Hepatitis C virus E2 envelope glycoprotein produced in Nicotiana benthamiana triggers humoral response with virus‐neutralizing activity in vaccinated mice

Abstract

Chronic infection with hepatitis C virus (HCV) remains a leading cause of liver-related pathologies and a global health problem, currently affecting more than 71 million people worldwide. The development of a prophylactic vaccine is much needed to complement the effective antiviral treatment available and achieve HCV eradication. Current strategies focus on increasing the immunogenicity of the HCV envelope glycoprotein E2, the major target of virus-neutralizing antibodies, by testing various expression systems or manipulating the protein conformation and the N-glycosylation pattern. Here we report the first evidence of successful production of the fulllength HCV E2 glycoprotein in Nicotiana benthamiana, by using the Agrobacterium-mediated transient expression technology. Molecular and functional analysis showed that the viral protein was correctly processed in plant cells and achieved the native folding required for binding to CD81, one of the HCV receptors. N-glycan analysis of HCV-E2 produced in N. benthamiana and mammalian cells indicated host-specific trimming of mannose residues and possibly, protein trafficking. Notably, the plant-derived viral antigen triggered a significant immune response in vaccinated mice, characterized by the presence of antibodies with HCV-neutralizing activity. Together, our study demonstrates that N. benthamiana is a viable alternative to costly mammalian cell cultures for the expression of complex viral antigens and supports the use of plants as cost-effective production platforms for the development of HCV vaccines.

Frontiers in the Standardization of the Plant Platform for High Scale Production of Vaccines

“…Among these, vaccines against influenza viruses H5N1, HAI-05, and H1N1 produced in N. benthamiana and an edible vaccine to treat the hepatitis B virus expressed in tobacco are under clinical trials [62][63][64][65]. More recently, the VP40 antigen of Zaire ebolavirus was produced in N. tobacum and maintained its antigenicity and the ability to induce immune response in mice [66]; a candidate therapeutic vaccine against papillomavirus, consisting of the oncoprotein E7 fused with a bacterial cell-penetrating peptide, was expressed in N. benthamiana [67]; a synthetic gene expressed in tobacco and encoding for a capsid protein of the food and mouth disease virus was able to stimulate the immunogenic response in rabbits [56]; a recombinant vaccine for classical swine fever virus was produced at a cost-effective large scale in N. benthamiana [57]; a full-length hepatitis C virus glycoprotein E2, correctly processed and folded, was expressed in N. benthamiana and induced immune response in vaccinated mice [68].…”

Challenges and Prospects of Plant-Derived Oral Vaccines against Hepatitis B and C Viruses

“…Plants as an ideal green factory have been exploited in the past three decades for the production of various biopharmaceuticals and vaccines for human and animal health as well as high value proteins [17][18][19][20][21].…”

“…With regard to vaccine production, plant-specific N-and O-glycosylation have various effects on recombinant antigens, increasing or attenuating their immunogenicity as compared to their mammalian cell counterparts [22,23]. Processing of complex transmembrane viral proteins, assembly of functional oligomers, and virus-like particles (VLPs) are also possible in plants [21,23]. Plants' components associated with VLPs may have adjuvant properties increasing the protein immunogenicity and thus preventing the use of additional adjuvants.…”

“…Another significant advantage of oral vaccination is the simplification of the immunization protocols, normally requiring multiple injectable doses to ensure sufficient protection, as it is the case for the HBV vaccine. An oral vaccine used as a booster (boost vaccine) after priming with an injectable vaccine was reported [21,25].…”

“…It is worth noting that while strategies to produce HCV proteins in plants have advanced a lot, the immunogenicity and virus neutralization to confirm correct structural and functional properties of the antigen and the efficacy of the immune response are mostly lacking. Our group has shown for the first time that the full length E2 can be expressed in plant-based platforms either as a E1E2 dimer or alone, while retaining the molecular and functional properties of the native antigen [18,21]. Importantly, plant produced HCV proteins induced a strong humoral immune response in vaccinated mice when used as a boost following parenteral immunization with the mammalian cell-derived antigen.…”