Immune responses in Atlantic salmon (Salmo salar) following protective vaccination against infectious salmon anemia (ISA) and subsequent ISA virus infection.

Abstract

Infectious salmon anemia (ISA) is an orthomyxoviral disease that has had devastating effects on farmed Atlantic salmon. ISA is still a disease resulting in continued loss of revenues and therefore development of effective vaccines is of great importance. Commercial vaccines against ISA are available, but the efficacy is poorly described. There is little information about vaccine-induced immune factors preventing ISA virus (ISAV) infection today. In this study we assessed the protective effects and immunogenicity of vaccines containing three different quantities of the inactivated ISAV antigen. Our findings indicated that immunization induced effective protection in Atlantic salmon with a relative percent survival (RPS) as high as 86. The level of protection was correlated to the amount of ISAV antigen in the vaccine, and fish immunized with high antigen amounts produced detectable ISAV-specific and neutralizing antibodies. While ISAV infection was detectable in non-vaccinated control fish challenged by cohabitation, no infection was detected in fish immunized with high antigen amounts. After challenge, transcriptional analysis of selected immune-related genes demonstrated activation of innate immune responses in ISAV-infected control fish, but not in vaccine protected fish. This study furthers the knowledge about vaccine efficacy and vaccine-induced immunity to ISAV challenge in Atlantic salmon.

Full text

Page 1

Vaccine
29 (2011) 6392–
6401
Contents
lists
available
at ScienceDirect
Vaccine
j ourna
l ho
me
pag
e:
www.elsevier.com/locate/vaccine
Immune
vaccination
infection
responses
in
Atlantic
salmon
(Salmo
salar) following
protective
against
Infectious
salmon
anemia
(ISA)
and
subsequent
ISA
virus
Astrid
Knut
Lauschera,∗,
Bjørn
Krossøyb,1,
Petter
Frostb,
Søren
Grovec,
Melanie
Königd,
Jon
Bohlina,
Falkc, Lars
Austbød,
Espen
Rimstada
aDepartment
bIntervet
cNorwegian
dDepartment
of Food
Safety
and
Infection
Biology,
The
Norwegian
School
of
Veterinary
Science,
P.O.
8146
Dep,
N-0033
Oslo,
Norway
Norbio
AS,
Thormøhlensgate
55,
N-5008
Bergen,
Norway
Veterinary
Institute,
P.O.
Box
750
Sentrum,
N-0106
Oslo,
Norway
of Basic
Science
and
Aquatic
Medicine,
The
Norwegian
School
of
Veterinary
Science,
P.O.
8146
Dep,
N-0033
Oslo,
Norway
a
r
t
i
c
l
e

i
n
f
o
Article
Received
Received
Accepted
Available online 7 May 2011
history:
2 March
2011
in revised
form
18
April
2011
19 April
2011
Keywords:
Infectious
ISAV
Vaccination
Antibody
Atlantic
salmon
anemia
response
salmon
a
b
s
t
r
a
c
t
Infectious
Atlantic
of
cacy
ISA
vaccines
immunization
high
immunized
While
tion
of
control
and
salmon
anemia
(ISA)
is
an
orthomyxoviral
disease
that
has
had
devastating
effects
on
farmed
salmon.
ISA
is still
a
disease
resulting
in
continued
loss
of
revenues
and
therefore
development
effective
vaccines
is
of
great
importance.
Commercial
vaccines
against
ISA
are
available,
but
the
effi-
is
poorly
described.
There
is
little
information
about
vaccine-induced
immune
factors
preventing
virus
(ISAV)
infection
today.
In
this
study
we
assessed
the
protective
effects
and
immunogenicity
of
containing
three
different
quantities
of
the
inactivated
ISAV
antigen.
Our
findings
indicated
that
induced
effective
protection
in
Atlantic
salmon
with
a
relative
percent
survival
(RPS)
as
as
86. The
level
of
protection
was
correlated
to
the
amount
of
ISAV
antigen
in
the
vaccine,
and
fish
with
high
antigen
amounts
produced
detectable
ISAV-specific
and
neutralizing
antibodies.
ISAV
infection
was
detectable
in
non-vaccinated
control
fish
challenged
by
cohabitation,
no
infec-
was
detected
in
fish
immunized
with
high
antigen
amounts.
After
challenge,
transcriptional
analysis
selected
immune-related
genes
demonstrated
activation
of
innate
immune
responses
in
ISAV-infected
fish,
but
not
in
vaccine
protected
fish.
This
study
furthers
the
knowledge
about
vaccine
efficacy
vaccine-induced
immunity
to
ISAV
challenge
in
Atlantic
salmon.
© 2011 Elsevier Ltd. All rights reserved.
1.
Introduction
Infectious
Atlantic
and
was
breaks
and
anemia
family
breaks
salmon
anemia
(ISA)
is
a systemic
disease
of
farmed
salmon
(Salmo
salar) that
has
had
impact
on
both
economy
productivity
of
the
Atlantic
salmon
aquaculture
industry.
ISA
first
described
in 1984
in Norway
and
since
then,
disease
out-
have
occurred
in Canada,
Scotland,
the
Faroe
Islands,
USA
Chile
[1–5]. The
causative
agent
of ISA is
the
infectious
salmon
virus
(ISAV),
the
prototype
virus
of
genus
Isavirus
in the
Orthomyxoviridae
[6–8]. Cumulative
mortality
in ISA
out-
varies
from
10%
to 95%
(reviewed
in [9]), but
intervention
∗Corresponding
E-mail
(B.
(S.
(J.
espen.rimstad@nvh.no
1Present
Norway.
author.
Tel.:
+47
22597420;
fax:
+47
22597309.
addresses:
astrid.lauscher@nvh.no
(A.
Lauscher),
bkrossoy@vaxxinova.no
Krossøy),
petter.frost@sp.intervet.com
(P.
Frost),
soren.grove@vetinst.no
Grove),
melanie.koenig@nvh.no
(M.
König),
jon.bohlin@nvh.no
Bohlin),
knut.falk@vetinst.no
(K.
Falk),
lars.austbo@nvh.no
(L.
Austbø),
(E.
Rimstad).
address:
Vaxxinova
Norway
AS,
Thormøhlensgate
55,
N-5008
Bergen,
by
allowed
ings
10%,
organs
In farmed
infectious
effective
be
vaccines.
extensively
nated
Little
by
responses
[13–16].
tral
For
cines
good,
fish
health
authorities
implies
that
outbreaks
are
normally
not
to run
to completion.
Clinical
signs
and
pathological
find-
in the
terminal
stage
are
severe
anemia
with
hematocrit
below
exophthalmia,
pale
gills,
ascites
and
hemorrhages
in several
suggesting
circulatory
failure
[5,9,10].
salmon,
vaccines
are
of
great
importance
to control
bacterial
diseases,
but
it
has
been
difficult
to develop
vaccines
against
viral
diseases
[11]. ISA
continues
to
a problem
in several
areas
despite
availability
of
commercial
The
protective
effects
of
these
vaccines
have
not
been
described.
However,
increased
survival
in ISAV
vacci-
salmon
has
been
reported
by
Jones
et al.
[12].
is known
about
the
mechanisms
that
induce
protection
immunization.
During
ISAV
infection
the
activation
of
innate
is evident
but
unable
to provide
sufficient
protection
Activation
of
adaptive
immunity
is assumed
to play
a cen-
role
for
host
survival
and
ISAV
clearance
[14,17].
influenza,
which
is also
caused
by
orthomyxoviruses,
vac-
based
on
inactivated
virus
antigens
are
known
to provide
but
not
long-lasting
protection.
Hemagglutinin
(HA)-specific
0264-410X/$
doi:10.1016/j.vaccine.2011.04.074
– see
front
matter ©

2011 Elsevier Ltd. All rights reserved.

Page 2

A.
Lauscher
et al.
/ Vaccine
29 (2011) 6392–
6401
6393
antibodies
can
titers
response
but
protection
in
serum
tance
to produce
strated
the
Gene
lar
However,
logical
be
transcripts
infection
ISAV.
The
taining
immune
immune
experimental
non-vaccinated
are
correlated
with
immunity
against
influenza
and
be
determined
by
measuring
hemagglutination
inhibition
in serum
after
immunization
[18,19]. Cell-mediated
immune
is
of
importance
in clearing
influenza
virus-infected
cells,
does
not
seem
to play
a crucial
role
in immunization-induced
[19]. Protection
against
ISA
was
partly
demonstrated
fish
that
were
exposed
to virus
by
intraperitoneal
injection
by
transfer
from
convalescent
fish,
and
explained
by the
impor-
of
humoral
factors
for
immunity
[17]. The
ability
of
salmon
neutralizing
antibodies
against
ISAV
has
been
demon-
in earlier
studies,
and
is presumed
to be
directed
against
hemagglutinin-esterase
(HE)
protein
[17,20,21].
transcription
analysis
by real-time
PCR
(qPCR)
is a popu-
method
for
the
characterization
of immune
responses
[22,23].
a correlation
between
detectable
mRNA
levels
and
bio-
activity
for
the
corresponding
protein
may
not
always
present
due
to post-transcriptional
regulations
[24]. Gene
in Atlantic
salmon
have
been
described
during
ISAV
[14,15,25],
but
so far
not
following
vaccination
against
aim
of
this
study
was
to test
the
efficacy
of
vaccines
con-
different
amounts
of
inactivated
ISAV
and
to examine
parameters
associated
with
protection
against
ISAV.
The
response
was
studied
after
vaccination
as
well
as
after
ISAV
infection
of vaccinated,
mock-vaccinated
and
fish.
2. Materials
and
methods
2.1.
Fish
Atlantic
fjorden,
months.
The
fish
fasted
by
for
On
salmon
(obtained
from
Urke
Fiskeoppdrett
AS,
Norangs-
Norway),
Aquagen
breed,
were
acclimatized
for
two
The
fish
were
kept
in a fresh
water
flow
system
at 12◦C.
water
conditions
were:
pH 6.6–6.9;
oxygen
> 65%
saturation;
density
< 40 kg/m3. The
fish
were
fed
according
to appetite
and
before
handling.
Prior
to handling,
all
fish
were
anesthetized
immersion
in Metacain
(Norsk
Medisinaldepot,
Oslo,
Norway)
a
period
of
2–5
min
(1 g/10
l water
buffered
with
1 g NaHCO3).
the
day
of
vaccination
the
average
weight
was
31
g.
2.2.
Vaccine
and
virus
The
ISAV
isolate
used
for
the
vaccine
preparation
was
Bremnes
98
infected
Inactivation
subsequent
tion
the
plates
diluted
up
monoclonal
mouse
stop
units
reading
formulated
(Seppic)
Vaccines
to
defined
4%
antigen
[26]. The
vaccine
antigen
was
produced
as
lysates
of
ISAV-
SHK-1
cells
inactivated
by
0.1%
formalin
at 4◦C for
7 days.
was
confirmed
by
inoculation
on
SHK-1
cells
in two
passages.
The
antigenic
content
of
the
antigen
prepara-
was
determined
by
an ELISA-based
antigenic
mass
assay
using
HE-specific
monoclonal
antibody
3H6F8
[20]. Briefly,
microtitre
were
coated
with
a rabbit
polyclonal
anti-ISAV
antibody
1:12,000
and
serial
dilutions
of antigen
(two-fold
diluted
from
antigen
dilution
1:200).
Antigen
was
then
detected
by
3H6F8
diluted
1:60
and
peroxidase
labeled
Rabbit
anti-
diluted
1:2500
using
standard
TMB
substrate
and
H2SO4to
the
reaction.
The
antigenic
strength
was
expressed
as
ELISA
with
the
antigen
dilution
(1280)
giving
an optical
density
(OD450 nm) of 1 as
the
Unit
definition/ml.
The
vaccine
was
as
a water-in-oil
emulsion
using
MontanideTMISA
763A
with
an
adjuvant/antigen
ratio
of 70/30
(weight/weight).
with
four
different
antigenic
strengths
were
made
in order
obtain
graded
vaccine
efficacy
[27]. The
graded
vaccines
were
as
ISAV
100%
(128
U/ml),
ISAV
20%
(25.6
U/ml),
and
ISAV
(5.1
U/ml).
As
mock
control
an
emulsion
with
PBS
instead
of
was
made.
2.3.
Experimental
design
and
samples
The
fish
were
randomly
picked
from
a reservoir
population
(n > 1500)
fish
were
tively,
(adjuvant
was
marked
experiments
60
induced
each
ity
handling.
(p.c.).
Cohabitant
natural
50
late
and
tion
trial
the
Serum,
ples
eight
ISAV
at
until
St.
tissues
lyzed.
for
ISAV
cause
fish
immunohistochemical
and
vaccinated
group
by
group
as
they
came
by hand
(60
per
group
in tank
I and
90 fish
per
group
in tank
II).
Groups
1–3
immunized
with
the
vaccine
ISAV
100%,
20%
and
4%,
respec-
while
fish
in groups
4 and
5 were
injected
with
mock
vaccine
control)
and
physiological
saline,
respectively.
One
dose
0.1
ml,
given
by
intraperitoneal
(i.p.)
injection.
The
groups
were
by
clipping
of
maxillae
and/or
adipose
fin.
Two
parallel
were
performed
in separate
tanks.
Tank
I, containing
vaccinated
fish
of
each
group,
was
used
for
monitoring
ISAV-
mortality,
while
tank
II, containing
90 vaccinated
fish
of
group,
was
used
for
sampling.
The
separate
tank
for
mortal-
monitoring
was
arranged
to exclude
influences
of
stress
due
to
Mortality
was
reported
daily
until
Day
45
post
challenge
infection
was
chosen
because
it better
imitates
the
route
of infection.
Six
weeks
post
vaccination
(wpv),
49 and
“shedder”
fish
infected
i.p.
with
0.1
ml
ISAV
of
a
Chilean
field
iso-
i.e.,
approximately
3 × 106TCID50/fish,
were
placed
into
tanks
I
II,
respectively.
The
infective
dose
of
ISAV
suitable
for
cohabita-
and
tank
parameters
had
been
determined
in a pre-challenge
using
the
same
population
density
and
water
flow
rate
as in
experiment.
heart,
head
kidney
and
gill
samples
were
taken.
Sam-
were
collected
from
eight
fish
before
vaccination,
and
from
fish
of each
group
at 3 and
6 wpv
but
prior
to challenge.
After
challenge,
samples
from
eight
fish
of
each
group
were
taken
0,
6,
9,
13
and
21
days
p.c.
(dpc).
Sera
were
stored
at
−80◦C
analysis
and
tissue
samples
were
stored
in RNAlater
(Sigma,
Louis,
MO,
USA)
at 4◦C and
processed
the
following
day.
Sera
and
of
the
eight
control
fish
taken
before
vaccination
were
ana-
Sera
from
sampling
at 6 wpv
and
6 and
21 dpc
were
analyzed
all
groups.
Tissues
of
all
samplings
were
analyzed
for
groups
100%,
adjuvant
control
and
saline.
To verify
that
ISA
was
the
of
death,
the
heart,
liver
and
kidney
of five
moribund
or dead
were
fixed
in 10%
buffered
formalin
for
histopathological-
and
(IHC)
examinations.
2.4.
ELISA
ELISA
was
used
to determine
ISAV-specific
antibodies
in salmon
sera.
mock-infected
CPE
PBS
at
until
Nunc)
ISAV-,
sodium
5%
added
salmon
(Amersham
of
temperature
bated
reactions
were
antigen
inate
six
non-immunized
respectively.
Antigens
for
the
ELISA
were
prepared
from
ISAV-infected
and
SHK-1
cells
in 75 cm2tissue
culture
flasks.
When
was
apparent,
cells
were
washed
twice
in PBS,
collected
in
using
cell
scrapers,
freeze-thawed
three
times,
centrifuged
16,000
× g for
15 min
to remove
debris,
and
stored
at
−80◦C
used.
Polystyrene
microtitre
plates
(Immuno-Plate
Maxisorp,
were
coated
overnight
at
4◦C with
100
?l/well
of
either
or
control
antigen
preparation
diluted
to 2.5
?g/ml
in 50 mM
carbonate
buffer,
pH 9.6.
After
washing
and
blocking
with
dry
milk
in 0.1%
PBS
Tween
20,
test
sera
diluted
1:200
were
and
incubated
at 4◦C overnight.
Polyclonal
rabbit
anti-
Ig and
subsequently
peroxidase
conjugated
anti-rabbit
Ig
Pharmacia,
Uppsala,
Sweden)
were
added
in dilutions
1:500
and
1:1000
in 0.1%
PBS
Tween
20,
and
incubated
at
room
for
60 min
and
45
min,
respectively.
Plates
were
incu-
with
o-phenylenediamine
dihydrochloride
substrate
in and
were
stopped
with
2.3
M H2SO4after
20 min.
Readings
measured
as
optical
density
(OD492 nm).
Values
of
the
control
were
subtracted
from
results
of
the
ISAV-antigen
to elim-
background
of
the
coating
antigen
itself.
Pools
of
sera
from
natural
ISAV-infected,
convalescent
fish
and
of
five
healthy,
fish
were
used
as
positive
and
negative
controls,
Both
controls
were
run
for
each
plate.
All
sera
were

Page 3

6394
A.
Lauscher
et al.
/ Vaccine
29 (2011) 6392–
6401
Table
Sequences
1
of
primer
and
probes
used
in real-time
PCR
analysis.
Gene

Sequence
5?→ 3?
GenBank
accession
no.
EF1?B
F-TGCCCCTCCAGGATGTCTAC
R-CACGGCCCACAGGTACTG
P-FAM-AAATCGGCGGTATTGG-MGBNFQ
F-CTACACAGCAGGATGCAGATGT
R-CAGGATGCCGGAAGTCGAT
P-FAM-CATCGTCGCTGCAGTTC-MGBNFQ
F-ACGCCTTGAAGAGCTGGATA
R-CTGGCTGGACTTGTGTCCTC
P-FAM-CCTGAAACCAGGAGTACTGATCGGGA-TAMRA
F-AGCAATGGCAGCATGATCAG
R-TGGTTGGTGTCCTCGTCAAAG
P-FAM-AGTGGTTCCAAACGTATGGCGAATACCTG-BHQ1
F-ACTGAAACGCTACTTCAAGAAGTTGA
R-GCAGATGACGTTTTGTCTCTTTCCT
P-FAM-TTGATGGGCTGGATGACTTTAGGA-TAMRA
F-AAGGGCTGTGATGTGTTTCTG
R-TGTACTGAGCGGCATTACTCC
P-FAM-TTGATGGGCTGGATGACTTTAGGA-MGBNFQ
F-GGAAGAGCACTCTGATGAGGACAG
R-CACCATGACTCCACTGGGGTAG
P-FAM-TCAGTGTCTCTGCTCCAGAAGACCCCCT-BHQ1
F-CCACCTGGAGTACACACCCAG
R-TTCCTCTCAGCCTCAGGCAG
P-FAM-TCCTGCATGGTGGAGCACATCAGC-BHQ1
F-GGAGGCCAGGAGTTCTTCTC
R-GGCTTGGGCTTCGTGACA
P-FAM-ACCCGGAGAAACTC-MGBNFQ
F-GCCCCTGAAGTCCAACGAC
R-AGGCTTCTCTCACTGCGTCC
P-FAM-CCGCACACTAGAGGGTCCACCACG-BHQ1
F-CTACAAGAGGGAGACCGGAG
R-AGGGTCACCGTATTATCACTAGTTT
P-FAM-TCCACAGCGTCCATCTGTCTTTC-BHQ1
F-CCTACAAGAGGGAGACCGA
R-GATGAAGGTGAAGGCTGTTTT
P-FAM-TGACTGACTGTCCATGCAGCAACACC-BHQ1
F-TGAACATCGCTGCTTCAAC
R-CCAGCACAGCACTGTCTCC
P-FAM-CACAGTTCTGATCAGGAGGTCTCTCCCA-BHQ1
F-GAATGTTTGGGACACGGAAG
R-TCACATATCTTGACATGAGTTACCC
P-FAM-CGCCGTCAGGCACGACAGCTT-BHQ1
F-GAATGTTTGGGACACGGAAG
R-GCTCAGTCAGTGGGATGTTCT
P-FAM-CGACAGCTTGGACAACAACACCGCAT-BHQ1
BG933897
ISAV
seg
8AY151791
RIG-Iab
FN178459
Viperin
NM001140939
IFN-?
AY216594
IFN-y
AY795563
MHCI
AF504022
MHCIIX70165
CD8b
AY693394
CD4-2aNM001128603
sIgM
Y12456.1
mIgM
S48658.1
IgD
AF141606.1
sIgT
GQ907004.1
mIgT
GQ907004.1
tested
determined
0.2
the
as
cut-off
≥9%
antibodies
in duplicates.
A titer
of
1:12,800
of
the
positive
control
was
by
end-point
titration
and
an absorbance
difference
of
between
ISAV-infected
and
control
cell
lysates
was
chosen
as
cut-off.
For
plate
normalization
the
test
sera
were
presented
a percentage
of
the
positive
control
run
on
the
same
plate.
The
is equal
to a percent
value
of
9 and
test
sera
with
a value
compared
to the
positive
control
reading
indicate
ISAV-specific
in sera.
2.5.
Neutralization
assay
(NA)
Neutralizing
activity
of
serum
was
measured
by
modified
NA
for
ISAV
lowing
96-well
bated
and
fixed
MAb
lar
were
relative
ISAV
[21,28]. In brief,
sera
were
diluted
two-fold,
mixed
with
(2.5
×102TCID50) and
incubated
at 4◦C overnight.
The
fol-
day
medium
was
added
and
each
mixture
dispersed
to
plates
with
SHK
cells
seeded
one
day
earlier
and
incu-
at
15◦C for
2 h.
After
the
addition
of
medium
with
2% FBS
incubation
at
15◦C for
three
days,
the
infected
cells
were
in 3.4%
paraformaldehyde
and
stained
with
the
ISAV
specific
3H6F8
[20]
and
Alexa
dye
488-labeled
anti-mouse
(Molecu-
Probes,
Carlsbad,
CA,
USA)
as
secondary
antibody.
The
results
read
in an inverted
epifluorescent
microscope
and
calculated
to virus-infected
control
wells.
Reciprocal
values
of
serum
dilutions
were
causing
a 50%
reduction
in the
number
of infected
cells
given
as
neutralizing
antibody
titer.
2.6.
RNA
extraction
and
cDNA
synthesis
RNA
was
extracted
using
the
RNeasy
96
Universal
Tissue
Kit
instructions,
quantified
Scientific,
genomic
(QuantiTect
ufacturer’s
(Qiagen,
Valencia,
CA,
USA)
according
to the
manufacturer’s
and
diluted
in RNase-free
water,
stored
at −80◦C and
spectrophotometrically
(Nanodrop
(ND-1000),
Thermo
Wilmington,
DE,
USA).
For
reverse
transcription
and
DNA
elimination,
1 ?g of
template
RNA
was
used
Reverse
Transcription
Kit,
Qiagen)
following
the
man-
recommendations.
cDNA
was
stored
at −20◦C until
use.
2.7.
Real-time
PCR
RT-qPCR
tem
system
well
kidney
were
the
was
performed
using
7900HT
Fast
Real-Time
PCR
sys-
(Applied
Biosystems,
Carlsbad,
CA,
USA)
or Mx
Pro
3000P
(Stratagene,
La Jolla,
CA,
USA)
to demonstrate
ISAV
RNA
as
as
transcripts
of
selected
immune
genes.
Tissue
of
heart,
head
and
gills
were
analyzed.
The
primer
and
probe
for
ISAV
RNA
obtained
from
Snow
et al.
[29]. The
tested
transcripts
were
retinoic
acid
inducible
protein
I (RIG-I);
the
virus
inhibitory

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29 (2011) 6392–
6401
6395
protein,
(Viperin)
I
(CD4);
sIgM);
immunoglobulin
the
ume
containing
cDNA
RNA.
for
60◦C for
used
ization
redesigned
endoplasmatic
reticulum
associated,
interferon
inducible
gene;
MHC
class
I (MHCI)
and
II (MHCII);
interferon
type
(IFN-?)
and
II (IFN-?);
cytotoxic
T cells
(CD8);
T helper
cells
membrane-bound
and
soluble
immunoglobulin
M
(mIgM;
immunoglobulin
D (IgD)
and
membrane-bound
and
soluble
T (mIgT;
sIgT).
Primer
and
probe
sequences
for
different
genes
are
listed
in Table
1.
To a
10
?l reaction
vol-
(TaqMan
Gene
Expression
Master
Mix,
Applied
Biosystems)
200
nM
primers
and
50
nM probe,
3 ?l of
1:16
diluted
were
added,
equivalents
to approximately
9 ng
of original
Reactions
were
run
in triplicates.
PCR
parameters
were
95◦C
10
min
followed
by 40
cycles
at 95◦C for
15 s,
58◦C for
15 s and
1 min.
To assess
PCR
efficiency,
template
dilutions
were
to generate
standard
curves
for
each
primer
pair.
For
normal-
of
transcription
levels
the
Elongation
Factor
1?B (EF1?B),
from
Olsvik
et al.
[30], was
used
as
reference
gene.
2.8.
Data
analysis
The
relative
percent
survival
(RPS)
was
calculated
by
the
following
group/cumulative
mortality
regression.
which
trol,
covariates
(global,
found
fit
the
was
similar
into
The
for
calculated
presented
(mean
but
group
For
log-transformed
sion
variable
interaction
explanatory
and
formula:
RPS
= (1 − cumulative
mortality
of
vaccinated
mortality
of
control
group
saline)
×100.
The
data
were
analyzed
using
Cox
proportional
hazard
The
model
was
fitted
with
a covariate
called
group,
consisted
of
the
following
categories:
saline,
adjuvant
con-
ISAV
4%,
ISAV
20%
and
ISAV
100%.
Proportionality
of the
was
assessed
using
both
a
Schoenfeld
residual
test
p ∼ 0.8)
and
a “log–log”
plot.
For
the
general
model
it was
that
R2= 0.2,
p < 0.001
(Wald
statistic),
and
the
validity
of the
was
confirmed
using
Cox-Snell
residuals.
It should
be
noted
that
shedder
fish
were
taken
out
of
the
model
and
that
the
model
left-truncated
to t = 15 dpc.
However,
significance
levels
were
for
the
vaccine
groups
when
the
shedder
fish
were
added
the
model
and
no left-truncation
carried
out.
threshold
cycles
(Ct)
in RT-qPCR
were
determined
manually
each
gene
and
tissue
and
set
equal
for
all
groups.
The
data
were
relative
to EF1?B.
Transcripts
of the
immune
genes
were
as
fold
expression
(=2−??Ct) [31]
relative
to Day
0 p.v.
of
eight
control
fish)
for
samples
collected
after
vaccination
prior
challenge,
and
to Day
0 p.c.
(mean
of eight
control
fish
of
saline)
for
samples
collected
after
challenge.
statistical
analysis
the
transcript
levels
of
each
gene
were
to decrease
the
variance
of
the
data.
Linear
regres-
was
subsequently
carried
out
for
each
gene
with
the
response
Y containing
log-transformed
transcript
levels
and
an
term
time
× group,
consisting
of
time
and
group,
as
an
variable.
The
following
regression
model
was
fitted
stratified
with
respect
to the
relevant
tissue:
log(YGENE) = a0+ a1time
a0represents
time
vaccination
and
carried
To
different
Wilcoxon–Mann–Whitney
hypothesis
normality
significant.
Association
expression
sion,
× group
+ ε
the
intercept,
a1is
the
estimated
coefficient
for
the
× group
factor
and
ε is the
error
term.
All
comparisons
after
but
prior
challenge
were
carried
out
against
time
0 wpv
the
eight
control
fish,
and
comparisons
after
challenge
were
out
against
time
0 dpc
and
group
saline.
compare
virus
RNA
levels
to the
covariate
representing
the
groups
on
13 and
21 dpc
for
each
tissue,
a
two-sample
signed
rank-sum
test
(under
the
null
of
no
true
shift
in location)
was
used
to the
non-
of
the
data.
p-Values
of
p ≤ 0.05
were
considered
as
measurements
between
ISAV
transcripts
and
gene
were
estimated
using
log-transformed
linear
regres-
i.e.:
log(YISAV) =
a0+
a1 log(Gene) +
ε
0
10
20
30
40
50
60
70
80
90
100
50 454035 3025 2015
Days post challenge
1050
shedders
saline
adjuvant control
ISAV 4%
ISAV 20%
ISAV 100%
Cumulative mortality
Fig.
lenge. Cumulative
vaccines
and
1. Mortality
curves
after
immunization
against
ISAV
and
subsequent
ISAV
chal-
mortality
of
shedder
fish,
three
groups
immunized
with
graded
(ISAV
100%,
ISAV
20%
and
ISAV
4%)
and
control
groups
(adjuvant
control
saline)
up to Day
45
post
ISAV
challenge
by cohabitation.
with
cept
expression
reported
tistical
(http://www.R-project.org).
YISAVrepresenting
the
level
ISAV
transcripts,
a0the
inter-
and
a1the
estimated
coefficient
for
the
log
transform
of gene
(Gene).
R2(coefficient
of
determination)
scores
were
as
measures
of
association/variance
explained.
All
sta-
analyzes
were
carried
out
using
the
statistical
program
R
3.
Results
3.1.
Mortality
Fig.
The
1 shows
cumulative
mortality
curves
for
tank
I.
mortality
of
shedder
fish
initiated
at 9 dpc
and
plateaued
at
saline
trol,
63.3%
declining
antigen,
ISAV
23.3
mortality
ISAV
groups
fered
(p < 0.005)
(p
ISAV
73.5%
on
20
dpc.
The
mortality
curve
of the
group
injected
with
was
almost
identical
with
that
of the
group
adjuvant
con-
and
on
45 dpc
the
cumulative
mortalities
were
71.7%
and
for
these
groups,
respectively.
The
vaccinated
groups
showed
cumulative
mortality
with
increasing
amounts
of ISAV
with
55.0%
for
ISAV
4%,
28.3%
for
ISAV
20%
and
10.0%
for
100%.
RPS
was
86.0
for
group
ISAV
100%,
60.5
for
ISAV
20%,
for
ISAV
4%
and
11.6
for
the
adjuvant
control.
The
cumulative
was
significant
lower
in group
ISAV
100%
than
in group
20%
(p < 0.02)
and
highly
significant
lower
than
in all
other
(p < 0.001).
The
cumulative
mortality
for
group
ISAV
20%
dif-
significantly
from
the
cumulative
mortality
of group
ISAV
4%
and
of
both
control
groups
(adjuvant
control
and
saline)
< 0.001).
There
were
no significant
differences
between
groups
4%,
adjuvant
control
and
saline.
3.2.
Detection
of
ISAV
RNA
The
earliest
time
point
ISAV
RNA
could
be detected
after
cohabi-
tation
On
saline
infected
and
the
few
shown
in
21
nificant
heart,
was
at
9 dpc
in one
gill
sample
of
the
adjuvant
control
group.
13
dpc,
one
fish
of
group
adjuvant
control
and
two
fish
of
group
were
positive
in all
tissues.
At 21
dpc,
the
number
of
ISAV-
fish
increased
considerably
in the
groups
adjuvant
control
saline.
At
this
time
ISAV
RNA
was
detectable
in most
fish
of
groups
adjuvant
control
and
saline
in all
tissues,
but
only
in
fish
from
group
ISAV
100%
(data
from
time
point
21 dpc
are
in Fig.
2).
Interestingly,
viral
load
appeared
to be
higher
the
adjuvant
control
group
than
in fish
injected
with
saline
at
dpc
(Fig.
2).
However
the
difference
was
not
statistically
sig-
(p ∼0.1,
p ∼
0.13
and
p ∼
0.17,
for
gills,
head
kidney
and
respectively).
In the
ISAV
100%
group,
significantly
lower

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6396
A.
Lauscher
et al.
/ Vaccine
29 (2011) 6392–
6401
Fig.
challenge.
RNA
2.
Viral
loads
analyzed
by
real-time
PCR.
Relative
load
of
ISAV
RNA
(y-axis)
and
the
number
of
ISAV
RNA
positive
fish
(listed
in each
column)
at 21
days
post
cohabitation
Tissues
of eight
fish
were
analyzed
from
each
of
the
group
saline,
adjuvant
control
and
ISAV
100%.
Real-time
PCR
was
used
to measure
the
relative
amount
of
ISAV
and
the
EF1?B was
chosen
as
reference
gene.
The
presented
data
were
log-transformed
values.
viral
group
Average
ney
and
kidney)
loads
were
detected
compared
to both
the
adjuvant
control
(p < 0.01)
and
the
saline
group
(p < 0.05)
at 21 dpc
(Fig.
2).
Ct-values
at 21
dpc
were:
36.3
(heart),
38.2
(head
kid-
and
gills)
in group
ISAV
100%;
27.0
(heart),
28.1
(head
kidney)
28.4
(gills)
in group
adjuvant
control;
32.2
(heart),
33.9
(head
and
32.7
(gills)
in group
saline.
3.3.
Serology
3.3.1.
ISAV-specific
ISAV-specific
eight
From
ISAV-specific
ISAV
weakly
of
tively.
for
antibodies
group
antibodies
antibodies
were
not
detected
by
ELISA
in any
of
the
control
fish
sampled
prior
to vaccination
(data
not
shown).
the
five
groups
examined
in the
course
of
the
experiment,
antibodies
were
only
observed
in the
ISAV
20%
and
100%
groups
(Fig.
3).
In the
ISAV
20%
group,
two
fish
were
to moderately
positive
for
ISAV-specific
antibodies
at each
the
two
first
serum
samplings
at 6 wpv
and
6 dpc,
respec-
In the
ISAV
100%
group,
the
majority
of
fish
were
positive
ISAV-specific
antibodies
at
all
samplings.
The
numbers
of
ISAV-
positive
fish
were
significantly
higher
(p < 0.001)
in the
ISAV
100%
than
in the
group
ISAV
20%
at all
samplings.
3.3.2.
Neutralizing
No
sampled
ISAV
detected
neutralizing
and
Table
antibodies
neutralizing
activity
could
be
detected
in the
control
fish
sera
prior
to vaccination
(data
not
shown).
In sera
from
groups
4%,
adjuvant
control
and
saline,
no
neutralizing
activity
was
in any
samples.
Although
the
titers
were
generally
low,
activity
was
detectable
in sera
from
groups
ISAV
100%
ISAV
20%
at the
serum
samplings
6 wpv,
and
6 and
21 dpc.
2 shows
results
of
the
NA.
3.4.
Immune
gene
transcript
profiles
After
vaccination
but
prior
to challenge,
no significant
differ-
ences
and
(data
in
gene
transcripts
were
observed
between
the
vaccinated
non-vaccinated
groups
at the
chosen
sampling
time
points
not
shown).
After
challenge,
variations
in the
transcript
levels
of
immune
genes
groups
related
to innate
immune
response
were
evident
between
and
samplings,
particularly
in head
kidney
and
heart
and
to
cant
were
(p
in
results
found
significant
kidney
and
IFN-? (p
not
Transcript
(MHCII,
consistent
sampling
some
extent
in gills.
In the
group
adjuvant
control
signifi-
up-regulations
(statistical
significance
p included
for
heart)
observed
at
21
dpc
in transcripts
of
RIG-I
(p < 0.001),
Viperin
< 0.001),
IFN-? (p < 0.001)
and
MHCI
(p < 0.001)
in all
tissues
and
heart
and
head
kidney
transcripts
of
IFN-?(p
< 0.02).
Fig.
4 shows
from
heart,
transcript
levels
for
head
kidney
and
gills
can
be
as
supplementary
material
in Figs.
6 and
7.
In the
saline
group
up-regulations
were
found
at 21
dpc
in heart
and
head
transcripts
of
RIG-I
(p < 0.001)
and
MHCI
(p < 0.004),
in heart
gills
transcripts
of
Viperin
(p < 0.007)
and
in heart
transcripts
of
< 0.001).
In contrast,
innate
response-related
genes
were
significantly
up-regulated
in the
group
ISAV
100%.
profiles
of
genes
related
to adaptive
responses
CD4,
CD8,
sIgM,
mIgM,
sIgT,
mIgT
and
IgD)
showed
no
alterations
in any
of
the
analyzed
groups
at
the
chosen
time
points
(data
not
shown).
Table
Neutralizing
2
serum
antibodies.
Group

Individual
fish

6 wpv

6 dpc

21
dpc
ISA
100%
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
8
1–8
1–8
1–8
–
8
–
–
4
–
–
8
–
–
8
–
8
–
–
4
2
2
–
8
–
–
–

16


–
–
–
8
–
4
2
–
–
8
8
4
–
–
–
–
16


8

16
16
16



ISA
20%
4
–
–
4
–
–
–
2
–
–
–







ISA
Adjuvant
Saline
4%
control


Reciprocal
infected
neutralizing
values
of
serum
dilutions
causing
a
50%
reduction
in
the
number
of
cells
are
given
as
neutralizing
antibody
titer.
“–”
denotes
no detectable
activity.

Page 6

A.
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et al.
/ Vaccine
29 (2011) 6392–
6401
6397
Fig.
and
results
3.
ISAV-specific
serum
antibodies.
ELISA
results
from
the
groups
ISAV
100%,
ISAV
20%,
ISAV
4%,
adjuvant
control
and
saline
at sampling
6 weeks
post
vaccination
and
6
21 days
post
challenge.
Data
are
presented
as
level
of
Ab
relative
to (%)
a positive
control
and
80%
equates
a
titer
of
1:3200.
A cut-off
of
0.2
(OD)
corresponds
to 9%
and
≥9%
indicate
presence
of
ISAV-specific
antibodies
in sera.
3.5.
immune
Correlation
between
ISAV
infection
and
transcripts
of
genes
The
significant
variations
in transcript
levels
of genes
related
to
innate
heart
were
heart):
MHCI
the
group
number
The
infection.
response
were
caused
by ISAV
infection.
In Fig.
5 (data
from
at 21 dpc)
it can
be
observed
that
the
up-regulated
transcripts
associated
with
detectable
ISAV
RNA
(R2value
included
for
Rig-I
(R2= 0.84),
Viperin
(R2= 0.93),
IFN-? (R2= 0.76)
and
(R2= 0.63).
Thus,
the
differences
found
in gene
transcripts
of
non-vaccinated
groups
but
not
in transcripts
of
the
vaccinated
ISAV
100%
were
caused
by a
high
number
contrary
to a low
of
ISAV
infected
individuals,
respectively.
transcript
levels
of
the
EF1?B
were
not
influenced
by
ISAV
4.
Discussion
In
this
study,
we
describe
the
efficacy
of
immunization
with
dif-
ferent
parameters
The
tivated
immunization.
had
inducing
saline
study
not
Corresponding
started
the
is
for
ity
loads
cated
period.
uncertainties
housing
ditions
effects.
amounts
of
inactivated
ISAV
and
the
observation
of
immune
following
vaccination
and
subsequent
ISAV
challenge.
results
show
that
the
RPS
after
immunization
with
inac-
ISAV
is
linked
to the
amount
of
antigen
used
in the
The
group
vaccinated
with
the
highest
antigen
dose
an RPS
of
86
and
demonstrated
that
vaccination
is capable
of
good
protection
against
ISA.
The
mortality
curves
of
group
or
adjuvant
control
were
similar,
demonstrating
that
in our
injection
of
the
MontanideTMISA 763A
adjuvant
alone
did
give
protection
six
weeks
after
vaccination.
with
the
pre-challenge
results,
shedder
fish
to die
on
Day
9 post
infection.
Among
the
cohabitants,
first
died
on
18 dpc
and
mortality
increased
from
24 dpc.
This
in accordance
with
the
10–20-day
incubation
period
described
experimental
induction
of
ISA
[32,33]. The
cumulative
mortal-
of
the
control
group
(saline),
71.7%,
as
well
as
obtained
viral
in the
saline
and
adjuvant
control
groups
at 21
dpc,
indi-
an effective
transmission
of
the
infection
during
the
trial
Performance
of single
tank
exposure
might
be
attended
by
due
to lack
of
replicate
groups,
but
on
the
other
hand,
of
all
groups
in the
same
tank
ensured
homogeneous
con-
for
group
comparison
and
excluded
differences
due
to tank
The
number
of
fish
with
detectable
ISAV
RNA
increased
rapidly
from
showing
detected
group
ing
with
speculated
ISAV
thus
In
specific
ISAV
present
production
of
dose
was
lower
was
four
groups
The
an
immunization.
This
is
antibodies
the
antibodies
immunization
antigens
trast,
Both
other
trating
Nevertheless,
had
of
ISAV
thus,
13
dpc
to 21
dpc
in the
adjuvant
control
and
saline
groups,
a progressive
ISAV
infection
in both
groups.
ISAV
RNA
was
in only
a
few fish
and
with
lower
loads
in the
ISAV
100%
compared
to the
adjuvant
control
and
saline
groups,
indicat-
impeded
replication
or clearance
of
virus.
This
is
in accordance
the
observed
mortalities
of the
respective
groups.
It can
be
that
the
existence
of
antibodies
found
in fish
of group
100%
could
have
blocked
the
initial
spread
of
the
virus
and
inhibited
viremia
and
infestation
of
target
cells.
a study
using
DNA
vaccination
Mikalsen
et al.
found
that
ISAV-
antibodies
could
be
detected
only
following
experimental
infection,
but
not
prior
to challenge
[28]. Conversely,
the
study
demonstrates
that
immunization
is able
to induce
the
of
ISAV-specific
serum
antibodies,
but
that
the
amount
antigen
used
in the
immunization
is pivotal.
The
highest
antigen
given
in immunization
led
to the
highest
protection
levels
and
found
to produce
ISAV-specific
antibodies
in
the
most
fish.
The
antigen
dose
(20%),
which
was
associated
with
lower
RPS,
only
capable
of
inducing
detectable
antibody
production
in
individuals.
Fish
in groups
ISAV
4%,
adjuvant
control
or saline
had
low
RPS,
and
did
not
produce
ISAV-specific
antibodies.
data
thus
seems
to suggest
that
ISAV-specific
antibodies
play
important
role
for
the
observed
protection
levels
induced
by
apparent
correlation
between
antibodies
and
protection
to a minor
degree
evident
when
focusing
on
the
fraction
of
that
have
virus
neutralizing
capacity.
Several
fish
in
ISAV
100%
and
ISAV
20%
groups
had
detectable
neutralizing
in sera
from
6 wpv
and
onwards.
This
indicates
that
with
a
high
dose
of
inactivated
virus
particles
as
induces
production
of
neutralizing
antibodies.
In con-
a
low
antigen
dose
did
not
induce
this
kind
of
response.
ISAV
100%
and
ISAV
20%
had
a much
higher
RPS
than
the
groups
for
which
no neutralizing
activity
was
found,
illus-
the
potential
effect
of
neutralizing
antibodies
on
protection.
not
all
tested
individuals
of
the
ISAV
100%
group
detectable
neutralizing
antibodies.
In addition,
the
number
fish
with
detectable
neutralizing
antibodies
was
lower
in the
100%
group
than
in the
ISAV
20%
group
at 6 wpv
and
prior
to challenge.
All
groups
were
kept
in the
same
tank,

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6398
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29 (2011) 6392–
6401
Fig.
100%,
4. Real-time
PCR
analyses
of
genes
related
to innate
immune
response
in heart.
Transcript
levels
of
IFN-?, RIG-I,
Viperin,
IFN-? and
MHCI
in heart
from
groups
ISAV
adjuvant
control
and
saline
post
cohabitation
challenge.
The
data
are
analyzed
by
RT-qPCR,
normalized
against
EF1?B and
log-transformed
??Ct
values.

Page 8

A.
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et al.
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29 (2011) 6392–
6401
6399
-0.5
0.0
0.5
1.0
1.5
-5
-4
-3
-2
-1
0
R2= 0.84
log10(RIG.I)
log10(ISAV)
0.00.5 1.0
-5
-4
-3
-2
-1
0
R2= 0.63
log10(MHCI)
log10(ISAV)
0.5
1.0
log10(IFNa)
1.5
-5
-4
-3
-2
-1
0
R2= 0.76
log10(ISAV)
-0.50.00.51.0 1.52.0
-5
-4
-3
-2
-1
0
R2= 0.93
log10(Viperin)
log10(ISAV)
Fig.
levels
R2depict
5.
Correlation
between
ISAV
and
transcript
levels
of
genes
related
to innate
immune
response.
The
individual
relative
loads
of
ISAV
are
plotted
against
??Ct
transcript
of
RIG-I,
MHCI,
IFN-? and
Viperin.
Heart
tissues
from
sampling
21 days
post
challenge
were
analyzed.
All
data
are
log-transformed
and
normalized
against
EF1?B.
The
the
coefficient
of
determination.
ensuring
tection
without
herd
generally
ever,
than
vided
using
quent
group,
found
no
tection
neutralizing
capacity,
get
by
[34],
of
tive
possess
and
may
the
endothelial
malian
importance
in
identical
exposure
to infectious
viruses.
Hence,
the
pro-
in individuals
in the
ISAV
100%
and
ISAV
20%
groups
detectable
neutralizing
antibodies
could
not
be
due
to
protection.
Titers
found
in ISAV
100%
and
ISAV
20%
were
low,
but
in accordance
with
other
findings
[21]. How-
the
neutralizing
antibody
titers
were
higher
in ISAV
100%
in ISAV
20%,
suggesting
that
higher
titers
may
have
pro-
better
protection
in the
present
study.
A previous
study
DNA
vaccination
with
the
HE
and
NP
genes
and
subse-
ISAV
exposure
showed
better
survival
in the
HE-vaccinated
but
the
RPS
was
relatively
low.
Neutralizing
response
was
earlier
in the
HE-vaccinated
group;
however,
in that
study
correlation
between
levels
of neutralizing
antibodies
and
pro-
was
found
at
the
end
of
the
study
(54
dpc)
[28]. Both
and
non-neutralizing
antibodies
may
have
antiviral
the
neutralizing
antibodies
by
blocking
infection
of
tar-
cells,
the
non-neutralizing
antibodies
by
opsonising
virus
and
forming
bulky
immune
complexes.
As
shown
by
Joseph
et al.
ISAV-specific
antibodies
may
even
mediate
ISAV
infection
macrophage-like
fish
cell
lines
in vitro, via
binding
to puta-
Fc
receptors.
Recently,
Atlantic
salmon
have
been
shown
to
putative
Fc receptors
for
both
IgG
(GenBank
BT060117)
IgE
(GenBank
BT058375)
[35]. While
salmon
macrophages
express
the
IgG
Fc receptor
[34]
thus
being
susceptible
to
antibody-mediated
enhancement,
this
is
less
likely
for
salmon
cells,
another
major
target
cell
for
ISAV,
since
mam-
endothelial
cells
do not
express
Fc? receptors
[36]. The
of
antibody-mediated
enhancement
for
ISAV
infections
vivo
thus
remains
speculative.
For
other
orthomyxoviruses,
e.g.,
influenza
A
viruses,
immunity
is
which
ization
compared
fusion
B viruses,
ent
The
izing
in
Passive
ered
Based
virus
activity
be
response
The
response
individual
trol
followed
during
ease
pattern-recognizing
and
cades
of
mediated
by humoral
responses
to the
viral
surface
antigens,
of
the
HA-specific
antibodies
are
most
important
for
neutral-
[19]. The
surface
antigens
are
organized
differently
in
ISAV
to the
influenza
A
virus.
While
both
receptor
binding
and
activity
are
associated
with
the
HA protein
in influenza
A and
these
functional
activities
are
associated
with
two
differ-
proteins
in ISAV,
the
HE
and
F protein,
respectively
[6,37–42].
monoclonal
antibody
3H6F8
that
recognizes
HE
has
a neutral-
effect,
and
it has
been
suggested
that
neutralizing
activity
salmon
serum
after
ISAV
infection
is directed
against
HE
[20].
transfer
experiments
using
sera
from
fish
that
had
recov-
from
ISAV-infection
have
demonstrated
partly
protection
[17].
on
the
comparative
knowledge
derived
from
the
influenza
HA
and
the
ISAV
HE,
and
correlation
between
neutralizing
and
protection
found
in our
experiment,
the
HE
protein
can
suggested
as
an effective
antigen
for
vaccine-induced
immune
against
ISA.
transcript
levels
of
genes
connected
to innate
immune
like
RIG-I,
Viperin,
IFN-? and
MHCI
were
induced
in the
fish
with
ISAV
infection,
i.e.,
in the
groups
adjuvant
con-
and
saline
at 21
dpc.
The
transcript
curves
of
these
genes
closely
the
load
of
ISAV.
Induction
of
IFN-? and
MHCI
ISAV
infection
but
lack
of
protective
effects
against
the
dis-
has
been
described
earlier
[13–16,43].
RIG-I
is a cytoplasmic
receptor
that
recognizes
intracellular
dsRNA
some
forms
of
ssRNA.
It initiates
downstream
signalling
cas-
and
activates
interferon
type
I responses
[44]. The
function
RIG-I
is
inhibited
by the
non-structural
(NS)
1
protein
of
the

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29 (2011) 6392–
6401
influenza
response
by
to
virus
response
cation,
vaccinated,
No
with
after
trasted
particular
discrepancy.
scription
likely
target
during
ing
not
detectable
been
no
lack
essary
delusive
vaccine-induced
In
cellular-mediated
been
fish
is
responses
to
limited
expression
for
Transcript
lymphocyte
ISAV-infected
Vaccine
mucosal
depending
of
represents
of
Combining
improving
[58,59]. A positive
tion
[12]. However,
salmon
of
vant
progress
A virus
[45–47].
Viperin
takes
part
in the
antiviral
host
[48]
and
is proposed
to affect
the
formation
of
lipid
rafts
interacting
with
farnesyl
diphosphate
synthase,
and
has
shown
inhibit
virus
release
from
the
plasma
membrane
in influenza
A
infections
[49]. Our
results
confirm
that
activation
of innate
was
dependent
upon
ISAV
infection
including
viral
repli-
but
no regulations
of
innate
responses
could
be
reported
in
protected
fish
after
viral
challenge.
significant
alterations
in gene
transcript
profiles
associated
adaptive
responses
could
be
detected
after
vaccination
or
challenge.
For
the
immunoglobulin
transcripts
this
was
con-
by the
evident
increase
in ISAV-specific
antibodies
seen
in ISAV
100%
fish.
There
can
be
several
reasons
for
this
In samples
from
lymphoid
tissues
the
basic
tran-
of
many
adaptive
immune
genes
is high
and
thus
more
to overshadow
any
induced
ISAV-specific
activity.
In infected
tissues,
affected
areas
may
be localized
and
hence
missed
sampling
and
analysis.
Furthermore,
it is apparent
that
tim-
for
sampling
is
crucial.
It is possible
that
the
sampling
did
overlap
the
time-interval
when
an increase
in transcripts
was
and
additionally
that
a higher
response
p.v.
could
have
observed
locally
at the
vaccination
site.
The
latter
also
includes
detectable
regulations
of
innate
responses
p.v.
In addition,
the
of
traceability
on
the
basis
of
mRNA
presence
does
not
nec-
imply
lack
of
protein
reactivity
[24]. Therefore
it could
be
to evaluate
the
role
of the
adaptive
immune
system
in
protection
on
the
basis
of
gene
transcript
levels.
studies
of
genetic
markers
of ISAV
infection,
activation
of
the
immune
response,
i.e.,
class
I MHC
pathway,
has
found
to be
a major
component
of
the
immune
response
in
[25,43]. However,
in experimentally
infected
fish
the
mortality
high,
often
over
90%,
and
therefore
difficult
to assess
if genetic
are
correlated
with
protection.
All
body
cells
are
able
express
MHCI,
while
the
number
of
cells
expressing
MHCII
is
[50], which
should
also
be
considered
when
evaluating
profile
results,
especially
in systemic
infections
like
ISA
which
the
ubiquitous
endothelial
cells
are
the
main
targets.
levels
of genes
related
to MHC
class
II pathway
and
B
were
not
observed
to change
in expression
studies
of
fish
[25,43].
protection
of
influenza
is inducible
by
systemic
and
responses,
but
measurable
parameters
differ
strongly
on
the
form
of administration
[51–53].
The
existence
a mucosal
immunity
in teleost
fish
is
described
[54–57]
but
still
a poorly
explored
field.
A possible
ISA
protective
effect
humoral
factors
on
mucosal
membranes
in salmon
is not
known.
vaccines
with
adjuvant
is a common
method
of
administration
and
modulation
of
the
immune
system
impact
of
an
oil-based
adjuvant
in combina-
with
ISAV
antigen
on
vaccine
efficacy
was
observed
earlier
negative
side-effects
of
oil-based
adjuvant
used
in
have
been
described
[60]. In the
present
study,
comparison
the
adjuvant
control
and
saline
groups
indicated
that
the
adju-
alone
had
slightly
negative
effects
regarding
the
virus
load
and
of
the
disease.
5.
Conclusions
The
results
of
the
present
study
provide
novel
basic
knowledge
about
could
that
induces
enced
immune
the
nature
of
vaccine-induced
protection
against
ISA
and
aid
future
vaccine
developments.
The
results
demonstrate
immunization
with
an
injectable,
inactivated
ISAV
vaccine
good
protection
in salmon.
The
protection
is highly
influ-
by
the
amount
of
antigen
administered.
Observations
of
responses
in vaccinated
and
non-vaccinated
fish
indicate
the
antibodies,
response-related
in
fish
immune-related
relevance
of humoral
response,
ISAV-specific
and
neutralizing
for
vaccine-induced
protection.
The
activation
of innate
genes
were
found
to correlate
with
ISAV
infection
non-immunized
fish,
but
the
protection
levels
of the
vaccinated
could
not
be
related
to transcript
profiles
of
a selection
of
genes
at
the
chosen
sampling
time
points.
Acknowledgements
This
study
was
funded
by
the
Norwegian
Research
Coun-
cil
Fauskanger
sample
Competing
peting
Author’s
main
imental
and
helping
ER,
the
(InNoVacc,
grant
183196/S40)
and
Intervet
Norbio.
Håvard
and
Sigrid
Lunde
are
acknowledged
for
assistance
in
collection.
interests: The
authors
declare
that
they
have
no com-
interests.
contributions: AL
wrote
the
manuscript
and
did
the
experimental
analysis.
ER,
AL,
BK
and
PF
designed
the
exper-
challenge.
BK,
SG and
MK
assisted
in practical
analysis.
JB
AL
carried
out
the
statistical
analysis.
KF
and
LA contributed
by
in establishment
of
some
of
the
laboratory
methods
used.
SG and
JB contributed
to writing.
All
authors
read
and
approved
final
manuscript.
Appendix
A.
Supplementary
data
Supplementary
the
data
associated
with
this
article
can
be
found,
in
online
version,
at doi:10.1016/j.vaccine.2011.04.074.
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