Preserved antigenicity of HIV-1 p24 produced and purified in high yields from plants inoculated with a tobacco mosaic virus (TMV)-derived vector

Abstract

Pérez-Filgueira, D. M.; Brayfield, B. P.; Phiri, S.; Borca, M. V.; Wood, Charles; and Morris, Thomas Jack, "Preserved antigenicity of HIV-1 p24 produced and purified in high yields from plants inoculated with a tobacco mosaic virus (TMV)-derived vector" (2004 AbstractProduction of structural proteins from foot-and-mouth disease virus (FMDV) and bovine herpes virus (BHV-1) in Nicotiana benthamiana through the use of a tobacco mosaic virus-based vector (TMV-30B) has been reported previously. The development of the TMV-30B-HISc vector, a new version that adds a C-terminal histidine (His) sequence to the foreign protein expressed is described. Coding sequences from the FMDV VPl protein and the core protein, p24, from a clade C HIV-1 isolate from Zambia were cloned into the new vector and infective RNAs were generated for each construct to inoculate N. benthamiana plants. His-tagged proteins were purified from inoculated leaves using immobilized metal affinity chromatography (IMAC) as detected by Coomassie blue staining and proteins were further characterized in Western blot assays using a commercial anti-6xHis mAb and specific polyclonal antisera for each protein. While yields obtained for the VPl-His protein after purification were similar to those in crude extracts obtained with the previous TMV-VPl vector, p24-His yields were 10-15 times higher than those of VPl-His. Twenty-five grams of TMV-p24-HISc inoculated leaves were processed to obtain 2.5 mg of isolated p24-His and the recombinant protein was inoculated in rabbits to test immunogenicity and antigenic integrity of the plant-produced p24-His. Animals developed a strong and specific humoral response to the p24-His after the first booster and immune sera was able to recognize the native p24 from a different clade expressed on the surface of the HIV-1 chronically infected HUT78/ARV T-cell line. Importantly, the recombinant p24-His proved its efficiency by confirming the serology of 117 samples previously tested by two rapid HIV-1 tests, thus representing an excellent alternative for production of highly specific diagnostic reagents for HIV endemic regions in the developing world.

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

“…However, the high number of HIV-1 subtypes and several other factors complicate the design of a successful vaccine (Young et al 2006;Walker and Burton 2008). For such a purpose, various HIV proteins have been expressed in plant cells using either transient or stable transformation (Zhang et al 2000(Zhang et al , 2002Pérez-Filgueira et al 2004;Karasev et al 2005;Obregon et al 2006;Shchelkunov et al 2006;Greco et al 2007;Marusic et al 2007; Barbante et al 2008;de Virgilio et al 2008;McCabe et al 2008;Zhou et al 2008).…”

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.…”

Use of Plant Viruses for Production of Plant-Derived Vaccines

“…Although not directly related to vaccine development, antigens were produced using the human immunodeficiency virus (HIV-1) p24 protein utilizing the TMV viral vector system (Pérez-Filgueira, Brayfield et al, 2004). Rabbits inoculated with the plant-produced protein developed a strong and specific humoral response to the p24 protein.…”