Vaccine potential of Nipah virus-like particles.

Abstract

Nipah virus (NiV) was first recognized in 1998 in a zoonotic disease outbreak associated with highly lethal febrile encephalitis in humans and a predominantly respiratory disease in pigs. Periodic deadly outbreaks, documentation of person-to-person transmission, and the potential of this virus as an agent of agroterror reinforce the need for effective means of therapy and prevention. In this report, we describe the vaccine potential of NiV virus-like particles (NiV VLPs) composed of three NiV proteins G, F and M. Co-expression of these proteins under optimized conditions resulted in quantifiable amounts of VLPs with many virus-like/vaccine desirable properties including some not previously described for VLPs of any paramyxovirus: The particles were fusogenic, inducing syncytia formation; PCR array analysis showed NiV VLP-induced activation of innate immune defense pathways; the surface structure of NiV VLPs imaged by cryoelectron microscopy was dense, ordered, and repetitive, and consistent with similarly derived structure of paramyxovirus measles virus. The VLPs were composed of all the three viral proteins as designed, and their intracellular processing also appeared similar to NiV virions. The size, morphology and surface composition of the VLPs were consistent with the parental virus, and importantly, they retained their antigenic potential. Finally, these particles, formulated without adjuvant, were able to induce neutralizing antibody response in Balb/c mice. These findings indicate vaccine potential of these particles and will be the basis for undertaking future protective efficacy studies in animal models of NiV disease.

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Vaccine Potential of Nipah Virus-Like Particles
Pramila Walpita1*, Jennifer Barr2, Michael Sherman3, Christopher F. Basler4, Linfa Wang2
1Departments of Microbiology and Immunology, Center for Biodefense and Emerging Infectious Disease, University of Texas Medical Branch, Galveston, Texas, United
States of America, 2Australian Commonwealth Scientific and Industrial Research Organization, Australian Animal Health Laboratory, Geelong, Victoria, Australia,
3Department of Biochemistry and Structural Biology, University of Texas Medical Branch, Galveston, Texas, United States of America, 4Department of Microbiology,
Mount Sinai School of Medicine, New York, New York, United States of America
Abstract
Nipah virus (NiV) was first recognized in 1998 in a zoonotic disease outbreak associated with highly lethal febrile
encephalitis in humans and a predominantly respiratory disease in pigs. Periodic deadly outbreaks, documentation of
person-to-person transmission, and the potential of this virus as an agent of agroterror reinforce the need for effective
means of therapy and prevention. In this report, we describe the vaccine potential of NiV virus-like particles (NiV VLPs)
composed of three NiV proteins G, F and M. Co-expression of these proteins under optimized conditions resulted in
quantifiable amounts of VLPs with many virus-like/vaccine desirable properties including some not previously described for
VLPs of any paramyxovirus: The particles were fusogenic, inducing syncytia formation; PCR array analysis showed NiV VLP-
induced activation of innate immune defense pathways; the surface structure of NiV VLPs imaged by cryoelectron
microscopy was dense, ordered, and repetitive, and consistent with similarly derived structure of paramyxovirus measles
virus. The VLPs were composed of all the three viral proteins as designed, and their intracellular processing also appeared
similar to NiV virions. The size, morphology and surface composition of the VLPs were consistent with the parental virus, and
importantly, they retained their antigenic potential. Finally, these particles, formulated without adjuvant, were able to
induce neutralizing antibody response in Balb/c mice. These findings indicate vaccine potential of these particles and will be
the basis for undertaking future protective efficacy studies in animal models of NiV disease.
Citation: Walpita P, Barr J, Sherman M, Basler CF, Wang L (2011) Vaccine Potential of Nipah Virus-Like Particles. PLoS ONE 6(4): e18437. doi:10.1371/
journal.pone.0018437
Editor: Patricia V. Aguilar, University of Texas Medical Branch, United States of America
Received December 11, 2010; Accepted March 7, 2011; Published April 6, 2011
Copyright: ? 2011 Walpita 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 a Developmental grant to PW from NIH/WRCE U54 AI057156-07. 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: prwalpit@utmb.edu
Introduction
Since it was first recognized in 1998, Nipah virus (NiV) has
caused several outbreaks in humans of encephalitic disease
associated with high lethality. In the first outbreak, which was in
Malaysia and Singapore, 265 humans became sick and some
,40% of them died. Epidemiological links pointed to human
contact with sick pigs in commercial piggeries, and the outbreak
was brought under control through culling of approximately ,1.1
million pigs [1,2,3,4]. Since then, the virus has re-emerged in
Bangladesh and neighboring India, starting in 2001, and between
then and now, has caused several smaller but even deadlier disease
outbreaks with case fatality rates ranging between 60 and 90%
[5,6,7,8]. Unlike the Malaysian outbreak, the route of transmission
in these outbreaks was considered to be bat-to-human via food
contaminated with bat saliva [9]. In some cases, nosocomial
transmissibility and person-to-person spread was also noted
[5,10,11,12]. An additional concern is that NiV is also potentially
an agent of agro-terror since the rate of transmission of this virus in
the pig population is close to 100% [13]. Effective vaccine and
therapies are needed to combat the threats posed by NiV.
NiV is a member of the genus Henipavirus in the subfamily
Paramyxovirinae, family Paramyxoviridae. It has several distinctive
genetic and biologic features [14,15,16,17,18] although its
morphology and genome organization is similar to that of other
members of the subfamily. NiV has six genes arranged in tandem,
39-N, P, M, F, G and L-59 [15,16]. The N, P and L are required
for reconstituting viral RNA polymerase activity, the matrix
protein M is required for particle formation and budding, and the
two surface glycoproteins G and F are required for attachment
and entry into the host cell [19,20]. EphrinB2 and B3 have been
identified as the NiV entry receptors [21,22,23]. After fusion of the
virus and the cell membrane, the viral ribonucleoprotein is
released in to the cell cytoplasm. Following transcription and
replication, the viral components migrate to the plasma membrane
for assembly and budding of progeny particles [24,25].
Two vaccination strategies for NiV disease prevention have
already been explored experimentally: A canarypox virus-based
vaccine vector approach was effective as veterinary vaccine [26],
and it is in the process of further development. The same
approach for human use vaccines is undergoing extensive
evaluation, largely for HIV and AIDS [27]. A soluble NiV G
protein approach has also shown promise [28,29]. However,
subunit approaches are in general less effective than particulate
immunogens, and can suffer from suboptimal presentation to the
immune system [30,31,32]. Immunogenicity in mice to NiV
glycoproteins has been reported recently using two vectored
approaches for gene delivery; one using Venezuelan equine
encephalitis virus replicons [33] and the other involving
inoculation of a mix of two complementing defective vesicular
stomatitis virus (VSVDG) vectors, one for expressing each of the
two NiV glycoproteins [34]. The latter approach is new and
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seems promising but its regulatory approval as human vaccine
might be problematic [34,35].
In this study we have explored the potential of NiV virus-like
particles (VLPs) as a vaccine. Plasmid-mediated expression of
selected viral proteins results in the spontaneous assembly and
release of VLPs. These particles make highly effective immuno-
gens because they possess several features of the authentic virus
such as their surface structure and dimensions [31,36]. They are
also safe because they do not contain any viral genetic material.
VLPs, where one or more of the constituent proteins serve as
immunogens (native VLPs), are particularly effective as vaccines
for infectious disease. The fact that two such vaccines [Gardasil
(Merck & Co) for human papillomavirus (HPV), and Sci-B-Vac
(SciGen) and Bio-HepB (GlaxoSimthKline) for Hepatitis B virus
(HBV)] have already been approved for human use, and many, for
non-enveloped and enveloped viruses [31,32,37,38,39,40,41,42]
are at various stages of development, attests to the desirability of
this approach for vaccine development.
The budding capacity of virus proteins as VLPs, the protein-
protein interactions that facilitate this process, and the central role
of M protein in VLP assembly and release has been described for
several paramyxoviruses such as Sendai virus (SeV), Newcastle
disease virus (NDV), respiratory syncytial virus (RSV), paramyxo-
virus simian virus 5 (PIV-5) and human parainfluenza virus type 1
(hPIV1) [20,43,44,45,46,47]: The efficiency of VLP formation in
virtually all these studies was based on M protein release in the
supernatant. NiV virus-like particles have also been described
[48]; the results of this study showed 1) that NiV G and F proteins
individually retained some budding capacity although it was far
less efficient than that of the M protein and 2) NiV N, M, F and G-
containing VLPs resembled the virus in some respects but differed
significantly from it with respect to ratio of VLP-incorporated F
protein; most of it was present in precursor F0form. Recently, the
vaccine potential of native VLPs of NDV [49] has been described:
these particles, composed of HN, F, M and NP proteins, had
several virus-like properties. However, since the F protein in this
formulation was modified by design to ablate the cleavage site, it
remained in its precursor form; consequently, the NDV VLPs
were non-fusogenic, and therefore incapable of inducing syncytia
formation.
Here we describe NiV VLPs composed of the two surface
glycoproteins G, and F, and the matrix protein M. The G and F
proteins were included because they mediate attachment and
entry into the host cell [50,51,52], both are major targets of
neutralizing antibodies, and both are major players in vaccine
induced protection [52,53,54]. NiV G and F together are also the
most effective as immunogens; this was elucidated in a canary pox
virus vector-based experimental protective efficacy study [26]. The
M protein was included in our formulation because it is required
for particle formation and release [20,25,45]. Under optimized
conditions, we were able to make substantial, quantifiable amounts
of NiV VLPs composed of these three NiV proteins. This has
allowed us to characterize their properties in detail to show that
they possessed many virus-like/vaccine desirable properties in vitro.
It has also allowed us to test for immunogenicity in vivo in Balb/c
mice; note that although NiV does not cause disease in these
animals, NiV proteins injected in them are known to induce robust
neutralizing antibody response [29,33,34]. Importantly, NiV-
specific mouse monoclonal antibodies are protective in the
hamster model of NiV disease [55].
In this study, careful assessment of immunogenicity has shown
for the first time, that these NiV VLPs are able to induce
neutralizing antibody response. We have also provided a detailed
methodology to optimize production of the VLPs for research
purposes. Beyond this, we have provided the first CryoEM study
of NiV VLPs and thus provide a careful assessment of their
morphology. We further demonstrate that NiV VLPs can trigger
‘‘fusion from without’’ upon addition to cells. To our knowledge
this is a first for an enveloped VLP. Finally, we have shown that
NiV VLPs activate innate immune signaling in ‘‘infected’’ cells
and provide a transcriptional profile of this response. Based on all
these attributes, NiV M, F and G-protein-containing VLPs show
promise as vaccine and will be the basis for undertaking future
protective efficacy studies in animal models of NiV disease.
Materials and Methods
Protein expression vectors, cells and viruses
NiV expression plasmids pCAGGS- G, F, and M are all under
the control of chicken beta actin promoter [56], and they were
constructed in the laboratory of one of the co-authors of this study
(CB) as described previously [20]. Human embryonic kidney 293
cells (ATCC, CRL-1573) and 293T cells (ATCC, CRL-11268)
were grown in Dulbecco’s minimum essential medium supple-
mented with10% fetal bovine serum (FBS) and penicillin and
streptomycin, and maintained in the same medium containing 2%
FBS. The minigenome that was used for optimizing VLP
formation has been described previously [57]. All the initial
minigenome-based optimization steps were done in BHK-T7 cells
(a gift from Dr. N. Ito). The same conditions were applicable to
produce VLPs in 293T cells and they were used throughout to
generate the VLPs used for the work described in this study.
Transfection
293T cells were grown in Dulbecco’s complete medium to
achieve semi-confluent (80–90% density) cell monolayers. The
cells were transiently transfected with the plasmids constructs using
the lipid reagent Lipofectamine 2000 according to the general
guidelines provided by the manufacturers’ instructions (Invitrogen
Inc). At 48 hrs post-transfection, the VLP-containing cell super-
natants (SUP) were harvested for concentration and purification of
the VLPs. Because of the fusogenic property of our VLPs, there
was widespread syncytia formation at this time point although the
cells were still adherent.
VLP harvest and purification
VLPs released in the transfected-cell SUP were harvested and
clarified by centrifugation at 3,500 rpm for 30 minutes at 4uC and
concentrated by sucrose density gradient centrifugation based on
previous descriptions [44,45,58]. Briefly, the clarified SUPs were
concentrated by ultracentrifugation through 20% sucrose cushion
in TN buffer (0.1 M NaCl; 0.05 M Tris-HCL, pH 7.4) at
200,0006 g for 8 hours at 4uC. The resulting VLP pellet in
,0.5 ml volume was purified on a discontinuous sucrose gradient
formed by layering 80%, 65%, 50% and 10% sucrose in TN
buffer. After centrifugation at 186,0006 g for 8 hours, the top
,1.5 ml of the gradient (which included the VLP-containing band
at the interface between the 10% and 50% sucrose layers) was
resuspended in 20% sucrose buffer and centrifuged once more at
160,0006g for one hour. The resulting pellet was resuspended in
20% sucrose solution in endotoxin-free TN bufffer and stored at
4uC for subsequent analysis. Supernatant of 293T cells transfected
with empty pCAGGS plasmid and processed similarly (referred to
as ‘‘mock’’ particles) served as negative control when needed.
VLP infectivity assay
Since the ratio of the protein expression plasmids used at
transfection and the time of harvest may have a bearing on the
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level of VLP formation, a minigenome-based VLP infectivity assay,
similar to those described previously [59,60] was used to determine
the relative concentrations of the constituent plasmids, and to
determine the kinetics of VLP formation for optimal production.
This assay provides only a comparative assessment of VLP
formation since it only accounts for VLPs that are able to
incorporate and passage minigenomes. However, based on the
assumption that the ratio of empty and minigenome-containing
VLPs will be equivalent in each reaction, the method provides an
indirect means to determine the optimal set of conditions for VLP
production as determined by VLP-incorporated minigenome-
encoded CAT enzyme activity. Briefly, the steps involved in the
VLP infectivity assay were 1) transfection of NiV minigenome
construct and co-transfection with full complement of the NiV
protein expression plasmids, N, P, L, M, F and G, using
Lipofectamine 2000. 2) following replication (48 hours post-
transfection), passage of equal volume of VLP-containing
transfected cell SUP on to fresh cells previously transfected with
N, P and L plasmids and 3), determination of CAT activity in the
VLP infected cells 48 hours later. Replication of the VLP-
incorporated incoming mingenomes based on reporter gene
activity indicates the level of particle formation and release, VLP
infectivity, and successful minigenome packaging.
CAT assays
FAST CAT Assay kit (Molecular Probes) was used according
the manufacturer’s instructions and allowed accurate quantifica-
tion of CAT enzyme levels over a wide linear range.
Electron Microscopy (EM)
VLPs were purified as described. The particles were adsorbed
on Formvar carbon coated copper grid by floating it on a drop of
VLP suspension for 15 minutes, the grids were blotted, and then
negatively stained with 2% aqueous uranyl acetate for viewing by
transmission electron microscopy.
Cryoelectron (CryoEM) microscopy
The VLPs were vitrified as reported previously [61] on holey
carbon film grids (C-flatTM, Protochips, Raleigh, North Carolina).
VLPs were imaged at 40,000x indicated magnification using a
4k64k slow-scan CCD camera (UltraScan 895, GATAN, Inc.,
Pleasanton, CA) using a low-dose imaging procedure.
Immunogold labeling
Unfixed VLPs were used for immunogold labeling to limit
antibody reactivity to the cell surface proteins. The particles were
adsorbed on formvar coated nickel grids, stained with NiV specific
primary antibody (hyper immune mouse ascites fluid, HMAF,
obtained from Dr. P. Rollin, CDC) diluted in buffer (1% BSA in
0.05 M tris buffer) rinsed in wash buffer (0.1% BSA in 0.05 M tris
buffer), stained with colloidal gold labeled goat anti-mouse
secondary antibody (Jackson ImmunoResearch Laboratories),
washed, and then negatively stained with 2% uranyl acetate for
viewing by EM.
VLP Protein concentration
The total protein concentration of the purified VLP prepara-
tions was measured by the BCA (Bicinchoninic Acid) method
(Thermo Scientific Laboratories).
Western blotting
VLP composition was determined by western blot analysis.
Briefly, purified VLPs resuspended in endotoxin free PBS were
lysed by resuspending them in equal amount of 2x SDS protein-
loading buffer and loaded into a 12% SDS-polyacrylamide gel
with a 4% stacking gel. 293T cell lysates processed similarly were
run in parallel as negative cell control. Following electrophoresis to
resolve the protein bands, and transfer to membrane, the blot was
incubated with NiV-specific HMAF primary antibody at a dilution
of 1:1000 dilution, overnight at 4uC, and HRP-conjugated anti-
mouse secondary antibody (from GE Healthcare) at a 1: 20,000
dilution for one hour at room temperature. The proteins were
revealed using western blot detection reagents according to
instructions provided by the manufacturer (GE Healthcare).
Protocol to immunize Balb/c mice
These studies were undertaken with the approval of the
Institutional Biosafety Committee (Protocol# #01/08-2010-1)
and the Institutional Animal Care and Use (IACUC) Committee
(Protocol # 0904028). Five to six week old female Balb/c mice
(Harlan Laboratories) were housed in microisolater cage for 4 days
in the Animal Resource Center at the University of Texas Medical
Branch before beginning the immunization protocol. Mice in
groups of five were immunized by subcutaneous inoculation of
four different concentrations of VLPs (1.75, 3.5, 7 or 14 mg/
mouse, referred to subsequently as treatment groups A through D
respectively) prepared just prior to use in sterile endotoxin free
PBS. No adjuvant was used. A group of five mice inoculated with
sterile endotoxin free PBS served as negative control group. Mice
in the four treatment groups (A through D) were boosted (6 mg/
mouse) on days 15 and 29; the negative control group received
PBS. Blood was collected from the submandibular vein of the
animals on days 21, 14, 21, 28 and 35; they were euthanized on
day 35.
VLP-induced immune response
Plaque Reduction neutralization test (PRNT).
dilutions of test sera were made in 50 ml cell culture medium.
Under biohazard level 4 conditions, each of the diluted sera were
mixed with 50 ml of NiV diluted to generate ,30 plaque forming
units and incubated for 30 min at 37uC. The pre-incubated virus-
antibody mix was added to Vero cell monolayers grown in 96 well
plates and incubated for 30 min at 37uC when the inoculum was
removed and replaced 150 ml of cell media. After incubation at
37uC for 24 h, the cells fixed in 100% ice-cold methanol and
staining by indirect immunofluorescence assay as follows: The
wells in the plate were blocked with BSA/PBS and stained with
rabbit sera raised against the G protein of HeV, and goat anti-
rabbit Alexa Fluor 488 conjugate (Invitrogen) diluted 1:1000 in
blocking buffer. Viral plaques were visualized and counted, and
neutralizing antibody titers were reported based on reduction in
plaque count by 50% relative to the untreated control (PRNT50).
Antibody levels measured by Immunofluorescence assay
(IFA).
For IFA, NiV-specific total antibody levels were measured
by using NiV G, F and M expressing 293T cells as target antigen.
Thirty six hours post-transfection, the cells were harvested, fixed in
paraformaldehyde, cytospun (Cytocentrifuge, Thermoscientific)
on glass slides to obtain monolayered preparations and then stored
at 4uC, and used as antigen within three weeks of preparation. On
the day of use, the slides were washed in PBS, permeabilized with
Triton-X-100 and blocked with BSA/PBS. After incubation with
two fold dilutions of the test sera, the cell monolayers were washed
and stained with Alexa fluor 488-conjugated goat anti-mouse
antibodyaccordingthemanufacturer’s
instructions. Negative and positive controls were run in parallel
with each batch.
Two-fold
(MolecularProbes)
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Gene expression profile by Real-time PCR
VLP-mediated transcriptional activation was tested for eighty
four genes involved in Toll-like receptor (TLR)-mediated signal
transduction using RT2Profiler PCR array (SABiosciences). The
96 well array format included mediators of TLR signaling
including adaptors and proteins that interact with the TLRs,
and members of NFKB, JNKp38, NF/IL6 and IRF signaling
pathways downstream of TLR signaling. Briefly, 293 cells grown
overnight in 60 mm dishes were exposed to 10 mg of purified
VLPs suspended in 1 ml of OPTI-MEM (Invitrogen). ‘‘Mock
particles’’ (see Methods, VLP harvest and purification) resus-
pended similarly and exposed to 293 cells served as negative
control. The inoculums were adsorbed on the cell monolayers for
3 hours at 37uC when additional 1.5 ml of OPTI-MEM was
added and the dishes further incubated. Twenty fours post VLP
exposure, total cell RNA was extracted according to the
manufacturer’s (SABiosciences) instructions. The integrity of the
RNA was verified by agarose gel electrophoresis and the same
concentration of total cell RNA from the VLP-stimulated and
‘‘mock’’ stimulated cells were used for gene expression profiling by
Real-time PCR using Eppendorf Mastercycler unit. The array
plate included positive and negative controls for quality assurance,
and three sets of housekeeping genes for normalization for data
analysis. The fold-change in gene expression in the VLP
stimulated 293 cells relative to the ‘‘mock’’ stimulated 293 cells
was calculated by the DDCt method according to the manufac-
turer’s instructions.
Results
Optimization of conditions for the production of NiV
VLPs
In preliminary studies it was found that co-expression of NiV G,
F and M proteins in 293T cells resulted in the formation of VLPs
that bud out into the transfected cell SUP and that they can be
harvested, concentrated and purified as described under Methods.
However, the VLP yield was low. To improve the efficiency of
VLP formation we proceeded to optimize the ratio of the three
expression plasmids used at transfection. We speculated that this
would be important based on the fact that a), during replication,
paramyxoviruses form a transcription gradient where the 39
proximal genes are transcribed more abundantly than the
successive downstream genes [52] and b), the stoichiometry of
interaction of the viral proteins has proved to be critical in
plasmid-driven minigenome and full-length rescue systems [62].
The importance of protein ratios for VLP formation was
alluded to in a previous NDV study where the expression plasmids
were co-transfected at ‘‘pre-determined concentrations’’ to
produce VLP-incorporated protein ratios analogous to those in
virus infected cells [49]. In a study by Patch el al [48], equivalent
amounts of NiV N, M, F and G were initially used to produce the
VLPs. In that study, VLPs were subsequently also made by
adjusting NiV expression plasmid concentrations by experimental
variations similarly to that in the NDV study [49]. The efficiency
of particle formation and budding in both these, and many other
paramyxovirus VLP formation systems was based on M protein
release [43,44,45,46,48].
We have chosen a minigenome-based functional assay, the VLP
infectivity assay (described under Materials and Methods), to
determine optimal expression plasmid ratios for efficient VLP
formation based on reporter gene readouts. Briefly, 293T cells
were transfected with plasmids as shown in Figure 1. For titrating
NiV G, F and M plasmids, increasing concentrations of either G,
or F or M expression plasmids were, in turn, co-transfected with
fixed concentrations of the other two plasmids. The minigenome
and N, P and L plasmids were transfected using a predetermined
ratio [57]. The VLP-containing cell SUP was harvested 48 hours
post-transfection, clarified by centrifugation, and equal volume
from each was passaged onto fresh cell monolayers (VLP-infected
cells) previously transfected with the core proteins required to
support the incoming packaged minigenomes. The VLP infected cells
were harvested 48 hours later and tested for optimal particle
production based on incoming minigenome-encoded CAT
activity. This time point was chosen because maximal VLP
formation was also found to be time dependent and optimal at
48 hours post-passage (data not shown). The reproducibility of the
results was verified in an independent repeat experiment. Results
presented in Figure 1 show that within the given range, and based
on the levels of minigenome-encoded CAT activity, varying the
concentrations of G, F and M plasmids had a bearing on VLP
formation. CAT activity in the VLP infected cells appeared
optimal in the boxed lanes 7 and 8 but further analysis to ensure
reporter activity in the linear range (data not shown) indicated that
the largest amount of minigenome-containing NiV VLPs were
produced when the cells were transfected with the NiV M, F and
G plasmid ratios of 3:1:1 as in lane 7. This ratio was used for
making all our VLP preparations.
Morphologic similarity between NiV VLPs and the
authentic virus
The optimized conditions were applied to transfect G, F and M
expression plasmids in 293T cells grown in 10 cm dishes. The
VLP-containing culture SUPs were harvested 48 hours later, and
concentrated and purified as described. Briefly, the clarified SUPs
were concentrated by ultracentrifugation through 20% sucrose
cushion, and then purified on a discontinuous sucrose gradient.
The VLP pellet was resuspended in TN buffer and viewed by EM
after negative staining. The result presented in Figure 2A shows a
VLP-containing band in the sucrose gradient. Viewing of the
negatively stained purified particles by transmission electron
microscopy (Figure 2B) showed numerous virus-like particles.
The size variation of these VLPs was consistent with the parental
virus: NiV is a pleomorphic virus ranging in size from 40–
1900 nm [63,64]; the sizes of the VLPs ranged from ,40–
500 nm. The particles also resembled authentic NiV morpholog-
ically, and this is seen more clearly in the magnified images
presented in Figure 2C; here, the fringe of the glycoproteins is
clearly visible on the VLP surface. An occasional VLP had what
appeared to be a double fringe (shown with an arrow), a feature
more frequently associated with Hendra rather than NiV virus
particles [64]. The image in Figure 2D is a cryoelectron
micrograph of one of our VLPs; the overall surface appearance
is virus-like, which is described as dense, ordered and repetitive
[31], and it shows the surface glycoproteins and their spatial
arrangement even more definitively.
Identification of NiV-specific proteins in the VLPs
To verify whether the NiV proteins were incorporated into the
VLPs as designed, purified particles were analyzed by western
blotting using NiV-specific mouse antibody, and HRP-conjugated
anti-mouse secondary antibody as described under Methods. The
right hand panel in the Figure 3 shows VLP-incorporated proteins
in two different preparations of NiV VLPs. The protein bands are
consistent in size to NiV proteins G, F0, F1 and M proteins
[17,48]. The relative amounts of the VLP-incorporated G and M
proteins appeared to be similar to that reported in NiV virions also
[17]. This was in spite of the fact that the viral proteins in that
study were revealed using rabbit sera raised against bacterially
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expressed Hendra virus proteins. However, the ratio of the VLP-
incorporated F1to F0was different from that in the virions. This
difference is more likely to be a reflection of timing and protein
turnover rather than the reagents used to reveal them since in a
previous study, pulse chase experiments have shown that similarly
to the VLP-incorporated F1to F0, intracellular cleavage of the
precursor NiV fusion protein by cathepsin L results in near equal
mix of mature fusogenic, and the precursor forms [65]. Absence of
the NiV-specific bands in two different 293T cell lysate
preparations processed similarly (and shown in the left hand panel
in Figure 3) confirms specificity of the VLP-incorporated proteins.
Immunoreactivity of VLP surface glycoproteins
The immunoreactivity of the VLP surface glycoproteins was
verified by staining purified unfixed VLPs by the immunogold
labeling technique using NiV-specific mouse antiserum and 6 nm
colloidal gold particle-conjugated goat anti-mouse secondary
antibody (Jackson ImmunoResearch Laboratories Inc). The
particles were viewed by EM after negative staining. The use of
unfixed particles assured that only the surface-exposed antigens
would be reactive. Numerous VLPs with the gold particles
decorating their surface were seen; Figure 2E shows two such
VLPs.
Inhibition of VLP-induced syncytia formation by
NiV-specific antibodies
Syncytium formation is a classical feature of NiV and other
paramyxovirus-induced cytopathology that can be blocked by
virus-specific neutralizing antibody. A similar observation was
made when 293 cells were ‘‘infected’’ with the NiV VLPs.
Briefly,the VLPswerepre-incubated
antibody, Junin virus (JV)-specific antibody, and with OPTI-
MEM I (Invitrogen Inc) medium only for one hour at 37uC
before inoculating onto near confluent 293 cell monolayers
grown overnight in 60 mm dishes. The inoculum was removed
after incubation for 3 hours at 37uC, replaced with OPTI-MEM
I, and the plates were further incubated overnight at 37uC
overnight. The monolayers were then viewed for the formation
of syncytia after staining with crystal violet. The results in
Figure 4 show that 293 cells exposed to NiV VLPs induced
syncytium formation and that this process was neutralized by
NIV-specific antibodies; prior incubation with the unrelated JV
antibodies failed to block this process.
with NiV-specific
NiV VLPs as immunogens in Balb/c mice
Mice in groups of five were inoculated subcutaneously with four
different concentrations of purified VLPs and boosted as described
Figure 1. The amount of M, F and G plasmids used at transfection has a bearing on the level of particle formation based on
minigenome-encoded reporter gene levels in VLP-infected cells. Cells were transfected with increasing concentrations of M, or F or G
expression plasmids (indicated by a triangle) while keeping the concentration of the other two plasmids fixed. They were co-transfected with the
previously optimized minigenome and N, P and L constructs [16,57]. Forty eight hours post transfection, the cell SUPs were clarified by centrifugation,
and same volume of SUP from each sample was used to infect new cell monolayers (VLP infected) which were transfected 24 hours previously with
the core plasmids N, P and L to support replication of the VLP-incorporated minigenome RNA; the VLP-infected cells were harvested 48 hours later
for reporter gene analysis. Panel A shows the plasmids transfected in each reaction. Panel B: shows minigenome-encoded CAT activity in VLP
infected cell monolayers. Lane 1 is a negative control. Absence of CAT activity in duplicate lanes 2 and 3 indicates that VLP formation, and
consequently VLP-incorporated minigenome transfer and expression, does not occur in the absence of M, F and G proteins. The results in lanes 4
through 15 shows that CAT levels varied in VLP infected samples depending on the concentration of M, F and G constructs used at transfection. Thus,
the amount of M, F and G plasmids used at transfection had a bearing on the level of particle formation, and the consequent CAT reporter gene
transfer and expression. CAT activity in the VLP infected reactions appeared optimal in the boxed lanes 7 and 8. Further analysis of CAT levels in the
linear range (data not shown) demonstrated that optimal VLP formation was achieved with the ratios of M, F and G expression plasmids of 3:1:1
(lane 7).
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under Methods. The negative control group of five mice were
inoculated with sterile endotoxin free PBS for each inoculation.
The mice were bled from the submandibular vein on the day
before primary inoculation, and then on days 14, 21, 28 and 35.
For initial evaluation, sera from each treatment group were
pooled, and the IFA method used to determine levels of NiV-
specific antibodies. The results in Figure 5A show that titers
(reciprocal of the highest serum dilution showing reactivity)
increased with time post primary inoculation, i.e., the highest
titers (1:2560) were seen on day 35. Titers also increased with VLP
dosage although by day 35, the three higher treatment groups
seemed to produce similar titers. As expected, the mice in the
negative control group remained nonresponsive.
All sera were tested individually by plaque reduction neutral-
ization method by doubling dilution of each sample (1:5 to 1:80) as
Figure 2. Co-expression of NiV proteins G, F and M results in
the formation substantial quantities of VLPs morphologically
resembling NiV virions. VLPs released in the transfected cell-
supernatant were harvested and purified as described under Methods,
and viewed by EM and cryoEM to evaluate their morphology. Under
optimized conditions, substantial amounts of VLPs were produced, (A)
shows VLP-containing band in the sucrose gradient. Negatively stained
sample in (B) show numerous well preserved VLPs. Selected VLPs which
were magnified (C) to show clearly the spikes of the glycoproteins
present on the VLP surface; an occasional particle had what appeared to
be a double fringe (shown with an arrow), a feature normally thought
to be associated with Hendra virus particles [9]. (D) Shows cryoelectron
micrograph of one of our VLPs. The glycoprotein spikes and their spatial
arrangement are seen here even more clearly. (E) Shows functional
assembly and immunoreactivity of NiV glycoproteins at the VLP surface.
Unfixed particles were stained by immunogold labeling technique
using NiV-specific polyclonal antibody and gold labeled secondary
antibody. Unfixed particles were used so that only the surface proteins
would be available for immunoreactivity. The Figure shows two VLPs
with gold-decorated proteins on the VLP surface.
doi:10.1371/journal.pone.0018437.g002
Figure 3. VLP-incorporated NiV proteins. The Figure shows
western blot analysis of NiV VLPs to verify their composition. The VLPs
were processed and analyzed by SDS-PAGE as described using
manufacturer’s instructions. VLP protein bands corresponding in size
to NiV proteins G, F0, F1and M were clearly visible.
doi:10.1371/journal.pone.0018437.g003
Figure 4. NiV VLP-induced syncytia in 293 cells is blocked by
prior treatment with NiV-specific antibody. NiV VLPs were pre-
incubated for one hour at 37uC with either NiV specific antibody or
Junin virus-(JV) specific antibody, or with OPTI-MEM I medium only
(untreated VLPs) before inoculating onto 293 cell monolayers grown
overnight in 60 mm dishes. The plates were incubated overnight at
37uC and stained with crystal violet. The results show VLP-mediated
formation of syncytia (a and b) that were blocked (c) when the VLPs
were pretreated with NiV-specific antiserum but not blocked (d) when
the VLPs were pre-treated with Junin virus-specific antibody. Images e
and f show uninfected 293 cells. Arrow points to syncytia.
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described under Methods. The results (Figure 5B) showed distinct
association between VLP dosage and the ability to mount a
neutralizing antibody response. Mice inoculated with the two
highest VLP doses (treatment groups C and D) were each able to
induce neutralizing antibodies by day 35. When samples from
mice receiving the two lower concentrations of VLPs (3.5 ug/dose
and 1.75 ug/dose, corresponding to treatment groups B and A
respectively) were similarly tested, 3 of 5 and 1 of 5 mice
respectively induced neutralizing antibody response; the titers
ranged from 1:5 to .1:80. As expected, the control mice did not
induce neutralizing response.
NiV VLP-induced activation of genes involved in
signaling innate immune response
A PCR array format (SABiosciences) was used to investigate
modulation in transcription profile of 84 genes involved in innate
immune responses to include TLR signaling family and members
of the downstream signaling pathways, NFKB, NF/IL6, IRF and
JNKp38. These genes represent key sensors of non-self that
signal, and ultimately shape the nature of innate immune
response that modulates the type and duration of adaptive
immune responses [66,67]. The differential expression of genes in
VLP-exposed 293 cells relative to the ‘‘mock’’ infected 293 cells
was measured by real-time PCR. Same concentration of total cell
RNA from the VLP-stimulated and the ‘‘mock’’ stimulated
control cells were used for first strand synthesis and Sybr green
PCR amplification of the relevant genes as described under
Methods. The integrity of RNA in each sample was confirmed by
gel electrophoresis (Figure 6A). Data representing the differential
transcription profile of VLP exposed vs. ‘‘mock’’ stimulated cells
is shown as a heat map (Figure 6B). A 4-fold cutoff threshold was
used to determine modulation in gene expression. We noted
significant VLP-stimulated up-regulation (89 fold and 7 fold) in
the expression of NFKB2 and TBK1 genes respectively. Close to
four fold (3.9 fold) up-regulation was noted also in IL-8 and
MAPK8 genes. NFKB2 and IL-8 are target genes in the
downstream NFKB pathway, and TBK1 which are in the IRF
and JNK/p38 pathways respectively.
Discussion
Using a minigenome-based functional assay, we have estab-
lished conditions (described under Results and shown in Figure 1)
that have allowed us to produce substantial quantities of NiV
VLPs to be able to undertake the studies described in this
manuscript. We have shown that these particles are functionally
assembled, biologically active and are able to induce innate
immune responses, and a neutralizing antibody response. Native
VLPs have been used to study various aspects of the virus lifecycle,
as carriers to deliver heterologous proteins for vaccination, and to
deliver small molecules for gene therapy purposes. Particularly
importantly, they have been used highly effectively as vaccines in
their native form [31,32,39,40,41].
No vaccine for NiV disease has been developed so far that
would be both safe and protective for humans. The two
vaccination strategies that have already been explored are the
canary pox-based vector approach [26] and soluble subunit
approach [28,29]. NiV vaccine by the former method is
undergoing development as a veterinary vaccine [26]. The same
approach is being evaluated for human use vaccines, mainly for
the prevention of HIV and AIDS [27]. The subunit approach has
limitations as already mentioned above [28,29,30,31,32]. One
particular challenge revealed by studies that tested a soluble NiV
G protein-based subunit vaccine formulated with adjuvant is the
potential difficulty of eradicating infection in the central nervous
system. In that study [28], live virus was present in the brain of one
cat, and viral RNA was present throughout the 21 day post-
challenge period in the brains of the remaining challenged
animals. A recently reported vaccination strategy [34] requires
simultaneous inoculation of two VSVDG vectors, one expressing
NiV G, and the other expressing NiV F proteins. It was of interest
to note that supernatants of cells co-infected with these two
defective viruses were infectious and could be passaged indefinitely
in the absence of VSV G trans-complementation. This vaccination
approach seems promising since self-propagated stock of these two
viruses induced robust neutralizing antibody response in mice.
However, potential pathogenicity of VSV-based vaccine vectors
remains a concern [34,35]. The potential of a recombination event
Figure 5. NiV VLP-induced immune response in Balb/c mice. NiV-specific antibody levels of serum samples from mice immunized
subcutaneously three times were measured by IFA and by Bio-Plex microsphere methods. Neutralizing antibody response was evaluated by PRNT50.
The experiments were done in duplicate. A: For evaluation by IFA, sera from each treatment group were pooled for analysis. The results show
serocoversion for each of the four treatment groups. In general, the titers increased progressively with time and with the VLP dose although by day
35, similar titers were seen with the three higher VLP doses. B: Shows neutralizing antibody titers (PRNT50) in sera from each mouse collected on the
stated days. Neutralizing antibodies were seen starting on day 28 after primary inoculation. The response was again clearly dose dependent; all mice
in the two highest treatment groups (C and D) showed neutralizing response by day 35. Such response was seen in 3 of 5 and 1 of 5 mice in the two
lower (B and A respectively) treatment groups.
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resulting in a single VSV vector virus expressing both these NiV
proteins is unlikely, but it may still be problematic for a human use
vaccine.
Native VLPs like the ones we have produced allow the viral
proteins to be presented to the immune system in the same
conformation as in the virion for effective B and T cell response
[31]. VLPs are particularly effective in producing a protective
antibody response because of their virus-like size range, their
particulate nature, and their virus-like dense, repetitive and
ordered surface structure [31,36]. The spacing of the antigenic
epitopes on the VLP is also optimal for B cell activation [31]: EM
analysis showed that our particles resembled the real virus in terms
of size and surface structure [63,64]. The image in Figure 2D is
the first elucidation of VLP structure of any paramyxovirus
imaged by CryoEM, and it provides a careful assessment of their
morphology; it alludes to a surface similar to that revealed for
measles virus by the same imaging technique (Dr. Elizabeth
Wright, Emory University). The proteins on the VLP surface are
clearly visible here; the average distance between the spikes was
9.13 nm and standard deviation was 1.72 nm. This is of interest
given that epitopes spaced between 5 and 10 nm are known to be
sufficient to drive optimal B cell activation [36].
NiV M, F, G and N protein-containing VLPs consistent in size
and morphology to the parental virus have also been reported in a
previous study which evaluated protein-protein interaction that
facilitate VLP formation [48]. However, in that study, most of the
particle-incorporated NiV F protein was predominantly in the
uncleaved precursor form. This finding is clearly distinct from ours
since our VLPs contained substantial amounts of cleaved F
protein, and this may have been related to ratios of the interacting
proteins expressed in 293T transfected cells. In a recent study of
NDV VLPs [49], the particle-incorporated proteins were reported
to have virus-like protein ratios, but the F protein remained in its
precursor form because the cleavage site required to produce the
fusion competent form was mutated by design. What effect a VLP-
incorporated non-fusogenic F protein may have, relative to the
fusogenic form, on the level and quality of VLP-induced immune
response is not clear at present since difference in immunogenicity
between fusion-competent and fusion-defective VLPs has not been
experimentally evaluated so far. However, a recent report suggests
that viral fusogenic membrane glycoproteins may enhance vaccine
potency [68].
Immunogold labeling of our unfixed NiV VLPs confirmed that
the surface proteins in our VLPs were functionally assembled and
they were biologically active (Figure 2E). We could deduce the
presence of biologically active G and F proteins on the VLP
surface by the fact that they were able to induce the formation of
syncytia in 293 cells (Figure 4); this is a process that requires the
interaction of both the surface glycoproteins, the attachment
protein G, and the fusion competent F protein, when they come in
contact with the cognate receptor-bearing cells. Formation of
syncytia or multinucleated cells in replication competent envel-
oped viruses, especially paramyxoviruses, is induced by a process
that is described as ‘‘fusion from within’’, and it can be blocked or
neutralized by prior treatment of the virus with specific antisera. In
contrast, ‘‘fusion from without’’ is induced by non-replicating
viruses at high multiplicities of infection, and it too can be blocked
by pretreatment with virus-specific antibodies ([69], and references
therein; [70]). Our non-replicating particles likewise induced
syncytia formation in 293 cells that could be neutralized with NiV-
specific antibodies (Figure 4). To our knowledge, this is the first
study describing fusion from without induced by VLPs of any
paramyxovirus, or any other enveloped viruses, although it has
been described for the VLPs of the non-enveloped rotavirus [71].
The mechanism(s) of fusion from without is not clear but two
models have been proposed [72,73]; one proposes that particles
connecting adjacent cells effectively promote fusion between them,
and the other is that when particles decorated with the surface
glycoproteins fuse with the target cell membrane, the glycoprotein
complexes diffuse freely in the lipid bilayer, and mimic fusion from
Figure 6. VLP-induced modulation in transcription profile of genes involved in signaling of innate immune response in 293 cells by
PCR Array. 293 cells were grown overnight in 60 mm dishes and were infected with 10 mg of purified VLPs suspended in OPTI-MEM (Invitrogen).
Mock infected cells served as negative control. The inoculum was adsorbed on the cell monolayers for 3 hours at 37uC when it was supplemented
with fresh OPTI-MEM and further incubated overnight when total cell RNA was extracted according to the manufacturer’s (SA Biosciences)
instructions. A: shows the integrity of the RNA used for this analysis. Note the ,2:1 ratio of 28S:18S which is a good indication of the integrity of the
RNA. Equal concentration of the RNA from the mock and VLP-exposed cells was used for expression profiling by RT2PCR Profiler PCR Array according
the manufacturer’s (SABiosciences) instructions. B: Shows the heat map, it is a visual illustration of the relative expression levels in the VLP-stimulated
vs. the ‘‘mock’’ stimulated control cells of the all the genes in the array: The four genes differentially expressed by a factor of ,4 fold or greater
(shown as red squares) are listed below the heat map.
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within. The type of VLP-induced syncytia formation and eventual
cell death is also not known. We are in the process of investigating
it.
Neutralizing antibody response is the critical correlate of
protection mediated by prophylactic vaccines [53,54] and native
VLPs promise to be highly effective prophylactic vaccines for
paramyxoviruses like NiV, and others like NDV and measles
where neutralizing immune response is known to play a pivotal
role in protection against disease [53,54,55,74]. Our VLPs were
highly effective immunogens, and all, especially in the three higher
treatment groups produced high levels of response by day 35
(Figures 5A). Importantly, NiV VLPs were able to induce
neutralizing antibodies. This response was clearly dose-dependent
(Figure 5B). All ten mice receiving a primary inoculation of 7 or
14 mg VLPs (subgroup C and D) were able to produce such
response; but even of those animals that received a first dose of
only 3.5 or 1.75 ug/mouse (treatment group B and A respectively),
3 of 5 and 1 of 5 produced neutralizing antibodies. Neutralization
antibody response was first seen on day 28, and increasing titers
were seen in some animals within a week of it; we believe that this
response, induced by our non-replicating and potentially safe
particles, formulated without adjuvant, compares favorably with the
levels of such response induced at an equivalent time point by
some replication competent pseudotype viruses [34].
Immunogenicity to native VLPs has been reported previously
for one other paramyxovirus namely NDV [49]. In that report,
immune response to NDV VLPs was evaluated by primary
inoculation of mice intraperitoneally with VLP concentrations
ranging between 10 and 40 mg, and a booster dose of 10 mg,
without adjuvant. NDV-specific titers by ELISA were high in each
mouse in each treatment group. Neutralizing antibody response to
20 and 40 mg of these particles was also detected.
The nature of innate immune response dictates the type and
duration of adaptive immune response [67,75]. The mechanism
by which NiV VLPs are recognized by host cells and trigger the
induction of innate immune response, and how this translates into
effective adaptive immunity is not known. Here we have taken the
first step (Figure 6) towards understanding this process. With the
experimental conditions as described, we observed VLP-induced
activation of some of the genes that are known to be involved in
the induction of an effective innate immune response [75]. Results
presented in the heat map in Figure 6 show that relative to the
‘‘mock’’ treated cells, NFKB2 gene (in the NFKB pathway) was
up-regulated 89 fold as a result of VLP exposure, and TBK1 (in
the IRF pathway) was 7 fold higher. In the light of these findings,
we are testing PCR array expression profiles of the same set of 84
genes in 293 and other cells at earlier and later time points to
identify their upstream effectors, and NiV VLP-responsive
signaling networks. In this respect, the murine system, with the
many available immunological reagents and knockout strains may
provide the best system to identify these host sensors. Currently
there is minimal information on live NiV infection-responsive cell-
signaling changes [76] and there is none on array-based
transcriptional alterations for comparative analysis. Likewise, it
has not been possible to compare the NiV VLP-induced
transcription modulation with those induced by other paramyxo-
virus VLPs since to our knowledge, such studies have not been
undertaken so far. Lastly, a growing number of reports point to
viral surface glycolproteins as relevant in host cell signaling and
triggering of innate immune response. We believe that particles
like NiV VLPs, with many virus-like properties (including their
surface glycoproteins organized to resemble the parental virus,
Figure 2D) would induce an effective innate immune response for
the promotion of the desired adaptive immunity [67,76].
Finally, as described above, our VLPs were highly effective as
immunogens, able to induce neutralizing antibody response in all
animals with primary inoculation of as little as 7 mg VLP protein
each. Fusogenic property of our VLPs may be critically relevant in
this regard in the light of recent findings, and would need to be
experimentally verified by comparing the potency of fusion-
competent and fusion-defective VLPs as vaccine [68].
In conclusion, we have been successful in producing substantial
quantities of NiV VLPs needed to characterize NiV VLPs, we
have demonstrated their many virus-like properties, and their
effectiveness as immunogens in Balb/c mice. These findings are
the basis on which we will be undertaking future challenge studies
in the hamster model of NiV disease [77].
Acknowledgments
We thank Dr. V. Popov and Julie Wen for their help and advice with
electron microscopy. Vishal Naik provided much valued technical
assistance. Joshua Lisinicchia, provided essential help at various stages of
the project.
Author Contributions
Conceived and designed the experiments: PW. Performed the experiments:
PW JB MS. Analyzed the data: PW JB MS. Contributed reagents/
materials/analysis tools: CB. Wrote the paper: PW. Reviewed the
manuscript and provided valuable comments and suggestions: CB LW.
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Virus-Like Particle-Based Nipah Virus Vaccine
PLoS ONE | www.plosone.org 10April 2011 | Volume 6 | Issue 4 | e18437