washing conditions are conditions of 1 x SSC,
0.1% SDS
at a temperature of 65 C; highly stringent conditions refer to a reduction in
SSC
concentration towards 0.3 x SSC.
Another embodiment of the invention relates to nucleic acid sequences encoding
an
180 kD protein or an immunogenic fragment of that protein comprising a nucleic
acid
sequence that has a identity of at least 70%, preferably 75%, more preferably
80% or
even 85%, 90%, 92%, preferably 94%, more preferably 95%, even more preferred
96%, 97%, 98%, 99% or even 100% in that order of preference identity with the
the
nucleic acid sequence depicted in SEQ ID NO: 3.
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Another embodiment of the invention relates to nucleic acid sequences encoding
an
100/85 kD protein or an immunogenic fragment of that protein comprising a
nucleic
acid sequence that has a identity of at least 70%, preferably 75%, more
preferably
80% or even 85%, 90%, 92%, preferably 94%, more preferably 95%, even more
preferred 96%, 97%, 98%, 99% or even 100% in that order of preference identity
with
the the nucleic acid sequence depicted in SEQ ID NO: 5.
Another embodiment of the invention relates to nucleic acid sequences encoding
an
79 kD protein or an immunogenic fragment of that protein comprising a nucleic
acid
sequence that has a identity of at least 70%, preferably 75%, more preferably
80% or
even 85%, 90%, 92%, preferably 94%, more preferably 95%, even more preferred
96%, 97%, 98%, 99% or even 100% in that order of preference identity with the
the
nucleic acid sequence depicted in SEQ ID NO: 7.
For the purpose of the invention, stringent conditions are those conditions
under
which a nucleic acid still hybridises if it has a mismatch of 30 %; i.e. if it
is 70 %
identical to the (relevant part of the) nucleotide sequence depicted in SEQ ID
NO: 1,
SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7.
Therefore, if a nucleic acid hybridises under stringent conditions to the
nucleic
acid having a nucleotide sequence depicted in SEQ ID NO: 1, SEQ ID NO: 3, SEQ
ID
NO: 5 or SEQ ID NO: 7, it is considered a nucleic acid according to the
invention.
In a preferred form of these embodiments, the invention relates to DNA
fragments
comprising a nucleic acid sequence encoding the 200 kD protein, the 180 kD
protein,
the 100/85 kD protein or the 79 kD protein or an immunogenic part of any of
said
proteins. A DNA fragment is a stretch of nucleotides that functions as a
carrier for a
nucleic acid sequence according to the invention. Such DNA fragments can e.g.
be
plasmids, into which a nucleic acid sequence according to the invention is
cloned.
Such DNA fragments are e.g. useful for enhancing the amount of DNA for use as
a
primer and for expression of a nucleic acid sequence according to the
invention, as
described below.
The nucleic acid sequence according to the present invention or the DNA
plasmid
comprising a nucleic acid sequence according to the present invention,
preferably
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operably linked to a transcriptional regulatory sequence, to be used in the
vaccine
according to the invention can be naked or can be packaged in a delivery
system.
Suitable delivery systems are lipid vesicles, iscoms, dendromers, niosomes,
polysaccharide matrices and the like, (see further below) all well-known in
the art.
Also very suitable as delivery system are attenuated live bacteria such as
Vibrio
species, and attenuated live viruses such as alphavirus vectors, as mentioned
above.
A more preferred form of this embodiment of the present invention relates to a
recombinant DNA molecule comprising a nucleic acid sequence encoding a 200 kD
protein, the 180 kD protein, the 100/85 kD protein or the 79 kD protein or an
immunogenic part of any of said proteins wherein the nucleic acid sequence is
placed
under the control of a functionally linked promoter. Such recombinants can be
obtained by means of e.g. standard molecular biology techniques.
(Maniatis/Sambrook (Sambrook, J. Molecular cloning: a laboratory manual, 1989.
ISBN 0-87969-309-6).
Still other forms of this embodiment relate to recombinant DNA molecules, live
recombinant carriers and host cells comprising a nucleic acids sequence as
described
above for use in a vaccine.
Another embodiment of the present invention relates to vaccines comprising
recombinant DNA molecules, live recombinant carriers and host cells comprising
a
nucleic acids sequence as described above.
Again another embodiment of the present invention relates to the use of a
nucleic acid
sequence, a DNA fragment, a recombinant DNA molecule, a Live Recombinant
Carrier or a host cell as described above, for the manufacturing of a vaccine
for
combating sea louse infections.
DNA vaccines can easily be administered through intradermal application e.g.
using a
needle-less injector. This way of administration delivers the DNA directly
into the
cells of the animal to be vaccinated. Amounts of DNA in the range between 10
pg and
1000 g provide good results. Preferably, amounts in the microgram range
between 1
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and 100 g are used. Alternatively, animals can be dipped in solutions
comprising e.g.
between 10 pg and 1000 g per ml of the DNA to be administered.
In a further embodiment, the vaccine according to the present invention
additionally
comprises one or more antigens derived from fish pathogenic organisms such as
sea
lice, micro-organisms and viruses, antibodies against those antigens or
genetic
information encoding such antigens.
Of course, such antigens can be e.g. other sea lice antigens. Such an antigen
can also
be an antigen selected from other fish pathogenic organisms, micro-organisms
and
viruses. Such organisms and viruses are preferably selected from the group of
aquatic
birnaviruses such as infectious pancreatic necrosis virus (IPNV), aquatic
nodaviruses
such as striped jack nervous necrosis virus (SJNNV), aquatic rhabdoviruses
such as
infectious haematopoietic necrosis virus (IHNV) and viral haemorrhagic
septicaemia
virus (VHSV), Pancreas Disease virus (SPDV) and aquatic orthomyxoviruses such
as
infectious salmon anaemia virus and the group of fish pathogenic bacteria such
as
Flexibacter columnaris, Edwardsialla ictaluri, E. tarda, Yersinia ruckeri,
Pasteurella
piscicida, Vibrio anguillarum, Aeromonas salmonicida and Renibacterium
salmoninarum
All vaccines according to the present invention comprise a pharmaceutically
acceptable carrier. A pharmaceutically acceptable carrier can be e.g. sterile
water or a
sterile physiological salt solution. In a more complex form the carrier can
e.g. be a
buffer.
Methods for the preparation of a vaccine comprise the admixing of the 200 kD
protein, the 180 kD protein, the 100/85 kD protein or the 79 kD protein or an
immunogenic part of any of said proteins and/or antibodies against that
protein or an
immunogenic fragment thereof, and/or a nucleic acid sequence and/or a DNA
fragment, a recombinant DNA molecule, a live recombinant carrier or host cell
according to the invention, and a pharmaceutically acceptable carrier.
Vaccines according to the present invention may in a preferred presentation
also
contain an immunostimulatory substance, a so-called adjuvant. Adjuvants in
general
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comprise substances that boost the immune response of the host in a non-
specific
manner. A number of different adjuvants are known in the art. Examples of
adjuvants
frequently used in fish and shellfish farming are muramyldipeptides,
lipopolysaccharides, several glucans and glycans and Carbopol(R) (a
homopolymer).
5 An extensive overview of adjuvants suitable for fish and shellfish vaccines
is given in
the review paper by Jan Raa (Reviews in Fisheries Science 4(3): 229-288
(1996)).
The vaccine may also comprise a so-called "vehicle". A vehicle is a compound
to
which the protein adheres, without being covalently bound to it. Such vehicles
are i.a.
bio-microcapsules, micro-alginates, liposomes and macrosols, all known in the
art.
10 A special form of such a vehicle, in which the antigen is partially
embedded in the
vehicle, is the so-called ISCOM (EP 109.942, EP 180.564, EP 242.380)
In addition, the vaccine may comprise one or more suitable surface-active
compounds
or emulsifiers, e.g. Span or Tween.
15 Often, the vaccine is mixed with stabilisers, e.g. to protect degradation-
prone proteins
from being degraded, to enhance the shelf-life of the vaccine, or to improve
freeze-
drying efficiency. Useful stabilisers are i.a. SPGA (Bovamik et al; J.
Bacteriology 59:
509 (1950)), carbohydrates e.g. sorbitol, mannitol, trehalose, starch,
sucrose, dextran
or glucose, proteins such as albumin or casein or degradation products
thereof, and
20 buffers, such as alkali metal phosphates. Preferably, the vaccines
according to the
invention are in a freeze-dried form. Freeze-dried proteins and DNA have a
much
longer shelf-live, especially at room temperature, than when they are in a
liquid form.
The process of freeze-drying as such is extensively known in the art.
In addition, the vaccine may be suspended in a physiologically acceptable
diluent.
It goes without saying, that other ways of adjuvating, adding vehicle
compounds or
diluents, emulsifying or stabilizing a protein are also embodied in the
present
invention.
Vaccines according to the invention that are based upon the 200 kD protein,
the 180
kD protein, the 100/85 kD protein or the 79 kD protein or an immunogenic part
of any
of said proteins can very suitably be administered in amounts ranging between
1 and
100 micrograms of protein per animal, although smaller doses can in principle
be
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used. A dose exceeding 100 micrograms will, although immunologically very
suitable, be less attractive for commercial reasons.
Vaccines based upon live attenuated recombinant carriers, such as the LRC-
viruses
and bacteria described above can be administered in much lower doses, because
they
multiply themselves during the infection. Therefore, very suitable amounts
would
range between 103 and 109 CFU/PFU for bacteria and viruses.
Many ways of administration, all known in the art can be applied. The protein-
based
vaccines according to the invention are preferably administered to the fish
via
injection, immersion, dipping or per oral. The administration protocol can be
optimized in accordance with standard vaccination practice. Preferably the
vaccine is
administered via immersion or per oral, especially in case of commercial aqua
culture
farms.
For oral administration the vaccine is preferably mixed with a suitable
carrier for oral
administration i.e. cellulose, food or a metabolisable substance such as alpha-
cellulose
or different oils of vegetable or animals origin. Also an attractive way of
administration is administration of the vaccine to high concentrations of live-
feed
organisms, followed by feeding the live-feed organisms to the target animal,
e.g. the
fish. Particularly preferred food carriers for oral delivery of the vaccine
according to
the invention are live-feed organisms which are able to encapsulate the
vaccine.
Suitable live-feed organisms include plankton-like non-selective filter
feeders
preferably members of Rotifera, Artemia, and the like. Highly preferred is the
brine
shrimp Artemia sp..
A very elegant way of administration would be the following: bacteria, yeast
cells or
any other cell in which the protein according to the invention has been
synthesised are
directly fed to plankton-like non-selective filter feeders preferably members
of
Rotifera, Artemia, and the like. The pharmaceutical composition so made, and
comprising i.e. those bacteria, yeast cells or any other cell ingested by
plankton-like
non-selective filter feeders can then be administered orally to the
crustaceans to be
protected against viral infection.
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The following examples are illustrative for the invention and should not be
interpreted
as limitations of the invention.
EXAMPLES
Example 1:
1. Vaccination and challenge
Vaccine preparation
Salmon lice eggs were hatched in incubators with flowing seawater (34,5%o ,
20uM
filtered). After development to the infectious copepodid stage, lice were
added to
tanks containing Atlantic salmon (S. salar) and left natural development.
Adult egg
producing L. salmonis (Ls) females were collected with forceps from
anaesthetized
fish and oocytes harvested by puncturing the gonade section. Water-soluble
proteins
were extracted by resuspending oocytes from 501ice in 2.5 ml of cold
sonication
buffer (50 mM Tris-HC1 pH 7.5, 50mM NaC1 and 1mM EDTA) and eggs were
disrupted by sonication using a micro ultrasonic cell disrupter. The sonicated
extract
was clarified by centrifugation (13000g for 20 min at 4 C), pellet and lipids
discarded
and the supematant stored at -20 C.
The supematant total protein content was analyzed by SDS-PAGE and Coomassie
staining and quantified relative to known quantities of BSA.
The Ls protein extract were diluted to app. 5mg/ml (BSA equivalents) with
ddHzO and
added 0,1% (voUvol) of 37% formaldehyde. A 5mg/ml BSA control antigen was
prepared simultaneously. Vaccines were prepared by emulsification of 27% (w/w)
of
antigen in 67% ISA763 oil (Seppic).
Vaccination and challenge experiment
50 Atlantic salmon pre-smolt (app 40g) were vaccinated by individually
intraperitonal
injection of 150u1 vaccine (approximately 200ug protein). A control group
(n=50) was
vaccinated with the control vaccine containing BSA (200ug/dose). All fish were
kept
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at 10 C in one circular 1200 L tank. Clipping of the adipose fin
differentiated the two
groups. Fish were kept on natural light and were adapted to salt water during
week 9-
11 post vaccination. The groups were challenged (15 weeks post vaccination) as
one
population, by adding approximately 2,600 infective copepodids to the tank,
following reduction in water volume and flow. The fish were maintained in the
thank
for two weeks for parasite development into the immobile physically attached
Chalimus stage, and then transferred to three different 250 L tanks; Tank 1
and 2
contained control and vaccinated fish, respectively (n=25) and tank 3
contained a
mixed group of vaccinated and control vaccinated fish (n=25+25).
The experiment was terminated 11 weeks post challenge, three weeks after the
first
egg string was observed on adult female lice. Vaccine effect was evaluated by
comparing prevalence (percentage of fish infected) and the abundance (number
of
lice/fish). Furthermore, fish-pathology (external wounds) was compared between
tank
1(control) and tank 2 (vaccine) only. All fish were anaesthetized before
handling and
sampling of animals were conducted in accordance with national legislation.
2. Identification of genes encoding proteins in the vaccine
The protein content of the vaccine antigen was analyzed by SDS-PAGE, using 10-
20% Linear Gradient Ready Gel (BioRad) and commercially prepared running
buffer
and Coomassie stain. Protein band A was excised from the gel and internal
amino acid
sequence analysis was performed by EuroSequence bv, essentially as described
by
Rosenfeld et al. (Annal. Biochem. 203:173-179 (1992)). This includes in-situ
tryptic
digestion of the protein band, extraction of the peptides and RP-HPLC
separation of
the generated fragments. On purified fragments, identification of the step-
wise
released PTH-amino acid (Hewick et al., J. Biol. Chem. 256: 7990-7997 (1981))
fragments was determined using an Applied Biosystems Mode1494 Procise
Sequencing system, on-line connected to an RP-HPLC unit.
The internal peptide sequences were back-translated and used to search an
internal
database of salmon louse ESTs for gene transcripts encoding the peptide
sequences.
Following this, overlapping ESTs clones were identified and, supplemented by
RACE
clones, full-length cDNA sequences were assembled using Vector NTI 9.1.0
(Invitrogen). Blast searches were performed to identify protein domains and to
indicate gene function.
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3. Evaluation of protein antigenicity
Immunization of rabbits with purified protein
The 200 kD protein, the 180 kD protein, the 100 and 85 kD proteins and the 79
kD
protein were purified by preparative SDS-PAGE using a Prep Cell mode1491
(BioRad) according to the manufacturer instructions. Purified protein was
analysed by
SDS-PAGE and Coomassie staining, and quantified relative to BSA as described
above. One rabbit was immunized with the purified 200 kD protein, one with the
purified 180 kD protein, one with the purified 100 D protein, one with the
purified 85
kD protein and one with the purified 79 kD protein by Eurogentec at their
facility
using their immunisation protocol (boost at day 14, 28 and 56).
Immunisation of Atlantic salmon with all vaccine antigens
Vaccine antigen from L. salmonis was prepared as described above (5mg/ml)
mixed
1:1 with Freud's complete adjuvant and vortexed until homogeneity. 5 Atlantic
salmon (app 250g) were immunised by intraperitoneal injection of 150u1 (app.
375ug
protein). Blood were collected 9 weeks post immunisation and left at 4 C
overnight
for coagulation. Antisera were aliquoted and stored at -20 C until used.
Rabbit antisera analysis
Rabbit anti-200 kD protein antiserum, anti-180 kD protein antiserum, anti-100
kD
protein antiserum, anti-85 kD protein antiserum and anti-79 kD protein
antiserum
were analysed by Western blotting (using a blot comprising all egg proteins)
and by
ELISA using single purified proteins as antigen.
Briefly, Western blotting was performed by SDS-PAGE as described above
followed
by blotting onto nitrocellulose (150V for 45 min using 25mM Tris, 192mM
glycin,
20% methanol and cooling) and blocked for 1 hour at room temperature using 3%
non-fat dry milk (Nestle) in TBS-Tween. Nitrocellulose was incubated with
rabbit
antiserum and pre-serum control (1:1000) for 2 hours at room temperature
followed
by 3 washes with TBS-Tween. Secondary, nitrocellulose was incubated with
horseradish peroxidase conjugated goat anti rabbit antibody (1:2000, BioRad)
at room
temperature for 1 hour. Following 3 washes with TBS-Tween and 1 with TBS,
colorimetric detection was performed using premixed HRP-4CN substrates
according
to the manufacturer (BioRad).
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Briefly, ELISA was performed by coating Nunc immunosorbent plate wells with
100u1 of the 200 kD protein, the 180 kD protein, the 100 kD protein, the 85 kD
protein or the 79 kD protein purified from eggs (using the egg 35 kD protein
as
control) diluted to app 2ug/ml in coating buffer (15mM NazCO3, 35mM NaHCO3, pH
5 9.6), and incubate at 4 C over night. Wells were then washed twice with PBS-
Tween,
blocked with 5% non-fat dry milk (Nestle) in PBS-Tween (200u1/well) for 1 hour
at
room temperature and washed again. Twofold dilutions of rabbit antiserum (from
1:2000) were incubated (100uUwell) for 2 hours at room temperature followed by
3
washes with PBS-Tween. Secondary, horseradish peroxidase conjugated goat-anti-
10 rabbit antibody (1:3000, BioRad) were added (100uUwe11) and incubated at
room
temperature for 1 hour. Following 4 washes with PBS-Tween and 1 with PBS,
colour
was developed for 20 minutes using 150u1 o-phenylenediamine dihydrocloride
(0,4
mg/ml in phosphate-citrate buffer pH 5 added fresh H202 to 0,012%) to each
well.
Reaction was stopped by adding 50u12,5N H2SO4 before absorbance was measured
at
15 492nm.
Salmon antisera analysis
Antisera from 5 Atlantic salmons immunised with the vaccine antigens were
analysed
by ELISA using either purified egg 200 kD protein, 180 kD protein, 100 kD
protein,
20 85 kD protein or 79 kD protein as antigen. Briefly, this was performed as
described
for rabbit antisera analysis but diluting the antisera from 1:25 and
incubating at 4 C
over night. Furthermore, an incubation step with rabbit anti-salmon-Ig 0206
(1:6000,
1 hour at room temperature) was added between the salmon antisera and the
horseradish peroxidase conjugated goat-anti-rabbit antibody.
4. Identification of homolog antigen in other copepod ectoparasites.
Comparison of homolog antigen in other copepod ectoparasites
The protein content of external egg strings of L. salmonis (lab strain),
Caligus curtus
(4th generation lab strain kept on cod) and Caligus rogercresseyi (collected
from
salmon on-growing farm in Chile) was compared. Water-soluble egg proteins were
extracted from the egg strings (15u1 buffer/cm egg string) using sonication as
described for vaccine preparation. The supernatant total protein content was
analyzed
as described above using SDS-PAGE followed by Coomassie staining and by
Western
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26
blotting as described above, using the rabbit anti L. salmonis egg 200 kD
protein, 180
kD protein, 100 kD protein, 85 kD protein or 79 kD protein antiserum.
Results
1. Vaccine experiment
No differences in fish weight, length and condition factor were observed
between the
experimental groups (tanks 1-3), demonstrating equal conditions between the
tanks.
Both prevalence and abundance of adult female lice was lower in tank 2
(vaccine)
compared to the tank 1(control). Prevalence and abundance of male lice did not
differ
significantly between the two tanks, indicating an increased mortality of
female lice in
the vaccinated group (Fig 1). Furthermore, 80% of the vaccinated fish (tank 2)
had no
pathology, while 80% of the control fish (tank 1) had erosion or wounds (Fig
2).
In tank 3 (mixed groups), prevalence and abundance did not vary significantly
between vaccinated and control fish (Fig 1). However, on both vaccinated and
control
fish a skew sex ratio was observed, with increased number of males for each
female,
unlike the typical l:l ratio as seen in tank 1(Table 1). A skew sex ratio of
lice has
never been observed in the lab before independent of density of fish. A skew
sex ratio
was also observed in tank 2 were abundance and prevalence demonstrate vaccine
effect. This indicates that lice in tank 3 have jumped between hosts and
therefore all
lice in tank 3 may have fed partly on vaccinated fish.
Table 1. Total number of salmon lice and sex ratio in experimental groups of
vaccinated Atlantic salmon
Separate tanks (tank 1 and 2) Mixed tank (tank 3)
Control Vaccine Control Vaccine
Male 72 48 40 41
Female 71 16 16 10
Ratio male/female 1,0 2,7 2,5 4,1
2. Identification of genes encoding proteins in the vaccine preparation
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SDS-PAGE of unfertilized eggs from L. salmonis revealed 5 major bands with
approximate molecular weight of 200 kD, 160 kD, 100 kD, 85 kD and 35K kD,
called
egg-band A-E respectively (Fig 3).
Three peptide sequences were obtained for egg protein A, the 200 kD protein
according to the invention (Table 2) and EST's encoding the peptide sequences
were
identified in an in-house salmon louse EST database. The full-length cDNA
sequence
was successfully assembled based on overlapping ESTs and RACE clones. An in
silico analysis of the egg-band A encoding cDNA sequence V6x (SEQ ID NO: 1),
revealed that it is a protein that possesses key features of a Vitelogenin
(Table 2).
Band A in the oocyte is approximately 200 kD, which correlates well with the
V6X
ORF and with Northern blot transcript analysis (data not shown).
Table 2. The peptide sequences obtained from isolated egg-band A and the
identified
gene that encodes egg-band A. Indications of protein-domains are also given.
Table 2.
Mw Internal peptide Identified cDNA / aa Signal Protein domain
isolated sequences Gene length peptide
protein
200 kD 1. YSPSYYGXAPXL V6x* 6,093* / Yes LPD-N + VWD
2. KDETLLEAFVSR 1,965
3. TXGNLFMEYPE
* See SEQ ID NO: 1 for cDNA sequence
Three peptide sequences were obtained for egg protein B, the 180 kD protein
according to the invention (Table 3) and EST's encoding the peptide sequences
were
identified in an in-house salmon louse EST database. The full-length cDNA
sequence
was successfully assembled based on overlapping ESTs and RACE clones. An in
silico analysis of the egg-band B encoding cDNA sequence Vlx (SEQ ID NO: 3),
revealed that it is a protein that possesses key features of a Vitelogenin.
Band B in the
oocyte is approximately 180 kD, which correlates well with the V6X ORF and
with
Northern blot transcript analysis (data not shown).
Table 3. The peptide sequences obtained from isolated egg-band B and the
identified gene
that encodes egg-band B. Indications of protein-domains are also given.
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Mw Internal peptide Identified cDNA / aa Signal Protein domain
isolated sequences Gene length peptide
protein
180 kD 1 GYGGEYSYXVIGS Vlx* 4,747* / Yes LPD-N + VWD
2 EGYLATGQFFEXD 1,521
3 TGLLPYWDIDPEI
Table 3.
* See SEQ ID NO: 3 for cDNA sequence
One peptide sequence was obtained for egg protein C and one peptide sequence
was
obtained for egg protein D (Table 4) and EST's encoding the peptide sequences
were
identified in an in-house salmon louse EST database. A full-length cDNA
sequence
was successfully assembled based on overlapping ESTs and RACE clones and
revealed that egg protein C and D are encoded by the same gene. According to
in silco
analysis this gene sequence encodes a protein that possesses key features of a
Vitelogenin. The molecular weight of the egg proteins and C and D together is
approximately 185 Kda, which correlates well with the open reading frame of
the
gene and with Northern blot transcript analysis. However, in the L. salmonis
oocyte
the protein encoded by this gene is apparently processed into one N-terminal
part
(band D) and one C-terminal part (band C).
Mw Internal peptide Identified cDNA / aa Signal Protein domain
isolated sequences Gene length peptide
protein
100 kD QGGSTLXSXMPY C%* 5,948* / Yes LPD-N + VWD
85 kD LLSGIPGLRPHFSGIG 1,903
Table 4.
* See SEQ ID NO: 5 for cDNA sequence
One peptide sequences was obtained for egg protein E (Table 5) and EST
encoding
the peptide sequence was identified in an in-house salmon louse EST database.
The
full-length cDNA sequence was successfully assembled based on overlapping ESTs
and RACE clones. According to in silco analysis the eggband E cDNA sequence
(SEQ ID NO: 7) encodes a protein of unknown function with molecular weight of
79
KDa. This correlates well with Northern blot transcript analysis (data not
shown),
indicating that egg-band E (79 kDa) has been processed. The ORF encoded
protein
has a signal peptide and 3 fasciclin (FAS 1) domains, an extracellular domain
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suggested to represent an ancient cell adhesion domain common to plants and
animals.
Table 5. The peptide sequences obtained from isolated egg-band E and the
identified
gene that encodes egg-band E. Indications of protein-domains are also given.
Mw Internal peptide Identified cDNA / aa Signal Protein domain
isolated sequences Gene length peptide
protein
79 kD GTWFTPGLISGQSVK Band E* 2,511* / Yes FAS1 x3
722
Table 5.
* See SEQ ID NO: 7 for cDNA sequence
3. Evaluation of protein antigenicity
Each of the purified egg protein A, B, C, D and E (Fig 4-7) used for the
immunization
of rabbits induced high levels of specific antibodies, as can be seen from
figure 8-11.
These antibodies bind only and specifically to either the 200 kD protein, the
180 kD
protein, the 100 kD protein, the 85 kD protein or the 79 kD protein on a
Western blot
of gels comprising all proteins as antigen (Fig 12-15).
The vaccine antigens (all egg proteins A-E) induced, when used together for
the
immunization of Atlantic salmon, production of antibodies against the purified
200
kD protein, the 180 kD protein, the 100 kD protein, the 85 kD protein or the
79 kD
protein egg-band, as can be clearly seen from figure 16-19.
Conclusion: it follows from the experiments above that each of the proteins
according
to the invention; the 200 kD protein, the 180 kD protein, the 100 kD protein,
the 85
kD protein or the 79 kD protein of the egg protein preparation are capable of
inducing
protection against sea lice, are highly immunogenic and are recognized by both
antibodies raised against them in rabbits and antibodies raised in fish
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5 4. Identification of homolog antigens in other copepod ectoparasites
SDS-PAGE and Coomassie total protein staining of egg-proteins from L.
salmonis,
Caligus curtus and Caligus rogercresseyi showed that all three species have a
similar
egg protein pattern (Fig 20a-23a). Western blotting of the same egg proteins
demonstrated that each of the rabbit anti L. salmonis egg-bands A, B, C, D and
E,
10 corresponding to the 200 kD, 180 kD, 100 kD, 85 kD and 79 kD proteins
specifically
bind to a homolog protein in both Caligus species (Fig 20b, 21b, 22b, 22c,
23b).
Conclusion: it can be concluded that protection on the basis of the 200 kD,
180 kD,
100 kD, 85 kD and 79 kD protein according to the invention is not only
feasible
against L. salmonis, but equally well against Caligus spp.
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Legend to the figures
Figure 1. Prevalence (A) and abundance (B) of adult salmon lice on vaccinated
Atlantic salmon smolt 11 weeks post challenge (26 weeks post vaccination).
Figure 2. Frequency and level of salmon lice induced external pathology on
vaccinated (tank 2) and control vaccinated (tank 1) Atlantic salmon, 11 weeks
post
challenge with salmon louse.
Figure 3. SDS-PAGE and Coomassie brilliant blue (total protein stain) analysis
of
vaccine antigen preparation.
Figure 4. SDS-PAGE and Coomassie brilliant blue (total protein stain) analysis
of
purified egg-band A (lane 2) and crude vaccine antigen preparation (lane 3).
Molecular weight standard is to the left (lane 1).
Figure 5. SDS-PAGE and Coomassie brilliant blue (total protein stain) analysis
of
purified egg-band B (lane 2) and crude vaccine antigen preparation (lane 3).
Molecular weight standard is to the left (lane 1).
Figure 6. SDS-PAGE and Coomassie brilliant blue (total protein stain) analysis
of
purified egg band C (lane 2), D (lane 5) and crude vaccine antigen preparation
(duplicated in lanes 3 and 6). Molecular weight standards are in lanes 1 and
4.
Figure 7. SDS-PAGE and Coomassie brilliant blue (total protein stain) analysis
of
purified egg-band E (lane 1) and crude vaccine antigen preparation (lane 3).
Molecular weight standard is in lane 2.
Figure 8. Level of antibodies against egg-band A protein, in antisera from
rabbits
immunised with egg-band A or egg-band E (control serum), analysed by ELISA.
ELISA plates were coated with purified egg-band A protein.
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Figure 9. Level of antibodies against egg-band B protein, in antisera from
rabbits
immunised with eggband B or egg-band E (controlserum), analysed by ELISA.
ELISA plates were coated with purified egg-band B protein.
Figure 10. Level of antibodies aginst egg proteins C and D (figure A and B,
respectively), in antisera from rabbits immunised with egg band C, D or egg
band E
(controlserum). Sera were analysed by ELISA with plates coated with purified
eggband C or D (figure A and B, respectively).
Figure 11. Level of antibodies against egg-band E protein, in antisera from
rabbits
immunised with egg-band E or egg-band D (control serum), analysed by ELISA.
ELISA plates were coated with purified egg-band E protein.
Figure 12. Antigen-specificity of rabbit anti egg-band A antiserum (A2)
analyzed by
Western blotting using all vaccine proteins as antigen. A3 was incubated with
pre-
serum. The vaccine antigen used in the Western blotting (egg-band A-E) is
shown in
lane B2, a total protein stain of the SDS-PAGE. Identical molecular weight
standards
are shown in lane Al and Bl.
Figure 13. Antigen-specificity of rabbit anti egg-band B antiserum (A2)
analyzed by
Western blotting using all vaccine proteins as antigen. A3 was incubated with
pre-
serum. The vaccine antigen used in the Western blotting (egg-band A-E) is
shown in
lane B2, a total protein stain of the SDS-PAGE. Identical molecular weight
standards
are shown in lane Al and Bl.
Figure 14. Antigen-specificity of rabbit anti egg band C and anti egg band D
antiserum (A2 and A3 respectively) analyzed by Western blotting using all
vaccine
proteins as antigen. A4 was incubated with pre-serum. The vaccine antigen used
in the
Western blotting (egg band A-E) is shown in lane B2, a total protein stain of
the SDS-
PAGE. Identical molecular weight standards are shown in lane Al and Bl.
Figure 15. Antigen-specificity of rabbit anti egg-band E antiserum (A2)
analyzed by
Western blotting using all vaccine proteins as antigen. A3 was incubated with
pre-
serum. The vaccine antigen used in the Western blotting (egg-band A-E) is
shown in
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33
lane B2, a total protein stain of the SDS-PAGE. Identical molecular weight
standards
are shown in lane Al and Bl.
Figure 16. Level of antibodies against egg-band A protein, in antisera from 5
Atlantic
salmon immunised with the vaccine antigens. ELISA plates were coated with
purified
egg-band A protein. Control sera are from un-immunised salmon.
Figure 17. Level of antibodies against egg-band B protein, in antisera from 5
Atlantic
salmon immunised with the vaccine antigens. ELISA plates were coated with
purified
egg-band B protein. Control sera are from un-immunised salmon.
Figure 18. Level of antibodies against egg band C protein (a) and egg band D
protein
(b), in antisera from 5 Atlantic salmon immunised with the vaccine antigens.
ELISA
plates were coated with purified egg band C protein (a) and purified egg band
D
protein (b). Control sera are from un-immunised salmon.
Figure 19. Level of antibodies against egg-band E protein, in antisera from 5
Atlantic
salmon immunised with the vaccine antigens. ELISA plates were coated with
purified
egg-band E protein. Control sera are from un-immunised salmon.
Figure 20. Analysis of egg protein A in Caligus curtus (C. c) and Caligus
rogercresseyi (C. r) compared to Lepeophtheirus salmonis (L. s).
(a) SDS-PAGE and Coomassie staining was performed for total protein analysis
of all
egg proteins. (b) Western blotting was performed using rabbit anti L. s egg
protein A
to analyze for cross binding to homologue gene products in Caligus. (c) Rabbit
pre-
serum was used as negative Western blotting control. Lanes labeled Std contain
identical molecular weight standards.
Figure 21. Analysis of egg proteins (A-E) in Caligus curtus (C. c) and Caligus
rogercresseyi (C. r) compared to Lepeophtheirus salmonis (L. s).
(a) SDS-PAGE and Coomassie staining was performed for total protein analysis
of all
egg proteins. (b) Western blotting was performed using rabbit anti L. s egg
protein B
to analyze for cross binding to homologue gene products in Caligus. (c) Rabbit
pre-
. a J
34
serum was used as negative Western blotting control. Lanes labeled Std contain
identical molecular weight standards.
Figure 22. Analysis of egg proteins C and D in Caligus curtus (C. c) and
Caligus
rogercresseyi (C. r), compared to Lepeophtheirus salmonis (L. s).
(a) SDS-PAGE and Coomassie staining was performed for total protein analysis
of all
egg proteins. Western blotting was performed using rabbit anti L. s egg
protein C (b)
and D (c) to analyze for cross binding to homologue gene products in Caligus.
(d)
Rabbit pre-serum was used as negative Western blotting control. Lanes labeled
Std
contain identical molecular weight standards.
Figure 23. Analysis of egg protein E in Caligus curtus (C. c) and Caligus
rogercresseyi (C. r) compared to Lepeophtheirus salmonis (L. s).
(a) SDS-PAGE and Coomassie staining was performed for total protein analysis
of all
egg proteins. (b) Western blotting was performed using rabbit anti L. s egg
protein E
to analyze for cross binding to homologue gene products in Caligus. (c) Rabbit
pre-
serum was used as negative Western blotting control. Lanes labeled Std contain
identical molecular weight standards.
SEQUENCE LISTING IN ELECTRONIC FORM
In accordance with Section 111(1) of the Patent Rules, this description
contains a sequence listing in electronic form in ASCII text format (file:
30339-128 Seq 20-MAR-08 vl.txt).
A copy of the sequence listing in electronic form is available from the
Canadian Intellectual Property Office.
CA 02624486 2008-03-31
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NOTE: Pour les tomes additionels, veillez contacter le Bureau Canadien des
Brevets.
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