Production of Japanese encephalitis virus-like particles in insect cells

ABSTRACTJapanese encephalitis virus (JEV) membrane (M) protein plays important structural roles in the processes of fusion and maturation of progeny virus during cellular infection. The M protein is anchored in the viral membrane, and its ectodomain is composed of a flexible N-terminal loop and a perimembrane helix. In this study, we performed site-directed mutagenesis on residue 36 of JEV M protein and showed that the resulting mutation had little or no effect on the entry process but greatly affected virus assembly in mammalian cells. Interestingly, this mutant virus had a host-dependent phenotype and could develop a wild-type infection in insect cells. Experiments performed on infectious virus as well as in a virus-like particle (VLP) system indicate that the JEV mutant expresses structural proteins but fails to form infectious particles in mammalian cells. Using a mouse model for JEV pathogenesis, we showed that the mutation conferred complete attenuationin vivo. The production of JEV neutralizing antibodies in challenged mice was indicative of the immunogenicity of the mutant virusin vivo. Together, our results indicate that the introduction of a single mutation in the M protein, while being tolerated in insect cells, strongly impacts JEV infection in mammalian hosts.IMPORTANCEJEV is a mosquito-transmitted flavivirus and is a medically important pathogen in Asia. The M protein is thought to be important for accommodating the structural rearrangements undergone by the virion during viral assembly and may play additional roles in the JEV infectious cycle. In the present study, we show that a sole mutation in the M protein impairs the JEV infection cycle in mammalian hosts but not in mosquito cells. This finding highlights differences in flavivirus assembly pathways among hosts. Moreover, infection of mice indicated that the mutant was completely attenuated and triggered a strong immune response to JEV, thus providing new insights for further development of JEV vaccines.

Download Full-text

Production of Japanese encephalitis virus-like particles using the baculovirus–insect cell system

Journal of Bioscience and Bioengineering ◽

10.1016/j.jbiosc.2012.06.012 ◽

2012 ◽

Vol 114 (6) ◽

pp. 657-662 ◽

Cited By ~ 22

Author(s):

Hideki Yamaji ◽

Maiko Segawa ◽

Masataka Nakamura ◽

Tomohisa Katsuda ◽

Miwa Kuwahara ◽

...

Keyword(s):

Japanese Encephalitis Virus ◽

Japanese Encephalitis ◽

Insect Cell ◽

Cell System ◽

Encephalitis Virus ◽

Virus Like Particles

Download Full-text

A Novel Recombinant Virus-Like Particles Displaying B and T Cell Epitopes of Japanese Encephalitis Virus Offers Protective Immunity in Mice and Guinea Pigs

Vaccines ◽

10.3390/vaccines9090980 ◽

2021 ◽

Vol 9 (9) ◽

pp. 980

Author(s):

Muhammad Naveed Anwar ◽

Chunying Jiang ◽

Di Di ◽

Junjie Zhang ◽

Shuang Guo ◽

...

Keyword(s):

Japanese Encephalitis Virus ◽

Japanese Encephalitis ◽

Guinea Pigs ◽

Neutralizing Antibodies ◽

Protective Immunity ◽

Vaccine Development ◽

Amino Acid Sequences ◽

Encephalitis Virus ◽

Potential Candidate ◽

Virus Like Particles

Virus-like particles (VLPs) are non-replicative vectors for the delivery of heterologous epitopes and are considered one of the most potent inducers of cellular and humoral immune responses in mice and guinea pigs. In the present study, VLP-JEVe was constructed by the insertion of six Japanese encephalitis virus (JEV) envelope protein epitopes into different surface loop regions of PPV VP2 by the substitution of specific amino acid sequences without altering the assembly of the virus; subsequently, the protective efficacy of this VLP-JEVe was evaluated against JEV challenge in mice and guinea pigs. Mice immunized with the VLP-JEVe antigen developed high titers of neutralizing antibodies and 100% protection against lethal JEV challenge. The neutralizing and hemagglutination inhibition (HI) antibody responses were also induced in guinea pigs vaccinated with VLP-JEVe. In addition, immunization with VLP-JEVe in mice induced effective neutralizing antibodies and protective immunity against PPV (porcine parvovirus) challenge in guinea pigs. These studies suggest that VLP-JEVe produced as described here could be a potential candidate for vaccine development.

Download Full-text

Genotype I of Japanese Encephalitis Virus Virus-like Particles Elicit Sterilizing Immunity against Genotype I and III Viral Challenge in Swine

Scientific Reports ◽

10.1038/s41598-018-25596-1 ◽

2018 ◽

Vol 8 (1) ◽

Cited By ~ 10

Author(s):

Yi-Chin Fan ◽

Jo-Mei Chen ◽

Jen-Wei Lin ◽

Yi-Ying Chen ◽

Guan-Hong Wu ◽

...

Keyword(s):

Japanese Encephalitis Virus ◽

Japanese Encephalitis ◽

Encephalitis Virus ◽

Genotype I ◽

Viral Challenge ◽

Virus Like Particles ◽

Sterilizing Immunity

Download Full-text

Stable High-Producer Cell Clone Expressing Virus-Like Particles of the Japanese Encephalitis Virus E Protein for a Second-Generation Subunit Vaccine

Journal of Virology ◽

10.1128/jvi.77.16.8745-8755.2003 ◽

2003 ◽

Vol 77 (16) ◽

pp. 8745-8755 ◽

Cited By ~ 36

Author(s):

Asato Kojima ◽

Atsushi Yasuda ◽

Hideki Asanuma ◽

Toyokazu Ishikawa ◽

Akihisa Takamizawa ◽

...

Keyword(s):

Japanese Encephalitis Virus ◽

Japanese Encephalitis ◽

Neutralizing Antibodies ◽

Second Generation ◽

Cell Clone ◽

Encephalitis Virus ◽

Rabbit Kidney ◽

Enzyme Linked Immunosorbent Assay ◽

Dilution Method ◽

Virus Like Particles

ABSTRACT We produced and characterized a cell clone (J12#26 cells) that stably expresses Japanese encephalitis virus (JEV) cDNA, J12, which encodes the viral signal peptide, premembrane (prM), and envelope (E) proteins (amino acid positions 105 to 794). Rabbit kidney-derived RK13 cells were transfected with a J12 expression plasmid, selected by resistance to marker antibiotics, and cloned by two cycles of a limiting-dilution method in the presence of antibiotics, a procedure that prevents the successful generation of E-producing cell clones. J12#26 cells secreted virus-like particles containing the authentic E antigen (E-VLP) into the culture medium in a huge enzyme-linked immunosorbent assay-equivalent amount (2.5 μg per 104 cells) to the internationally licensed JE vaccine JE-VAX. E-VLP production was stable after multiple cell passages and persisted over 1 year with 100% expressing cells without detectable cell fusion, apoptosis, or cell death, but was suspended when the cells grew to 100% confluency and contact inhibition occurred. Mice immunized with the purified J12#26 E-antigen without adjuvant developed high titers of neutralizing antibodies for at least 7 months and 100% protection against intraperitoneal challenge with 5 × 106 PFU of JEV when examined according to the JE vaccine standardization protocol. These results suggest that the recombinant E-VLP antigen produced by the J12#26 cell clone is an effective, safe, and low-cost second-generation subunit JE vaccine.

Download Full-text