Setting up a platform for plant-based influenza virus vaccine production in South Africa.

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.

An inhibition enzyme-linked immunosorbent assay for the detection of antibody to Rift Valley fever virus in humans, domestic and wild ruminants.

This paper describes the development and validation of an inhibition ELISA based on gamma-irradiated tissue culture-derived antigen for the detection of antibody to Rift Valley fever virus (RVFV) in humans, domestic and wild ruminants. Validation data sets derived from field-collected sera in Africa (humans=1367, cattle=649, goats=806, sheep=493, buffalo=258, camels=156) were categorized according to the results of a virus neutralisation test. In addition, individual sera from 93 laboratory workers immunized with inactivated RVF vaccine, 136 serial bleeds from eight sheep experimentally infected with wild-type of RVFV, and 200 serial bleeds from 10 sheep vaccinated with the live-attenuated strain of the virus, were used to study the kinetics of RVFV antibody production under controlled conditions. At cut-off values selected at 95% accuracy level by the two-graph receiver operating characteristic analysis the ELISA sensitivity ranged from 99.47% (humans) to 100% (sheep, buffalo, camels). The specificity ranged from 99.29% (sheep) to 100% (camels). Compared to virus neutralisation and haemagglutination-inhibition tests, the ELISA was more sensitive in detection of the earliest immunological responses in experimentally infected and vaccinated sheep. Our results demonstrate that the ELISA format reported here can be used as a safe, robust and highly accurate diagnostic tool in disease-surveillance and control programmes, import/export veterinary certification, and for monitoring of the immune response in vaccinees.

Production of H5N1 Influenza Virus Matrix Protein 2 Ectodomain Protein Bodies in Tobacco Plants and in Insect Cells as a Candidate Universal Influenza Vaccine.

The spread of influenza A viruses is partially controlled and prevented by vaccination. The matrix protein 2 ectodomain (M2e) is the most conserved sequence in influenza A viruses, and is therefore a good potential target for a vaccine to protect against multiple virus subtypes. We explored the feasibility of an M2e-based universal influenza A vaccine candidate based on the highly pathogenic avian influenza A virus, H5N1. A synthetic M2e gene was human- and plant-codon optimized and fused in-frame with a sequence encoding the N-terminal proline-rich domain (Zera(®)) of the γ-zein protein of maize. Zera(®)M2e was expressed transiently in Nicotiana benthamiana and Sf21 baculovirus/insect cell expression systems, and Zera(®)M2e protein bodies (PBs) were successfully produced in both expression systems. The plant-produced Zera(®)M2e PBs were purified and injected into Balb/c mice. Western blot analysis using insect cell-produced Zera(®)M2e PBs and multiple tandem M2e sequences (5xM2e) fused with the avian influenza H5N1 transmembrane and cytosolic tail (5xM2e_tHA) confirmed the presence of M2e-specific antibodies in immunized mice sera. The immunogenicity of the Zera(®)M2e indicates that our plant-produced protein has potential as an inexpensive universal influenza A vaccine.