Rational design of thermostable vaccines by
engineered peptide-induced virus self-
biomineralization under physiological conditions
Guangchuan Wanga,b, Rui-Yuan Caob, Rong Chenc, Lijuan Mod, Jian-Feng Hanb, Xiaoyu Wangb,e, Xurong Xue,
Tao Jiangb, Yong-Qiang Dengb, Ke Lyuc, Shun-Ya Zhub, E-De Qinb, Ruikang Tanga,e,1, and Cheng-Feng Qinb,1
aCenter for Biomaterials and Biopathways, andeQiushi Academy for Advanced Studies, Zhejiang University, Hangzhou 310027, China;bDepartment of
Virology, State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, China;cKey Laboratory
of Molecular Virology and Immunology, Institute Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200025, China; anddBiomedical Center,
Sir Run Run Shaw Hospital, Hangzhou 310016, China
Edited by Bernard Roizman, University of Chicago, Chicago, IL, and approved March 22, 2013 (received for review January 5, 2013)
The development of vaccines against infectious diseases represents
one of the most important contributions to medical science. However,
vaccine-preventable diseases still cause millions of deaths each year
due to the thermal instability and poor efficacy of vaccines. Using the
human enterovirus type 71 vaccine strain as a model, we suggest
a combined, rational design approach to improve the thermostability
and immunogenicity of live vaccines by self-biomineralization. The
biomimetic nucleating peptides are rationally integrated onto the
capsid of enterovirus type 71 by reverse genetics so that calcium
phosphate mineralization can be biologically induced onto vaccine
surfaces under physiological conditions, generating a mineral exte-
rior. This engineered self-biomineralized virus was characterized in
detail for its unique structural, virological, and chemical properties.
Analogous to many exteriors, the mineral coating confers some new
properties on enclosed vaccines. The self-biomineralized vaccine can
be stored at 26 °C for more than 9 d and at 37 °C for approximately
1 wk. Both in vitro and in vivo experiments demonstrate that this
engineered vaccine can be used efficiently after heat treatment or
ambient temperature storage, which reduces the dependence on
a cold chain. Such a combination of genetic technology and biomi-
neralization provides an economic solution for current vaccination
programs, especially in developing countries that lack expensive
genetic engineering|vaccine design|shell
of the most successful medical interventions ever developed
by humans (1, 2). Live attenuated viruses provide an effective
vaccination strategy devised since the beginning of the vacci-
nology era, and vaccines against polio, smallpox, measles, ru-
bella, yellow fever, Japanese encephalitis, and influenza, for
example, have been used to immunize billions of children and
adults worldwide. However, there are still more than 17 million
deaths caused by infectious diseases every year, accounting for
25% of all deaths worldwide (3, 4). Most of the deaths are caused
by vaccine-preventable diseases and the underutilization of vac-
cines. This is especially true for the poorest countries, because
most vaccines are sensitive to heat and the cold chain is very dif-
ficult to maintain in countries with a minimal infrastructure. The
poor efficacy and instability of vaccine products lead to incomplete
immunization and a rapid loss of potency during storage and de-
livery, which severely limits the coverage of current vaccination
programs. For example, ∼50% of vaccine products are finally
discarded due to poor thermostability (5). Refrigeration is essential
for vaccines to maintain their quality. However, keeping vaccines
at low temperatures is difficult and expensive. The cold chain
consumes ∼80% of the total cost of vaccination programs (3, 6),
and it is not always reliable. The situation is even worse in de-
veloping countries and in the least-developed countries, where, due
to the lack of extensive and reliable refrigeration infrastructure,
over half of all deaths are caused by infectious diseases. Improving
he development of vaccines against infectious diseases is one
the efficacy and thermostability of vaccines is of great importance
and has been highlighted as a Grand Challenge in Global Health
by the Gates Foundation (www.grandchallenges.org).
New advances in genetic and materials sciences have created
the possibility of improving and stabilizing vaccine products.
Stabilizers, such as deuterium oxide, proteins, MgCl2, and non-
reducing sugars, have been introduced to produce stabilized
formulations of vaccines (7–9). By using reverse genetic tech-
physicochemical properties (10–12). In nature, biomineralization
is adopted by many organisms to improve their performance in
harsh environments. For example, living organisms, such as dia-
toms (13), mollusks (14), eggs, and some plants (15), have de-
veloped biomineral exteriors that play a protective role. It should
be noted that egg cells with mineral shells are extremely ther-
mostable and can be stored under ambient conditions. Un-
fortunately, most living organisms, including vaccines, cannot
generate such mineral shells due to the lack of biomineralization-
related proteins, which usually play a key role in the control of
biomineralization. Among biogenic minerals, calcium phosphate
(CaP), the major component of bones and teeth, is of interest
because of its unique biocompatibility (16), and it is now used as
a transfer agent and adjuvant (17). CaP represents an excellent
mineral shell candidate to stabilize vaccines. Recently, we have
shown that a protective CaP shell can be introduced onto some
virus surfaces in the presence of a high concentration of calcium
ions (18, 19). However, the biomineralization process requires
specific treatments and only occurs with selected premade viruses
stocks. A natural virus cannot induce biomineralization by itself
during its normal life cycle. In contrast, biomineralization in
organisms is generally controlled using biomacromolecules in
a mild biological system, and some polypeptides and macro-
molecules have been used to induce biomineralization (20–24).
For example, several peptides have been genetically incorporated
into the M13 bacteriophage and plant viruses as nucleators to
direct nanomaterial synthesis (25, 26) or as specific nucleators
for CaP mineralization in living systems (23, 27–29). Inspired by
this achievement, we propose to design rationally an engineered
virus carrying the selected biomimetic peptides that control the
Human enterovirus type 71 (EV71) is a typical nonenveloped
icosahedral virus belonging to the Picornaviridae family (30).
Author contributions: G.W., R.T., and C.-F.Q. designed research; G.W., R.-Y.C., L.M., J.-F.H.,
X.W., Y.-Q.D., and S.-Y.Z. performed research; R.C. contributed new reagents/analytic
tools; G.W., R.C., L.M., X.X., T.J., K.L., E.-D.Q., R.T., and C.-F.Q. analyzed data; and G.W.,
R.T., and C.-F.Q. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1To whom correspondence may be addressed. E-mail: email@example.com or rtang@zju.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
www.pnas.org/cgi/doi/10.1073/pnas.1300233110PNAS Early Edition
| 1 of 6
Picornaviruses cause acute diseases in humans and animals, in-
cluding polio, hepatitis A, and foot-and-mouth disease. Live at-
tenuated picornavirus vaccines are widely used with high efficacy.
In this study, we protected the EV71 vaccine strain with an egg-
shell-like exterior, which is spontaneously formed under physio-
logical conditions due to the engineered CaP biomineralization
peptide. The biomineralized engineered vaccine exhibits overall
improved thermostability and immunogenicity, and it can be used
efficiently even after a week’s storage at room temperature. Thus,
by the genetically induced self-biomineralization of the vaccine.
Genetic Engineering. To endow a virus with the capacity of self-
biomineralization, we aimed to display nucleating peptides on
the virion surface to enhance its capacity to initiate CaP miner-
alization. Among nucleating peptides, two types of representative
nucleators, a phosphate chelating agent (N6p) or calcium che-
lating agents (NWp and W6p), were selected. N6p is a reported
CaP-binding peptide identified through phage display, and its
binding effect is thought to be due to phosphate-chelating
domains (SVKRGTSVG and VGMKPSP) (31). NWp is derived
from the N-terminal 15-residue fragment of salivary statherin,
a CaP high-affinity protein, and it has been used in biomimetic
mineralization (32). W6p is an acidic analog of the amino-ter-
minal 15-residue fragment of salivary statherin and core motifs of
dentin matrix protein 1, an acidic protein that can trigger CaP
formation in vitro by binding calcium ions (33). The site between
amino acids 100 and 101 of the β-(BC)-loop of viral protein 1
(VP1) (Fig. 1A) has been demonstrated to be an appropriate
insertion site for the placement of an engineered peptide on the
viral surface (34, 35). The coding nucleotides of the above-men-
tioned nucleating peptides (Fig. 1A) were cloned into the in-
fectious full-length cDNA of the attenuated EV71 strain A12
using standard DNA recombination technology (36). The engi-
neered viruses carrying nucleating peptides were recovered by
transfecting in vitro-transcribed RNAs into human rhabdomyo-
sarcoma (RD) cells, and they were named EV71-N6, EV71-NW,
and EV71-W6. DNA sequencing and indirect fluorescence assay
(IFA) results (Figs. S1 and S2) confirmed that the nucleating
peptides were successfully engineered into the viral genome.
These genetically modified viruses exhibited similar growth pat-
terns to the parental EV71 (Fig. 1B) and formed similar small
plaques in RD cells (Fig. 1C), indicating that the incorporated
the Phyre2 (Protein Homology/Analogy Recognition Engine 2)
server showed that the insert peptide protruded from the natural
VP1 proteins, and 60 copies of the nucleating peptides were pre-
dicted to be uniformly displayed on the surface of EV71 without
affecting the original structure of the EV71 virion (Fig. 1D).
Self-Biomineralization. The engineered viral vaccines were in-
cubated in calcium-enriched DMEM (calcium concentration of
5.5 mM) to evaluate their biomineralization capacity. Usually,
viral particles can only be concentrated by ultracentrifugation
under complicated conditions. When the viral particles were
mineralized, they could be readily separated and concentrated
by normal-speed centrifugation (18,000 × g for 10 min) due to
the high relative gravity of the attached mineral phase. Thus, the
biomineralization efficacy could be estimated by the ratio of viral
particles in the supernatant and precipitate using plaque assays
or quantitative RT-PCR (qRT-PCR) assays. For native EV71,
few infectious viral particles were precipitated and most virions
remained in the supernatant. As expected, native EV71 could not
induce effective biomineralization (Fig. 2A). There was no sig-
nificant difference between EV71 and EV71-N6, meaning that the
integrated N6p peptide failed to induce vaccine biomineralization.
However, the engineered EV71-NW and EV71-W6 viruses
demonstrated enhanced biomineralization capacity. For EV71-
W6, over 90% of infectious virions could be separated by cen-
trifugation due to mineral incorporation (Fig. 2A). Dot blot
assays revealed that the biomineralized EV71-W6 could barely
be detected using EV71-specific antibodies. This experimental
result implied that the capsid proteins were shielded by the
precipitated mineral phase. In contrast, only some EV71-NW
proteins and negligible quantities of EV71-N6 proteins were
shielded by minerals due to their insufficient biomineralization
(Fig. 2A, Upper).
SEM (Fig. 2B) and transmission EM (Fig. 2C) revealed that
biomineralized EV71-W6 had a diameter of ∼40 nm. The core-
shell structure of the biomineralized vaccine was confirmed: The
particle core was a 30-nm negatively stained (by phosphotungstic
acid) virion (Fig. 2C, Inset), and it was surrounded by a 5- to
10-nm inorganic layer. Energy dispersive X-ray spectroscopy
showed that the inorganic layer was CaP (Fig. 2B, Inset), and X-
ray diffraction indicated that the mineral phase was amorphous
(Fig. S3). The biomineralized EV71-W6 with the amorphous CaP
coating was called EV71-W6-CaP. Importantly, the mineral ex-
terior was stable at physiological pH > 7.0 but could be dissolved
from themineral coatingwas also demonstrated by dot blotassays
biomedical applications because slightly acidic conditions are
frequently involved in the intracellular processing of internalized
particles. We hypothesized that the mineral shell would not in-
fluence the vaccine’s antigenicity, which was demonstrated by our
following in vitro and in vivo assessments.
It should be emphasized that the serial passage of EV71-W6 in
RD cells did not cause any loss of the W6p coding sequence. In
our experiment, the coding sequence of W6p was still genetically
stable after passaging 10 times, according to the sequencing
peptides. (A) EV71 genome and the insertion site of the β-(BC)-loop of VP1.
(B) Plaque morphologies of parental EV71 and engineered viruses in RD cells.
(C) One-step growth curves of parental EV71 and engineered viruses in RD
cells [multiplicity of infection (MOI) = 0.1]. (D) Homology modeling of the
mutant viral protein. EV71 capsid proteins VP1, VP2, and VP3 are shown in
cyan, yellow, and orange, respectively. The inserted peptides are marked
with blue, and 60 copies are uniformly distributed on the surface of the
engineered EV71 virion. These peptides may induce in situ biomineralization
to form a CaP mineral exterior (gray) for the vaccine.
Design and characterization of engineered EV71 carrying nucleating
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results of the viral genome (Fig. S2), and the progeny EV71-W6
virus could still be efficiently self-biomineralized (Fig. 2D).
Thus, self-biomineralization became an inheritable trait of
EV71-W6, and future generations retained the inheritable CaP
shell, which was attributed to the combination of genetic engi-
neering and biomineralization.
Enhanced Immunogenicity. EV71-W6-CaP and EV71 caused sim-
ilar cytopathic effects and plaque morphologies (Fig. 3A) in RD
cells. One-step growth curves revealed similar growth patterns.
The titer of EV71-W6-CaP was slightly higher than that of
EV71-W6 at 12 h postinfection (Fig. S5). The results from IFA
with EV71-specific antibodies showed that EV71-W6-CaP was
infectious; the viral antigens were expressed as in EV71-W6 and
parental EV71 (Fig. 3B, green), and the cell nucleus was stained
by DAPI (Fig. 3B, blue). The expression of viral antigens was
higher after CaP mineralization at the early stage in RD cells
(Fig. 3B). Such infection enhancement was more obvious in Vero
cells (approximately onefold higher) and THP-1 cells (greater
than twofold higher). Furthermore, the biomineralization treat-
ment did not enhance virulence in animals, and EV71-W6-CaP
remained nonvirulent to suckling mice. These results revealed
that CaP biomineralization did not alter either viability or the
original attenuation of virus. It follows that a biomineralized
vaccine could be used directly in biomedical applications.
The immunogenicity of the biomineralized vaccine was also
evaluated in vivo. The results revealed that at 4 wk post-
immunization, EV71-W6-CaP induced ∼3.5-fold higher serum
IgG (Fig. 3C) and almost twofold higher neutralization antibody
titers (Fig. 3D) in mice than native EV71 did. EV71-W6 induced
similar levels of IgG and neutralizing antibodies as EV71. The
results indicated that it is the biomineralized CaP exterior, rather
than the genetic engineering itself, that contributed to the
immunogenicity enhancement of vaccines. Nevertheless, the CaP
shell formation was induced by the integrated W6p.
Improved Thermostability. EV71-W6-CaP, EV71-W6, and paren-
tal EV71 were incubated at room temperature (26 °C) for
varying numbers of days, and their subsequent infectivities were
titrated by plaque assays. These data are represented in a loga-
rithmic scale as a function of the storage time. EV71-W6 and
parental EV71 shared similar thermal inactivation kinetics, with
inactivation rate constants of 0.25246 d−1and 0.26011 d−1,
respectively (Fig. 4A). They exhibited ∼1-log10plaque-forming
unit (PFU; 90%), 2-log10PFU (99%), and 4-log10PFU (99.99%)
losses after 3, 6, and 14 d of storage, respectively. According to
indices of the World Health Organization requirement for vac-
cine efficacy (37), no more than a 1-log10reduction of the initial
titer is permitted. Therefore, the room temperature storage of
EV71 or EV71-W6 should be less than 3 d, and the genetic
engineering alone did not alter viral thermostability in our case.
After the combination of self-biomineralization, the resulting
EV71-W6-CaP exhibited a significantly slower inactivation rate
(0.09921 d−1) and its storage could be prolonged to more than
9 d at 26 °C. In tropical areas, ambient temperature is always
>30 °C or even >40 °C, such that an examination of the vaccine
storage period at high temperatures is also of importance. Ac-
celerated degradation tests with samples subjected to physio-
logical temperature (37 °C) and tropical zone temperature (42 °C)
were performed to estimate vaccine potency loss. CaP miner-
alization reduced the inactivation kinetics of the engineered
vaccine EV71-W6 nearly threefold, according to its inactivation
rate constants (Fig. 4 B and C). At 37 °C, EV71-W6, similar to
parental EV71, lost ∼1 log10PFU at 44 h, whereas EV71-W6-
CaP lost only 1 log10PFU at 168 h (Fig. 4B). At 42 °C, EV71-
W6-CaP was viable even after storage for 9 h, whereas the
storage times of EV71-W6 and parental EV71 should be less
than 2.5 h (Fig. 4C). However, the same treatment could only
improve the thermostability of native EV71 by less than 1.4-fold
due to its poor biomineralization capacity (Fig. S6). These results
indicated that CaP biomineralization greatly improved vaccine
thermostability and the improvement was directly related to
The immunogenicity of the heat-treated vaccines was then
verified in animals. All vaccines were incubated at 37 °C for 5 d,
and groups of BALB/c mice were then s.c. immunized. EV71-
W6-CaP induced a similar amount of neutralization antibodies
after storage, whereas a significant (∼75%) decrease in EV71-spe-
cific IgG titers (Fig. 5A) and a more than 50% decrease in neu-
was determined. Because the frequencies of IFN-γ–secreting cells
were commonly examined to determine the T-cell response, the
levels of EV71-specific IFN-γ–secreting splenocytes induced by
native and biomineralized vaccines were determined by enzyme-
linked immunospot assays. The stored EV71-W6-CaP could in-
duce high frequencies of EV71-specific IFN-γ–secreting cells in
mice, and no significant decrease was observed compared with the
fresh virus without storage (Fig. 5C). However, the same storage
resulted in a significant ∼40% decrease in the frequencies of
EV71-W6 and parental EV71 (Fig. 5C). Collectively, the geneti-
cally induced biomineral shell significantly improved the thermo-
stability of the vaccine to the point that it could be viable for use in
a vaccine program even after a week’s storage at ambient tem-
engineered vaccines could be less dependent on the cold chain.
By genetically engineering potential nucleating peptides onto the
surfaces of live viruses, we rationally designed a self-biomineralized
virus with improved thermostability and immunogenicity. This
CaP biomineralization capacity was given to EV71-W6 by ge-
netically engineering a CaP nucleating peptide onto the viral
After biomineralization, the viral surface proteins were immunologically
detected by dot blot assays to indicate the stealth conditions by the CaP
exterior. (A, Lower) Following spontaneous mineralization and centrifuga-
tion, the biomineralization efficacy was determined by examining the in-
fectious viral particles in the supernatant and pellet using plaque assays;
error bars represent SDs (n ≥ 4, Student’s paired t test, one-tailed, **P <
0.01). (B) SEM images of CaP-mineralized EV71-W6; the inserted energy
dispersive X-ray spectroscopy shows the presence of Ca and P on the vaccine
surfaces. (Scale bar: 100 nm.) (C) Transmission EM images of biomineralized
EV71-W6. (Inset) Image shows that each particle contains a negatively
stained vaccine. (Scale bar: 100 nm.) (D) Biomineralization capacity of
progeny EV71-W6 was determined by quantifying viral RNA using qRT-PCR
assays; error bars represent SDs (n ≥ 3).
Self-biomineralization of genetically engineered vaccines. (A, Upper)
Wang et al.PNAS Early Edition
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capsid. Sixty copies of the engineered peptide provide CaP nu-
cleation sites on the vaccine surface because they can concen-
trate calcium ions from incubation solutions, increasing CaP
supersaturation around the vaccine and leading to localized
mineralization. In this study, EV71-W6 and EV71-NW displayed
improved CaP biomineralization capacity, whereas EV71-N6 did
not exhibit significantly increased biomineralization. This result
indicates that cationic calcium ion-chelating peptide motifs,
rather than phosphate ion-chelating motifs, can efficiently initi-
ate CaP mineralization, which is also in accordance with the
previous understanding that anionic peptides are more effective
in eliciting CaP biomineral generation (23, 33, 38, 39). That
EV71-W6 displayed better self-biomineralization capacity than
EV71-NW may due to the higher density of anionic Glu and Asp
amino acids in W6p. It has been documented that Glu and Asp
are the most active residues for inducing biomineralization in
nature (40, 41). In comparison to W6p, NWp cannot initiate CaP
mineralization efficiently because the Ser residues in NWp are
replaced by Asp and Glu in W6p. The abundant Asp and Glu
residues make W6p more active in CaP mineralization. This
phenomenon reveals the relationship between biomimetic pep-
tides and biomineralization controls. We conclude that the bio-
mineralization capacity of living organisms can be regulated by
the genetic engineering of nucleating peptides. Again, our study
confirms the importance of Glu and Asp residues in CaP bio-
mineralization proteins. Compared with our previous work on
the biomineralization of purified adenovirus by incubation with
calcium ions and phosphate titration and the biomineralization
of enveloped viruses in a high-calcium environment, this genetic
strategy confers to the virus an automatic self-biomineralization
capacity under physiological conditions, which benefits uniform
formation of a reliable mineral exterior.
phologies of EV71, EV71-W6, and EV71-W6-CaP in RD
cells. (B) Indirect immunological fluorescence of re-
covered virus in Vero cells, RD cells (MOI = 0.1), and
THP-1 cells (MOI = 1) at 12 h postinfection; the cell
nuclei were stained by DAPI (blue). (Magnification: B,
40×.) (C) EV71-specific serum IgG titers of mice (n ≥ 5)
at 4 wk postimmunization. (D) Serum neutralization
antibody titers of mice (n ≥ 5) at 4 wk post-
immunization were determined
neutralization assays (Student’s paired t test, one-
tailed, *P < 0.05).
Biological characterizations. (A) Plaque mor-
infectivity is represented in a logarithmic scale as a function of incubation time (n ≥ 4); error bars represent SDs. The calculated average inactivation rate constants
for EV71, EV71-W6, and EV71-W6-CaP, as shown in the figures, were KEV71= 0.25246 d−1, KEV71-W6= 0.26011 d−1, and KEV71-W6-CaP= 0.09921 d−1at 26 °C; KEV71=
0.01590 h−1, KEV71-W6= 0.01639 h−1, and KEV71-W6-CaP= 0.00594 h−1at 37 °C; and KEV71= 0.29896 h−1, KEV71-W6= 0.31073h−1, and KEV71-W6-CaP= 0.11117 h−1at 42 °C.
In vitro tests of virus thermostability. Thermal-inactivation kinetics were determined at 26 °C (A), 37 °C (B), or 42 °C (C). The remaining percentage of
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The spontaneous release of the enclosed vaccine from the
mineral shell is key to ensure the availability of biomineralized
vaccine. The CaP biomineral phase exhibits its advantage in
protection and release due to its pH-dependent stability. It has
been found that the mineral exterior is stable under extracellular
conditions, because the pH of body fluids is ∼7.4 (42). However,
when the biomineralized vaccine is taken up by cells, the in-
ternalization process is often accompanied by acidic conditions,
such as endosomes, which can induce the spontaneous degrada-
tion of the CaP exterior. After biomineralization, the CaP shell
will not hinder the infectivity of vaccines, and the biomineralized
virus shares similar replication and growth patterns with the native
virus (Fig. 3A and Fig. S5). The entry of the biomineralized virus
into host cells may be more like the noninfectious virus-like par-
ticles that have differential uptake by dendritic cells and other
antigen-presenting cells. Our results reveal that higher virus titers
and viral protein expression are observed in EV71-W6-CaP–
infected cells at the early stage of infection, and the enhancement
of infectivity is more obvious in viral receptor-deficient cells (Fig.
3B), which is in accord with our previous findings (19). This in-
fectivity enhancement is restricted to the primary infection when
the virus possesses mineral shells; therefore, it may favor the ef-
ficacy of vaccines without elevating their virulence. Additionally,
because the CaP exterior can shelter the vaccine and help the
vaccine escape direct clearance, the eliciting of IFN-α and IFN-β
in EV71-W6-CaP–infected cells is delayed (Fig. S7).
The vaccine with a protective shell, EV71-W6-CaP, exhibits an
ideal thermostability,extending its storage time by nearlythreefold.
It can be understood by means of the isolation of enclosed vaccine
from destructive external adverse factors by means of the sponta-
neously biomineralized CaP exterior, which may protect viral pro-
teins from thermal inactivation by insulating the direct interactions
between water solutions and vaccines (14). Furthermore, an at-
tachmentofthemineral phase can stabilizethe protein andsubunit
structures on vaccine surfaces by means of the direct binding effect
betweenCaPandbiomineralizationpeptide.If the immunogenicity
enhancement conferred by this CaP biomineralization is taken into
consideration, the total efficacy of the vaccine can be significantly
improved by this modification. The self-biomineralized vaccines
exhibit higher efficacies in vivo even after storage at ambient tem-
peratures, which simplifies their application.
Developing regions lacking extensive, reliable refrigeration
infrastructures are particularly vulnerable to vaccine failure,
which, in turn, increases the burden of disease. It is estimated
that at least 3 million children die of infectious diseases that are
preventable with presently available but underused vaccines, and
the death toll of vaccine-preventable meningitis, pneumonia, and
diarrheal disease remains a major cause of death in Africa (4).
Therefore, the maintenance of vaccine efficacy without a cold
chain has the potential to extend immunity against deadly dis-
eases to the world’s poorest communities. Development of a ro-
bust vaccine is promising for a wide variety of vaccines. Inspired
by nature, we engineered nucleating peptides into vaccines by
reverse genetics to produce self-biomineralized vaccines with
a mineral exterior as a proof of concept. The exterior confers
improved thermal stability and immunogenicity to the enclosed
vaccine. Our results indicate that under a wide range of ambient
temperatures, the biomineralized vaccine can be guaranteed for
use in a health clinic without any access to refrigeration for at
least 1 d, which is often the situation in clinics located in tropical
climates with unreliable cold chains. Most reconstituted vaccines
lose potency rapidly and must be used within a few hours (9, 37),
and the vaccine exposure time out of the cold chain should be
kept as short as possible. This achievement means that we may
be able to create a thermostable vaccine in a liquid formulation
without freeze-drying and reconstitution before injection. Fur-
thermore, current immunization programs in resource-limited
areas are greatly hampered by the terminal distribution of vac-
cines from local storage centers to immunization clinics or mo-
bile on-site utilization. The biomineralized vaccine described in
our study will no doubt gain additional exposure time. Also,
temperature monitor studies have indicated that the deviation
from ideal storage temperature is inevitable (43) and that oc-
casional that cold chain breakdown cases occur. The enhanced
thermostability of a vaccine is thereby supposed to reduce its
validity loss during storage and transportation. Robust liquid
vaccines produced with less dependence on cold chains can
provide benefits by reducing waste, ensuring potency, and de-
creasing financial cost, which are highly helpful to expand the
global coverage of vaccination programs (44, 45).
Unlike previous attempts, we combined biomimetic minerali-
zation and genetic engineering to produce self-biomineralized
vaccines, and the resulting vaccines have inheritable performance
improvements. Such biomineralization capacity resulting from
genetic engineering is inheritably stable, and our study shows that
the mineralization efficacy and improved thermostability can be
maintained after serial passages. Vaccine biomineralization can
be spontaneously induced under biological conditions after ge-
netic modification so that the improvement is costless, which
allows pharmaceutical companies to manufacture thermostable
vaccines economically. Furthermore, the strategy combines the
advantages of genetic engineering and biomineralization, and it is
easily reproduced. Currently, most viral protective antigens and
virion structures have been resolved so that they can readily be
genetically reconstructed. Furthermore, pseudovirus particles
and other biological products can also be modified to display
a specific nucleating peptide that will confer biomineralization
capacity onto the product. In general, analogous improvement by
genetically induced self-biomineralization can be used in more
viral vaccines. This strategy may hold significant promise for vac-
cination programs by providing a feasible path toward large-scale
thermostable vaccine production, with advantages that include be-
ing fast, inexpensive, and easily adapted to any other live vaccines.
However, as with any other new technologies, the manufacturing
process and safety profile should be carefully evaluated before the
use of these thermostable vaccines in clinical practice.
after 5 d of storage at 37 °C (B). Antibody titers were determined at 4 wk postimmunization (n ≥ 5); error bars represent SDs. (C) Frequencies of EV71-specific INF-
γ–secreting splenocytes in the immunized mice before and after storage. Splenocytes were isolated from the immunized mice at 2 wk postimmunization and
subjected to enzyme-linked immunospot assay (n ≥ 3); error bars represent SDs (Student’s paired t test, one-tailed, *P < 0.05, **P < 0.01). SFC, spot forming cells.
Animal tests of stored vaccines. EV71-specific IgG titers (A) and neutralizing antibody responses induced in mice by EV71, EV71-W6, and EV71-W6-CaP
Wang et al. PNAS Early Edition
| 5 of 6
We used a combination of genetic engineering and biominer-
alization techniques to produce a thermostable vaccine. The self-
directed biomineralized vaccine can be used efficiently after
storage at ordinary temperatures, which significantly increases the
efficacy of immunization systems and lowers the cost of vaccine
delivery and storage. We suggest that this simple attempt can be
developed into a general strategy for vaccine improvement to as-
sist in the execution and expansion of immunization programs
globally, especially for the poorest countries.
Materials and Methods
More detailed descriptions of the experimental procedures and data analysis
are provided in SI Materials and Methods.
Construction and Recovery of Recombinant Vaccines. The coding nucleotides
of nucleating peptides N6p (SVKRGTSVGMKPSPRP), NWp (DSSEEKFLRRIGRFG),
and W6p (RWRLEGTDDKEEPESQRRIGRFG) were cloned into the β-(BC)-loop
of a full-length infectious cDNA clone of EV71-attenuated strain A12 using
the fusion PCR technique as previously described (36). The recombinant
plasmids carrying the biomimetic peptides were linearized and used as
templates for in vitro transcription, which was performed using the Ribo-
MAX Large Scale Production System (Promega) according to the manu-
facturer’s protocols. The yield and integrity of transcripts were analyzed by
gel electrophoresis under nondenaturing conditions. RNA transcripts (5 μg)
were transfected with Lipofectamine 2000 (Invitrogen) into RD cells grown
in 60-mm-diameter culture dishes. Supernatants were then harvested at ∼3–
5 d posttransfection, when typical cytopathic effects were observed. The
supernatants were then clarified by low-speed centrifugation. All the plas-
mids mentioned above were confirmed by DNA sequencing.
Vaccine Biomineralization. Vaccine solutions (106–108pfu/mL) in DMEM sup-
plemented with 1–4 mM calcium chloride were incubated at 37 °C for 6 h.
The biomineralized viruses were separated by centrifugation at 16,000 × g
for 10 min. The biomineralization efficacy was determined by measuring the
percentage of viral RNA and infectious particles in the deposit by qRT-PCR
and plaque assays, respectively. Calcium chloride, at 3.7 mM, was used as the
optimized concentration throughout our experiments.
Animal Assessments. Animal experiments were approved by and performed
in strict accordance with the guidelines of the Animal Experiment Com-
mittee of the State Key Laboratory of Pathogen and Biosecurity, Ministry
of Science and Technology of the People’s Republic of China. Groups of
BALB/c mice were s.c. immunized with fresh or stored EV71, EV71-W6, or
EV71-W6-CaP, respectively. Sera were collected at 14 and 28 d post-
infection. All samples had the same initial titers before storage (200 μL,
106pfu/mL). Serum IgG and neutralization antibodies were detected by
ELISA and microneutralization assays, respectively.
ACKNOWLEDGMENTS. We thank Dr. Guang Tian and Ge Yan for technical
assistance with transmission EM and Dr. Qi-Bin Leng (Institute Pasteur of
Shanghai), Dr. Pei-Yong Shi (Novartis Institute for Tropical Diseases), and Dr. Ben
Wang (Zhejiang University) for helpful discussions. This work was supported by
the Fundamental Research Funds for the Central Universities, the National Basic
Research Project of China (Grant 2012CB518904), the National Natural Science
Foundation of China (Grants 81000721, 91127003, and 21201150), and the Bei-
jing Natural Science Foundation (Grant 7122129). C.-F.Q. was supported by the
Beijing Nova Program of Science and Technology (Grant 2010B041).
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| www.pnas.org/cgi/doi/10.1073/pnas.1300233110Wang et al.
Wang et al. 10.1073/pnas.1300233110
SI Materials and Methods
Cells and Virus. Human rhabdomyosarcoma (RD) cells, human
THP-1 cells, and African green monkey Vero cells were cultured
in DMEM (Invitrogen) supplemented with 10% (vol/vol) FBS at
37 °C in an incubator with 5% (vol/vol) CO2. Human enterovirus
type 71 (EV71) strain A12 was isolated from a patient with hand-
foot-and-mouth disease and was identified as genotype C4. In
vitro and in vivo characterization demonstrated A12 was non-
virulent in suckling mice with ideal attenuated characteristics (1).
The virus stock was prepared and titrated in RD cells, and it was
stored at −70 °C until use.
IndirectImmunofluorescenceAssay.RD cells at 80–90% confluence
were infected with EV71, EV71-W6, or EV71-W6-calcium
phosphate (CaP) at the desired multiplicity of infection (MOI),
and 12–24 h postinfection, the infected cells were fixed with
acetone at −20 °C for 30 min. The infected cells were then
washed with PBS and incubated with mice antisera against EV71
or calcium chelating agent W6p peptide for 1 h, followed by
three washes with PBS, and they were then incubated with Alexa
Fluor 488-conjugated goat anti-mouse IgG as the secondary
antibody for 30 min. The fluorescence was detected after wash-
ing with PBS. DAPI was added, and the cells were incubated at
room temperature for 5 min to stain the nuclei.
Plaque Assays. Ninety percent confluent RD cells in a 12-well plate
were infected with 400 μL of serial 10-fold viral dilutions. After 1 h
of adsorption, the infected cells were washed and incubated for
3 d with DMEM containing 2% (vol/vol) FBS and 1% (wt/vol)
low-melting-point agarose. The cells were fixed with 4% formal-
dehyde and stained with crystal violet solution [1% (wt/vol) crystal
violet, 0.85% (wt/vol) NaCl, 2% (wt/vol) formaldehyde].
One-Step GrowthCurves.Viral growth curves in RD and Vero cells
were determined in a 24-well plate with parental EV71, EV71-
W6, and EV71-W6-CaP, respectively, at the desired MOI. The
supernatants were collected at intervals, and the titers were ex-
amined by plaque assays as previously described.
EMCharacterizations.The viral solutions were added onto carbon-
coated copper transmission electron microscopy (TEM) grids
(400 mesh; Agar Scientific) by dip-coating, and the samples
were then dried at room temperature before observation. The
samples were also stained with phosphotungstic acid. TEM
observations were performed using a JEM-1200EX microscope
(JEOL). SEM and energy dispersive X-ray spectroscopy anal-
yses were performed using a JSM-35CF microscope (JEOL).
Samples were prepared by dispersing 50 μL of solution on the
surface of silica specimen stubs. They were dried at 30 °C for
at least 24 h and were sputter-coated with gold before the ex-
Thermal Stability Tests. EV71-W6-CaP, EV71-W6, and parental
EV71 were incubated at 26 °C, 37 °C, and 42 °C, respectively, and
samples were collected periodically. The remaining infectivity
was determined by plaque assays as previously described (2).
One-Step Quantitative Real-Time RT-PCR Method. Viral RNA was
extracted using an RNA Purelink RNA mini kit (Ambion) fol-
lowing the manufacturer’s instructions. The obtained RNA was
quantified by one-step quantitative real-time RT-PCR using
a One Step PrimeScript RT-PCR Kit (Takara) according to the
manufacturer’s instructions with EV71-specific forward primer
(5′-GGCCATTTATGTGGGTAACTTTAGA-3′), reverse primer
(5′-CGGGCAATCGTGTCACAAC-3′), and probe (5′-FAM-
quantification of RNA was calculated according to the standard
curve, which was generated by serially diluting an RNA solution of
determined titer in sterile water.
Dot Plot Assays. For immunological detection of EV71 coat pro-
teins, 106plaque-forming units each of bare EV71-W6 and EV71-
W6-CaP were spotted onto a 100% methane-activated PVDF
membrane (Millipore) and the membrane was then air-dried at
room temperature. Nonspecific binding sites were blocked using
5% skim milk in PBS. The membrane was then probed with
a polyclonal antibody specific to EV71, followed by an alkaline
phosphatase-conjugated horse anti-mouse antibody. Both anti-
bodies were diluted in blocking solution (0.05% BSA). Signals
were generated by the addition of 5-bromo-4-chloro-3-isdolyl
phosphate/nitro blue tetrazdiom (BCIP/NBT).
Neurovirulence Tests.The neurovirulence of EV71, EV71-W6, and
EV71-W6-CaP was examined as previously described (3). Briefly,
the rescued virus was diluted and then intracranially inoculated
into the 3-d-old BALB/c suckling mice (n = 5 per group). All
mice were monitored daily for clinical symptoms and death until
14 d after inoculation.
ELISA. Detection of serum IgG antibodies against EV71 was
performed by indirect ELISA using 96-well, flat-bottomed plates
(Costar) coated with EV71 control strain A12 diluted 1:100 in
0.1 M carbonate/bicarbonate buffer (pH 9.6) overnight at 4 °C.
After blocking with 5% skim milk powder in PBS and Tween 20
(PBST), plates were incubated with serially diluted sera in du-
plicate wells for 1 h at 37 °C. Peroxidase-conjugated horse anti-
mouse IgG at a dilution of 1:5,000 was then added and incubated
for 0.5 h at 37 °C, followed by the substrate BCIP/NBT. Plates
were washed with PBST (pH 7.2) three times with an interval of
5 min after each reaction. Absorbance of the color developed
was determined at 492 nm and corrected for background with
PBS control group sera.
Microneutralization Assays. Mice serum was serially twofold di-
luted in DMEM starting at a ratio of 1:8. A 50-μL virus sus-
pension at 100-fold the 50% tissue culture infective dose was
mixed with 50 μL of sera, and the mixtures were incubated at 37
°C for 1 h. The mixtures were then added to 60% confluent
Vero cells and incubated for 7 d. Appropriate serum, virus, and
cell control was included in the test. The end-point titers were
calculated using the Reed–Muench method (4) as previously
IFN-γ Enzyme-Linked Immunospot Assays. IFN-γ enzyme-linked
immunospot assays of a mouse set (BD Biosciences) were per-
formed according to the manufacturer’s instructions (6, 7). Briefly,
96-well filtration plates were coated overnight at 4 °C with
IFN-capture monoclonal antibody, followed by washing and
blocking with RPMI-1640 medium containing 1% L-glutamine
and 10% FBS for 2 h at room temperature. Splenocytes (5 × 105
cells per well) were then added in RPMI-1640, with the sub-
sequent addition of native EV71 as the stimulation antigen, and
cultured for 20 h at 37 °C under 5% CO2. After washing with
water once and washing with PBST three times, plates were in-
cubated with biotinylated IFN-γ detection antibody at room
temperature for 2 h, followed by three washes with PBST and 1 h
Wang et al. www.pnas.org/cgi/content/short/13002331101 of 5
of incubation with streptavidin HRP at room temperature. Spots
were revealed using an AEC substrate reagent set (BD Bio-
science) at room temperature and were counted with an Im-
munospot reader (Cellular Technology).
Statistical Analysis. The statistical significance of differences in
antibody titers among different groups was determined by the
paired Student t test using SPSS software (IBM). Results with
error bars are expressed as the SDs.
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with nucleating peptide-specific polyantibodies (Lower). The green fluorescence indicates the successful expression of engineered peptides on the viral surface,
and the blue fluorescence represents the nucleus stained by DAPI. (Magnification: 40×.)
Indirect immunological fluorescence of parental EV71 and recovered engineered viruses on Vero cells with EV71-specific polyantibody (Upper) or
extracted and sequenced, and the results showed that it was relatively stable.
Genetic stability of the engineered peptide’s coding sequence. After 10 generations, the viral RNA of passage 1, 4, 7, and 10 progeny viruses was
Wang et al. www.pnas.org/cgi/content/short/13002331102 of 5
amorphous. A.U., arbitrary unit; 2q, two theta.
X-ray diffraction analysis of self-biomineralized viral particles. The pattern indicated the formation of a CaP mineral phase around the vaccine was
collected, diluted in Tris-buffered saline (TBS) solution of different pH, and incubated for 0.5 h. The amount of released vaccine was estimated by quantifying
the viral RNA in the supernatant by the quantitative RT-PCR method after centrifugation (16,000 × g for 10 min); square represents percentage of released
EV71-W6 vaccines at indicated pH. (B) Dot-blotting assays of biomineralized or demineralized EV71 and EV71-W6. At physiological pH 7.4, the CaP bio-
mineralized exterior was rather stable and viral antigens could not be detected by EV71-specific antibodies, whereas the CaP exterior was demineralized at pH
less than 6.5 and the viral antigens were detectable again.
(A) pH-sensitive release of enclosed EV71-W6 from EV71-W6-CaP particles. A total of 107plaque-forming units of EV71-W6-CaP in DMEM were
Wang et al. www.pnas.org/cgi/content/short/13002331103 of 5
Fig. S5. One-step growth curves of EV71-W6-CaP or bare EV71-W6 on RD cells at MOI = 0.1 (n ≥ 3); error bars represent SDs.
was represented in a logarithmic scale as a function of storage time (n ≥ 3); error bars represent SDs. The calculated average inactivation rate constants
for EV71 and EV71-CaP, as shown in the figure, were KEV71= 0.0159 h−1and KEV71-CaP= 0.01147 h−1at 37 °C and KEV71= 0.29896 h−1and KEV71-CaP= 0.24567 h−1
at 42 °C.
In vitro test of virus thermostability. Thermal-inactivation kinetics were determined at 37°C (A) or 42 °C (B). The remaining percentage of infectivity
Wang et al. www.pnas.org/cgi/content/short/1300233110 4 of 5
postinfection; error bars represent SDs (n ≥ 3, Student’s paired t test, one-tailed, **P < 0.01).
Expression levels of IFN-α (A) and IFN-β (B) on RD cells activated by EV71-W6 and EV71-W6-CaP were measured by quantitative RT-PCR at intervals
Wang et al. www.pnas.org/cgi/content/short/13002331105 of 5