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

D. M. Pérez-Filgueira, B. P. Brayfield, S. Phiri, M. V. Borca, C. Wood, T. J. Morris

Research output: Contribution to journalArticlepeer-review

30 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)201-208
Number of pages8
JournalJournal of Virological Methods
Volume121
Issue number2
DOIs
StatePublished - Nov 2004

Keywords

  • Diagnosis
  • HIV
  • Immune response
  • Plant expression
  • TMV-vector

ASJC Scopus subject areas

  • Virology

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