title: Preserved antigenicity of HIV-1 p24 produced and purified in high yields from plants inoculated with a tobacco mosaic virus (TMV)-derived vector.
abstract: Production 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.
authors.affiliation: School of Biological Sciences, University of Nebraska, Lincoln, NE 68588-0118, USA. mperez@inia.es
authors.forename: D M
authors.initials: DM
authors.lastname: Pérez-Filgueira
authors.forename: B P
authors.initials: BP
authors.lastname: Brayfield
authors.forename: S
authors.initials: S
authors.lastname: Phiri
authors.forename: M V
authors.initials: MV
authors.lastname: Borca
authors.forename: C
authors.initials: C
authors.lastname: Wood
authors.forename: T J
authors.initials: TJ
authors.lastname: Morris
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000097:blood / Q000276:immunology; D000956:Antigens, Viral / Q000276:immunology*; D003001:Cloning, Molecular; D005260:Female; D005822:Genetic Vectors / Q000378:metabolism; D016655:HIV Core Protein p24 / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology*; D006679:HIV Seropositivity; D015497:HIV-1; D006639:Histidine; D006801:Humans; D007106:Immune Sera; D018515:Plant Leaves / Q000378:metabolism; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis; D013045:Species Specificity; D014026:Nicotiana / Q000378:metabolism* / Q000821:virology; D014027:Tobacco Mosaic Virus / Q000235:genetics* / Q000378:metabolism
chemical_list: D000914:Antibodies, Viral; D000956:Antigens, Viral; D016655:HIV Core Protein p24; D007106:Immune Sera; D011994:Recombinant Proteins; D006639:Histidine