Epitope Presentation of Dengue Viral Envelope Glycoprotein Domain III on Hepatitis B Core Protein Virus-Like Particles Produced in Nicotiana benthamiana

Abstract

Dengue fever is currently ranked as the top emerging tropical disease, driven by increased global travel, urbanization, and poor hygiene conditions as well as global warming effects which facilitate the spread of Aedes mosquitoes beyond their current distribution. Today, more than 100 countries are affected most of which are tropical Asian and Latin American nations with limited access to medical care. Hence, the development of a dengue vaccine that is dually cost-effective and able to confer a comprehensive protection is ultimately needed. In this study, a consensus sequence of the antigenic dengue viral glycoprotein domain III (cEDIII) was used aiming to provide comprehensive coverage against all four circulating dengue viral serotypes and potential clade replacement event. Utilizing hepatitis B tandem core technology, the cEDIII sequence was inserted into the immunodominant c/e1 loop region so that it could be displayed on the spike structures of assembled particles. The tandem core particles displaying cEDIII epitopes (tHBcAg-cEDIII) were successfully produced in Nicotiana benthamiana via Agrobacterium -mediated transient expression strategy to give a protein of ∼54 kDa, detected in both soluble and insoluble fractions of plant extracts. The assembled tHBcAg-cEDIII virus-like particles (VLPs) were also visualized from transmission electron microscopy. These VLPs had diameters that range from 32 to 35 nm, presenting an apparent size increment as compared to tHBcAg control particles without cEDIII display (namely tEL). Mice immunized with tHBcAg-cEDIII VLPs showed a positive seroconversion to cEDIII antigen, thereby signifying that the assembled tHBcAg-cEDIII VLPs have successfully displayed cEDIII antigen to the immune system. If it is proven to be successful, tHBcAg-cEDIII has the potential to be developed as a cost-effective vaccine candidate that confers a simultaneous protection against all four infecting dengue viral serotypes.

Efficient Production of Chimeric Hepatitis B Virus-Like Particles Bearing an Epitope of Hepatitis E Virus Capsid by Transient Expression in Nicotiana benthamiana

“…The chimeric particle diameter ranged from 28 to 38 nm, and the surface appeared to be rather “knobbly” ( Figure 5 , panel b). The “knobbly” surface morphology is expected for HBcAg core particles displaying a heterologous sequence on their surface [ 34 , 42 ]. This indicates that HEV ORF2 551–607 aa were presented on the surface of HBcAg spikes.…”

“…The hepatitis B capsid protein (HBcAg) has been shown to be capable of self-assembly into VLPs when expressed in a number of eukaryotic heterologous systems including mammalian cells [ 27 ], plants [ 28 , 29 , 30 ], yeast [ 31 ], insect cells [ 32 ], and Xenopus oocytes [ 33 ]. VLPs composed of HBcAg are safe and potent vaccine delivery systems [ 34 , 35 ]. HBcAg consists of 183 amino acids with an N-terminal assembly domain (1–149 aa) and a C-terminal arginine-rich domain (34 aa) required for the packaging of nucleic acid [ 36 ].…”

Rapid High-Yield Transient Expression of Swine Hepatitis E ORF2 Capsid Proteins in Nicotiana benthamiana Plants and Production of Chimeric Hepatitis E Virus-Like Particles Bearing the M2e Influenza Epitope

“…Therefore, we compared the performance of two expression systems, i.e., the -pEAQ vector based on genetic elements of the CPMV genome, which we were unable to replicate in plant cells, and PVX-based self-replicating pEff vector. Both systems have been successfully used for the expression of a number of recombinant proteins in plants [37][38][39][40][41]. The first two-four days post infiltration of the plant leaves with pEff vector showed an accumulation of higher level of the HEV ORF2 proteins compared with pEAQ-HT vector expression of the same proteins.…”

Hepatitis B core-based virus-like particles: A platform for vaccine development in plants

“… Pathogen Epitope(s) Expression system Site of epitope insertion Ref. Dengue virus cEDIII N. benthamiana MIR [ 68 ] H1N1 Influenza A virus matrix protein 2 E. coli MIR [ 188 ] H7N9 Influenza long alpha-helix (LAH) E. coli MIR [ 189 ] Mycobacterium Tuberculosis (Tuberculosis) Culture filtrate protein 10 (CFP 10) E. coli MIR [ 190 ] – Hepatitis B Core Antigen E. coli MIR [ 86 ] Nicotiana benthamiana GGS sequence Influenza virus M2e E. coli MIR [ 191 ] Influenza virus M2e N. benthamiana N-terminal [ 192 ] Dengue virus EDIII-2 E. coli MIR [ 193 ] Dengue virus EDIII E. coli MIR [ …”

“…After the primary study by Clarke et al in 1987 [ 65 ], different epitopes and antigens have been introduced into HBc protein for vaccine development ( Table 6 ). The immunogenic epitope of the virus was fused to the N-terminus end of the HBc sequence [ [66] , [67] , [68] , [69] ]. This VLP has been used to produce HBV vaccine in yeast [ 70 , 71 ] and mammalian cells (CHO) [ 20 ].…”