ABSTRACT
Malaria transmission blocking (TB) vaccines (TBVs) directed against proteins expressed on the sexual stages of Plasmodium parasites are a potentially effective means to reduce transmission. Antibodies induced by TBVs block parasite development in the mosquito, and thus inhibit transmission to further human hosts. The ookinete surface protein P25 is a primary target for TBV development. Recently, transient expression in plants using hybrid viral vectors has demonstrated potential as a strategy for cost-effective and scalable production of recombinant vaccines. Using a plant virus-based expression system, we produced recombinant P25 protein of Plasmodium vivax (Pvs25) in Nicotiana benthamiana fused to a modified lichenase carrier protein. This candidate vaccine, Pvs25-FhCMB, was purified, characterized and evaluated for immunogenicity and efficacy using multiple adjuvants in a transgenic rodent model. An in vivo TB effect of up to a 65% reduction in intensity and 54% reduction in prevalence was observed using Abisco-100 adjuvant. The ability of this immunogen to induce a TB response was additionally combined with heterologous prime-boost vaccination with viral vectors expressing Pvs25. Significant blockade was observed when combining both platforms, achieving a 74% and 68% reduction in intensity and prevalence, respectively. This observation was confirmed by direct membrane feeding on field P. vivax samples, resulting in reductions in intensity/prevalence of 85.3% and 25.5%. These data demonstrate the potential of this vaccine candidate and support the feasibility of expressing Plasmodium antigens in a plant-based system for the production of TBVs, while demonstrating the potential advantages of combining multiple vaccine delivery systems to maximize efficacy.
Subject(s)
Malaria Vaccines/immunology , Malaria, Vivax/prevention & control , Protozoan Proteins/immunology , Adjuvants, Immunologic/administration & dosage , Animals , Chromobox Protein Homolog 5 , Female , Immunization, Secondary , Mice, Inbred BALB C , Plants, Genetically Modified , Plasmodium vivax , Recombinant Proteins/immunology , Nicotiana , Vaccines, Subunit/immunology , Vaccines, Synthetic/immunologyABSTRACT
The coding region of the licB gene from Clostridium thermocellum was truncated at the 3' end. The modified lichenase encoded by the construct (LicBM2) retained the most important properties of the enzyme - its high activity and thermostability. LicBM2 consists of the catalytic domain and part of the Pro-Thr-box. We demonstrated the application of the licBM2 gene as a reporter system for prokaryotic (Escherichia coli) and eukaryotic (Saccharomyces cerevisiae and mammalian) cells by expressing it either as a transcriptional fusion with selected promoters or as a translational fusion with the E. coli uidA gene. The assays available for LicB activity are sensitive, accurate and simple, and can be used for the analysis of various gene fusion systems or for screening of transformants.
Subject(s)
Clostridium/enzymology , Clostridium/genetics , Genes, Reporter , Glycoside Hydrolases/genetics , Animals , Artificial Gene Fusion , Base Sequence , DNA, Bacterial/genetics , Enzyme Stability , Escherichia coli/genetics , Eukaryotic Cells , Gene Expression , Genes, Bacterial , Genetic Vectors , PC12 Cells , Prokaryotic Cells , Rats , Recombinant Fusion Proteins/genetics , Recombinant Proteins/genetics , Saccharomyces cerevisiae/genetics , TemperatureABSTRACT
The main properties (pH and temperature range, stability, substrate specificity) of the modified cellulase CelE (endo-beta-1,4-glucanase) from Clostridium thermocellum have been analyzed with the goal of its expression in plants. The modified enzyme is similar to plant cellulases. Deletions in the N-terminus of the enzyme do not affect its biochemical properties. Based on the present investigation, we conclude that the modified beta-1,4-glucanase CelEM1, when expressed in plants, will be a good model to study the role of cellulases in plants.
Subject(s)
Carboxymethylcellulose Sodium/metabolism , Cellulase/genetics , Cellulase/metabolism , Clostridium/enzymology , Glucans/metabolism , Sequence Deletion/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cellulase/chemistry , Clostridium/genetics , Computer Simulation , Enzyme Activation , Gene Expression/genetics , Gene Expression Regulation, Bacterial/genetics , Gene Expression Regulation, Plant/genetics , Models, Genetic , Mutation/genetics , Plants/enzymology , Plants/geneticsABSTRACT
Major properties (pH and temperature optimum, stability) of lichenase (beta-1,3-1,4-glucanase) deletion variants from Clostridium thermocellum were comparatively studied. The deletion variant LicBM2 was used to create hybrid bifunctional proteins by fusion with sequences of the green fluorescent protein (GFP) from Aequorea victoria. The data show that in hybrid proteins both GFP and lichenase retain their major properties, namely, GFP remains a fluorescent protein and the lichenase retains activity and high thermostability. Based on the results of this investigation and results that have been obtained earlier, the use of the deletion variants of lichenase and the bifunctional hybrid proteins as reporter proteins is suggested.
