| Research Article |
Open Access |
|
| Evaluation of Different Strategies for Post-Exposure Treatment of Ebola
Virus Infection in Rodents |
| Jason S. Richardson1#, Gary Wong1,3#, Stéphane Pillet1, Samantha Schindle1, Jane Ennis2, Jeffrey Turner2, James E. Strong1,3 and Gary P.
Kobinger1,3,4* |
| 1Special Pathogens Program, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba, Canada |
| 2Defyrus Incorporated, Toronto, Ontario, Canada |
| 3Department of Medical Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada |
| 4Department of Immunology, University of Manitoba, Winnipeg, Manitoba, Canada |
| #The authors contributed equally to the work. |
| *Corresponding author: |
Dr. Gary P. Kobinger, PhD
Special Pathogens Program,
National Microbiology Laboratory
Public Health Agency of Canada, 1015 Arlington
Street
Winnipeg, MB, R3E 3R2,Canada
Tel: 204-784-5923
Fax: 204-789-2140
E-mail: gary_kobinger@phac-aspc.gc.ca |
|
| |
| Received August 31, 2011; Accepted October 18, 2011; Published October 20,
2011 |
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| Citation: Richardson JS, Wong G, Pillet S, Schindle S, Ennis J, et al. (2011)
Evaluation of Different Strategies for Post-Exposure Treatment of Ebola Virus
Infection in Rodents. J Bioterr Biodef S1:007. doi:10.4172/2157-2526.S1-007 |
| |
| Copyright: © 2011 Richardson JS, et al. This is an open-access article distributed
under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited. |
| |
| Abstract |
| |
| Zaire Ebola virus (ZEBOV) is a pathogen that causes severe hemorrhagic fever in humans and non-human
primates. There are currently no licensed vaccines or approved treatments available against ZEBOV infections. The
goal of this work was to evaluate different treatment strategies in conjunction with a replication deficient, recombinant
human adenovirus serotype 5 (Ad-CAGopt)-based vaccine expressing the Zaire Ebola virus glycoprotein (ZGP) in
Ebola infected mice and guinea pigs. |
| |
| Guinea pigs were treated with Ad-CAGoptZGP in combination with different treatment strategies after challenge
with guinea pig adapted-ZEBOV (GA-ZEBOV). B10.BR mice were used to further characterize efficacy and immune
responses following co-administration of Ad-CAGoptZGP with the most effective treatment: AdHu5 expressing
recombinant IFN-α (hereafter termed DEF201) after challenge with a lethal dose of mouse adapted-ZEBOV (MAZEBOV). |
| |
| In mice, DEF201 treatment was able to elicit full protection against a lethal dose of MA-ZEBOV when administered
30 minutes after infection. In guinea pigs the Ad-CAGoptZGP and DEF201 combination therapy elicited full
protection when treated 30 minutes post-exposure and were a superior treatment to Ad-CAGoptZGP supplemented
with recombinant IFN-α protein. Further analysis of the immune response revealed that addition of DEF201 to Ad-
CAGoptZGP enhances the resulting adaptive immune response against ZGP. The results highlight the importance of
the innate immune response in the prevention of ZEBOV pathogenesis and support further development of the Ad-
CAGoptZGP with DEF201 treatment combination for post-exposure therapy against ZEBOV infection. |
| |
| Introduction |
| |
| Ebola virus (EBOV) is a member of the family Filoviridae. They
are enveloped, single-stranded, negative-sense RNA viruses, which
cause severe hemorrhagic fever and are associated with highly lethal
infections in humans. Several strains of EBOV have been identified to
date including the Zaire, Sudan, Bundibugyo, Ivory Coast and Reston
Ebola virus [1]. The most aggressive strain identified is the Zaire Ebola
virus (ZEBOV) with mortality rates reported to be as high as 90% [2].
Outbreaks of EBOV infection have occurred sporadically in Africa in the
past and caused substantial fatalities within the affected communities.
Several factors including high lethality rates in humans, the ease of in
vitro propagation and the potential for aerosol dissemination make
EBOV a causative agent for biological warfare [3]. While significant
progress has been made in understanding the pathogenesis of EBOV
infection there is still no clinically approved EBOV vaccine or treatment
available. Thus, the development of an effective post-exposure therapy
is considered a high priority despite the limited impact of EBOV on the
human population worldwide. |
| |
| Over the years many candidate vaccine platforms have been
evaluated for their efficacy against ZEBOV. These include: naked or
lipid encapsulated DNA [4,5], virus-like particle preparations (VLPs)
[6-9], Vesicular stomatitis virus, strain Indiana (VSV) [10-14], Human
parainfluenza virus 3 (HPIV-3) [15-17], vaccinia [18], Venezuelan
equine encephalitis virus (VEEV) and replication-deficient human
adenovirus serotype 5 (AdHu5) vectors [4,19]. Among these strategies,
the VSV-based ZEBOV vaccine demonstrated 50% survival of NHPs
when administered 30 minutes post-ZEBOV infection [10]. In another
study, NHPs treated with encapsulated siRNA targeting the ZEBOV RNA polymerase resulted in complete protection when administered
30 minutes after ZEBOV infection followed by additional siRNA
administration on days 1 through 6 [20]. It has also been reported
that administration of recombinant nematode anticoagulant protein
c2 (rNAPc2), a potent inhibitor of tissue factor-initiated blood
coagulation, protected 33% of infected rhesus macaques [21]. |
| |
| Macrophages and dendritic cells (DCs) are important components
of the innate immune system and known to be the primary early
targets for EBOV infection [22]. Upon infection of macrophages with
ZEBOV, sustained cytokine and chemokine production was observed
but with little or no interferon-alpha (IFN-α) response in vitro [23].
ZEBOV-infected DCs also did not produce IFN-α [24], but contrary
to macrophages, infected DCs do not become fully activated and hence
do not secrete pro-inflammatory cytokines, upregulate co-stimulatory
molecules or properly stimulate T-cells [25]. This lack of stimulation results in a poor adaptive immune response. EBOV infection results in
the suppression of a normal stimulation of the host interferon response
through VP35 and VP24 viral proteins. VP35 has been shown to
block IFN-α/β production by small ubiquitin-like modifier (SUMO)-
ylation of interferon regulatory factor 7 (IRF-7) [26] and inhibition of
the IRF-3 kinases, namely IKK-ε and TBK-1 [27]. VP24 can interact
with host cell importin-α proteins which prevent the nuclear import
and accumulation of phosphorylated STAT-1 [28]. Since the STAT-1
transcription factor is utilized by both IFN-α/β and IFN-γ signaling
pathways, the presence of VP24 inhibits cellular responses to both
Type I and II IFN [28]. As a result the dysregulated innate immune
response becomes ineffective at limiting and clearing viral infection,
promotes non-productive inflammation as well as negatively impacting
the potency of subsequent specific adaptive immune responses. The
robust non-specific inflammatory response is suspected to contribute
to the progression to shock-like symptoms, coagulation abnormalities
and multiple organ failure ultimately causing a fatal outcome to the
infected host. Modulating the innate immune system during the
early stages of EBOV infection may therefore improve host immune
defenses and lead to a more positive outcome. Bolus administration of
recombinant interferon was shown to delay EBOV replication in vitro
[29] and induce survival in infected mice [30] but this strategy was not
successful in NHPs [29]. However, constitutive in situ production of
IFN-α via a replication deficient adenovirus has demonstrated strong
antiviral efficacy against SARS [31] and Yellow Fever Virus [32]. |
| |
| The present study compares several treatment strategies, some
which modulate the innate immune system and investigates whether
these treatments are able to enhance survival against a lethal ZEBOV
challenge in mice and guinea pigs. The strategies evaluated include:
isopropanol, dextrose, azithromycin, CD40 ligand (CD40L), AdHu5-
iMYD88.CD40 with the AP1903 dimerization drug, recombinant
IFN-α protein (rIFN-α), and an adenovirus expressing recombinant
IFN-α (DEF201). |
| |
| Materials and Methods |
| |
| Construction and production of adenoviral vectors |
| |
| Molecular clones of E1-deleted human adenovirus serotype
5 vectors (AdHu5) expressing ZEBOV glycoprotein (ZGP) were
generated and called Ad-CAGoptZGP as described previously [42].
The authenticity of each vector was confirmed by sequencing and the
recombinant virus was rescued by transfecting the linearized DNA
into HEK 293 cells maintained in Dulbecco’s modified Eagle’s medium
(DMEM) supplemented with 1% penicillin, 1% streptomycin, 1%
L-glutamine, 1% sodium pyruvate, and 10% fetal bovine serum. Largescale
infections (5 x 108 cells) were initiated from positive transfectants
and purified by cesium chloride as previously described [43]. Genome
structures of vectors were analyzed by restriction digestions of viral
DNA and compared with those of the molecular clones as previously
described [43]. Particle number and infectivity (IFU) of vectors were
determined by standard optical density and immunodetection of the
AdHu5 hexon protein respectively, following infection of HEK 293
cells with limiting dilutions of each vector preparation according to
the recommendations by the manufacturer (Adeno-X rapid titer kit,
Clontech, Mountain View, CA). Several Ad-CAGoptZGP preparations
were generated and quantified for both infectious particle and total
particle number. Preparations with a ratio of at least 1:200 infectious to
total particle were used in this study. AdHu5 expressing recombinant
IFN-α (DEF201) were provided by Defyrus Incorporated, ON and
manufactured as described [44]. |
| |
| Animal models, vaccination and challenge |
| |
| Groups of 5 or 10 B10.BR mice (Jackson Laboratory, ME) were
challenged by intraperitoneal (I.P.) injection with 1000 x LD50 of
mouse-adapted ZEBOV strain Mayinga [45]. They were then treated
by intramuscular (I.M.) injection with 1 x 107 infectious particles of
either empty recombinant adenoviral vector diluted to a total of 500μl
volume with PBS or DEF201 via the I.M. or I.N. routes 30 minutes
post challenge. The mice were then weighed every day for 14 days and
monitored for clinical signs of disease for 30 days following challenge. |
| |
| Hartley breed of guinea pigs (Charles River, QC) were challenged
by I.P. injection of 100 x LD50 of guinea pig-adapted ZEBOV strain
Mayinga [46]. 30 minutes post challenge they were either treated I.M.
with 2 x 108 infectious particles of DEF201, or by I.M. vaccination
with 1 x 1010 infectious particles of Ad-CAGoptZGP, with or without
treatment of a subcutaneous (S.C) injection of 2g/Kg of isopropanol (6
hours post-challenge), S.C. injection of 5% dextrose solution dissolved
in water, S.C. injection of azithromycin (20mg/Kg on day 1, 10mg/Kg
on days 2-5 post challenge), mixed I.M. injection of 2 x 1010 infectious
particles of AdHu5-iMYD88.CD40, mixed I.M. injection of 0.44μg/Kg
rIFN-α, mixed I.M. injection of CD40L, or mixed I.M. injection of 2 x
108 infectious particles of DEF201. Guinea pigs were weighed every day
for 16 days and monitored for clinical signs of disease. |
| |
| All animal procedures and scoring sheets were approved by the
Institutional Animal Care Committee at the National Microbiology
Laboratory (NML) of the Public Health Agency of Canada (PHAC)
according to the guidelines of the Canadian Council on Animal Care.
All infectious work was performed in the ‘Biosafety Level 4’ (BSL4)
facility at NML, PHAC. |
| |
| Neutralizing antibody assay |
| |
| Sera harvested from rodents 28 days post vaccination were
inactivated at 56°C for 45 minutes. Twofold serial dilutions of each
sample (1:10, 1:20, 1:40, etc, in 50 ml of DMEM) was mixed with equal
volume of ZEBOV encoding the eGFP reporter gene (100 transducing
units/well) and incubated at 37°C for 60 minutes. The mixture was then
transferred onto sub-confluent VeroE6 cells in 96-well flat-bottomed
plates and incubated for 90 minutes at 37°C in 5% CO2. Control wells
were infected with equal amounts of ZEBOV-eGFP with or without
the addition of serum. 100 ml of DMEM supplemented with 20% FBS
was then added to each well and plates were incubated at 37°C in 5%
CO2 for 48 hr. Dilutions were scored as positive for the presence of
NAbs when there was a greater than a 50% reduction in the number of
cells expressing eGFP in a field of view using a fluorescent microscope.
The highest serum dilution scoring positive for NAb was recorded
and neutralization titers were reported as the reciprocal of this
dilution. Positive and negative control sera used in this experiment
were harvested from mice or guinea pigs 28 days post immunization
with a VSV vaccine expressing ZGP, or naïve mice or guinea pig
sera respectively. All infectious in vitro work was performed in the
biocontainment level 4 laboratory of the NML, PHAC. |
| |
| ZGP-specific IgG ELISA |
| |
| Immulon 2 HB 96 well flat bottom MicroTiter ELISA Plates
(Thermo Scientific) were coated overnight with 50μl/well of 1 μg/
ml His-ZEBOV-GP capture antigen diluted in PBS [47]. Plates were
washed three times with wash buffer (PBS 0.1% TWEEN 20) and
then blocked for 90 min with blocking buffer (5% skim milk powder/
PBS/0.2% Tween 20) at 37°C [47]. Plates were washed three times
with wash buffer. Sera collected from either mice or guinea pigs were inactivated at 56°C for 45 minutes. Serum was diluted at 1:50
in blocking buffer and 50μl of dilution was added to each well and
incubated for 60 min at 37°C. The plates were washed three times
with wash buffer. A secondary antibody for mouse (horseradish
peroxidase (HRP)-conjugated rat anti-mouse antibody to mouse IgG)
(Jackson Laboratories) or for guinea pig (HRP-conjugated goat antiguinea
pig antibody to guinea pig IgG), (KPL) or for NHPs (HRPconjugated
Goat anti-Human antibody to IgG) (KPL) was added to the
plate and then incubated for 60 min at 37°C Horseradish peroxidase
substrate (3% hydrogen peroxide solution with ABTS [2,2’-azinobis(3-
ethylbenzthiazolinesulfonic acid)]) was then added and incubated at
room temperature for 30 min. The plates were read using a VMax
Kinetic ELISA Microplate Reader (Molecular Devices) and the data
was analyzed using CellMaxPro software for the detection of ZGPspecific
IgG antibodies. The data is reported as the optical density
measured by absorbance at 405nm. Positive and negative control sera
used in this experiment were harvested from mice or guinea pigs 28
days post immunization with a VSV vaccine expressing ZGP, or naïve
mice or guinea pig sera respectively. |
| |
| Flow cytometry analysis |
| |
| The frequency of CD4+ or CD8+ cells producing IFN-γ TNF-α and IL-2 was assessed by flow cytometry. Mouse splenocytes were isolated
day 10 post Ad-CAGoptZGP vaccination by harvesting the spleens
and grinding spleen tissues. Splenocytes were seeded at 2 x 106 cells
per well in DMEM media supplemented with 10% FBS 1% penicillin,
1% streptomycin, 1% L-glutamine, 1% non-essential amino acids, 1%
sodium pyruvate, 1% HEPES buffer, 5 × 10−3 M 2-mecaptoethanol.
Cells were stimulated for 5 hours with 5μg/ml of the peptide carrying
the immunodominant MHC class I epitope of ZGP (TELRTFSI) for
mice with the H-2k haplotype [48] in the presence of 1μl/ml of the
protein transport inhibitor GolgiPlug (BD Biosciences). Splenocytes
were then stained with PacBlue-conjugated rat anti-mouse CD4
and PerCp-Cy5.5-conjugated rat anti-mouse CD8 antibodies (BD
Biosciences), followed by a 20 minute incubation in Cytofix/Cytoperm
(BD Biosciences). Intracellular cytokines were detected after staining
with PE-conjugated anti-mouse IFN- γ, PECy7-conjugated anti-mouse TNF-α (BD Biosciences), and APC-conjugated anti-mouse IL-2 diluted
in PermWash buffer (BD Biosciences). At least 300 000 events were
analysed using the 17-color flow cytometer (LSR II Flow Cytometer,
BD Biosciences). |
| |
| Results |
| |
| Complete survival was previously observed in mice administered
Ad-CAGoptZGP 30 minutes after infection with 1000 x LD50 of MAZEBOV
[33]. However, the same strategy resulted in delayed time to
death with only 1 survivor out of 3 guinea pigs after challenge with 100
x LD50 of GA-ZEBOV in a pilot experiment (Figures 1A and 1B). In an
attempt to improve the survival rate, several treatments were evaluated
in guinea pigs when administered 30 minutes after lethal challenge
with GA-ZEBOV. Selected strategies hypothesized to improve survival
included modulators of the innate immune response; fluids or an
antibiotic each evaluated in conjunction with the Ad-CAGoptZGP
vaccine. Guinea pigs were first infected with 100 x LD50 of GA-ZEBOV,
followed 30 minutes later by a single intramuscular (I.M.) injection of 1 x 1010 infectious forming units (IFU) of Ad-CAGoptZGP per animal in
conjunction with either dextrose, AdHu5-iMYD88.CD40 with dimer
drug, CD40L, rIFN-α, DEF201 or 6 hours later with isopropanol,
or azithromycin from days 1-5 post-challenge. Control guinea pigs
were injected I.M. with PBS. Infection of control guinea pigs resulted
in rapid weight loss combined with 100% mortality by day 7 post
infection. Delays to time of death were observed for the isopropanol
treatment (day 12), dextrose (day 12), azithromycin (day 10), AdHu5-
iMYD88.CD40 with the dimer drug (day 15) and CD40L (day 11) but
survival was not observed for these treatment groups (Figures 2A and
2B). Treatment of guinea pigs with Ad-CAGoptZGP in combination
with rIFN-α resulted in 2 of 3 survivors where surviving animals
demonstrated moderate clinical symptoms with on average a maximum
weight loss of 11% (Figures 2A and 2B). When DEF201 was mixed
with the Ad-CAGoptZGP vaccine and injected I.M. into guinea pigs,
complete protection (6 survivors out of 6) was observed with little to
no weight loss or clinical symptoms (Figures 1A and 1B). Interestingly,
treatment with DEF201 alone did not provide protection in guinea pigs
against a lethal challenge with GA-ZEBOV (data not shown). |
| |
|
Figure 1: Survival curves and weight loss post challenge in vaccinated guinea
pigs with or without DEF201 treatment. A) Protective efficacy of Ad-CAGoptZGP
with or without DEF201 in female Hartley guinea pigs and B) their
associated weight loss curves. Groups of 3 or 6 guinea pigs were challenged
intraperitoneally with 100 x LD50 of guinea pig-adapted Zaire Ebola virus and
treated intramuscularly with Ad-CAGoptZGP at 1 x 1010 IFU per animal in conjunction
with the specified treatment 30 minutes post-challenge. Data represents
percentage survival or percentage weight loss respectively. |
|
| |
|
Figure 2: Survival curves and weight loss post challenge in vaccinated guinea
pigs with various treatments. A) Protective efficacy of Ad-CAGoptZGP with various
treatments in female Hartley guinea pigs and B) their associated weight
loss curves. Groups of 3 or 6 guinea pigs were challenged intraperitoneally
with 100 x LD50 of guinea pig-adapted Zaire Ebola virus and treated intramuscularly
with Ad-CAGoptZGP at 1 x 1010 IFU per animal in conjunction with the
specified treatment either 30 minutes or 6 hours post-challenge. Data represents
percentage survival or percentage weight loss respectively. |
|
| |
| Survival and weight loss in mice following Ebola virus
challenge and treatment |
| |
| There are currently only limited reagents available for studying the
immune response in guinea pigs. Therefore mice were used to better
define the additive effect of DEF201, either directly or indirectly, to
Ad-CAGoptZGP. Thirty minutes after challenge with 1000 x LD50 of
MA-ZEBOV, B10.BR mice were administered with either a single I.M.
or intranasal (I.N.) dose of 1 x 107 IFU of DEF201 per animal. Control
B10.BR mice were administered PBS. An additional control consisting
of mice administered replication-deficient AdHu5 vector not
expressing any transgene (AdHu5-empty) I.M. was added to evaluate
the contribution the vector alone since AdHu5 particles were shown to
modulate the innate immune response [34]. Infection of PBS control
mice resulted in rapid weight loss and 100% mortality between days
6 and 8 post-challenge. In contrast, complete survival was observed
in MA-ZEBOV challenged mice following treatment with either I.M.
or I.N. administration of 1 x 107 IFU of DEF201 with no weight loss
or clinical symptoms (Figure 3A). Interestingly, treatment of B10.BR
mice with AdHu5-empty at the same dose resulted in 40% survival.
Surviving mice displayed signs of illness with an average maximum
weight loss of 17% (Figure 3B). |
| |
|
Figure 3: Survival curves and weight loss post challenge in mice. A) Protective
efficacy of Ad-CAGoptZGP in B10.BR mice and B) their associated weight
loss curves. Groups of 5 or 10 mice were challenged intraperitoneally with
1000 x LD50 of mouse-adapted Zaire Ebola virus and treated intramuscularly
or intranasally with 1 x 107 IFU DEF201 per animal, or intramuscularly with
AdHu5-empty at 1 x 107 IFU per animal 30 minutes post-challenge. Data represents
percentage survival or percentage weight loss respectively. |
|
| |
| Immune responses in mice following treatment |
| |
| The increased protection observed in guinea pigs with the addition
of DEF201 to Ad-CAGoptZGP could be due to an independent antiviral
effect from the expression of IFN-α directly on ZEBOV, and/or
that IFN-α further improves the adaptive immune response generated
by Ad-CAGoptZGP. To address this last possibility, cellular and
humoral immune responses were evaluated in the mouse model. The
flow cytometry analysis revealed that vaccination with Ad-CAGoptZGP
primed a T-cell response characterized by functional CD8+ effector
cells producing IFN-γ and TNF-α after restimulation with the
immunodominant ZGP peptide, and the absence of these cytokines in
CD4+ cells (Figure 4, middle and left panels respectively). A significant
amount of those CD8+/IFN-γ/TNF-α+ cells also produced IL-2 (Figure
4, right panel). Co-treatment with DEF201 increased the proportion of
the CD8+/IFN-γ+ cells to 5.42% from 2.82% and CD8+/IFN-γ/IL-2+
cells to 0.42% from 0.25% with Ad-CAGoptZGP alone (Figure 4, lower
panel). As expected, no ZGP-specific response was observed in cells
from mice treated with DEF201 alone. |
| |
|
Figure 4: Functional expression of cytokines in mouse splenocytes from each indicated treatment groups after ex vivo stimulation with the immunodominant ZEBOV
GP peptide. Lymphocytes were gated according to the expression of CD4 or CD8 (upper panel). CD4+ and CD8+ cells were then analysed for the expression of
IFN-γ, TNF-α and IL-2 (lower panels). Numbers in the grid in the upper right quadrant represent the percent of cells in each quadrant. |
|
| |
| The humoral immune response was analyzed on mice serum
harvested at 28 days post treatment via total anti-ZGP IgG ELISAs and
neutralizing antibody (NAb) assays. Significant levels of total anti-ZGP
IgG antibodies were detected for both the Ad-CAGoptZGP with and
without DEF201 treatment groups, with corresponding A405 values
at 1.31±0.03 and 1.24±0.02 respectively. Background levels of IgG
were observed for the DEF201 treatment group with an A405 value
of 0.19+0.01 (Figure 5A). Neutralizing assays revealed that sera from
DEF201 treated B10.BR mice did not have detectable NAb levels on
day 28, where the limit of detection was set at 10 reciprocal dilutions.
Significant levels of NAb were detected from sera of mice treated with the
Ad-CAGoptZGP with or without DEF201, with average NAb titers of
42±13 or 23±6 reciprocal dilutions respectively on day 28 (Figure 5B). |
| |
|
Figure 5: Humoral immune responses in vaccinated mice with or without
DEF201 treatment. A) Serum ZEBOV GP-specific IgG ELISAs. The harvested
serum samples were also tested for the presence of IgG antibodies against
ZEBOV GP at a 1:50 dilution ratio. The data is reported as the optical density
measured by absorbance at 405nm. B) Serum ZEBOV GP-specific neutralizing
antibody response. Groups of 3 to 6 mice were vaccinated and treated
with or without DEF201, where serum was harvested 28 days post-vaccination.
Serum neutralizing antibody levels were reported as the reciprocal of the
dilution where greater than 50% viral neutralization was observed. |
|
|
| |
| Humoral immune responses in guinea pigs following
treatment |
| |
| The humoral immune response was also analyzed on guinea pig
serum harvested 28 days post vaccination via total anti-ZGP IgG ELISAs
and neutralizing antibody (NAb) assays. Significant levels of total anti- ZGP IgG antibodies were detected from sera of animals treated with
Ad-CAGoptZGP with or without DEF201, with corresponding A405
values of 2.40±0.09 or 2.17±0.06 respectively (Figure 6A). Similarly,
significant levels of NAb were detected in the serum of guinea pigs
treated with Ad-CAGoptZGP alone or in combination with DEF201
with corresponding values at 20±12 and 45±21 respectively (Figure 6B). |
| |
|
Figure 6: Humoral immune responses in vaccinated guinea pigs with or without
DEF201 treatment. A) Serum ZEBOV GP-specific IgG ELISAs. The harvested
serum samples were also tested for the presence of IgG antibodies
against ZEBOV GP at a 1:50 dilution ratio. The data is reported as the optical
density measured by absorbance at 405nm. B) Serum ZEBOV GP-specific
neutralizing antibody response. Groups of 4 guinea pigs were vaccinated and
treated with or without DEF201, where serum was harvested 28 days postvaccination.
Serum neutralizing antibody levels were reported as the reciprocal
of the dilution where greater than 50% viral neutralization was observed. |
|
| |
| Discussion |
| |
| EBOV viruses are among the most deadly infectious agents in
humans and it is thought that their virulence is in part due to their
capacity to suppress and evade host IFN responses [1]. Consequently,
it was hypothesized that productive modulation of the innate immune
response with interferon could suppress EBOV replication in vivo
sufficiently enough to allow the immune system to mount a protective
adaptive response. |
| |
| Several strategies which modulate the innate immune response
were investigated in this study. Isopropanol administration has been
shown to have immunosuppressive properties that prevent toxic shock
[35], a common suspected occurrence in EBOV human cases. Dextrose administration induces a temporary state of acute hyperglycemia
which causes endothelial progenitor cells to adapt a pro-inflammatory
phenotype and enhance naïve T-cell activation [36]. Azithromycin is a
macrolide antibiotic with the ability to suppress the pro-inflammatory
activity of macrophages and is used to treat chronic inflammatory
diseases [37]. Since loss of intestinal integrity is common in advanced
EBOV infection, it also has the added benefit of preventing the normal
intestinal flora from causing further infection after traversing the
gastrointestinal barrier. CD40 ligand (CD40L) can partially mimic
CD4+ T-cell activation of the CD40 receptor on DCs which aids in the
maturation of immature DCs [36]. AdHu5-iMYD88.CD40 treatment in
combination with the AP1903 dimerization drug targets the sustained
activation of DCs and results in higher antigen-specific CD8+ T-cell
responses [36]. IFN-α has been shown to be a clinically effective general
immunotherapeutic antiviral in the past, where it is responsible for
priming the host innate immune response and then transitioning into
an effective adaptive immune response by regulating various cytokines
and their receptors [38]. Furthermore, administration of recombinant
IFN-α was shown to prevent death in EBOV-infected mice but not in
nonhuman primates thus far [29]. |
| |
| Protection against ZEBOV infection was demonstrated in mice
following I.M. and I.N. administration with DEF201. Surprisingly partial protection was observed within the AdHu5-empty control
group, where 2 of 5 mice survived despite being challenged with a
uniformly lethal dose of MA-ZEBOV. This suggests that the AdHu5
vector itself also contributes to the stimulation of a protective immune
response against ZEBOV. Indeed, the AdHu5 vector has been shown
to promote both transduced and bystander DC maturation as well as
stimulate Type I IFN production [39]. |
| |
| While protection was not observed with the isopropanol, dextrose,
azithromycin, CD40L, and AdHu5-iMYD88.CD40 treatments on
guinea pigs, delays in the occurrence of clinical symptoms and median
time to death were observed for all these treatments, suggesting that
they participated in delaying ZEBOV-induced pathogenesis. It is
possible that further optimization of some of these interventions could
lead to better outcome and in turn be useful notably for improving patient management. In all aspects, the present study further support
the concept that IFN-α is important early in EBOV infection with
regards to survival. This was also a conclusion of previous work, which
showed that I.M. administration of IFN-α2β protein into cynomolgus
macaques was able to delay viremia and death from lethal ZEBOV
infection by several days [29]. Unfortunately, recombinant IFN-α has
a short half-life of 8 hours in vivo [40] and can be extremely costly.
Effective treatment of EBOV infection may require sustained levels
of IFN-α for days in order to increase efficacy and give more time to
the adaptive immunity to mount a protective response. DEF201 can
produce constitutively high levels of IFN-α for days in a cost efficient
manner. In addition to higher and more sustained expression of IFN-α,
the AdHu5 particles themselves resulted in improved survival in mice
when administered 30 minutes after lethal MA-ZEBOV infection.
The addition of DEF201 to the Ad-CAGoptZGP vector resulted in
improved survival in comparison to recombinant IFN-α together with
Ad-CAGoptZGP. The lack of survival in guinea pigs administered only
DEF201 further highlights the enhanced protection of the combination
therapy. |
| |
| Analysis of the resulting T- and B-cell immune response following
Ad-CAGoptZGP administration with DEF201 showed overall
elevated cellular and humoral responses relative to Ad-CAGoptZGP
alone. CD8+ cells primed by the Ad-CAGoptZGP vaccination were
functional according to their ability to produce IFN-γ and TNF-α
after ex vivo restimulation. The presence of a significant proportion of
these cells producing IL-2 is indicative of resting memory T cells [41].
Therefore, DEF201 treatment could contribute to a better clearance
of the virus and also promote the establishment of memory cells.
However, the positive effect observed with DEF201 alone also indicates
that the antiviral state induced by added expression of IFN-α plays a
role in controlling or delaying EBOV replication. Interestingly, even
only delaying replication is likely to be helpful in providing more time
for the generation of a protective immune response. Taken together
the results further emphasize the role of IFN-α and the innate immune
response in early ZEBOV infection. Future work will need to evaluate
the combination of DEF201 with Ad-CAGoptZGP as a treatment to
nonhuman primates already infected with a lethal dose of ZEBOV. |
| |
| Acknowledgements |
| |
| Financial support was received from the following sources: The Public Health
Agency of Canada and the Chemical, Biological, Radiological or Nuclear Research
and Technology Initiative (grant #CRTI-06-0218RD and CRTI-09-453TD awarded
to G.P.K.). G.W. is the recipient of a Doctoral Research Award from CIHR. The
authors would like to thank Heinz Feldmann, Andrea Marzi and Xiangguo Qiu for
providing reagents. The authors would also like to thank David Spencer and Maria
Croyle for their experimental discussion and input. The authors would also like to
thank Geoff Soule, Max Abou, Kaylie Tran, Gregg Schumer, Akeel Baig, Jason
Gren and Shane Jones for their technical support. |
| |
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