High-level HIV-1 Nef transient expression in Nicotiana benthamiana using the P19 gene silencing suppressor protein of Artichoke Mottled Crinckle Virus

Abstract

BackgroundIn recent years, different HIV antigens have been successfully expressed in plants by either stable transformation or transient expression systems. Among HIV proteins, Nef is considered a promising target for the formulation of a multi-component vaccine due to its implication in the first steps of viral infection. Attempts to express Nef as a single protein product (not fused to a stabilizing protein) in transgenic plants resulted in disappointingly low yields (about 0.5% of total soluble protein). In this work we describe a transient expression system based on co-agroinfiltration of plant virus gene silencing suppressor proteins in Nicotiana benthamiana, followed by a two-step affinity purification protocol of plant-derived Nef.ResultsThe effect of three gene silencing viral suppressor proteins (P25 of Potato Virus X, P19 of either Artichoke Mottled Crinckle virus and Tomato Bushy Stunt virus) on Nef transient expression yield was evaluated. The P19 protein of Artichoke Mottled Crinckle virus (AMCV-P19) gave the highest expression yield in vacuum co-agroinfiltration experiments reaching 1.3% of total soluble protein, a level almost three times higher than that previously reported in stable transgenic plants. The high yield observed in the co-agroinfiltrated plants was correlated to a remarkable decrease of Nef-specific small interfering RNAs (siRNAs) indicating an effective modulation of RNA silencing mechanisms by AMCV-P19. Interestingly, we also showed that expression levels in top leaves of vacuum co-agroinfiltrated plants were noticeably reduced compared to bottom leaves. Moreover, purification of Nef from agroinfiltrated tissue was achieved by a two-step immobilized metal ion affinity chromatography protocol with yields of 250 ng/g of fresh tissue.ConclusionWe demonstrated that expression level of HIV-1 Nef in plant can be improved using a transient expression system enhanced by the AMCV-P19 gene silencing suppressor protein. Moreover, plant-derived Nef was purified, with enhanced yield, exploiting a two-step purification protocol. These results represent a first step towards the development of a plant-derived HIV vaccine.

Plant-based vaccines against viruses

“…The other chimaeric L1s did not form distinct particles, and elicited significantly weaker humoral immune responses for the same dose of protein. The L1:L2 [108][109][110][111][112][113][114][115][116][117][118][119][120] particle formation was in contrast to repeated previous expression of this and the other chimaeras in insect cells, where only loose aggregates of capsomers were formed for L1:L2 [108][109][110][111][112][113][114][115][116][117][118][119][120] [95,96].…”

“…All chimaeras were very highly expressed, with yields of up to 1.2 g/kg plant tissue. The L1 chimaera containing L2 amino acids 108-120 (L1:L2 [108][109][110][111][112][113][114][115][116][117][118][119][120] ) was the most successful candidate in that it assembled into small VLPs (~30 nm), and elicited anti-L1 and anti-L2 responses in IM immunised mice, and immune sera neutralised homologous HPV-16 and heterologous HPV-52 pseudovirions. The other chimaeric L1s did not form distinct particles, and elicited significantly weaker humoral immune responses for the same dose of protein.…”

“…As could be expected, HIV vaccines were an early target for plant expression studies, with a variety of targets: indeed, HIV-1 p24 capsid protein was expressed successfully in transgenic tobacco, albeit at low yield (0.35% of TSP), as long ago as 2002 [107]; the suitability of transgenic maize as a production platform for oral delivery of HIV vaccines in seed extracts was tested using SIV major surface glycoprotein gp130 [108]; a novel gp41-derived molecule incorporating a CTB fusion as polymerising agent and adjuvant was produced via agroinfiltration-mediated expression in N benthamiana [109] and shown to produce mucosal and serum antimembrane proximal region (MPR) antibodies in mice after mucosal prime-systemic boost immunisation [110]; a Tat monomer was produced in spinach via rTMV as a potential oral vaccine [111]; epitopes derived from HIV-1 Gag and Env were fused to HBsAg and expressed as VLPs in transgenic tomato fruit [112]; various Gag-derived antigens (p24, p41, p55) were produced in transgenic tobacco and via rTMV in N benthamiana for investigation of their utility as CTL-inducing immunogens by our group [113]; HIV-1 Nef was produced in N benthamiana using an agroinfiltration-mediated transient expression system [114]. The field was comprehensively reviewed recently [115], so this review will be limited discussion of major advances, and more recent work.…”

Plant heat shock protein 70 as carrier for immunization against a plant-expressed reporter antigen

“…By this approach, when the vacuum was released, the pressure difference forced the bacterial suspension through the stomata into the mesophyll where it transferred the genes to plant cells. All the leaves except the apical ones (previously demonstrated to have a decreased expression efficiency; Lombardi et al 2009) were sampled.…”

High-level transient expression of the N-terminal domain of IpaD from Shigella dysenteriae in four plant species transformed with different construct configurations

“…Transient-based gene expression has been reported as a fast, simple, and cost-effective technique and seems to be unaffected by chromosomal positional effects (Lombardi et al 2009). This makes transient expression adequate for confirming gene product quality and function before committing to the expense of transgenic plants (Rodríguez et al 2005).…”