An Envelope Domain III–based Chimeric Antigen Produced in Pichia pastoris Elicits Neutralizing Antibodies Against All Four Dengue Virus Serotypes

Etemad, Behzad; Batra, Gaurav; Raut, Rajendra; Dahiya, Satinder; Khanam, Saima; Swaminathan, Sathyamangalam; Khanna, Navin

doi:10.4269/ajtmh.2008.79.353

Cited by 77 publications

(44 citation statements)

References 57 publications

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“…When expressed in Pichia pastoris, the tetravalent fusion protein linking the EDIII domains of the four dengue virus serotypes has already been reported to elicit neutralizing antibodies against all four serotypes (Batra et al 2007;Etemad et al 2008). Although, the plantderived recombinant proteins are required to undergo separate and independent evaluations regarding immunogenicity, the outcome of such studies is expected to be very similar.…”

Section: Discussionmentioning

confidence: 99%

“…Since the main target group for a dengue vaccine are the relatively poor people in developing countries, the ideal dengue vaccine should be affordable, heat-stable (i.e., independent of an uninterrupted cooling chain) and easy to administer. Recombinant dengue virus EDIII protein-based antigens have been previously expressed in E.coli (Khanam et al 2006;McDonald et al 2009;Simmons et al 1999;Srivastava et al 2000;Tripathi et al 2008Tripathi et al , 2011Zhao et al 2014), in yeast (Arora et al 2013;Batra et al 2010a;Cardoso et al 2013;Etemad et al 2008;Nguyen et al 2015), in insect cells (Ivy et al 2000) and also via nuclear transformation in plants (Kim et al 2013;Martinez et al 2010;Saejung et al 2007). In the present study, the dengue virus envelop protein domain III-based tetravalent fusion protein (EDIII-1-4) and the monovalent forms (EDIII-1, EDIII-3 and EDIII-4) have been expressed in tobacco chloroplasts.…”

Section: Discussionmentioning

confidence: 99%

“…Recombinant antigens based on EDIII have been produced using bacteria, yeast, insect cells and plants (Batra et al 2010b;Cardoso et al 2013;Clements et al 2010;Etemad et al 2008;Martinez et al 2010;Saejung et al 2007). Importantly, a recombinant fusion protein linking the EDIII domain of the four dengue viruses as a tetravalent antigen (EDIII-1-4) was able to elicit neutralizing antibodies against all four serotypes (Batra et al 2007;Etemad et al 2008).…”

Section: Introductionmentioning

confidence: 99%

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Production of dengue virus envelope protein domain III-based antigens in tobacco chloroplasts using inducible and constitutive expression systems

et al. 2016

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Dengue fever is a disease in many parts of the tropics and subtropics and about half the world's population is at risk of infection according to the World Health Organization. Dengue is caused by any of the four related dengue virus serotypes DEN-1, -2, -3 and -4, which are transmitted to people by Aedes aegypti mosquitoes. Currently there is only one vaccine (Dengvaxia Ò ) available (limited to a few countries) on the market since 2015 after half a century's intensive efforts. Affordable and accessible vaccines against dengue are hence still urgently needed. The dengue envelop protein domain III (EDIII), which is capable of eliciting serotype-specific neutralizing antibodies, has become the focus for subunit vaccine development. To contribute to the development of an accessible and affordable dengue vaccine, in the current study we have used plant-based vaccine production systems to generate a dengue subunit vaccine candidate in tobacco. Chloroplast genome engineering was applied to express serotypespecific recombinant EDIII proteins in tobacco chloroplasts using both constitutive and ethanol-inducible expression systems. Expression of a tetravalent antigen fusion construct combining EDIII polypeptides from all four serotypes was also attempted. Transplastomic EDIIIexpressing tobacco lines were obtained and homoplasmy was verified by Southern blot analysis. Northern blot analyses showed expression of EDIII antigen-encoding genes. EDIII protein accumulation levels varied for the different recombinant EDIII proteins and the different expression systems, and reached between 0.8 and 1.6 % of total cellular protein. Our study demonstrates the suitability of the chloroplast compartment as a production site for an EDIII-based vaccine candidate against dengue fever and presents a Gateway Ò plastid transformation vector for inducible transgene expression.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Production of dengue virus envelope protein domain III-based antigens in tobacco chloroplasts using inducible and constitutive expression systems

et al. 2016

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“…[17][18][19] In this study, we cloned the EIII gene into pQE30-Xa expression vector and the expression of the recombinant EIII protein in E. coli XL1-Blue MRF′ was chemically induced with IPTG. SDS-PAGE analyses of the protein profiles demonstrated that recombinant EIII protein expression was optimal at approximately 4-5 h post-IPTG induction at 37°C.…”

Section: Resultsmentioning

confidence: 99%

Dengue Envelope Domain III-Peptide Binding Analysis <i>via</i> Tryptophan Fluorescence Quenching Assay

Baharuddin

Hassan

Othman

et al. 2014

CHEMICAL & PHARMACEUTICAL BULLETIN

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In the efforts to find an anti-viral treatment for dengue, a simple tryptophan fluorescence-screening assay aimed at identifying dengue domain III envelope (EIII) protein inhibitors was developed. Residue Trp391 of EIII was used as an intrinsic probe to monitor the change in fluorescence of the tryptophan residue upon binding to a peptide. The analysis was based on the electron excitation at 280 nm and fluorescence emission at 300-400 nm of EIII, followed by quenching of fluorescence in the presence of potential peptidic inhibitors coded DS36wt, DS36opt, DN58wt and DN58opt. The present study found that the fluorescence of the recombinant EIII was quenched following the binding of DS36opt, DN58wt and DN58opt in a concentration-dependent manner. Since the λ max for emission remained unchanged, the effect was not due to a change in the environment of the tryptophan side chain. In contrast, a minimal fluorescence-quenching effect of DS36wt at 20 and 40 µM suggested that the DS36wt does not have any binding ability to EIII. This was supported by a simple native-page gel retardation assay that showed a band shift of EIII domain when incubated with DS36opt, DN58wt and DN58opt but not with DS36wt. We thus developed a low-cost and convenient spectrophotometric binding assay for the analysis of EIII-peptide interactions in a drug screening application.Key words dengue virus type 2; envelope protein; tryptophan assay; fluorescence spectroscopy More than 2.5 million people are at risk of dengue infections in over 100 countries, with several hundred thousand cases of life threatening dengue hemorrhagic fever (DHF)/ dengue shock syndrome (DSS) occurring annually.1) Currently, there is no commercially available vaccine or antiviral agent against dengue virus (DENV) infections. In the efforts towards developing an anti-viral treatment for dengue, a simple tryptophan fluorescence quenching screening assay aimed at identifying dengue EIII protein inhibitors was developed.DENV is a positive-sense, single stranded RNA virus belonging to the Flaviviridae family. The DENV consists of four antigenically distinct serotypes (serotypes 1-4). The glycosylated envelope (E) protein is a type 1 integral membrane protein, and makes up the major structural protein present on the surface of the mature dengue virions. It plays an important role in viral infection via virus attachment to cell-surface receptors and mediates viral-cell membrane fusion. The E protein is the major target for protective antibodies against dengue.2) It has been demonstrated that the mature DENV E protein in the pre-fusion state forms homodimers in an antiparallel manner (head to tail orientation), which transforms to its trimeric form in the post-fusion state.2) The stability and function of the DENV E protein during conformational transition from dimeric to trimeric form is controlled by the change in pH. Each monomer is folded in three distinct domains, with the central domain (domain I) bordered by the immunoglobulin-like C-terminal domain (domain III) and the dimer...

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“…Other laboratories have established high-yield expression systems for EPs of JEV or West Nile virus using the mammalian cell lines COS-1 (5, 15), RK-13 (24,43), and CHO-K1 (52). In addition to mammalian cells, bacterial (2,42,54,57), yeast (7,8,38), and insect (23,36,48,51,56) cells have been used to produce flavivirus vaccine candidates; most of these studies have focused on a truncated E protein or domain III of the E protein. These vaccine candidates were able to induce neutralizing antibodies and/or protection in animals.…”

Section: Discussionmentioning

confidence: 99%

Evaluation of Extracellular Subviral Particles of Dengue Virus Type 2 and Japanese Encephalitis Virus Produced bySpodoptera frugiperdaCells for Use as Vaccine and Diagnostic Antigens

Kuwahara

Konishi

2010

Clin Vaccine Immunol

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An Envelope Domain III–based Chimeric Antigen Produced in Pichia pastoris Elicits Neutralizing Antibodies Against All Four Dengue Virus Serotypes

Cited by 77 publications

References 57 publications

Production of dengue virus envelope protein domain III-based antigens in tobacco chloroplasts using inducible and constitutive expression systems

Production of dengue virus envelope protein domain III-based antigens in tobacco chloroplasts using inducible and constitutive expression systems

Dengue Envelope Domain III-Peptide Binding Analysis <i>via</i> Tryptophan Fluorescence Quenching Assay

Evaluation of Extracellular Subviral Particles of Dengue Virus Type 2 and Japanese Encephalitis Virus Produced bySpodoptera frugiperdaCells for Use as Vaccine and Diagnostic Antigens

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