Browsing by Author "Maclean, James"

Now showing 1 - 3 of 3
  • Thumbnail Image
    Item
    Open Access
    Expression of HIV-1 antigens in plants as potential subunit vaccines
    (BioMed Central Ltd, 2008) Meyers, Ann; Chakauya, Ereck; Shephard, Enid; Tanzer, Fiona; Maclean, James; Lynch, Alisson; Williamson, Anna-Lise; Rybicki, Edward
    BACKGROUND: Human 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. RESULTS: Both 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. CONCLUSION: Transient 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.
  • Thumbnail Image
    Item
    Open Access
    Expression of HPV-16 L2 in plants
    (2008) Pereira, Roman; Rybicki, Ed; Becker, Inga; Maclean, James
    This study demonstrates high level expression of L2, 25 mg.kg⁻¹of leaf material, is achievable. Interestingly, expression was best when coded for by a mammalian codon-optimized form of the L2 gene as opposed to the wildtype or plant codon-optimized (plantized) genes. Moreover, real time PCR revealed limited levels of transcript when coded for by the plantized gene in comparison to the other genes. A set of vectors which target the protein to the cytoplasm, the endoplasmic reticulum (ER), chloroplast or apoplastic space were Expression of HPV -16 L2 in Plants used and it was found that targeting of the protein had no effect on its expression levels.
  • Thumbnail Image
    Item
    Open Access
    Setting up a platform for plant-based influenza virus vaccine production in South Africa
    (BioMed Central Ltd, 2012) Mortimer, Elizabeth; Maclean, James; Mbewana, Sandiswa; Buys, Amelia; Williamson, Anna-Lise; Hitzeroth, Inga; Rybicki, Edward
    BACKGROUND:During a global influenza pandemic, the vaccine requirements of developing countries can surpass their supply capabilities, if these exist at all, compelling them to rely on developed countries for stocks that may not be available in time. There is thus a need for developing countries in general to produce their own pandemic and possibly seasonal influenza vaccines. Here we describe the development of a plant-based platform for producing influenza vaccines locally, in South Africa. Plant-produced influenza vaccine candidates are quicker to develop and potentially cheaper than egg-produced influenza vaccines, and their production can be rapidly upscaled. In this study, we investigated the feasibility of producing a vaccine to the highly pathogenic avian influenza A subtype H5N1 virus, the most generally virulent influenza virus identified to date. Two variants of the haemagglutinin (HA) surface glycoprotein gene were synthesised for optimum expression in plants: these were the full-length HA gene (H5) and a truncated form lacking the transmembrane domain (H5tr). The genes were cloned into a panel of Agrobacterium tumefaciens binary plant expression vectors in order to test HA accumulation in different cell compartments. The constructs were transiently expressed in tobacco by means of agroinfiltration. Stable transgenic tobacco plants were also generated to provide seed for stable storage of the material as a pre-pandemic strategy. RESULTS: For both transient and transgenic expression systems the highest accumulation of full-length H5 protein occurred in the apoplastic spaces, while the highest accumulation of H5tr was in the endoplasmic reticulum. The H5 proteins were produced at relatively high concentrations in both systems. Following partial purification, haemagglutination and haemagglutination inhibition tests indicated that the conformation of the plant-produced HA variants was correct and the proteins were functional. The immunisation of chickens and mice with the candidate vaccines elicited HA-specific antibody responses. CONCLUSIONS: We managed, after synthesis of two versions of a single gene, to produce by transient and transgenic expression in plants, two variants of a highly pathogenic avian influenza virus HA protein which could have vaccine potential. This is a proof of principle of the potential of plant-produced influenza vaccines as a feasible pandemic response strategy for South Africa and other developing countries.