assessment of a vesicular stomatitis virus–based vaccine by use of the mouse model of ebola virus...

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S404 JID 2007:196 (Suppl 2) Jones et al. SUPPLEMENT ARTICLE Assessment of a Vesicular Stomatitis Virus–Based Vaccine by Use of the Mouse Model of Ebola Virus Hemorrhagic Fever Steven M. Jones, 1,2,3 Ute Stro ¨ her, 1,3 Lisa Fernando, 1 Xianggou Qiu, 1 Judie Alimonti, 1 Pasquale Melito, 1 Mike Bray, 4 Hans-Dieter Klenk, 5 and Heinz Feldmann 1,3 1 Special Pathogens Program, National Microbiology Laboratory, Public Health Agency of Canada, 2 Department of Immunology and 3 Department of Medical Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada; 4 Biodefense Clinical Research Branch, National Institutes of Health, Bethesda, Maryland; 5 Institute of Virology, Philipps-University, Marburg, Germany Background. In humans and nonhuman primates, Ebola virus causes a virulent viral hemorrhagic fever for which no licensed vaccines or therapeutic drugs exist. In the present study, we used the mouse model for Ebola hemorrhagic fever to assess the safety and efficacy of a vaccine based on a live attenuated vesicular stomatitis virus expressing the Zaire ebolavirus (ZEBOV) glycoprotein. Methods. Healthy mice were given the vaccine in various doses, decreasing from to 2 plaque-forming 4 2 10 units (pfu), with both systemic and mucosal vaccination routes used. Mice were challenged with to lethal 3 6 10 10 doses of mouse-adapted ZEBOV. Severely immunocompromised mice were injected with pfu, which is 10 5 2 10 times greater than the immunization dose normally used, to test vaccine safety. Results. Two plaque-forming units of the vaccine protected against lethal challenge, and mucosal immunization was found to be as protective as systemic injection. The replicating vaccine was never detected in the immunized animals, nor were there clinical signs after immunization. Immunization of severely immunocompromised mice with 200,000 pfu of vaccine resulted in no clinical symptoms. Conclusions. Our data suggest that the vaccine is highly potent and safe and that it very rapidly induces “sterile” immunity in mice. The potential for mucosal delivery, if confirmed in nonhuman primates, makes it an excellent candidate for mass immunization during outbreaks or in the event of intentional release. Zaire ebolavirus (ZEBOV) causes highly virulent hem- orrhagic fever in humans, with a fatality rate approach- ing 90% [1]. ZEBOV is listed on the Centers for Disease Control and Prevention’s “A” list as a pathogen that can be considered to be a bioterrorism threat [2]. No antiviral therapy or licensed vaccine exists for this dev- astating disease [3]. It has been shown that Ebola virus Potential conflicts of interest: none reported. Presented in part: XIIth International Congress of Virology, 27 July to 1 August 2002, Paris, France (abstract V-291); XIIIth International Congress of Virology, 23– 28 July 2005, San Francisco, California (abstract 89-V); Filoviruses: Recent Advances and Future Challenges, International Centre for Infectious Diseases Symposium, Winnipeg, Manitoba, Canada, 17–19 September 2006. Financial support: Public Health Agency of Canada; Canadian Institute of Health Research (CIHR; grant MOP 43921 to H.F.). Supplement sponsorship is detailed in the Acknowledgments. Reprints or correspondence: Dr. Steven M. Jones, 1015 Arlington St., Winnipeg, Manitoba, Canada R3E 3R2 ([email protected]). The Journal of Infectious Diseases 2007; 196:S404–12 2007 by the Infectious Diseases Society of America. All rights reserved. 0022-1899/2007/19610S2-0040$15.00 DOI: 10.1086/520591 (EBOV) is infectious after oral and ocular exposure of nonhuman primates (NHPs) [4], as well as after aerosol exposure [5]. Therefore, an effective ZEBOV vaccine should be capable of rapidly inducing both systemic and mucosal immunity, because the intentional release of ZEBOV would probably result in mucosal infection by small-particle aerosol dispersion [3, 6]. EBOV vaccine research has been reviewed extensively [1, 7–9]; in general, many of the approaches reviewed resulted in protective efficacy in rodent models but failed to protect primates [8]. Studies of immunization of NHPs by use of a DNA prime/adenovirus boost approach [10] and, subsequently, a single dose of ad- enovirus [11] were highly successful. In both of these studies [10, 11], the genes for the ZEBOV glycoprotein (GP) and nucleoprotein (NP) were expressed as the virus-specific immunogens. There are, however, limi- tations associated with these approaches. First, the ini- tial vaccination strategy involved a very lengthy DNA prime/adenovirus boost protocol [10], and, although at Aston University on September 6, 2014 http://jid.oxfordjournals.org/ Downloaded from

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S404 • JID 2007:196 (Suppl 2) • Jones et al.

S U P P L E M E N T A R T I C L E

Assessment of a Vesicular Stomatitis Virus–BasedVaccine by Use of the Mouse Model of Ebola VirusHemorrhagic Fever

Steven M. Jones,1,2,3 Ute Stroher,1,3 Lisa Fernando,1 Xianggou Qiu,1 Judie Alimonti,1 Pasquale Melito,1 Mike Bray,4

Hans-Dieter Klenk,5 and Heinz Feldmann1,3

1Special Pathogens Program, National Microbiology Laboratory, Public Health Agency of Canada, 2Department of Immunology and 3Departmentof Medical Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada; 4Biodefense Clinical Research Branch, National Institutesof Health, Bethesda, Maryland; 5Institute of Virology, Philipps-University, Marburg, Germany

Background. In humans and nonhuman primates, Ebola virus causes a virulent viral hemorrhagic fever forwhich no licensed vaccines or therapeutic drugs exist. In the present study, we used the mouse model for Ebolahemorrhagic fever to assess the safety and efficacy of a vaccine based on a live attenuated vesicular stomatitis virusexpressing the Zaire ebolavirus (ZEBOV) glycoprotein.

Methods. Healthy mice were given the vaccine in various doses, decreasing from to 2 plaque-forming42 � 10units (pfu), with both systemic and mucosal vaccination routes used. Mice were challenged with to lethal3 610 10doses of mouse-adapted ZEBOV. Severely immunocompromised mice were injected with pfu, which is 1052 � 10times greater than the immunization dose normally used, to test vaccine safety.

Results. Two plaque-forming units of the vaccine protected against lethal challenge, and mucosal immunizationwas found to be as protective as systemic injection. The replicating vaccine was never detected in the immunizedanimals, nor were there clinical signs after immunization. Immunization of severely immunocompromised micewith 200,000 pfu of vaccine resulted in no clinical symptoms.

Conclusions. Our data suggest that the vaccine is highly potent and safe and that it very rapidly induces“sterile” immunity in mice. The potential for mucosal delivery, if confirmed in nonhuman primates, makes it anexcellent candidate for mass immunization during outbreaks or in the event of intentional release.

Zaire ebolavirus (ZEBOV) causes highly virulent hem-

orrhagic fever in humans, with a fatality rate approach-

ing 90% [1]. ZEBOV is listed on the Centers for Disease

Control and Prevention’s “A” list as a pathogen that

can be considered to be a bioterrorism threat [2]. No

antiviral therapy or licensed vaccine exists for this dev-

astating disease [3]. It has been shown that Ebola virus

Potential conflicts of interest: none reported.Presented in part: XIIth International Congress of Virology, 27 July to 1 August

2002, Paris, France (abstract V-291); XIIIth International Congress of Virology, 23–28 July 2005, San Francisco, California (abstract 89-V); Filoviruses: RecentAdvances and Future Challenges, International Centre for Infectious DiseasesSymposium, Winnipeg, Manitoba, Canada, 17–19 September 2006.

Financial support: Public Health Agency of Canada; Canadian Institute of HealthResearch (CIHR; grant MOP 43921 to H.F.). Supplement sponsorship is detailed inthe Acknowledgments.

Reprints or correspondence: Dr. Steven M. Jones, 1015 Arlington St., Winnipeg,Manitoba, Canada R3E 3R2 ([email protected]).

The Journal of Infectious Diseases 2007; 196:S404–12� 2007 by the Infectious Diseases Society of America. All rights reserved.0022-1899/2007/19610S2-0040$15.00DOI: 10.1086/520591

(EBOV) is infectious after oral and ocular exposure of

nonhuman primates (NHPs) [4], as well as after aerosol

exposure [5]. Therefore, an effective ZEBOV vaccine

should be capable of rapidly inducing both systemic

and mucosal immunity, because the intentional release

of ZEBOV would probably result in mucosal infection

by small-particle aerosol dispersion [3, 6].

EBOV vaccine research has been reviewed extensively

[1, 7–9]; in general, many of the approaches reviewed

resulted in protective efficacy in rodent models but

failed to protect primates [8]. Studies of immunization

of NHPs by use of a DNA prime/adenovirus boost

approach [10] and, subsequently, a single dose of ad-

enovirus [11] were highly successful. In both of these

studies [10, 11], the genes for the ZEBOV glycoprotein

(GP) and nucleoprotein (NP) were expressed as the

virus-specific immunogens. There are, however, limi-

tations associated with these approaches. First, the ini-

tial vaccination strategy involved a very lengthy DNA

prime/adenovirus boost protocol [10], and, although

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VSV Ebola Vaccine Trials in Mice • JID 2007:196 (Suppl 2) • S405

the accelerated vaccination strategy [11] provided protection

within 28 days after immunization with a single dose of ade-

novirus vaccine, there remain concerns related to the significant

rate of preexisting immunity to adenoviruses in humans (up

to 40%–50%) [12–14] and the high dose of recombinant ad-

enovirus required for protection [15–17]. Furthermore, the im-

munization route for both of these vaccines was intramuscular

injection, which is less favorable for rapid mass vaccination

than either oral or intranasal delivery.

Recombinant vesicular stomatitis virus (VSV) has potential

as a vaccine vector because it is capable of high-titer growth

in vitro and is relatively easy to genetically manipulate. Fur-

thermore, it induces strong cellular and humoral immune re-

sponses in vivo [18, 19] and has the potential to elicit both

mucosal and systemic immunity [20]. There is a low incidence

of mostly asymptomatic or mild human infections leading to

a negligible level of preexisting immunity in the world popu-

lation [21]. The efficacy of recombinant VSVs as vaccine vectors

against several infectious agents has been demonstrated else-

where [21–25], including in a study demonstrating the pro-

tective efficacy of live attenuated VSV expressing the HIV en-

velope and core proteins in rhesus monkeys challenged with a

highly pathogenic simian HIV (SHIV89.6P) [26].

We previously described the generation of recombinant live

attenuated vaccines for Ebola, Marburg, and Lassa viruses caus-

ing hemorrhagic fever [27], and we showed that these vaccines

can protect NHPs from otherwise lethal challenge [28–30]. Very

recently, we showed that postexposure administration of the

MARV vaccine protects all NHPs against lethal infection [31]

and that 50% of animals survive when the EBOV vaccine is

given after challenge [32].

Although the NHP is indeed the reference standard model for

efficacy testing of EBOV vaccines, here we demonstrate that,

contrary to previous reports, the mouse can be extremely useful

as an alternative model for testing candidate vaccines for EBOV.

We demonstrated that protection in mice [33] required only a

single dose of as little as 2 pfu. Immunization just 7 days before

an otherwise lethal challenge conferred complete specific pro-

tection, and the vaccine did not result in disease, even in severely

immunocompromised NOD-SCID mice. Importantly, protec-

tion could also be achieved for the first time by mucosal im-

munization, which makes the vaccine vector easy to administer

and, therefore, a promising candidate for mass immunization.

MATERIALS AND METHODS

Vaccine vectors and viruses. The recombinant VSV express-

ing the GP of Zaire EBOV Mayinga (ZEBOV-May) was gen-

erated as recently described elsewhere [27], by use of the in-

fectious clone for VSV Indiana serotype [27]. In brief, the

appropriate open reading frame for the ZEBOV GP was gen-

erated using polymerase chain reaction (PCR), cloned into the

VSV genomic vectors lacking the VSV GP gene, sequence con-

firmed, and rescued using the method described elsewhere [27,

34]. The virus was designated “VSVDG/ZEBOVGP.” Mice were

challenged using a mouse-adapted (MA) variant (9 passages in

mice) derived from ZEBOV-May (MA-ZEBOV-May) [33].

Animal studies. Female BALB/c mice, 5–6 weeks of age, were

purchased from Charles Rivers (Quebec, Canada). Mice (in

groups of 5) were routinely immunized intraperitoneally at 2

sites with pfu of VSVDG/ZEBOVGP (total dose, pfu).4 410 2 � 10

Control animals were immunized with either recombinant wild-

type VSV (rVSVwt) (given in the same dose and by the same

route) or PBS. Mice were challenged intraperitoneally 28 days

after immunization with LD50 of MA-ZEBOV-May.310

The vaccine dose remained constant, independent of the im-

munization route used; however, the volume administered was

altered as follows: for the intranasal route, 20 mL was split between

both nostrils; for the intramuscular route, 100 mL was split be-

tween 2 sites; for the subcutaneous route, 100 mL was admin-

istered in the scruff of the neck; and, for the oral route, 100 mL

was administered by oral gavage (number of animals/group, 10).

To determine the minimum effective vaccine dose stock, VSVDG/

ZEBOVGP was repeatedly diluted 10-fold in unsupplemented

Dulbecco’s modified Eagle medium to attain desired immuni-

zation doses decreasing from to 2 pfu.42 � 10

All animals were weighed for the first 11 days after vaccination

and challenge and were kept 3 times longer than the length of

time to the death of the last control animal (i.e., at least 28 days),

to confirm that protection was complete and that death was not

merely delayed by immunization. NOD-SCID mice ( ) thatn p 5

were 6–8 weeks of age were immunized intraperitoneally at 2

sites with pfu (total dose, pfu) of VSVDG/ZEBOVGP5 510 2 � 10

or VSVDG/MARVGP or were left untreated. Because of the need

for sterile handling conditions, NOD-SCID mice were not

weighed every day; however, they were carefully observed every

day for 14 days and had their clinical symptoms scored.

All animal work was performed according to “Animal Use

Document H-01-001 and H-04-019,” approved by the Animal

Care Committee, Canadian Science Centre for Human and An-

imal Health (Winnipeg, Manitoba), according to the guidelines

of the Canadian Council on Animal Care (Ottawa, Ontario), and

was performed in the biosafety level 4 facility at the National

Microbiology Laboratory.

Virus detection. RNA was isolated from blood and organs

by use of the appropriate RNA isolation kits from Qiagen (Mis-

sissauga, Ontario, Canada). For the detection of VSV, we used

a reverse-transcription (RT)–PCR assay targeting the matrix

gene (nt 2355–2661; GenBank accession no. NC_001560).

ZEBOV RNA was detected using a primer pair targeting the L

gene (nt 13349–13622; GenBank accession no. AF 272001). Live

virus was titrated by means of a TCID50 assay performed on

Vero E6 cells obtained from blood and select tissue samples.

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Figure 1. Protective efficacy of 1-dose vs. 2-dose immunization regi-mens. Mice were immunized intraperitoneally with either 2 doses (solidline with triangles) of pfu of VSVDG/ZEBOVGP (recombinant42 � 10vesicular stomatitis virus in which the native glycoprotein [GP] gene hasbeen deleted and functionally replaced with the Zaire ebolavirus Mayinga[ZEBOV-May] GP gene) given 28 days apart or a single dose (dashed linewith triangles) or were mock immunized with recombinant wild-type ve-sicular stomatitis virus (rVSVwt) (dashed line with squares) and challengedwith LD50 of mouse-adapted ZEBOV Mayinga 28 days after the final310immunization. Weight was recorded for 11 days after immunization andchallenge, and surviving animals were monitored for at least 3 timeslonger than the length of time to the death of the last control animal.

Immune responses. IgG and IgM antibodies against ZE-

BOV were quantified using a modification of an ELISA [35]

using purified virus-like particles [36] as an antigen and horse-

radish peroxidase–conjugated, isotype-specific antibodies to de-

tect mouse antibodies to EBOV. A standard neutralization assay

(PRNT80) was performed as previously described, by measuring

VSVDG/ZEBOVGP plaque reduction in a constant virus:serum

dilution format in a modification of a protocol described else-

where [37, 38]. For this assay, a standard amount of VSVDG/

ZEBOVGP (∼100 pfu) was incubated with serial 2-fold dilu-

tions of the serum sample for 60 min. The mixture was used

to inoculate Vero E6 cells for 60 min. Cells were overlayed with

an agar medium and incubated for 3 days, and plaques were

counted for 48 h after crystal violet staining.

T cell depletion. Mice were immunized intraperitoneally

once with the standard dose of the VSVDG/ZEBOVGP vaccine.

Challenge with LD50 of MA-ZEBOV-May was performed310

28 days after immunization. Mice were injected on days 3 and

1 before challenge and subsequently every 3 days with 100 mL

(0.5 mg/dose) of anti-CD8 antibody (YTS 169.4), in a manner

similar to a protocol described elsewhere [39]. Depletion of

CD8 T cells was confirmed by flow cytometry performed using

antibody that binds to an epitope on CD8 T cells different from

the epitope used for depletion.

Passive transfer of immune serum. On days 1 and 28, mice

were immunized intraperitoneally with a standard dose of

VSVDG/ZEBOVGP vaccine or rVSVwt (in the same dose and

by the same route), and age-matched vaccine-naive animals

were left untreated. Sixty days after the final immunization, the

mice were killed by exposure to terminal anesthesia, and blood

was removed by cardiac puncture. Serum samples obtained

from the 3 groups of mice were injected intraperitoneally into

vaccine-naive mice (0.5 mL/animal) 24 h before challenge with

LD50 of MA-ZEBOV-May.310

RESULTS

Provision of complete protection against EBOV challenge with

106 LD50 given in a single-dose vaccination. For initial effi-

cacy testing, groups of 5 mice were immunized either once (on

day 0) or twice (on days 0 and 14) with rVSVwt or VSVDG/

ZEBOVGP. Mice were challenged 28 days after initial immu-

nization with LD50 of MA-ZEBOV-May. Untreated control310

animals and animals immunized with rVSVwt rapidly lost

weight and died. However, mice immunized with VSVDG/

ZEBOVGP were completely protected against MA-ZEBOV-May

infection, regardless of the number of immunizing doses re-

ceived (figure 1).

To define the absolute upper limit of protection by increasing

the challenge dose in 10-fold increments, mice were immunized

with either rVSVwt or VSVDG/ZEBOVGP on day 0 and were

challenged with MA-ZEBOV-May in the range of to3 610 10

LD50 28 days later. All control animals died after having lost

up to 18% of their body weight in 6 days. In contrast, the

animals immunized with VSVDG/ZEBOVGP were all protected

against MA-ZEBOV-May infection, regardless of the challenge

dose (figure 2A).

Because EBOV is transmitted by close contact and blood

contamination, it was essential to demonstrate that immuni-

zation prevents the establishment of viremia as well as severe

disease or death. Mice were immunized with either rVSVwt or

VSVDG/ZEBOVGP and were challenged 28 days later. Each

day after challenge, 3 mice from each treatment group were

killed, and blood samples were obtained for EBOV isolation

and detection of EBOV-specific RNA by RT-PCR. Animals im-

munized with rVSVwt remained without viremia for 2 days

after challenge but then rapidly developed viremia, which

reached a maximum level of TCID50 before death (6 days710

after infection). At no time after challenge did the mice im-

munized with VSVDG/ZEBOVGP have detectable viremia (fig-

ure 2B).

Protection against EBOV infection provided by very low

doses of vaccine and in the absence of detectable vaccine

replication. Although efficacy is an important concern after

immunization, the safety of the vaccine and the potential for

serious side effects are as important. In general, lower doses of

vaccine are preferred. We determined the minimum dose of

VSVDG/ZEBOVGP required for complete protection of mice.

Animals were immunized with 10-fold dilutions of the vaccine,

starting at pfu and decreasing to 2 pfu, and they un-42 � 10

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Figure 2. Protective efficacy and induction of “sterile” immunity of immunization with a single dose of VSVDG/ZEBOVGP (recombinant vesicularstomatitis virus in which the native glycoprotein [GP] gene has been deleted and functionally replaced with the Zaire ebolavirus Mayinga [ZEBOV-May] GP gene). Mice were immunized intraperitoneally with pfu of VSVDG/ZEBOVGP and were challenged 28 days thereafter with increasing42 � 10doses of mouse-adapted ZEBOV-May (MA-ZEBOV-May). Weight was recorded for a minimum of 11 days after challenge, and surviving animals weremonitored for at least 3 times longer than the length of time to the death of the last control animal. A, Vaccine resulted in protection against highchallenge doses. Animals mock immunized with recombinant wild-type vesicular stomatitis virus (rVSVwt) were not protected from lethal challengewith LD50 (group 1). They rapidly lost weight and died by day 6 after infection with MA-ZEBOV-May. In contrast, mice immunized with VSVDG/210ZEBOVGP and subsequently challenged with increasing doses of MA-ZEBOV-May (group 2, LD50; group 3, LD50; group 4, LD50; and group3 4 510 10 105, LD50 ) did not lose any weight, and all survived. Each group consisted of 5 animals. B, Vaccination induced sterile immunity. Each day for 6610days after challenge, 3 animals from each treatment group were killed to determine levels of viremia. In mice immunized with VSVDG/ZEBOVGP(black bars), no viremia was detectable. In contrast, in rVSVwt-immunized controls (gray bars), infectious MA-ZEBOV-May was detectable by day 3after challenge, and viremia rapidly increased until day 6 (3 animals/group each day). The limit of detection was 2.3 log10 TCID50 and is denoted bythe dotted line. Please note that reverse transcription–polymerase chain reaction also failed to detect MA-ZEBOV-May in blood and tissues samplesobtained from the immunized mice (data not shown).

derwent challenge with LD50 28 days later. All immunized310

animals were protected from the lethal challenge, even at an

ultra-low immunization dose (figure 3A). Because replication—

not only the initial dose—may affect safety, we attempted to

determine the extent of VSV replication in immunized animals.

Immunized and control mice were culled each day between

days 1 and 7 and then every 7 days after immunization until

day 28. Blood, spleen, liver, and kidney samples were obtained

for viral nucleic acid detection and virus isolation. We were

unable to detect virus by means of either RT-PCR or virus

isolation at any time after immunization with either rVSVwt

or VSVDG/ZEBOVGP (data not shown).

Provision of complete protection against systemic challenge

by means of mucosal immunization. The intraperitoneal

route of immunization is safe and relatively easy to perform

under experimental conditions with rodents in high contain-

ment, but it is not a practical route for immunization of hu-

mans. To investigate the effect of different immunization routes

on efficacy, mice were immunized with pfu of VSVDG/42 � 10

ZEBOVGP or rVSVwt by the intramuscular and intranasal

routes. After 28 days, groups of animals were challenged with

10-fold increasing doses of MA-ZEBOV-May (between and310

LD50). All mice were completely protected, with no evidence610

of differences in clinical symptoms or weight changes noted in

any of the mice immunized with VSVDG/ZEBOVGP (figure

3B). All vaccine-naive control or rVSVwt control immunized

mice died within 6 days. Intranasal immunization significantly

increases the potential for rapid mass immunization. The oral

route is the only route with greater potential than the intranasal

route for mass vaccination of susceptible populations. Mice

were immunized with the VSVDG/ZEBOVGP or rVSVwt vac-

cine twice (on days 0 and 28), and animals were challenged 28

days later with MA-ZEBOV-May. All control mice died within

4 days of infection, whereas the immunized animals were pro-

tected (figure 3C).

Cellular and humoral immunity. When human volunteers

cannot ethically be used to test effectiveness of vaccination

strategies, as in the case of EBOV infection, it is possible, under

US Food and Drug Administration regulations, to use animal

models. This requires that immune correlates of protection be

determined in the animal model and that similar immune re-

sponses can be detected in human volunteers in phase 1 or

phase 2 clinical trials. The mechanism for protection against

EBOV infection has not been definitively determined for any

of the animal models; however, there is evidence suggesting

that different pathological mechanisms are important in ro-

dents and primates [8, 40, 41]. To determine the role of an-

tibody and cytotoxic T cell responses in mice, we performed

T cell depletion and serum transfer studies (table 1). Complete

depletion of cytotoxic T cells by use of a specific antibody

against CD8 T cells did not compromise protection, indicating

that, in immunized mice, CD8+ cytotoxic T cells are not an

absolute requirement for protection. Passive transfer of im-

mune serum 60 days after immunization resulted in 80% pro-

tection, but transfer of vaccine-naive or rVSVwt immune serum

failed to provide protection from lethal challenge with MA-

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Figure 3. Protective efficacy depending on dose and route of immunization. Animals were immunized intraperitoneally with decreasing doses ofthe vaccine, as well as by different routes. The challenge was performed with increasing doses of mouse-adapted Zaire ebolavirus Mayinga (MA-ZEBOV-May) 28 days thereafter. A, Ultra-low-dose immunization did not compromise protective efficacy. Mice immunized with recombinant wild-typevesicular stomatitis virus (rVSVwt) (group 1) lost weight and died by day 6 after challenge. In contrast, mice immunized with VSVDG/ZEBOVGP(recombinant vesicular stomatitis virus in which the native glycoprotein [GP] gene has been deleted and functionally replaced with the Zaire ebolavirusMayinga GP gene) in decreasing doses (group 2, pfu; group 3, pfu; group 4, pfu; group 5, pfu; group 6, pfu)4 3 2 1 02 � 10 2 � 10 2 � 10 2 � 10 2 � 10all survived infection with no evidence of clinical disease or weight loss after challenge. Each group consisted of 5 animals, and challenge wasperformed with LD50 of MA-ZEBOV-May. B, Mucosal immunization was as effective as systemic injection. Mice (5 animals/group) were immunized310with a dose of pfu of rVSVwt (group 2) or VSVDG/ZEBOVGP by intraperitoneal injection (group 3), intramuscular injection (group 4), or intranasal42 � 10instillation (group 5). Vaccine-naive controls were left untreated (group 1). Subsequently, mice were challenged with LD50 of MA-ZEBOV-May 28610days after immunization. All rVSVwt-immunized and vaccine-naive control mice displayed clinical symptoms, lost weight, and died. In contrast, VSVDG/ZEBOVGP-immunized mice did not show any clinical symptoms or weight loss. All animals survived, irrespective of challenge dose or route ofimmunization. C, Oral immunization is also effective. Mice (10 animals/group) were immunized with 2 doses of pfu of rVSVwt (group 1) or42 � 10VSVDG/ZEBOVGP (group 2) by oral gavage on days 0 and 28. Subsequently, mice were challenged with LD50 of MA-ZEBOV-May 28 days after310immunization. All rVSVwt-immunized mice displayed clinical symptoms, lost weight, and died within 4 days. In contrast, VSVDG/ZEBOVGP-immunizedmice did not show any clinical symptoms or weight loss.

ZEBOV-May (table 1). Antibody is sufficient to protect mice

from infection, a finding that concurs with results published

elsewhere [42, 43].

After showing that passive transfer of immune serum results

in protection (table 1), we further characterized the humoral

immune response. We used a quantitative ELISA to determine

the concentration of ZEBOV GP–specific antibodies in the im-

munized mice and a plaque reduction neutralization test

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Table 1. Correlates of immunity in BALB/c mice (5 animals/group).

Survivalrate

at day 28,a

%Time to death,

mean days

CD8 T cell depletion 100 NA

No T cell depletion 100 NA

Passive transfer

Of VSVDG/ZEBOVGP immune serum 80 10b

Of rVSVwt immune serum 0 5.0

Of vaccine-naive serum 0 5.33

NOTE. NA, not applicable; rVSVwt, recombinant wild-type vesicular sto-matitis virus; VSVDG/ZEBOVGP, recombinant vesicular stomatitis virus in whichthe native glycoprotein (GP) gene has been deleted and functionally replacedwith the Zaire ebolavirus Mayinga GP gene.

a After challenge.b .n p 1

(PRNT80) to detect neutralizing antibodies. Neutralizingantibody

titers detected by PRNT80 were equivocal, despite the clear pro-

tection afforded by passive immunization (table 1). ELISA data

(table 2) showed that (1) within 7 days of immunization, the

mean concentration of GP-specific IgM was 1.3 mg/mL, whereas

that of IgG2a was 152 ng/mL; and (2) by the time of challenge

in most studies (28 days after immunization), IgG2a levels had

increased to a mean of 781 ng/mL. However, IgM levels had

decreased to 66 ng/mL. The levels of the other IgG isotypes

remained negligible throughout the study (data not shown).

Rapid immunity and long-term protection. The induction

of rapid immunity from challenge is essential if the VSVDG/

ZEBOVGP vaccine vector is to be useful in natural outbreaks

or in the event of an intentional release. To determine the

minimum time required for protective immunity to develop,

mice were immunized at 7-day intervals starting 28 days before

challenge. All VSVDG/ZEBOVGP-immunized animals treated

between days 28 and 7 before challenge were completely pro-

tected from lethal challenge (figure 4A). In contrast, all of the

rVSVwt control animals died of the infection (figure 4B).

Rapid immunity is an important characteristic of an effective

vaccine, but the longevity of protection is also very important.

To test this, we immunized groups of mice with a single dose

of pfu of rVSVwt or VSVDG/ZEBOVGP vaccine. The42 � 10

mice were challenged 3, 6, or 9 months later, along with age-

matched vaccine-naive controls. At each time point, all

VSVDG/ZEBOVGP-vaccinated animals were completely pro-

tected (data not shown).

Infection of NOD-SCID mice with VSV vaccines. Im-

munization of the NOD-SCID mice with 10 times the normal

dose of vaccine given to healthy mice appeared to result in a

self-limiting, asymptomatic infection. At no time were clinical

signs, such as ruffling, hunching, or lack of mobility, observed,

and the infected animals were indistinguishable from the un-

treated control mice (data not shown).

DISCUSSION

In the present study, we utilized the mouse model for ZEBOV

infection to characterize and evaluate the suitability of a re-

combinant live attenuated vaccine candidate (i.e., VSVDG/

ZEBOVGP). The mouse was the only feasible animal model

for such extensive studies [33]. When used appropriately, the

mouse model provides a very useful tool for screening vaccine

candidates and is the only practical tool available for fully ex-

ploring the limits of protection afforded by a vaccine strategy.

Indeed, virtually all observations made in the mouse model

have been confirmed in subsequent studies of NHPs [28, 29,

31, 32].

Animals can be protected from a challenge dose of 1 million

LD50, which is the highest dose ever used in a study of protection

from ZEBOV. This protection was noted even when the vaccine

was delivered mucosally. Importantly, oral and intranasal im-

munization with VSVDG/ZEBOVGP provided protection from

a systemic infection with ZEBOV. This is significant for 2 rea-

sons. First, these are the routes of choice for mass vaccination.

Second, these routes may stimulate mucosal as well as systemic

immunity. Any deliberate attempt to release ZEBOV is likely

to involve the generation of aerosols or contamination of food

supplies. After such an event, protection at the mucosal surfaces

would be important in reducing the dose of virus entering the

body. Because natural infections may also occur by the oral

route or by ocular inoculation, mucosal immunity could be

important in containing transmission in both natural and de-

liberate outbreaks.

We have shown that CD8+ T cells are not required for the

protection of mice and that passive transfer of serum results

in significant specific protection. Further studies involving both

the mouse and NHP models will be required to identify possible

immune correlates of protection. Our data on CD8 T cell de-

pletion contrasts with recently published data, which showed

that (1) after subcutaneous injection of mice with MA-ZEBOV-

May, CD8 T cells were essential for protection, and (2) under

certain experimental conditions, they are sufficient for protec-

tion [44]. ZEBOV infection is an extremely acute disease in

mice, with rapid progression to death (mode time to death, 6

days). CD8 T cell responses generally require 7–12 days to reach

maximum efficiency, and early induction of potent antibody

responses are likely to be essential for effective vaccination.

Despite equivocal in vitro neutralization data, passive transfer

of immune serum to vaccine-naive mice provides protection,

indicating that either neutralizing antibodies are generated or

alternative antibody dependent immune mechanisms are more

important in protection; antibody responses are required and

sufficient for protection. We purposely maintained the actively

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Table 2. IgM and IgG2a antibody response kinetics after immunization of 6 mice.

Antibody, day afterimmunization

Serum antibody level, ng/mL

Mouse 1 Mouse 2 Mouse 3 Mouse 4 Mouse 5 Mouse 6 Mean � SD

IgM7 1321.4 1355.3 1567.5 1181.3 990.4 … 1238.2 � 214.214 341.4 448.9 299.0 387.4 403.7 … 376.1 � 57.828 23.8 60.3 39.8 167.3 15.6 93.9 66.8 � 56.7

IgG2a7 162.9 151.1 140.0 175.0 133.2 … 152.5 � 16.914 336.5 486.5 1059.6 1404.9 691.7 … 795.9 � 435.328 613.0 958.4 671.2 1154.1 509.0 780.8 781.1 � 238.7

Figure 4. Time required to develop protective immunity. Groups of 5 mice were immunized with either recombinant wild-type vesicular stomatitisvirus (rVSVwt) (dotted lines) or VSVDG/ZEBOVGP (recombinant vesicular stomatitis virus in which the native glycoprotein [GP] gene has been deletedand functionally replaced with the Zaire ebolavirus Mayinga [ZEBOV-May] GP gene) (solid lines) on the following days before challenge with mouse-adapted ZEBOV-May (MA-ZEBOV-May) ( LD50): day 28 (A and B; groups 2 and 6), day 21 (A and B; groups 3 and 7), day 14 (A and B; groups 4310and 8), and day 7 (A and B; groups 5 and 9). Vaccine-naive mice (group 1) were not treated before challenge. All mice immunized with the VSVDG/ZEBOVGP vaccine were completely and specifically protected when immunized as soon as 7 days before challenge (A and B).

immunized mice for 60 days after immunization, to preclude

the transfer of nonspecific immune mediators, resulting from

infection with VSV, in serum.

There has been significant debate over the safety of the VSV

live attenuated recombinant viral platform, compared with the

adenovirus alternative. This debate has focused mainly on the

possibility that VSV infection will lead to significant patholog-

ical findings in immunocompromised individuals. To assess this

possibility, we used the most severely immunocompromised

animal model. The NOD-SCID mouse has no functional T or

B cell responses and is considered to have a nonleaky phenotype

(i.e., T cell and B cell functions are not partially restored over

time, as is seen with some SCID mouse strains). These mice

completely controlled the VSV infection, even when given 10

times the normal vaccine dose. These vaccines apparently are

safe as well as efficacious. In a recent study, toxicological as-

sessment of VSV-based HIV vaccine vectors showed that they

were indeed attenuated in NHPs, and this finding should clear

the way for phase 1 clinical trials in humans [45].

Furthermore, the adenovirus-based ZEBOV vaccine required

the delivery of – particles for effective immunization of10 1210 10

mice and NHPs, respectively [11]. In contrast, mice can be

protected with doses as low as 2 pfu, and NHPs can be protected

with pfu [29] with the VSVDG/ZEBOVGP. The safety of710

adenovirus vectors has been called into question by the death

of 1 person from systemic inflammatory response syndrome

in a gene therapy trial. The steep toxicity curve for replication-

defective adenovirus vectors and the substantial subject-to-sub-

ject variation in host responses to systemically administered

vectors are important [46]. We believe that reducing the initial

dose of vaccine will help to minimize possible adverse effects.

ZEBOV causes the most virulent form of viral hemorrhagic

fever, and control of an epidemic resulting from a maleficent

act or a natural outbreak would be greatly assisted by a vaccine

capable of rapidly inducing strong immunity and an application

route that allows for mass vaccination. The DNA/adenovirus

vaccine and a single-dose adenovirus vaccine delivered the GP

and NP from ZEBOV and induced protective immunity in the

cynomolgus macaque model [10, 11]. There are limitations with

these strategies—in particular, with the length of the immu-

nization scheme associated with the initial DNA prime/ade-

novirus boost protocol [10] and the fact that the preexisting

immunity associated with the accelerated vaccine [11] might

limit the utility of these approaches. The VSV platform has

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advantages, compared with the existing successful vaccination

approaches, including the very low incidence of immunity to

VSV in human populations; the lack of the VSV GP, which is

the main target for preexisting neutralizing antibodies; the ease

of vaccine production; the short time to immunity; and the

potential for mucosal delivery and postexposure treatment.

Last, the potential for use as a mucosal vaccine, particularly by

the oral route of administration, may offer some hope of pro-

tecting the great apes, which are currently under threat from

ZEBOV [47, 48].

Acknowledgments

We thank Daryl Dick, Friederike Feldmann, Allen Grolla, and AndreaPaille for technical assistance and assistance with animal care. We also thankAllison Groseth and Toni Marchione for assistance in preparation of themanuscript. We are grateful to Thomas Geisbert (US Army Medical Re-search Institute of Infectious Diseases, Fort Detrick, Frederick, Maryland),for support and discussions; John Rose (Yale University, New Haven, Con-necticut), for kindly providing the vesicular stomatitis virus infectious clonesystem; and Kent HayGlass (Immunology Department, University of Man-itoba, Winnipeg, Manitoba, Canada), for kindly providing the antibodyused for T cell depletion.

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 Institutesof 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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