Abstract
BackgroundHuman immunodeficiency virus type 1 (HIV-1) has infected more than 40 million people worldwide, mainly in sub-Saharan Africa. The high prevalence of HIV-1 subtype C in southern Africa necessitates the development of cheap, effective vaccines. One means of production is the use of plants, for which a number of different techniques have been successfully developed. HIV-1 Pr55Gag is a promising HIV-1 vaccine candidate: we compared the expression of this and a truncated Gag (p17/p24) and the p24 capsid subunit in Nicotiana spp. using transgenic plants and transient expression via Agrobacterium tumefaciens and recombinant tobamovirus vectors. We also investigated the influence of subcellular localisation of recombinant protein to the chloroplast and the endoplasmic reticulum (ER) on protein yield. We partially purified a selected vaccine candidate and tested its stimulation of a humoral and cellular immune response in mice.ResultsBoth transient and transgenic expression of the HIV antigens were successful, although expression of Pr55Gag was low in all systems; however, the Agrobacterium-mediated transient expression of p24 and p17/p24 yielded best, to more than 1 mg p24/kg fresh weight. Chloroplast targeted protein levels were highest in transient and transgenic expression of p24 and p17/p24. The transiently-expressed p17/p24 was not immunogenic in mice as a homologous vaccine, but it significantly boosted a humoral and T cell immune response primed by a gag DNA vaccine, pTHGagC.ConclusionTransient agroinfiltration was best for expression of all of the recombinant proteins tested, and p24 and p17/p24 were expressed at much higher levels than Pr55Gag. Our results highlight the usefulness of plastid signal peptides in enhancing the production of recombinant proteins meant for use as vaccines. The p17/p24 protein effectively boosted T cell and humoral responses in mice primed by the DNA vaccine pTHGagC, showing that this plant-produced protein has potential for use as a vaccine.
Highlights
Human immunodeficiency virus type 1 (HIV-1) has infected more than 40 million people worldwide, mainly in sub-Saharan Africa
We investigated the usefulness of codon optimisation and of targeting recombinant proteins to the endoplasmic reticulum (ER) and chloroplasts as a means of improving yields, and investigated whether transient expression via recombinant tobamoviruses or Agrobacterium tumefaciens produced better yields than the Agrobacterium-engineered transgenic system
Despite the low levels of recombinant protein expression, samples of crude plant sap from N. benthamiana infected with the pBSGngag recombinant tobacco mosaic virus (TMV) RNA that were immunotrapped with HIV-1 p17 antiserum and viewed under the electron microscope, showed the presence of virus-like particles (VLPs) approximately 110 to 120 nm in diameter
Summary
Human immunodeficiency virus type 1 (HIV-1) has infected more than 40 million people worldwide, mainly in sub-Saharan Africa. HIV-1 subtype C is the predominant HIV in southern Africa; South African isolates of this subtype have been used in the development of candidate DNA and other virally- and bacterially-vectored and subunit vaccines for South Africa under the auspices of the South African AIDS Vaccine Initiative (SAAVI) [3,4,5]. DNA vaccines are expensive to manufacture, as are recombinant viral and subunit vaccines produced via cell culture. This makes the proposition costly and possibly unfeasible, for the poorer developing countries where HIV infection is prevalent. There is a strong need to develop vaccine expression systems which have the potential to produce vaccines at low cost for these countries
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