Perspective
Evaluation in Nonhuman
Primates of Vaccines against Ebola Virus
Thomas W. Geisbert,* Peter Pushko,* Kevin Anderson,* Jonathan
Smith,* Kelly J. Davis,* and Peter B. Jahrling*
*U.S. Army Medical Research Institute of Infectious Diseases,
Fort Detrick, Maryland, USA
Ebola virus
(EBOV) causes acute hemorrhagic fever that is fatal in up to 90%
of cases in both humans and nonhuman primates. No vaccines or
treatments are available for human use. We evaluated the effects
in nonhuman primates of vaccine strategies that had protected
mice or guinea pigs from lethal EBOV infection. The following
immunogens were used: RNA replicon particles derived from an attenuated
strain of Venezuelan equine encephalitis virus (VEEV) expressing
EBOV glycoprotein and nucleoprotein; recombinant Vaccinia virus
expressing EBOV glycoprotein; liposomes containing lipid A and
inactivated EBOV; and a concentrated, inactivated whole-virion
preparation. None of these strategies successfully protected nonhuman
primates from robust challenge with EBOV. The disease observed
in primates differed from that in rodents, suggesting that rodent
models of EBOV may not predict the efficacy of candidate vaccines
in primates and that protection of primates may require different
mechanisms.
Ebola virus (EBOV) and Marburg virus (MBGV), which make
up the family Filoviridae, cause severe hemorrhagic disease
in humans and nonhuman primates, killing up to 90% of those infected.
EBOV was first recognized in the former Zaire in 1976. Subsequently,
outbreaks have been documented in Sudan, Gabon, the former Zaire,
Côte d’Ivoire, and Uganda (1-3). In addition to
the African outbreaks, the species Reston Ebola virus, which
may be less pathogenic for humans, was isolated from cynomolgus
monkeys imported from the Philippines to the United States (4).
Although outbreaks of EBOV have been self-limiting, the lack of
an effective vaccine or therapy has raised public health concerns
about these emerging pathogens.
In early attempts to develop a vaccine against EBOV, guinea pigs
or nonhuman primates were vaccinated with formalin-fixed or heat-inactivated
virion preparations. Results from these studies were inconsistent:
Lupton et al. (5) partially protected guinea pigs
against EBOV, while Mikhailov et al. (6) achieved
complete protection of four of five hamadryad baboons by vaccinating
them with an inactivated EBOV vaccine. However, other studies suggested
that inactivated EBOV did not induce sufficient immunity to reliably
protect hamadryl baboons against a lethal challenge (7).
Conventional strategies of attenuating viruses for use as human
vaccines have not been pursued for EBOV because of concerns about
reversion to a wild-type form. However, the possibility of following
this strategy by using newly developed infectious clones of EBOV
may now be feasible (8).
Recent efforts have focused on the use of recombinant DNA techniques
to stimulate cytotoxic T-lymphocyte responses. Vaccinating guinea
pigs with plasmids against EBOV nucleoprotein (NP), soluble glycoprotein,
or glycoprotein (GP) elicited humoral and cellular immune responses
against these gene products but only partially protected them against
lethal challenge (9). However, results of this
study were difficult to interpret because all the guinea pigs were
killed 10 days after EBOV challenge, which is within the expected
survival time for untreated animals (8-14 days) (10).
In 2000, Sullivan et al. (11) reported protection
of cynomolgus monkeys from EBOV infection by injecting them with
naked-DNA GP, followed by an adenovirus-expressing GP booster. Results
of this study document the feasibility of vaccination against EBOV.
However, these results require confirmation and further evaluation,
as a low dose (6 PFU) was used for the challenge. Other studies
reported a protective effect of EBOV vaccination with a low infective
challenge dose (10 50% lethal doses [LD50]) (7);
however, all vaccinated animals in these dosing studies died after
receiving higher infective doses (100 and 1,000 LD50),
which may more accurately mimic natural or nosocomial exposures.
Our efforts to develop a vaccine against EBOV focused on several
potential vaccine candidates. First, we used Venezuelan equine
encephalitis virus (VEEV) replicon particles (VRP) expressing
EBOV genes known to protect guinea pigs and mice from EBOV disease
(10); VRP expressing MBGV genes also protected
guinea pigs and cynomolgus monkeys against MBGV (12).
Second, we used a recombinant Vaccinia virus (VACV) system
expressing EBOV GP and demonstrated that this vector protected guinea
pigs from EBOV hemorrhagic fever (13). A third
strategy used encapsulated, gamma-irradiated EBOV particles in liposomes
containing lipid A (14); and the fourth approach
evaluated vaccination with a concentrated, gamma-irradiated whole-virion
preparation. None of these approaches, which successfully protected
rodents from lethal infection, were protective for cynomolgus or
rhesus macaques challenged with EBOV.
Materials
and Methods
Cynomolgus macaques (Macaca fascicularis) or rhesus macaques
(M. mulatta) weighing 4 to 6 kg were used. For vaccine studies
with VEE replicons, EBOV GP or NP genes were introduced into the
VEEV RNA as described (10). Groups of three cynomolgus
macaques were vaccinated with VRP that expressed EBOV GP, EBOV NP,
a mixture of EBOV GP and EBOV NP, or a control antigen (influenza
hemagglutinin) that has no effect on EBOV immunity. Animals were
vaccinated by subcutaneous injection of 107 focus-forming
units of VRP in a total of 0.5 mL at one site. Vaccinations were
repeated 28 days after the first injection and 28 days after the
second.
In conducting research with animals, the investigators followed
the Guide for the Care and Use of Laboratory Animals prepared by
the Committee on Care and Use of Laboratory Animals of the Institute
of Laboratory Animal Resources, National Research Council (1996).
The animal facilities and animal care and use program of the U.S.
Army Medical Research Institute of Infectious Diseases are accredited
by the Association for Assessment and Accreditation of Laboratory
Animal Care International.
For vaccine studies using primates, we adapted the optimal immunization
regimens determined from the rodent studies. For the vaccine based
on recombinant VACV, the EBOV GP gene was inserted into a VACV transfer
vector plasmid, and recombinant VACV expressing EBOV GP were isolated
as reported (13). Three cynomolgus macaques were
injected subcutaneously with the EBOV GP-expressing VACV vector.
Injections were repeated at 28 and 53 days after the first
injection.
For vaccine studies with inactivated EBOV whole-virion preparation,
viral particles were concentrated from Vero cell culture fluids
by ultracentrifugation in a sucrose density gradient. The infectivity
titer of the preparation was approximately 8.0 log10
PFU/mL. The preparation was inactivated by exposure to 60Co
gamma rays (6 x 106 rads). The absence of residual infectivity
was proven by exhaustive testing for residual infectivity in assays
in Vero cells (15,16). Two cynomolgus monkeys
and two rhesus monkeys were injected subcutaneously with a 50-µg
dose of the gamma-irradiated virion preparation in RIBI adjuvant
(Corixa, Hamilton, MT). As a further check on complete viral inactivation,
blood samples taken from the monkeys 3 and 5 days after they received
the vaccine were free of infectious viremia. Injections were repeated
at days 7 and 35 after the initial injection.
For vaccine studies using a liposome formulation, three cynomolgus
monkeys were vaccinated with gamma-irradiated virus encapsulated
in liposomes containing lipid A, as described for previous studies
in mice (14). Animals received 1.0 mL of the liposome
preparation by intravenous injections that were repeated at 28 and
55 days after the initial vaccination. Four macaques (two cynomolgus
and two rhesus) served as unvaccinated controls for the VACV, gamma-inactivated
virion, and liposome studies.
Anti-EBOV neutralizing antibody titers were monitored by measuring
plaque reduction in a constant virus:serum dilution format (15).
All macaques received intramuscular injections in the leg with 1,000
PFU of the Zaire subtype of EBOV, which was isolated from a human
patient in 1995 (16). Blood was obtained from
all monkeys under Telazol anesthesia (Fort Dodge Laboratories, Fort
Dodge, IA) at 2- or 3-day intervals postinfection to determine infectious
viremia, neutralizing antibody titers, and standard hematologic
and clinical pathology parameters. All terminally ill monkeys were
killed and necropsied for pathologic examination. Virus infectivity
assays on plasma and tissue homogenates were done by forming plaques
on Vero cell monolayers as described (15,16).
Tissues were immersion fixed in 10% neutral-buffered formalin and
processed for histopathologic and immunohistochemical characteristics
as described (17-19). Replicate sections of spleen
were stained with phosphotungstic acid hematoxylin to demonstrate
polymerized fibrin. Sections of spleen from five EBOV-infected guinea
pigs and five mice from previous studies (20,21)
were similarly stained for polymerized fibrin. Portions of selected
tissues from 11 monkeys were also immersion fixed in 4% formaldehyde
and 1% glutaraldehyde and processed for transmission electron microscopy
according to conventional procedures (17-19).
Results
Serologic Response
Prechallenge EBOV neutralization titers were measured for the 26
nonhuman primates used in this study (Table 1).
Although all vaccinated animals seroconverted by immunoglobulin
G enzyme-linked immunosorbent assay, neutralizing antibody (PRNT50)
titers were very low. Only one macaque vaccinated with VRP-expressed
EBOV GP had detectable neutralizing antibody. The marginal PRNT
did not preclude challenge of the monkeys; however, in previous
studies, similar results were obtained when cynomolgus macaques
were vaccinated with the VRP expressing MBGV genes, yet the animals
were protected from lethal disease (12).
Challenge of Vaccinated
Monkeys with EBOV
All animals, including the four untreated macaques, were challenged
with 1,000 PFU of EBOV. Timing of challenge varied because of differences
in the optimal immunization regimens determined by preliminary testing
in rodents. VRP-vaccinated animals were challenged 49 days after
the third vaccine dose. At postchallenge day 3, all animals became
ill; two animals from each vaccination group (i.e., GP, NP, GP +
NP, influenza HA) died on day 6, and the remaining animals died
on day 7 (Table 2). VACV GP-inoculated macaques
were challenged 45 days after the third vaccine dose, EBOV liposome-vaccinated
animals 35 days after the third vaccine dose, and macaques vaccinated
with the gamma-irradiated whole-virion preparation 43 days after
the third vaccine dose. Again, all animals except one rhesus macaque,
which received the gamma-irradiated virion preparation, became ill
on the third day after challenge. Two cynomolgus macaques vaccinated
with the gamma-irradiated virion preparation, one VACV-GP animal,
and one untreated cynomolgus macaque died on postchallenge day 6
(Table 2). The two remaining VACV-GP animals
died at day 7 after challenge, as did two of the animals vaccinated
with the EBOV liposome preparation and the remaining untreated cynomolgus
macaque. The untreated rhesus macaques died on days 8 and 9 postchallenge;
one rhesus vaccinated with the gamma-irradiated virion preparation
died on day 9, and the other survived challenge. The remaining animal
vaccinated with the EBOV liposome preparation died 11 days after
challenge. The rhesus macaque that survived challenge did not become
ill during the study and had a PRNT50 values >320
at day 26 postchallenge and 80 at days 26, 61, 99, and 902 postchallenge.
Histopathologic Examination
Conventional histopathologic and electron microscopic examination
of lymphatic tissues, liver, and gastrointestinal tract showed no
differences in lesions between the vaccinated animals and the unvaccinated
EBOV-infected controls. Depletion and necrosis or apoptosis were
noted in all lymphoid germinal centers in spleen, peripheral, and
mesenteric lymph nodes, as described in other studies (17-19).
The spleen had copious deposits of fibrin throughout the red pulp,
as well as abundant karyorrhectic cellular debris. By electron microscopy,
widespread bystander lymphocyte apoptosis was a prominent feature
in all the lymphatic tissues examined. Fibrin and fibrinocellular
thrombi were also prominent in the submucosa of the gastrointestinal
tract and in hepatic sinusoids, again consistent with well-documented
findings (17,18).
We also evaluated retrospectively EBOV-infected rodent tissues
in parallel. Although sites of infection and morphologic changes
between guinea pigs, mice, and nonhuman primates had many similarities,
the lack of fibrin thrombi in spleen and visceral vasculature was
particularly striking in the EBOV-infected mice (Figure).
Fibrin deposition was seen in guinea pigs as reported (20),
but fibrin deposits and thrombi were considerably less prevalent
compared with deposits in nonhuman primates (Figure).
Lymphocyte apoptosis was also less frequently observed by electron
microscopy in rodent lymphatic tissues than in nonhuman primates.
EBOV was demonstrated in liver, spleen, kidney, lung, adrenal gland,
and lymph nodes of all necropsied monkeys by immunohistochemistry,
electron microscopy, or virus infectivity titration.
Discussion
Our results indicate that rodent models of EBOV hemorrhagic fever
do not consistently predict efficacy of candidate vaccines in nonhuman
primates, perhaps because the disease course in rodents differs
from that reported in human and nonhuman primates (17-19,22,23).
Mice do not have the hallmark disseminated intravascular coagulation
(DIC) found in end-stage lesions of humans and nonhuman primates.
Viremia and widespread tissue dissemination are much more apparent
in nonhuman primates than in guinea pigs (20).
In addition, guinea pigs have less DIC than do nonhuman primates.
Lymphocyte apoptosis was not reported to be a prominent feature
of EBOV infection in mice or guinea pigs (20,21)
but was a consistent feature of disease in humans (24)
and nonhuman primates (19). Clinical disease and
related pathologic features in nonhuman primates infected with EBOV
appear to more closely resemble those described in human EBOV hemorrhagic
fever (22,23). Other studies have shown inconsistencies
between rodent and nonhuman primate models of human hemorrhagic
disease in the protective efficacy of candidate vaccines. For example,
guinea pigs were protected from Lassa virus by VACV recombinants
expressing the viral nucleoprotein (25,26); however,
this vaccination strategy failed to protect rhesus macaques (27).
The effort to develop an EBOV vaccine began after the initial identification
of EBOV in 1976, but 25 years later the goal remains elusive. Attempts
to develop killed-virus vaccines against EBOV hemorrhagic fever
have had inconsistent results (5-7). Recent progress
in genetic vaccination strategies has demonstrated that immunity
can be achieved against a low dose of EBOV. While protection against
any lethal challenge dose of EBOV is a remarkable achievement, we
have set the bar somewhat higher than 6 PFU, since a laboratory
exposure through a needlestick and infected blood would likely entail
a dose of at least 1,000 PFU. Therefore, our priority is to empirically
develop a vaccine that protects against at least 1,000 PFU rather
than to initiate an exhaustive investigation of protective immune
mechanisms. We were encouraged by the demonstrated success of the
VEEV replicon vector expressing MBGV glycoprotein in protecting
cynomolgus macaques from challenge with homologous MBGV (12).
No MBGV-neutralizing activity was observed at >1:20 dilutions
in prechallenge sera of any of the MBGV GP VRP-vaccinated macaques
(12), yet these animals did not become viremic,
showed no signs of disease, and survived challenge. Historically,
Filovirus-neutralizing antibodies have been difficult to
demonstrate in vitro (15); while the presence
of neutralizing antibodies is desirable, it is neither sufficient
nor necessary to clear viral infection (16). Unfortunately,
the VEEV replicon strategy that was successfully employed for MBGV
in cynomolgus macaques and for EBOV in mice and guinea pigs (10)
did not protect cynomolgus macaques from EBOV disease. These differences
observed between EBOV and MBGV may result from differences in the
course of infection. Specifically, the mean day of death for untreated
cynomolgus monkeys experimentally infected intramuscularly with
1,000 PFU of EBOV (Zaire subtype) is 6.3 (n=15; data not shown),
while the mean day of death for cynomolgus monkeys infected intramuscularly
with a comparable dose of MBGV (Musoke isolate) is 9.1 (n=8; data
not shown). Thus, macaques infected with MGBV have nearly three
more days to mount an effective immune response against the challenge
virus than macaques infected with EBOV (Zaire). Clearly, other variables,
including differences observed between EBOV (Zaire) and MBGV with
respect to GP gene expression (28), tropism, and
host cell responses, may contribute to differences in disease pathogenesis
and outcome of infections.
The induction of humoral and cytotoxic T-lymphocyte responses to
EBOV NP and GP has been demonstrated in guinea pigs, although the
relative contributions of these responses to immune protection are
unclear (9). Moreover, transfer of EBOV immune
serum in rodent and nonhuman primate models provided inconsistent
results. Passive transfer of immune serum from VRP-vaccinated animals
did not protect guinea pigs or mice against lethal challenge (10);
however, transfer of hyperimmune equine immune globulin (which had
high EBOV neutralization titers) to guinea pigs protected them against
disease (16,29). Passive treatment
of cynomolgus monkeys with the equine immune globulin delayed death
but did not ultimately protect the monkeys against lethal EBOV hemorrhagic
fever (16,29). In contrast,
hamadryl baboons were protected against lethal EBOV challenge by
passive treatment with the equine immune globulin and the use of
a lower challenge dose (30). These results suggest
that cell-mediated effector mechanisms may play a more important
role in protection than do humoral responses. Nonetheless, the role
of humoral immunity is in fact supported by studies showing consistent
delay in death or protection of primates therapeutically treated
with EBOV-neutralizing antibodies (16,29,30).
We conclude that, although rodent models are useful as preliminary
screens for candidate vaccines and therapeutic treatments, nonhuman
primates likely provide a more useful and definitive model for EBOV
hemorrhagic fever in humans. Furthermore, differences in disease
pathology between rodent and nonhuman primate models of EBOV suggest
that protection of primates may require different protective mechanisms.
Acknowledgments
The authors thank Denise Braun and Joan Geisbert for expert technical
assistance.
Dr. Geisbert is chief of the Electron Microscopy Department, Pathology
Division, at the U.S. Army Medical Research Institute of Infectious
Diseases. His research interests include the pathology and pathogenesis
of hemorrhagic fever viruses.
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Table
1. Prechallenge neutralization titers of Ebola virus (EBOV)-vaccinated
monkeys |
|
Nonhuman primate species |
No. of animals
|
Vector
|
Antigen
|
Neutralization titersa
|
|
Cynomolgus
|
3
|
Replicon
|
GP
|
0, 0, 0
|
Cynomolgus
|
3
|
Replicon
|
NP
|
0, 0, 0
|
Cynomolgus
|
3
|
Replicon
|
GP + NP
|
0, 0, 10
|
Cynomolgus
|
3
|
Replicon
|
Influenza HA
|
0, 0, 0
|
Cynomolgus
|
3
|
Vaccinia
|
GP
|
10, 20, 20
|
Cynomolgus
|
3
|
Liposome
|
Inactivated virion
|
20, 40, 80
|
Cynomolgus
|
2
|
|
Inactivated virion
|
10, 20
|
Rhesus
|
2
|
|
Inactivated virion
|
10b, 20
|
Cynomolgus
|
2
|
None
|
|
0, 0
|
Rhesus
|
2
|
None
|
|
0, 0
|
|
aImmunoglobulin G enzyme-linked
immunosorbent assay, neutralizing antibody (PRNT50)
All vaccinated monkeys seroconverted by enzyme-linked
immunosorbent assay before challenge.
bAnimal survived challenge.
GP, glycoprotein; NP, nucleoprotein.
|
Table
2. Challenge of vaccinated monkeys with Ebola virus (EBOV) |
|
NHP Species
|
Vector
|
Antigen
|
Survival/total
|
Viremic/total
|
Day of deatha
|
|
Cynomolgus
|
Replicon
|
GP
|
0/3
|
3/3
|
6, 6, 7
|
Cynomolgus
|
Replicon
|
NP
|
0/3
|
3/3
|
6, 6, 7
|
Cynomolgus
|
Replicon
|
GP + NP
|
0/3
|
3/3
|
6, 6, 7
|
Cynomolgus
|
Replicon
|
Influenza HA
|
0/3
|
3/3
|
6, 6, 7
|
Cynomolgus
|
Vaccinia
|
GP
|
0/3
|
3/3
|
6, 7, 7
|
Cynomolgus
|
Liposome
|
Inactivated virion
|
0/3
|
3/3
|
7, 7, 11
|
Cynomolgus
|
|
Inactivated virion
|
0/2
|
2/2
|
6, 6
|
Rhesus
|
|
Inactivated virion
|
1/2
|
2/2
|
9
|
Cynomolgus
|
None
|
|
0/2
|
2/2
|
6, 7
|
Rhesus
|
None
|
|
0/2
|
2/2
|
8, 9
|
|
aNumber of days after challenge
with 1,000 PFU of EBOV.
NHP, nonhuman primate; GP, glycoprotein; NP, nucleoprotein.
|
|