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200-L Scale Serum-Free Microcarrier Bioreactor System Produced Inactivated
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Enterovirus 71 Vaccine Provides Cross-protective Efficacy in Human SCARB2
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transgenic mice
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Chia-Ying Wu a, Yi-Wen Lin b Chia-Ho Kuoa, Wan-Hsin Liua, Hsiu-Fen Taia,
Chien-Hung Pana, Ching-Chuan Chang a, Chung-Cheng Liu ac, Yen-Hung Chow b*
Juine-Ruey Chena*
a
Adimmune Corporation, Taichung, Taiwan
b
Institute of Infectious Disease and Vaccinology, National Health Research Institutes,
Zhunan, Miaoli County, Taiwan
C
Enimmune Corporation, Taichung, Taiwan
*Corresponding authors (J.-R.C. and Y.-H.C.)
Mailing address:
No. 3, Sec., 1, Tanxing Road, Tanzi Dist., Taichung City 42743, Taiwan. Tel.:
886-4-2538-1220 ext. 3601; Fax:
886-4-2538-2105;E-mail:JR_Chen@adimmune.com.tw; juinerueychen@gmail.com
(Juine-Ruey Chen)
Author contributions: C.-Y.W., Y.-H.C., C.-C.C., C.-H.C., C.-C.L., and J.-R.C.
designed research; C.-Y.W., Y.-W.L., C.-H.K., H.-F.T., C.-Y.C., and Y.-T.C.
performed research; C.-Y.W., C.-C.C., and J.-R.C. analyzed data; and C.-Y.W.,
C.-C.L., Y.-H.C., and J.-R.C. wrote the paper.
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Abstract
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Occurrence of enterovirus epidemics over the past 15 years in the Asia-Pacific
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region has raised serious public health concerns in young children. Co-circulation of
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several sub-lineages of enterovirus 71 (EV71) with other serotype coxsackievirus A
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viruses caused several outbreaks with fatalities, highlights the urgent need to develop
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a broadly protective or multivalent vaccine. Here, we produced the EV71(E59)
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vaccine using a scalable microcarrier bioreactor processing up to 200L with
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serum-free medium and demonstrated that can be a feasible alternative technology to
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meet surge in demand. In this study, the EV71 vaccine with different formulations
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were evaluated by immunogenicity; cross-protective immunity against the other
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sublineage of EV71 viruses and suppression of immunopathology in an
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hSCRAB2-transgenic (Tg) mouse model. In contrast to placebo group, all of Tg mice
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immunized either with alum or squalene-based formulations provided fully protection
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against distinct sublineages of EV71 virus. Vaccine-treated mice did not shown any
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degree of neurological paralysis symptom as well as damage or pathogenic-induced
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inflammatory in limb muscle tissue after challenges. In addition, significant viral
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clearance in spinal cords and suppression of indicative pro-inflammatory gene
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expression in the central nervous system and muscle tissue correlated with the
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survival of vaccinated mice. These findings provide evidence that inactivated EV71
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vaccine produced by employing scalable bioreactor platform is a promising vaccine
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candidate affording protective efficacy against lethal EV71 viruses.
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1. Introduction
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Over the last decade, more than 7 million cases of hand, foot, and mouth disease
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(HFMD) accompanying with 2000 associated deaths have been reported globally [1,
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2]. Among widespread epidemics caused by enterovirus, Enterovirus 71 (EV71) is the
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most prevalent caused HFMD symptom and developed a poliomyelitis-like paralysis
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in young children leading to severe neurological disorders and illnesses such as
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aseptic meningitis, brain stem encephalitis and even death [1, 3-9]. Since 1997,
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several EV71 outbreaks with high fatality rates occurred in Asian countries has raised
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a serious public health concern in children in the Asia-Pacific region (Malaysia in
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1997, Taiwan in 1998 and 2001 and China in 2008) [3, 4]. So far, effective antiviral
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drug or approved vaccine for infants and young children against EV71 disease are not
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available. Since the experience on eradicating the poliovirus was successfully
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achieved by vaccination, EV71 vaccine is now considered as the first priority of
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candidate to be developed against human enteroviruses.
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Recently, several formalin-inactivated whole virus EV71 vaccines either prepared
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by roller-bottle or cell-factory technologies with serum-containing or in an
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animal-origin free condition for clinical trials are conducted in several companies and
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organizations in Asia [10]. Three inactivated EV71 vaccines with a C4 sublineage of
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EV71 virus which is prevalent in mainland have been independently developed and
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evaluated in China. More than 30,000 young children and infants were enrolled in
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three individual vaccination programs and recently the positive results of phase III
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clinical traits have announced [11-14]. Additionally, Vaccine R&D Center of the
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National Health Research Institutes (NHRI) firstly launched the EV71 vaccine human
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phase I clinical trial in 2010 in Taiwan and Inviragen Pte Ltd of Singapore also
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completed the phase I clinical trial [15].
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Beyond the prevalent EV71 epidemics caused by infection of five genetic
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lineages (genotypes B3, B4, C1, C2, C4) among different countries, there are other
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serotype enterovirus such as coxsackievirus A virus (CAV) found frequently to
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co-circulate with EV71 in the Asia Pacific region [16-19]. In addition, the recent
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reports indicated the EV71 vaccine candidate failure to elicit cross-neutralizing
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antibody responses against CAV16 in humans and antisera raised against bivalent
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EV71/CAV16 vaccine cannot cross-neutralized other CAV strains [15, 20]. It
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highlights that the development of a multivalent vaccine provided protection against
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EV71 and CAV strains are desirable and warrant controlling of epidemics in Asia. In
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this study, we developed a large-scale serum-free bioreactor platform for upstream
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process improvement which is suggested promising to produce sufficient quantities of
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vaccine [20, 21]. Herein, we successfully established and optimized a 200L
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serum-free microcarrier bioreactor system to prepare EV71 vaccine in CGMP facility,
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which might provide cost-effective manufacturing process for other enterovirus
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vaccine production. We also investigated the immunogenicity of EV71 vaccine with
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various formulations, cross-protective efficacy, and evaluated the immune-associated
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inflammatory response with vaccination in hSCRAB2-transgenic mice. The results of
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survival in animal immunized with vaccine followed by viral challenge, histological
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staining, pro-inflammatory gene expression profile in muscle tissue and central
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neuron system provide comprehensive evidences for the safety of EV71 vaccine and
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its ability to cross-protect against lethal-infection of distinct subgenotype of EV71
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viruses.
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2. Materials and Methods
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2.1 Cell and virus banks
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The master Vero cell stocks were obtained by technology transfer from National
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Health Research Institutes (NHRI), Taiwan. The working Vero cells bank (WCB)
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were delegated and established by BioReliance (UK) following CGMP guidelines for
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the manufacture of biopharmaceutical products [22]. The CGMP-certificated working
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virus bank (WVB) (EV71: E59 strain) were purchased from NHRI and described
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detailed in previously study [23].
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2.2 Production scale-up of Vero cells and EV71 virus
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One vial of WCB was thawed and cultured in a T-flask using VP-SFM (GIBCO)
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media. The cells were detached by the treatment of TrypLE™ Select (GIBCO) and
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transferred to the roller bottles (Corning). The 8 roller bottles harvested cells were
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seeded into 4x3L spinner flasks containing 3g/L of microcarrier (Cytodex-1, GE).
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Vero cells were grown on Cytodex-1 and stirred at 25-40 rpm in 37℃, 5% CO2
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incubator. After the TrypLE™ Select treatment, the mixture of cells and microcarriers
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was transferred to a 50L Bioreactor (Sartorius) operated under the conditions of 30
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rpm, 37℃, pH 6.8-7.2, DO 30%. To keep high cell density over microcarriers to
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facilitate cell attachment in serum-free condition, cell culture was maintained with
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25L of VP-SFM and then fully filled after cell adherence. When the cell density
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exceeded 1.0 × 106 cells/mL, the microcarriers were allowed to settle by stopping the
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rotation and the culture medium was removed. After TrypLE™ Select treatment, the
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mixture of cells and microcarriers was transferred to a 200L Bioreactor (Sartorius)
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and cultured under the control of 20 rpm, 37℃, pH 6.8-7.2, DO 30% until the cell
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density reached 1 × 106 cells/mL. For the EV71 virus production, one vial of the
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WVB thawed in VP-SFM was inoculated into the bioreactor at a multiplicity of
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infection (MOI) of 10^-4 and the stir-tank bioreactor was continuously operated at 20
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rpm for 1 h to facilitate the adsorption of EV71 to the cells. After that, the cells/virus
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culture was fully filled with VP-SFM up to 200L and further incubated for another
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9-10 days. The optimal endpoint of EV71 culture reflecting the plateau level of virus
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growth was evaluated by in-house prepared sandwich ELISA method along with
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observation of the inoculums until a 95% cytopathic effect. The virus titers were
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determined by tissue culture infectious dose (TCID50) assay based on Vero cells,
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according to the Reed–Muench method [24].
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2.3 Virus purification and Inactivation of Immunogen
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The
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293C8HP13AFFF) and 0.45/0.22μm filter (Sartopore2, 5445307H7--00) to remove
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the cell debris, the filtrate was concentrated to about 1/50 of the original volume by
virus
culture
supernatant
was
clarified
by
4μm
filter
(Sartoclear,
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100kDa TFF (Sartorius, 3021466807E-SG) and remove the low-molecular weight
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impurities. The final concentration was passed through the iron exchange column and
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then treated with Benzonase to remove the host cell DNA. The purified virus solution
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was applied on to a 2–55% sucrose density gradient and centrifuged at 40,000 rpm, 4
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℃, for 30 min by using Core (H) rotor in the HimacCC40 ultracentrifuge (Hitachi
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Koki). Fractions were collected from the centrifuge tube and measured for sucrose
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concentration and specific EV71-unit (U/mL) by in-house sandwich ELISA. The virus
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was dialyzed against PBS by 100kDa TFF and finally sterilized with 0.22μm filter.
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For virus inactivation, a final formalin concentration of 0.025% was used and the
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mixture was incubated at 37 ◦C for 6 days. After dialysis with PBS, the inactivated
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virus preparation was confirmed for loss of infectivity in human rhabdomyosarcoma
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(RD) cells as described previously [25].
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2.4 Electron microscopy of EV71 vaccine.
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1-2 g of purified EV71 vaccine was placed on a 200-mesh copper grid. The
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specimen on the grid was stained with 1% uranyl acetate. Micrographs were obtained
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by electronmicroscopy (FEG-TEM, FEI Tecnai G2 TF20 S-TWIN).
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2.5 Vaccine Formulation and Animal Immunization
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To prepare formulated vaccine, each dose of EV71 antigens was mixed with
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300μg/mL of AlPO4 (ADJU-PHOS ®, BRENNTAG) or AddaVAX in sterile
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phosphate-buffered saline PBS. AddaVAX (Invivogen) is an oil-in-water emulsion
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with a formulation similar to MF59 adjuvant (Norvatis). Groups of BALB/c mice
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were intramuscularly immunized twice at day 0 and 14, and further bleed at day 28
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post-immunization. The hSCRAB2-transgenic (Tg) mice subcutaneously immunized
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twice as shown in Fig. 3A were subjected to collect the antisera at day 12 or 14 from
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birth. The White New Zealand rabbits (3-month-old) were immunized twice
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intramuscularly at 21 days apart with 2 µg of AlPO4-EV71 vaccine and bleed at day
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35 post-immunization.
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2.6 Microneutralization
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Virus-neutralizing titers of mock and post-vaccination mice and rabbit sera were
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determined in a modified plaque reduction assay [26]. Briefly, each serum diluted
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from 1:8 to 1:2048 was co-incubated with equal volume of distinct viruses at a
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multiplicity of infection (MOI) of 1. The serum/virus mixtures were incubated at
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37°C and 5% CO2 for 1 h. Vero cell monolayers (95% confluence), prepared in 96
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well plates, were infected with 100 µL/well (in duplicate) of the mixture and viral
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adsorption was allowed for 1.5 h in the incubator. After incubation, plates were
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immediately overlaid with 1.25% Avicel RC591 (A gift kindly provided by FMC
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BioPolymer), prepared in 1× M199 with 2% FBS. Plates were incubated at 37°C in a
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5% CO2 incubator for 20 h to allow plaque formation. Viral plaques were stained with
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Mab979 antibody (Chemicon International, Inc.), and the resulting plaques were
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counted by eyes under the microscope. Microneutralization titers were calculated
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from the average of triplicate sample wells by extrapolating the inverse dilution of
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serum that produced a 100% reduction of virus by comparing the total number of
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plaques revealed in PBS serum and vaccination serum samples.
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2.7 Virus Challenge
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Clinically isolated strains of EV71, Tainan/5746/98 (C2) (GenBank: AF304457.1),
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and N3340-TW-02 (C4) strains (GenBank: EU131776.1) was propagated in Vero
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cells and the Tg mice were bred and maintained as previously described [27]. To
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evaluate vaccine efficacy, Tg mice were vaccinated subcutaneously with various
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EV71 vaccines or PBS at day 1 and day 8 after birth. 3 × 106 pfu of EV71 5746 (C2)
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strain or 1 × 106 pfu of EV71 3340 (C4) strain were individually injected
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subcutaneously at day 14 or day 12. Mice were observed for body weight change and
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neurological symptom development for 15 days post-infection. Groups of mice were
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sacrificed to collect tissues for Real-time RT-PCR and immune-histological
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experiments at day 6 post-infection. All animal experiments in this study were
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evaluated and approved by the Institutional Animal Care and Use Committee of
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Adimmune Corporation and NHRI.
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2.8 ELISpot Assay
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Spleens harvested from mock-treated and vaccinated Tg mice at day 13 or day 15
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after birth were used to isolate splenocytes for ELISpot assays (R & D Systems,
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Minneapolis, MN, USA). The detailed procedures are performed according to
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previous study [27].
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2.9 HE Stain and Immunohistochemical Staining
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The tissues from sacrificed Tg mice were fixed with 10% buffered formalin
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(Sigma-Aldrich) and then embedded in paraffin (Sigma-Aldrich), sectioned, and
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stained with hematoxylin and eosin (H&E). For detection of virus particle in tissues,
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the procedure of immunohistochemical staining was described as previously [27].
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Bright field microscopy pictures were taken at 200X magnification.
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2.10 Real-time RT-PCR
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Tissues from specific organs were homogenized to extract RNAs. The cDNA was
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synthesized by using a HiScript I Reverse Transcriptase (Bionovas) and subjected to
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quantitative PCR analysis (the LightCyclerH480 SYBR Green Real-Time PCR
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system) with primer pairs specific to target genes as shown in Table 1. CXCL10,
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TNF-α and Mouse β-actin were detected by KAPA SYBR FAST qPCR kit following
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the instrument. To determine the expression of IFN-γ, Taqman assay was performed
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with specific primers and the probe No.69 from Universal Probe Library (Cat. No.
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04688686001, Roche). The relative expression of the target gene normalized to
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Mouse β-actin gene was calculated and described as previous study [27].
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2.11 Statistical analysis
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The significant differences between vaccine groups was statistically computed
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applying t-test using GraphPad Prism software, Version 6.0.
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3. Results
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3.1 The development of 200L-scale EV71 virus culture by serum-free bioreactor
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technology
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To prepare high-density Vero cells with large-scale bioreactor for EV71 virus
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infection, the cell culture was initially amplified from one vial to a 200L fermenter by
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using the serum-free media VP-SFM and TrypLE™ Select (Invitrogen) in a fully
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animal-component free condition. The typical profile of cell growth is
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representatively shown in Fig. 1A. The certificated Vero cells were expanded
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gradually through T- flasks, roller bottles, spinner flasks, and a 50L fermenter. After
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that, the detached cells/microcarriers mixture was inoculated into 200L at an initial
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cell density of 1 × 105 cells/mL. The culture process in a 200L fermenter, a maximal
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Vero cell density level up to 1 × 106 cells/mL was achieved after 6 days
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post-inoculation (Fig.1A, C-a). Base on the metabolism profile daily (glucose,
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glutamine, and lactate concentration), the nutrient adding was conducted at day 6 and
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8 after initiation of 200L cell culture to maintain optimal condition (Fig. 1B). Cell
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infection was performed at day 6 after 200L culture with an MOI of 10^-4 and the
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virus production reached a maximal virus titer equal to 1 × 107TCID50/mL for another
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10-days culture exhibiting 95% cytopathic effect (Fig. 1A and C-b).
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3.2 Purification, characterization, and inactivation of EV71 vaccine
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The 200L virus culture were harvested and concentrated by TFF system as described
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in section of Materials and Methods. Further, concentrated virus culture was purified
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by sucrose density gradient ultracentrifugation. The viral existing fractionations were
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analyzed by sucrose density, total protein, ELISA, host cell protein (HCP) and
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SDS-PAGE (Fig. 2A and B). Content of EV71 particle were measured by in-house
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established sandwich-ELISA assay and revealed the distribution of EV71 purified
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particles on fractions of 11-15 (Fig. 2A). In parallel detection of fractions by
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SDS-PAGE with sliver staining, the EV71 antigens were observed from the fraction 5
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to 14. The infectious particles (F-particle) and empty particles (E-particle) of EV71
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were separated by sucrose-density fractionations and distributed on the fractions 5-10
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and 11-14, respectively (Fig. 2B). To investigate the characters between two types of
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EV71 viral particles, the formalin treated F-particles and the E-particles of EV71 were
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individually observed by electron microscopy (Fig. 2C). TEM results revealed that
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large-scale prepared Vero cell-derived EV71 particle were approximately 30-35 nm
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diameters (Fig. 2C). The physical appearance in the TEM analysis of F-particles and
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E-particles was consistent with previous study [22]. Based on three batches of
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purification, the E-particles and F-particles with a ratio of 6:4 contribute to total
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vaccine antigens. To validate the inactivation process of EV71, the samples collected
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from 5 to 14 fractions were pooled and treated with formalin solution (4000:1) at
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37◦C for 6 days. By analyzing with TCID50 and ELISA assays, after two days
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formalin-treatment, EV71 viral particles were completely lost infectivity (data not
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shown) and SDS-PAGE results demonstrated the vaccine antigen pattern before and
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after inactivation for 6 days (Fig. 2D). Consistent with previous study, with formalin
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treatment, the viral antigens were more and more cross-linked and shift to high
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molecular weight on SDS-PAGE (Fig. 2D, a vs. b). The major antigens of VP0 and
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VP2 were detected by western blot analysis with specific antibody (Mab979,
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Millipore) in prepared vaccine bulk (Fig. 2D-c).
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3.3 Immune response to EV71 vaccine in two species of animals
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For the immune response against EV71 infections, vaccine-induced humoral and
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cell-mediated immune responses both play significant roles in activating adaptive
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immunity [28]. To address the vaccine effectiveness, we investigated the humoral and
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cell-mediated immune responses elicited by various formulations in adult BALB/c
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mice, rabbits, and infant Tg mice. Groups of BALB/c mice (six-to-eight weeks old,
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n=5) were immunized with various formula of 1 g EV71 E59 vaccine derived from
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B4 subgenotype as indicated. At day 28 post-vaccination, the mice sera and prepared
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splenocytes were applied to determine viral-neutralization titers and cell-mediated
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immune responses. The results showed the squalene-based EV71 vaccine elicited
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highest viral-neutralization mean titer (1:16384) against EV71 E59(B4) strain
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compared with AlPO4-formulated vaccine (1:4096), without adjuvants (1:1024), and
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PBS (<1:8). In contrast to group vaccinated with vaccine without adjuvant, both
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splenocytic IL-4 and INF- were significantly observed on adult BALB/c mice
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immunized with AddaVAX- or AlPO4-adjuvanted EV71 vaccine (Fig. 4A and B). To
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exam whether the adjuvanted-EV71 vaccines offer stronger immune response and
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provide better cross-protective immunity against heterotypic strains, the rabbits and
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Tg mice were vaccinated as immune protocol (Fig. 3A). Antisera were collected and
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tested for cross-strain viral-neutralization assay (Fig. 3B). Both species of animal
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revealed that antibodies raised against EV71 E59(B4) vaccine were specifically
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neutralized the EV71 E59 B4) and 3340 (C4) strains but did not show obviously in
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vitro neutralization with 5746(C2) strain. With either 2 or 6 g adjuvanted-EV71 E59
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vaccine, the antisera from immunized rabbits or Tg mice achieved protective GMT
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titer ranging from 1:69~1:512 against homotype B4 strain and exhibited significant
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cross-neutralizing activities against subgenotype 3340(C4) virus (GMT titer:
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1:45~1:327). However, 80% of Tg mice vaccinated with AlPO4-EV71 vaccine failed
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to induce neutralizing antibody positive to subgenotype 5746(C2) strain (GMT titer 
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1:8). In AddaVAX-EV71 group, only 60% of Tg mice developed weak
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cross-neutralizing antibody responses (GMT titer = 1:8) against C2 strain. Consistent
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with the results of Tg mice, the rabbits vaccinated with AlPO4-formula exhibited low
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levels of cross-reactive GMT titer (1:8~1:16) against C2 strain.
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We next to investigate vaccine formula induced cellular immune response in Tg
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mice. After vaccination, T-helper cell responses were evaluated by mouse IL-4 (Th-2)
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and IFN-γ (Th-1) ELISpot analyses. We found that secretion of splenocytic IL-4 by
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stimulated with heterologous 5746 (C2) or 3340 (C4) virions was significantly
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observed on Tg mice immunized with AlPO4- or AddaVAX-adjuvanted EV71 E59
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(B4) vaccine compared with mock group (Fig. 4C and E). However, significant IFN-γ
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spots were recorded only in group of AddaXAV-adjuvanted EV71 vaccine either with
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heterologous 5746(C2) or 3340(C4) virus stimulation (Fig. 4D and F). It illustrated
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that EV71-AlPO4 formulation induced a majority of Th2-mediated humoral immunity
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while the squalene-based adjuvant elicited balanced Th1/Th2 immune response with
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EV71 vaccine.
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3.4 Vaccine protects infant human SCARB2-Tg mice from heterologous EV71
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infection and inhibits paralysis symptoms
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To evaluate the efficacy of EV71 vaccine with different formulations, the
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vaccinated-Tg mice were challenged with EV71 5746(C2) or 3340(C4) strain as
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previously [27]. In contrast to placebo group (PBS), the mice were 100% protected by
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all tested vaccines with daily monitoring for survival rate, body weight change, and
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viral-induced neurological symptoms (Fig. 5A and B). All mice continually gained
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weight after EV71 infections through the experimental period except the mice without
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vaccinated vaccine (PBS) (Fig. 5C and D). In addition, mice in placebo group (PBS)
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induced obvious NCS syndrome as early as on 2-3 dpi and then peaked on 5-6 dpi
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depending on distinct viral infection (Fig. 5E and F). Representative photographs
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showed that the PBS-received mice exhibited serious hind limb paralysis symptom at
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6 dpi, but no apparent symptom appeared in vaccinated mice (Fig. 6). To verify
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whether the vaccines could effectively prevent EV71 infection-induced pathogenesis,
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the experiments were performed by hematoxylin and eosin and immunohistochemical
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(IHC) staining. The results showed clear muscle fibers and well-organized in
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uninfected mice with/without vaccination (Fig. 7A-D, Mock) and vaccinated mice
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with infections at 6 dpi (Fig. 7F-H and J-K). The inflammatory cells infiltrated into
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the muscle tissues and the destructed structures were only observed in EV71-infected
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mice without vaccination (Fig. 7E and I). The complete clearance of viral antigen in
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brainstem was only observed in mice vaccinated with 6μg of AlPO4-EV71 or 2μg of
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AddaVAX-EV71 vaccine following EV71 infections (Fig. 8). Viral antigens were
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obviously detected in numerous brain stem of mice without vaccination after both
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viral infections and few occasionally appeared in brain stem of mice with 2μg
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AlPO4-EV71 vaccination by EV71 5746(C2) infection (Fig. 8, arrows).
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3.5 Vaccine treatment significantly suppressed expression of pro-inflammatory
cytokines and chemokine in tissues of EV71-infected Tg mice
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The observations of a strong inflammatory cytokine activation in the plasma and
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cerebrospinal fluid of patients according to disease severity of EV71 infection have
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been reported [29-31]. Previous study demonstrated that the secretion of CXCL10,
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CCL3, TNF-α, and IL-6, correlated to the severity of neurologic pathogenesis induced
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by EV71 challenge was confirmed in hSCARB2-Tg mice model [27]. Here, we
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measured the expression of pro-inflammatory indicators in the tissues of vaccinated
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Tg mice followed by EV71 challenge to evaluate the vaccine in preventing
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inflammatory cytokine pathogenesis. As shown in Figs. 9 and 10, upregulation of
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CXCL10 and TNF in the CNS and muscle tissues in the placebo group of transgenic
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mice were observed after the infection of EV71 5746 (C2) or 3340 (C4). Compared to
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the unvaccinated group (PBS), the mice immunized with AlPO4- or AddaVAX-EV71
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vaccine significantly or slightly suppressed the expression level of CXCL10, TNF,
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and IFN- after virus challenge (Figs 9 and 10). Interestingly, 2g of AddaVAX-EV71
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vaccine showed as more potent as 6 g of AlPO4-EV71 vaccine to reduce
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pro-inflammatory gene expression in mice issues after C2 virus challenge, proved that
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AddaVAX-formula provided the substantial alleviation of viral-induced neurologic
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pathogenesis (Fig. 9). The similar scenario was also observed in C4 virus challenge
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(Fig. 10), suggesting the balance of Th1/Th2 immune response induced by both
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vaccines provides a substantial role for EV71 cross-strain protection (Figs. 4 and 10).
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4. Discussion
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The prevalence of hand-foot-mouth disease (HFMD) caused by EV71 and
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coxsackievirus A (CAV) infection has become serious threat to public health in Asia.
375
In the past 15 years, the quick evolutionary of EV71 has increased the occurrence of
376
epidemics and outbreaks with high mortality rate ranging from 10 to 25.7% in Asia
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country [32, 33]. In addition, co-circulating with EV71 and CAV16 caused major
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evolutionary forces leading to emergence of genetic variants has accounted for the
379
recurrent HFMD outbreaks in China [16, 19]. Due to a lack of effective therapy,
380
controlling EV71 epidemics has mainly focused on the research and development of
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EV71 vaccines. So far, the reports of three EV71 vaccines completing phase III trials
382
represent a good safety profile and provide a vaccine efficacy of 90% against
383
EV71-associated diseases, indicating the formalin-inactivated whole virus is an
384
effective vaccine candidate [10].
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However, face the threats from continuous co-circulating and co-infection by EV71
386
and CAV, the challenge to licensure of EV71 vaccines is now necessary to fully
387
assess the degree of widely-protective immunity for subgenotypic viruses prevalent in
388
other epidemic countries. Recently, several reports have underscored the importance
389
and urgency of developing bivalent or multivalent of vaccine to control
390
HFMD-related epidemics and outbreaks [20, 34-37]. To meet the surge requirements
391
of vaccine production, an alternative upstream process by implementing the
392
microcarrier/bioreactor platform should be developed and established [20]. Here, we
393
firstly demonstrated a manufacturing system for a Vero cell-derived inactivated EV71
394
vaccine at a 200L scale with bioreactor technology in a fully animal-component free
395
condition (Figs. 1 and 2) and evaluated the protective efficacy of vaccine in a
396
well-established animal model of human SCRAB2-transgenic (Tg) mouse [27]. The
397
advantage of using microcarrier/bioreactor technology is considered as easily
398
expanding the adherent Vero cells for virus culture that could be scaled-up feasibly to
399
allow vaccine production at a several thousand liter scale with less labor-intensive and
400
easier to maintain batch-to-batch consistency than roller-bottle or cell-factory
401
technology. Additionally, amplification of the virus in a serum-free condition avoids
402
concerns associated with bovine serum such as infectious agent included and
403
simplifies the bioprocess to remove impurity during the downstream purification
404
process [22].
405
In this report, we confirmed the quality and immunological properties of
406
large-prepared cGMP vaccine as judged by SDS-PAGE analysis, TEM morphology
407
observation, viral-neutralization, and ELISpot assay (Figs. 2, 3, and 4). The antigens
408
obtained from the 200L-scale of manufacture were highly purified and its structural
409
morphology was comparable with those of antigen derived from 5L-scale production
410
[22]. The specification of inactivated EV71 vaccine bulk was confirmed to fulfill the
411
draft proposed by Center for Drug Evaluation (CDE) in Taiwan (data not shown). To
412
optimize the formulation of EV71 E59 (B4) vaccine, we prepared squalene-based and
413
AlPO4 adjuvanted EV71 vaccines and tested their immunogenicity in rabbit and Tg
414
mice. We observed that the antisera from vaccinated rabbits and Tg mice all exhibited
415
stronger neutralizing capability against homotypic B4 and heterotypic 3340(C4) virus
416
instead of 5746(C2) virus. It consists to the previous results shown in monkey (Liu,
417
C.C., et al., PLoS ONE, 2014, Vol. 9, e106756) and in human phase I clinical trial
418
(Chou, A.H., et al., PLoS ONE, 2013, Vol. 8, e79783) immunized with inactivatged
419
EV71 E59 vaccine. The compatible results might provide the supporting information
420
to bridge the next application of phase II clinical trial using this bioreactor platform
421
[38]. Additionally, ELISpot results indicated the EV71 vaccine with AddaVAX
422
formulation induced Th1 and Th2 balance immunity other than Th2 response
423
induced by the conventional AlPO4 adjuvant, that is a potential account for its
424
wide-spectrum protective capability observed in the study of EV71 challenge via the
425
significant suppression of EV71-caused neurological symptoms in Tg mice (Figs. 4, 5,
426
8, 9, and 10).
427
We also evaluates the potential cross-protection efficacy of AddaVAX and AlPO4
428
adjuvanted EV71 vaccines , the Tg mice immunized with both vaccines were fully
429
protected from two different sub-strains of virus challenge; developing no
430
neurological paralysis symptom, and gained body weight gradually for 15 days
431
post-infection, whereas the placebo group with no vaccine treatment exhibited serious
432
paralysis in hind limb and subsequently all die before 9 days post-challenge (Figs. 5
433
and 6). Moreover, the scenario of EV71 causing the muscle tissue damage,
434
appearance of viral antigen in CNS compartments, and highly-regulated of
435
pro-inflammatory cytokine expression was comprehensively compromised by 2g
436
AddaVAX-EV71 and 6g AlPO4-EV71vaccine (Figs. 7, 8, 9, and 10). They suggest
437
that the squalene-based emulsion may be a suitable adjuvant offering better
438
dose-sparing effect and higher potency against distinct EV7 viruses than the
439
conventional alum adjuvant. Taken together, these results evidence that the
440
inactivated EV71 vaccine is successfully produced by a serum-free scaleable
441
microcarrier/bioreactor technology. It
442
the infection of distinct EV71 strains circulating in other country. Further trials in
443
humans are warranted to fully elucidate the vaccine potency and optimal formulation
444
toward controlling the widespread EV71 epidemics and HFMD-related outbreaks.
445
affords the cross-protective immunity against
446
Acknowledgments
447
This study was financially supported by Adimmune Corporation and a Ministry of
448
Economic Affairs grant (101-EC-17-A-20-I1-0054). The funders had no role in study
449
design, data collection and analysis, decision to publish, or preparation of the
450
manuscript. We especially thank Adimmune Corporation Chairman Dr. Chi-Hsien
451
Chan and Chief Financial Officer Che-Wei Chang for their intensive support this
452
research work and lead a team to develop the innovative EV71 vaccine in Taiwan. We
453
also thank the Electron Microscopy Core Facilities of Academia Sinica for TEM
454
technical support of this study and the materials of EV71 strains provided by Dr.
455
Chia-Chyi Liu from The Institute of Infectious Disease and Vaccinology, National
456
Health Research Institutes, Zhunan, Miaoli County, Taiwan. The authors thank for
457
their excellent technical assistance Kun-Tang Chiang, Hsin-Chuan Wang, Chien-Hua
458
Chen, Shao-Fu Wu, Yu-Min Liu, Yu-Lun Ho, Yen-Ling Chang, Kuo-Ming Lee,
459
Ching-Yuan Chang, Yung-Tsung Chen, Chiung-Wen Wang, Yu-Jung Chen,
460
Kai-Cheng Chuang, Bo-Lun Su, Yun Jang, and Shen-Fung Chuang.
461
Figure legend
462
Fig. 1. Scale-up of Vero cells culture and EV71 E59(B4) virus production in a 200L
463
fermenter. (A) Profile of Vero cells growth and EV71 amplification during a 200L
464
fermenter process. (B) Metabolism profile by monitoring the contents of glucose,
465
glutamine and lactate in a 200L cell/EV71 virus culture. (C) Photomicrographs of
466
Vero cells on microcarriers at 6 days after the start of culture in 200L fermenter for
467
initial EV71 inoculation (a) and at 16 days as the maxima of virus production to be
468
harvested (b).
469
470
Fig. 2. The purification and characterization of EV71 virus. (A) Purification of EV71
471
by sucrose
472
fractionation analysis by sucrose percentage (Sucrose), contents of total protein (TP)
473
and host cell protein (HCP), and EV71-specific unit (ELISA). (B) Definition of viral
474
particles distributed in fractions by using SDS-PAGE. (C) Electromicrographs of
475
EV71 particles by transmission electron microscopy. (a) Fraction 10 represented as
476
full-particle (F-particle) with infectious capability and had solid particle structure; (b)
477
Fraction 13 referred to as empty particle (E-particle) with defective particle structure;
478
and (c) Pooled viral particles for the vaccine bulk preparation. (D) Quality
479
confirmation of EV71 pooled viral particles before and after formalin-inactivation by
density
ultracentrifugation
using
zonal
ultracentrifugation
and
480
using SDS-PAGE and western blot analysis with specific monoclonal antibody
481
(Mab979). The molecule weight of VP0, 1, 2, and 3 are as indicated. The black
482
triangle is symbolic as the amounts of loading antigen from high to low.
483
484
Fig. 3. Antibody response to EV71 vaccine in hSCRAB2 transgenic (Tg) mice and
485
rabbits. (A) The immune and challenge schedule design for evaluating EV71 vaccine
486
efficacy in Tg mice and rabbits. Groups of Tg mice receiving vaccine or PBS twice
487
were assigned to challenge with EV71 5746(C2) or 3340(C4) virus, monitoring of
488
developed pathological symptom, and tissues collection for histological and gene
489
expression analyses. (B) Measurement of the vaccine-induced neutralizing antibody
490
titers against EV71 viruses from the rabbits and Tg mice. The used vaccine formulas
491
are as labeled.
492
493
Fig. 4. T helper cell responses in vaccinated mice. The level of IL-4 and IFN-γ
494
spot-forming cells in spleens of mice was determined by ELISpot assay. Mice groups
495
(n = 5) were immunized with various dosage of AddaVAX-EV71, AlPO4-EV71,
496
EV71 alone, and PBS as indicated. Splenocytes from PBS or vaccine-treated mice
497
were counted and stimulated in vitro with heat-inactivated whole viruses of EV71
498
E59(B4), 5746(C2), or 3340(C4) strain, respectively. The cells were stimulated with
499
ConA (2.5 µg/mL) as a positive control for IFN-γ ELISpot assay or were mock
500
stimulated as negative control (w/o) for both ELISpot assays. Bars represent means ±
501
SEM of spot counts in triplicate wells. The P value between the vaccination and PBS
502
control group or two vaccination groups is labeled.
503
504
Fig. 5. Vaccine protection against lethal-dose challenge of subgenotypes of EV71
505
virus. Survival rate of hSCARB2 Tg mice challenged with 1×106 pfu of C2 strain (A)
506
or 1×106 pfu of C4 strain (B) after vaccination. Mice body weight change after EV71
507
C2 (C) or C4 (D) virus challenge. Scores of central neuron system (CNS)-like hind
508
limb paralysis caused by EV71 C2 (E) or C4 (F) viral infection. The severity of
509
central nervous system (CNS) syndromes was scored from 0 to 5 using the following
510
criteria; for scoring CNS diseases; 5= severe front and rear limb paralysis (LP) and no
511
movement, 4 =moderate 2 rear LP and hesitant movement, 3 = one rear LP with
512
bending legs, 2 = mild rear limb bended, 1 = slightly rear limb bended, 0 =normal
513
movement. LP is defined as the rigidness of mouse legs which are hesitate to move.
514
The Tg mice receiving various vaccine formulas are indicated as symbols. *: p＜0.05,
515
**：p＜0.01, and ***: p＜0.001.
516
517
Fig. 6. Neurological symptoms in hSCARB2 Tg mice at 6 days after lethal challenge
518
with EV71 C2 or C4 strain. (A-B) Tg mice receiving with PBS, (C-D) 2µg EV71
519
E59(B4) vaccine with AddaVAX, 2µg (E-F) and 6µg (G) EV71 E59(B4) vaccine with
520
AlPO4.
521
522
Fig. 7. The features of pathological changes in muscles of vaccinated hSCARB2 Tg
523
mice with EV71 challenge. The haematoxylin and eosin (H&E) staining of muscle
524
tissues collected from mice vaccinated with indicated vaccine formulations or PBS
525
without EV71 challenge were shown in Panels (A-D) as control group. At 6 days
526
post-infection with EV71 C2 or C4, the resulting images by H&E staining were
527
shown in Panels (E-H) and (I-K), respectively. The used vaccine formulations and
528
challenged viruses are labeled.
529
530
Fig. 8. In situ detection of EV71 distribution in brain stem of vaccine-pretreated
531
hSCARB2 Tg mice after EV71 challenge. The uninfected Tg mice either pre-treated
532
with PBS or vaccines were used as the negative control and the images of
533
immunohistochemistry (IHC) staining against Mab979 antibody were shown in
534
Panels (A-D). The vaccinated and unvaccinated Tg mice infected with EV71 C2 or
535
C4 were sacrificed on day 6 post-infection and the waxed sections of brain stem
536
stained with IHC were representatively shown twice in Panels (E-R) as labeled. All
537
pictures were taken at 200X magnification. Viral particles in the sections are indicated
538
with arrows.
539
540
Fig. 9. Expression profile of pro-inflammatory cytokines in the CNS compartments
541
and muscle tissue of vaccine-pretreated hSCARB2 Tg mice by EV71 C2 challenge.
542
The vaccinated Tg mice were subcutaneously infected with 3×106 pfu of EV71
543
5746(C2) strain and RNA was extracted from the brainstem, spinal cord, and muscle
544
at 6 days post-infection. Quantitative RT-PCR analysis was conducted to quantify
545
CXCL10, TNF-α, and IFN-γ genes. Tg mice that unvaccinated and uninfected were
546
set as the negative control. The number of PCR cycles required for fluorescent
547
detection of target genes was calculated and presented as the relative expression after
548
normalization with the internal control of β-actin expression from the same tissue.
549
Mock (M)：uninfected mice group；Virus (V)：EV71 5746(C2) infected mice group.
550
A schematic representation of the target gene expression and the statistical average
551
from 5 mice per group is shown. Unpaired student t test with Welch’s correction was
552
used for statistical analysis. The used vaccine formulas are as labeled. Significant
553
difference between each group was shown as *: p＜0.05, **：p＜0.01, and ***: p＜
554
0.001.
555
556
Fig. 10. Expression profile of pro-inflammatory cytokines in the CNS compartments
557
and muscle tissue of vaccine-pretreated hSCARB2 Tg mice by EV71 C4 challenge.
558
The vaccinated Tg mice were subcutaneously infected with 1×106 pfu of EV71
559
3340(C4) strain and RNA extracted from the brainstem, spinal cord, and muscle at 6
560
days post-infection were subjected to quantitative RT-PCR analysis. Mock (M)：
561
uninfected mice group；Virus (V)：EV71 3340(C4) infected mice group. A schematic
562
representation of the target gene expression and the statistical average from 5 mice per
563
group is shown. Unpaired student t test with Welch’s correction was used for
564
statistical analysis. The used vaccine formulas are as labeled. Significant difference
565
between each group was shown as *: p＜0.05, **：p＜0.01, and ***: p＜0.001.
566
567
568
569
570
571
572
573
574
575
576
577
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Table 1. List of the sequence of primer pairs specific to target genes.
Mouse CXCL10
F
CTCTCTCCATCACTCCCCTTTAC
R
ACTTAGAACTGACGAGCCTGAGC
Mouse TNF-α
F
TCTCATGCACCACCATCAAGGACT
R
TTGCACCTCAGGGAAGAATCTGGA
Mouse IFN-γ
F
TCAAAAGAGTTCCTTATGTGCCTA
R
TACGAGGACGGAGAGCTGTT
Mouse actin
679
F
ACCAACTGGGACGACATGGAGAAA
R
TAGCACAGCCTGGATAGCAACGTA
F：Forward primer； R：Reverse primer