JOURNAL OF VIROLOGY, Nov. 2004, p. 12471–12479
Vol. 78, No. 22
Modified Vaccinia Virus Ankara Immunization Protects against Lethal
Challenge with Recombinant Vaccinia Virus Expressing
Lewis H. McCurdy, John A. Rutigliano, Teresa R. Johnson, Man Chen, and Barney S. Graham*
Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of
Health, Bethesda, Maryland
Received 19 February 2004/Accepted 7 July 2004
Recent events have raised concern over the use of pathogens, including variola virus, as biological weapons.
Vaccination with Dryvax is associated with serious side effects and is contraindicated for many people, and the
development of a safer effective smallpox vaccine is necessary. We evaluated an attenuated vaccinia virus,
modified vaccinia virus Ankara (MVA), by use of a murine model to determine its efficacy against an
intradermal (i.d.) or intranasal (i.n.) challenge with vaccinia virus (vSC8) or a recombinant vaccinia virus
expressing murine interleukin-4 that exhibits enhanced virulence (vSC8-mIL4). After an i.d. challenge, 15 of
16 mice who were inoculated with phosphate-buffered saline developed lesions, one dose of intramuscularly
administered MVA was partially protective (3 of 16 mice developed lesions), and the administration of two or
three doses of MVA was completely protective (0 of 16 mice developed lesions). In unimmunized mice, an i.n.
challenge with vSC8 caused a significant but self-limited illness, while vSC8-mIL4 resulted in lethal infections.
Immunization with one or two doses of MVA prevented illness and reduced virus titers in mice who were
challenged with either vSC8 or vSC8-mIL4. MVA induced a dose-related neutralizing antibody and vaccinia
virus-specific CD8?-T-cell response. Mice immunized with MVA were fully protected from a low-dose vSC8-
mIL4 challenge despite a depletion of CD4?cells, CD8?cells, or both T-cell subsets or an antibody deficiency.
CD4?- or CD8?-T-cell depletion reduced the protection against a high-dose vSC8-mIL4 challenge, and the
depletion of both T-cell subsets was associated with severe illness and higher vaccinia virus titers. Thus, MVA
induces broad humoral and cellular immune responses that can independently protect against a molecularly
modified lethal poxvirus challenge in mice. These data support the continued development of MVA as an
alternative candidate vaccine for smallpox.
The continued threat of bioterrorism has led to concern over
the reemergence of smallpox. Global immunity against poxvi-
ruses has declined over the last 20 years, as routine immuni-
zation against smallpox ceased in the early 1980s following the
declaration of smallpox eradication by the World Health Or-
ganization. The consequences of a deliberate smallpox release
would be great, as mortality for nonimmune persons has been
reported to be near 30% (10).
While historical experience supports the efficacy of replica-
tion-competent vaccinia virus immunization against natural
smallpox, the current Food and Drug Administration (FDA)-
licensed vaccine Dryvax presents some safety concerns. Previ-
ous studies have described a significant rate of serious compli-
cations following Dryvax administration, including death, in 1
per 1 million vaccinees (26, 27). This rate could be even higher
if mass vaccination were instituted today because of the large
and growing number of persons for whom Dryvax is contrain-
dicated. This has led to hesitation within the medical commu-
nity for the use of widespread vaccination (9, 33). As a result,
there have been renewed investigations into the development
of a safer second-generation smallpox vaccine.
Previous strategies for the development of safe smallpox
vaccines have focused on less virulent vaccinia virus strains,
including recombinant vaccinia viruses with selected deletions
of virulence genes or insertions of proinflammatory cytokines
(13, 14, 34, 40) and empirically attenuated live vaccinia virus
strains (21, 25, 29, 30, 37). The modified vaccinia virus Ankara
(MVA) was attenuated by ?570 passages in chicken embryo
fibroblasts, resulting in the loss of approximately 15% of its
parent genome, including several host range genes (2, 29, 31).
MVA is consequently unable to replicate effectively in mam-
malian cells, which reduces the risk of dissemination and trans-
mission (8, 35). In addition, MVA no longer encodes many of
the soluble inhibitors of cytokine and chemokine function as
well as other factors that play a role in immune evasion (1, 6,
39). However, epitopes that are known to elicit neutralizing
antibodies are conserved (16, 32, 44), and recently three hu-
man CD8?cytotoxic T lymphocyte (CTL) epitopes restricted
to HLA-A*0201 have been identified that are present in MVA,
the Copenhagen strain of vaccinia virus, and variola virus (11,
41). Thus, MVA can induce significant vaccinia virus-specific
immune responses that are unmodified by normal vaccinia
virus immune evasion mechanisms.
Earlier work with MVA demonstrated its safety and its abil-
ity to protect against the development of poxvirus infections
in several animal models (22, 30, 43). Recently, MVA immu-
nization has been shown to provide protection against a pul-
monary vaccinia virus challenge (4, 11). With the threat of
bioterrorism and the potential for exposure to genetically ma-
* Corresponding author. Mailing address: Vaccine Research Cen-
ter/NIAID/NIH, 40 Convent Dr., MSC 3017, Building 40, Room 2502,
Bethesda, MD 20892-3017. Phone: (301) 594-8468. Fax: (301) 480-
2771. E-mail: firstname.lastname@example.org.
at NIH Library on November 10, 2008
nipulated weaponized smallpox, the ability of a new vaccine to
protect against pathogens with enhanced virulence may be
necessary. Type 2 cytokines have been shown to diminish CTL
activity in vivo and to inhibit viral clearance (2, 12, 24, 42). The
coexpression of interleukin-4 (IL-4) in the presence of vaccinia
virus infection results in the downregulation of type 1 cyto-
kines, reduces cytolytic activity, and delays viral clearance (2, 3,
24, 36). The demonstration of potent immunity and in vivo
protection by novel second-generation vaccines against vac-
cinia virus strains with enhanced virulence would lend further
support to the development of a new approach to smallpox
We sought to evaluate the comparative efficacies of MVA
and a replication-competent vaccinia virus strain, vSC8, against
both intradermal and pulmonary challenges of vaccinia virus in
a mouse model. MVA immunization elicited both humoral and
cellular immunity equivalent to that elicited by replication-
competent vaccinia virus and protected mice from the illness
associated with poxvirus challenge, including a lethal intranasal
challenge with a recombinant vaccinia virus, vSC8-mIL4. MVA
immunization reduced viral replication in the lung tissue and
reduced the pathology associated with vaccinia virus pulmo-
nary infections. After the selective depletion of B- and T-cell
subsets, mice immunized with MVA retained sufficient immu-
nity for protection, suggesting that the immunologic correlate
of protection from vaccinia virus is complex and redundant.
MATERIALS AND METHODS
Viruses and cells. The vaccinia virus strain vSC8 expressing ?-galactosidase
was provided by Bernie Moss (National Institutes of Health [NIH], Bethesda,
Md.). Viral stocks were grown in BSC40 cells and purified as previously de-
scribed (23). A vaccinia virus expressing murine IL-4 (vSC8-mIL4) was con-
structed from wild-type vSC8 as described previously (24). Modified vaccinia
virus Ankara was provided by Therion Biologics (Cambridge, Mass.). BSC40
cells were obtained from Shiu-Lok Hu (University of Washington, Seattle) and
were maintained in minimum essential medium (MEM) supplemented with 10%
fetal calf serum, glutamine, and antibiotics. P815 cells were obtained from
Jonathan Yewdell (NIH) and were maintained in RPMI medium supplemented
with 10% fetal calf serum, glutamine, and antibiotics.
Mice. Six- to 8-week-old pathogen-free BALB/c mice were purchased from
Charles River Laboratories (Raleigh, N.C.). Six- to 8-week-old mice in a
C57BL/6 genetic background that lacked mature B cells (B6.12952-Igh-6tmlCgn
mice) were purchased from Jackson Laboratory (Bar Harbor, Maine). Age-
specific C57BL/6 mice purchased from Jackson Laboratory served as a control
strain for the B-cell-deficient mice. All mice were housed and cared for in
accordance with the Guide for the Care and Use of Laboratory Animals prepared
by the Committee on Care and Use of Laboratory Animals of the Institute of
Laboratory Animal Resources, National Research Council.
Mouse immunization and challenge. Mice were immunized intramuscularly
with a 0.1-ml volume in each thigh with MVA, the vSC8 vaccinia virus, or
phosphate-buffered saline (PBS). MVA and vSC8 were administered at a dose of
2 ? 106PFU per vaccination. As indicated for each experiment, groups of mice
received single or multiple doses of MVA, with each dose separated by 2 weeks.
Intradermal and intranasal challenges were performed 4 weeks after the last
immunization. Intradermal challenges were performed by the administration of
a 50-?l volume of vSC8 intradermally at the base of the tail (2 ? 106PFU). Mice
were observed daily for lesion formation at the site of challenge for 14 days.
For intranasal challenges, mice were anesthetized intramuscularly with xyla-
zine-ketamine and then infected with vSC8 or vSC8-mIL4 in a 100-?l volume.
The mice were weighed and observed for illness daily, as previously described
In vivo replication of vaccinia virus. Subgroups of mice were sacrificed on days
4, 8, and 12. The right lung tissue was removed, weighed, and immediately
quick-frozen in minimum essential medium supplemented with 10% fetal bovine
serum (10% MEM). Briefly, the samples were quick-thawed at 37°C and main-
tained at 4°C prior to being individually ground with a mortar and pestle. Serial
10-fold dilutions of clarified supernatants were used to infect subconfluent
monolayers of BSC40 cells in triplicate in 12-well plates. After 1 h, the plates
were covered in 0.75% methylcellulose in 10% MEM and incubated at 37°C. The
cells were fixed with formalin 2 days after infection and stained with 2% crystal
violet–40% methanol, and plaques were counted under a dissecting microscope.
Data are presented as geometric mean log10PFU per gram of lung at dilutions
that produced more than three plaques per well.
Vaccinia virus neutralization assay. Retro-orbital bleeding of mice was per-
formed on days 14 and 28 postimmunization. Blood samples were centrifuged at
5,000 rpm for 5 min in a Sorvall Fresco Biofuge microcentrifuge, and the sera
were collected. Serum samples were diluted 1:10 in PBS and stored at ?20°C.
Each sample was thawed and diluted two- or fourfold in a 96-well plate. An equal
volume of vSC8 vaccinia virus (2 ? 105PFU/well) was added to each well. A
negative control and standard vaccinia immune globulin positive control (ob-
tained from Hana Golding, FDA, Bethesda, Md.) were used in each assay. The
plates were incubated overnight at 37°C, and then 50 ?l of each sample was
added to a subconfluent monolayer of BSC40 cells, with each sample tested in
triplicate. After 1 h at room temperature, the cells were overlaid with 10% MEM
containing 0.75% methylcellulose. The plates were incubated at 37°C for 48 h
and then fixed with 3.7% formaldehyde prior to staining with 0.2% crystal
violet–40% methanol. Each well was then evaluated for the number of plaques
under a dissecting microscope, and the 60% plaque reduction value was deter-
mined and expressed as the log2reciprocal neutralization titer. Cumulative data
are given as mean titers ? standard deviations.
Intracellular cytokine assay. Mice were sacrificed 14 and 28 days after their
last immunization, and their spleens were harvested. Lymphocytes were isolated
by centrifugation on a Ficoll-Hypaque cushion (specific gravity, 1.09) at room
temperature, washed twice, and resuspended in RPMI containing 10% fetal
bovine serum. P815 cells were infected for 6 h with vSC8 at a multiplicity of
infection of 3 to 5 in serum-free RPMI at 37°C. After incubation, vaccinia
virus-infected and uninfected P815 cells were added to 2 ? 106lymphocytes at a
1:10 ratio. The cells were costimulated with monoclonal anti-mouse CD28 and
anti-mouse CD49d antibodies. Phorbol myristate acetate (0.2 ?g/5 ml) and
ionomycin (2 ?g/5 ml) served as positive controls. After 1 h, brefeldin A (Pharm-
ingen, San Diego, Calif.) was added to each sample at 3.5 ?g/5 ml. The samples
were incubated for 12 to 16 h at 37°C. The cells were then fixed, permeabilized
(Cytofix/Cytoperm; Pharmingen), and stained with anti-gamma interferon
(IFN-?) (clone XMG1.2), anti-CD3e (clone 145-2C11), anti-CD4a (clone RM4-
5), and anti-CD8a (clone 53-6.7) monoclonal antibodies. After being washed, the
cells were resuspended in staining buffer and analyzed in a FACSCalibur instru-
ment (BD Biosciences, San Jose, Calif.). Analyses of samples were performed
with FlowJo software (TreeStar Inc., Ashland, Oreg.). All antibodies were pur-
chased from Pharmingen.
Antibody depletion. Mice were injected intraperitoneally with an antibody
specific to either CD4 (clone GK 1.5) or CD8 (clone 2.43), with both of these
antibodies, or with an isotype control antibody (clone HB 151), as previously
described (19). On days ?2 and ?1 and on the day of challenge, mice received
200 ?g of the indicated antibody intraperitoneally. On day 7, the remaining mice
received an additional injection of 500 ?g of antibody. Mice from each depletion
group were sacrificed on day 11 postchallenge to measure the frequencies of
CD3-, CD4-, and CD8-positive splenocytes by flow cytometry. Relative to isotype
control-treated mice, the depleted mice had only 3.2% of their original CD4?T
cells and 2.8% of the CD8?T cells remaining.
Statistical analysis. Data were maintained in a Paradox database. Determi-
nations of neutralization antibody titers were performed with SAS statistical
software (SAS Institute Inc., Cary, N.C.) as previously described (24). Weight
loss and neutralizing antibody titers were analyzed by analyses of variance with
Kruskal-Wallis and Wilcoxon rank sum tests. Comparisons were made between
individual experiments by statistical modeling, and trend analysis was performed
by the general linear model method of the SAS software. Virus titers and T-cell
responses were compared by Tukey’s t test analysis in Sigma Stat 2.0 (SPSS Inc.,
MVA immunization protects against intradermal challenge
with vaccinia virus. The intradermal inoculation of vaccinia
virus results in a characteristic development of lesions at the
site of the inoculation. After vaccinia virus immunization of
humans, the lesions progress from papules to pustules or ves-
icles prior to the formation of scabs. BALB/c mice were im-
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munized intramuscularly with 2 ? 106PFU of MVA or vSC8
given as one, two, or three doses at 2-week intervals. Four
weeks after immunization, the mice were challenged with vSC8
intradermally at the base of the tail, and the development of
lesion formation was compared with that in PBS-inoculated
controls. For control mice, single or multiple lesions were
noted at the site of inoculation beginning on day 6. Although
the numbers and sizes of the lesions varied, pox lesions were
noted for nearly all animals (94%) (Table 1). A single immu-
nization with MVA or vSC8 provided a partial yet significant
reduction in the numbers of lesions compared to the control
animals (P ? 0.05) (Table 1). After two or three doses of
MVA, complete protection from the formation of pox lesions
was noted. These data suggest that a single dose of MVA elicits
an immune response equivalent to that against a single dose of
replication-competent vaccinia virus when given intramuscu-
larly and that immunity improves with multiple injections.
MVA immunization protects against illness and reduces
viral burden in mice challenged intranasally with vaccinia
virus. After demonstrating the protection from intradermal
challenge of MVA immunization, we next immunized mice
with MVA or vSC8 and challenged them intranasally with
vSC8 at 107PFU. The challenge volume of 100 ?l is known to
deposit at least 70% of the inoculum directly in the lung (18).
Mice were immunized with either one or two doses of MVA
prior to the challenge, as two doses demonstrated complete
protection in the intradermal challenge model. After the chal-
lenge, the mice were observed daily for illness and weight loss.
PBS-inoculated mice developed weight loss beginning on day 4
(Fig. 1A). On day 6, control mice suffered a peak weight loss of
nearly 25% and then gradually recovered by day 12. In sharp
contrast, all immunized mice were protected from significant
weight loss. Lungs were harvested on days 4, 8, and 12 after the
challenge, and vaccinia virus titers were determined (Fig. 1B).
The viral titers reflected the illness of the PBS-inoculated mice:
the vaccinia virus titers per gram of lung were 7.52 ? 0.50 and
6.99 ? 0.26 log10on days 4 and 8, respectively. The vaccinia
virus titers were significantly reduced on day 4 for all groups of
immunized mice compared to those in PBS-inoculated mice (P
? 0.05), but there were detectable virus titers on day 4 in mice
that had received a single MVA immunization. By day 8, there
was no detectable vaccinia virus in any immunized mouse.
Thus, clinical protection was achieved after a single-dose im-
munization with MVA and correlated directly with a reduction
in the overall viral burden and the rapid clearance of vaccinia
MVA induces both humoral and cellular immune responses.
Vaccinia virus neutralization was performed by use of a plaque
reduction assay with samples collected 2 and 4 weeks after
immunization. A dose-dependent humoral response was elic-
ited by MVA immunization (Fig. 2A). The peak response was
seen on day 14 and persisted to at least day 28. A single dose
FIG. 1. Mice immunized with MVA demonstrate absence of weight loss and reduced vaccinia virus titers in the lungs after an intranasal
challenge with vSC8. Mice were immunized with vSC8 or one or two doses of MVA or were inoculated with PBS and then were challenged 4 weeks
later with intranasal vSC8. (A) PBS-inoculated mice demonstrated weight loss that peaked on days 6 and 7. MVA- and vSC8-immunized mice were
protected from weight loss and clinical illness. (B) Lungs were harvested on days 4 and 8, and vaccinia virus titers were determined and expressed
as log10PFU per gram of lung tissue. The limit of detection for this assay was 2.0 log10PFU/g. On day 4, there was a significant decline in vaccinia
virus titer in MVA-immunized mice compared to PBS-inoculated mice. By day 8, there was a complete clearance of vaccinia virus from the lungs
of mice that were immunized with MVA.
TABLE 1. MVA immunization protects against intradermal
Frequency of lesion
formation (no. of mice
with lesions/total no.
of mice [%])b
PBS.................................................................................... 15/16 (94)
MVA ................................................................................. 3/16 (19)*
MVA-MVA...................................................................... 0/16 (0)*
MVA-MVA-MVA........................................................... 0/16 (0)*
VSC8................................................................................. 2/8 (25)*
aMice were immunized with PBS, vSC8, or single or multiple doses of MVA
and then challenged intradermally at the base of the tail 4 weeks after the last
immunization with vSC8. Mice were observed daily for pox lesion formation.
Data are a combination of two experiments and demonstrate a significant de-
crease in lesion formation in all immunized mice. There was partial protection in
mice that received a single injection of MVA, and complete protection was
observed for mice immunized with two or three doses of MVA.
b*, P ? 0.05.
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of MVA elicited a response equivalent to that seen with intra-
muscular vSC8. The administration of multiple doses of MVA
boosted the antibody response, with a significant enhancement
of neutralization seen after three doses on days 14 and 28
compared to that with a single dose (P ? 0.05).
Similarly, the magnitude of the cellular response was depen-
dent on the number of MVA immunizations. Twenty-eight
days after immunization, splenocytes were isolated, stimulated
with vaccinia virus, and evaluated for intracellular IFN-? pro-
duction by flow cytometry. The percentage of vaccinia virus-
specific CD8?CD3?T cells was determined for mice receiving
PBS, vSC8, or MVA (Fig. 2B). A higher frequency of vaccinia
virus-specific CD8?T cells was elicited by the three-dose reg-
imen of MVA than by either one or two doses of MVA (P ?
MVA protects against lethal vSC8-mIL4 intranasal chal-
lenge. In order to determine the efficacy of MVA immuniza-
tion against a vaccinia virus strain with enhanced virulence, we
challenged mice intranasally with a recombinant vaccinia virus
encoding murine IL-4, vSC8-mIL4. As with the vSC8 parent
strain, mice immunized with one or two doses of MVA were
protected from the development of clinical illness, as measured
by weight loss (Fig. 3A). Nonimmunized mice lost weight rap-
idly, and all mice died or were euthanized because of extreme
illness by day 8 after the challenge. Nonimmunized mice dem-
onstrated significantly higher vaccinia virus titers in the lungs
than did MVA-immunized mice (Fig. 3B). Lung histopathol-
ogies were evaluated to compare the inflammatory processes in
immunized versus nonimmunized mice. MVA-immunized
mice showed only a minimal infiltrate on day 8, while there was
a profound alteration of the lung architecture in nonimmu-
nized mice, with severe mononuclear cell peribronchiolar and
interstitial infiltrates, alveolar edema and exudate, and epithe-
lial necrosis (Fig. 4).
T-cell depletion reduces efficacy of MVA immunization.
Groups of mice were depleted of CD4?cells, CD8?cells, or
both by the use of specific antibodies prior to an intranasal
challenge with 107PFU of vSC8-mIL4. PBS-inoculated mice
developed significant illness and mortality by day 8 after the
challenge (Fig. 5A). MVA-immunized mice that received an
isotype control antibody or mice that were depleted of
CD4?cells, CD8?cells, or both had no significant weight
loss. In mice that were depleted of both CD4?and CD8?
cells, there was a higher vaccinia virus titer in the lungs on
day 4 after the challenge than those for the other groups of
MVA-immunized mice (P ? 0.05) (Fig. 5B). On day 8,
vaccinia virus was still detectable in the lungs of CD4- and
CD8-depleted mice, while all other MVA-immunized mice
had cleared the vaccinia virus. To better define the roles of
the CD4?and CD8?T cells in this setting, we challenged
other groups of mice that had been immunized with two
doses of MVA with a higher dose (108PFU) of vSC8-mIL-4
intranasally after differential T-cell subset depletion. With
the higher-titer vaccinia virus challenge, the MVA vaccine-
induced immunity was partially mitigated by either CD4?-
or CD8?-T-cell depletion alone (Fig. 6B), but illness was
most severe when both T-cell subsets were depleted prior to
the challenge (Fig. 6A). The most severe illness was associ-
ated with higher residual vaccinia virus titers on both days 4
and 8 in mice that were depleted of both T-cell subsets.
These data indicate that both CD4?and CD8?vaccine-
induced T-cell responses are important for controlling vac-
cinia virus replication and illness.
FIG. 2. MVA elicits dose-dependent humoral and cellular immune responses. After immunization with PBS, vSC8, or MVA, mice were
assessed for an immune response by plaque neutralization and intracellular IFN-? production. (A) On days 14 and 28 after the last immunization,
neutralizing antibody titers were determined and expressed as reciprocal log2serum dilutions resulting in 60% plaque reduction. Data represent
means from two independent experiments, resulting in data for 10 mice per group. The lines between bars indicate values that are significantly
different (P ? 0.05). (B) Twenty-eight days after immunization, splenocytes were harvested, and intracellular IFN-? production in vaccinia
virus-specific T cells was measured by flow cytometry. The data are for five mice in each group from one representative experiment of four total
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Mice lacking mature B cells. In order to assess the contribu-
tion of the humoral immune response to vaccine-induced immu-
nity, we immunized mice lacking mature B cells, B6.12952-Igh-
6tmlCgn(B6.12952) mice, with one or two doses of MVA prior to
a challenge with vSC8-mIL4. Neutralization assays were per-
formed on day 28 postimmunization with B6.12952 and C57BL/6
mice. C57BL/6 mice immunized with MVA demonstrated the
presence of neutralizing antibodies, while there were no detect-
FIG. 3. MVA immunization provides clinical and virologic protection after an intranasal challenge with vSC8-mIL4. Mice were immunized with
PBS, vSC8, or one or two doses of MVA and then challenged 4 weeks after immunization with intranasal vSC8-mIL4. (A) PBS-inoculated mice
demonstrated a rapid decline in weight loss that resulted in death. MVA-immunized mice were protected from weight loss and clinical illness.
(B) Lungs were harvested on days 4 and 8, and vaccinia virus titers in the lungs were determined and expressed as log10PFU per gram of lung
tissue. The limit of detection for this assay was 2.0 log10PFU/g. Similar to the case for the vSC8 challenge, there was a significant decline in viral
titer in MVA-immunized mice compared to PBS-inoculated mice on day 4, with a complete clearance of vaccinia virus by day 8.
FIG. 4. Lung histopathology in mice challenged with vSC8-mIL4. Mice were sacrificed on day 8 after the vSC8-mIL4 challenge, lungs were fixed
in 10% formalin phosphate and embedded in paraffin, and thin sections were stained with hematoxylin and eosin. The images were obtained with
a Zeiss Axioplan microscope using a 20? objective. (A) Lung from a mouse immunized intradermally with Dryvax prior to vSC8-mIL4 challenge;
(B) lung from a mouse immunized with one dose of MVA prior to vSC8-mIL4 challenge; (C and D) lungs from mice challenged with vSC8-mIL4
subsequent to PBS inoculation. Both Dryvax and MVA immunization provided significant protection from the extensive peribronchiolar and
interstitial inflammation, alveolar exudates and edema, and epithelial necrosis seen in the lungs of unimmunized mice.
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FIG. 5. T-cell-depleted mice that are immunized with MVA are partially protected from a vaccinia virus intranasal challenge. Mice were
immunized with two doses of MVA and then challenged 4 weeks after immunization with 107PFU of intranasal vSC8-mIL4. Prior to challenge,
the mice were depleted of CD4?, CD8?, or both CD4?and CD8?T cells on days ?2, ?1, and 0, and repeat depletion was done on day 7.
(A) Changes in baseline weight were determined daily. The depletion of either CD4?or CD8?T cells did not result in weight loss in mice receiving
the lower-titer challenge. Compared to PBS-inoculated controls, all MVA-immunized mice recovered to their baseline weight by day 8. (B) Vac-
cinia virus titers in the lungs were determined on days 4 and 8 after the challenge. Clinical illness corresponded to vaccinia virus titers in
MVA-immunized mice. MVA-immunized double-depleted mice had significantly higher vaccinia virus titers on days 4 and 8 than did MVA-
immunized mice treated with the isotype control or an antibody to either CD4 or CD8.
FIG. 6. Mice were immunized with two doses of MVA and then challenged intranasally 4 weeks after immunization with 108PFU of
vSC8-mIL4. Prior to challenge, the mice were depleted of CD4?, CD8?, or both CD4?and CD8?T cells on days ?2, ?1, and 0, and repeat
depletion was done on day 7. (A) Changes in baseline weight were determined daily. After the high-titer challenge, the depletion of either CD4?
or CD8?T cells resulted in modest weight loss, despite the prior immunization with MVA. The depletion of both CD4?and CD8?T cells resulted
in a more severe illness, suggesting that both T-cell subsets contribute to immunity. (B) The vaccinia virus titers in the lungs were determined on
days 4 and 8 after challenge and demonstrated the same patterns as those seen in the weight loss curves and were also consistent with the patterns
seen after the lower-dose vSC8-mIL4 challenge. Mice depleted of both CD4?and CD8?T cells had more residual virus on days 4 and 8 than the
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able neutralizing antibodies in the B6.12952 mice (Fig. 7A).
MVA-immunized mice showed no decline in weight after the
challenge, while PBS-inoculated B6.12952 and C57BL/6 mice
showed significant weight reductions that peaked on day 7 (Fig.
7B). The vaccinia virus lung titers measured on day 4 postinfec-
tion revealed a significant decline in titer in the B6.12952 mice
that received one or two doses of MVA compared to those in
PBS-inoculated B6.12952 mice (Fig. 7C). However, when com-
pared to that in C57BL/6 MVA-immunized mice, the degree of
vaccinia virus inhibition was significantly lower (P ? 0.05). Both
B6.12952 and C57BL/6 mice that were immunized with MVA
cleared the virus from the lungs by day 8, while PBS-inoculated
support a significant role for the humoral immune response in
protection against vaccinia virus, particularly in controlling the
peak of vaccinia virus replication, but mice deficient in neutraliz-
ing antibodies were eventually able to clear the virus without
significant clinical illness.
Following the anthrax attacks in the fall of 2001, discussions
about the potential use of other virulent pathogens as biolog-
ical weapons have increased. The threat of a smallpox out-
break elicits great concerns because of its acute and significant
morbidity and mortality rates and the lack of significant im-
munity against poxviruses among civilians born after 1970,
despite a large vaccination campaign effort among the military
and medical first responders (17). Discussions about the wide-
spread use of the present FDA-approved vaccine Dryvax have
resulted in much debate in the medical community. While the
vaccine is efficacious, the complications associated with Dryvax
make the development of a safer second-generation smallpox
vaccine a medical priority.
For the present study, we evaluated the efficacy and immune
response of a highly attenuated vaccinia virus, MVA, by use of
a mouse model. Investigations of this vaccinia virus strain be-
FIG. 7. B-cell-deficient mice challenged with vSC8-mIL4 after MVA immunization are protected from weight loss but demonstrate higher
vaccinia virus titers in the lungs than MVA-immunized C57BL/6 mice. B6.12952 mice lacking mature B cells were immunized with PBS or one or
two doses of MVA and then challenged intranasally 4 weeks after immunization with vSC8-mIL4. C57BL/6 mice served as controls. (A) C57BL/6
mice had significant neutralizing antibody responses postchallenge, while there was no detectable antibody for B6.12952 mice. (B) After the
challenge with vSC8-mIL4, PBS-inoculated mice demonstrated a rapid decline in weight while MVA-immunized mice lacked clinical illness despite
the absence of neutralizing antibodies. (C) Vaccinia virus titers in the lungs were determined and expressed as log10PFU per gram of lung tissue.
The viral burden in MVA-immunized B6.12952 mice was significantly less than that in PBS-inoculated mice, but they had a higher vaccinia virus
titer on day 4 than that in C57BL/6 mice with normal humoral immunity.
VOL. 78, 2004 MVA PROTECTS AGAINST mIL-4-EXPRESSING VACCINIA VIRUS12477
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gan in the late 1950s after attenuation of the parent strain
CVA in chicken embryo fibroblasts. Since that time, MVA has
been evaluated in several animal models as well as in humans
(22, 30, 43). During the 1970s, more than 120,000 people were
immunized with MVA (37). Reports reflect a good safety pro-
file for MVA, including safe administration to immunocom-
promised animals (38, 43). However, the specific determinants
of MVA-induced immunity compared with those induced by
replication-competent vaccinia virus have only recently been
studied (5, 45).
We demonstrated the ability of a single dose of MVA to
protect against both intradermal and intranasal challenges with
vaccinia virus. In particular, we demonstrated that MVA im-
munization can protect against a lethal respiratory challenge
with a molecularly modified recombinant vaccinia virus ex-
pressing murine IL-4. While previous studies have shown a
dose-related effect following single-dose escalation (4, 11), we
showed that a multidose schedule of MVA may enhance the
overall immune response and provide added protection against
vaccinia virus replication. For both the intradermal and intra-
nasal models, virologic protection, as judged by the presence or
absence of pox lesions, and vaccinia virus titers in the lungs
improved in mice that received two or three immunizations
(Table 1 and Fig. 1). While clinical illness was reduced in all
MVA-immunized mice after an intranasal challenge, the vac-
cinia virus titers in the lungs were higher for mice that received
a single immunization. This enhanced protection correlated
with the magnitude of both the humoral and cellular immune
responses, as judged by the neutralization activity and intra-
cellular IFN-? production in vaccinia virus-specific T cells,
respectively (Fig. 2). In mice receiving multiple immunizations,
the immune responses were enhanced, suggesting that strate-
gies to optimize the protection of humans against poxviruses
such as variola virus and monkeypox virus may necessitate a
multidose immunization regimen. Although it was not evalu-
ated in these studies, the route of immunization is also impor-
tant. For example, MVA was administered intramuscularly in
our studies, and the responses compared favorably to those
induced by replication-competent vaccinia virus given by the
intramuscular or intradermal route. However, when vaccinia
virus is given by intraperitoneal injection, much higher fre-
quencies of CD4?- and CD8?-T-cell responses can be
achieved in splenocytes (20). Therefore, additional exploration
of the route of poxvirus immunization may be warranted.
With the threat of bioterrorism, there is concern not only
about the potential release of virulent pathogens, but also
about the purposeful manipulation of pathogens in an effort to
augment their virulence. Importantly, we have demonstrated
that MVA immunization can protect against a lethal pulmo-
nary challenge with a molecularly modified recombinant vac-
cinia virus expressing murine IL-4. Prior studies have demon-
strated the importance of cytokine balance in the pathogenesis
of viral, bacterial, and parasitic diseases. The Th1 cytokine
IFN-? plays a key role in the control of vaccinia virus infection
(24). Disruption of the Th1-Th2 balance has been shown to
adversely affect viral clearance and protection from dissemina-
tion. As previously described (24, 36), we demonstrated that
the virulence of vaccinia virus is enhanced in the setting of
excess IL-4 production, with a prolonged elevation of lung viral
titers and death in mice infected with vSC8-mIL4 (Fig. 3).
Despite this enhanced virulence, MVA immunization pro-
tected mice from a lethal pulmonary challenge with vSC8-
mIL4 and eliminated clinical illness and weight loss. The ability
to protect against a lethal molecularly modified vaccinia virus
lends further support to MVA as a candidate vaccine against
While they are effective against vaccinia virus challenges, the
determinants of immunity elicited by MVA and other vaccinia
viruses have not been clearly defined. The immunization of
human subjects with Dryvax has been shown to elicit both
humoral and cellular immunity (15). Historical reports suggest
that both arms of the immune system are relevant to protection
from smallpox. Investigations of villages during outbreaks of
smallpox correlated the vaccine take and antibody response
with immunity (28). More recent reports point to the impor-
tance of the cellular immune response for containing dissem-
inated vaccinia virus (7). It becomes important in the evalua-
tion of novel vaccines to dissect the role of each component of
the immune response in order to demonstrate similar patterns
of response to both replication-competent and attenuated vac-
Recent work suggested that selected human CD8?CTL
epitopes are conserved between various poxviruses, including
MVA and variola virus (11, 41). The selective depletion of
either CD4?or CD8?T cells alone prior to vSC8-mIL4 chal-
lenge resulted in minimal weight loss and viral replication,
which suggests that neither cell type is required for the con-
tainment of vaccinia virus replication (Fig. 4). The depletion of
both CD4?and CD8?T cells resulted in weight loss, which
corresponds to enhanced viral replication and delayed clear-
ance, but the weight loss was minimal. While this demonstrates
the importance of cellular immunity for vaccinia virus clear-
ance, it suggests that T cells are not the sole factor in the
defense against poxvirus infection and that the immune system
has evolved redundant mechanisms to control this important
class of pathogens.
Contributions from the humoral immune response following
immunization are also likely to be important for protection
against vaccinia virus infection. We demonstrated a dose-de-
pendent increase in the antibody response to MVA immuni-
zation, which correlated with protection. However, in the ab-
sence of neutralizing antibodies there was minimal illness in
this murine model from an intranasal vaccinia virus challenge
(Fig. 5). While MVA-immunized B-cell-deficient mice demon-
strated significant viral replication in the lungs on day 4 after
the intranasal challenge, they lacked signs of clinical illness and
cleared the vaccinia virus by day 8. As for many other virus
infections, we believe that the data support an important role
for vaccine-induced antibodies in protection from infection,
but effective T-cell responses also appear to be important for
protection against severe poxvirus-induced disease. These data
are consistent with recent studies that evaluated the immune
determinants of protection against the WR strain of vaccinia
virus (5) and that indicated that vaccine-induced protection
from poxviral challenge should not be restricted to a single arm
of the immune response, but should include a combination of
both cellular and humoral immune responses.
We demonstrated a robust immune response following
MVA immunization in a murine model. MVA not only pro-
tected mice from a standard vaccinia virus challenge, but it was
12478MCCURDY ET AL.J. VIROL.
at NIH Library on November 10, 2008
also able to prevent illness and limit viral replication after the
administration of a lethal molecularly modified strain of vac-
cinia virus. These data support the further development of
MVA as a stand-alone vaccine against smallpox and infections
caused by other orthopoxviruses.
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