Transiently Expressed Mistletoe Lectin II in Nicotiana benthamiana Demonstrates Anticancer Activity In Vitro

Mistletoe (Viscum album) extracts have been used as alternative and complementary therapeutic preparations in multiple cancers for decades. Mistletoe lectins (ML-I, ML-II, and ML-III) are considered to be the main anticancer components of such preparations. In the present study, ML-II was transiently expressed in Nicotiana benthamiana using the pEAQ-HT expression system. Expression levels of up to 60 mg/kg of the infiltrated plant tissue were obtained, and a three-fold increase was achieved by adding the endoplasmic reticulum (ER) retention signal KDEL to the native ML-II sequence. The native protein containing His-tag and KDEL was purified by immobilized metal affinity chromatography (IMAC) and gel filtration. We found that the recombinant ML-II lectin was glycosylated and retained its carbohydrate-binding activity. In addition, we demonstrated that plant produced ML-II displayed anticancer activity in vitro, inhibiting non-small cell lung cancer H460 and A549 cells with EC50 values of 4 and 3.5 µg/mL, respectively. Annexin V-448A and PI double staining revealed that cell cytotoxicity occurred via apoptosis induction. These results indicate that ML-II transiently expressed in N. benthamiana plants is a promising candidate as an anticancer agent, although further optimization of production and purification methods is required to enable further in vitro testing, as well as in vivo assays

Progress in the Production of Virus-Like Particles for Vaccination against Hepatitis E Virus

Hepatitis E virus (HEV), a pathogen that causes acute viral hepatitis, is a small icosahedral, quasi-enveloped, positive ssRNA virus. Its genome has three open reading frames (ORFs), with ORF1 and ORF3 encoding for nonstructural and regulatory proteins, respectively, while ORF2 is translated into the structural, capsid protein. ORF2 is most widely used for vaccine development in viral hepatitis. Hepatitis E virus-like particles (VLPs) are potential vaccine candidates against HEV infection. VLPs are composed of capsid subunits mimicking the natural configuration of the native virus but lack the genetic material needed for replication. As a result, VLPs are unable to replicate and cause disease, constituting safe vaccine platforms. Currently, the recombinant VLP-based vaccine Hecolin® against HEV is only licensed in China. Herein, systematic information about the expression of various HEV ORF2 sequences and their ability to form VLPs in different systems is provided

Plant-Derived Lectins as Potential Cancer Therapeutics and Diagnostic Tools

Cancer remains a global health challenge, with high morbidity and mortality, despite the recent advances in diagnosis and treatment. Multiple compounds assessed as novel potential anticancer drugs derive from natural sources, including microorganisms, plants, and animals. Lectins, a group of highly diverse proteins of nonimmune origin with carbohydrate-binding abilities, have been detected in virtually all kingdoms of life. These proteins can interact with free and/or cell surface oligosaccharides and might differentially bind cancer cells, since malignant transformation is tightly associated with altered cell surface glycans. Therefore, lectins could represent a valuable tool for cancer diagnosis and be developed as anticancer therapeutics. Indeed, several plant lectins exert cytotoxic effects mainly by inducing apoptotic and autophagic pathways in malignant cells. This review summarizes the current knowledge regarding the basis for the use of lectins in cancer diagnosis and therapy, providing a few examples of plant-derived carbohydrate-binding proteins with demonstrated antitumor effects

Lectins as Promising Therapeutics for the Prevention and Treatment of HIV and Other Potential Coinfections

Human immunodeficiency virus-acquired immunodeficiency syndrome (HIV/AIDS) remains a global health problem. Current therapeutics specifically target the viral pathogen at various stages of its life cycle, although complex interactions between HIV and other pathogenic organisms are evident. Targeting HIV and concomitant infectious pathogens simultaneously, both by therapeutic regimens and in prevention strategies, would help contain the AIDS pandemic. Lectins, a ubiquitous group of proteins that specifically bind glycosylated molecules, are interesting compounds that could be used for this purpose, with demonstrated anti-HIV properties. In addition, potential coinfecting pathogens, including other enveloped viruses, bacteria, yeasts and fungi, and protozoa, display sugar-coated macromolecules on their surfaces, making them potential targets of lectins. This review summarizes the currently available findings suggesting that lectins should be further developed to simultaneously fight the AIDS pandemic and concomitant infections in HIV infected individuals

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

The core antigen of hepatitis B virus (HBcAg) is capable of self-assembly into virus-like particles (VLPs) when expressed in a number of heterologous systems. Such VLPs are potential carriers of foreign antigenic sequences for vaccine design. In this study, we evaluated the production of chimeric HBcAg VLPs presenting a foreign epitope on their surface, the 551–607 amino acids (aa) immunological epitope of the ORF2 capsid protein of hepatitis E virus. A chimeric construct was made by the insertion of 56 aa into the immunodominant loop of the HBcAg. The sequences encoding the chimera were inserted into the pEAQ-HT vector and infiltrated into Nicotiana benthamiana leaves. The plant-expressed chimeric HBcHEV ORF2 551–607 protein was recognized by an anti-HBcAg mAb and anti-HEV IgG positive swine serum. Electron microscopy showed that plant-produced chimeric protein spontaneously assembled into “knobbly” ~34 nm diameter VLPs. This study shows that HBcAg is a promising carrier platform for the neutralizing epitopes of hepatitis E virus (HEV) and the chimeric HBcAg/HEV VLPs could be a candidate for a bivalent vaccine

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

The Hepatitis E virus (HEV) is a causative agent of acute hepatitis, mainly transmitted by the fecal-oral route or zoonotic. Open reading frame (ORF) 2 encodes the viral capsid protein, which is essential for virion assembly, host interaction, and inducing neutralizing antibodies. In this study, we investigated whether full-length and N- and C-terminally modified versions of the capsid protein transiently expressed in N. benthamiana plants could assemble into highly-immunogenic, virus-like particles (VLPs). We also assessed whether such VLPs can act as a carrier of foreign immunogenic epitopes, such as the highly-conserved M2e peptide from the Influenza virus. Plant codon-optimized HEV ORF2 capsid genes were constructed in which the nucleotides coding the N-terminal, the C-terminal, or both parts of the protein were deleted. The M2e peptide was inserted into the P2 loop after the residue Gly556 of HEV ORF2 protein by gene fusion, and three different chimeric constructs were designed. Plants expressed all versions of the HEV capsid protein up to 10% of total soluble protein (TSP), including the chimeras, but only the capsid protein consisting of aa residues 110 to 610 (HEV 110–610) and chimeric M2 HEV 110–610 spontaneously assembled in higher order structures. The chimeric VLPs assembled into particles with 22–36 nm in diameter and specifically reacted with the anti-M2e antibody