JOURNAL OF VIROLOGY, May 2010, p. 5314–5328
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 84, No. 10
Preclinical Studies of a Modified Vaccinia Virus Ankara-Based HIV
Candidate Vaccine: Antigen Presentation and Antiviral Effect?
Samantha Brandler,1,4Alice Lepelley,1Marion Desdouits,2Florence Guivel-Benhassine,1
Pierre-Emmanuel Ceccaldi,2Yves Le ´vy,3,4Olivier Schwartz,1,4†* and Arnaud Moris1,4†*
Unite ´ Virus et Immunite ´1and Unite ´ d’Epide ´miologie et Physiopathologie des Virus Oncoge `nes,2Institut Pasteur,
URA CNRS 3015, Paris, France; INSERM U955, AP-HP, Groupe Henri-Mondor Albert-Chenevier,
Immunologie Clinique, Creteil, France3; and ANRS HIV Vaccine Network (AHVN)4
Received 4 November 2009/Accepted 22 February 2010
Poxvirus-based human immunodeficiency virus (HIV) vaccine candidates are currently under evaluation in
preclinical and clinical trials. Modified vaccinia virus Ankara (MVA) vectors have excellent safety and
immunogenicity records, but their behavior in human cell cultures remains only partly characterized. We
studied here various virological and immunological aspects of the interactions of MVA-HIV, a vaccine candi-
date developed by the French National Agency for AIDS Research (ANRS), with primary human cells. We
report that MVA-HIV infects and drives Gag expression in primary macrophages, dendritic cells (DCs), and
epithelial and muscle cells. MVA-HIV-infected DCs matured, efficiently presented Gag, Pol, and Nef antigens,
and activated HIV-specific cytotoxic T lymphocytes (CTLs). As expected with this type of vector, infection was
cytopathic and led to DC apoptosis. Coculture of MVA-HIV-infected epithelial cells or myotubes with DCs
promoted efficient Gag antigen major histocompatibility complex class I (MHC-I) cross-presentation without
inducing direct infection and death of DCs. Antigen-presenting cells (APCs) infected with MVA-HIV also
activated HIV-specific CD4?T cells. Moreover, exposure of DCs to MVA-HIV or to MVA-HIV-infected
myotubes induced type I interferon (IFN) production and inhibited subsequent HIV replication and transfer
to lymphocytes. Altogether, these results show that MVA-HIV promotes efficient MHC-I and MHC-II presen-
tation of HIV antigens by APCs without facilitating HIV replication. Deciphering the immune responses to
MVA in culture experiments will help in the design of innovative vaccine strategies.
Early efforts to develop an anti-human immunodeficiency
virus (anti-HIV) vaccine focused on the generation of HIV-
specific humoral responses. For instance, recombinant gp120
was shown to rapidly induce neutralizing antibodies, which
displayed a limited capacity to control replication of primary
viral isolates (38, 41) and did not confer protection from in-
fection (15, 48). However, recent studies identified new classes
of neutralizing antibodies that may form the basis for future
humoral immunity-based vaccine strategies (24, 53, 59). Vac-
cines promoting HIV-specific cellular responses have also been
studied extensively for the past 2 decades. Multiple lines of
evidence suggest that T cells participate in the control of
HIV-1 replication. During acute infection, expansion of HIV-
specific CD8?T cells (HS CTLs) before the appearance of
neutralizing antibodies is associated with decreased viremia
(60). Resistance to disease progression correlates with the de-
tection of Gag-specific CTLs and with the presence of partic-
ular HLA alleles, such as HLA-B57 and -B27 (32, 33). Viral
escape mutants are found in infected individuals (31), under-
scoring the selection pressure exerted by CTLs. In addition,
CD8?T-cell depletion experiments in nonhuman primates
(NHP) established the importance of these cells in controlling
viral replication during acute and chronic phases of infection
In NHP, vaccination with attenuated simian immunodefi-
ciency virus (SIV) mutants confers near-complete protection
from homologous challenge, providing proof of concept that
development of an HIV vaccine is feasible (62). However, for
obvious safety reasons, attenuated HIV strains are not cur-
rently under consideration. Recombinant viral vectors encod-
ing HIV proteins or peptide strings, based on viruses such as
poxviruses, adenoviruses, vesicular stomatitis virus, and mea-
sles virus, represent promising alternatives. These viruses in-
fect various cells and tissues. In animals and human volunteers,
they elicit specific cellular and humoral responses to foreign
genes inserted in the vector genome. Moreover, their natural
adjuvant effect may lead to secretion of appropriate cytokines
and chemokines. Each type of viral vector induces distinct
qualities of T-cell and antibody responses, depending on pa-
rameters such as viral tropism, fitness, and interplay with the
innate immune system (2, 18, 25, 50).
Recombinant poxviruses, such as vaccinia virus (VACV),
canarypox virus, and modified vaccinia virus Ankara (MVA),
are among the most-studied vectors. The recent Thai phase III
clinical trial combined, in a prime-boost strategy, a canarypox
vector expressing HIV Gag, Pro, and Env antigens (Ags) and
a trimeric recombinant gp120 protein. Preliminary results
demonstrated a partial but significant protection against HIV
infection in vaccinated individuals. This raises considerable
interest in characterizing other poxviruses as vaccine candi-
dates. MVA is a nonpersisting vector with excellent safety
* Corresponding author. Mailing address for Arnaud Moris:
INSERM UMRS945, Infection et Immunite ´, UPMC, 91 Bd. de l’Ho ˆpital,
75641 Paris Cedex 13, France. Phone: 33 1 40 77 99 10. Fax: 33 1 40 77
97 34. E-mail: firstname.lastname@example.org. Mailing address for Olivier
Schwartz: Institut Pasteur, Unite ´ Virus et Immunite ´, URA CNRS
3015, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France. Phone: 33 1
45 68 83 53. Fax: 33 1 40 61 34 65. E-mail: email@example.com.
† O.S. and A.M. contributed equally as co-senior authors.
?Published ahead of print on 10 March 2010.
records. It is being considered for vaccination against infec-
tious diseases, including HIV infection, malaria, and influenza,
and against cancers (for a review, see reference 19). In pre-
clinical studies, vaccination with an MVA vector encoding
HIV-1 Gag linked to a string of CTL epitopes (so called MVA-
HIVA), used alone or in combination with DNA in a prime-
boost strategy, induced HS T cells (21, 63). Moreover, vacci-
nation of NHP with MVA encoding SIV Gag, Pol, Nef, and
Env induced a robust polyfunctional SIV-specific T-cell re-
sponse leading to reduced viral replication and prolonged sur-
vival upon simian/human immunodeficiency virus (SHIV)
challenge (40). Mucosal vaccination of NHP with MVA en-
coding SIV Gag, Pol, and Env also led to good control of SHIV
replication, with a 2-log reduction in viral loads (1). The route
of MVA inoculation influences the immunogenicity of the vec-
tor and the quality of the immune response (20, 37). MVA has
been studied in cell culture experiments and in animal models
(5, 10, 11, 35). MVA was generated by 500 passages of the
Ankara strain of vaccinia virus, leading to deletion of multiple
viral genes (39). It replicates in BHK cells and chicken embryo
fibroblasts but does not perform a full replication cycle in other
cell lines or primary cells (5). Viral expression is limited to
early gene products and to inserted foreign genes. MVA vec-
tors generally display a broad tropism, infecting a large panel
of cell lines and antigen-presenting cells (APCs), and induce
rapid cell death by apoptosis. Dendritic cells (DCs) are impor-
tant targets of MVA infection (7, 13, 29). Upon MVA infec-
tion, DC morphology, gene expression profiles, and matura-
tion state are modified (23). A comparison of canarypox virus
and MVA vectors revealed differences in infectivity and anti-
gen production by DC cultures that may influence immunoge-
nicity in vivo (64). The pathways mediating innate immune
sensing were recently characterized and shown to involve Toll-
like receptor 2 (TLR2) to TLR6, the cytosolic sensor MDA5,
and the NALP3 inflammasome (10). Much less is known about
the sensitivity of primary epithelial and muscle cells to MVA
infection. These cells are likely some of the first cells, along
with DCs, that may be targeted by the vector after mucosal or
intramuscular vaccination. The ability of MVA-infected cells
to present or cross-present antigens to human CD4?and
CD8?virus-specific lymphocytes, as well as the susceptibility of
MVA-exposed DCs to subsequent HIV infection, remain
In the present work, we describe and characterize an MVA-
Gag-Pol-Nef (MVA-HIV) vaccine candidate developed by the
French National Agency for AIDS Research (ANRS). We first
studied the tropism of MVA-HIV by using a panel of human
primary cells and immortalized cell lines. We report that
MVA-HIV preferentially infects APCs, myotubes, and epithe-
lial cells. MVA-HIV infection is cytopathic. Infected cells
present Gag-, Pol-, and Nef-derived epitopes and directly ac-
tivate HS CD8?cells. MVA-HIV-infected APCs also stimu-
late HS CD4?T-cell clones. In addition, HIV antigens ex-
pressed by MVA-HIV-infected muscle and epithelial cells are
cross-presented by DCs to stimulate HS CTLs. Finally, we
show that exposure of DCs to MVA induces an antiviral state
that inhibits HIV replication and transfer to T cells. Our re-
sults strongly suggest that the highly immunogenic properties
of MVA vector are not associated with an enhancement of
MATERIALS AND METHODS
MVA-HIV vaccine candidate. MVA-HIV was developed in partnership with
the ANRS. The recombinant virus (MVATG17401) was manufactured by Trans-
ge `ne (Strasbourg, France) according to standard procedures (39). The recombi-
nant HIV antigens include the full-length codon-optimized sequence of gag
(encoding amino acids [aa] 1 to 512) fused with fragments from pol (encoding aa
172 to 219, 325 to 383, and 461 to 519) and nef (encoding aa 66 to 147 and 182
to 206) from the Bru/Lai isolate (Los Alamos database accession number
K02013). To enhance further Gag expression, poly(C), poly(G) (longer than 3
nucleotides), and poly(GC) (longer than 8 nucleotides) motifs within gag were
replaced. Using PCR, the gag-pol-nef sequence was then introduced into the
transfer plasmid pTG17401. The recombinant virus vaccine (MVATG17401)
expressing the gag-pol-nef fusion of HIV-1 was then produced by homologous
recombination between pTG17401 and the virus (MVATGN33). The gag-pol-nef
sequences were inserted into the excision III site of MVA by cloning using
standard procedures (39). Briefly, after transfer of the plasmid to MVATGN33,
selection of MVATG17401 clones was carried out by successive subcloning.
Characterization was carried out on 100 clones after 6 passages. The structure
and expression profiles of the clones were determined based on PCRs and
Western blotting. By use of specific primers, the absence of the wild-type (WT)
virus was shown in 100% of clones. Expression of HIV recombinant protein is
under the control of the early-late vaccinia virus promoter p5HR (51).
MVA infection. Cells were infected in serum-free medium for 1 h at the
indicated multiplicity of infection (MOI) (ranging from 0.1 to 10), washed three
times with serum-free medium, and resuspended in the appropriate medium for
further use. MVA infection was monitored using anti-Gag p24 antibodies and
Western blotting, fluorescence-activated cell sorting (FACS), or confocal micros-
copy. When required, virus was UV inactivated for 15 min by use of a 312-nm
UV bulb positioned 4 cm over the sample (Fisher Bioblock-Scientific).
Cells. Clinical-grade DCs were prepared using a VacCell processor as de-
scribed previously (43). Peripheral blood mononuclear cells (PBMCs) were cul-
tured for 7 days in serum-free medium (Invitrogen) with 500 U/ml granulocyte-
macrophage colony-stimulating factor (GM-CSF; Gentaur) and 50 ng/ml
interleukin-13 (IL-13; Peprotech, Tebu-bio), and DCs were isolated by elutria-
tion. Alternatively, DCs were generated using 1,000 U/ml IL-4 (R&D) and 100
ng/ml GM-CSF (Gentaur) as described previously (44). Both isolation proce-
dures yielded immature DCs (CD1a?MHC-I?MHC-II?CD64?CD83?
CD80lowCD86lowcells). HeLa cells, 293T cells, and primary human cells (Hsk-
789.Sk cells, A549 cells, BEAS-2B cells, and MRC5 cells) were grown in Dul-
becco’s modified Eagle’s medium (DMEM) with 10% heat-inactivated fetal
bovine serum (FBS). Hsk-789.Sk cells are primary human skin cells (ATCC 7518
[American Type Culture Collection]). MRC5 cells (ATCC CCL-171) are pri-
mary human fetal lung epithelial cells. A549 cells (ATCC CCL-185) are human
malignant alveolar type II pneumocytes, and BEAS-2B cells (ATCC CRL-9609)
are simian virus 40 (SV40)-transformed human airway epithelial cells. PBMCs
were isolated from the blood of healthy donors by Ficoll centrifugation. CD4?T
lymphocytes were isolated by negative selection, using magnetic beads (Miltenyi
Biotec), and were cryopreserved. Where stated, CD4?T lymphocytes were
activated by phytohemagglutinin (PHA) and grown with recombinant human
IL-2 (rhIL-2) for 7 days. HD420 and Bre cells are Epstein-Barr virus (EBV)-
immortalized B cells (44). Jurkat T cells, MT4C5 cells, CEM cells, HUT cells,
PBMCs, primary lymphocytes, and monocytes were grown in RPMI medium
with 10% FBS. CD14?monocytes were isolated from PBMCs by positive selection,
using magnetic beads (Miltenyi Biotec). To allow differentiation of macrophages
(M?), monocytes were cultured for 7 days in RPMI 1640 medium with 5% human
AB? serum, 5% FBS, and rhuM-CSF (12.5 ng/ml; Promokine). CHQ5 primary
myoblasts, originally isolated from the quadriceps of a 5-day-old infant (9), were
cultured in Ham’s F10 medium with 20% heat-inactivated FBS and 50 ?g/ml gen-
tamicin. Myotubes were differentiated from myoblasts after 5-day cultures in
DMEM supplemented with 50 ?g/ml gentamicin, 100 ?g/ml transferrin, and 10
?g/ml insulin (9). CTLs specific for Gag (recognizing the HLA-A*02-restricted SL9
epitope p17 [aa 77 to 85]) (43), Pol (recognizing the HLA-A*02-restricted IV9
epitope [aa 476 to 484]; a kind gift from Florence Buseyne, Institut Pasteur) (43),
and Nef (recognizing the HLA-A*11-restricted QK10 epitope [aa 73 to 82]; a kind
gift from Miche `le Fe ´vrier, Institut Pasteur) (16) and Gag-specific CD4?T-cell
clones (44) were cultured as described previously (16, 43, 44).
ELISPOT assays. (i) Direct presentation. Stimulator cells were exposed to
MVA-HIV, UV-inactivated MVA-HIV, or cognate peptides (1 ?g/ml) as a positive
control, washed, and incubated for 5 h in RPMI medium containing 10% FBS to
allow virus internalization. Stimulator cells were then cocultured for at least 8 h with
HS T-cell clones. Gamma interferon (IFN-?) production was measured using
enzyme-linked immunospot (ELISPOT) assays as described previously (43).
VOL. 84, 2010 MVA-HIV: FROM ANTIGEN PRESENTATION TO HIV REPLICATION5315
(ii) Cross-presentation. Donor cells were infected with MVA-HIV for 1 h at
an MOI of 1 or incubated with 1 ?g/ml of SL9 peptide, washed extensively,
seeded for 5 h, and then cocultured with DCs for 1 h. Donor and stimulator cells
(DCs) were then cocultured for at least 8 h with HS T-cell clones, and IFN-?
production was measured by ELISPOT assays.
Surface and intracellular staining. Cell surface staining was performed at 4°C
for 30 min, using anti-HLA-A2 (BB7.2-fluorescein isothiocyanate [BB7.2-FITC];
BD Pharmingen), anti-CD4, anti-CD8 (BD Pharmingen), anti-CD14 (BD Pharmin-
gen), anti-CD19 (BD Pharmingen), anti-CD56 (Becton Dickinson), anti-CD86–
phycoerythrin (anti-CD86–PE) (BD Pharmingen), anti-CD80–allophycocyanin
(anti-CD80–APC) (BD Pharmingen), anti-CD83–FITC (BD Pharmingen), anti-
major histocompatibility complex class I–FITC (anti-MHC I–FITC) (W6.32-FITC;
Sigma), and anti-DC-SIGN (120507-APC/-PE) (R&D) monoclonal antibodies
(MAbs). Anti-HIV Gag (KC57-FITC; Beckman-Coulter) MAb was used to detect
infected cells. Briefly, cells were fixed for 10 min with phosphate-buffered saline
(PBS) plus 4% paraformaldehyde, washed, and permeabilized for 15 min in PBS
containing 0.1% bovine serum albumin (BSA) and 0.05% saponin prior to Ab
staining. Isotype-matched MAbs were used as negative controls. Samples were
analyzed by flow cytometry, using a FACSCalibur (Becton Dickinson) or FacsCanto
(Becton Dickinson) flow cytometer with CellQuest or FacsDIVA software.
Confocal imaging. Myotubes or DCs were infected with MVA-HIV (MOI of
1 or 0.1, respectively) for 6, 15, or 24 h and fixed in 4% paraformaldehyde (10
min on ice). Cells were then stained with anti-desmin rabbit polyclonal Ab
(Abcam) and goat anti-rabbit–DyLight-547 (Pierce) or with rhodamine-phalloi-
din (4 U/ml; Molecular Probes) and anti-HIV Gag (KC57-FITC; Beckman-
Coulter) Abs in PBS-5% BSA plus 0.01% saponin. Samples were analyzed by
confocal microscopy on a Zeiss LSM510 instrument.
Cell viability assays. (i) 7-AAD staining. 7-Aminoactinomycin D (7-AAD)–
APC (BD Pharmingen) was used according to the manufacturer’s instructions. In
brief, cell pellets were resuspended in 100 ?l PBS (107cells/ml), and 5 ?l of
7-AAD was added to the cell suspension. Cells were incubated for 10 min on ice
in the dark. As a positive control, cells were treated with actinomycin D (1 ?g/ml,
12 h, 37°C) (Sigma). Samples were analyzed by flow cytometry.
(ii) Trypan blue exclusion. Myotubes were harvested from plates by use of
PBS plus 1% EDTA and were incubated with trypan blue (5 min, room tem-
perature). Trypan blue-negative cells (viable cells) were counted in a Bu ¨rker
Analysis of HIV replication in DCs. (i) HIV infection. The R5-tropic virus
HIVNLAD8was produced as described previously (44).
(ii) DCs directly exposed to MVA-HIV. DCs were exposed to MVA-HIV for
1 h, washed extensively, loaded with HIVNL-AD8at a low viral inoculum (1 ng of
p24/ml) or high viral dose (100 ng of p24/ml) with DEAE-dextran (10 ?g/ml;
Sigma) for 2 to 3 h at 37°C, washed, and seeded in 96-well plates. HIV replication
was measured using a Gag p24 enzyme-linked immunosorbent assay (ELISA;
NEN, Perkin-Elmer Life Sciences). Alternatively, DCs exposed to MVA-HIV
and loaded with a low HIV inoculum (1 ng/ml) were cocultured with autologous
PHA-activated CD4?T cells at a 1/1 ratio in 96-well plates. As a control,
PHA-activated T cells alone were infected with HIVNLAD8(1 ng/ml of p24),
washed, and seeded in 96-well plates with rhIL-2, and HIV replication was
DCs exposed to MVA-infected cells. A total of 2 ? 105HeLa cells or myotubes
seeded in 6-well plates were infected with MVA-HIV for 1 h, washed extensively,
rested for 5 h to allow MVA-HIV internalization, washed, and cocultured with
2 ? 106DCs. After overnight incubation, DCs were harvested by gentle pipet-
ting, infected with HIVNLAD8(high dose of 100 ng p24/ml and low dose of 1 ng
p24/ml) for 2 h, and then seeded in 96 well-plates at 106cells/ml. HIV production
in culture supernatants was measured using Gag p24 ELISA. Alternatively, DCs
exposed to MVA-HIV-infected cells and loaded with a low HIV inoculum (1 ng
p24/ml) were cocultured with autologous PHA-activated CD4?T cells at a 1/1
ratio in 96-well plates. As a control, PHA-activated T cells alone were infected
with HIVNLAD8(at the indicated concentrations), washed, and seeded in 96-well
plates with rhIL-2, and HIV replication was measured using Gag p24 ELISA.
IFN-? detection. IFN-? secretion was quantified using the reporter cell line
HL116 (a kind gift from Sandra Pellegrini, Institut Pasteur, France). HL116 cells
carry the luciferase gene under the control of the IFN-inducible 6-16 promoter
(57). HL116 cells were grown in DMEM supplemented with 10% FBS and
hypoxanthine-aminopterin-thymidine (HAT) (50 mM). A total of 20,000 HL116
cells, plated in a 96-well plate 24 h prior to the assay, were incubated for 7 h with
the desired culture supernatants or standards containing a titration of human
IFN-?2a (Immunotools). Cells were then lysed (luciferase cell culture lysis re-
agent [5?]; Promega), and luciferase activity was measured using luciferase assay
reagent (Promega). Samples were analyzed using a Perkin Elmer Wallac 1420
instrument. IFN levels are expressed as equivalent IFN-?2a concentrations.
Western blot analysis of MVA-HIV-infected cells. HeLa cells were infected
with MVA, MVA-HIV (MOI of 0.1 for 1 h at 37°C), or HIVNL4-3pseudotyped
with vesicular stomatitis virus glycoprotein (VSV-G) (100 ng of p24/ml for 3 h at
37°C). Sixteen or 36 h after infection with MVA, MVA-HIV, or HIVNL4-3, cells
were lysed in PBS–1% Triton X-100 (Sigma) supplemented with protease inhib-
itors (Roche). Fifteen micrograms of protein lysate was analyzed by SDS-PAGE,
using 4 to 12% NuPage gels (Invitrogen). The blots were probed with an anti-
Gag p24 MAb (25A) (46). Horseradish peroxidase (HRP)-conjugated goat anti-
IgG (Amersham) was used as secondary antibody. Peroxidase activity was re-
vealed using chemiluminescence (Pierce).
The MVA-HIV vaccine candidate from the ANRS. The
MVA-HIV vaccine candidate was designed by the ANRS. It
encodes an HIV polyprotein from the clade B isolate Bru,
composed of the full-length Gag protein fused to three Pol and
two Nef fragments (see Materials and Methods for further
details). Upon MVA-HIV infection of HeLa cells, the expected
-immunity/supplemental-material). Additional species with lower
molecular masses, likely corresponding to processed forms of the
polypeptide, were also revealed (http://www.pasteur.fr/ip/easysite
stained with anti-Gag MAbs confirmed the expression of HIV
proteins (Fig. 1).
MVA-HIV infects APCs and muscle and epithelial cells. We
examined the susceptibility of various cell types to MVA-HIV
infection. We first studied whether MVA-HIV infects and
drives the expression of the recombinant protein in hemato-
poietic cells (Fig. 1). To this end, PBMCs were incubated with
MVA-HIV at an MOI of 1. After 15 h, cells were stained with
an anti-Gag MAb and analyzed by FACS. We observed a weak
infection of whole PBMCs (3% Gag?cells) (Fig. 1A). Double
staining indicated that NK cells, CD4?or CD8?T lympho-
cytes, and B cells were marginally infected by MVA-HIV
(?1% Gag?cells), whereas CD14?monocytes represented
the majority of Gag-expressing cells (Fig. 1B). Purified CD14?
monocytes, as well as monocyte-derived M? and DCs, were
highly susceptible to MVA infection (Fig. 1A, left panel). In
DCs, confocal microscopy analysis showed a cytoplasmic and
membrane-bound localization of Gag (Fig. 1C), resembling the
pattern observed in HIV-infected cells. Gag expression was
rapid in M? and DCs. It was detected as soon as 3 h postin-
fection (p.i.) and increased from 7 to 15 h p.i. (32 and 62% Gag?
cells for M? and DCs, respectively) (Fig. 1D). In contrast to
primary lymphocytes, immortalized B- and T-cell lines were gen-
erally efficiently infected by MVA-HIV (Fig. 1A, right panel).
Overall, these results indicate that primary APCs are partic-
ularly sensitive to MVA infection, driving rapid expression of
the vaccine antigens.
Current protocols for MVA vaccination include intramuscu-
lar, intradermal, and intramucosal applications. We thus ex-
amined the sensitivity of muscle cells and of epithelial cells to
MVA-HIV infection. Primary muscle fibers (myotubes) de-
rived from satellite muscle cells (myoblasts) were incubated
with MVA-HIV. Infection was monitored with anti-Gag MAbs
by use of confocal microscopy, since these cells are too large
and adherent to be processed by flow cytometry. Remarkably,
at 15 h p.i., up to 70% of myotubes expressed Gag (Fig. 1E and
5316BRANDLER ET AL.J. VIROL.
FIG. 1. MVA-HIV preferentially infects APCs and vaccination target cells. (A) Gag expression in primary cells (left) or in cell lines (right) after
MVA-HIV infection at the indicated MOI. At 15 h p.i., cells were stained for intracellular HIV Gag and analyzed by flow cytometry. DCs were
differentiated in the presence of IL-4 or IL-13. The data represent the means ? standard deviations (SD) for at least three independent experiments.
(B) PBMCs were infected with MVA-HIV (MOI ? 1). At 15 h p.i., cells were stained for cell surface markers and intracellular HIV Gag and analyzed
by flow cytometry. Uninfected PBMCs and isotype-matched Abs were used as negative controls (not shown). T cells, CD8?and CD4?cells; NK cells,
CD56?cells; monocytes, CD14?cells; B cells, CD19?cells. The percentages of Gag?cells in the corresponding cell subsets are shown. The data are
representative of three independent experiments. (C) DCs were infected with MVA-HIV (MOI ? 0.1), and HIV Gag expression was further analyzed
by immunofluorescence and confocal microscopy. Green, HIV Gag staining; red, actin-phalloidin-PE. NI, not infected. (D) Kinetics of HIV Gag
cells 15 h after MVA-HIV infection (MOI ? 1), assessed using flow cytometry and immunofluorescence for myotubes. The data represent the means ?
SD for at least three independent experiments. (F) Myotubes were infected with MVA-HIV (MOI ? 1), and HIV Gag expression was analyzed by
immunofluorescence and confocal microscopy. Green, HIV Gag staining; red, anti-desmin MAb to identify myotubes. NI, not infected.
F). The epithelial cell lines 293T, HeLa, A549 (a pulmonary
alveolar cell line), and BEAS-2B (a bronchial cell line), as well
as primary lung fibroblasts (MRC5 cells), were efficiently in-
fected, with 50 to 90% Gag-expressing cells (Fig. 1E). These
results confirmed that MVA-HIV has a very large tropism,
including relevant primary cells (myotubes and epithelial cells)
that may be encountered by MVA upon injection.
MVA-HIV-infected cells present Gag, Nef, and Pol antigens.
We asked whether MVA-HIV-infected APCs present MHC-I-
and MHC-II-restricted HIV-derived epitopes. To this end,
DCs, M?, monocytes, or B cells were exposed to MVA-HIV
for 1 h, washed, and seeded for 5 h to allow MVA internaliza-
tion and antigen expression. Antigen presentation was re-
vealed by overnight coculture of infected cells with HIV-spe-
cific (HS) CD8?or CD4?T-cell clones and was quantified by
IFN-? ELISPOT assay (Fig. 2). We first analyzed MHC-I an-
tigen presentation by using the HS CD8 T-cell clone EM40-
F21, derived from an HIV-infected patient and recognizing an
FIG. 2. MVA-HIV-infected cells directly present antigens to HS CTLs. (A) Experimental procedure. APCs were loaded (1 h) with MVA-HIV,
UV-inactivated MVA-HIV (at the indicated MOI), or peptide (1 ?g/ml) or mock treated and then were washed, seeded for 5 h to allow HIV
antigen expression, and cocultured for at least 8 h with HS T cells. T-cell activation was monitored by IFN-? ELISPOT assay. (B) The CTL clone
EM40-F21, specific for Gag, was used to test the capacity of diverse MVA-HIV-infected cell types to present HIV Gag-derived antigens.
(C) MVA-HIV-infected APCs present Pol- and Nef-derived epitopes, leading to CTL activation. The CTL lines IV9 and P1, specific for Pol aa
476 to 484 and Nef aa 73 to 82, respectively, were cocultured with autologous B-cell lines, and T-cell activation was monitored by IFN-? ELISPOT
assay. Background IFN-? production by target cells alone was subtracted and was at least three times lower than that with HS T cells. For each
panel, data are means ? SD for triplicates and are representative of at least 2 independent experiments. Percentages of HIV Gag?APCs,
determined by flow cytometry at the end of the coculture period, are indicated.
5318 BRANDLER ET AL.J. VIROL.
immunodominant HLA-A*0201-restricted epitope from Gag
p17 (SL9) (43). MVA-HIV-infected APCs induced a dose-
dependent activation of EM40-F21 (Fig. 2B). At a high MOI,
IFN-? production by EM40-F21 was equivalent to that induced
by the cognate peptide, added exogenously (Fig. 2B). MVA-
HIV-infected DCs and M? efficiently activated the HS CTL
clone EM40-F21, whereas whole PBMCs, primary monocytes,
primary T cells, and HeLa cells were less potent (Fig. 2B).
Furthermore, using Pol (aa 476 to 484)- and Nef (aa 73 to
82)-specific CTL lines restricted by HLA-A*0201 and HLA-
A*11, respectively (16, 43), we demonstrated that MVA-HIV-
infected B cells and DCs also present Pol and Nef epitopes
(Fig. 2C and data not shown).
The HS CD4 T-cell clones F12 and IV-9 recognize two
Gag-derived epitopes (aa 271 to 290 and aa 331 to 350, respec-
tively) of p24, presented by HLA-DR?*01 (44). MHC-II-re-
stricted antigen presentation was studied using autologous pri-
mary monocyte-derived DCs or B-cell lines (HD420 and Bre).
MVA-HIV-infected DCs and B cells activated F12 and IV-9
cells (Fig. 3). The HD420 B-cell line also expresses HLA-A2.
We verified that HD420 activated the HS CD8 clone EM40-
F21 upon MVA-HIV infection (not shown). Therefore, MVA-
HIV-exposed APCs activate both HS CD4?and CD8?T cells.
Importantly, cells infected with wild-type MVA (not express-
ing any HIV antigens) did not stimulate EM40-F21 or HS
CD4?T-cell clones (not shown). UV-inactivated MVA-HIV
lost viral infectivity (measured by the lack of appearance of
Gag?cells) and did not induce viral antigen presentation by
DC and B cells (Fig. 2 and 3). Therefore, activation of EM40-
F21 and HS CD4?T-cell clones was not due to the presenta-
tion of incoming Gag antigens that may have been carried over
with MVA-HIV virions. In agreement with these observations,
the epithelial cell line HeLa-A2, which does not perform an-
tigen cross-presentation (not shown), activated EM40-F21
cells when they were infected with MVA-HIV (Fig. 2B).
Altogether, these data demonstrate that APCs infected with
MVA-HIV present HIV-derived epitopes through MHC-I and
MHC-II molecules. Gag Ags are derived from neo-synthesized
proteins. The capacity of MVA-HIV-infected cells to present
Ag is not limited to professional APCs, thus highlighting the
immunogenic potential of the vaccine candidate.
MVA-HIV stimulates DC maturation. Upon Ag capture,
immature DCs (imDCs) differentiate into a mature stage and
acquire specific functions allowing Ag-specific T-cell activa-
tion. We investigated the effect of MVA-HIV on DC matura-
tion. imDCs were exposed to MVA-HIV or treated with me-
dium or lipopolysaccharide (LPS) as a control. DC maturation
was analyzed by FACS 24 h later. As expected, LPS fully
matured DCs, with an upregulation of CD83, the costimulatory
molecules CD80 and CD86, and MHC-I and -II molecules
(Fig. 4A). Interestingly, exposure of DCs to MVA-HIV also
led to cell maturation (Fig. 4A). Only a fraction (30%) of the
cells exhibited an intermediate maturation phenotype (see
CD83 staining in Fig. 4A).
DCs interact with infected cells to acquire antigens or to
induce inflammation. We thus examined if MVA-infected cells
induced DC activation. To this end, HeLa cells were exposed
to MVA-HIV, washed thoroughly, incubated for 6 h to allow
degradation of residual viral input and viral replication, and
then cocultured with DCs. As expected, control, uninfected
HeLa cells did not affect DCs (Fig. 4A). In contrast, MVA-
HIV-infected HeLa cells induced partial DC maturation, char-
FIG. 3. MVA-HIV-infected cells directly present antigens to HS CD4?T cells. As in Fig. 2, MVA-HIV-infected DCs and B cells were used
to activate the HS CD4?T-cell clone F12, which is specific for the Gag p24(271–290) amino acid sequence. MVA-HIV-infected B cells were used
to stimulate the IV-9 clone, which is specific for Gag p24(331–350). T-cell activation was monitored by IFN-? ELISPOT assay. Background IFN-?
production by target cells alone was subtracted and was at least three times lower than that with HS T cells. For each panel, data are means ?
SD for triplicates and are representative of at least 2 independent experiments. Percentages of HIV Gag?APCs, determined by flow cytometry
at the end of the coculture period, are indicated.
VOL. 84, 2010 MVA-HIV: FROM ANTIGEN PRESENTATION TO HIV REPLICATION5319
FIG. 4. Effects of MVA-HIV on DC maturation and cell viability. (A) MVA-HIV promotes DC maturation. DCs were mock treated or infected
with MVA-HIV (MOI of 1 for 1 h), washed, and incubated for 24 h in cell culture medium. As a positive control, DC maturation was induced
using LPS (1 ?g/ml; Sigma). For the coculture experiments, HeLa cells were infected with MVA-HIV (1 h), washed extensively, seeded for 6 h
to allow MVA-HIV internalization, and cocultured with DCs for 24 h. DCs were then harvested, stained with the indicated Abs, and analyzed by
5320 BRANDLER ET AL.J. VIROL.
acterized by a slight upregulation of CD83 and CD80 and a
pronounced expression of CD86 (Fig. 4A). This maturation
might be induced by the presence of apoptotic or dead MVA-
HIV-infected HeLa cells or by the release of soluble factors by
infected cells, as previously reported for canarypox virus-in-
fected cells (27). DCs were apparently not infected after con-
tact with MVA-HIV-infected HeLa cells, as assessed by the
absence of Gag?DCs (not shown).
Taken together, these results indicate that direct infection of
DCs with MVA-HIV allowed full maturation of the APCs,
whereas contact with MVA-infected cells induced an interme-
diate maturation phenotype.
MVA-HIV induces apoptosis of infected cells. MVA is a
cytopathic virus that induces apoptosis of infected cells (7, 23,
29, 64). We analyzed the viability of DCs, monocytes, B cells,
and HeLa cells following MVA-HIV infection. Cells were in-
fected at two MOIs, and viability was monitored by FACS,
using 7-AAD (Fig. 4B and data not shown). As a positive
control for induction of apoptosis, cells were treated with ac-
tinomycin D. At 24 h post-MVA-HIV infection with the high
MOI, a significant proportion of the cells underwent apoptosis
(30% of 7-AAD?cells [Fig. 4B] or cells expressing active
caspase-3 [not shown]). Interestingly, apoptosis was associated
with direct infection of the cells, since coculture of DCs with
MVA-HIV-infected HeLa cells did not induce significant DC
mortality (Fig. 4B). Moreover, UV-treated MVA-HIV did not
promote cell death (not shown).
We also studied the fate of muscle fibers after MVA-HIV
infection (Fig. 4C). At 24 h p.i., 50% of infected cells were
dying or dead, as measured by trypan blue exclusion. Confocal
imaging indicated that infected fiber cells expressed Gag anti-
gens at 6 h p.i. and then displayed, at 24 h p.i., membrane blebs
and fragmented nuclei, well-known markers of apoptosis
These results confirm and extend previous works by demon-
strating that DCs as well as muscle fibers undergo apoptotic
death upon MVA-HIV infection.
HIV-antigens expressed by MVA-HIV-infected cells are
cross-presented by DCs. DCs display a unique capacity to take
up antigens from apoptotic cells, leading to MHC-I-restricted
cross-presentation. In vaccinated individuals, DCs will proba-
bly encounter MVA-infected cells. We asked whether MVA-
HIV-infected cells might represent a source of antigen for
cross-presentation to HS CTLs. For this purpose, HLA-A*02-
negative cells (so-called donor cells) were exposed to MVA-
HIV, washed extensively to remove unbound virus, and culti-
vated for 5 h to allow infection, potential internalization or
degradation of incoming virions, and expression of HIV anti-
gens (Fig. 5A). Infected cells were then cocultivated with
HLA-A2*02?DCs. Whatever the donor cells, we did not de-
tect any Gag?DCs, suggesting that no infectious MVA-HIV
was transferred to DCs in the cocultures (not shown). Cross-
presentation was then revealed in an IFN-? ELISPOT assay
after overnight coculture with HLA-A*02-restricted EM40-
F21 CTLs. Primary skin (Hsk-789.Sk) and lung (MRC5) fibro-
blasts, primary myoblasts or myotubes, and a bronchiolar ep-
ithelial cell line (A549) were selected as donor cells, since they
lack HLA-A*02 expression when tested by FACS (not shown).
Accordingly, when infected by MVA-HIV, these HLA-A*02?
donor cells did not directly activate EM40-F21 (Fig. 5). In
contrast, coculture of these MVA-HIV-infected donor cells
with HLA-A2?DCs led to Gag antigen presentation and
EM40-F21 stimulation. A dose-response analysis indicated
that a ratio of 0.5 to 0.05 infected donor cell for 1 target DC
was sufficient to activate EM40-F21 (Fig. 5B). Cross-presenta-
tion was detected with all donor cells tested (Fig. 5B). Notably,
coculture of uninfected donor cells with HLA-A2?DCs did
not lead to HS CTL activation (Fig. 5B, DC ? donor mock
bars). HIV antigens expressed by MVA-HIV-infected HeLa
cells were also cross-presented by HLA-A2?DCs (Fig. 5B).
Hence, HeLa cells are able to perform both direct (see our
results with a HeLa-A2?derivative cell line [Fig. 2]) and in-
direct (cross-presentation [Fig. 5]) activation of HS CTLs.
Altogether, these results underline the immunogenic poten-
tial of MVA-HIV. Infected primary muscle fiber cells and
primary fibroblasts likely represent an important source of
antigens. Activation of immune cells by MVA-HIV may occur
by different means. Infected cells may directly present antigens
or be processed by DCs to activate CTLs.
MVA-HIV-infected DCs do not efficiently spread HIV. DCs
are natural targets of HIV infection and efficiently transmit the
virus to T lymphocytes. We analyzed the interactions that may
exist between MVA infection of DCs and HIV replication.
DCs were exposed to MVA-HIV for 1 h and then infected with
HIV-1 at two MOIs. HIV replication was then monitored by
measuring Gag p24 in culture supernatants by ELISA (Fig. 6A
and B). In control DCs, HIV replication reached 90 and 13 ng
p24/ml at day 10 p.i., at high and low MOIs, respectively (Fig.
6B). Interestingly, after DC exposure to MVA-HIV, HIV rep-
lication was barely detected (about 2 ng/ml at the high MOI)
HIV is known to exploit the capacity of DCs to form inti-
mate contacts with T cells to enhance its replication and to
spread (44, 45, 56, 61). We thus examined the effect of MVA-
FACS to monitor DC maturation. Histograms represent DC maturation within viable cells. Shaded histograms, isotype-matched Ab controls; gray
lines, mock-treated imDCs; dark lines, DCs treated with the indicated stimuli. The data are representative of three independent experiments. (B)
MVA-HIV induces apoptosis of infected cells. DCs and HeLa cells were infected with MVA-HIV at the indicated MOI (1 h), washed three times,
and seeded in cell culture medium. Coculture experiments were performed as described for panel A. At the indicated time points, DCs exposed
to MVA-HIV or MVA-HIV-infected HeLa cells were harvested and cell viability analyzed using 7-AAD and FACS. As a positive control for the
induction of cell death, cells were treated with actinomycin D (Act-D; 1 ?g/ml) for 12 h. Cell mortality is expressed as the percentage of 7-AAD?
cells (cells were gated to exclude cellular debris). Data are means ? SD for 3 independent experiments. (C) Primary myotubes were treated as
in panel B, and cell viability was analyzed using trypan blue exclusion. Cell mortality is expressed as the percentage of trypan blue-positive cells.
Data are means ? SD for duplicates and are representative of 2 independent experiments. (D) Primary myotubes used for panel C were stained
intracellularly for HIV Gag and using actin-phalloidin-PE and were analyzed by confocal microscopy. Data are representative of 3 independent
VOL. 84, 2010 MVA-HIV: FROM ANTIGEN PRESENTATION TO HIV REPLICATION5321
HIV on HIV replication within DC–T-cell cocultures. To this
end, DCs were exposed to MVA-HIV, loaded with HIV-1 (at
a low MOI [1 ng p24/ml]), and then incubated with PHA-
activated autologous CD4?T cells (Fig. 6C and D). At this low
MOI, HIV replicated poorly in DCs or in activated T cells
cultured separately (with a peak of 25 and 48 ng of p24/ml at
day 11 in DCs and T cells alone, respectively). In contrast,
robust replication occurred in DC–T-cell cocultures, with a
FIG. 5. MVA-HIV-infected cells are cross-presented by DCs to HS CTLs. (A) Cross-presentation experimental procedure. HLA-A2?donor cells
were mock treated or infected with MVA-HIV (MOI ? 1, 1 h), washed, seeded for 5 h to allow Gag expression, and cocultured with HLA-A2?DCs
control, HLA-A2?DCs were directly infected with MVA-HIV (MOI ? 1) or loaded with the SL9 peptide (1 ?g/ml). Importantly, in the absence of DCs,
HLA-A2?donor cells infected with MVA-HIV (MOI ? 1) cannot stimulate HS CTLs. Mock-treated or MVA-infected donor cell-to-DC ratios are
indicated (donor cell/DC). Background IFN-? production induced by uninfected cells and IFN-? production by target cells alone (at least three times
5322 BRANDLER ET AL.J. VIROL.
peak of 450 ng of p24/ml. Remarkably, when DCs were preex-
posed to MVA-HIV, there was a dramatic decrease of HIV
replication in DC–T-cell cocultures (21 ng of p24/ml) (Fig. 6D).
Thus, MVA-HIV-infected DCs do not allow efficient HIV
replication and transmission to T cells. This may be due in
large part to the proapoptotic effects of MVA-HIV, which will
reduce the number of viable DCs (Fig. 4). The fraction of dying
DCs may also release apoptotic compounds that hamper HIV
replication in bystander DCs, or MVA-HIV-exposed DCs may
express antiviral factors (23).
MVA-HIV-infected myotubes inhibit HIV replication in DC–
T-cell cocultures. We then examined whether MVA-HIV-in-
fected bystander cells affect HIV replication in DCs and trans-
fer to T cells. To this end, DCs were exposed to control or
FIG. 6. Exposure to MVA-HIV inhibits HIV replication in DC and DC–T-cell cocultures. (A) Impact of MVA-HIV on HIV replication in DCs.
DCs were mock treated or incubated with MVA-HIV (1 h, MOI ? 1), washed, and loaded with HIVNL-AD8(2 h, high and low doses of 100 and
1 ng of HIV Gag p24/ml/106cells, respectively), and HIV replication was monitored. (B) Kinetics of HIV replication in DC culture supernatants,
assessed by HIV Gag p24 ELISA. (C) Impact of MVA-HIV on HIV transfer. DCs were treated as in panel A (HIVNL-AD8, 2 h, 1 ng of HIV Gag
p24/ml/106cells) and cocultured with activated autologous CD4?T cells (1 DC/1 T cell). (D) Kinetics of HIV replication in DC–T-cell culture
supernatants, assessed by HIV Gag p24 ELISA. As controls, DCs were cultured separately (DC alone) and CD4?T cells were directly infected
with the same HIV dose (T alone), and HIV replication was monitored. For each panel, data are means ? SD for duplicates and are representative
of at least 3 independent experiments. d, day.
VOL. 84, 2010MVA-HIV: FROM ANTIGEN PRESENTATION TO HIV REPLICATION5323
MVA-HIV-infected HeLa cells (Fig. 7A and B). Viability of
DCs was not altered by contact with MVA-HIV-infected cells
(Fig. 4). DCs were then exposed to two MOIs, and HIV rep-
lication was followed by measuring p24 release. MVA-HIV-
infected HeLa cells inhibited HIV replication in DCs, whereas
this was not the case with mock-treated HeLa cells (Fig. 7B).
We then tested the effect of MVA-HIV-infected cells on
HIV spread in DC–T-cell cocultures. HeLa cells or primary
FIG. 7. Exposure to MVA-HIV-infected cells inhibits HIV replication in DCs and DC–T-cell cocultures. (A) Impact of MVA-HIV-infected cells on
HIV replication in DCs. HeLa cells were infected with MVA-HIV (1 h, MOI ? 1) (HeLa-MVA) or mock infected (HeLa), washed extensively, seeded
(5 h) to allow MVA-HIV infection, and cocultured with DCs (1 HeLa cell for 10 DCs). After overnight (ON) coincubation, DCs were harvested by gentle
pipetting and infected with HIVNL-AD8(2 h, high and low doses of 100 and 1 ng of HIV Gag p24/ml/106cells, respectively), and HIV replication was
monitored. (B) Kinetics of HIV replication in DC culture supernatants, assessed by HIV Gag p24 ELISA. Data are means ? SD for duplicates and are
representative of 2 independent experiments. (C) Impact of MVA-HIV-infected cells on HIV transfer. HeLa cells or primary myotubes were infected
with MVA-HIV or mock infected and were cocultured with DCs as described for panel A. After overnight coincubation, DCs were harvested, infected
with HIVNL-AD8(2 h, 1 ng of HIV Gag p24/ml/106cells), washed, and cocultured with activated autologous CD4?T cells (1 DC to 1 T cell) in 96-well
plates at 2 ? 106cells/ml. (D) Kinetics of HIV replication in DC–T-cell culture supernatants, assessed by HIV Gag p24 ELISA. As controls, DCs were
cultured separately (DC) and CD4?T cells were directly infected with the same HIV dose (T), and HIV replication was monitored. Data are means ?
SD for duplicates and are representative of 3 independent experiments.
5324 BRANDLER ET AL.J. VIROL.
myotubes were infected with MVA-HIV for 1 h, washed ex-
tensively to remove unbound virus, cultivated for 5 h to allow
degradation of incoming virions, and seeded with DCs. Cells
were loaded with HIV for 2 h to allow viral capture by DCs
and were then cocultured with activated CD4?T cells, as
outlined in Fig. 7C. A representative experiment with HeLa
cells is depicted in Fig. 7D (left panel). In DC–T-cell cocul-
tures in the absence of HeLa cells, HIV replication reached
350 ng of p24/ml, and the addition of mock-treated HeLa
cells did not alter HIV replication. In sharp contrast, HIV
replication was almost fully abrogated when MVA-HIV-
infected HeLa cells were present. Similar results were ob-
tained when MVA-HIV-infected myotubes were preadded
to DC–T-cell cultures instead of HeLa cells (Fig. 7D, right
panel). Exposure to MVA-HIV-infected cells did not induce
apoptotic death of lymphocytes within DC–T-cell cocultures
in contact with MVA-HIV or MVA-HIV-infected cells, HIV
does not efficiently replicate in DCs or spread to lymphocytes.
DCs exposed to MVA-HIV-infected myotubes secrete IFN-?.
Type I IFN and other IFN-inducible genes may be responsible
for the anti-HIV state mediated by MVA-HIV or MVA-HIV-
infected cells in DCs. MVA is indeed known to trigger IFN-?
production in different cell types, such as MRC5 cells and DCs
(5, 23). Interestingly, neither HeLa cells nor myotubes pro-
duced detectable levels of bioactive IFN-? when directly in-
fected by MVA-HIV (Fig. 8). The situation was different with
DCs. When these cells were infected with MVA-HIV or were
cocultivated with MVA-HIV-infected HeLa cells or myotubes,
they released significant levels of IFN-?, in the range of those
obtained with Sendai virus (SeV), a well-characterized type I
IFN inducer (Fig. 8).
These results show that DCs produce type I IFN when they
encounter MVA-HIV-infected cells. This may restrict HIV
replication and transfer in DCs.
The design of efficient candidate HIV vaccines requires a
better knowledge of the mechanisms inducing appropriate im-
mune responses. Attenuated viral vectors naturally stimulate
systemic and mucosal immunity and represent attractive anti-
HIV vaccine candidates. In the present work, we studied the
capacity of an MVA vector coding for HIV antigens to infect
primary human cells, to activate HS T-cell immunity, and to
inhibit replication and transmission of HIV-1.
MVA is an attenuated strain of vaccinia virus that exhibits
very limited replication in most mammalian cells (39). Tradi-
tional application routes include intramuscular and subcutane-
ous immunization, and alternative strategies targeting mucosal
immunity are currently being developed (8, 30). We screened
a panel of human primary and immortalized cells of various
origins for susceptibility to MVA. We showed that muscle
(primary myotubes and myoblasts) and epithelial cells are sen-
sitive to MVA-HIV, driving the expression of the vaccine an-
tigen. MVA infects primary lung fibroblast and alveolar and
bronchial epithelial cell lines. Overall, we demonstrated that
MVA infects primary cells relevant for intramuscular (myo-
tubes), subcutaneous (epithelial cells), and intranasal (lung
cells) immunization routes. We further characterized the fate
of MVA-infected cells. As previously described for HeLa cells
(23), we demonstrated that MVA-HIV induces apoptotic cell
death, with morphological changes noticeable at 6 h postexpo-
sure, in most cell types tested. MVA is known to trigger early
immigration of leukocytes at the site of infection (35). We thus
tested the capacity of MVA-HIV to infect human PBMCs.
Confirming previous work (52), we showed that among
PBMCs, monocytes are the main targets of the vector. More-
over, monocyte-derived DCs and M? are particularly sensitive
to MVA-HIV infection, suggesting that this may also be the
case for primary blood DCs. As previously reported for murine
cells (36), MVA-HIV induces phenotypic maturation of imma-
ture DCs, as well as apoptosis of a significant fraction of in-
FIG. 8. DCs infected with MVA-HIV or exposed to MVA-HIV-infected cells secrete IFN-?. DCs, HeLa cells, and myotubes were directly
infected with MVA-HIV (1 h, MOI ? 1), washed, and cultured for 20 h, and IFN-? release in the culture supernatants was monitored.
Alternatively, HeLa cells or myotubes (2 ? 105plated in 6-well plates) were infected with MVA-HIV (1 h, MOI ? 1), washed extensively, seeded
for 5 h to allow MVA-HIV internalization, and cocultured with DCs (2 ? 106). After overnight coculture, supernatants were assessed for IFN-?.
As a positive control, DCs were incubated with 4 U of hemagglutinin of SeV (55). IFN-? was quantified using a bioassay (HL116 reporter cell line)
and is expressed in IFN-?2a concentration equivalents. For each sample, a serial dilution was performed and submitted to the bioassay. Data for
one dilution are presented and are representative of 3 (left) and 2 (right) independent experiments.
VOL. 84, 2010 MVA-HIV: FROM ANTIGEN PRESENTATION TO HIV REPLICATION5325
fected DCs. Interestingly, in contact with MVA-infected cells,
DCs undergo phenotypic maturation without apoptosis. Hence,
DCs attracted to the site of injection are potential targets for
MVA infection and most likely undergo maturation, suggesting
that MVA-HIV-infected DCs might efficiently prime CTL re-
sponses in vivo.
MVA-HIV was designed to induce HIV-specific T-cell re-
sponses. It is known that in naïve individuals, MVA primes
CTL responses to the vector (vaccination to smallpox ) and
to desired transgenes (HIV or tumor antigens [21, 54]). We
assessed the impact of the vaccine candidate on T-cell activa-
tion in a cell culture system. We demonstrated that MVA-
HIV-infected cells (monocytes, macrophages, and DCs) effi-
ciently activate HIV Gag CTLs. Epitopes derived from the Nef
and Pol segments of the HIV polyprotein are also presented by
MVA-HIV-infected cells. Our results confirm and extend pre-
vious works showing that DCs infected with an MVA coding
for a tumor antigen activate tumor-specific CTLs (12). They
suggest that DCs are in part responsible for the transgene-
specific responses generated in animal models and human vac-
MVA-infected cells can directly present pathogen- or tu-
mor-derived antigens to Ag-specific T cells. Additionally, frag-
ments of infected cells may be engulfed by DCs, leading to
cross-presentation of Ags to CTLs. Here we characterized the
mechanisms of MVA-HIV antigen presentation leading to
CTL activation. We observed that various MVA-HIV-infected
cells, including HeLa-A2?cells (which are unable to perform
cross-presentation), directly present HIV antigens to HS
CTLs. Moreover, MVA induces apoptotic cell death as early as
6 h postinfection (Fig. 3) (23). In DCs, this time scale allows
activation of CTL clones in culture but might not be sufficient
to prime T-cell responses in vivo. Accordingly, in a mouse
model, MVA-infected DCs did not prime T-cell responses,
whereas injection of MHC-deficient MVA-infected cells led to
Ag-specific T-cell priming (17). This study and others suggest
that cross-presentation dictates the immunogenicity of MVA
vaccination (22, 36). In line with these observations, we dem-
onstrated that primary human fibroblasts, myoblasts, and myo-
tubes infected with MVA-HIV are cross-presented by DCs to
HS CTLs. Therefore, DCs recruited to the site of MVA inoc-
ulation might sample MVA-infected dying cells and then ma-
ture and migrate to lymph nodes to activate Ag-specific T cells.
Priming, expansion, and maintenance of CD8?T-cell re-
sponses require CD4?T-cell help (4, 14). We showed that
MVA-HIV-infected APCs potently activated HS CD4?T
AIDS vaccine development is facing multiple challenges.
The choice of immunogens and adjuvants able to induce
broad-spectrum and long-lasting memory and protection is of
paramount importance. Vaccination should be safe and well
tolerated and should not enhance susceptibility to infection or
pathogenesis. The Merck/NIH trial using an attenuated recom-
binant adenovirus 5 (rAd5) expressing HIV antigens as a vec-
tor was recently stopped based on the observation that the
vaccine did not provide protection (3). Even worse, initial
analysis of preclinical data indicated a significant trend toward
increased HIV-1 acquisition among vaccinees who had high
preexisting antibody titers to Ad5. A plausible explanation is
that rAd5 vectors complexed to antibodies bind to Fc recep-
tors, induce DC maturation, and favor HIV replication and
spread to T cells (42, 47). However, further analysis of the
preclinical outcomes of the STEP study, presented at the AIDS
Vaccine 2009 meeting, suggested that the initial weak associ-
ation of Ad5 seropositivity with increased HIV acquisition was
no longer valid, except perhaps among uncircumcised men (6).
Whatever the outcomes, this study highlighted the importance of
preexisting immunity to vaccine vectors as a potential risk asso-
ciated with vaccination. Vaccine-induced enhancement of viral
infection is a major problem observed not only with lentiviruses
but also with flaviviruses, coronaviruses, and paramyxoviruses
Preexisting immunity to MVA is rarely observed in the pop-
ulation because of the end of smallpox vaccine campaigns after
the 1970s. This should reduce the risk of adverse effects gen-
erated by the presence of anti-MVA antibodies. On the other
hand, the activation of CD4?T cells induced by vaccination
may create a milieu facilitating HIV infection. HIV is indeed
known to exploit the capacity of DCs to form intimate contacts
with T cells to spread to activated CD4?T cells (44, 45, 56, 61).
However, we demonstrated here that MVA-HIV-infected DCs
do not allow HIV replication and transfer to lymphocytes.
More importantly, exposure of DCs to MVA-HIV-infected
epithelial cells and myotubes also inhibited HIV spread in
DC–T-cell cultures. The underlying mechanism remains to be
elucidated but probably involves type I IFN activity. We indeed
showed that DCs infected with MVA or encountering MVA-
infected cells produced significant levels of type I IFNs. IFN-?
enhances the expression of innate cellular factors, such as
APOBEC3G, Trim5?, and tetherin, that restrict different steps
of the viral life cycle. Exogenously added IFN-? also inhibits
HIV replication (28) and limits HIV cell-to-cell spread (58).
Determining further whether IFNs directly impact HIV repli-
cation and spread will require further investigation with block-
ing anti-IFN or anti-IFN-receptor antibodies. A recent report
demonstrated that in addition to IFNs, MVA-infected DCs
secrete cytokines and chemokines, including RANTES, mac-
rophage inflammatory protein 1? (MIP-1?), IL-6, and tumor
necrosis factor (TNF) (10). It is tempting to speculate that
IFNs and some of these cytokines create an antiviral state in
DC–T-cell cultures, limiting further HIV infection. It will be
worthwhile to determine more precisely whether MVA-HIV-
infected HeLa cells or myotubes release additional cytokines
or chemokines or other components, such as apoptotic debris,
which may impact the sensitivity of DCs to HIV infection. This
observation has important implications in vivo. Hopefully, the
probability that MVA-vaccinated humans simultaneously or
rapidly encounter HIV is not very high. However, these results
suggest that exposure to HIV shortly after MVA-HIV vacci-
nation might not facilitate HIV infection.
In sum, our work reveals novel aspects of the interaction
between MVA and primary human cells, which is of interest
for understanding poxvirus infection and for improving poxvi-
rus-based vaccine approaches. This preclinical study also shows
that the MVA-HIV vector displays important characteristics
expected to induce HIV-specific cellular responses in vacci-
nees. It should be used in clinical trials in the near future,
either alone or in a prime-boost regimen with other immuno-
gens (such as lipopeptides or DNA).
5326 BRANDLER ET AL.J. VIROL.
We thank Anne de Saunie `re, Helen K. W. Law, and the Centre
d’Immunologie Humaine for assistance and Florence Buseyne,
Miche `le Fe ´vrier, Yves Rivie `re, and the NIH AIDS Research and
Reference Reagent Program for kind gifts of reagents. We acknowl-
edge V. Mouly and the Human Cell Culture Platform of the Myology
Institute, Paris, for providing the myoblasts.
S.B. is a fellow of the Agence Nationale de Recherche sur le SIDA
(ANRS). This work was supported by grants from the ANRS HIV
Vaccine Network (AHVN) and the Institut Pasteur.
The authors have no conflicting financial interests.
S.B. designed and performed experiments, A.L., M.D., F.G.-B., and
P.-E.C. performed experiments and provided material, Y.L. supervised
the study, and O.S. and A.M. designed experiments, supervised the
study, and wrote the paper.
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