Fragment of Japanese encephalitis virus envelope protein produced in Escherichia coli protects mice from virus challenge

doi:10.1006/mpat.2001.0442

Microbial Pathogenesis

Volume 31, Issue 1, July 2001, Pages 9-19

https://doi.org/10.1006/mpat.2001.0442 Get rights and content

Abstract

A fragment from the N-terminal part (E_A) and a fragment from the C-terminal part (E_B) of the envelope (E) protein of Japanese encephalitis virus (JEV) was synthesized in Escherichia coli. These two fragments were overlapping with each other by nine amino acids, however, they were not cross-reacting with each other at the antisera level. Both E_Aand E_Bare antigenic by themselves when injected into mice, but when tested against sera from mice, rabbit, swine and human that had been immunized or naturally infected with JEV, E_Bacted as a better antigen than E_Aby ELISA assays. E_Balso proved to be a better immunogen in protection against lethal JEV infection than E_A. The protection appears to be correlated with the neutralizing titres of the anti-JEV sera. The response elicited by E_Bis a Th1 response and the antibody produced contained higher neutralizing titre than E_Afragment. The major difference between E_Aand E_Bfragments is the solubility during expression inE. coli , while E_Bfragment is soluble, E_Awas isolated from the insoluble inclusion bodies. Therefore the antigenicity and immunogenicity expressed by the E_Bfragment may probably be due to its proper folding to assume a correctly assembled form during expression in E. coli, a quality that is important for a protein to qualify as a good vaccine candidate.

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A trans-complemented chimeric CSF-JE virus replicon was constructed using an infectious cDNA clone of the CSF virus (CSFV) Alfort/187 strain. The CSFV E2 gene was deleted, and a fragment containing the region encoding a truncated envelope protein (tE, amino acid 292–402, domain III) of JE virus (JEV) was inserted into the resultant plasmid, pA187delE2, to generate the recombinant cDNA clone pA187delE2/JEV-tE. Porcine kidney 15 (PK15) cells that constitutively express the CSFV E2p7 proteins were then transfected with in vitro-transcribed RNA from pA187delE2/JEV-tE. As a result, the chimeric CSF-JE virus replicon particle (VRP), rv187delE2/JEV-tE, was rescued. In a mouse model, immunization with the chimeric CSF-JE VRP induced strong production of JEV-specific antibody and conferred protection against a lethal JEV challenge. Pigs immunized with CSF-JE VRP displayed strong anti-CSFV and anti-JEV antibody responses and protection against CSFV and JEV challenge infections. Our evidence suggests that E2-complemented CSF-JE VRP not only has potential as a live-attenuated non-transmissible vaccine candidate against CSF and JE but also serves as a potential DIVA (Differentiating Infected from Vaccinated Animals) vaccine for CSF in pigs. Together, our data suggest that the non-transmissible chimeric VRP expressing foreign antigenic proteins may represent a promising strategy for bivalent DIVA vaccine design.
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Citation Excerpt :
Various other types of JE vaccine are now at advanced stages of development or already available on the international markets. Although still at the preliminary stage of preclinical studies, research on the development of recombinant JE vaccines using E or NS1 protein is ongoing in several laboratories; the results of such studies are briefly summarized here [17–25]. Chia et al. have shown that a sub-fragment of the E protein produced in E. coli was protective to mice when 25 μg of the antigen was used in combination with Complete Freund's adjuvant (CFA) or Incomplete Freund's adjuvant (IFA) [24].
Ectodomain of Japanese encephalitis virus (JEV) E protein [domains I through III (D1–3), domains I and II (D1–2) and domain III (D3)] and the nonstructural protein 1 (NS1) were expressed in Escherichia coli, and administered to BALB/c mice via the intranasal (i.n.) route. The E protein, but not the NS1, induced JEV-specific serum IgG with virus-neutralization capacity in vitro. When mice were lethally challenged with JEV, i.n. immunization with D1–3, D1–2, D3, or a mouse brain-derived formalin-inactivated JE vaccine conferred complete protection, while an 80% protection rate was observed in the NS1 immunized mice. Cytokine analysis of the cervical lymph nodes of mice i.n. immunized with D1–3 or NS1 revealed antigen-specific IL-2 and IL-17 responses, but no IFN-γ T cell response, were observed. This study demonstrates for the first time the i.n. vaccine efficacy of the E. coli-expressed recombinant JEV proteins.
Enhancement of humoral and cellular immunity in mice against Japanese encephalitis virus using a DNA prime-protein boost vaccine strategy
2010, Veterinary Journal
A synthetic multi-epitope gene containing critical epitopes of the Japanese encephalitis virus (JEV) envelope gene was cloned into both prokaryotic and eukaryotic expression vectors. The recombinant plasmid and purified recombinant protein (heterologously expressed in Escherichia coli) were used as immunogens in a mouse model. The results indicate that both the recombinant protein and the DNA vaccine induce humoral and cellular immune responses. Neutralising antibody titres in mice in the pcDNA-TEP plus rEP group increased considerably relative to mice immunised using either pcDNA-TEP or rEP alone (P < 0.05). Furthermore, the highest levels of interleukin (IL)-2, interferon-γ and IL-4 were induced following priming with the DNA vaccine and boosting with the recombinant protein. Together these findings demonstrate that a DNA-recombinant protein prime-boost vaccination strategy can produce high levels of antibody and trigger significant T cell responses in mice, highlighting the potential value of such an approach in the prevention of JEV infection.
Cloning and Sequence Analysis of the Full-Length Genome of Japanese encephalitis virus Strain SXBJ07 Isolated from Swine
2009, Agricultural Sciences in China
A virus strain, showing cytopathic effect in BHK-21 cell, was isolated from swine brains in Shaanxi Province, China, in 2007. The isolate was confirmed as Japanese encephalitis virus (JEV) by immunofluorescence assay (IFA) and reverse transcription polymerase chain reaction (RT-PCR), and named SXBJ07. The complete nucleotide and deduced amino acid sequences of the JEV strain SXBJ07 were determined. Its single open reading frame has a total of 3 432 amino acid residues. An extensive E gene based phylogenetic analysis was performed, the result showed that SXBJ07 strain belongs to genotype I. Comparison of the SXBJ07 genomic sequence with those of the 24 fully sequenced JEV strains in published databases showed nucleotide homology ranging from 99.0 to 83.7%; amino acid homology ranged from 99.8 to 94.8%. Compared SXBJ07 with SA14-14-2 strain, the current live vaccine strain in China, the homology of amino acid in envelope gene was 97.0%; and there were amino acid substitutions in 13 sites of the active domains of E protein (E1-E411).
Evaluation of murine bone marrow-derived dendritic cells loaded with inactivated virus as a vaccine against Japanese encephalitis virus
2009, Vaccine
Japanese encephalitis (JE) is a serious infectious disease in southern and eastern Asia. Design and development of safer and more efficacious vaccines against Japanese encephalitis virus (JEV) is a high-priority target in the world. Dendritic cells (DCs) are the most potent professional antigen-presenting cells (APCs) playing a central and unique role in the generation of primary T-cell responses, and are considered attractive “live adjuvants” for vaccination and immunotherapy against cancer and infectious diseases. In this study, mouse bone marrow-derived dendritic cells (bmDCs were generated and stimulated with inactivated JEV in vitro. BALB/c mice were immunized with stimulated bmDCs and then challenged with JEV wild-type strain. The neutralization antibody, interferon gamma (IFN-γ), tumor necrosis factors alpha (TNF-α) or interleukin-6 (IL-6), and virus-specific CD8+ cytotoxic T-lymphocyte (CTL) levels, as well as survival rates, were analyzed and compared with inactivated vaccine and DCs control groups. The results demonstrated that intravenous (i.v.) injection of 2 × 10⁵ JEV-pulsed bmDCs into each mouse produced notable levels of JEV-specific neutralizing antibodies and higher levels of CD8+ CTL, IFN-γ and TNF-α compared with JEV-inactivated vaccine. Furthermore, stimulated bmDCs could elicit a highly protective efficacy (90%) against JEV challenge. It suggests that stimulated bmDCs can be considered as an attractive “live adjuvant” for vaccination against JEV infection.
Selection of immunodominant fragments from envelope gene for vaccine against Japanese encephalitis virus in DNA priming-protein boosting protocols
2005, Microbial Pathogenesis
Fragmentation of E gene of JEV into smaller fragments, none of the fragments either in plasmids form or in recombinant protein form can induce optimal protection against the virus infection. It is only when DNA priming–protein boosting strategies are used then the N-terminal E_A and the C-terminal E_B showed full protection against JEV as those induced by commercial vaccine, provided both fragments are preceded in the N-terminal by a signal peptide M₁₅ derived from C-terminal of prM gene in JEV genome. When the subfragments of E_A: E_A1 and E_A2 and E_B: E_B1 and E_B2 are tested, only E_A1 subfragment can replace E_A in protein boosting to induce optimal protection against JEV, E_A2, E_B1, E_B2 in plasmid or protein forms are not. Therefore, along the E gene (978–2330 bp) N-terminal, E_A1 (978–1580 bp) and C-terminal E_B (1851–2330 bp) are the most effective in inducing immunity against JEV but not the middle fragment E_A2 (1518–1877 bp) (see Fig. 1 for orientation of E_A1, E_A2 and E_B in E gene). Under the notion that molecular complexity determines the outcome of immune response of the host, E_B being shorter, simpler in molecular structure and can be easily expressed in soluble form in E. coli (as opposed to insoluble E_A1), E_B probably will be the choice as a candidate vaccine to protect the host against JEV infection.

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^f1: Author for correspondence. E-mail: [email protected]. edu.tw

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Regular ArticleFragment of Japanese encephalitis virus envelope protein produced in Escherichia coli protects mice from virus challenge

Abstract

Virology

Virology

Vaccine

Virology

Mol Biochem Parasitol

Virology

Adv Immunol

Virology

Virology

FlaviÍviridae

Intervirology

Flavivirus genome organization, expression and replication

Ammu Rev Microbiol

Biological activities of the structural proteins of Japanese encephalitis virus

Acta Virologica

Immunogenicity of tick-borne encephalitis virus glycoÍprotein fragments: epitope-specific analysis of antibody response

J Gen Virol

Protection of mice against Japanese encephalitis virus by passive administration with monoclonal antibodies

J Immunol

Molecular characterization of a neutralizing domain of the Japanese encephalitis virus structural glycoprotein

J Gen Virol

Regular Article
Fragment of Japanese encephalitis virus envelope protein produced in Escherichia coli protects mice from virus challenge