Immunological solutions for treatment and prevention of porcine reproductive and respiratory syndrome (PRRS)

Abstract

Vaccination is the principal means used to control and treat porcine reproductive and respiratory syndrome virus (PRRSV) infection. An array of PRRS vaccine products is available in various regions of the world. However, despite extensive efforts, little progress has been made to improve efficacy since the first introduction of a live, attenuated vaccine in 1994 in the USA. Key limitations include: (a) uncertainty about the viral targets of protective immunity that prevents a research focus on individual viral structures and proteins, and frustrates efforts to design novel vaccines; (b) inability to establish clear immunological correlates of protection that requires laborious in vivo challenge models for evaluation of protection against challenge; and (c) the great genetic diversity of PRRSV which requires that challenge experiments be interpreted cautiously since it is not possible to predict how immunological protection against one isolate will translate to broadly cross-protective immunity. Economically significant levels of cross-protection that are provided to a variety of field isolates still cannot assure that effective protection will be conferred to isolates that might emerge in the future. In addition to these substantial barriers to new PRRSV vaccine development, there are enormous gaps in our understanding of porcine immunological mechanisms and processes that provide immunity to PRRSV infection and memory responses for long-term protection. Despite these impediments, we should be confident that progress will be made. Sequencing of the swine genome is providing a rich source of primary knowledge of gene structure and transcriptional regulation that is certain to reveal important insights about the mechanisms of anti-PRRSV immunity, and continued efforts to unravel the details of the interaction of PRRSV with pigs will lead to new insights that overcome the current limitations in the field.

Full text

Vaccine
29 (2011) 8192–
8204
Contents
lists
available
at
SciVerse
ScienceDirect
Vaccine
j
ourna
l
ho
me
pag
e:
www.elsevier.com/locate/vaccine
Review
Immunological
solutions
for
treatment
and
prevention
of
porcine
reproductive
and
respiratory
syndrome
(PRRS)
Michael
P.
Murtaugha,∗,
Marika
Genzowb
aDepartment
of
Veterinary
and
Biomedical
Sciences,
University
of
Minnesota,
1971
Commonwealth
Avenue,
St.
Paul,
MN,
USA
bBoehringer
Ingelheim
Animal
Health
GmbH,
Ingelheim
am
Rhein,
Germany
a
r
t
i
c
l
e
i
n
f
o
Article
history:
Received
2
July
2011
Received
in
revised
form
31
August
2011
Accepted
6
September
2011
Available online 17 September 2011
Keywords:
Vaccine
Animal
virus
Immunity
a
b
s
t
r
a
c
t
Vaccination
is
the
principal
means
used
to
control
and
treat
porcine
reproductive
and
respiratory
syn-
drome
virus
(PRRSV)
infection.
An
array
of
PRRS
vaccine
products
is
available
in
various
regions
of
the
world.
However,
despite
extensive
efforts,
little
progress
has
been
made
to
improve
efficacy
since
the
first
introduction
of
a
live,
attenuated
vaccine
in
1994
in
the
USA.
Key
limitations
include:
(a)
uncer-
tainty
about
the
viral
targets
of
protective
immunity
that
prevents
a
research
focus
on
individual
viral
structures
and
proteins,
and
frustrates
efforts
to
design
novel
vaccines;
(b)
inability
to
establish
clear
immunological
correlates
of
protection
that
requires
laborious
in
vivo
challenge
models
for
evaluation
of
protection
against
challenge;
and
(c)
the
great
genetic
diversity
of
PRRSV
which
requires
that
challenge
experiments
be
interpreted
cautiously
since
it
is
not
possible
to
predict
how
immunological
protection
against
one
isolate
will
translate
to
broadly
cross-protective
immunity.
Economically
significant
levels
of
cross-protection
that
are
provided
to
a
variety
of
field
isolates
still
cannot
assure
that
effective
protection
will
be
conferred
to
isolates
that
might
emerge
in
the
future.
In
addition
to
these
substantial
barriers
to
new
PRRSV
vaccine
development,
there
are
enormous
gaps
in
our
understanding
of
porcine
immuno-
logical
mechanisms
and
processes
that
provide
immunity
to
PRRSV
infection
and
memory
responses
for
long-term
protection.
Despite
these
impediments,
we
should
be
confident
that
progress
will
be
made.
Sequencing
of
the
swine
genome
is
providing
a
rich
source
of
primary
knowledge
of
gene
structure
and
transcriptional
regulation
that
is
certain
to
reveal
important
insights
about
the
mechanisms
of
anti-PRRSV
immunity,
and
continued
efforts
to
unravel
the
details
of
the
interaction
of
PRRSV
with
pigs
will
lead
to
new
insights
that
overcome
the
current
limitations
in
the
field.
© 2011 Elsevier Ltd. All rights reserved.
Contents
1.
Introduction
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2.
Immunological
facts
that
need
to
be
re-examined
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.8193
2.1.
PRRSV
evades
host
defenses
through
suppression
of
innate
immunity?
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2.2.
Antibody-dependent
enhancement
(ADE)
of
PRRS?
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.8194
2.3.
Persistent
infection?.
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.8194
2.4.
Neutralizing
antibody
is
essential
for
protective
immunity? . .
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.8195
3.
Efficacy
of
vaccines
against
PRRS
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.8195
3.1.
Homologous
protection
can
appear
absolute
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.8196
3.2.
Heterologous
protection
is
variable
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4.
Scientific
constraints
to
PRRSV
vaccine
development
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.8198
4.1.
Basic
porcine
immunology
and
the
immunological
response
to
PRRSV
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.8198
4.2.
Absence
of
anamnestic
responses
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4.3.
Host
age-dependent
resistance
to
infection
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.8199
∗Corresponding
author.
E-mail
address:
murta001@umn.edu
(M.P.
Murtaugh).
0264-410X/$
–
see
front
matter ©
2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.vaccine.2011.09.013

M.P.
Murtaugh,
M.
Genzow
/
Vaccine
29 (2011) 8192–
8204 8193
4.4.
Requirement
for
live,
replicating
immunogen
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.8199
4.5.
Genetic
diversity
of
the
viral
target
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.8200
Acknowledgements
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.8200
References
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.8201
1.
Introduction
Porcine
reproductive
and
respiratory
syndrome
(PRRS)
emerged
as
a
widespread
reproductive
and
respiratory
disease
of
swine
in
the
late
1980s.
Recognition
of
a
new
disease
agent
in
swine,
which
is
the
leading
source
of
animal
protein
in
the
human
diet,
stim-
ulated
extensive
efforts
to
identify
the
causative
agent
[1,2].
The
viral
etiology
of
PRRS
was
determined
in
1990
to
1991,
primarily
through
epidemiological
methods
in
Europe
leading
to
the
discov-
ery
and
isolation
of
Lelystad
virus,
and
through
fulfillment
of
Koch’s
postulates
in
the
USA,
that
firmly
established
porcine
reproduc-
tive
and
respiratory
syndrome
virus
(PRRSV)
as
the
sole
causative
agent
of
PRRS
[3,4].
Subsequent
nucleic
acid
sequencing
demon-
strated
the
existence
of
two
markedly
different
genotypic
forms
of
PRRSV
in
Europe
(type
1)
and
North
America
(type
2)
that
showed
immunological
cross-reactivity
[5–8].
Identification
of
the
causative
agent
was
crucial
to
control
of
the
viral
disease.
Viruses
provide
the
raw
material
for
vaccines,
and
vaccines
are
essential
for
control
of
disease.
In
contrast
to
bacteria
and
parasites,
whose
genetically
encoded
biochemistry
and
physi-
ology
provide
ample
targets
for
design
of
selective
drugs
to
control
and
eliminate
infection,
viruses,
especially
RNA
viruses,
provide
few
specific
targets
since
they
use
host
biochemical
and
cellular
processes
to
accomplish
their
life
cycles.
Vaccination,
in
which
the
host
immune
system
is
stimulated
to
produce
effector
and
mem-
ory
responses
to
specific
pathogens,
is
a
highly
effective
tool
for
treatment
and
prevention
of
viral
infections.
Vaccination
was
discovered
and
implemented
for
disease
con-
trol
long
before
the
underlying
mechanisms
of
immunity
were
deduced.
Attenuation
of
virulent
pathogens
by
cultivation
in
non-
native
conditions
and
the
development
of
adjuvants
provided
tools
to
broaden
the
useful
range
of
vaccines,
while
fundamental
immunological
research
revealed
the
mechanistic
bases
of
pro-
tective
immunity.
Modern
vaccine
development
and
application
is
based
on
established
immunological
principles
of
innate
and
adaptive
immunity.
Stimulation
of
innate
antiviral
interferons
and
cytokines
mobilizes
antigen
presenting
cells
and
activates
B
and
T
lymphocytes.
The
environment
of
antigen
stimulation
induces
pathways
of
lymphocyte
development
directed
toward
intracellu-
lar
or
extracellular
pathogens
according
to
the
Th1-Th2
paradigm.
Antigen-specific
lymphocyte
maturation
yields
both
effector
and
memory
lymphocytes
that
are
primed
to
respond
to
future
expo-
sure
by
the
same
pathogen.
Recent
excellent
reviews
of
PRRSV
immunology
and
vaccinol-
ogy
expanded
on
previously
identified
knowledge
and
gaps
in
knowledge
that
may
be
relevant
for
the
development
of
more
effective
vaccines
[9,10].
Meanwhile,
new
findings
have
raised
questions
related
to
common
assumptions
about
the
interaction
of
PRRSV
with
pigs,
and
about
the
efficacy
of
immunological
pro-
tection
against
PRRS.
Here
we
review
evidence
indicating
that
the
paradigms
which
guide
vaccine
development
for
PRRS
control
and
prevention
need
to
be
revised,
and
identify
key
limitations
in
the
scientific
knowledge
of
pig
immunology
and
PRRSV
biology
that
constrain
vaccine
development
efforts.
2.
Immunological
facts
that
need
to
be
re-examined
It
has
been
said
that
vaccines
have
been
produced
to
all
of
the
easy
pathogens,
such
that
remaining
needs
represent
more
complicated
host-pathogen
interactions
which
require
a
more
sophisticated
understanding
for
successful
vaccine
development.
Biological
factors
in
the
host-pathogen
interaction
can
present
substantial
barriers
to
conventional
vaccine
development.
For
example,
bovine
viral
diarrhea
virus
(BVDV)
infection
in
utero
prior
to
acquisition
of
immunocompetence
results
in
failure
to
immuno-
logically
recognize
antigens
as
foreign,
absence
of
an
adaptive
immune
response,
and
lifelong
infection.
Constraints
to
vaccine
development
against
BVDV
relate
to
presence
of
the
virus
in
the
fetus
when
immunocompetence
is
developed,
thus
resulting
in
the
inability
to
recognize
viral
proteins
as
foreign
[11,12].
In
the
case
of
HIV,
infection
of
CD4+
T
cells
leads
to
depletion
of
helper
T
cells,
while
an
innate
APOBEC3
activity
helps
facilitate
rapid
viral
evolution
that
outpaces
antigen-specific
immune
responses
[13].
Loss
of
helper
functions
that
are
central
to
induction
of
adaptive
immunity,
in
combination
with
host-assisted
genetic
shifts
that
facilitate
ongoing
emergence
of
variant
viruses
resistant
to
neutral-
izing
antibodies,
presents
a
formidable
challenge
to
AIDS
vaccine
development.
PRRSV
infects
macrophages
and
dendritic
cells,
whose
abilities
to
secrete
inflammatory
and
immunomodulatory
cytokines,
and
to
present
antigens
to
T
cells,
are
essential
for
the
induction
of
effec-
tive
adaptive
immune
responses.
The
virus
infects
pigs
of
all
ages,
but
disease
occurs
primarily
in
late
gestation
sows,
manifested
as
death,
abortions
and
weak-born
piglets
that
fail
to
survive
to
wean-
ing;
and
in
young
and
growing
pigs,
in
which
it
causes
respiratory
disease,
associations
with
secondary
viral
and
bacterial
infections,
reduced
growth
rates,
and
death.
The
virus
shows
extensive
genetic
variation
within
both
type
1
and
type
2
forms,
with
periodic
emer-
gence
of
more
severe
disease
phenotypes
that
may
or
may
not
be
associated
with
novel
genotypes
[14–18].
Early
immunological
studies
of
PRRSV
interaction
with
pigs
gave
the
perception
that
PRRSV
subverted
porcine
innate
and
adaptive
immune
responses,
that
it
resulted
in
persistent
infections,
and
that
its
genetic
variation
contributed
to
vaccine
failures.
More
recent
findings
suggest
the
possibility
of
alternative
explanations
for
the
early
observations,
or
lead
to
different
conclusions
altogether.
The
following
sections
address
generally
accepted
facts
about
PRRSV
immunity
that
are
incorrect,
need
to
be
re-considered,
or
may
not
apply
to
PRRSV
isolates
that
now
circulate
in
major
swine
produc-
ing
regions
of
the
world.
2.1.
PRRSV
evades
host
defenses
through
suppression
of
innate
immunity?
Van
Reeth
and
colleagues
at
Ghent
University
in
Belgium
first
showed
the
absence
of
a
classical
innate
antiviral
immune
response,
characterized
by
interferon
and
inflammatory
cytokine
production,
to
PRRSV
infection
[19].
Recent
data
showing
that
PRRSV
nonstruc-
tural
protein
1
(nsp1)
blocks
activation
of
transcription
factors
necessary
for
interferon
induction
provide
a
mechanistic
expla-
nation
[20–22].
However,
other
data
suggest
a
more
complicated
situation.
Respiratory
infection
by
type
1
PRRSV,
especially
the
pro-
totypical
Lelystad
virus,
is
mild
and
levels
of
viremia
are
relatively
low
compared
to
type
2
PRRSV
isolates
[23–28].
Therefore,
it
is
possible
that
inflammatory
responses
in
the
lung
are
an
indicator
of
disease,
as
was
originally
proposed,
rather
than
an
indicator
of
strong
induction
of
immunity
[19].
Type
2
PRRSV
field
isolates
vary
in
ability
to
suppress
or
enhance
interferon
production,
suggesting

8194 M.P.
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/
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29 (2011) 8192–
8204
that
the
innate
immune
response
to
PRRSV
infection
is
complex
and
capable
of
producing
a
strong
innate
response
[29,30].
In
addi-
tion,
induction
of
antibody
responses
to
either
PRRSV
proteins
or
to
an
irrelevant
protein
antigen
is
not
retarded
by
PRRSV
infec-
tion.
The
response
to
multiple
viral
proteins
occurred
in
the
same
time
course
as
antibody
responses
to
the
foreign
antigen,
keyhole
limpet
hemocyanin,
whether
the
virus
and
protein
antigen
were
present
in
the
same
animal
or
in
separate
animals
[31].
On
bal-
ance,
it
appears
that
PRRSV
nonstructural
proteins
can
interfere
in
interferon
signaling
pathways
in
model
cell
culture
systems,
that
the
innate
immune
response
to
PRRSV
is
influenced
by
virus
strain,
and
that
the
adaptive
immune
response
is
not
delayed.
Thus,
innate
immunity
to
PRRSV
infection,
as
presently
characterized,
may
not
be
relevant
to
the
adaptive
immune
response
to
PRRSV
infection.
It
is
possible
that
the
innate
and
adaptive
response
to
highly
viru-
lent
PRRSV
strains
is
fundamentally
different
than
the
response
to
strains
of
low
virulence.
There
is
a
great
need
to
more
fully
char-
acterize
the
innate
anti-viral
immune
response
to
PRRSV
infection
by
virulent
strains
that
are
circulating
in
major
swine-producing
regions
of
the
world,
to
characterize
the
innate
response
in
porcine
macrophages,
and
to
assess
their
relevance
to
anti-PRRSV
immu-
nity
in
swine.
Immunosuppressive
cytokine
expression,
including
IL-10,
IL-4,
and
transforming
growth
factor
␤(TGF␤)
induced
in
response
to
PRRSV
also
has
been
proposed
to
explain
immune
evasion
and
secondary
infections.
The
proposal
is
based
primarily
on
in
vitro
studies,
or
assessments
of
mRNA
expression,
or
both
[32–40].
The
interpretation
of
these
studies
depends
on
assumptions
that
mRNA
expression
predicts
protein
expression
and
secretion,
and
that
the
behavior
of
isolated
cell
populations
predicts
cytokine
fluctuations
in
vivo.
These
assumptions
are
not
well
established
in
the
pig
[41,42].
Analysis
of
IL-10
levels
in
serum
over
time
in
pigs
of
var-
ious
ages
infected
with
virulent
or
attenuated
PRRSV
showed
no
correlation
between
IL-10
levels
and
viremia
[43].
An
aberrant
immune
bias
toward
a
Th2-type
response
also
could
be
responsible
for
an
inefficient
immune
response
to
PRRSV,
if
a
Th1-type
response
were
protective
[40,44].
However,
the
appli-
cation
of
the
Th1-Th2
paradigm
to
PRRSV
immunity
in
pigs
is
challenging.
The
model
is
based
on
cytokine
expression
patterns,
specifically
interferon
␥
(IFN␥)
and
IL-4,
in
antigen-specific
CD4+
helper
T
cells.
In
the
porcine
model,
investigators
rely
primarily
on
assessment
of
IFN␥
alone
or
in
combination
with
IL-10
in
bulk
lym-
phocyte
preparations.
The
consequent
limitations
of
this
approach
have
been
analyzed
previously,
and
seriously
weaken
conclusions
that
PRRSV
might
induce
a
Th2
response
that
impairs
anti-PRRSV
immunity
[45].
2.2.
Antibody-dependent
enhancement
(ADE)
of
PRRS?
Viruses
enter
permissive
cells
via
receptor-mediated
uptake
using
molecular
interactions
between
viral
envelope
constituents
and
cell
surface
molecules.
Viruses
that
infect
macrophages
also
are
able
to
enter
permissive
cells
via
cell-surface
receptors
that
bind
the
Fc
region
of
immunoglobulin
molecules
that
are
com-
plexed
to
viral
surface
antigens
[46].
For
example,
dengue
virus
may
exhibit
increased
pathogenicity
in
the
presence
of
low
lev-
els
of
specific
antibody
using
this
mechanism
[47].
Since
PRRSV
infects
macrophages,
and
since
anti-PRRSV
neutralizing
antibody
levels
frequently
appear
to
be
quite
low,
it
is
reasonable
to
specu-
late
that
the
presence
of
antibodies
might
enhance
PRRSV
infection.
ADE
is
reported
to
be
a
feature
of
PRRSV
infection
[48,49].
Even
though
ADE
is
an
attractive
concept
in
PRRS
pathogenesis,
it
has
not
been
shown
to
play
a
role
in
the
interaction
of
PRRSV
with
pigs
in
the
field.
There
has
never
been
a
confirmed
report
of
PRRS
disease
being
more
severe
immediately
following
administration
of
live,
attenuated
vaccines,
even
though
revaccination
and
ther-
apeutic
vaccination
in
the
face
of
a
PRRSV
outbreak
are
widely
practiced
in
the
field.
Numerous
PRRSV
challenge
experiments
have
been
reported
from
around
the
world
[50–52].
None
of
these
studies
report
more
severe
disease
following
challenge
of
immune
animals.
Titration
of
neutralizing
antibody
levels
in
swine
serum
does
not
show
evidence
of
increased
viral
growth;
instead,
anti-
body
inhibits
infection
in
a
dose-dependent
fashion
[53].
Studies
of
neutralization
specificity
comparing
simian
cells
and
porcine
alve-
olar
macrophages
revealed
no
evidence
of
enhanced
infection
of
macrophages
[54].
Thus,
the
relevance
of
ADE
to
PRRSV
infection
of
immune
swine
is
questionable
and
need
not
be
a
consideration
in
attenuated
virus
vaccine
development.
2.3.
Persistent
infection?
Viral
infections
of
animals
are
commonly
controlled
in
a
mat-
ter
of
weeks.
In
pigs,
sterilizing
immunity
to
influenza
is
achieved
in
10–14
days,
and
to
foot-and-mouth
disease
virus
in
14–21
days
[55–57].
Early
studies
showed
that
PRRSV
infection
was
prolonged
compared
to
other
viral
diseases
of
swine,
with
viremia
consistently
lasting
for
28
to
42
days.
However,
uninfected
sentinels
introduced
into
groups
of
recovered,
nonviremic
pigs
at
later
times
consis-
tently
became
infected,
showing
that
infection
persisted
beyond
the
viremic
period
[58].
Presence
of
PRRSV
was
demonstrated
in
experimental
test
groups
up
to
186
days,
and
intermittently
in
indi-
vidual
pigs
up
to
251
days
[59,60].
Thus,
it
was
concluded
that
PRRSV
infection
was
persistent
in
pigs.
Predictive
modeling
fur-
ther
suggested
that
persistence
in
farrow-to-finish
herds
would
be
maintained
for
periods
of
hundreds
of
days
or
more,
the
duration
increasing
with
increased
herd
size,
increased
contact
between
dif-
ferent
age
groups
and
increased
re-introduction
of
infectious
gilts
[61].
Thus,
the
trend
to
larger
herd
sizes
in
recent
decades
may
have
strengthened
the
assumption
of
PRRSV
persistence
[14].
The
con-
clusion
that
PRRSV
was
a
persistent
infection
was
consistent
with
evidence
that
the
innate
immune
response
was
compromised,
and
that
B-
and
T-cell
responses
were
weak.
Moreover,
persistent
infection
appeared
to
be
a
common
feature
of
other
arteriviruses,
especially
lactate
dehydrogenase
elevating
virus
(LDV)
of
mice
and
equine
arteritis
virus
(EAV)
[62,63].
The
mechanism(s)
of
persistence
have
not
been
elucidated
in
these
cases.
There
is
no
evidence
of
latent
infection,
or
immunological
escape
leading
to
recurrent
or
periodic
waves
of
viremia.
PRRSV
infection,
with
its
finite
period
of
viremia,
is
distinct
from
LDV
infection,
in
which
viremia
appears
to
be
life-long
[64].
Thus,
per-
sistent
infection
became
a
firmly
established
tenet
of
PRRSV
biology
despite
distinct
differences
from
LDV
and
EAV,
or
a
known
mech-
anism
of
action.
Because
PRRSV
is
cytopathic
for
macrophages
in
vitro
it
is
likely
that
the
same
is
true
for
in
vivo
infections.
If
so,
nascent
virus
would
have
to
move
from
the
lytic
cell
to
one
or
more
susceptible
cells
or
infection
would
end.
To
explain
why
infections
are
eventually
cleared
it
may
be
hypothesized
that
the
successful
transfer
of
nascent
virus
from
infected
cells
to
additional
susceptible
cells
is
less
than
100%
and
therefore
infection
is
slowly,
but
eventually,
terminated–with
the
actual
time
for
any
individ-
ual
depending
on
the
extent
of
initial
infection
and
the
relative
efficiency
of
that
individual’s
immune
response.
In
recent
years,
veterinarians
have
shown
that
PRRSV
is
com-
pletely
eliminated
from
large
sow
herds
of
hundreds
to
thousands
of
animals
using
a
procedure
known
as
herd
closure
[65–67].
All
animals
in
an
infected
herd
are
exposed
to
vaccine
or
virulent
virus,
then
the
herd
is
closed
for
a
minimum
of
200
days,
during
which
time
no
new
animals
are
introduced.
After
this
time,
naïve
gilts
and
sows
do
not
become
infected
when
added
to
the
herd,
over
periods
of
time
exceeding
2
years,
and
piglets
weaned
from
farrowed
sows
are
negative
for
PRRSV.
The
complete
absence
of
viral
shedding
and
transmission
in
large
populations
of
animals
over
extended

M.P.
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/
Vaccine
29 (2011) 8192–
8204 8195
periods
of
time
in
multiple
independent
cases
proves
that
the
immune
response
produces
sterilizing,
complete
immunity
to
PRRSV.
This
observation
is
fundamentally
important
to
immuno-
logical
control
of
PRRSV.
Since
the
immune
system
of
swine
is
able
to
completely
eliminate
PRRSV,
comprehensive
immunologi-
cal
protection
is
possible.
Future
research
should
be
focused
on
elucidation
of
the
mech-
anisms
of
protective
immunity.
Vaccine
development
can
then
be
focused
on
the
means
of
enhancing
the
rate
at
which
protection
is
achieved
and
increasing
the
breadth
of
protection
across
a
wider
spectrum
of
PRRSV
genetic
diversity.
2.4.
Neutralizing
antibody
is
essential
for
protective
immunity?
Neutralizing
antibodies
play
a
critical
role
in
immunological
control
of
a
wide
variety
of
viral
infections
in
general,
and
are
believed
to
be
crucial
for
anti-PRRS
immunity
as
well
[68,69].
How-
ever,
a
variety
of
studies
show
that
PRRSV
viremia
is
often
resolved
before
neutralizing
antibodies
are
detected
[9,70],
and
PRRSV
can
be
isolated
from
blood
of
pigs
that
have
neutralizing
antibodies
[71].
Pigs
that
resolve
acute
infection
do
not
always
develop
neu-
tralizing
antibodies,
and
animals
lacking
neutralizing
antibodies
are
resistance
to
re-infection
[33].
Passive
immunization
with
neu-
tralizing
antibodies
was
shown
to
prevent
PRRSV
viremia,
but
it
did
not
prevent
infection
of
lymph
nodes
[53,68].
Because
of
these
observations,
it
is
clear
that
porcine
neutralizing
antibodies,
as
they
are
now
characterized,
are
not
essential
for
immunity
to
PRRSV,
and
their
precise
role
in
clearance
of
primary
viremia
and
prevention
of
reinfection
is
uncertain.
To
better
understand
the
role
of
neutralizing
antibodies
in
pro-
tection
against
PRRSV,
it
will
be
necessary
to
address
various
inconsistencies
that
hinder
progress,
and
to
address
key
central
questions
in
viral
targets
of
the
porcine
immune
response.
A
central,
unanswered
question
in
PRRSV
immunity
is
the
viral
tar-
gets
of
humoral
and
cell-mediated
immunity
that
elicit
protection
against
primary
infection
and
memory
against
future
infection.
With
respect
to
neutralizing
antibodies,
several
groups
claim
that
the
target
is
a
highly
conserved
amino
acid
sequence
in
ectodomain
region
of
envelope
glycoprotein
5
(GP5)
[72–76].
If
true,
then
viruses
containing
this
motif
in
GP5
would
be
equally
suscepti-
ble
to
neutralization.
And
if
neutralizing
antibodies
were
essential
for
protective
immunity,
then
immunity
against
any
PRRSV
should
provide
broadly
effective
cross-protection
against
other,
similar
PRRSV
isolates.
This
logic
conflicts
with
the
widely
held
assumption
that
lack
of
cross-protection
is
a
major
constraint
in
development
of
PRRSV
vaccines.
This
apparent
contradiction
between
predic-
tion
and
observation
needs
to
be
resolved.
A
second
difficulty
is
that
immunization
with
GP5
is
reported
to
exacerbate,
rather
than
prevent,
clinical
disease
following
challenge
[77].
It
might
be
argued
that
a
neutralization
motif
exists
in
the
ectodomain
of
GP5,
but
glycosylation
masks
the
epitope.
N-linked
glycosylation
in
this
region
of
GP5
appears
to
be
highly
variable
based
on
deduced
amino
acid
sequence
predictions,
but
the
concept
has
not
been
subjected
to
rigorous
experimentation.
Experiments
to
directly
test
the
hypothesis
that
glycosylation
masks
a
key
neu-
tralization
epitope,
and
that
removal
of
the
epitope
will
lead
to
more
effective
control
of
PRRSV
infection,
have
been
inconclusive
[78–81].
In
one
example,
genetic
elimination
of
putative
glycosyla-
tion
sites
increased
the
susceptibility
of
hypoglycosylated
virions
to
neutralization
by
serum
derived
from
pigs
infected
with
wild-type
virus
[78].
The
finding
suggests
that
fully
glycosylated
GP5
stim-
ulates
production
of
antibodies
that
neutralize
virions
containing
it
poorly,
but
neutralize
virions
containing
hypoglycoslylated
GP5
efficiently.
Conversely,
infection
of
pigs
with
hypoglycosylated
viri-
ons
elicited
higher
neutralizing
titers
to
wild-type
virions
than
did
infection
with
the
wild-type
virions
themselves
[78].
Regardless
of
how
these
results
are
interpreted,
their
relevance
to
resolution
of
primary
PRRSV
infection
is
questionable,
since
the
glycosylation
status
of
the
virus
had
no
effect
on
clinical
or
virological
outcomes
in
infected
pigs
[78].
A
potentially
confounding
factor
in
mutational
studies
of
glyco-
sylation
is
the
presence
in
ORF5
of
a
second,
functional
ORF,
ORF5a,
that
overlaps
the
sequence
encoding
the
hypervariable
region
and
N44
in
type
2
PRRSV
[82,83].
Thus,
genetic
manipulation
of
GP5
gly-
cosylation
sites
may
inadvertently
disrupt
the
functional
properties
of
a
non-target
protein.
No
one
to
date
has
offered
a
convincing
model
to
explain
how
GP5
glycosylation
patterns
acting
to
mask
a
key
neutralization
epi-
tope
in
immunizing
and
challenge
viruses
would
account
for
the
outcomes
of
PRRS
protection
studies.
Alternatively,
one
report
of
masking
epitopes
invoked
a
sequence
in
GP5
that
is
predicted
to
reside
in
the
signal
peptide,
which
is
cleaved
prior
to
incorporation
of
GP5
into
virion
envelopes
[76].
Since
a
key
portion
of
the
pro-
posed
site
would
not
be
present
in
virions,
it
is
not
likely
to
exert
a
masking
effect.
Even
if
neutralizing
antibodies
play
a
central
role
in
anti-PRRSV
immunity,
their
target
may
not
be
GP5.
Genetic
experiments
show
that
GP5
is
not
required
for
macrophage
permissiveness,
suggesting
that
antibodies
directed
to
GP5
may
not
block
key
sites
for
PRRSV
binding
to
macrophages
[84].
Various
laboratories
have
shown
that
neutralizing
antibodies
are
directed
to
GP2,
GP3,
and
GP4
[85–88],
and
that
complexes
of
minor
envelope
proteins
GP2,
GP3,
and
GP4
are
essential
for
infection
of
permissive
cells
[89].
Glycan
addition
to
GP2
and
GP3
is
required
for
infectious
virus
production,
and
glycosylation
of
any
three
of
the
four
sites
in
GP4
is
necessary
for
recovery
of
infectious
PRRSV
[90,91].
However,
the
relevance
of
minor
envelope
glycoprotein
N-
linked
glycosylation
and
neutralizing
antibodies
to
anti-PRSV
immunity
in
pigs
remains
to
be
established.
For
example,
neu-
tralizing
anti-GP4
antibodies
that
are
highly
strain
specific
do
not
support
an
essential
role
for
neutralizing
antibodies
in
immunolog-
ical
control
of
PRRS,
especially
for
cross-protective
immunity
that
underlies
vaccine
efficacy
[86].
Similarly,
variation
in
minor
enve-
lope
glycoprotein
glycosylation
has
been
associated
with
variation
in
induction
of
and
sensitivity
to
neutralizing
antibodies
in
a
limited
set
of
specific
PRRSV
isolates
[87,90,92].
However,
the
significance
of
these
observations
to
the
control
and
prevention
of
PRRSV
in
all
of
its
glorious
genetic
diversity
still
needs
to
be
determined.
Toward
this
end,
identification
of
the
viral
structures
that
elicit
protective
immunity
in
pigs,
and
factors
that
modulate
the
effi-
cacy
of
protection
in
vivo,
is
essential
to
rational
development
of
immunological
tools
to
prevent
and
control
PRRS.
Secondly,
in
vitro
and
in
vivo
experimental
models
must
be
developed
to
elucidate
mechanisms
of
protective
immunity
and
cross-protection
that
are
generally
applicable
to
the
diversity
of
field
viruses
that
cause
dis-
ease
in
pigs
(Fig.
1).
3.
Efficacy
of
vaccines
against
PRRS
Many
vaccines
have
been
produced
to
combat
PRRSV
(Table
1).
They
include
products
containing
live
virus
derived
by
cell
cul-
ture
attenuation
of
virulent
field
isolates
(e.g.
Ingelvac®PRRS
MLV
and
Porcilis®PRRS),
inactivated
preparations
of
attenuated
PRRSV
strains
(e.g.
Progressis®and
PRRomiSe®),
inactivated
preparations
of
virulent
isolates
expanded
by
in
vitro
cell
culture
for
use
in
the
same
herd
(autogenous
vaccines),
inactivated
preparation
of
multi-
ple
virulent
isolates
enriched
with
viral
antigens
(e.g.
MJPRRS),
and
subunit
vaccines
expressing
selected
proteins
(e.g.
PRRSV-RS).
Doc-
umentation
of
the
immunological
properties
and
capacities
to
elicit
protective
immunity
has
only
been
established
for
a
few
of
these
products.
The
majority
have
not
been
evaluated
scientifically
in
any

8196 M.P.
Murtaugh,
M.
Genzow
/
Vaccine
29 (2011) 8192–
8204
Table
1
Vaccines
against
PRRSV.
Name
Description
Year
of
introduction
Manufacturer
Comments
Literature
citationsa
Ingelvac
PRRS
MLV
Cell-culture
attenuated
form
of
type
2
strain
VR-2332
1994
Boehringer
Ingelheim
Vetmedica
Also
known
as
RespPRRS,
RespPRRS
Repro
37
Ingelvac
PRRS
ATP
Cell-culture
attenuated
form
of
type
2
strain
JJ1884
2004
Boehringer
Ingelheim
Vetmedica
6
PRIMEPAC
PRRS
Cell-culture
attenuated
form
of
a
type
2
strain
1996
Schering
Plough
Discontinued
in
2000
1
Suvaxyn
PRRS Inactivated
form
of
type
1
strain
Olot
No
information
available Fort
Dodge
Animal
Health Discontinued
2
Amervac
PRRS
Attenuated
type
1
strain
MLV
VP064
BIS
No
information
available
Laboratories
Hipra
Piglet
vaccine
1
Suipravac
PRRS
Inactivated
type
1
strain
No
information
available
Laboratories
Hipra
Sow
vaccine
0
Porcilis
PRRS
Attenuated
type
1
DV
strain,
adjuvanted
with
Diluvac
Forte
2000
Intervet
8
Pyrsvac-183 Inactivated
type
1
strain
All-183,
adjuvanted
in
oil
2000
SYVA
Laboratories
1
Progressis
(Ingelvac
PRRS
KV)
Inactivated
type
1
strain
2000
Merial
Sow
vaccine
only,
only
in
Europe
2
PRRomiSe
Inactivated
form
of
type
2
strain
VR2402
No
information
available
Intervet
Discontinued
0
Autogenous
vaccines
Inactivated
field
isolates
No
information
available
Newport
Laboratories
and
others
US
only
3
MJPRRS Inactivated
type
2
strains
in
cell
homogenate
2008 MJ
Biologics Recommended
for
use
in
previously
exposed
animals,
US
only
0
PRRSV
RS
AAV-type
2
GP5/6
vectored
vaccine
2009
Sirrah
Bios
US
only
0
BSL-PS
100
MLV
No
information
available
Bestar
Laboratories
Pte
Ltd.
Singapore
0
BSL-PS
100
Killed
vaccine
No
information
available
Bestar
Laboratories
Pte
Ltd.
Singapore
0
SuiShot
PRRS Killed
vaccines No
information
available ChoongAng
Vaccine
Laboratories
Co.
Ltd
South
Korea
0
Suivac
PRRS-IN Killed
vaccine
VD-E1,
VD-E2,
VD-A1
No
information
available
Dyntec
spol.
S.r.o.
Czech
Republic
0
Suivac
PRRS-Ine
Killed
vaccine
VD-E1,
VD-E2
No
information
available
Dyntec
spol.
S.r.o.
Czech
Republic
0
??
MLV
No
information
available
Kaketsuken
Japan
0
,,PRRS
vaccine“
Killed
vaccine
NVDC-JXA1
No
information
available
Qilu
Animal
Health
Products
Factory
China
0
“PRRS
Vaccine,
Live
(R98
Strain)”
Lan
Yi
Ling
MLV
R98
strain
No
information
available
Qilu
Animal
Health
Products
Factory
China
0
ImmunoPRRS
Avian
immuno-globulins
2007
Iasa
Mexico
0
PPV
and
PRRS
Inactivated
emulsified
vaccine
No
information
available
Narvac
R&D
Russia
0
ARRIAH
Killed
vaccine
No
information
available
FGI
Russia
0
aLiterature
citations:
Publications
identified
in
PubMed
or
Journal
of
Swine
Health
and
Production
using
as
search
terms
all
of
the
names
of
each
product
listed
in
“Name”
and
“Comments.”
respect,
as
indicated
by
peer-reviewed
publications
(Table
1).
There
is
a
pressing
need
for
rigorous
evaluation
of
protective
capacity
of
untested
vaccine
products
against
challenge
of
pigs
with
current,
virulent
field
viruses.
Fig.
1.
Steps
in
establishment
of
a
foundation
for
understanding
immunological
mechanisms
of
PRRSV
protection
and
their
relevance
to
PRRSV
control
in
the
field.
Attenuated
virus
vaccines
produce
infection
of
lung
and
lym-
phoid
tissues,
maintain
infection
for
periods
of
time
equivalent
to
virulent
viruses,
are
transmissible
to
contact
pigs,
and
are
immuno-
logically
protective,
i.e.
eliminate
or
reduce
infection
and
disease
qualitatively
and
quantitatively
[93,94].
Killed
vaccines
may
reduce
levels
of
viremia
in
some
pigs,
but
are
not
demonstrated
to
show
consistent
benefit
against
infection
or
disease
in
a
respiratory
model
[95,96].
Killed
virus
vaccination
improved
the
percentage
of
pigs
weaned
in
a
reproductive
PRRS
model,
but
did
not
improve
overall
reproductive
performance
[97].
At
this
point
in
time,
it
appears
that
live
PRRSV
replication
is
required
to
provide
immuno-
logical
protection
against
PRRSV
infection.
Of
concern,
then,
is
the
nature
of
this
protection
in
swine
populations
at
risk
of
PRRSV
infection
and
how
it
might
be
enhanced
to
achieve
more
com-
plete
protection.
The
following
sections
present
issues
that
affect
the
design
and
interpretation
of
studies
that
assess
vaccine-based
protection
against
PRRS
infection
and
disease.
3.1.
Homologous
protection
can
appear
absolute
In
the
1990s,
Lager
and
colleagues
showed
that
two
pigs
inoculated
with
a
type
2
PRRSV
were
resistant
to
rechallenge
603
days
later
[98].
Since
PRRSV
is
completely
cleared
in
about
200
days,
these
studies
are
assumed
to
have
demonstrated
true,

M.P.
Murtaugh,
M.
Genzow
/
Vaccine
29 (2011) 8192–
8204 8197
lymphocyte-dependent,
immunological
memory.
Subsequently,
a
variety
of
experimental
studies
in
respiratory
and
reproductive
dis-
ease
models
of
type
1
and
type
2
PRRSV
infection
showed
that
preventive
vaccination
with
attenuated
PRRSV
or
inoculation
with
virulent
field
virus
provides
a
high
level
of
protection
against
challenge
with
the
same
or
nearly
the
same
virus
[27,99–101].
However,
homologous
protection,
including
protection
by
virulent
virus
exposure,
was
not
absolute
in
all
cases
[27,100].
Active
PRRSV
infection
is
prolonged
in
pigs,
persisting
for
long
periods
of
time
after
viremia
has
ceased
[59,60,102–105].
Thus,
vir-
ulent
challenges
following
immunization,
even
though
carried
out
after
viremia
ceased,
were
usually
performed
before
the
primary
infection
with
attenuated
or
virulent
viruses
had
been
resolved.
Thus,
the
protective
response
that
was
observed
may
have
been
due
to
existing,
active
immunity
rather
than
to
a
memory
response.
At
the
time
of
these
studies,
the
significance
of
prolonged
infection
in
the
absence
of
viremia
was
not
fully
appreciated.
In
the
interpreta-
tion
of
challenge
results,
presence
of
virus
in
lymphoid
tissues
was
usually
not
assessed,
and
even
if
it
was,
there
were
no
tests
to
quan-
titatively
or
qualitatively
distinguish
challenge
virus
from
vaccine
or
inoculation
virus.
For
these
reasons,
apparently
solid
immunity
rendered
by
vaccines
against
the
same
strain
and
closely
related
viruses
may
not
have
been
complete,
and
may
have
been
due
to
ongoing
resistance
to
the
primary
infection.
If
homologous
protec-
tion
is
not
absolute,
as
appears
to
be
the
case,
then
it
is
important
to
resolve
the
limits
of
PRRSV
protection
using
relevant
field
iso-
lates
that
are
significant
causes
of
disease
today,
and
experimental
conditions
that
are
optimized
for
protective
immunity.
3.2.
Heterologous
protection
is
variable
For
PRRS,
immune
protection
against
a
spectrum
of
PRRSV
strains
is
paramount
due
to
the
extreme
genetic
diversity
and
rapid
evolution
of
PRRSV.
Thus,
vaccines
need
to
provide
broadly
cross-
reactive
protection
against
challenge
with
genetically
dissimilar,
heterologous,
strains.
Despite
occasion
reports
to
the
contrary
[106,107],
there
is
scant
evidence
that
immunization
with
non-
replicating
PRRSV
or
subunits
by
themselves
affords
homologous
or
heterologous
protection
[95,97,108,109].
By
contrast,
controlled
studies
show
that
live,
attenuated
PRRSV
immunization
under
a
variety
of
conditions
substantially
reduces
clinical
signs
and
lesions
induced
by
genetically
unrelated,
virulent
field
viruses
[24,50,93–95,110–117].
The
cross-protective
benefit
of
attenuated
PRRSV
vaccination
that
is
observed
in
experimental
studies
also
is
observed
in
the
field
[51,118–121].
Even
though
attenuated
PRRSV
strains
are
demonstrated
to
pro-
vide
solid
protection
against
PRRSV
infection
and
disease
caused
by
genetically
divergent
field
isolates,
there
are
many
other
instances
in
which
vaccinated
swine
herds
have
experienced
PRRS
outbreaks,
occasionally
with
recovery
and
characterization
of
the
causative
virulent
virus
[122,123].
It
is
a
widespread
assumption
that
vac-
cines
should
prevent
infection
with
field
viruses;
however,
the
claims
granted
for
vaccines
are
that
vaccination
will
reduce
the
clinical
impact
of
the
disease,
not
that
they
will
prevent
a
future
infection.
The
reasons
for
true
vaccine
failures,
excluding
trivial
explanations
related
to
improper
storage
and
application,
are
not
obvious.
They
are
not
due
to
emergence
of
immunologically
resis-
tant
strains,
since
such
strains
have
not
been
described
at
this
time.
Nor
do
they
appear
to
be
the
result
of
viral
attenuation.
Controlled
inoculation
of
pigs
with
on-farm
virulent
strains,
a
cottage
industry
in
the
United
States,
also
fails
to
provide
consistent
long-term
pro-
tection
to
swine
herds.
In
addition,
controlled
exposure
itself
may
give
negative
clinical
outcomes
[124].
There
are
a
multitude
of
potential
sources
for
the
variation
observed
in
cross-protection
efficacy.
Breed
and
age
variation
in
susceptibility
to
PRRSV
infection
may
affect
the
level
of
immunity
achieved
by
attenuated
vaccines
that
require
growth
for
efficacy
[43,125–129].
Strain
differences
in
attenuated
vaccines
may
give
rise
to
variation
in
the
level
or
quality
of
cross-protective
immu-
nity,
especially
if
high
levels
of
attenuation
result
in
poor
growth
in
pigs
[112].
The
genetic
and
biological
diversity
in
challenge
viruses
surely
has
an
impact
on
disease
outcomes.
However,
the
relationship
between
immunological
similarity
between
a
vaccine
strain
and
virulent
field
challenge
virus
that
might
predict
cross-
protective
efficacy,
and
genetic
relatedness
of
the
two
viral
isolates
is
not
known.
While
genetic
relatedness
is
easy
to
measure,
its
immunological
relevance
is
unknown;
and
immunological
corre-
lates
of
protection
that
might
be
used
to
predict
protection
are
likewise
unknown.
Various
challenge
models
are
used
to
evaluate
cross-protective
immunity.
The
growing
pig
model
is
widely
used
in
studies
using
type
2
PRRSV
isolates
due
to
convenience
and
the
reproducible
induction
of
disease
sequelae,
including
clinical
signs,
reduced
weight
gain,
and
histopathological
lesions,
that
are
fairly
well
cor-
related
with
intensity
of
infection
measured
by
viremia
[28,43,130].
Thus,
in
protection
studies
there
is
a
good
correlation
between
severity
of
disease
and
level
of
viremia
[94,131].
The
same
rela-
tionship
also
is
observed
in
pregnant
sow
studies
[93,132].
An
important
corollary
is
that
low
levels
of
viremia
produce
little
or
no
disease,
particularly
in
the
growing
pig
model.
Thus,
attenuated
vaccines
that
produce
low
levels
of
viremia
as
compared
to
viru-
lent
viruses
are
largely
avirulent
[28,43].
In
addition,
highly
virulent
viruses
elicit
low
levels
of
viremia
and
low
levels
of
disease
in
pre-
viously
immunized
animals
[111,116].
Since
the
level
of
viremia
is
determined
by
viral
production
in
lung
and
lymphoid
tissue
sites
of
viral
replication,
it
suggests
for
type
2
PRRSV
that
the
amount
of
virus
circulating
in
blood
is
proportional
to
the
amount
of
virus
at
sites
in
the
respiratory
and
reproductive
tracts
where
disease
manifestations
occur.
In
contrast
to
a
consistent
link
between
viremic
load
and
virulence
in
type
2
PRRSV,
the
relationship
between
viremia
and
virulence
in
type
1
PRRSV
is
more
complicated.
Type
1
field
viruses
typically
appear
to
reach
lower
levels
of
viremia
in
growing
pigs
and
sows
than
do
type
2
PRRSV
isolates
[23–26,28,43,51,97,113,114,133].
Respiratory
disease
due
to
type
1
PRRSV
in
growing
pigs
may
be
inapparent,
and
appears
associ-
ated
with
low
levels
of
viremia
[19,133].
Perhaps
for
this
reason,
the
relationship
between
viremia
and
clinical
disease
in
stud-
ies
of
immune
protection
is
confusing.
Clinical
signs
have
been
reported
in
the
presence
or
absence
of
detectable
virus
[25],
and
higher
levels
of
virus
infection
elicited
in
protection
studies
may
not
produce
clinical
signs
[114].
By
contrast,
heterologous
vaccine
protection
against
PRRS
disease
has
been
observed
in
the
absence
of
a
substantial
effect
on
viremia
[24,51].
The
range
of
discordant
observations
between
viremia
and
disease
in
type
1
PRRSV
het-
erologous
protection
studies
suggests
that
the
amounts
of
PRRSV
present
in
respiratory
and
reproductive
tract
sites
of
disease
are
not
as
tightly
linked
as
in
type
2
PRRSV.
It
also
appears
to
be
more
diffi-
cult
to
establish
reproducible
models
of
PRRS
disease
using
type
1
viral
strains
since
the
disease
manifestations
are
substantially
less
severe
than
those
caused
by
type
2
field
isolates
[28,94,134–137].
Taken
together,
experimental
studies
and
field
research
demon-
strate
that
attenuated,
replicating
PRRSV
administration
reduces
infection
and
disease
due
to
virulent
PRRSV
infection.
The
biological
limitations
and
constraints
on
the
degree
of
heterologous
protec-
tion
that
is
achieved
need
to
be
elucidated.
Once
identified,
they
can
be
overcome.
The
ability
of
pigs
to
eliminate
a
PRRSV
infec-
tion
has
been
demonstrated
conclusively
by
the
practice
of
herd
closure,
showing
that
the
immune
response
to
primary
infection
is
completely
effective
and
sterilizing
[67].
However,
the
mechanisms
of
immune
protection
that
are
essential
to
ward
off
future
infec-
tion
with
genetically
distinct
PRRSV
isolates,
and
their
molecular

8198 M.P.
Murtaugh,
M.
Genzow
/
Vaccine
29 (2011) 8192–
8204
targets
on
the
virus
or
expressed
in
infected
cells
are
still
unknown.
Answers
to
these
issues
will
go
a
long
way
toward
achieving
reliable
immunological
protection
against
PRRS.
4.
Scientific
constraints
to
PRRSV
vaccine
development
Natural
elimination
of
primary
PRRSV
infection
by
pigs,
which
is
demonstrated
by
herd
closure,
establishes
that
the
porcine
immune
system
is
able
to
completely
eliminate
PRRSV
infection.
This
critical
observation
removes
PRRSV
from
the
list
of
pathogens
that
estab-
lish
permanent,
persistent
infections
that
cannot
be
successfully
controlled
immunologically.
Although
PRRSV
is
not
in
the
category
of
persistent
disease
agents
that
present
formidable
challenges
to
control
by
vaccination,
it
nevertheless
has
demonstrated
that
sub-
stantial
barriers
must
be
overcome.
Principal
barriers
include
an
inadequate
understanding
of
basic
porcine
immunology
and
the
immunological
response
to
PRRSV,
age-dependent
differences
in
host
response
to
infection,
the
current
need
for
a
live,
replicating
virus
to
produce
an
effective
vaccine,
and
the
genetic
diversity
of
PRRSV
itself.
The
following
sections
expand
on
these
constraints
to
PRRSV
vaccine
development.
4.1.
Basic
porcine
immunology
and
the
immunological
response
to
PRRSV
Current
concepts
of
pig
immunology
and
immunity
to
infec-
tious
disease
are
based
primarily
on
analogy
to
murine
and
human
systems.
The
approach
has
demonstrated
that
swine
are
simi-
lar
to
humans
and
mice
at
the
comparative
genomic
level.
They
express
similar
cytokines,
Toll-like
receptors
(TLRs),
interferons,
and
other
effectors
of
innate
immunity,
generate
immunoglobu-
lin
and
T-cell
receptor
diversity
in
similar
ways,
and
appear
to
have
similar
lymphocyte
developmental
pathways.
However,
the
detailed
molecular
and
cellular
pathways
by
which
swine
execute
immune
responses
appear
to
vary
in
ways
that
could
be
critical
to
successful
responses
to
swine
pathogens,
including
PRRSV
(e.g.
[45,138]).
Cytokine
expression
early
in
the
infectious
process
may
be
instrumental
in
effective
induction
of
adaptive
immunity
and
memory
responses.
For
this
reason,
lack
of
interferon
␣
production
is
assumed
to
reduce
the
overall
immune
response.
Further,
IL-10,
possibly
acting
in
a
Th2-type
fashion,
is
believed
to
direct
adap-
tive
immune
responses
toward
ineffective
antibody
responses.
These
concepts
have
not
been
validated
by
in
vivo
experiments
with
PRRSV
isolates.
In
contrast,
lack
of
interferon
␣
production
was
associated
with
absence
of
type
1
PRRSV
respiratory
disease
[19,139],
and
type
2
PRRSV
was
shown
to
induce
interferon
␣
pro-
duction
[29,30].
Increased
levels
of
IL-10
in
serum
of
growing
pigs
infected
with
type
2
PRRSV
appears
to
be
a
result
of
infection,
and
high
levels
of
IL-10
are
found
in
adult
swine
independently
of
any
PRRSV
exposure
[43].
Thus,
roles
of
cytokines
and
interferons
in
innate
immunity
to
PRRSV
need
to
be
more
clearly
elucidated.
With
respect
to
methods
that
might
improve
vaccine
efficacy,
inclusion
of
interferons
or
interferon-inducing
substances
may
be
deleterious
to
the
efficacy
of
live
vaccines
since
more
potent
innate
immunity
could
suppress
vaccine
virus
replication,
thus
reducing
the
amount
of
antigen
available
for
activation
and
proliferation
of
PRRSV-specific
lymphocytes
[140].
Such
a
result
was
observed
in
a
type
2
highly
virulent
PRRSV
heterologous
protection
study
[116].
Addition
of
IFN␣
or
poly
ICLC
treatments
at
the
time
of
vaccination
did
not
improve
protection
against
virulent
MN184
challenge
[116].
The
gross
pathological
average
lung
involvement
was
numerically
worse
and
the
number
of
severely
affected
pigs
was
increased
in
groups
treated
with
IFN␣
or
poly
ICLC
(Table
2).
There
is
virtually
nothing
known
about
the
role
of
T-cells,
including
regulatory
T
cells,
in
anti-PRRSV
immunity.
T-cell
recog-
nition
of
viral
structural
proteins
has
been
reported
[141–143],
but
there
are
no
established
models
to
examine
lymphocyte
prolifera-
tive
responses
that
are
required
for
expansion
of
antigen-specific
T-cells,
or
to
investigate
cytotoxic
T
cell
functions
that
mediate
recognition
and
killing
of
virally
infected
cells.
At
this
time
there
is
limited
evidence
that
T-cells
play
a
significant
role
in
anti-PRRSV
immunity,
even
though
cytotoxic
T
cell
function
is
a
standard
feature
of
adaptive
immune
responses
to
a
wide
variety
of
viral
infections
[37,144–147].
Specific
knowledge
of
porcine
T-cell
func-
tion
and
mechanisms
of
action
in
response
to
viral
infection
of
all
types
is
lacking,
due
primarily
to
the
absence
of
tools
and
methods
for
selection
and
culture
of
antigen-specific
T
cells.
The
develop-
ment
of
methods
for
antigen-specific
T
cell
culture
would
greatly
facilitate
a
better
understanding
of
adaptive
immunity
to
PRRSV
specifically,
and
to
all
porcine
viruses
in
general.
There
is
great
opportunity
to
gain
novel
insights
into
porcine
anti-viral
immu-
nity
here,
since
swine
have
a
unique
subpopulation
of
circulating
CD4+
CD8+
double-positive
T-cells
that
is
not
present
in
other
mammalian
species
[148].
They
are
proposed
to
represent
mem-
ory
T-cells,
but
this
concept
has
not
yet
been
pursued
with
respect
to
specific
viral
pathogens
of
swine.
Memory
lymphocyte
responses
are
the
key
to
vaccine-based
protection.
Antigen-specific
B-
and
T-cells
proliferate
into
differen-
tiated
helper
and
effector
cells
that
respond
to
the
primary
infection
Table
2
Effect
of
immunization
on
protection
against
challenge
with
virulent
PRRSV
strain
MN184.
Growing
pigs
were
challenged
55
days
after
primary
immunization
and
sacrificed
14
days
later.
Gross
lung
lesion
scores
(%
lung
involvement)
is
shown
for
each
pig
and
by
group.
Symbol
(†)
indicates
that
the
animal
died
before
the
study
ended.
Detailed
methods
were
described
previously
[116].
Summary
statistics
were
obtained
with
permission
from
Elsevier,
and
individual
animal
data
were
provided
by
E.
Vaughn,
Boehringer
Ingelheim
Vetmedica
(Ames,
IA).
Vaccine Ingelvac
MLV
GP5
peptides
None
None
Adjuvant
None
IFN␣
Poly
ICLC
GP5
peptides
Cholera
toxin
None
None
Challenge MN184
None
Individual
animal
lung
scores
at
necropsy
(†
pig
died)
0
1
68
†
84
5
0
3
†
1
0
27
7
0
0
6
6
72
86
0
0
1
5
16
43
†
55
1
0
43
4
60
77
49
2
9
0
26
56
58
74
0
1
7
9
17
15
48
0
0
22
3
18
16
86
0
58
63
4
3
37
35
0
32 1
1
4
9
5
0
Mean 10.4
16.4
13.8
30.3
45.4
36.4
0.3
#
>
10% 2
3
3
6
8
6
0

M.P.
Murtaugh,
M.
Genzow
/
Vaccine
29 (2011) 8192–
8204 8199
or
vaccination,
and
into
memory
and
long-lived
plasma
cells
that
are
maintained
for
extended
periods
of
time,
ready
to
respond
rapidly
to
a
new
infection.
Information
on
memory
T-cells
in
swine
is
not
available.
However,
infection
of
pigs
with
PRRSV
results
in
substantial
populations
of
memory
B-cells
in
diverse
lymphoid
tissues
[31].
Memory
B-cells
are
produced
to
both
structural
and
nonstructural
PRRSV
proteins,
populate
spleen,
tonsil,
and
regional
lymph
nodes,
but
not
bone
marrow,
in
contrast
to
the
major
site
of
memory
B-cell
residence
in
the
mouse
[149,150].
Memory
cells
are
highly
significant
since
they
facilitate
rapid,
antigen-specific,
and
effective
neutralization
and
cytotoxic
effector
responses
that
control
infection
before
overt
disease
occurs.
The
presence
of
an
anamnestic
response
is
generally
assumed
to
indicate
that
pre-
vious
vaccination
has
successfully
elicited
memory
B-cells
that
provide
a
state
of
protection
against
future
challenge.
Absence
of
an
anamnestic
response
indicates
failure
of
vaccine
to
induce
mem-
ory.
However,
in
the
case
of
PRRSV,
classical
anamnestic
responses
to
re-challenge
of
immune
animals
does
not
occur.
4.2.
Absence
of
anamnestic
responses
A
common
approach
to
vaccine
development
is
to
test
initial
hypotheses
in
a
model
organism
to
select
for
antigen
formulations
that
induce
memory
immune
responses
that
may
be
relevant
to
in
vivo
protection
against
challenge
with
virulent
organisms.
The
classic
indicator
of
a
strong
memory
response
is
an
anamnestic
antibody
response
[151].
However,
in
the
case
of
PRRSV,
challenge
of
immune
animals
usually
elicits
little
if
any
change
in
antibody
levels
[99].
Re-challenge
of
vaccinated
animals
with
genetically
unrelated
viruses
elicits
insignificant
changes
in
antibody
lev-
els
that
are
within
animal-to-animal
variation
(Murtaugh
et
al.,
unpublished
data).
In
retrospect,
previous
PRRSV
vaccine
challenge
studies
were
conducted
at
various
times
after
viremia
had
ended,
but
infection
was
still
present
in
lymph
nodes
and
tonsil.
There-
fore,
the
response
to
re-infection
may
have
been
accomplished
with
existing
effector
cells,
so
that
memory
cell
activation
may
not
have
occurred.
As
a
result,
there
possibly
may
be
only
two
studies
of
anti-PRRSV
immune
memory
in
which
the
challenge
virus
was
given
at
a
time
when
all
virus
had
been
cleared
from
the
body
such
that
there
was
no
antigenic
stimulation
of
actively
secreting
plasma
cells
[93,98].
However,
three
independent
experiments
now
show
that
complete
or
nearly
complete
protection
against
PRRSV
re-
challenge
occurs
in
the
absence
of
a
classical
anamnestic
antibody
response
(manuscript
in
preparation).
These
findings
confirm
that
porcine
anti-PRRSV
immunity
does
not
conform
to
standard
mod-
els
of
innate
and
adaptive
immunity,
and
that
these
models
have
limited
usefulness
in
guiding
development
of
new
vaccines
against
PRRSV.
The
biological
conclusion
here
is
that
immunity
to
re-infection
is
profound,
such
that
a
wave
of
infection,
producing
antigen
for
presentation
to
memory
cells,
does
not
occur
following
re-
challenge.
Absence
of
viremia
supports
this
conclusion
[99].
The
mechanism
responsible
for
limited
or
negligible
PRRSV
replication
following
re-infection
might
be
antibody-mediated
viral
neutral-
ization.
This
mechanism
does
not
explain
how
neutralizing
activity
in
serum
could
prevent
infection
of
alveolar
macrophages
follow-
ing
intranasal
administration,
although
low
levels
of
neutralizing
activity
may
be
present
in
the
lung
[85].
Nor
does
it
explain
sup-
pression
of
re-infection
in
animals
without
detectable
neutralizing
activity
in
serum
[116].
These
observations
indicate
that
the
hall-
mark
of
immunological
memory,
an
anamnestic
response,
is
not
present
in
the
memory
response
to
PRRSV,
and
so
is
not
useful
in
interpreting
vaccine
efficacy
experiments.
The
absence
of
standard,
surrogate
predictors
of
immune
efficacy
is
a
major
constraint
that
complicates
vaccine
development.
4.3.
Host
age-dependent
resistance
to
infection
Pigs
acquire
innate
resistance
to
PRRSV
infection
with
age
as
indicated
by
substantially
reduced
duration
of
viremia
and
viral
loads
in
blood
in
finishing
pigs
and
adults
as
compared
to
weaned
pigs
[43].
The
reduced
levels
of
viremia
appear
to
be
due
to
innate
resistance
of
macrophages
to
viral
infection,
and
are
not
associ-
ated
with
circulating
IL-10
levels,
which
also
increase
with
age
[43,152].
Immune
responses,
determined
by
antibody
responses
to
PRRSV
proteins,
were
not
affected
by
age,
indicating
that
viral
infection
in
lung
and
lymphoid
tissues
must
have
been
sufficient
to
elicit
robust
immune
responses.
Nevertheless,
the
apparently
reduced
antigenic
load
reflected
in
low
or
unmeasurable
viremia
observed
in
adult
sows
suggests
that
memory
immune
responses
that
provide
vaccine-based
protection
against
re-challenge
may
show
greater
variation
within
large
populations
of
immunized
swine,
resulting
in
a
proportion
of
animals
with
low
levels
of
immu-
nity.
This
point
is
evident
in
the
variation
in
lung
pathology
shown
in
Table
2,
among
pigs
within
each
of
the
eight
treatment
groups
of
a
vaccination
study.
Regardless
of
the
average
within-group
degree
of
lung
pathology,
every
group
had
at
least
one
animal
with
severe
lung
pathology
(>50%
involvement)
and
at
least
one
animal
with
mild
or
no
lung
lesions
(<10%
lung
involvement)
(Table
2).
These
data,
which
are
typical
of
the
range
of
responses
that
are
commonly
observed
within
experimental
groups
exposed
to
the
identical
type
2
PRRSV
and
well-controlled
for
other
variables,
indi-
cate
that
extensive
variation
is
present
in
swine,
be
it
genetic,
epigenetic,
nonclinical
virological
or
microbiological
infection,
or
other,
that
influences
individual
responses
to
PRRSV
infection.
Elu-
cidation
of
these
factors
is
essential
to
establishing
a
mechanistic
basis
of
PRRSV
immunity
that
predicts
solid
protection.
The
late-gestation
sow
is
a
critical
at-risk
population
for
PRRSV
infection.
Even
in
the
face
of
stronger
innate
resistance,
many
PRRSV
isolates,
especially
type
1
isolates,
cause
reproductive
dis-
ease
more
readily
and
with
greater
severity
than
respiratory
disease
[153,154].
Furthermore,
transmission
of
PRRSV
from
sows
to
newborn
piglets
may
maintain
active
infection
in
a
herd.
For
these
reasons,
age-dependent
resistance
to
infection
places
extra
demands
on
vaccine
viruses,
whose
attenuation
for
growth
in
pigs
is
exacerbated
in
older
animals
by
innate
resistance.
The
phenomenon
of
innate,
age-dependent
PRRSV
resistance
raises
many
questions.
Resistance
may
be
due
to
changes
in
viral
binding
and
internalization
activities
that
reduce
macrophage
permissiveness
to
infection,
or
to
increased
cellular
resistance
mechanisms
due
to
expression
of
TLR’s
or
other
surveillance
molecules
that
sense
the
presence
of
viral
RNA
[155].
Research
in
this
area
may
lead
to
the
discovery
of
novel
mechanisms
of
innate
anti-viral
immunity
in
swine.
Further
elucidation
of
cellular
infec-
tion
processes
and
pathways
of
immune
stimulation
in
the
pig
may
lead
to
new
avenues
for
PRRSV
vaccine
engineering
to
overcome
age-dependent
PRRSV
resistance.
4.4.
Requirement
for
live,
replicating
immunogen
At
this
time,
solid
immunological
protection
against
PRRSV
infection
can
be
achieved
only
by
immunization
with
live,
replicat-
ing
PRRSV.
Attenuated
PRRSV
vaccines
induce
antibody
responses
indistinguishable
from
virulent
viruses
and
provide
high
levels
of
protection
against
re-infection
with
virulent
PRRSV,
measured
both
by
reduced
levels
of
infection
and
reduced
levels
of
disease.
These
observations
are
consistently
observed
in
experimental
studies
as
well
as
in
field
trials,
as
discussed
above.
Likewise,
there
is
little
evi-
dence
that
killed
PRRSV
vaccines,
subunit
vaccines,
and
other
types
of
vaccine
formulations
provide
substantial
protection
against
PRRSV
challenge
outside
of
experimental
studies
[106,156–161].
Overcoming
the
need
for
a
replicating
immunogen
is
a
significant

8200 M.P.
Murtaugh,
M.
Genzow
/
Vaccine
29 (2011) 8192–
8204
hurdle
in
the
development
of
improved
vaccines
against
PRRSV.
Current
live
PRRSV
vaccines
are
transmissible
to
susceptible
pigs.
While
transmission
of
vaccine
virus
to
non-vaccinated
members
of
a
population
may
be
desirable
under
many
circumstances,
main-
tenance
of
replicating
PRRSV
in
swine
populations
provides
the
opportunity
for
mutation
and
recombination
events
that
have
the
potential
for
reversion
to
virulence
[162].
To
date
there
is
little
evi-
dence
that
attenuated
PRRSV
vaccines
can
become
re-established
in
swine
producing
regions
of
the
world
in
a
virulent
form
[163].
Nevertheless,
the
possibility
of
reversion
to
virulence
will
always
exist
in
a
replicating
immunogen,
so
that
development
of
non-
replicating
PRRSV
vaccines
that
are
efficacious
is
desirable.
4.5.
Genetic
diversity
of
the
viral
target
Antigenic
variation
in
key
immunological
targets
mitigates
the
effectiveness
of
structurally-dependent
antibody
and
T-cell
receptor-dependent
mechanisms
of
viral
recognition
and
control.
Antigenic
variation
is
driven
in
many
instances
by
immunological
selection,
manifested
as
cycles
of
recurrent
infection
in
individu-
als
due
to
the
appearance
of
variants
that
escape
immune
control,
and
are
reined
in
by
a
new
wave
of
antibodies,
as
exemplified
by
equine
anemia
virus
[164].
In
other
cases
such
as
influenza,
genetic
variation
occurs
by
processes
of
genetic
drift
and
genetic
shift
in
which
escape
mutants
arise
in
time
and
space
among
pop-
ulations
[165,166].
Thus,
vaccines
are
evaluated
on
an
annual
basis
to
contain
strains
that
are
expected
to
circulate
in
a
given
influenza
season.
PRRSV
shows
extensive
genetic
and
antigenic
diversity.
New
type
2
strains
with
notable
increases
in
virulence
have
appeared
at
regular
intervals
since
1996
in
North
America
and
China.
There
have
been
multiple
attempts
to
dissect
PRRSV
genetically
to
identify
crit-
ical
determinants
of
virulence
and
immune
protection;
all
have
failed
[167–171].
The
failure
of
hypothesis-driven
research
to
iden-
tify
targets
of
protective
immunity
has
foiled
efforts
to
determine
molecular
and
cellular
mechanisms
of
protection
against
PRRSV.
A
second
approach
is
to
look
for
escape
mutants
that
are
resistant
to
immunity
induced
by
previous
infection
or
vaccination.
Mutants
that
can
escape
neutralizing
antibody
responses
to
GP4
have
been
described
[87],
but
a
virus
that
escapes
anti-PRRSV
immunity
has
never
been
identified.
The
inability
to
associate
genetic
variation,
or
specific
genetic
variants,
with
escape
from
immunity,
is
dou-
bly
frustrating
because
genetic
variation
surely
must
contribute
to
variation
in
the
effectiveness
of
vaccine-derived
protection.
Nev-
ertheless,
relevant
genetic
changes
that
influence
susceptibility
or
resistance
to
pre-existing
immunity
have
not
been
identified,
mak-
ing
it
difficult
to
mount
a
systematic
examination
of
the
problem.
Serum
cross-neutralization
is
commonly
used
to
define
immunologically
related
groups
of
viruses
in
which
exposure
to
any
one
provides
solid
immunity
to
all
others
in
the
group.
At
this
time,
no
system
of
PRRSV
classification
has
successfully
pre-
dicted
clusters
of
immunologically
related
strains
that
exhibit
cross-protection
within
the
group,
and
reduced
protection
outside
the
group.
Serum
neutralization
appears
unlikely
to
provide
a
use-
ful
basis
for
classification
of
cross-protective
PRRSV
isolates
since
induction
of
neutralizing
antibodies
is
not
a
prominent
feature
of
the
response
to
PRRSV
infection
or
of
protective
immunity
[116].
Genetic
similarity,
especially
phylogenetic
analysis,
is
widely
used
to
ascertain
strain
relatedness
and
to
discern
evolutionary
trends.
However,
the
results
do
not
predict
immunological
relatedness,
and
are
usually
limited
to
analysis
of
small
regions
of
the
genome,
especially
GP5,
which
accounts
for
approximately
4%
of
the
coding
capacity
of
the
genomic
RNA.
GP5
is
the
most
logical
target
of
immunological
protection
since
it
appears
to
be
critical
for
host
cell
infection,
and
it
is
the
most
commonly
described
target
of
neutralizing
antibodies.
However,
immunization
with
vectored
vaccines
that
express
GP5
or
GP5
pep-
tides
does
not
provide
solid
protection
against
infection
and
in
two
cases,
one
each
for
type
1
and
type
2
PRRSV
challenge,
exacerbated
disease
(Table
2)
[77,116].
This
result
might
be
due
to
conforma-
tional
or
structural
differences
in
recombinant
proteins,
but
it
is
also
possible
that
immunity
is
dependent
on
other
factors.
For
exam-
ple,
replacement
of
EAV
GP5
with
PRRSV
GP5
did
not
enable
EAV
to
infect
porcine
macrophages,
indicating
that
GP5
does
not
medi-
ate
infection
by
itself
and
thus
may
not
be
a
critical
neutralization
target
[84].
A
lack
of
biochemical
and
molecular
knowledge
of
viral
proteins
responsible
for
cell
infection
has
been
a
major
constraint
on
the
design
of
improved
vaccines.
Fortunately,
emerging
research
on
minor
envelope
proteins
indicates
that
they
play
a
critical
role
in
infection
of
permissive
cells
and
may
be
targets
of
viral
neu-
tralization
[85,86,89,90,92].
While
this
information
may
lead
to
more
productive
vaccine-development
research,
an
effector
role
for
neutralizing
antibodies
remains
difficult
to
define
[172].
Since
all
attenuated
vaccines
are
based
on
field
isolates
whose
exact
nucleotide
sequence
is
no
longer
present
in
the
field,
pro-
tection
is
based
on
immunity
to
genetically
dissimilar
viruses.
This
“heterologous”
protection
varies
from
extensive
to
limited,
as
dis-
cussed
previously.
The
problem
of
identifying
viral
determinants
of
immune
cross-protection
is
greatly
exacerbated
by
the
distribution
of
genetic
variation
broadly
across
the
entire
viral
genome,
and
the
inability
to
associate
specific
genetic
regions
with
effective
cross-
protection.
This
problem
is
made
more
difficult
since
natural
or
artificial
selection
pressures
driving
viral
evolution
have
not
been
identified.
There
are
no
confirmed
examples
of
viral
mutants
that
have
escaped
immunological
control.
There
are
no
verified
exam-
ples
of
re-emergence
of
viral
infection
in
immune
animals
that
have
been
studied
experimentally
even
though
PRRSV
outbreaks
occur
in
immunized
herds.
The
absence
of
these
examples
is
a
signifi-
cant
limitation
since
genomic
comparisons
that
might
reveal
key
mutations
cannot
be
performed.
Ongoing
evolution
of
the
virus
in
the
apparent
absence
of
clear
selective
pressure
provides
little
guidance
for
predicting
future
needs.
In
summary,
uncertainty
about
the
viral
targets
of
protective
immunity
prevents
a
research
focus
on
individual
viral
struc-
tures
and
proteins,
and
frustrates
efforts
to
design
novel
vaccines.
Inability
to
establish
clear
immunological
correlates
of
protection
requires
that
laborious
in
vivo
challenge
models
be
used
to
evaluate
protection
against
challenge.
The
great
genetic
diversity
of
PRRSV
means
that
challenge
experiments
must
be
interpreted
cautiously
since
it
is
not
possible
to
predict
how
immunological
protec-
tion
against
one
isolate
will
translate
to
broadly
cross-protective
immunity.
Even
demonstration
of
economically
significant
lev-
els
of
cross-protection
to
a
variety
of
field
isolates
cannot
assure
that
effective
protection
will
be
conferred
to
isolates
that
might
emerge
in
the
future.
In
addition
to
these
substantial
barriers
to
new
PRRSV
vaccine
development,
there
are
enormous
gaps
in
our
under-
standing
of
porcine
immunological
mechanisms
and
processes
that
provide
immunity
to
PRRSV
infection
and
memory
responses
for
long-term
protection.
Despite
these
impediments,
we
should
be
confident
that
progress
will
be
made.
Sequencing
of
the
swine
genome
is
providing
a
rich
source
of
primary
knowledge
of
gene
structure
and
transcriptional
regulation
that
is
certain
to
reveal
important
insights
about
the
mechanisms
of
anti-PRRSV
immu-
nity,
and
continued
efforts
to
unravel
the
details
of
the
interaction
of
PRRSV
with
pigs
will
lead
to
new
insights
that
overcome
the
current
limitations
in
the
field.
Acknowledgements
We
thank
an
anonymous
reviewer
whose
detailed
comments
improved
the
review,
and
Sergey
Kukushkin,
Boehringer
Ingelheim
LLC
for
assistance
in
assembling
Table
1.
We
apologize
to
authors

M.P.
Murtaugh,
M.
Genzow
/
Vaccine
29 (2011) 8192–
8204 8201
whose
contributions
to
PRRS
vaccinology
may
have
been
inad-
vertently
and
unintentionally
missed.
MPM
declares
no
conflicts
of
interest.
MG
is
an
employee
of
Boehringer
Ingelheim
Animal
Health,
a
veterinary
biologics
company
that
manufactures
vaccines
for
prevention
of
PRRS.
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