Background: During a global influenza pandemic, the vaccine requirements of developing countries can surpass\r\ntheir supply capabilities, if these exist at all, compelling them to rely on developed countries for stocks that may not\r\nbe available in time. There is thus a need for developing countries in general to produce their own pandemic and\r\npossibly seasonal influenza vaccines. Here we describe the development of a plant-based platform for producing\r\ninfluenza vaccines locally, in South Africa. Plant-produced influenza vaccine candidates are quicker to develop and\r\npotentially cheaper than egg-produced influenza vaccines, and their production can be rapidly upscaled. In this\r\nstudy, we investigated the feasibility of producing a vaccine to the highly pathogenic avian influenza A subtype\r\nH5N1 virus, the most generally virulent influenza virus identified to date. Two variants of the haemagglutinin (HA)\r\nsurface glycoprotein gene were synthesised for optimum expression in plants: these were the full-length HA gene\r\n(H5) and a truncated form lacking the transmembrane domain (H5tr). The genes were cloned into a panel of\r\nAgrobacterium tumefaciens binary plant expression vectors in order to test HA accumulation in different cell\r\ncompartments. The constructs were transiently expressed in tobacco by means of agroinfiltration. Stable transgenic\r\ntobacco plants were also generated to provide seed for stable storage of the material as a pre-pandemic strategy.\r\nResults: For both transient and transgenic expression systems the highest accumulation of full-length H5 protein\r\noccurred in the apoplastic spaces, while the highest accumulation of H5tr was in the endoplasmic reticulum. The\r\nH5 proteins were produced at relatively high concentrations in both systems. Following partial purification,\r\nhaemagglutination and haemagglutination inhibition tests indicated that the conformation of the plant-produced\r\nHA variants was correct and the proteins were functional. The immunisation of chickens and mice with the\r\ncandidate vaccines elicited HA-specific antibody responses.\r\nConclusions: We managed, after synthesis of two versions of a single gene, to produce by transient and transgenic\r\nexpression in plants, two variants of a highly pathogenic avian influenza virus HA protein which could have vaccine\r\npotential. This is a proof of principle of the potential of plant-produced influenza vaccines as a feasible pandemic\r\nresponse strategy for South Africa and other developing countries.
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