Selection and characterization of vaccine strain for Enterovirus 71 vaccine development

Chang, Jui-Yuan; Chang, Cheng-Peng; Tsai, Hutchinson Hau-Pong; Lee, Chen-Dou; Lian, Wei-Cheng; Ih-Jen-Su,; Sai, I-Hsi; Liu, Chia-Chyi; Chou, Ai-Hsiang; Lu, Ya‐Jung; Chen, Ching‐Yao; Lee, Pi-Hsiu; Chiang, Jen-Ron; Chong, Pele

doi:10.1016/j.vaccine.2011.11.087

Cited by 57 publications

(50 citation statements)

References 20 publications

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“…In several laboratories, neonatal mice have been used as models for studying EV71 pathogenesis and for the development of vaccines and antiviral drugs (17,18,(45)(46)(47). However, EV71 infection in the neonatal mouse model is significantly different from that in humans as the major viral replication sites in the mouse system include muscle and adipose tissues.…”

Section: Discussionmentioning

confidence: 99%

Transgenic mouse model for the study of enterovirus 71 neuropathogenesis

Fujii¹,

Nagata

Sato

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

139

150

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Enterovirus 71 (EV71) typically causes mild hand-foot-and-mouth disease in children, but it can also cause severe neurological disease. Recently, epidemic outbreaks of EV71 with significant mortality have been reported in the Asia-Pacific region, and EV71 infection has become a serious public health concern worldwide. However, there is little information available concerning EV71 neuropathogenesis, and no vaccines or anti-EV71 drugs have been developed. Previous studies of this disease have used monkeys and neonatal mice that are susceptible to some EV71 strains as models. The monkey model is problematic for ethical and economical reasons, and mice that are more than a few weeks old lose their susceptibility to EV71. Thus, the development of an appropriate small animal model would greatly contribute to the study of this disease. Mice lack EV71 susceptibility due to the absence of a receptor for this virus. Previously, we identified the human scavenger receptor class B, member 2 (hSCARB2) as a cellular receptor for EV71. In the current study, we generated a transgenic (Tg) mouse expressing hSCARB2 with an expression profile similar to that in humans. Tg mice infected with EV71 exhibited ataxia, paralysis, and death. The most severely affected cells were neurons in the spinal cord, brainstem, cerebellum, hypothalamus, thalamus, and cerebrum. The pathological features in these Tg mice were generally similar to those of EV71 encephalomyelitis in humans and experimentally infected monkeys. These results suggest that this Tg mouse could represent a useful animal model for the study of EV71 infection.picornavirus | neurotropism | viral receptor E nterovirus 71 (EV71) is a human enterovirus species A of the genus Enterovirus within the Picornaviridae family, and it is known to be one of the causative agents of hand-foot-and-mouth disease (HFMD) (1, 2). HFMD is generally considered to be a mild exanthematous disease. However, in some infants and young children, after a few days of prodromal illness, HFMD caused predominantly by EV71 can be complicated by neurological manifestations, including ataxia, tremor, myoclonus, polio-like paralysis, encephalomyelitis, cardiopulmonary failure, and death (3, 4). In humans with fatal EV71 encephalomyelitis, inflammation and viral antigens in neurons were observed mainly in the spinal cord, brainstem, hypothalamus, cerebellar dentate nucleus, and cerebrum (5, 6). Since the 1970s, HFMD outbreaks with significant mortality have been reported throughout the world, including in Bulgaria in 1975 [44 deaths (7) Appropriate animal models are needed to better understand EV71 neuropathogenesis and to facilitate the development of effective vaccines and drugs. EV71-infected cynomolgus monkeys developed neurological complications similar to those observed in human cases, including ataxia, tremor, and flaccid paralysis (11-15), and pathological lesions were observed in the spinal cord, brainstem, cerebellar dentate nucleus, and other parts of the brain (14,15). However, the use of monkeys to mod...

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Section: Discussionmentioning

confidence: 99%

Transgenic mouse model for the study of enterovirus 71 neuropathogenesis

Fujii¹,

Nagata

Sato

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

139

150

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“…Master and working Vero cell and virus (E59 strain) seed banks were established following cGMP guidelines, characterized to fulfill the requirements for the manufacture of biological products by BioReliance (UK), and reported in our previous study [19].…”

Section: Methodsmentioning

confidence: 99%

“…EV71 virus stock was produced using the roller bottle technology, and purified by an AKTA Pilot liquid chromatography system purchased from GE Healthcare (USA) equipped with Sepharose Fast Flow 6 gel, and reported in our previous studies [10], [19].…”

Section: Methodsmentioning

confidence: 99%

Pilot Scale Production of Highly Efficacious and Stable Enterovirus 71 Vaccine Candidates

et al. 2012

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BackgroundEnterovirus 71 (EV71) has caused several epidemics of hand, foot and mouth diseases (HFMD) in Asia and now is being recognized as an important neurotropic virus. Effective medications and prophylactic vaccine against EV71 infection are urgently needed. Based on the success of inactivated poliovirus vaccine, a prototype chemically inactivated EV71 vaccine candidate has been developed and currently in human phase 1 clinical trial.Principal FindingIn this report, we present the development of a serum-free cell-based EV71 vaccine. The optimization at each step of the manufacturing process was investigated, characterized and quantified. In the up-stream process development, different commercially available cell culture media either containing serum or serum-free was screened for cell growth and virus yield using the roller-bottle technology. VP-SFM serum-free medium was selected based on the Vero cell growth profile and EV71 virus production. After the up-stream processes (virus harvest, diafiltration and concentration), a combination of gel-filtration liquid chromatography and/or sucrose-gradient ultracentrifugation down-stream purification processes were investigated at a pilot scale of 40 liters each. Although the combination of chromatography and sucrose-gradient ultracentrifugation produced extremely pure EV71 infectious virus particles, the overall yield of vaccine was 7–10% as determined by a VP2-based quantitative ELISA. Using chromatography as the downstream purification, the virus yield was 30–43%. To retain the integrity of virus neutralization epitopes and the stability of the vaccine product, the best virus inactivation was found to be 0.025% formalin-treatment at 37°C for 3 to 6 days. Furthermore, the formalin-inactivated virion vaccine candidate was found to be stable for >18 months at 4°C and a microgram of viral proteins formulated with alum adjuvant could induce strong virus-neutralizing antibody responses in mice, rats, rabbits, and non-human primates.ConclusionThese results provide valuable information supporting the current cell-based serum-free EV71 vaccine candidate going into human Phase I clinical trials.

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“…Production of bulk EV-71 vaccines in roller bottles/bioreactor using a serum-free media and its purification by Sepharose Fast Flow 6 gel was reported by Liu et al [28] where the average recovery was about 50%, as is the case with our results with a slight (5%) increase in recovery. A 50% recovery of EV-71 during the purification by gel-filtration chromatography was also reported by Chang et al [5] but they suggested that the chromatographic process needs to be improved due to the inconsistent results obtained.…”

Section: Discussionmentioning

confidence: 59%

“…The most effective way to control the disease caused by EV-71 is by vaccination and thus arises the need for the development of new vaccines [4]. As inactivated polio vaccine elicits long term protection against the virus, this strategy might be efficacious for chemically inactivated EV-71 as a vaccine candidate [5]. In recent years, several researchers [4,6,7] have shown that inactivated EV-71 (heat or formalin inactivation) induces a strong, viral-neutralizing antibody response in animal models, thus protecting them against a lethal EV-71 challenge.…”

Section: Introductionmentioning

confidence: 99%

Concentration and purification of enterovirus 71 using a weak anion-exchange monolithic column

Venkatachalam

Szyporta

Kiener

et al. 2014

Virol J

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BackgroundEnterovirus 71 (EV-71) is a neurotropic virus causing Hand, Foot and Mouth Disease (HFMD) in infants and children under the age of five. It is a major concern for public health issues across Asia-Pacific region. The most effective way to control the disease caused by EV-71 is by vaccination thus a novel vaccine is urgently needed. Inactivated EV-71 induces a strong, virus-neutralizing antibody response in animal models, protecting them against a lethal EV-71 challenge and it has been shown to elicit cross-neutralizing antibodies in human trials. Hence, the large-scale production of purified EV-71 is required for vaccine development, diagnosis and clinical trials.MethodsCIM® Monolith columns are single-piece columns made up of poly(glycidyl methacrylate co-ethylene dimethacrylate) as support matrix. They are designed as porous channels rather than beads with different chemistries for different requirements. As monolithic columns have a high binding capacity, flow rate and resolution, a CIM® DEAE-8f tube monolithic column was selected for purification in this study. The EV-71 infected Rhabdomyosarcoma (RD) cell supernatant was concentrated using 8% PEG 8000 in the presence of 400 mM sodium chloride. The concentrated virus was purified by weak anion exchange column using 50 mM HEPES + 1 M sodium chloride as elution buffer.ResultsHighly pure viral particles were obtained at a concentration of 350 mM sodium chloride as confirmed by SDS-PAGE and electron microscopy. Presence of viral proteins VP1, VP2 and VP3 was validated by western blotting. The overall process achieved a recovery of 55%.ConclusionsEV-71 viral particles of up to 95% purity can be recovered by a single step ion-exchange chromatography using CIM-DEAE monolithic columns and 1 M sodium chloride as elution buffer. Moreover, this method is scalable to purify several litres of virus-containing supernatant, using industrial monolithic columns with a capacity of up to 8 L such as CIM® cGMP tube monolithic columns.

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Selection and characterization of vaccine strain for Enterovirus 71 vaccine development

Cited by 57 publications

References 20 publications

Transgenic mouse model for the study of enterovirus 71 neuropathogenesis

Transgenic mouse model for the study of enterovirus 71 neuropathogenesis

Pilot Scale Production of Highly Efficacious and Stable Enterovirus 71 Vaccine Candidates

Concentration and purification of enterovirus 71 using a weak anion-exchange monolithic column

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