Recombinant Measles Virus Vaccine Expressing the Nipah Virus Glycoprotein Protects against Lethal Nipah Virus Challenge.
ABSTRACT Nipah virus (NiV) is a member of the genus Henipavirus, which emerged in Malaysia in 1998. In pigs, infection resulted in a predominantly non-lethal respiratory disease; however, infection in humans resulted in over 100 deaths. Nipah virus has continued to re-emerge in Bangladesh and India, and person-to-person transmission appeared in the outbreak. Although a number of NiV vaccine studies have been reported, there are currently no vaccines or treatments licensed for human use. In this study, we have developed a recombinant measles virus (rMV) vaccine expressing NiV envelope glycoproteins (rMV-HL-G and rMV-Ed-G). Vaccinated hamsters were completely protected against NiV challenge, while the mortality of unvaccinated control hamsters was 90%. We trialed our vaccine in a non-human primate model, African green monkeys. Upon intraperitoneal infection with NiV, monkeys showed several clinical signs of disease including severe depression, reduced ability to move and decreased food ingestion and died at 7 days post infection (dpi). Intranasal and oral inoculation induced similar clinical illness in monkeys, evident around 9 dpi, and resulted in a moribund stage around 14 dpi. Two monkeys immunized subcutaneously with rMV-Ed-G showed no clinical illness prior to euthanasia after challenge with NiV. Viral RNA was not detected in any organ samples collected from vaccinated monkeys, and no pathological changes were found upon histopathological examination. From our findings, we propose that rMV-NiV-G is an appropriate NiV vaccine candidate for use in humans.
- SourceAvailable from: M Sadman Sakib[Show abstract] [Hide abstract]
ABSTRACT: study aims to design epitope-based peptides for the utility of vaccine development by targeting glycoprotein G and envelope protein F of Nipah virus (NiV) that, respectively, facilitate attachment and fusion of NiV with host cells. Using various databases and tools, immune parameters of conserved sequence(s) from G and F proteins of different isolates of NiV were tested to predict probable epitope(s). Binding analyses of the peptides with MHC class-I and class-II molecules, epitope conservancy, population coverage, and linear B cell epitope prediction were analyzed. Predicted peptides interacted with seven or more MHC alleles and illustrated population coverage of more than 99% and 95%, for G and F proteins, respectively. The predicted class-I nonamers, SLIDTSSTI and EWISIVPNF, superimposed on the putative decameric B cell epitopes, were also identified as core sequences of the most probable class-II 15-mer peptides GPKVSLIDTSSTITI and EWISIVPNFILVRNT. These peptides were further validated for their binding to specific HLA alleles using in silico docking technique. Our in silico analysis suggested that the predicted epitopes, either GPKVSLIDTSSTITI or EWISIVPNFILVRNT, could be a better choice as universal vaccine component against NiV irrespective of different isolates which may elicit both humoral and cell-mediated immunity.Advances in Bioinformatics 05/2014;
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ABSTRACT: The impact of morbilliviruses on both human and animal populations is well documented in the history of mankind. Indeed, prior to the development of vaccines for these diseases, morbilliviruses plagued both humans and their livestock that were heavily relied upon for food and motor power within communities. Measles virus (MeV) was responsible for the death of millions of people annually across the world and those fortunate enough to escape the disease often faced starvation where their livestock had died following infection with rinderpest virus (RPV) or peste des petits ruminants virus (PPRV). Canine distemper virus has affected dog populations for centuries and in the past few decades appears to have jumped species, now causing disease in a number of non-canid species, some of which are been pushed to the brink of extinction by the virus. During the age of vaccination, the introduction and successful application of vaccines against rinderpest and measles has led to the eradication of the former and the greater control of the latter. Vaccines against PPR and canine distemper have also been generated; however, the diseases still pose a threat to susceptible species. Here we review the currently available vaccines against these four morbilliviruses and discuss the prospects for the development of new generation vaccines.Vaccine 04/2014; · 3.77 Impact Factor
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ABSTRACT: Nipah virus (NiV), a zoonotic pathogen causing severe respiratory illness and encephalitis in humans, emerged in Malaysia in 1998 with subsequent outbreaks on an almost annual basis since 2001 in parts of the Indian subcontinent. The high case fatality rate, human-to-human transmission, wide-ranging reservoir distribution and lack of licensed intervention options are making NiV a serious regional and potential global public health problem. The objective of this study was to develop a fast-acting, single-dose NiV vaccine that could be implemented in a ring vaccination approach during outbreaks. In this study we have designed new live-attenuated vaccine vectors based on recombinant vesicular stomatitis viruses (rVSV) expressing NiV glycoproteins (G or F) or nucleoprotein (N) and evaluated their protective efficacy in Syrian hamsters, an established NiV animal disease model. We further characterized the humoral immune response to vaccination in hamsters using ELISA and neutralization assays and performed serum transfer studies. Vaccination of Syrian hamsters with a single dose of the rVSV vaccine vectors resulted in strong humoral immune responses with neutralizing activities found only in those animals vaccinated with rVSV expressing NiV G or F proteins. Vaccinated animals with neutralizing antibody responses were completely protected from lethal NiV disease, whereas animals vaccinated with rVSV expressing NiV N showed only partial protection. Protection of NiV G or F vaccinated animals was conferred by antibodies, most likely the neutralizing fraction, as demonstrated by serum transfer studies. Protection of N-vaccinated hamsters was not antibody-dependent indicating a role of adaptive cellular responses for protection. The rVSV vectors expressing Nipah virus G or F are prime candidates for new 'emergency vaccines' to be utilized for NiV outbreak management.Vaccine 03/2014; · 3.49 Impact Factor
Recombinant Measles Virus Vaccine Expressing the
Nipah Virus Glycoprotein Protects against Lethal Nipah
Misako Yoneda1*, Marie-Claude Georges-Courbot3, Fusako Ikeda1, Miho Ishii1, Noriyo Nagata4,
Frederic Jacquot3, Herve ´ Raoul3, Hiroki Sato1, Chieko Kai1,2*
1Laboratory Animal Research Center, Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, Japan, 2International Research Center for Infectious
Diseases, Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, Japan, 3Institut National de la Sante et de la Recherche Me ´dicale, Laboratory P4 INSERM
Jean Me ´rieux, Lyon, France, 4National Institute of Infectious Diseases, Department of Pathology Tokyo, Japan
Nipah virus (NiV) is a member of the genus Henipavirus, which emerged in Malaysia in 1998. In pigs, infection resulted in a
predominantly non-lethal respiratory disease; however, infection in humans resulted in over 100 deaths. Nipah virus has
continued to re-emerge in Bangladesh and India, and person-to-person transmission appeared in the outbreak. Although a
number of NiV vaccine studies have been reported, there are currently no vaccines or treatments licensed for human use. In
this study, we have developed a recombinant measles virus (rMV) vaccine expressing NiV envelope glycoproteins (rMV-HL-G
and rMV-Ed-G). Vaccinated hamsters were completely protected against NiV challenge, while the mortality of unvaccinated
control hamsters was 90%. We trialed our vaccine in a non-human primate model, African green monkeys. Upon
intraperitoneal infection with NiV, monkeys showed several clinical signs of disease including severe depression, reduced
ability to move and decreased food ingestion and died at 7 days post infection (dpi). Intranasal and oral inoculation induced
similar clinical illness in monkeys, evident around 9 dpi, and resulted in a moribund stage around 14 dpi. Two monkeys
immunized subcutaneously with rMV-Ed-G showed no clinical illness prior to euthanasia after challenge with NiV. Viral RNA
was not detected in any organ samples collected from vaccinated monkeys, and no pathological changes were found upon
histopathological examination. From our findings, we propose that rMV-NiV-G is an appropriate NiV vaccine candidate for
use in humans.
Citation: Yoneda M, Georges-Courbot M-C, Ikeda F, Ishii M, Nagata N, et al. (2013) Recombinant Measles Virus Vaccine Expressing the Nipah Virus Glycoprotein
Protects against Lethal Nipah Virus Challenge. PLoS ONE 8(3): e58414. doi:10.1371/journal.pone.0058414
Editor: Maria G. Masucci, Karolinska Institutet, Sweden
Received October 4, 2012; Accepted February 4, 2013; Published March 14, 2013
Copyright: ? 2013 Yoneda 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.
Funding: This work was supported by grants in aid from the Ministry of Education, Science, Culture and Sports of Japan. The funders had no role in study design,
data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com (MY); firstname.lastname@example.org (CK)
Nipah virus (NiV) is a member of the genus Henipavirus, within
the family Paramyxoviridae. This virus emerged in Malaysia in
1998, resulting in predominantly nonlethal respiratory disease in
pigs. However, in humans, there were 105 deaths. In the first
outbreak, pigs were the amplifying host [1,2] and they were
probably infected through fruits contaminated by secretions and/
or body fluids from bats . In Malaysia, a higher prevalence of
infection was found among pig farmers, pork sellers and army
personnel involved in the culling of pigs. However, person-to-
person transmission was not apparent at the time. NiV has re-
emerged in Bangladesh and India, with the total number of
reprted cases exceeding 300, with 161 fatalities . These new
outbreaks have occured in patients who have never come in
contact with pigs, therefore it is suspected that the infection is
being directly transmitted from fruit bats, and person-to-person
transmission has been noted in some cases in Bangladesh [5,6,7,8].
NiV infection causes a severe acute encephalitic syndrome or a
severe respiratory disease with high mortality in humans [2,9–13].
Although many patients eventually recover fully, some develop
neurological manifestations several months after recovery from
acute non-encephalitic or asymptomatic infection. There are four
countries where NiV outbreaks have occurred, or are currently in
progress. The habitat of fruit bats, the natural hosts of NiV
infection, is widely distributed in the world  from Australia,
South-East and South Asia to west coast of Africa. To prevent
further outbreaks, it is necessary that effective vaccines and
therapies are developed.
A number of NiV vaccine studies have been conducted in which
the NiV envelope proteins F (fusion) and G (glycoprotein), were
chosen for vaccine development based on existing knowledge
regarding immunity to other paramyxoviruses. Vaccinia virus-
expressed recombinant NiV F and G proteins have been shown to
be immunogenic, and can induce protective immune responses in
hamsters . Canarypox virus-based vaccine vectors carrying
genes encoding NiV F or G proteins induce neutralizing
antibodies in pigs and prevent viral shedding during NiV challenge
. However, there are currently no vaccines or treatments
licensed for human use.
Our aim was to develop an affordable, live-attenuated MV
vaccine that could be used in areas where NiV outbreaks are
PLOS ONE | www.plosone.org1March 2013 | Volume 8 | Issue 3 | e58414
occurring. MV-based vaccines induce life-long immunity after one
or two low-dose inoculations [16–18]. The MV vector can stably
express proteins derived from other infectious viruses, inducing
strong and long-term humoral and cellular immune responses,
even if there is preexisting immunity to MV [19–21].
In the present study, we evaluated whether African green
monkeys were a suitable animal model for NiV infection. We also
examined the efficacy of vaccination in the monkey model as well
as a previously established hamster model, with recombinant MV
vectors expressing the G protein of NiV.
Materials and Methods
The majority of this work was performed in a biosafety level 4
(BSL4) laboratory at INSERM, Lyon, France and carried out in
strict accordance with the French ‘‘Comite ´ National de Re ´flexion
Etihque sur l’Expe ´rimentation Animale’’: All animal experiments
were approved by Comite ´ re ´gional d’ethique pour l’expe ´rimen-
tation animale Rhone Alpes (Permit Number: 0236, 324,
P4_2010_004). All surgery was performed under sodium pento-
barbital anesthesia, and all efforts were made to minimize
Animals and Housing
African green monkeys (n=8) were studied, then euthanized
under anesthesia at the end of experimental period. Monkeys were
individually housed in cages, and had free access to food and
water. They were given pellet, fruits and confectionery once a day.
During the experiment, toys such as mirrors, balls and rings
especially designed for monkeys, were provided.
We also examined hamsters (n=30), which were euthanized
under anesthesia at the completion of the experiment.
Cells and Viruses
The NiV we used was propagated in Vero cells grown in
Dulbecco’s minimal essential medium (DMEM) supplemented
with 5% fetal calf serum (FCS), L-glutamine, penicillin and
streptomycin at 37uC/5% CO2. Virus titration was conducted by
assessing the 50% tissue culture infectious dose (TCID50) in 96-
well plates. To generate a recombinant MV (rMV) expressing the
NiV G protein, we used replication competent MV-based vectors
(pMV-HL, HL strain; pMV-Ed, Edmonston B strain) [22,23]. The
NiV G cDNA was amplified from pNiV(6+)  by using
following primers, NipG-SacI-F, 59-GAGCTCATGCCGGCA-
GAAAACAAGAA-39 (SacI site in italic); and reverse primer
CATTGCTCTGGTA-39 (FseI site in italics; additional two
nucleotides for rule of six in boldface). The intergenic region
between the N and P junction was amplified by using the following
primers. NP-F, GGCCGGCCTCCAATATTCTA (FseI site in
GAATTGTTATTA (SacI site in italics). The PCR products were
cloned into pGEM-T Easy (Promega, Madison, WI, USA). The
NiV G fragment was inserted downstream of the N-P intergenic
region, followed by digestion by SacI. Finally, the fragment of NiV
G connected to the N-P intergenic region was cloned into the FseI
site of the pMV-HL and pMV-Ed, and the resulting clones were
used to rescue the infectious recombinant MVs expressing NiV G
protein (rMV-HL-G and rMV-Ed-G).
NiV Infection of African Green Monkeys
Young adult African green monkeys (Chlorocebus aethiops;
n=4), weighing 4–5 kg were caged individually. All animals
were anesthetized and inoculated with 16106or 16108TCID50
of NiV by intraperitoneal or intranasal and per os routes.
Animals were anesthetized for clinical examination, tempera-
ture, weight, blood draws and nasal and oral swabs on days 0,
2, 4, 7, 9, 11, 14, 16, 18, 21, 24. Animals were sacrificed when
they reached a moribund state, or showed symptoms of
irreversible disease (15% weight loss, or no intake of food and
Immunization and Challenge
For protection studies, 10 8-week-old golden hamsters were
immunized intraperitoneally with 26104TCID50of the recom-
binant MVs at 0 and 21 dpi. Hamsters were challenged 1 week
after the second immunization. Two African green monkeys were
immunized subcutaneously with 16105TCID50of the recombi-
nant MV-Ed-G on 0 and 28 dpi. Monkeys were challenged 2
weeks after the second immunization.
Measurement of Antibody Titres
The titer of antibodies against the NiV G protein (anti-NiV
G) in the monkey sera was determined using an indirect
enzyme-linked immunosorbent assay (ELISA). Ninety-six-well
microtitre plates were coated with a 2 mg/ml solution of
purified NiV G protein diluted in coating buffer (0.1 M
carbonate-hydrogen carbonate buffer, pH9.6) overnight. Unoc-
cupied sites in wells were blocked with 300 ml of 0.8% Block
Ace (Dainihonseiyaku, Osaka, Japan) in PBS at room temper-
ature for 3 h, washed with PBS containing 0.05% Tween 20
(PBS-T). Monkey sera (100 mL) were serially diluted 2-fold
(1:100 to 1:12800) and added to duplicate wells. After 2 h
incubation at 4uC, the wells were washed with PBS-T and
incubated for 1 h with 100 ml of horseradish peroxidase (HRP)-
conjugated goat anti-monkey-IgG (1:1000 dilution; CAPPEL).
Following a final wash with PBS-T, 100 ml of peroxidase
substrate (Bio-Rad Laboratories) was added to each well and the
absorbance at 655 nm measured 30 min later.
Quantitative Real-time PCR (qPCR) Analyses
Tissue and swab samples were homogenized in 500 ml of
Trizol reagent (Ambion) and total RNA extracted in accordance
with the instructions of the manufacturer. First strand cDNA
was synthesized using total RNA and random primers. The
qPCR assays were carried out on an ABI Prism 7900HT
(Applied Biosystems, USA) using SYBR Premix Ex TaqII
(Takara, Japan). Specific Ribosomal Protein L13A (RPL13A)
was used as an internal control. Data were analyzed with
Sequence Detection Systems version 1.7a software (Applied
calculated using the threshold cycle time (Ct), the first cycle
number at which emitted fluorescence
standard deviation (SD) of base-line emission as measured in
the cycles of PCR. A standard curve was generated using
known cDNA concentrations (10-fold dilution from 10 ng ,
1 pg/reaction). Normalized results were expressed as the ratio
of NiV N RNA to RPL13A RNA.
exceeds 10X the
Tissues were processed by routine histological methods and
sections of tissue were stained with hematoxylin and eosin and
examined for histopathological changes. Separate sections were
stained using immunohistochemical techniques with a rabbit
polyclonal antiserum against the NiV nucleoprotein.
Recombinant Vaccine for Nipah Virus Infection
PLOS ONE | www.plosone.org2March 2013 | Volume 8 | Issue 3 | e58414
rMV Vaccine Expressing the NiV G Protein
Recombinant viruses expressing the NiV G protein were
generated and rescued using vectors based on the HL (pMV-
HL) and Edmonston (pMV-Ed) strains [22,23]. The rescued
viruses were tested for the expression of G protein using infected
cells. B95a and Vero cells were infected with the rMVs (rMVs,
rMV-HL-G and rMV-Ed-G) and the expression of NiV G was
examined by immunofluorescence. The NiV G protein was well
expressed in rMV-HL-G- or rMV-Ed-G-infected cells (Fig. 1A).
We compared the in vitro growth characteristics of the recombi-
nant viruses with those of the parental virus (Fig. 1B). rMV-HL-G
grew well and had a growth rate similar to that of the parental
rMV-HL virus. In contrast, the rMV-Ed-G had lower maximum
titers than its parental virus. The sequence of the inserted NiV G
gene in recovered viruses was checked and no substitutions were
Vaccination of Hamsters with rMV Expressing NiV G
Protects against a Lethal Infection
Hamsters are not fully susceptible to MV infection, but are
highly susceptible to NiV infection. In our preliminary experi-
ments, antibodies against MV were observed to increase in the
sera from hamsters 3 weeks after intraperitoneal inoculation with
MV, although the hamsters did not exhibit any symptoms of
infection. In the present study, 8-week-old golden hamsters were
intraperitoneally immunized with 26104TCID50of rMV-HL-G
or rMV-Ed-G. Three weeks later, antibodies against NiV G, as
measured by ELISA, were observed in the sera of all animals
inoculated with rMV-HL-G (1:400), and in most of animals except
one with rMV-Ed-G. All the animals were boosted with the same
dose of the rMVs and then challenged 1 week after the second
immunization. The ELISA titer was expressed as reciprocal of the
dilution factor. Serum antibody titers increased in all hamsters
well; in 9 rMV-HL-G-vaccinated hamsters; .1:1600 and in 1;
1:800, and in 9 rMV-Ed-G vaccinated hamsters; .1:1600 and 1;
1:200 at the challenge day. In the NiV hamster model,
intraperitoneal inoculation of NiV induces fatal encephalitis 7 to
10 days later [24,25]. When unimmunized control hamsters were
challenged intraperitoneally with 103TCID50/animal of NiV,
90% died (Fig. 2). However, all hamsters vaccinated with rMV-
HL-G or rMV-Ed-G showed complete protection. During the
observation period (14 days after the challenge), all hamsters
immunized with the recombinant MVs showed no clinical
symptoms of the disease and survived.
Nipah Virus Infection in African Green Monkeys
Intraperitoneal inoculation of one monkey with 16106TCID50
NiV and one monkey with 16108TCID50NiV resulted in death
within 7 days. Body weights began to decrease by 2 dpi (Fig. 3),
and clinical signs including severe depression, reduced ability to
move, and reduced food ingestion were observed from 5 dpi.
Inoculation with NiV by intranasal and oral routes was also tested
in the monkeys, to mimic natural infection in humans. Although a
combined intranasal and oral inoculation was not lethal for
monkeys, it caused similar clinical illness beginning at 9 dpi, and
progressed at a slower rate than when the intraperitoneal route
was used. Monkeys were seriously moribund at around 14 dpi. In
particular, the monkey inoculated with 16108TCID50of virus by
the intranasal and oral route lost 10% of its weight in 14 days. The
monkey inoculated with 16106TCID50NiV by the same route
lost 6% of its weight in 14 days. Blood biochemistry results for
both monkeys showed an increase in both the total number of
white blood cells and the lymphocyte/monocyte ratio by around
14 dpi (data not shown).
To assess the extent of NiV replication and tissue tropism in
monkeys, the relative quantity of NiV RNA was measured in
swabs and tissue samples. In the monkeys inoculated with NiV
intraperitoneally and euthanized at 7 dpi, viral RNA was detected
at variable levels in the tissues of many organs (Fig. 4). The highest
relative levels of viral RNA were detected in the lung tissue of the
monkey inoculated with 16108TCID50NiV, and in the spleen of
monkey inoculated with 16106TCID50NiV. Viral RNA was not
detected in tissues from monkeys inoculated with NiV by
intranasal and oral routes. These monkeys had recovered from
infection and were euthanized at 24 dpi for tissue collection.
Histopathological changes observed in the infected monkeys are
shown in Table 1. In the monkeys inoculated with NiV
intraperitoneally, advanced lesions were found in many abdominal
organs and the lungs. Confluent consolidations with serum protein
in lung alveoli and pulmonary congestion with edema were
observed in both monkeys inoculated by the intraperitoneal route.
In their spleen, severe hemorrhage, necrosis and lyphocyte
depletion were oserved. These histopathological changes were
similar to those observed in human cases. Virus antigen was also
observed in vascular endothelial cells of small vein and capillaries
in the lung, and in the follicular area in spleen. In monkeys
inoculated with NiV via the intranasal and oral routes and
euthanized at 24 dpi, follicular hyperplasia was seen in the spleens
and tonsils of both monkeys. However, severe pathological
changes in lung were observed only in the monkey inoculated
with 16108TCID50NiV. There were no apparent changes in
brain samples from of any of the monkeys.
Application of the rMV Vaccines for Monkeys
Two monkeys were subcutaneously immunized with 16105
TCID50rMV-Ed-G. Four weeks later, they were boosted with the
same dose of rMV-Ed-G. The vaccinations well induced antibody
responses against NiV G protein. One monkey showed antibody
response at 14 dpi, and both monkeys showed high serum
antibody titers 1 week after the second immunization (Table 2).
The vaccinated and unvaccinated monkeys were challenged
introperitoneally with 16105TCID50 NiV 1 week after the
second immunization. The unvaccinated monkeys showed a
decrease in rectal temperature 13 days after challenge, and
clinical signs of illness; this was not observed in the vaccinated
monkeys (Fig. 5). The vaccinated monkeys did not show any
clinical illness prior to euthanasia. Their organ samples, taken 21
days after NiV challenge, were tested for presence of viral RNA by
qPCR. Viral RNA was detected only in the brain and liver of an
unvaccinated monkey (T+ SV085 ).
We also examined these monkeys for histophathological
changes. In the lungs of unvaccinated monkeys, severe congestion
was widely observed in addition to the accumulation of blood
plasma in alveoli simiar to those with previous experiment (Fig. 6).
Perivascular edema and lymphocytic infiltration around vessels
were also observed. In the brain, perivascular cuffing and an
accumulation of glial and foam cells were observed in the cerebral
cortex. There were no lesions observed in the tissues of vaccinated
NiV is a zoonotic virus that has recently emerged in Malaysia. It
has a broad host range and can cause severe respiratory illness and
encephalitis with high mortality in humans . Despite several
previous NiV vaccine studies, there are still no licensed vaccines
Recombinant Vaccine for Nipah Virus Infection
PLOS ONE | www.plosone.org3 March 2013 | Volume 8 | Issue 3 | e58414
Figure 1. Recombinant measles virus expressing NiV G protein. Two strains of rMV expressing the NiV G protein were generated; rMV-HL-G
and rMV-Ed-G. (A) rMV-HL-G-infected B95a cells and rMV-Ed-G-infected Vero cells were stained with anti-NiV G polyclonal antibody and analyzed by
phase contrast microscopy and immunofluorescence. Cells infected with empty vectors served as controls. (B) B95a cells were infected with rMV-HL
or rMV-HL-G. Vero cells were infected with rMV-Ed or rMV-Ed-G. Infections were conducted at a multiplicity of infection (MOI) of 0.1 TCID50/cell. Cells
and supernatants were collected at the indicated time points for determination of virus titer. Error bars indicate means 6 the standard deviation (SD)
from three experiments.
Recombinant Vaccine for Nipah Virus Infection
PLOS ONE | www.plosone.org4March 2013 | Volume 8 | Issue 3 | e58414
for human use. Efficacy studies in a non-human primate model are
required for the development and approval of a new vaccine or
antiviral for use in humans. Recently, African green monkey have
been shown to be a highly pathogenic model for NiV infection
. Pathogenicity of NiV in these monkeys was demonstrated
through intratracheal and oral inoculation. When we started to
test susceptibility of African green monkeys to NiV, there was no
report available. We compared infections resulting from inocula-
tion via intraperitoneal or intranasal/oral routes. Our findings
show that intraperitoneal inoculation induces a more severe
manifestation than intranasal and oral inoculation. However, the
intranasal/oral route, which mimics the more natural infection
route for humans, did cause severe illness in two infected monkeys
by 9–14 dpi. Symptoms were consistent with human NiV
infection, and the monkeys became moribund, although they
eventually recovered. The progression of illness clinically is similar
to human cases. Histopathological tests suggested that when
administered via an intraperitoneal route, lymphoid organs
including spleen and lung were the main target organs of virus
propagation. The cause of death was severe respiratory distress
resulting from hemorrhage and edema in the lungs, and monkeys
died at 7 dpi, which is well before the viral infection could have
advanced to the cerebral region of the brain. Although intranasal/
oral inoculation also made monkeys ill, we found no evidence of
virus propagation or pathological changes, possibly because the
samples were taken at the end of the experiment (24 dpi) after the
monkeys had recovered. Intranasal/oral inoculation is more
natural route for human infection, and induced symptoms in
monkeys similar to those observed in humans. However, we
decided to use intraperitoneal route for the NiV challenge after
immunization with our recombinant MV vaccine because it
caused more severe illness and resulted in an earlier death.
A canarypox virus-based vaccine vector has been shown to be
effective as a vaccine against NiV-associated disease in veterinary
vaccine . The canarypox virus does not replicate in
mammalian cells, although it can infect and produce viral
proteins. Thus, it is able to eliminate the safety concerns that exist
for vaccinia virus vectors. For human use, approaches employing
soluble subunit vaccines, virus-like particles, vaccinia virus vectors
or complementing defective vesicular stomatitis virus vectors have
been explored previously [14,28,29,30,31]. Although they seem
promising as vaccines, their efficacy might be problematic because
these replication-defective vectors cannot induce long-term
Live-attenuated measles vaccines have been used since the
1960 s worldwide because they are highly effective and safe.
Because MV is an RNA virus, with no DNA intermediates during
replication, MV genome does not integrate into host genome.
These characteristics make live-attenuated MV vaccines an
attractive candidate vector to provide safe and effective immunity
against various pathogens. In particular, it induces strong cellular
immunity and the effects are long term. For these reasons, many
recombinant MVs expressing antigenic proteins of other infectious
diseases are under development. It could be argued that the
widespread vaccination for measles could result in inactivation of
the recombinant MV vector before there is a chance that the NiV
G protein can be expressed and induce protective immunity. We
examined the antibody responses induced by rMV-Ed-G and
rMV-HL-G in MV-seropositive monkeys. Both recombinant MVs
could induce specific antibodies; in particular, two inoculation
with rMV-Ed-G induced a high titer of a antibodies (1:12800; data
not shown). Therefore, it appears that our rMV vaccines are
available for people that have been exposed to MV vaccines
In this study, we have demonstrated that recombinant live-
attenuated MVs are effective at preventing the onset of symptoms
typical of NiV infection. Immunization with recombinant MVs
expressing NiV glycoproteins perfectly protected hamsters against
Figure 2. Survival curves of hamsters inoculated with NiV.
Hamsters (n=10) were immunized intraperitoneally with 26104TCID50
of rMV-HL-G or rMV-Ed-G, and boosted with the same dose 3 weeks
after the first immunization. Unimmunized hamsters were inoculated
with phosphate-buffered saline (PBS). Four weeks after the first
immunization, hamsters were challenged intraperitoneally with 26103
TCID50NiV. The survival of each group was observed for 14 days after
Figure 3. Body weights of Nipah virus-infected monkeys. Each
monkey was inoculated with 108or 106TCID50of NiV via intraperitoneal
(A) or intranasal and oral routes (B). Monkeys were examined every 2–3
days, and body weights recorded. Levels were standardized, with the
weight at the first day of the experiment set as 1.
Recombinant Vaccine for Nipah Virus Infection
PLOS ONE | www.plosone.org5 March 2013 | Volume 8 | Issue 3 | e58414
a lethal dose upon challenge, although rMV-Ed-G induced NiV-
specific IgG antibody level was low in a small number of hamsters.
The antibody response is considered to be an essential component
of protection against NiV encephalitis [14,28,29]; however,
cellular immunity might play an important role in eradicating
NiV infection. We tested two MV vectors, based on our previous
Figure 4. Virus replication in Nipah virus-infected monkeys. Virus replication was determined in tissues (A) and oral and nasal swabs (B) of
NiV-infected animals by qPCR. (A) Tissue samples of monkeys infected via the intraperitoneal (IP) route were collected at 7 dpi, while samples from
monkeys infected via intranasal (IN)+per os (PO) route were collected at 24 dpi. (B) Nasal and pharynx swab samples were collected every 2 days. All
samples were measured in triplicate, and error bars represent the standard error of the mean (SEM).
Recombinant Vaccine for Nipah Virus Infection
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experiences where we have observed that rMV-HL-based vaccines
sometimes elicit a stronger effect than rMV-Ed-based vaccines.
Both rMV-HL-G and rMV-Ed-G induced well protective effect in
hamsters against NiV challenge.
The HL strain is isolated from patient and still possesses weak
first licensed vaccine in United States in 1963, and further
attenuated vaccine derived from the Edmonston strain is widely
adopted in the world. Aiming at early practical use, we tested the
monkeys did not show any symptoms of NiV infection. We used a
lower titer (105TCID50) of NiV for challenge in this experiment, to
observe symptoms of infected monkeys for a slightly longer period
than those with 106TCID50. This dose did not induce a lethal
pathology, even in unvaccinated individuals. However, histopath-
ological and clinical observations of monkeys indicated that those
challenged with NiV did suffer from a severe illness, with
unimmunized monkeys found to also have lesions in their brains.
frominfection.Further, NiVchallenge causedpathologicalchanges
in the brain, which has been widely documented in human cases.
This observation might be due to slow spreading of the virus in
animals challenged with a lower dose.
NiV is highly virulent and has a broad host range, causing
respiratory and neurological symptoms that often lead to
encephalitis. The rate of mortality in humans range from 40–
92% [8,32,33]. To date, no vaccine for NiV disease has been
Table 1. Pathological findings in organ samples of NiV infected monkeys.
IP, 10‘6 IP, 10‘8 INPO, 10‘6 INPO, 10‘8
LiverCongestion, focal necrosis, and
slight infiltration of neutrophils
in the sinusoids.
necrosis with hemorrhage
No histopathological changes
KidneyEndothelial syncytia in large to
middle sized blood vessels.
SpleenSyncytial cells in germinal center.
Follicular necrosis with
Lymphocyte depletion and
necrotic germinal center.
Follicular hyperplasia, Follicular hyperplasia, VA (-)
LungConfluent consolidation with
serum protein in alveoli.
Confluent consolidation with
serum protein in alveoli.
Focal consolidation with serum
protein in alveoli. VA (6, Blood
Lymph NodeLymphocyte depletion Lymphocyte depletionNoneNone, VA (2)
Tonsil Lymphocyte depletion Lymphocyte depletionFollicular hyperplasia, Follicular hyperplasia, VA (+,
Table 2. Vaccination with rMV-Ed-G induced well antibody
responses in monkeys.
d0d7 d14d21 d28d35
NDND ND NDND ND
ND NDNDND NDND
Ed 8192ND ND64003200 1600 3200
Ed 8358 NDND ND NDND1600
Monkeys were immunized with rMV-Ed-G twice on d0 and d28. Antibody levels
were measured by ELISA. Shadowed columns represent the samples which
showed positive response. T+: unimmunized. ND: Not detected (,1:100).
Figure 5. Body temperature of monkeys after NiV challenge.
The rectal temperature of unimmunized (upper) monkeys or monkeys
immunized with 105TCID50of rMV-Ed-G was recorded from 4 days
before the NiV challenge until the end of the experiment. T+ B8144 and
T+ SV085 were unimmunized monkeys. Ed B8358 and Ed B8192 were
monkeys immunized with rMV-Ed-G before virus challenge.
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Figure 6. Histopathology of monkey tissues. Lung and brain samples from unvaccinated monkeys (T +B8144, T+ SV085) and vaccinated
monkeys (Ed B8358, Ed 8192) were stained with hematoxylin and eosin. 1006magnification. The lungs of T+ B8144 and T+ SV085 showed severe
congestion, infiltration of neutrophils and accumulation of blood plasma in the alveoli. Their brains showed perivascular cuffing (black arrow; SV085)
and an accumulation of glial (white arrow; SV 085) and foam cells (white arrow in; B8144) in the cerebral cortex. No lesions were observed in tissues
from Ed B8358 or Ed B8192.
Recombinant Vaccine for Nipah Virus Infection
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developed that is both safe and protective in humans. Our
recombinant MV-Ed-G vaccine has the potential to elicit long-
term immunity against both MV and NiV in children and adults
located in endemic areas. Therefore we believe it is an effective
vaccine candidate for human use. We were only able to use two
monkeys for vaccination in this study, as the costs of non-human
primate and spaces for animal experimentation in BSL4 facility
were prohibitive. Further studies in greater number of monkeys
will be necessary to validate the safety and efficacy of our vaccine
The authors would like to thank Laura Barrot, Damaris Cornec, Audrey
Vallve, Aurelie Duthey, Ste ´phanie Mundweiler and Ste ´phane Mely for
their assistance in the BSL4 laboratory. Without their valuable contribu-
tions, this study could not have been undertaken.
Conceived and designed the experiments: MY CK. Performed the
experiments: MY MCGC FI MI NN FJ. Analyzed the data: MY HS
CK. Contributed reagents/materials/analysis tools: MI HS HR. Wrote the
paper: MY CK.
1. Chua KB, Bellini WJ, Rota PA, Harcourt BH, Tamin A, et al. (2000) Nipah
virus: a recently emergent deadly paramyxovirus. Science 288: 1432–1435.
2. Chua KB, Goh KJ, Wong KT, Mamarulzaman A, Tan PS, et al. (1999) Fatal
encephalitis due to Nipah virus among pig-farmers in Malaysis. Lancet 354,
3. Hooper P, Zaki S, Daniels P, Middleton D (2001) Comparative pathology of the
diseases caused by Hendra and Nipah viruses. Microbes Infect 3,315–322.
4. Kai C, Yoneda M (2011) Henipavirus Infections – An Expanding Zoonosis from
Fuit Bats. J Disaster Research, in press.
5. Chadha MS, Comer JA, Lowe L, Rota PA, Rollin PE, et al. (2006) Nipah virus –
associated encephalitis outbreak, Siliguri, India. Emerg Infect Dis 12, 235–240.
6. ICDDR B (2004) Person-to-person transmission of Nipah virus during outbreak
in Faridpur District, 2004. Health Science Bulletin 2, 5–9.
7. Lo MK, Rota PA (2008) The emergence of Nipah virus, a highly pathogenic
paramyxovirus. J Clin Virol 43, 396–400.
8. Luby SP, Hossain MJ, Gurley ES, Ahmed BN, Banu S, et al. (2009) Recurrent
zoonotic transmission of Nipah virus into humans, Bangladesh, 2001–2007.
Emerg Infect Dis 15, 1229–1235.
9. Goh KJ, Tan CT, Chew NK, Tan PS, Kamarulzaman A, et al. (2000) Clinical
features of Nipah virus encephalitis among pig farmers in Malaysia. N Engl J Med
10. Chua KB, Lam SK, Goh KJ, Hooi PS, Ksiazek TG, et al. (2001) The presence
of Nipah virus in respiratory secretions and urine of patients during outbreak of
Nipah virus encephalitis in Malaysia. J Infect 42, 40–43.
11. Hsu VP, Hossain MJ, Parashar DU, Ali MM, Ksiazek TG, et al. (2004) Nipah
virus encephalitis reemergence, Bangladesh. Emerg Infect Dis 10, 2082–2087.
12. Paton NI, Leo YS, Zaki SR, Auchus AP, Lee KE, et al. (1999) Outbreak of
Nipah-virus infection among abattoir workers in Singapore. Lancet 354, 1253–
13. Wong KT, Shieh WJ, Kumar S, Norain K, Abdullah W, et al. (2002) Nipah
virus pathology working group. Nipah virus infection: pathology and
pathogenesis of an emerging paramyxoviral zoonosis. Am J Pathol 161, 2153–
14. Guillaume V, Contamin H, Loth P, Georges-Courbot MC, Lefeuvre A, et al.
(2004) Nipah virus: vaccination and passive protection studies in a hamster
model. J Virol 78, 834–840.
15. Weingartl HM, Berhane Y, Caswell JL, Loosmore S, Audonnet JC, et al. (2006)
Recombinant Nipah virus vaccines protect pigs against challenge. J. Virol 80,
16. Griffin D (2001) Measles virus. Fields virology. 4thed. Vol 2. Philadelphia:
Lippincott-Raven Publishers. 1401–1441.
17. Hilleman M (2001) Current overview of the pathogenesis and prophylaxis of
measles with focus on practical implications. Vaccine 20, 651–665.
18. Ovsyannikova I, Dhiman N, Jacobson R, Vierkant R, Poland G (2003)
Frequency of measles virus-specific CD4+ and CD8+ T cells in subjects
seronegative or highly seropositive for measles vaccine. Clin. Diagn Lab
Immunol 10(3), 411–416.
19. Despre `s P, Combredet C, Frenkiel M, Lorin C, Brahic M, et al. (2005) Live
measles vaccine expressing the secreted form of the West Nile virus envelope
glycoprotein protects against West Nile virus encephalitis. J Infect Dis, 191(2),
20. Guerbois M, Moris A, Combredet C, Najburg V, Ruffie C, et al. (2009) Live
attenuated measles vaccine expressing HIV-1 Gag virus like particles covered
with gp160DeltaV1V2 is strongly immunogenic. Virology, 388, 191–203.
21. Lorin C, Mollet L, Delebecque F, Combredet C, Charneau P, et al (2004) A
single infection of recombinant measles vaccine expressing HIV-1 clade B
envelope glycoproteins induces neutralizing antibodies and cellular immune
responses to HIV. J Virol 78(1), 146–157.
22. Terao-Muto Y, Yoneda M, Seki T, Watanabe A, Tsukiyama-Kohara K, et al.
(2008) Heparin-like glycosaminoglycans prevent the infection of measles virus in
SLAM-negative cell lines. Antiviral Res. 80(3), 370–376.
23. Radecke F, Spielhofer P, Schneider H, Kaelin K, Huber M, et al. (1995) Rescue
of measles viruses from cloned DNA. EMBO J, 14(23), 5773–5784.
24. Yoneda M, Guillaume V, Ikeda F, Sakuma Y, Sato H, et al. (2006)
Establishment of a Nipah virus rescue system. Proc Natl Acad Sci USA.,
25. Wong KT, Grosjean I, Brisson C, Blanquier B, Fevre-Montange M, et al. (2003)
A golden amster model for human acute Nipah virus infection. Am J Pathol,
26. Lo MK, Rota PA (2008) The emergence of Nipah virus, a highly pathogenic
paramyxovirus. J Clin Virol, 43(4), 396–400.
27. Geisbert TW, Daddario-DiCaprio KM, Hickey AC, Smith MA, Chan YP, et al.
Development of an acute and highly pathogenic nonhuman primate model of
Nipah virus infection. PLoS One, 18;5(5), e10690, 2010.
28. McEachern JA, Bingham J, Crameri G, Green DJ, Hancock TJ, et al. (2008) A
recombinant subunit vaccine formulation protects against lethal Nipah virus
challenge in cats. Vaccine, 26, 3842–3852.
29. Mungall BA, Middleton D, Crameri G, Bingham J, Halpin K, et al. (2006)
Feline model of acute nipah virus infection and protection with a soluble
glycoprotein-based subunit vaccine. J Virol, 80, 12293–12302.
30. Walapita P, Barr J, Sherman M, Basler CH, Wang LF (2011) Vaccine potential
of Nipah virus-like particles. PLoS One, 6(4), e18437.
31. Chattopadhyay A, Rose JK (2011) Complementing defective viruses expressing
separate paramyxovirus glycoproteins provide anew vaccine vector approach.
J Virol, 85(5), 2004–2011.
32. Tan CT, Wong KT (2003) Nipah encephalitis outbreak in Malaysia. Annals of
the Academy of Medicine, Singapore 32, 112–117.
33. Lam SK (2003) Nipah virus-a potential agent of bioterrorism? Antiviral Res, 57,
Recombinant Vaccine for Nipah Virus Infection
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