Successful vaccination of immune suppressed recipients using Listeria vector HIV-1 vaccines in helminth infected mice

Abstract

Vaccines for HIV, malaria and TB remain high priorities, especially for sub-Saharan populations. The question is: will vaccines currently in development for these diseases function in populations that have a high prevalence of helminth infection? Infection with helminth parasites causes immune suppression and a CD4+ Th2 skewing of the immune system, thereby impairing Th1-type vaccine efficacy. In this study, we conduct HIV vaccine trials in mice with and without chronic helminth infection to mimic the human vaccine recipient populations in Sub-Saharan Africa and other helminth parasite endemic regions of the world, as there is large overlap in global prevalence for HIV and helminth infection. Here, we demonstrate that Listeria monocytogenes functions as a vaccine vector to drive robust and functional HIV-specific cellular immune responses, irrespective of chronic helminth infection. This observation represents a significant advance in the field of vaccine research and underscores the concept that vaccines in the developmental pipeline should be effective in the target populations.

Full text

Vaccine
31 (2013) 2050–
2056
Contents
lists
available
at
SciVerse
ScienceDirect
Vaccine
j
ourna
l
ho
me
pag
e:
www.elsevier.com/locate/vaccine
Successful
vaccination
of
immune
suppressed
recipients
using
Listeria
vector
HIV-1
vaccines
in
helminth
infected
mice
Lisa
M.
Shollenbergera,
Cac
Buia,
Yvonne
Patersonb,
Kelsey
Allena,
Donald
Harna,∗
aDepartment
of
Infectious
Diseases
and
Center
for
Tropical
and
Emerging
Global
Diseases,
University
of
Georgia,
501
DW
Brooks
Drive,
Athens,
GA
30602-7387,
USA
bDepartment
of
Microbiology,
University
of
Pennsylvania,
3610
Hamilton
Walk,
Philadelphia,
PA
19104-6076,
USA
a
r
t
i
c
l
e
i
n
f
o
Article
history:
Received
5
September
2012
Received
in
revised
form
22
January
2013
Accepted
19
February
2013
Available online 5 March 2013
Keywords:
Chronic
helminth
infection
Endemic
schistosomiasis
Vaccine
efficacy
HIV
vaccine
Listeria
monocytogenes
a
b
s
t
r
a
c
t
Vaccines
for
HIV,
malaria
and
TB
remain
high
priorities,
especially
for
sub-Saharan
populations.
The
question
is:
will
vaccines
currently
in
development
for
these
diseases
function
in
populations
that
have
a
high
prevalence
of
helminth
infection?
Infection
with
helminth
parasites
causes
immune
suppression
and
a
CD4+
Th2
skewing
of
the
immune
system,
thereby
impairing
Th1-type
vaccine
efficacy.
In
this
study,
we
conduct
HIV
vaccine
trials
in
mice
with
and
without
chronic
helminth
infection
to
mimic
the
human
vaccine
recipient
populations
in
Sub-Saharan
Africa
and
other
helminth
parasite
endemic
regions
of
the
world,
as
there
is
large
overlap
in
global
prevalence
for
HIV
and
helminth
infection.
Here,
we
demonstrate
that
Listeria
monocytogenes
functions
as
a
vaccine
vector
to
drive
robust
and
functional
HIV-specific
cellular
immune
responses,
irrespective
of
chronic
helminth
infection.
This
observation
represents
a
significant
advance
in
the
field
of
vaccine
research
and
underscores
the
concept
that
vaccines
in
the
developmental
pipeline
should
be
effective
in
the
target
populations.
© 2013 Elsevier Ltd. All rights reserved.
1.
Introduction
Malaria,
tuberculosis
and
HIV-1
remain
tremendous
disease
burdens
for
much
of
the
world’s
population
and
vaccines
for
these
diseases
are
desperately
needed.
Recently
there
has
been
modest
success
in
HIV-1
vaccine
clinical
trials,
and
as
the
greatest
burden
of
HIV
is
found
in
Sub-Saharan
Africa,
it
is
therefore
likely
that
future
HIV-1
vaccine
trials
will
be
performed
in
those
countries
[1,2].
Globally,
in
2010,
34
million
people
were
living
with
HIV-1/AIDS,
with
68%
of
these
infected
individuals
residing
in
Sub-Saharan
Africa.
Prevalence
of
HIV-1
exceeds
20%
in
some
southern
African
countries
[3].
In
addition,
with
pronounced
geographic
coincidence,
a
large
percentage
of
these
populations
are
infected
with
one
or
more
parasitic
worms,
with
prevalence
rates
surpassing
50%
in
some
countries
[4].
Chronic
helminthiasis
causes
systemic
Th2
bias-
ing
and
IL-10
mediated
immune
suppression
[5–8].
Therefore
it
is
likely
that
helminth
infected
vaccine
recipients
will
have
compro-
mised
vaccine
specific,
cell
mediated
immune
responses
[4,6–11].
The
current
trend
of
HIV
vaccine
design
places
more
emphasis
on
eliciting
cytotoxic
T
lymphocytes
(CTL),
rather
than
humoral
Abbreviation:
SEA,
soluble
egg
antigens
from
Schistosoma
mansoni.
∗Corresponding
author.
Tel.:
+1
706
542
4569;
fax:
+1
706
542
5771.
E-mail
addresses:
lisaluvsciens@gmail.com
(L.M.
Shollenberger),
ctbui88@uga.edu
(C.
Bui),
yvonne@mail.med.upenn.edu
(Y.
Paterson),
kwallen10@gmail.com (K.
Allen),
dharn@uga.edu
(D.
Harn).
responses
even
though
both
arms
of
the
immune
response
are
likely
needed
for
an
effective
vaccination
strategy
[12].
In
this
regard,
sev-
eral
studies
have
suggested
that
the
effectiveness
of
HIV-1
vaccines
designed
to
induce
cell
mediated
immunity
will
be
based
on
both
the
quantity
and
quality
of
the
virus-specific
CD4+
and
CD8+
T
cell
responses
[1,13,14].
Therefore,
tremendous
effort
has
been
devoted
to
studies
on
the
design
of
prime-boost
vaccines
that
induce
strong
and
durable
HIV-1
specific
CD4+
and
CD8+
T
cell
responses
in
naïve
recipients
[12,13].
Because
of
the
negative
impact
of
helminth
infection
on
vaccine
efficacy,
we
believe
it
is
critical
that
can-
didate
vaccines
be
evaluated
in
helminth
infected
recipients
to
insure
they
are
capable
of
driving
the
desired
vaccine
specific
responses.
For
example,
efficacy
of
vaccines
for
tetanus
and
tuberculosis
were
shown
to
be
compromised
in
helminth
infected
recipients
liv-
ing
in
helminth
endemic
sites
[15–17].
Similarly,
the
only
study
to
examine
HIV-specific
CTL
responses
in
the
context
of
systemic
Th2
biasing
due
to
chronic
helminth
infection,
demonstrated
that
mice
chronically
infected
with
the
helminth
parasite
Schistosoma
man-
soni
were
not
able
to
mount
significant
HIV-1
vaccine-specific
CTL
responses
post-vaccination,
even
when
the
vaccine
was
enhanced
[18].
This
observation,
taken
together
with
other
studies
exam-
ining
virus-specific
immune
responses
in
helminth
infected
mice
suggests
that
helminth
infection
will
pose
a
significant
problem
for
the
development
of
HIV-1
vaccines
designed
to
induce
viral-
specific
Th1
type
CD4+
and
cytotoxic
CD8+
T
cell
responses.
A
goal
of
our
research
is
to
find
vaccine
vectors
able
to
drive
significant
0264-410X/$
–
see
front
matter ©
2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.vaccine.2013.02.037

L.M.
Shollenberger
et
al.
/
Vaccine
31 (2013) 2050–
2056 2051
HIV-specific
immune
responses
in
helminth
infected
recipients,
despite
the
immune
status
of
the
individual.
Having
effective
vac-
cine
vectors
will
facilitate
development
of
an
HIV-1
vaccine
that
functions
in
a
real
world,
helminth
endemic
setting.
To
address
the
deleterious
effect
of
immune
system
biasing
and
suppression
on
vaccine
efficacy,
de-worming
strategies
have
been
employed
to
increase
vaccine
efficacy,
but
treatment
must
be
repeated,
often
yearly,
as
re-infection
is
both
common
and
frequent
[19].
The
approach
presented
here
is
to
utilize
a
live
vaccine
vec-
tor,
Listeria
monocytogenes,
to
induce
rigorous
T
cell-based
HIV-1
vaccine-specific
immune
responses
in
helminth
infected
recipients
as
proof
of
concept,
as
viral
and
bacterial
vectors
are
known
to
induce
substantial
CTL
responses
[20].
Further,
Listeria
has
been
shown
to
overcome
pre-existing
Th2
biasing
and
reverse
vaccine
specific
immune
responses
to
third
party
antigens
from
Th2
to
Th1
type
[21].
Highlighting
both
the
promising
results
achievable
using
Listeria
as
well
as
the
continuing
acceptance
of
this
pathogen
as
a
vaccine
vector,
the
US
Department
of
Defense
has
recently
solicited
research
proposals
using
L.
monocytogenes
as
a
vaccine
vector.
The
ability
of
Listeria
to
strongly
augment
CD4+
and
CD8+
anti-viral
T
cell
responses
makes
it
an
ideal
candidate
as
a
vaccine
vector
for
HIV;
Listeria
produces
strong
mucosal
and
systemic
innate
and
adaptive
immune
responses
[22–24]
and
can
be
delivered
orally
to
target
the
mucosa,
a
major
immune
compartment
of
the
host
where
HIV
infection
is
initially
established
[22,24–26].
Listeria
lives
within
phagosomal
and
intracytoplasmic
com-
partments,
facilitating
delivery
of
HIV
antigens
to
exogenous
and
endogenous
antigen
processing
and
presentation
pathways
as
well
as
to
the
MHCII
pathway
via
cross
presentation
[27–29].
Unlike
ade-
novirus
or
vaccinia
vectors,
where
pre-existing
immunity
hampers
priming
of
immune
responses
and
repeated
delivery
of
exogenous
antigens
[30–33],
pre-existing
immunity
to
Listeria
does
not
neg-
atively
impact
the
ability
of
Listeria
to
be
administered
repeatedly
with
no
diminishment
in
the
response
to
HIV
antigens
[34–37].
Listeria
vaccines
induce
robust
immune
responses
in
macaques
and
partially
protect
against
challenge
[24,38].
Listeria
has
also
been
used
successfully
as
a
vector
to
induce
protective
immu-
nity
to
other
pathogens
including
influenza,
Francisella
tularensis,
herpes
simplex
virus
type
1,
Leishmania
major
and
human
papil-
lomavirus
[39–43],
resulting
in
induction
of
systemic
immune
responses.
In
the
event
immune
compromised
patients
were
to
exhibit
overgrowth
of
attenuated
Listeria
vectors,
these
bacteria
remain
antibiotic
susceptible.
The
overgrowth
scenario
has
not
been
observed
in
ongoing
therapeutic
cancer
vaccine
clinical
tri-
als.
Therefore,
we
believe
Listeria
is
an
ideal
vaccine
vector,
able
to
repeatedly
boost
the
CD4+
and
CD8+
T
cell
responses
to
HIV
antigens.
2.
Materials
and
methods
2.1.
Biological
reagents
The
HIV
vaccine,
an
attenuated
strain
of
L.
monocytogenes
expressing
the
HIV-1
IIIB
gag
protein
(Lm-gag)
[44],
and
the
control
strain,
which
expresses
the
E7
oncoprotein
of
the
human
papillo-
mavirus
16
(Lm-E7)
[45],
were
grown
in
BHI
supplemented
with
streptomycin.
S.
mansoni
(PR
strain)
infected
Biomphalaria
glabrata
snails
were
provided
by
the
NIAID
Schistosome
Reagent
Program.
Infectious
cercariae
were
obtained
by
exposing
infected
snails
to
direct
light.
Female
BALB/c
mice
at
5–7
weeks
old
were
purchased
from
Harlan
Laboratories,
housed
in
specific
pathogen-free
condi-
tions
and
allowed
to
acclimate
for
1
week
prior
to
manipulation.
All
animal
work
was
performed
in
accordance
with
institutional
policy
and
approved
by
the
institutional
animal
care
and
use
committee.
2.2.
Helminth
infection
of
mice
Six
to
eight-week
old
female
BALB/c
mice
were
infected
by
intraperitoneal
injection
of
35–50
infectious
cercariae
of
S.
mansoni.
Helminth
infection
was
verified
by
indirect
ELISA
for
the
presence
of
anti-egg
and/or
anti-worm
antibodies
in
sera
collected
10
weeks
post
infection
(wpi).
2.3.
Vaccination
of
mice
Age-matched
female
BALB/c
mice
were
primed
i.p.
with
0.2
LD50
(or
0.1
LD50,
as
indicated)
Lm-gag
vaccine
(106CFU),
control
Lm-
E7
vaccine
(matched
CFU
dose)
or
left
unvaccinated.
Mice
were
boosted
2
weeks
after
the
prime
in
an
identical
manner.
2.4.
ELISA
Two
weeks
post
last
vaccination
(wplv),
splenocytes
were
har-
vested
and
plated
at
1.5
million
cells
per
well
in
48-well
plates
in
complete
media
(RPMI-1640)
supplemented
with
FBS,
penicillin,
streptomycin,
amphotericin
B
and
␤-mercaptoethanol.
Cells
were
stimulated
with
25
␮g/ml
Schistosome
soluble
egg
antigen
(SEA)
or
1
␮g/ml
concanavalin
A
(conA)
for
72
h,
supernatants
harvested
then
analyzed
for
levels
of
IFN␥,
IL-4
and
IL-10
by
ELISA
(Becton
Dickinson).
2.5.
ELISpot
Two
wplv,
splenocytes
were
harvested
and
plated
at
300
K
and
150
K
cells
per
well
in
IFN␥
ELISpot
plates
(BD).
The
spleno-
cytes
were
re-stimulated
in
the
presence
of
media,
20
␮M
specific
CTL
peptide
(H2-Kd-restricted,
AMQMLKETI
from
HIV-1
IIIB
gag
protein),
20
␮M
irrelevant
peptide
(H2-Kd-restricted,
TYQRTRALV
from
influenza
A/PR/8/34
nucleoprotein),
20
␮M
specific
helper
peptide
(Class
II-restricted,
NPPIPVGEIYKRWIILGLNK
from
HIV-1
IIIB
gag
protein)
or
1
␮g/ml
conA
(as
a
positive
control).
Pep-
tides
were
synthesized
by
Biosynthesis,
Inc.
at
greater
than
95%
purity
and
reconstituted
in
DMSO
prior
to
dilution
in
media.
After
incubation
for
20
h,
ELISpots
were
performed
according
to
man-
ufacturer’s
instructions
and
enumerated
using
an
Immunospot
analyzer
(C.T.L.).
2.6.
Flow
cytometry
Splenocytes
were
incubated
in
the
presence
of
PMA,
Iono-
mycin
and
GolgiStop
(monensin)
for
6
h.
Surface
markers
were
stained
with
␣-CD8,
␣-CD4,
␣-CD25
antibodies
(BD)
and/or
HIV
gag
tetramer
(H2-Kd+AMQMLKETI,
Beckman
Coulter)
then
cells
were
fixed.
After
membrane
permeabilization,
intracellular
mark-
ers
were
stained
using
␣-IFN␥,
␣-IL-4
(BD)
or
␣-FoxP3
antibodies
(eBiosciences)
and
analyzed
by
flow
cytometry.
To
detect
HIV-
specific
CTLs
at
14
wplv,
splenocytes
were
stained
with
an
HIV
gag
tetramer
and
an
␣-CD8
antibody
(BD).
To
evaluate
CD8+
central
and
effector
memory
compartments
at
14
wplv,
splenocytes
were
stained
with
␣-CD8,
␣-CCR7
and
␣-CD62L
antibodies
(BD).
Live
cells
(as
indicated
by
using
a
LIVE/DEAD
fixable
dye,
Invitrogen)
were
acquired
and
analyzed
using
an
LSRII
flow
cytometer
running
FACS
Diva
(BD).
2.7.
In
vivo
cytotoxic
T
lymphocyte
(CTL)
assay
To
prepare
target
cells,
splenocytes
from
naïve,
syngeneic
mice
were
fluorescently
labeled
with
10
␮M
green
(Vybrant
CFDA
SE
Cell
Tracer
Kit)
or
20
␮M
violet
(CellTrace
Violet
Cell
Proliferation
Kit)
fluorescent
dye,
according
to
manufacturer’s
instructions
(Invitro-
gen).
Cells
were
then
pulsed
for
2
h
with
20
␮M
irrelevant
or
specific

2052 L.M.
Shollenberger
et
al.
/
Vaccine
31 (2013) 2050–
2056
CTL
peptide,
respectively.
Targets
were
mixed
and
twelve
million
cells
were
injected
intravenously
per
vaccinated
animal.
After
20
h,
splenocytes
were
collected
and
analyzed
by
flow
cytometry
for
target
recovery.
3.
Results
and
discussion
3.1.
Vaccination
strategy
in
a
murine
model
of
helminth
infection
To
address
the
impact
of
Th2
biasing
on
vaccine
efficacy,
we
test
vaccines
in
animals
chronically
infected
with
helminth
par-
asites.
Cytokine
biasing
caused
by
chronic
schistosomiasis
was
assessed
in
mice
in
the
absence
of
vaccination
with
Listeria
vectors
(Fig.
1A).
For
unvaccinated
mice,
no
cytokines
were
detected
after
media
stimulation
or
upon
SEA
stimulation,
as
expected,
since
these
mice
had
no
prior
exposure
to
schistosomal
antigens.
A
decrease
in
the
IFN␥:IL-4
ratio
correlates
with
an
increase
in
Th2
bias-
ing,
which
is
shown
upon
conA
stimulation
in
a
time-dependent
manner.
Th2
biasing
increases
with
duration
of
infection,
shown
by
a
97.2%
decrease
in
the
IFN␥:IL-4
ratio
of
conA
stimulated
cells
(between
uninfected
to
18
wpi).
At
18
wpi,
the
cytokine
ratio
is
significantly
different
from
both
uninfected
mice
and
mice
at
7
wpi,
showing
mice
become
increasingly
Th2
biased
with
time.
In
mice
and
humans,
cytokine
switching
from
Th1
to
Th2
begins
approximately
6–7
wpi,
which
is
coincident
with
egg
deposition
by
adult
worms.
Previous
studies
have
used
animals
at
8–10
wpi
as
a
model
of
helminth
infection.
However,
in
our
studies,
vacci-
nation
is
postponed
until
12
weeks
after
infection
with
S.
mansoni
to
ensure
the
mice
have
fully
developed
Th2
biasing
(Fig.
1B)
and
to
eliminate
ambiguity
regarding
its
status
as
a
chronic
infection.
The
vaccine
is
then
administered
intraperitoneally
in
a
prime-boost
strategy
with
2
weeks
between
vaccinations
and
2
weeks
between
the
last
vaccination
and
the
analysis
of
vaccine-specific
immune
responses.
To
examine
whether
Listeria
vaccinated
mice
with
chronic
helminth
infection
would
also
exhibit
Th2
biasing,
splenocytes
were
harvested
from
age-matched,
Listeria
vaccinated
mice
with
or
without
schistosome
infection
at
16
weeks,
then
stimulated
ex
vivo
for
72
h
with
SEA
(antigen-specific
stimulation)
or
conA
(non-specific
stimulation)
and
cytokine
levels
measured
by
ELISA
(Fig.
1C–F).
At
16
wpi,
Th2
biasing
of
Schistosome
infected
mice
increases
relative
to
uninfected
when
both
groups
have
been
vac-
cinated
with
Listeria
vectors,
shown
by
a
78%
decrease
in
the
IFN␥:IL-4
ratio
upon
conA
stimulation
(Fig.
1C).
As
shown
in
Fig.
1D,
IFN␥
is
produced
by
cells
from
helminth
infected
mice
in
response
to
both
SEA
and
conA,
however
is
reduced
compared
to
cells
from
uninfected
mice
stimulated
with
conA.
IL-4
and
IL-10
levels
are
markedly
increased
in
splenocytes
from
Schistosome
infected
mice
relative
to
uninfected
mice
(Fig.
1E–F).
These
results
indicate
the
helminth
infected
mice
are
immune
suppressed
in
addition
to
Th2
biased.
Taken
together,
these
results
indicate
helminth
infected,
Listeria
HIV-1
vaccinated
mice
are
Th2
biased
and
immune
sup-
pressed,
as
indicated
by
a
reduction
in
the
IFN␥:IL-4
ratio
and
increases
in
IL-10
levels.
Th2
biasing
is
coincident
with
schistosome
infection,
as
shown
by
a
reduction
in
IFN␥:IL-4
ratio
upon
conA
stimulation,
whether
the
mice
are
naïve
(Fig.
1A)
or
Lm-gag
vaccinated
(Fig.
1C–E).
Fur-
ther,
helminth
infected,
Listeria
vaccinated
mice
are
also
immune
suppressed,
shown
by
relative
increases
in
IL-10
levels
com-
pared
to
uninfected
mice
(Fig.
1F).
Therefore,
the
schistosome
infection
employed
here
is
a
good
model
for
testing
vaccine
effi-
cacy
in
the
context
of
chronic
helminth
infection,
as
it
exhibits
characteristics
similar
to
real
world
chronic
helminth
infec-
tion.
3.2.
Listeria
vector
vaccines
induce
functional
HIV-specific
T
cell
responses
despite
chronic
helminth
infection
Two
wplv,
gag-specific
responses
were
assessed
by
IFN␥
ELISpot
assay
by
re-stimulation
of
splenocytes
in
the
presence
of
specific
CTL
peptide
or
specific
helper
peptide.
As
shown
in
Fig.
2,
Lm-gag
induces
antigen-specific
vaccine
responses
toward
immunodom-
inant
CTL
(Fig.
2A)
and
helper
(Fig.
2B)
epitopes
during
chronic
helminth
infection.
Further,
varying
the
vaccine
dose
and
regimen
does
not
alter
the
vaccine
response
to
the
immunodominant
CTL
(Fig.
2C)
or
CD4+
helper
(Fig.
2D)
epitopes.
For
vaccination
of
ani-
mals
with
chronic
Schistosomiasis,
the
vaccine
dose
was
lowered
to
0.1
LD50 or
the
schedule
was
altered
to
eliminate
the
boost,
result-
ing
in
a
prime
only
vaccine
strategy.
No
significant
differences
were
observed
when
comparing
the
responsive
groups.
These
results
suggest
the
Listeria
vector
is
a
potent
inducer
of
the
immune
sys-
tem
for
generation
of
vaccine-specific
responses
in
the
context
of
pre-existing
chronic
helminth
infection
without
the
need
for
drug
intervention
(anthelminthics)
prior
to
vaccination
and
there
exists
ample
room
for
modification
of
vaccine
dose
and
regimen.
To
validate
that
the
responses
measured
by
ELISpot
assays
result
from
functional
effector
cells,
an
in
vivo
CTL
assay
was
performed
(Fig.
2E).
Shown
by
a
lack
of
specific
killing
in
the
Lm-E7
vacci-
nated
(control)
mice,
non-specific
effects
of
the
Listeria
vector
itself
are
not
a
cause
for
the
observed
killing.
No
significant
difference
was
observed
between
Lm-gag
vaccinated
groups
with
and
with-
out
chronic
schistosomiasis,
indicating
the
vaccine
response
is
as
effective
in
helminth
infected
mice
as
in
uninfected
mice.
Levels
of
killing
are
modest,
perhaps
owing
to
the
multitude
of
proteins
expressed
by
Listeria,
resulting
in
a
small
percentage
of
the
host’s
immune
responses
specific
to
the
HIV
IIIB
gag
protein.
However,
clear
gag-specific
killing
is
demonstrated
in
groups
vaccinated
with
Lm-gag,
indicating
the
IFN␥+
CD8+
T
cells
are,
indeed,
functional
CTLs
generated
by
Listeria
vectored
vaccines
in
a
Th2
environment.
3.3.
T
cell
compartment
composition
is
independent
of
chronic
helminth
infection
To
further
characterize
the
composition
of
T
cell
compartments
in
vaccinated
mice
with
and
without
chronic
helminth
infection,
splenocytes
were
harvested
2
wplv
and
analyzed
by
flow
cytome-
try.
Upon
polyclonal
stimulation
with
PMA
and
Ionomycin,
CD4+
helper
T
cells
secreted
IFN␥
or
doubly
secreted
IFN␥
and
IL-4,
but
very
few
cells
produced
IL-4
without
IFN␥
(Fig.
3A).
The
differ-
ences
between
helminth
infected
and
uninfected
mice
that
were
vaccinated
with
Listeria
vectors
were
not
statistically
significant
(p
>
0.05),
consistent
with
the
antigen-specific
data
shown
in
Fig.
2.
Additionally,
as
shown
in
Fig.
3B,
the
regulatory
T
cell
compartment
(CD4+CD25±FoxP3±)
was
also
unaltered
in
these
animals.
When
evaluating
the
CD8+
compartment
for
molecular
specificity
and
IFN␥
production,
there
were
no
significant
differences
between
the
groups.
However,
there
was
an
observed
(not
significant)
increase
in
IFN␥
producing
non-gag-specific
(tetramer
negative)
CD8+
T
cells
in
the
vaccinated,
helminth
infected
group.
Likely,
this
resulted
from
either
increased
antigen
exposure
in
helminth
infection
or
an
increase
in
the
variability
of
the
response.
To
reconcile
these
results
with
the
overall
increase
in
IL-4
and
IL-10
levels
after
ex
vivo
stimulation
seen
in
Fig.
1,
several
pos-
sibilities
exist,
such
as
T
cell
plasticity,
increased
expression
of
cytokines
by
the
same
number
of
producers,
IL-4
can
originate
from
non-T
cells
during
helminth
infection
[46]
and
IL-10
can
come
from
T
cell
compartments
not
investigated
in
this
paper
(CD4+CD25−FoxP3−IL10+)
[47].
Nevertheless,
the
composition
of
the
T
cell
compartments
in
Listeria
vaccinated
mice
is
independent
of
pre-existing
chronic
helminth
infection.

L.M.
Shollenberger
et
al.
/
Vaccine
31 (2013) 2050–
2056 2053
Fig.
1.
Vaccination
strategy
in
a
murine
model
of
helminth
infection.
(A)
To
verify
Th2
biasing
of
helminth
infected
mice,
splenocytes
from
uninfected
mice
(open
bars)
or
schistosome
infected
mice
at
7
wpi
(gray
bars)
or
18
wpi
(solid
bars)
were
stimulated
for
with
SEA
or
conA
ex
vivo
and
supernatants
assayed
for
IFN␥and
IL-4
expression
by
ELISA.
Results
from
two
independent
experiments
are
pooled
and
plotted
as
the
mean
+
SEM
of
the
IFN␥:IL-4
ratio.
(B)
In
order
to
assess
vaccine-specific
responses
in
helminth
infected
mice,
6–8-week
old
female
BALB/c
mice
were
infected
with
Schistosoma
mansoni
or
left
naïve
and
helminth
infection
was
verified
by
ELISA
at
10
weeks
post
infection
(wpi).
Twelve
wpi,
age-matched
mice
were
primed
with
Listeria
vector
HIV-1
vaccine
(Lm-gag),
control
Listeria
vector
HPV
vaccine
(Lm-E7)
or
left
unvaccinated.
Mice
were
boosted
2
weeks
after
the
prime
in
an
identical
manner.
Vaccine
responses
were
evaluated
two
or
more
weeks
post
last
vaccination
(wplv).
(C–F)
Splenocytes
were
harvested
at
16
weeks
post
Schistosome
infection
from
Listeria
gag
vaccinated,
age
matched
mice
that
were
uninfected
(open
bars)
or
schistosome
infected
(solid
bars).
Splenocytes
were
stimulated
with
SEA
or
conA
ex
vivo
and
supernatants
assayed
for
IFN␥,
IL-4
and
IL-10
expression
by
ELISA.
Pooled
results
from
3
independent
experiments
are
plotted
as
the
mean
+
SEM
of
the
IFN␥:IL-4
ratio
(C)
and
absolute
IFN␥
(D),
IL-4
(E)
and
IL-10
values
(F)
(n
=
16–19).
Responding
groups
were
analyzed
by
ANOVA
with
Bonferroni
post
test.
*p
<
0.05,
***p
<
0.001,
****p
<
0.0001.
Fig.
2.
Listeria
vector
vaccines
induce
functional
HIV-specific
T
cell
responses
in
helminth
(schistosome)
infected
mice.
Naïve
or
chronic
schistosome
infected
mice
were
vaccinated
with
HIV
gag
(Lm-gag),
a
control
vector
(Lm-E7)
or
left
unvaccinated.
Splenocytes
were
collected
2
wplv
and
immune
responses
to
the
immunodominant
CTL
(A)
and
CD4+
helper
(B)
epitopes
were
assayed
by
IFN␥
ELISpot.
Vaccine
dose
(0.1
LD50)
and
regimen
(P
=
prime
only,
P
+
B
=
prime
and
boost)
were
altered
in
schistosome
infected
mice
and
vaccine-specific
responses
against
the
CTL
(C)
and
CD4+
helper
(D)
epitopes
were
evaluated.
(A–D)
Experiments
were
performed
in
triplicate,
pooled
and
shown
as
mean
+
SEM.
Total
numbers
of
mice
per
group
are
shown
and
statistical
analyses
of
responding
groups
were
performed
using
two-way
ANOVA
with
a
Bonferroni
post-test.
(E)
To
confirm
functionality
of
the
CTL
responses
shown
in
panel
A,
an
in
vivo
CTL
assay
was
performed.
Results
from
individual
mice
are
plotted
and
the
groups
that
showed
specific
killing
were
compared
by
unpaired,
two-tailed
t-test
analysis.

2054 L.M.
Shollenberger
et
al.
/
Vaccine
31 (2013) 2050–
2056
Fig.
3.
Comparable
T
cell
compartments
exist
2
weeks
post
last
Listeria
vector
HIV
gag
vaccination
independent
of
chronic
helminth
infection.
Naïve
(open
bars)
or
chronic
schistosome
infected
(solid
bars)
mice
were
vaccinated
with
Lm-gag
using
the
standard
prime-boost
method.
Splenocytes
were
collected
2
wplv
and
assayed
by
flow
cytometry
for
Th1
and
Th2
cells
(A),
T
regulatory
cells
(B)
or
specific
CTLs
by
tetramer
staining
(C).
Percentages
(upper
panel)
and
total
numbers
of
the
cells
in
the
spleen
(lower
panel)
are
shown.
Results
(n
=
6)
are
shown
as
mean
+
SEM.
Statistical
analysis
was
performed
using
two-way
ANOVA
with
a
Bonferroni
post-test.
Fig.
4.
Listeria
vectors
drive
HIV-specific
cell-mediated
vaccine
responses
that
are
durable
in
mice
chronically
infected
with
helminth
parasites.
(A
and
B)
Splenocytes
from
uninfected
(open
bars)
and
schistosome-infected
(hatched
bars)
mice
were
collected
at
various
times
post
last
vaccination
and
immune
responses
to
the
immunodominant
CTL
(A)
and
CD4+
helper
(B)
epitopes
were
assayed
by
IFN␥
ELISpot,
shown
as
mean
+
SEM
and
analyzed
by
unpaired,
two-tailed
t-test.
n
=
5–6
per
group.
(C
and
D)
Splenocytes
harvested
from
uninfected
and
schistosome-infected
mice
14
wplv
were
analyzed
by
flow
cytometry
to
confirm
molecular
specificity
to
the
HIV
gag
immunodominant
epitope
(C)
and
evaluate
CD8+
memory
compartments
(D).
Total
splenocytes
were
stained
with
␣-CD8
antibody
and
gag-tetramer
or
memory
markers
and
live
cells
were
acquired
and
analyzed
within
the
CD8+
population.
Groups
are
compared
using
unpaired,
two-tailed
t-test
analysis.
n
=
5–6
per
group.
*p
<
0.05,
**p
<
0.01,
****p
<
0.0001.

L.M.
Shollenberger
et
al.
/
Vaccine
31 (2013) 2050–
2056 2055
3.4.
Listeria
vectors
generate
HIV-specific
cell
mediated
vaccine
responses
that
are
durable
in
pre-existing
chronic
helminth
infection
To
evaluate
the
duration
of
the
vaccine-specific
cell-mediated
immune
responses,
uninfected
or
Schistosome-infected
mice
were
analyzed
several
months
after
the
last
vaccination
(Fig.
4A
and
B).
Although
differences
were
seen
for
helper
responses
at
early
time
points
of
this
experiment
(not
normal),
no
significant
differences
were
found
for
CTL
responses,
indicating
the
effector
(and/or
effec-
tor
memory)
cell
response
to
the
vaccine
is
unchanged
over
time
between
the
groups
and
the
helper
response
equalizes
with
time.
These
results
suggest
the
vaccine-specific
cell
mediated
immune
responses
generated
by
the
Listeria
vaccine
vectors
are
durable
and
independent
of
pre-existing
chronic
helminth
infection.
Therefore,
we
hypothesize
only
the
kinetics
of
memory
generation
is
affected
by
chronic
helminth
infection
and
not
the
magnitude
of
the
resul-
tant
memory
pool.
Tetramer
staining
at
14
wplv
was
used
to
verify
the
IFN␥
responses
measured
by
ELISpot
arise
from
antigen-specific
CD8+
T
cells
(Fig.
4C).
Consistent
with
the
ELISpot
data,
no
significant
dif-
ference
was
found
when
comparing
tetramer-positive
populations
between
vaccinated
groups
with
and
without
chronic
helminth
infection.
Further,
CD8+
memory
populations
were
evaluated
using
flow
cytometry.
As
shown
in
Fig.
4D,
there
was
a
higher
percentage
of
CD8+
central
memory
(CCR7+CD62L+)
populations
at
14
wplv
in
uninfected
mice
than
helminth
infected
animals,
coincident
with
a
lower
CD8+
effector
memory
and
effector
(CCR7−CD62L−)
com-
partment.
These
results
suggest
the
similar
values
seen
at
14
wplv
from
the
IFN␥
ELISpot
assays
were
a
combined
result
from
CTLs
and
the
CD8+
TCM and
TEM memory
pools.
4.
Conclusions
Vaccine
development
should
take
into
consideration
underly-
ing
diseases
present
in
recipient
populations
that
may
suppress
vaccine-specific
immune
responses.
Therefore,
it
is
critical
to
test
candidate
vaccines
in
animal
models
that
represent
as
closely
as
possible,
characteristics
of
the
intended
target
human
population.
Specifically,
vaccines
for
HIV,
TB
and
malaria
need
to
be
effective
in
helminth
infected
and
uninfected
individuals.
The
results
pre-
sented
here
demonstrate
the
existence
of
vaccine
vectors
capable
of
overcoming
helminth
infection
without
the
need
to
administer
anthelminthics
prior
to
vaccination.
Remarkably,
Listeria
vectored
vaccines
are
able
to
induce
robust
and
durable
CTL
and
Th1
vaccine
responses
in
helminth
infected,
immune
suppressed
populations.
This
represents
an
important
step
toward
our
goal
of
induc-
ing
vaccine-specific
immunity
in
helminth
infected,
Th2
biased
and
immune
suppressed
populations.
Further,
our
results,
taken
together
with
other
studies,
suggest
that
attenuated
Listeria
vector
vaccines
might
be
effective
on
a
global
scale.
We
hope
these
stud-
ies
encourage
others
who
are
developing
global
health
vaccines
to
validate
vaccine
efficacy
in
helminth
infected
recipients.
Acknowledgements
The
authors
would
like
to
thank
the
NIAID
Schistosomiasis
Resource
Center
for
biological
reagents
as
well
as
the
University
of
Georgia
College
of
Veterinary
Medicine
Cytometry
Core
Facility.
The
authors
would
also
like
to
thank
Lindsay
Nyhoff
for
techni-
cal
assistance
and
Drs.
Balazs
Rada
and
Wendy
Watford
for
critical
review
of
this
manuscript.
Conflicts
of
interest:
A
patent
has
been
filed
on
behalf
of
the
authors
LS,
YP
and
DH
for
use
of
Listeria
vectored
vaccines
in
helminth
endemic
areas.
CB
and
KA
have
no
competing
financial
interests
to
declare.
YP
discloses
that
she
has
a
financial
inter-
est
in
Advaxis,
Inc.,
a
vaccine
and
therapeutic
company
that
has
licensed
or
has
an
option
to
license
all
patents
from
the
University
of
Pennsylvania
that
concern
the
use
of
Listeria
or
listerial
products
as
vaccines.
Funding
sources:
Supported
by
grants
NIH-AI-071883
and
NIH-
AI-078787
awarded
to
DAH.
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