Chimeric Hepatitis B core antigen virus-like particles displaying the envelope domain III of dengue virus type 2
© Arora et al.; licensee BioMed Central Ltd. 2012
Received: 1 May 2012
Accepted: 13 July 2012
Published: 13 July 2012
Dengue is a global public health problem for which no drug or vaccine is available. Currently, there is increasing interest in developing non-replicating dengue vaccines based on a discrete antigenic domain of the major structural protein of dengue viruses (DENVs), known as envelope domain III (EDIII). The use of bio-nanoparticles consisting of recombinant viral structural polypeptides, better known as virus-like particles (VLPs), has emerged as a potential platform technology for vaccine development. This work explores the feasibility of developing nanoparticles based on E. coli- expressed recombinant Hepatitis B virus core antigen (HBcAg) designed to display EDIII moiety of DENV on the surface.
We designed a synthetic gene construct encoding HBcAg containing an EDIII insert in its c/e1 loop. The fusion antigen HBcAg-EDIII-2 was expressed in E. coli, purified to near homogeneity using Ni+2 affinity chromatography and demonstrated to assemble into discrete 35–40 nm VLPs by electron microscopy. Competitive ELISA analyses showed that the EDIII-2 moieties of the VLPs are accessible to anti-EDIII-2-specific monoclonal and polyclonal antibodies, suggesting that they are surface-displayed. The VLPs were highly immunogenic eliciting high titer anti-EDIII-2 antibodies that were able to recognize, bind and neutralize infectious DENV based on ELISA, immunofluorescence and virus-neutralization assays.
This work demonstrates that HBcAg-derived nanoparticles can serve as a useful platform for the display of DENV EDIII. The EDIII-displaying nanoparticles may have potential applications in diagnostics/vaccines for dengue.
Dengue is a mosquito-borne viral disease prevalent in more than a hundred tropical and sub-tropical countries placing about 2.5 billion of the global population at risk, causing 50–100 million infections and ~12,500 deaths each year . There are four serotypes of dengue viruses (DENV-1, -2, -3 and −4), belonging to the family Flaviviridae, each of which can cause dengue disease . The tremendous challenges being faced in the development of live viral dengue vaccines  has spurred a keen interest in new generation non-replicating subunit vaccines [3, 4]. In this context, a discrete domain of the viral envelope protein has been identified as a potential subunit vaccine candidate [3–5]. This domain known as envelope domain III (EDIII), is exposed and accessible on the virion surface , contains multiple type- and subtype-specific neutralizing epitopes  and is implicated in host receptor binding .
One way to augment the vaccine potential of EDIII would be to display it in multiple copies on the surface of a nanoparticle carrier. Naturally occurring bio-nanomaterials, by virtue of their biocompatibility and biodegradability, are emerging as preferred platforms for biomedical applications . Viruses are naturally occurring nanoparticles whose particulate nature with repetitive coat protein architecture and pathogen associated molecular patterns make them a potentially valuable platform for vaccine development. A subclass of viral nanoparticles can be generated in heterologous expression systems exploiting the propensity of several recombinant viral coat proteins to self-assemble. These genome-free viral nanoparticles which eliminate the infectious biohazard inherent in the naturally occurring viral nanoparticles are more popularly known as virus-like particles (VLPs) [9, 10]. The 183 amino acid (aa) residue Hepatitis B virus core antigen (HBcAg) which can assemble into VLPs is a well-documented carrier of foreign antigens from several different pathogens  and HBcAg-based VLP vaccine candidates for malaria and influenza are being evaluated in clinical trials [12, 13]. Of relevance to our study is the reported capacity of HBcAg to accommodate foreign inserts (in the size range of 100–260 aa residues) in its surface-exposed c/e1 loop, with retention of its VLP-forming ability. This loop which contains the major B-cell epitopes ‘c’ and ‘e1’ is also known as the major immunodominant region of the HBcAg VLPs . Optimal immunogenicity of the foreign insert is ensured by the concomitant removal of these major HBcAg immunodominant epitopes. Interestingly, many of these chimeric VLPs have been produced using E. coli expression hosts [14–20]. In this study, we report the design, creation and characterization of chimeric nanoparticles containing DENV-2 EDIII (herein indicated as EDIII-2) inserted into the c/e1 loop of HBcAg. Further we show that these chimeric VLPs display EDIII-2 on the surface and elicit antibodies specific to DENV-2.
In conclusion, this work has demonstrated the feasibility of using an E. coli expression system to produce chimeric nanoparticles using a fusion antigen comprising of the HBcAg polypeptide with EDIII-2 inserted into its surface-exposed c/e1 loop. It has shown further that the EDIII-2 moiety is displayed on the surface of the chimeric nanoparticle and is able to induce the production of specific antibodies capable of binding and neutralizing the infectivity of DENV-2. This work provides the basis for us to envisage next generation HBcAg-derived mosaic nanoparticles that display the EDIII domains of not one, but all four DENV serotypes. Such nanoparticles could have potentially interesting and possibly useful diagnostic and vaccine potential.
Baby hamster kidney
Dengue virus type 2
Envelope domain III, EDIII-2, Envelope domain III of dengue virus type 2
Enzyme-linked immunosorbent assay
Hepatitis B virus core antigen
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
SS and NK acknowledge the funding support received from the Department of Biotechnology, Government of India. UA was the recipient of a research fellowship from the Department of Biotechnology, Government of India.
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