Vector replication and expression of HIV-1 antigens by the HIV/AIDS vaccine candidate MVA-B is not affected by HIV-1 protease inhibitors.

Abstract

MVA-B is an attenuated poxvirus vector expressing human immunodeficiency virus type 1 Env, Gag, Pol, and Nef antigens from clade B, and is considered a promising HIV/AIDS vaccine candidate. Recently, a phase I clinical trial in human healthy volunteers has shown that MVA-B is safe and highly immunogenic, inducing broad, polyfunctional, and long-lasting CD4(+) and CD8(+) T cell responses to HIV-1 antigens, with preference for effector memory T cells; and it also triggers the induction of specific antibodies to Env in most of the vaccines. While MVA recombinants expressing HIV-1 antigens are being used or plan to use in therapeutic clinical trials, little is known on the effect of HIV-1 highly active antiretroviral therapy in MVA life cycle. To define this role, here we have evaluated in established cell cultures and human dendritic cells to what extent different HIV-1 protease inhibitors affect virus replication and expression of HIV-1 antigens during MVA-B infection. The results obtained revealed that the most commonly used HIV-1 protease inhibitors (atazanavir, ritonavir, and lopinavir) had no effect on MVA-B virus growth kinetics, even at higher concentrations than those normally used on HAART. Furthermore, expression of gp120 and the fused Gag-Pol-Nef polyprotein in permissive and non-permissive cells infected with MVA-B were also not affected. These findings are relevant information for the therapeutic use of MVA-B as an HIV-1/AIDS vaccine.

Full text

Page 1

Virus
Research
167 (2012) 391–
396
Contents
lists
available
at
SciVerse
ScienceDirect
Virus
Research
jo u r n al
hom
epa
ge:
www.elsevier.com/locate/virusres
Short
Vector
candidate
communication
replication
and
expression
of
HIV-1
antigens
by
the
HIV/AIDS
vaccine
MVA-B
is
not
affected
by
HIV-1
protease
inhibitors
Juan
María
García-Arriazaa,
Pilar
Arnáeza,
José
Luis
Jiménezb, Carmen
E.
Gómeza,
Ángeles
Mu˜ noz-Fernándezb,
Mariano
Estebana,∗
aCentro
bLaboratorio
Nacional
de
Biotecnología,
Consejo
Superior
de
Investigaciones
Científicas
(CSIC),
Madrid,
Spain
Inmuno-Biología
Molecular,
Plataforma
de
Laboratorio,
Hospital
General
Universitario
Gregorio
Mara˜ nón,
Madrid,
Spain
a
r
t
i
c
l
e

i
n
f
o
Article
Received
Received
Accepted
Available online 30 May 2012
history:
13
March
2012
in revised
form
22 May
2012
23
May
2012
Keywords:
MVA-B
HIV-1
Vaccine
Protease
inhibitors
a
b
s
t
r
a
c
t
MVA-B
and
phase
inducing
preference
in
use
in
dendritic
of
HIV-1
kinetics,
of
MVA-B
as
is
an
attenuated
poxvirus
vector
expressing
human
immunodeficiency
virus
type
1
Env,
Gag,
Pol,
Nef
antigens
from
clade
B,
and
is considered
a
promising
HIV/AIDS
vaccine
candidate.
Recently,
a
I clinical
trial
in
human
healthy
volunteers
has
shown
that
MVA-B
is
safe
and
highly
immunogenic,
broad,
polyfunctional,
and
long-lasting
CD4+and
CD8+T
cell
responses
to
HIV-1
antigens,
with
for
effector
memory
T
cells;
and
it
also
triggers
the
induction
of
specific
antibodies
to
Env
most
of
the
vaccines.
While
MVA
recombinants
expressing
HIV-1
antigens
are
being
used
or
plan
to
in
therapeutic
clinical
trials,
little
is
known
on
the
effect
of HIV-1
highly
active
antiretroviral
therapy
MVA
life
cycle.
To
define
this
role,
here
we
have
evaluated
in
established
cell
cultures
and
human
cells
to
what
extent
different
HIV-1
protease
inhibitors
affect
virus
replication
and
expression
HIV-1
antigens
during
MVA-B
infection.
The
results
obtained
revealed
that
the
most
commonly
used
protease
inhibitors
(atazanavir,
ritonavir,
and
lopinavir)
had
no
effect
on
MVA-B
virus
growth
even
at
higher
concentrations
than
those
normally
used
on
HAART.
Furthermore,
expression
gp120
and
the
fused
Gag-Pol-Nef
polyprotein
in
permissive
and
non-permissive
cells
infected
with
were
also
not
affected.
These
findings
are
relevant
information
for
the
therapeutic
use
of
MVA-B
an
HIV-1/AIDS
vaccine.
© 2012 Elsevier B.V. All rights reserved.
AIDS
is
a pandemic
caused
by the
human
immunodeficiency
virus
ple
(WHO
1
antiretroviral
However,
vaccine
the
Among
combination
a
B/E)
about
vaccine
three
2009),
cine
poxvirus
(HIV),
and
it is
estimated
that
actually
around
34 million
peo-
are
living
with
HIV-1
with
about
1.8
million
deaths
in 2010
report).
There
is
no cure
for
the
infection,
although
HIV-
infected
patients
can
control
the
viral
load
under
highly
active
therapy
(HAART),
which
inhibits
virus
replication.
the
finding
and
development
of
a safe
and
efficacious
against
HIV-1
is one
of
the
main
objectives
to try
to cure
disease,
prevent
HIV-1
infection,
and
limit
viral
transmission.
all
the
HIV/AIDS
vaccine
candidates
actually
developed,
a
of
recombinant
canarypox-HIV-1
vector
(ALVAC)
plus
recombinant
HIV-1
envelope
gp120
subunit
protein
(AIDSVAX
evaluated
in a phase
III
clinical
trial
(RV144)
in Thailand
in
16,000
volunteers
at risk
of
infection,
remains
to be
the
only
protocol
that
showed
some
degree
of
efficacy
(31.2%),
after
years,
in reducing
HIV-1
infection
rates
(Rerks-Ngarm
et
al.,
providing
for
the
first-time
evidence
that
an
HIV/AIDS
vac-
can
prevent
HIV-1
infection
and
highlighting
that
improved
vectors
should
be
considered
as
components
of
a future
∗Corresponding
E-mail
author.
Tel.:
+34
91 5854553;
fax:
+34
91 5854506.
address:
mesteban@cnb.csic.es
(M.
Esteban).
effective
ated
promising
1
vectors
and
in
2011;
2011;
et
induce
strong,
responses.
candidate
expressing
polyprotein
2007).
broad,
T cell
et
lar
(from
cell
HIV/AIDS
vaccine.
Among
poxviruses,
the
highly
attenu-
modified
vaccinia
virus
Ankara
(MVA)
strain
is one
of
the
most
vectors
to be
used
as
an
effective
vaccine
against
HIV-
(Esteban,
2009;
Pantaleo
et
al.,
2010).
A great
number
of
MVA
expressing
different
HIV-1
antigens
have
been
generated
tested
in different
animal
models
and
in several
clinical
trials
humans
(Cebere
et
al.,
2006;
Currier
et
al.,
2010;
Goepfert
et
al.,
Gomez
et al.,
2011a;
Goonetilleke
et al.,
2006;
Keefer
et al.,
Kutscher
et
al.,
2010;
Ramanathan
et al.,
2009;
Sandstrom
al.,
2008;
Vasan
et
al.,
2010),
showing
that
they
are
safe,
able
to
high
levels
of
expression
of
HIV-1
antigens,
and
to trigger
broad,
polyfunctional
and
durable
HIV-1-specific
immune
In our
lab
we
have
constructed
an
HIV/AIDS
vaccine
termed
MVA-B
(Fig.
1A),
based
in a recombinant
MVA
Env,
as
monomeric
gp120,
and
the
codon-optimized
Gag-Pol-Nef
(GPN)
of
HIV-1
from
clade
B (Gomez
et
al.,
We
have
previously
shown
that
MVA-B
induced
in mice
polyfunctional
and
durable
HIV-1
specific
CD4+and
CD8+
immune
responses
(with
a bias
for
CD8+) (Garcia-Arriaza
al.,
2010,
2011;
Gomez
et
al.,
2007,
2009).
In macaques,
a
simi-
MVA
construct
but
expressing
gp120
(from
SHIV89.6P) and
GPN
SIVmac239) induced
strong
specific
HIV/SIV
CD4+and
CD8+T
immune
responses
and
trigger
protection
following
challenge
0168-1702/$
http://dx.doi.org/10.1016/j.virusres.2012.05.020
– see
front
matter ©

2012 Elsevier B.V. All rights reserved.

Page 2

392
J.
García-Arriaza
et al.
/ Virus
Research
167 (2012) 391–
396
A
MVA-B
A
B
Cen tral con served region
E P
Left te rmina l
C
regi on
Right termina l
regi on
NM K
F
DHJLGO I
1 E+08 1.E+08
B
J2R
sE/L
promoter
Gag-Po l-Nef (IIIB
)
gp120

(BX08 )
R-KTL-KT
1.E+05
1.E+06
1.E+07
Titer (PFU/ml)
0 h
24 h
1.E+04
lopinavir / ritonavi
(80%-20%)

r
MVA-B Mock
10
µg/ml
1
µg/ml
0,1
µg/ml
atazanavir
100
nM
10
nM
1
nM
ritonavir
10
µM
1
µM
0,1
µM
atazana

(75%-25%)
vir / ritona

vir
10
µM
0.1
µM
1
nM
Fig.
regions
as
within
plaque
(5
in
solution
treated
virus
corresponding
times
from
1.
Effect
of
HIV-1
PIs
on
the
viral
growth
of
MVA-B.
(A)
Scheme
of
MVA-B
genome
map.
The
different
regions
are
indicated
by capital
letters.
The
right
and
left
terminal
are
shown.
Below
the
map,
the
deleted
or fragmented
genes
from
MVA,
and
compared
to the
original
strain
Chorioallantois
vaccinia
virus
Ankara
(CVA),
are
depicted
black
boxes.
The
HIV-1
Gag-Pol-Nef
(from
isolate
IIIB)
and
gp120
(from
isolate
BX08)
clade
B sequences
driven
by the
synthetic
early/late
(sE/L)
virus
promoter
inserted
the
TK
viral
locus
(J2R)
are
indicated.
MVA-B
was
grown
in CEF
cells,
purified
through
two
36%
(w/v)
sucrose
cushions
in 10
mM
Tris–HCl
pH 9,
and
titrated
by
immunostaining
assay,
as
previously
described
(Ramirez
et al.,
2000).
(B)
Viral
growth
kinetics
of
MVA-B
in the
presence
of
HIV-1
PIs.
Monolayers
of BHK-21
cells
× 105cells),
were
treated
overnight
with
different
concentrations
of
HIV-1
PIs
lopinavir/ritonavir,
atazanavir,
ritonavir,
and
atazanavir/ritonavir;
and
incubated
at 37◦C
a 5%
CO2atmosphere
with
fresh
Dulbecco’s
modified
Eagle’s
medium
(DMEM)
containing
10%
fetal
calf
serum
(FCS).
HIV-1
PIs
were
dissolved
in DMSO
to obtain
a stock
(stock
solutions
of
lopinavir/ritonavir,
atazanavir,
and
ritonavir
were
10
mg/ml,
100
mM,
and
100
mM,
respectively).
As
controls,
cells
were
either
left
untreated
or
with
a comparable
concentration
of
DMSO,
but
without
HIV-1
PI.
Next
day,
cells
were
washed
and
infected
at 5 PFU/cell
with
MVA-B
or
mock-infected.
Following
adsorption
for
60
min
at 37◦C,
the
inoculum
was
removed
and
cells
were
incubated
at 37◦C in a 5% CO2atmosphere
with
fresh
DMEM
containing
2%
FCS
and
the
concentrations
of
lopinavir/ritonavir,
atazanavir,
ritonavir,
and
atazanavir/ritonavir.
At 0 and
24
h post-infection,
cells
were
collected,
freeze-thawed
three
and
briefly
sonicated.
Viral
titer
was
determined
by plaque
immunostaining
assay
with
anti-WR
antibodies,
as
previously
described
(Ramirez
et al.,
2000).
Data
are
one
experiment
representative
of
two
experiments.
with
MVA-B
different
to
DCs
functional
ation,
against
et
a phase
in
ated,
long-lasting
preference
responses
et
As a new
ration
entering
infected
order
Furthermore,
mon
can
pathogenic
strain
SHIV89.6P(Mooij
et
al.,
2008).
Furthermore,
induced
in human
dendritic
cells
(DCs)
the
expression
of
molecules
that
act
as
regulators
of
immune
responses
HIV-1
antigens
(Guerra
et
al.,
2010).
Moreover,
MVA-B-infected
co-cultured
with
autologous
T lymphocytes
induced
a highly
HIV-1-specific
CD8+T cell
responses
including
prolifer-
secretion
of
different
cytokines,
and
strong
cytotoxic
activity
autologous
HIV-1-infected
CD4+T lymphocytes
(Climent
al.,
2011).
Based
on
all
these
previous
results,
MVA-B
entered
in
I clinical
trial
(RISVAC02)
in 30 human
healthy
volunteers
Spain
and
the
results
showed
that
MVA-B
was
safe,
well
toler-
and
highly
immunogenic,
inducing
broad,
polyfunctional,
and
CD4+and
CD8+T cell
responses
to HIV-1
antigens,
with
for
effector
memory
T cells;
and
also
induced
antibody
to Env
in 95%
of
volunteers
(Garcia
et al.,
2011;
Gomez
al.,
2011b).
step
to move
forward
to support
further
explo-
of
MVA-B
as
an HIV-1
vaccine
candidate,
MVA-B
is now
into
a phase
I clinical
trial
in Spain
with
30 HIV-1
patients
on
HAART,
with
suppressed
viral
replication,
in
to evaluate
the
therapeutic
benefit
of
this
HIV/AIDS
vaccine.
it have
been
reported
that
one
of
the
most
com-
HIV-1
protease
inhibitors
(PIs)
use
on
HAART,
atazanavir,
significantly
reduce
heterologous
HIV-1
Gag
expression
by
recombinant
blocking
signaling
et
the
the
pared
in
are
HAART:
nation
which
tion
inhibit
Mayers
tories,
Reyataz
75%
centrations
in cell
trol,
dimethyl
virus
PIs
canarypoxvirus
ALVAC
expressing
HIV-1
antigens,
the
activation
of
phosphoinositide-3-OH
kinase
(PI3K)
pathway
in poxvirus-infected
human
monocytes
(Hu
al.,
2009).
Therefore,
we
performed
this
study
to determine
if
HIV-1
PIs
used
on
HAART
could
affect
MVA-B
replication
and
expression
of
HIV-1
antigens
gp120
and
GPN.
First,
we
com-
the
growth
of
MVA-B
in the
permissive
cell
line
BHK-21
the
presence
of
different
concentrations
of
HIV-1
PIs
which
currently
given
to HIV-1
infected
people
as
part
of
their
lopinavir/ritonavir
(Kaletra,
Abbott
Laboratories,
Combi-
of
lopinavir/ritonavir
mixed
in a relative
proportion
of
4:1,
means
80%
lopinavir–20%
ritonavir,
50%
effective
concentra-
(EC50) = 0.04–0.18
?g/ml;
the
drug
concentration
required
to
HIV-1
viral
replication
by
50%),
atazanavir
(Reyataz,
Bristol
Squibb,
EC50= 2–5 nM),
ritonavir
(Norvir,
Abbott
Labora-
EC50= 0.04
?M)
and
atazanavir/ritonavir
(a combination
of
+ Norvir
mixed
in a relative
proportion
of
3:1,
which
means
atazanavir–25%
ritonavir).
At
24
h post-infection,
all
the
con-
of
HIV-1
PIs
tested
did
not
affect
MVA-B
replication
culture,
as
the
virus
titers
were
similar
(Fig.
1B).
As
a con-
treatment
of
BHK-21
cells
with
comparable
concentrations
of
sulfoxide
(DMSO),
but
without
HIV-1
PIs,
did
not
alter
yields
(data
not
shown).
It should
be
noted
that,
the
HIV-1
were
added
to BHK-21
cells
at a total
concentration
equal
to or

Page 3

J. García-Arriaza
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al.
/ Virus
Research
167 (2012) 391–
396
393
Fig.
concentrations
legend
post-infection
(ARP
respectively.
Gag
fixed
Gag
FlowJo
overnight
MVA-B
total
2.
Effect
of
HIV-1
PIs
on
the
expression
of
HIV-1
gp120
and
GPN
by MVA-B.
Monolayers
of
BHK-21
cells
(A)
or
HeLa
cells
(B and
C),
pretreated
overnight
with
different
of
HIV-1
PIs
lopinavir/ritonavir,
atazanavir,
ritonavir,
and
atazanavir/ritonavir,
were
mock-infected
or
infected
with
MVA-B
at
5 PFU/cell,
as
described
in
of
Fig.
1.
(A and
B)
Expression
of
HIV-1
gp120
and
GPN
proteins
by
MVA-B
in the
presence
of
HIV-1
PIs
was
determined
at 24
h post-infection
(A)
or 24
h and
48
h
(B)
by western
blot,
using
rabbit
polyclonal
anti-gp120
antibody
against
IIIB
(Centro
Nacional
de Biotecnología;
1:3000)
or
polyclonal
anti-gag
p24
serum
432,
NIBSC,
Centralized
Facility
for
AIDS
reagent,
UK;
1:3000)
followed
by
anti-rabbit-HRP
(Sigma;
1:5000)
to evaluate
the
expression
of
gp120
and
GPN
proteins,
?-Actin
was
determined
in BHK-21
cells
as
a loading
control.
Data
are
from
one
experiment
representative
of
two
experiments.
(C)
FACS
analysis
of
HIV-1
p24
antigen
expression
in HeLa
cells
infected
with
MVA-B
in the
presence
of
HIV-1
PIs.
At 24
h post-infection
cultures
were
harvested
by
treating
with
trypsin,
cells
were
and
permeabilized
using
cytofix/cytoperm
solution
(Pharmigen
Inc.)
according
to manufacturer’s
instructions.
To test
HIV-1
antigen
expression
we
used
anti-HIV-1
antibody
conjugated
to phycoerythrin
(clone
KC57,
Beckman
Coulter).
A total
of
100,000
events
were
acquired
on
a LSR
II FACS
(Beckton
Dickinson)
and
analyzed
using
software
(FlowJo).
(D)
FACS
analysis
of
HIV-1
p24
Gag
antigen
expression
in moDCs
infected
with
MVA-B
in the
presence
of
HIV-1
PIs.
Briefly,
moDCs
were
pretreated
with
different
concentrations
of
HIV-1
PIs
lopinavir/ritonavir,
atazanavir,
ritonavir,
and
atazanavir/ritonavir.
Next
day,
moDCs
were
mock-infected
or
infected
with
at 5 PFU/cell
for
8 h in the
continuous
presence
of
different
concentrations
of
HIV-1
PIs.
At
8 h post-infection
cells
were
harvested
and
processed
similarly
as
in (C).
A
of
100,000
events
were
acquired
on
a LSR
II
FACS
(Beckton
Dickinson)
and
analyzed
using
FlowJo
software
(FlowJo).

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et al.
/ Virus
Research
167 (2012) 391–
396
A
8080
100
120
ction of p24)
% infection (produc
00
20
40
60
BB
lopinavir / ritonavi
(80%-20%)

r
Control
10
µg/ml
1
µg/ml
0,1
µg/ml
atazanavir
100
nM
10
nM
1
nM
ritonavir
10
µM
1
µM
0,1
µM
atazana

(75%-25%)
vir / ritona

vir
10
µM
0.1
µM
1
nM
100
µg/ml
10000
nM
60
80
100
120
on (luciferase)
% infectio
0
20
40
Control
1010
µg/ml
11 0 10,1
µg/ml
100 100
nM
1010
nM
1 1
nM
1010
µM
11
µM
0 1 0,1
µM
1010
µM
0 1 0.1
µM
11
nM
100100
µg/ml
1000010000
nM
lopinavir / ritonavi
(80%-20%)

r
µg/ml
atazanavirritonaviratazana

(75%-25%)
vir / ritona

vir
Fig.
from
10%
counted
medium
and
in
NL4-3-LUC)
presence
luciferase
3.
Inhibition
of
HIV-1
replication
by
HIV-1
PIs.
(A)
PBMCs
from
human
healthy
HIV-1-seronegative
donors
were
provided
by
the
HIV
HGM
BioBank
and
were
isolated
whole
blood
by Ficoll-Hypaque
density
gradient
centrifugation
(Pharmacia
Fine
Chemicals,
Uppsala,
Sweden),
resuspended
in RPMI
1640
medium
supplemented
with
FCS,
and
then
were
stimulated
with
phytohemagglutinin
(PHA)
for
2 days
(2 ?g/ml),
as
previously
described
(Garcia-Merino
et al.,
2009).
After
that,
cells
were
washed,
and
they
were
infected
with
HIV-1
NL4-3for
2 h at 37◦C at a MOI
of
10
ng of
virus
per
106PBMCs,
washed
three
times
and
incubated
in a 24-well
plate
with
RPMI
plus
5% serum,
20
IL-2
units
in presence
of
different
concentrations
of
HIV-1
PIs
(as
detailed
in legend
of
Fig.
1).
At day
3 post-infection,
supernatant
was
collected
assayed
for
viral
concentration
using
the
HIV-1
core
protein
p24
ELISA
assay
according
to kit
protocol
(Innogenetics,
Zwijnaarde,
Belgium).
(B)
The
T cell
line
MT-2,
grown
RPMI
1640
supplemented
with
10%
FCS,
1% penicillin–streptomycin
and
2 mM
l-glutamine,
was
infected
with
a VSV-G
pseudotyped
single-round
HIV-1
virus
(VSV-HIV
for
2 h at 37◦C at a MOI
of
10
ng
of
virus
per
106MT2
cells.
Cells
were
then
washed
3 times
and
laid
in a 24-well
plate
with
RPMI
medium
plus
5%
serum
in
of
different
concentrations
of
HIV-1
PIs
(as
detailed
in legend
of
Fig.
1).
At
day
2,
cells
were
recovered,
lysed
and
the
infectivity
was
determined
by
measuring
activity.
Cells
treated
with
ritonavir
1000
?M and
atazanavir/ritonavir
1000
?M were
dead.
higher
trations
the
permissive
Next,
expression
the
presence
infection
HIV-1
or
in
mined
(up
to 250
times)
than
the
EC50; and
none
of
these
concen-
were
able
to inhibit
MVA-B
virus
replication.
Summing
up,
presence
of
HIV-1
PIs
does
not
alter
MVA-B
replication
in virus
cells.
we
analyzed
if the
same
HIV-1
PIs
could
affect
the
of
HIV-1
antigens
gp120
and
GPN
by
MVA-B.
Thus,
permissive
cell
line
BHK-21
was
infected
with
MVA-B
in the
of
different
concentrations
of
HIV-1
PIs,
and
at 24
h post-
analysis
by
western
blot
revealed
that
MVA-B
expresses
antigens
gp120
and
GPN
at the
same
level
in the
presence
in the
absence
of
HIV-1
PIs
(Fig.
2A).
Since
MVA-B
is inoculated
humans,
where
the
virus
life
cycle
is
restricted,
we
next
deter-
the
expression
of
HIV-1
antigens
gp120
and
GPN
by
MVA-B
in
that
PIs,
post-infection
ment
DMSO,
lar
higher
GPN
HIV-1
HeLa
concentrations
harvested,
the
non-permissive
human
HeLa
cell
line.
The
results
revealed
treatment
of HeLa
cells
with
different
concentrations
of
HIV-1
did
not
abrogate
gp120
and
GPN
expression
levels
at
24
or
48 h
(Fig.
2B).
Similar
results
were
observed
after
treat-
of
BHK-21
or HeLa
cells
with
a comparable
concentration
of
but
without
HIV-1
PIs
(data
not
shown).
Significantly,
simi-
as
for
BHK-21
cells,
concentrations
of
HIV-1
PIs
up to 250
times
the
EC50were
not
capable
to inhibit
expression
of
gp120
and
by
MVA-B.
Moreover,
to provide
a better
quantification
of
the
antigens
expressed
by
MVA-B
in the
presence
of
HIV-1
PIs,
cells
were
infected
with
MVA-B
in the
presence
of
different
of
HIV-1
PIs;
and
at
24
h post-infection
cells
were
stained
for
HIV-1
p24
Gag
protein,
and
the
frequency
of

Page 5

J. García-Arriaza
et
al.
/ Virus
Research
167 (2012) 391–
396
395
Gag-expressing
sorting
HIV-1
ing
p24
confirm
cells,
cells
of
uous
harvested,
Gag-expressing
sorting
cells,
centage
the
(Fig.
commonly
antigens
To
cation
peripheral
donors
then
inhibited
2
and
(VSV-G)-pseudotyped
HIV
showed
growth
of
culture,
Different
HAART
(Cosma
2008;
them
of
sist
HIV-1
1
processing
into
final
tease
producing
are
they
cells,
receiving
activated
was
poxvirus
and/or
2009).
virion
is
regulation
of
the
Interestingly,
ing
canarypox
antigens
icantly
cells
was
quantified
by
fluorescent
activated
cell
(FACS)
analysis.
The
results
revealed
that
the
presence
of
PIs
did
not
alter
significantly
the
percentage
of
cells
express-
HIV-1
p24
Gag,
with
around
70%
of
the
cells
expressing
HIV-1
Gag
under
all
conditions
analyzed
(Fig.
2C).
Furthermore,
to
these
results,
we
performed
a
new
study
in human
primary
a more
relevant
cell
line.
Thus,
monocyte-derived
dendritic
(moDCs)
were
treated
overnight
with
different
concentrations
HIV-1
PIs,
and
then
infected
with
MVA-B
for
8 h in the
contin-
presence
of
HIV-1
PIs;
and
at 8 h post-infection
cells
were
stained
for
HIV-1
p24
Gag
protein,
and
the
frequency
of
cells
was
quantified
by
fluorescent
activated
cell
(FACS)
analysis.
Similarly
to the
results
obtained
in HeLa
the
presence
of
HIV-1
PIs
did
not
alter
significantly
the
per-
of
moDCs
expressing
HIV-1
p24
Gag,
with
around
80%
of
cells
expressing
HIV-1
p24
Gag
under
all
conditions
analyzed
2D).
Summing
up,
these
findings
established
that
the
HIV-1
PIs
used
in HAART,
do not
affect
to the
expression
of
HIV-1
by
MVA-B
in BHK-21,
HeLa,
and
moDCs.
compare
the
effects
of
HIV-1
PIs
observed
on
MVA-B
repli-
with
the
effects
of
the
same
PIs
on
HIV-1
replication,
blood
mononuclear
cells
(PBMCs)
obtained
from
healthy
were
treated
with
different
concentrations
of
HIV-1
PIs,
and
infected
with
HIV-1.
At
all
doses
analyzed,
HIV-1
PIs
tested
HIV-1
growth
in cell
culture
(Fig.
3A).
In addition,
MT-
cells
were
treated
with
different
concentrations
of
HIV-1
PIs,
then
infected
with
a vesicular
stomatitis
virus
glycoprotein
HIV-1
vector,
expressing
luciferase
(VSV-
NL4-3-LUC).
After
measuring
luciferase
activity,
the
results
also
that
all
the
HIV-1
PIs
tested
inhibited
the
pseudovirus
in cell
culture
(Fig.
3B).
Summing
up,
all
the
concentrations
HIV-1
PIs
tested
were
able
to inhibit
HIV-1
replication
in cell
while
have
no effect
on
the
replication
of
MVA-B.
phase
I clinical
trials
in HIV-1
infected
people
on
using
MVA-based
HIV/AIDS
vaccines
have
been
realized
et
al.,
2003;
Dorrell
et
al.,
2006,
2007;
Greenough
et
al.,
Harrer
et
al.,
2005;
Ondondo
et al.,
2006).
However,
none
of
studied
the
effect
of
HAART
on
the
replication
and
expression
HIV-1
antigens
by the
MVA
vector.
Current
HAART
regimens
con-
of
a combination
of
three
or
four
antiretroviral
drugs,
including
PIs
that
target
viral
assembly
by
inhibiting
the
activity
of
HIV-
protease,
an
enzyme
which
is critical
in the
post-translational
of
the
polyprotein
products
of
Gag
and
Gag-Pol
genes
the
functional
core
proteins
and
viral
enzymes
required
for
assembly
of
new
virions.
Thus,
the
inhibition
of
HIV-1
pro-
by the
PIs
prevents
cleavage
of
the
Gag-Pol
polyprotein,
immature,
non-infectious
virions.
Although
HIV-1
PIs
designed
to inhibit
HIV-1
protease,
it have
been
reported
that
can
also
inhibit
PI3K
signaling
pathway
activation
in tumor
at
serum
concentrations
routinely
achieved
in HIV-1
patients
HAART
(Gupta
et
al.,
2005).
PI3K
signaling
pathway
is
in poxvirus
infected
cells,
playing
an important
role,
as
it
shown
that
its
inhibition
by
specific
PI3K
inhibitors
reduced
replication,
blocked
or
delayed
the
expression
of
early
late
poxviral
genes,
and
induced
apoptosis
(Soares
et al.,
Furthermore,
PI3K
signaling
pathway
regulated
vaccinia
morphogenesis
(McNulty
et
al.,
2010;
Soares
et
al.,
2009)
and
one
of
the
most
critical
cellular
signaling
pathways
involved
in
of
poxvirus-mediated
foreign
gene
expression
at levels
protein
and
mRNA
synthesis
in non-permissive
cells,
and
also
at
level
of
viral
DNA
replication
in permissive
cells
(Hu
et
al.,
2009).
HIV-1
PI
atazanavir
can
inhibit
PI3K
cellular
signal-
pathway
activated
in human
primary
monocytes
infected
by
and,
as
a consequence,
the
expression
of
heterologous
(as
GFP
or
HIV-1
Gag)
by
ALVAC
recombinant
was
signif-
reduced
(Hu
et
al.,
2009).
Moreover,
it is
remarkable
that
some
of
herpes
2011).
multiple
as
poxvirus
more
PIs,
et
murine
have
processing
topes,
T cell
no
nized
HAART
vaccines
such
study
and
MVA
permissive
ent
HAART.
analyzed,
replication
quest
candidate,
on
might
evaluation
HIV-1
PIs,
as
nelfinavir,
could
inhibit
the
in vitro
replication
some
DNA
viruses,
as
Kaposi’s
sarcoma-associated
herpesvirus,
simplex
virus,
and
human
cytomegalovirus
(Gantt
et
al.,
Therefore,
PIs
could
affect
the
biology
of poxviruses
acting
on
enzymes
and
factors
encoded
or activated
by
poxviruses,
PI3K
signaling
pathway,
which
may
result
in an
inhibition
of
replication
and
expression
of
foreign
antigens.
Further-
and
interestingly,
it has
been
published
that
some
HIV-1
such
as
ritonavir,
could
inhibit
the
proteasome
activity
(Andre
al.,
1998;
Kelleher
et
al.,
2001),
affecting
immune
responses.
In a
model,
with
lymphocytic
choriomeningitis
virus
(LCMV)
it
been
shown
that
this
inhibition
of
the
proteasome
affects
the
of
MHC
class
I-restricted
cytotoxic
T lymphocyte
epi-
therefore
altering
antigen
presentation
and
antigen-specific
responses
(Andre
et
al.,
1998).
However,
others
have
reported
effect
of
ritonavir
on
the
processing
of
four
commonly
recog-
HIV-1
CTL
epitopes
(Kelleher
et
al.,
2001).
Thus,
PIs
present
on
may
negatively
impact
the
efficacy
of
poxvirus
vector-based
and
should
be
carefully
considered
when
administering
vaccines
to HIV-1
infected
people
on
HAART.
Therefore
to
this
phenomenon,
here
we
have
analyzed
the
replication
the
expression
of
the
foreign
antigens
gp120
and
GPN
by
an
vector-based
HIV-1
vaccine
(MVA-B)
during
virus
infection
of
and
non-permissive
cells,
under
the
presence
of
differ-
HIV-1
PIs,
commonly
used
in HIV-1
infected
patients
receiving
Our
results
showed
that
none
of
the
different
HIV-1
PIs
and
none
of
the
concentrations
tested,
could
inhibit
the
of
MVA-B
and
the
expression
of
gp120
and
GPN.
In the
for
an
effective
therapeutic
strategy
of
an HIV/AIDS
vaccine
it is
important
to establish
the
consequences
of
HAART
the
biology
of
the
poxvirus
vector
used
for
immunization,
which
limit
the
benefit
of
vaccination.
Our
results
support
further
of
MVA-B
as
an HIV-1
therapeutic
vaccine.
Acknowledgments
Blood
samples
from
human
healthy
HIV-1-seronegative
donors
were
Spanish
ported
RIS
contract
Programa
nidad
kindly
provided
by
the
HIV
HGM
BioBank,
integrated
in the
AIDS
Research
Network
(RIS).
This
investigation
was
sup-
by grants
from
(SAF2008-02036
and
FIPSE
to ME),
RED
RD06/0006/0035
and
RD06/0006/0032.
JG-A
was
supported
by
from
the
Ministry
of
Health
of
Spain.
JLJ
is supported
by
de
Investigación
de
la Consejería
de Sanidad
de
la Comu-
de
Madrid.
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