High-level expression of the HIV-1 Pr55gag polyprotein in transgenic tobacco chloroplasts

Abstract

Plants have been recognized as a promising production platform for recombinant pharmaceutical proteins. The human immunodeficiency virus Gag (Pr55(gag)) structural polyprotein precursor is a prime candidate for developing a HIV-1 vaccine, but, so far, has been expressed at very low level in plants. The aim of this study was to investigate factors potentially involved in Pr55(gag) expression and increase protein yield in plant cells. In transient expression experiments in various subcellular compartments, the native Pr55(gag) sequence could be expressed only in the chloroplast. Experiments with truncated subunits suggested a negative role of the 5'-end on the expression of the full gene in the cytosol. Stable transgenic plants were produced in tobacco by Agrobacterium-mediated nuclear transformation with protein targeted to plastids, and biolistic-mediated plastid transformation. Compared to the nuclear genome, the integration and expression of the gag transgene in the plastome resulted in significantly higher protein accumulation levels (up to 7-8% TSP, equivalent to 312-363 mg/kg FW). In transplastomic plants, a 25-fold higher protein accumulation was obtained by translationally fusing the Pr55(gag) polyprotein to the N-terminus of the plastid photosynthetic RbcL protein. In chloroplasts, the Pr55(gag) polyprotein was processed in a pattern similar to that achieved by the viral protease, the processing being more extended in older leaves of mature plants. The Gag proteins produced in transgenic plastids were able to assemble into particles resembling VLPs produced in baculovirus/insect cells and E. coli systems. These results indicate that plastid transformation is a promising tool for HIV antigen manufacturing in plant cells.

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.…”

“…The most successful expression of full-length Pr 55 Gag precursor polyprotein to date was done with subtype B HIV-1 Gag in transplastomic tobacco, with yields of enveloped~100 nm diameter VLPs of up to 400 mg/kg biomass [117]. This was close to 10 000-fold more than a previous best by our group, with VLPs produced in transgenic tobacco [81], and represents a viable yield for a product with serious vaccine potential: recent evidence that lack of progression to AIDS is linked to strong CTL responses to Gag [118][119][120] reinforces the need for vaccines that can target such responses, and Pr 55 Gag VLPs are potent elicitors of CTL, especially when used as a protein boost to a heterologous prime in primate models [121,122].…”

Genetic Transformation in Tomato: Novel Tools to Improve Fruit Quality and Pharmaceutical Production

“…Major advantages of plant-made pharmaceuticals include low cost of production, higher scale-up capacity and lack of risk of contamination with mammalian pathogens. Several antigenic proteins have been produced in plant, examples are plant-made vaccines against smallpox, HIV and HPV (Human Papilloma Virus) (Lenzi et al, 2008;Rigano et al, 2009;Scotti et al, 2009). In addition, transgenic plants can represent a suitable vehicle for oral delivery of pharmaceuticals since the plant cell wall protects the recombinant antigen in the harsh condition of stomach and intestine (Sharma et al, 2008a).…”

Production of vaccines for treatment of infectious diseases by transgenic plants

“…The great genetic variety among HIV-1 subtypes, high mutation rate and biological properties of HIV-1 regulatory proteins by avoiding immune response (Cueno et al, 2010) significantly worsen the process of developing an efficient vaccine. Various HIV proteins have been expressed in plant systems so far: HIV-1 p24 in tobacco Perez-Filgueira et al, 2004b) and A. thaliana (Lindh et al, 2014), HIV 1 P55 Gag polyprotein in tobacco chloroplasts (Scotti et al, 2009) and tobacco plants both in full length and truncated form (Meyer et al, 2008), HIV-1 Tat in potato (Kim and Langridge, 2004a) and tomato (Cueno et al, 2010), HIV-1 Nef in tobacco (Marusic et al, 2007) and N. benthamiana (Lombardi et al, 2009;Circelli et al, 2010), HIV-1 CA capsid protein VLPs in Lycium barbarum (Du et al, 2004), HIV-1/HBV fusion protein has been expressed in tobacco Guetard et al, 2008) and A. thaliana . Tat regulatory element of the simian-HIV-1 virus (SHIV 89.6 Tat) has also been expressed in potato , fused to cholera toxin subunit B.…”