A plant-produced Pfs25 VLP malaria vaccine candidate induces persistent transmission blocking antibodies against Plasmodium falciparum in immunized mice.

Malaria transmission blocking vaccines (TBVs) are considered an effective means to control and eventually eliminate malaria. The Pfs25 protein, expressed predominantly on the surface of the sexual and sporogonic stages of Plasmodium falciparum including gametes, zygotes and ookinetes, is one of the primary targets for TBV. It has been demonstrated that plants are an effective, highly scalable system for the production of recombinant proteins, including virus-like particles (VLPs). We engineered VLPs (Pfs25-CP VLP) comprising Pfs25 fused to the Alfalfa mosaic virus coat protein (CP) and produced these non-enveloped hybrid VLPs in Nicotiana benthamiana plants using a Tobacco mosaic virus-based 'launch' vector. Purified Pfs25-CP VLPs were highly consistent in size (19.3±2.4 nm in diameter) with an estimated 20-30% incorporation of Pfs25 onto the VLP surface. Immunization of mice with one or two doses of Pfs25-CP VLPs plus Alhydrogel® induced serum antibodies with complete transmission blocking activity through the 6 month study period. These results support the evaluation of Pfs25-CP VLP as a potential TBV candidate and the feasibility of the 'launch' vector technology for the production of VLP-based recombinant vaccines against infectious diseases.

A single component two-valent LcrV-F1 vaccine protects non-human primates against pneumonic plague.

Yersinia pestis continues to pose a threat as a potential biological weapon and is recognized by public health experts as a re-emerging infectious disease. Therefore there is great interest in developing a safe and effective vaccine. Vaccines against plague containing both the Fraction 1 (F1) and V antigens of Y. pestis have shown promise in protecting animal models against pneumonic plague, the deadliest form of the disease. Here we report on a plague vaccine consisting of the F1 and LcrV antigens fused to a single carrier molecule, the thermostable enzyme lichenase from Clostridium thermocellum, and expressed in and purified from Nicotiana benthamiana plants. When administered to Cynomolgus Macaques this purified plant-produced vaccine induced high titers of serum IgG, mainly of the IgG1 isotype, against both F1 and LcrV. These immunized animals were subsequently challenged and the LcrV-F1 plant-produced vaccine conferred complete protection against aerosolized Y. pestis.

Transient protein expression in three Pisum sativum (green pea) varieties.

The expression of proteins in plants both transiently and via permanently transformed lines has been demonstrated by a number of groups. Transient plant expression systems, due to high expression levels and speed of production, show greater promise for the manufacturing of biopharmaceuticals when compared to permanent transformants. Expression vectors based on a tobacco mosaic virus (TMV) are the most commonly utilized and the primary plant used, Nicotiana benthamiana, has demonstrated the ability to express a wide range of proteins at levels amenable to purification. N. benthamiana has two limitations for its use; one is its relatively slow growth, and the other is its low biomass. To address these limitations we screened a number of legumes for transient protein expression. Using the alfalfa mosaic virus (AMV) and the cucumber mosaic virus (CMV) vectors, delivered via Agrobacterium, we were able to identify three Pisum sativum varieties that demonstrated protein expression transiently. Expression levels of 420 +/- 26.24 mg GFP/kgFW in the green pea variety speckled pea were achieved. We were also able to express three therapeutic proteins indicating promise for this system in the production of biopharmaceuticals.

Peptide-based candidate vaccine against respiratory syncytial virus.

We engineered a 21-mer peptide representing amino acids 170-190 of the respiratory syncytial virus (RSV) G protein as a fusion with the Alfalfa mosaic virus (AlMV) coat protein (CP), produced recombinant AlMV particles presenting this peptide (VMR-RSV) on their surfaces and tested the immunogenicity in vitro in human dendritic cells and in vivo in non-human primates. Significant pathogen-specific immune responses were generated in both systems: (i) human dendritic cells armed with VMR-RSV generated vigorous CD4+ and CD8+ T cell responses; (ii) non-human primates that received these particles responded by mounting strong cellular and humoral immune responses. This approach may validate the use of a novel RSV vaccine delivery vehicle in humans.

The expression of a mammalian proteinase inhibitor, bovine spleen trypsin inhibitor in tobacco and its effects on Helicoverpa armigera larvae.

The cDNA for bovine spleen trypsin inhibitor (SI), a homologue of bovine pancreatic trypsin inhibitor (BPTI), including the natural mammalian presequence was expressed in tobacco using Agrobacterium tumefaciens-mediated transformation. Stable expression required the N-terminal targeting signal presequence although subcellular localization was not proven. SI was found to exist as two forms, one coinciding with authentic BPTI on western blots and the second marginally larger due to retention of the C-terminal peptide. Both were retained on a trypsin-agarose affinity gel and had inhibitory activity. Newly emergent leaves contained predominantly the large form whereas senescent leaves had little except the fully processed form present. Intermediate-aged leaves showed a gradual change indicating that a slow processing of the inhibitor peptide was occurring. The stability of SI was shown by the presence of protein at high levels in completely senescent leaves. Modifications to the cDNA (3' and 5' changes and minor codon changes) resulted in a 20-fold variation in expression. Expression of modified SI in transgenic tobacco leaves at 0.5% total soluble protein reduced both survival and growth of Helicoverpa armigera larvae feeding on leaves from the late first instar. In larvae surviving for 8 days, midgut trypsin activity was reduced in SI-tobacco fed larvae, while chymotrypsin activity was increased. Activities of leucine aminopeptidase and elastase-like chymotrypsin remained unaltered. The use of SI as an insect resistance factor is discussed.