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S430 •JID 2007:196 (Suppl 2) •Warfield et al.
SUPPLEMENT ARTICLE
Ebola Virus-Like Particle–Based Vaccine Protects
Nonhuman Primates against Lethal Ebola Virus
Challenge
Kelly L. Warfield,
a
Dana L. Swenson,
a
Gene G. Olinger, Warren V. Kalina, M. Javad Aman,
b
and Sina Bavari
US Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland
Background. Currently, there are no licensed vaccines or therapeutics for the prevention or treatment of
infection by the highly lethal filoviruses, Ebola virus (EBOV) and Marburg virus (MARV), in humans. We previously
had demonstrated the protective efficacy of virus-like particle (VLP)–based vaccines against EBOV and MARV
infection in rodents.
Methods. To determine the efficacy of vaccination with Ebola VLPs (eVLPs) in nonhuman primates, we
vaccinated cynomolgus macaques with eVLPs containing EBOV glycoprotein (GP), nucleoprotein (NP), and VP40
matrix protein and challenged the macaques with 1000 pfu of EBOV.
Results. Serum samples from the eVLP-vaccinated nonhuman primates demonstrated EBOV-specific antibody
titers, as measured by enzyme-linked immunosorbent assay, complement-mediated lysis assay, and antibody-
dependent cell-mediated cytotoxicity assay. CD44
+
T cells from eVLP-vaccinated macaques but not from a naive
macaque responded with vigorous production of tumor necrosis factor–aafter EBOV-peptide stimulation. All 5
eVLP-vaccinated monkeys survived challenge without clinical or laboratory signs of EBOV infection, whereas the
control animal died of infection.
Conclusion. On the basis of safety and efficacy, eVLPs represent a promising filovirus vaccine for use in
humans.
During the past 20 years, owing to advances in molec-
ular biology and in the understanding of basic virology,
scientists have been able to develop subunit vaccines
based on virus-like particles (VLPs). VLPs for many
viruses have been developed and are based on the
knowledge that expression of specific viral structural
proteins results in the self-assembly of particles that
morphologically resemble the authentic virus [1]. Some
Potential conflicts of interest: K.L.W., D.L.S., M.J.A., and S.B. hold patent rights
to Ebola and Marburg virus-like particle–based vaccines. G.G.O. and W.V.K.: none
reported.
Presented in part: Filoviruses: Recent Advances and Future Challenges,
International Centre for Infectious Diseases Symposium, Winnipeg, Manitoba,
Canada, 17–19 September 2006.
Financial support: Defense Threat Reduction Agency. Supplement sponsorship
is detailed in the Acknowledgments.
Opinions, interpretations, conclusions, and recommendations are those of the
authors and are not necessarily endorsed by the US Army.
a
K.L.W. and D.L.S. contributed equally to this work.
b
Present affiliation: Integrated BioTherapeutics Inc., Frederick, Maryland.
Reprints or correspondence: Dr. Sina Bavari or Dr. Kelly Warfield, US Army
Medical Research Institute of Infectious Diseases, 1425 Porter St., Fort Detrick,
MD 21702 (sina.bavari@us.army.mil or kelly.warfield@us.army.mil).
The Journal of Infectious Diseases 2007;196:S430–7
This article is in the public domain, and no copyright is claimed.
0022-1899/2007/19610S2-0043
DOI: 10.1086/520583
of the many advantages of using VLPs as vaccines include
(1) their similar morphology to the live enveloped or
nonenveloped viruses from which they are derived; (2)
a strong safety profile, since they are nonreplicating; (3)
no concerns regarding viral vector or preexisting anti-
vector immunity; (4) the fact that they can be generated
in large quantities by use of mammalian or insect cell
lines; (5) their ability to generate innate, humoral, and
cellular immunity; and (6) the fact that they have been
safely and effectively administered in humans [1–7].
We and others have demonstrated previously the
generation of enveloped Ebola VLPs (eVLPs) in mam-
malian and insect cell-expression systems [8–13]. VLPs
containing glycoprotein (GP) and VP40 derived from
Ebola virus (EBOV) have been used successfully to vac-
cinate rodents [2, 13–17]. Both BALB/c and C57BL/6
mice have been protected against a range of challenge
doses (∼10–1,000 pfu or ∼300–30,000 LD
50
) by means
of dose-dependent eVLP vaccination in the presence or
absence of adjuvant [2, 14, 15]. Addition of saponin-
derived QS-21 or RIBI adjuvant to the eVLP-vaccine
regimen allows administration of a decreased dose of
the vaccine and completely protects mice and guinea
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VLP-Based Vaccine in Nonhuman Primates •JID 2007:196 (Suppl 2) •S431
pigs against challenge, even after only 1 inoculation (authors’
unpublished data) [18]. eVLP vaccination completely prevents
viremia and clinical symptoms after EBOV challenge in rodents
but does not induce sterile immunity, as evidenced by an ex-
pansion of immune responses to viral proteins not present in
the vaccine, after challenge [2, 14, 15, 18]. However, the ques-
tion of whether eVLPs would be able to protect nonhuman
primates against EBOV infection has remained. Therefore, the
goal of the current study was to determine whether eVLPs
would be viable vaccine candidates for the protection of pri-
mates against lethal EBOV challenge.
MATERIALS AND METHODS
Generation and characterization of eVLPs. 293T-derived
eVLPs containing EBOV GP, nucleoprotein (NP), and VP40
were prepared essentially as described elsewhere [9, 14, 16, 17].
Total protein concentrations in the vaccine preparations were
determined in the presence of detergent, by use of a Bradford
protein assay (BioRad). Expression of GP, NP, and VP40 in
each vaccine preparation was verified by Western blot analysis
[9, 17, 18]. eVLPs were processed and imaged via electron
microscopy, as described elsewhere [9, 14, 16, 17]. Endotoxin
levels in all eVLP preparations used in this study were !0.03
endotoxin units/mg, as determined by the Limulus amebocyte
lysate test (Invitrogen).
Animals. In testing before the start of this study, the cy-
nomolgus macaques used were found to be antibody negative
for filovirus, simian T cell leukemia virus–1, simian immu-
nodeficiency virus, and herpes B virus. The eVLP-vaccinated
monkeys received 3 intramuscular injections, at 42-day inter-
vals, of ∼1.0 mL of sterile saline containing 250 mg of eVLPs
and 0.5 mL of RIBI adjuvant (Corixa). Blood samples were
obtained from the femoral vein of monkeys under anesthesia.
Female cynomolgus macaques of 3–4 kg in weight were chal-
lenged by intramuscular injection of ∼1000 pfu of Zaire EBOV
(ZEBOV; 1995 outbreak strain) [19]. Viremia was determined
by means of a traditional plaque assay [20]. Hematology and
kidney- and liver-associated enzymes were assessed as described
elsewhere [21]. For ethical reasons, the use of relevant historical
control animals was required by the Laboratory Animal Care
and Use Committee of the US Army Medical Research Institute
of Infectious Diseases (Fort Detrick, MD), to reduce the num-
ber of nonhuman primates needed in these studies. For this
reason, the control monkey in the current study was not treated,
so that, for the data analysis, results for this monkey could be
combined with those for 23 historical control animals chal-
lenged with the same seed stock of ZEBOV at the same dose
and via the same route.
All EBOV-infected animals and their samples were handled
under maximum containment in a biosafety level 4 laboratory
at the US Army Medical Research Institute of Infectious Dis-
eases (Fort Detrick, MD). Research was conducted in compli-
ance with the Animal Welfare Act and other federal statutes
and regulations relating to animals and experiments involving
animals and adhered to principles stated in the Guide for the
Care and Use of Laboratory Animals [22]. The facility where
this research was conducted is fully accredited by the Associ-
ation for Assessment and Accreditation of Laboratory Animal
Care International (Rockville, MD).
Total antibody responses against EBOV. Levels of EBOV-
specific antibodies were determined from serum or plasma sam-
ples by use of ELISA, as described elsewhere [14]. Antibody
titers were defined as the reciprocal of the highest dilution
giving a net optical density value ⭓0.2.
Determination of complement-mediated lysis by EBOV GP–
specific antibody. Antibodies recognizing viral antigens on
infected cells can bind complement via their Fc region and can
initiate activation of the complement cascade, resulting in kill-
ing of the virus-infected cell. To determine whether eVLP vac-
cination generated antibodies capable of inducingcomplement-
mediated lysis, Vero cells were infected at an MOI of 10 with
EBOV GP–expressing Venezuelan equine encephalitis virus
(VEE) replicons, produced as described elsewhere [23], and
were incubated in a humidified, 5% CO
2
incubator at 37Cfor
16 h. After incubation, Vero cells were removed from the plate
by means of trypsinization and were labeled with 100 mCi of
51
Cr for 1 h. Cells were washed 3 times in RPMI 1640 without
fetal bovine serum (FBS) and were resuspended to 100,000cells/
mL in RPMI 1640 containing 10% FBS. Next, 100 mLofVero
cells were plated in each well of a 96-well plate containing
various dilutions of plasma samples from eVLP-vaccinated and
control monkeys. Then, low-endotoxin guinea pig complement
was added, at a final dilution of 1: 20, and samples were in-
cubated for 3 h. The amount of radioactivity released into the
supernatants was determined with a g-radiation counter. Spon-
taneous lysis was measured in VEE replicon–expressing
EBOV GP–infected cells [23] with complement added but no
antibodies present, and total lysis was measured in Vero cells
incubated with 1% Triton X-100. Percentage of specific
lysis was calculated as [(experimental release⫺spontaneous
.
release)/(maximum release ⫺spontaneous release)]⫻10 0
Antibody-dependent cell-mediated cytotoxicity (ADCC)
assay. ADCC occurs when virus-specific antibodies coat tar-
get infected cells and make them vulnerable to killing during
coculture with other cytolytic cells. To determine whether an-
tibodies stimulated by eVLP vaccination are capable ofinducing
ADCC, Vero cells were infected with VEE replicon–expressing
ZEBOV GP for 16 h and were labeled with 100 mCi of
51
Cr, as
described above. Labeled Vero cells were plated in a 96-well
plate (10,000 cells/well). Primate plasma from previously vac-
cinated or naive (negative control) animals was added at various
dilutions to labeled, GP-expressing Vero cells. Purified effector
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S432 •JID 2007:196 (Suppl 2) •Warfield et al.
Figure 1. Antibody responses in nonhuman primates vaccinated with
Ebola virus-like particles (eVLPs) containing glycoprotein (GP), nucleopro-
tein, and VP40. Cynomolgus macaques were vaccinated 3 times, at 6-
week intervals, with 250 mg of eVLPs in RIBI adjuvant. A, Total anti–
Ebola virus (EBOV) antibody titers for individual animals (VLP1–5), as
determined by ELISA. B, Induction of complement-mediated lysis indicated
in plasma samples from eVLP-vaccinated macaques. EBOV GP–expressing
Vero cells were incubated with guinea pig complement and the indicated
dilutions of plasma. The percentage lysis was determined as compared
with that in untreated cells. C, Results of lysis of EBOV GP–expressing
target cells by an antibody-dependent cell-mediated cytotoxicity (ADCC)
assay. Target cells incubated with plasma from eVLP-vaccinated macaques
or an unvaccinated animal (naive) and human effector cells (effector-to-
target cell ratio, 40:1) showed various levels of ADCC antibody titers.
Error bars indicate SDs.
cells (peripheral blood mononuclear cells [PBMCs]), resus-
pended in RPMI 1640 with 10% FBS, were added to antibody-
coated target cells at the following effector-to-target cell (ET)
ratios: 1: 10, 1: 20, 1:40, and 1:80. Optimization of the assay
was determined by choosing the ET ratio that produced the
least background in wells with no antibody or with plasma
from unvaccinated animals. Each plate was incubated for 4 h
at 37C in the presence of 5% CO
2
. After 4 h, centrifugation
of each plate was done at 250 gto pellet the cells, 50 mLof
supernatant was removed, and
51
Cr released into supernatant
was quantified by use of a g-radiation counter. Spontaneous
lysis was measured in VEE replicon–expressing EBOV GP–in-
fected cells [23] with effector cells added but no antibodies
present, and total lysis was measured in Vero cells incubated
with 1% Triton X-100. Percentage of specific lysis was calculated
as [(experimental release ⫺spontaneous release)/(maximum
.release ⫺spontaneous release)]⫻10 0
T cell responses to EBOV peptides in eVLP-vaccinated non-
human primates. Epitopes recognized by circulating CD4
+
and CD8
+
T cells were determined as described elsewhere [15,
24], with several minor modifications. In brief, EBOV-specific
responses were analyzed by culturing PBMCs with 1–5 mgof
overlapping 15-residue peptides representing EBOV GP, NP, or
VP40 (Mimotopes) or 1 mg/mL staphylococcal enterotoxin B
(SEB) in complete RPMI 1640 containing 10 mg/mL brefeldin
A. After 18 h of culture, the cells were stained with anti-CD44,
-CD8, or -CD4 (Pharmingen) in brefeldin A. After cell-surface
staining, cells were fixed in 1% formaldehyde, permeabilized
with saponin, and stained with anti–tumor necrosis factor
(TNF)–aphycoerythrin (Pharmingen). Samples with an in-
crease in the frequency of TNF-a–positive cells of 12-fold above
background, as assessed by no peptide stimulation or by re-
sponse to an irrelevant peptide (Lassa virus N: RPLSA-
GVYMGNLSSQ), were considered to be positive.
RESULTS
Humoral responses to EBOV infection, after eVLP vaccination.
To determine whether eVLP vaccination elicits humoral re-
sponses in nonhuman primates, total antibody responses in
blood from the eVLP-vaccinated macaques were determined,
by ELISA using irradiated whole EBOV virions, immediately
before each vaccination and before challenge (figure 1A). Total
EBOV-specific antibodies in the eVLP-vaccinated macaques
rose 3–10-fold after the first vaccination and plateaued after 2
vaccinations (figure 1A).
Neutralizing titers were observed in the eVLP-vaccinated ma-
caques, consistent with our results in previous studies of rodents
[14, 16], with 80% plaque reduction/neutralization titers rang-
ing from 1:20 to 1:160 (data not shown). Additional studies
of antibody function revealed that eVLP vaccination of non-
human primates elicited both complement-mediated lysis and
ADCC (figure 1Band 1C). Although the interaction between
antibodies and antigen provides the specificity of the response,
the complement system is likely to provide the actual protective
response by destroying antigen-coated cells. Antibodies from
the eVLP-vaccinated animals were able to induce complement-
mediated lysis of cells expressing EBOV GP, in a dilution-de-
pendent manner (figure 1B). ADCC caused by EBOV GP–
specific antibodies coating target infected cells, making them
vulnerable to killing during coculture with other cytolytic cells,
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VLP-Based Vaccine in Nonhuman Primates •JID 2007:196 (Suppl 2) •S433
Figure 2. Vigorous CD44
+
T cell response to Ebola virus (EBOV) glycoprotein (GP) and nucleoprotein (NP) peptides, in nonhuman primates vaccinated
with Ebola virus-like particles (eVLPs). Peripheral blood leukocytes from eVLP-vaccinated cynomolgus macaques were collected 10 days after vaccination
and were used ex vivo for identification of peptides that induced intracellular tumor necrosis factor (TNF)–ain CD4
+
/CD44
+
or CD8
+
/CD44
+
T cells.
Percentages of TNF-a–producing cells that were 12-fold higher than the background percentage (no peptide or irrelevant peptide) were considered
to be positive. Representative responses to EBOV GP and NP peptides in an eVLP-vaccinated animal (VLP4) and a naive control animal are shown.
SEB, staphylococcal enterotoxin B.
also was observed when serum samples from the eVLP-vac-
cinated monkeys were used (figure 1C).
Cellular immune responses of eVLP-vaccinated nonhuman
primates. Representative flow-cytometry data are shown for
the naive control animal and for monkey VLP4, at 10 days
after their third vaccination (figure 2). Although this particular
eVLP-vaccinated monkey appeared to have the lowest total
antibody titers, as assessed by ELISA (figure 1A), it demon-
strated strong T cell responses to EBOV-peptide stimulation
(figures 2 and 3). In figure 2, we plotted responses from both
the naive and the eVLP-vaccinated monkeys and also show
TNF-aproduction in T cells stimulated overnight with SEB,
irrelevant peptide, or EBOV-specific peptides. The SEB-stim-
ulated cells were used as a positive control for T cell activation
and cytokine secretion, while several wells with no peptide or
an irrelevant peptide (such as Lassa virus N [15, 24]) were
included as negative controls, to determine the assay back-
ground (figure 2). Interestingly, specific pools from the majority
of the eVLP-vaccinated macaques were recognized, such as GP
pools 9 and 10 and NP pools 3, 4, and 11 (figure 3Aand 3B).
However, epitopes within the VP40 matrix protein were not
strongly recognized in samples from the eVLP-vaccinated an-
imals (figure 3C).
Protection of nonhuman primates against EBOV infection,
by eVLP vaccination. All 5 eVLP-vaccinated monkeys and
the single naive control monkey were challenged with ∼1000
pfu of ZEBOV at 4 weeks after the last vaccination (figure 4A).
As typically observed in cynomolgus macaques, the control
monkey developed clinical and laboratory signs of EBOV in-
fection beginning 4–5 days after challenge and died from the
disease 6 days after challenge (figures 4 and 5). Results for a
cohort of 23 naive control monkeys infected with the same
challenge virus via the same means are shown in figure 4A, to
indicate the normal course of disease and the mean time to
death after EBOV challenge. The eVLP-vaccinated monkeys
were completely protected against disease after lethal EBOV
challenge. We observed no signs of clinical disease, such as rash,
anorexia, or weight loss, in any of the eVLP-vaccinated mon-
keys. In addition, viremia was not detected by standard plaque
assay at any of the time points when measurements were taken
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S434 •JID 2007:196 (Suppl 2) •Warfield et al.
Figure 3. Cellular immune responses of nonhuman primates vaccinated
with Ebola virus-like particles (eVLPs) to Ebola virus (EBOV)–based pep-
tides. Peripheral blood mononuclear cells from eVLP-vaccinated animals
(VLP1–5) were collected 10 days after vaccination and were used ex vivo
for identification of peptides from EBOV glycoprotein (GP; A), nucleoprotein
(NP; B), and VP40 (C) that induced CD4
+
/CD44
+
or CD8
+
/CD44
+
T cells
expressing tumor necrosis factor (TNF)–a. Percentages of TNF-a–pro-
ducing cells that were 12-fold higher than the background percentage
(dashed line) were considered to be positive. All cells were tested against
duplicate pools of peptides, and responses to 1 set are shown here.
Figure 4. Protection of nonhuman primates vaccinated with Ebola vi-
rus-like particles (eVLPs) containing glycoprotein, nucleoprotein,andVP40.
Cynomolgus macaques ( ) were vaccinated 3 times with eVLPs innp5
RIBI adjuvant. A, Survival after challenge with 1000 pfu of Zaire Ebola
virus (ZEBOV), assessed 4 weeks after the last vaccination. For the pur-
poses of representation on the Kaplan-Meier plot, results for the control
monkey in the current study were combined with those for 23 historical
control animals challenged with the same seed stock of ZEBOV, at the
same dose and via the same route. B, Virus titers in the plasma of ZEBOV-
challenged eVLP-vaccinated monkeys were determined by use of a stan-
dard plaque assay. The laboratory values for the naive control monkey
are shown from an unscheduled blood sample at 6 days after challenge,
obtained immediately before euthanasia. C, Rectal temperature after chal-
lenge. Data are presented as the mean value for the eVLP-vaccinated
animals and the single control animal. Error bars indicate SDs.
(figure 4B). We did observe a mild elevation (2F–3F) in core
body temperature in 2 eVLP-vaccinated animals on day 8 after
challenge, although this increase was transient and not typical
of the high fever usually observed in filovirus-infected monkeys
(figure 4C).
We observed only minor hematologic and liver-enzyme
changes in the eVLP-vaccinated monkeys after EBOV challenge
(figure 5). Liver enzymes, such as aspartate transaminase, al-
kaline phosphatase, and alanine transaminase, were not altered
after EBOV infection of the eVLP-vaccinated monkeys (figure
5A–5C). The numbers of circulating platelets did not decrease
in the eVLP-vaccinated monkeys after EBOV challenge (figure
5D). Not surprisingly, total white blood cell (WBC) count and
percentage of lymphocytes in the blood were slightly more
variable in all the animals after challenge (figure 5Eand 5F).
As expected, the naive control animal in this experiment ex-
hibited an increase in total WBC count, with a concomitant
decrease in the percentage of circulating lymphocytes (figure
5Eand 5F).
DISCUSSION
Numerous vaccine approaches have been used against lethal
filoviral infections, including classic vaccine preparations of in-
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VLP-Based Vaccine in Nonhuman Primates •JID 2007:196 (Suppl 2) •S435
Figure 5. Clinical laboratory results for nonhuman primates vaccinated with Ebola virus-like particles (eVLPs) containing glycoprotein, nucleoprotein,
and VP40. Cynomolgus macaques ( ) were vaccinated 3 times with eVLPs in RIBI adjuvant. Liver function was assessed by analysis of serumnp5
samples from eVLP-vaccinated monkeys after challenge. Levels of aspartate transaminase (AST; A), alkaline phosphatase (ALP; B), and alanine
transaminase (ALT; C) were measured at the indicated time points. Platelet count (D), total white blood cell (WBC) count (E), and percentage of
lymphocytes (F) in blood also were assessed after challenge and are presented as the mean (SD) of the eVLP-vaccinated animals and the single
control animal. The laboratory values for the naive control monkey are shown from an unscheduled blood sample at 6 days after challenge, obtained
immediately before euthanasia.
activated or avirulent virus [25, 26]; viral vectors such as ad-
enovirus, VEE, paramyxovirus, vaccinia, and vesicular stoma-
titis virus that encode protective filoviral proteins [24, 26–29],
DNA [27, 30–32]; and other subunit vaccines [2, 26, 33]. In
this study, we have shown that VLPs are a promising vaccine
candidate for protection of nonhuman primates against lethal
EBOV infection. Some of the advantages of VLPs as filovirus
vaccines include presentation of antigen in its native form, an
excellent safety profile, no interference by a vector backbone,
lack of problems related to vector immunity, and induction of
strong antibody and T cell responses.
In our studies, we have found high levels of antibodies in
the serum of eVLP-vaccinated nonhuman primates, using 4
different assays. Not only did the vaccinated animals develop
high levels of total EBOV-specific antibodies, as determined by
ELISA, but they also developed antibodies that were highly
active in vitro. These antibodies induced virus-neutralizing
(data not shown), ADCC, and complement-mediated lysis ac-
tivity (figure 1Band 1C). Data suggest that our VLP vaccines
induced EBOV-specific antibodies that are multifunctional. On
the basis of our studies of nonhuman primates thus far (figure
1 and authors’ unpublished data), whether total levels of an-
tibodies correlate with protection against filovirus challenge is
not clear. We had shown previously that, although B cells are
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S436 •JID 2007:196 (Suppl 2) •Warfield et al.
absolutely required for eVLP-mediated protection in mice,
transfer of serum from eVLP-vaccinated mice to naive recip-
ients did not confer protection against EBOV infection, but
this result may have been dependent on the amount of antibody
transferred [15]. However, the role of B cells and antibodies in
protection against filoviral infection needs further investigation,
and their necessity for the protection of nonhuman primates
against EBOV infection is uncertain [34–38].
The eVLP-vaccinated monkeys developed strong T cell re-
sponses to EBOV epitopes, as assessed by intracellular cytokine
(TNF-a) staining of peptide-stimulated PBMCs. More re-
sponses to GP and NP were observed than to the VP40 matrix
protein (figure 3). Although responses to the peptides varied
among the monkeys, certain epitopes were recognized by more
than 1 monkey. Unfortunately, at this time, the reagents that
are available to describe the T cell responses of cynomolgus
macaques are limited, and further characterization of these re-
sponses may be required [39]. The importance of T cell re-
sponses in VLP-mediated protection against EBOV infection
has been demonstrated by use of knockout mice: CD4
+
knock-
out mice were protected only partially against EBOV infection,
and CD8
+
T cells were absolutely required [15]. On the basis
of our studies of mice, induction of both EBOV-specific an-
tibodies, to impede early viral infection and replication, and
cytotoxic T cells, to destroy virus-infected cells, was found to
be necessary for immunity and protection against EBOV in-
fection. Future studies of nonhuman primates and clinicaltrials
with humans will be required, to identify correlates of immunity
for VLPs and filoviral infection.
Proper glycosylation and presentation of viral proteins, as
well as vaccine dose, are critical factors for successful filovirus
vaccines [14, 27, 30, 33, 40]. We previously have shown that
eVLP-mediated protection against EBOV challenge is dose de-
pendent but that the addition of adjuvant can help reduce the
VLP dose and the number of injections required to mitigate
protection [14–16, 18, 41]. Kinetic studies of antibody titers
and T cell responses in eVLP-vaccinated nonhuman primates
indicated that 3 doses of VLPs did not appear to boost the
gross immune responses, compared with the responses ob-
served after 2 vaccines (figure 1 and data not shown). None-
theless, future studies are required in order to refine the VLP
vaccination schedule, dose, and requirement for adjuvant in
nonhuman primates. Studies of mice and guinea pigs have
indicated that protection after only 1 VLP vaccination is an
achievable goal [2, 18]. Currently, we also are examining the
role of a variety of adjuvants in enhancing VLP responses.
The eVLPs provided robust protection in thevaccinated non-
human primates. Significant clinical or laboratory signs of
EBOV infection, including detectable viremia, anorexia, or con-
siderably elevated levels of liver enzymes, were not found in
the eVLP-vaccinated monkeys. However, eVLP vaccination did
not induce sterile immunity against this lethal EBOV challenge.
A half-log increase in total anti-EBOV antibody titers was ob-
served in the eVLP-vaccinated monkeys at 28 days after chal-
lenge (figure 1A). Furthermore, we observed a broadening of
the T cell repertoire in the eVLP-vaccinated monkeys after
EBOV challenge. This was demonstrated by the recognition of
T cell epitopes in proteins not included in the vaccine, such as
VP24 and VP35, in lymphocytes after challenge but not before
challenge (data not shown). This observation was not surpris-
ing, since we previously had noted an increase in EBOV-specific
antibodies and the development of cytotoxic T lymphocytes
recognizing VP24 and VP35 in eVLP-vaccinated rodents after
challenge [14, 15, 18]. Since the vaccinated nonhuman primates
were completely protected against clinical signs and symptoms
of EBOV infection, the induction of sterile immunity by an
EBOV vaccine candidate does not seem to be critical.
Although VLPs produced in mammalian cells are highly im-
munogenic, we currently are exploring alternative strategies for
vaccine production, to accelerate the development process to-
ward the use of eVLPs and Marburg VLPs (mVLPs) in humans.
We and others have successfully generated eVLPs and mVLPs
in a baculovirus expression system [13, 41]. Both insect cell–
derived eVLPs and mVLPs were able to mature and activate
human myeloid dendritic cells (data not shown) [41]. Fur-
thermore, mice vaccinated with insect cell–derived eVLPs sur-
vived lethal challenge with mouse-adapted EBOV, suggesting
that the baculovirus-derived eVLPs are as effective as those
produced in mammalian cells [41].
Since our current data indicate that eVLPs are highly im-
munogenic in monkeys and stimulate virus-specific humoral
and cellular responses, our next major goal is to demonstrate
the efficacy of mVLPs against Marburg virus (MARV) infection
[16] and also of a mixture of eVLPs and mVLPs as a pan-
filovirus vaccine against both EBOV and MARV infections [18].
In addition, we currently are planning preclinical experiments
in preparation for future clinical trials with humans. Besides
use as a vaccine, VLPs also are being used to dissect innate
immune responses to filoviruses, with the goal of developing
immunotherapeutics for the treatment of EBOV and MARV
infections [42], as well as a safe surrogate model for the ex-
amination of filoviral replication, entry, and assembly [2].
Acknowledgments
We especially would like to thank M. A. Fernandez, as well as D. K.
Reed, S. Van Tongeren, K. Kuehl, A. Pace, N. Posten, J. Smith, C. Rice,
and J. Stockman, for excellent technical assistance.
Supplement sponsorship. This article was published as part of a sup-
plement entitled “Filoviruses: Recent Advances and Future Challenges,”
sponsored by the Public Health Agency of Canada, the National Institutes
of Health, the Canadian Institutes of Health Research, Cangene, CUH2A,
Smith Carter, Hemisphere Engineering, Crucell, and the International Cen-
tre for Infectious Diseases.
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VLP-Based Vaccine in Nonhuman Primates •JID 2007:196 (Suppl 2) •S437
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