Transiently Transfected Mammalian Cell Cultures: An Adaptable and Effective Platform for Virus-like Particle-Based Vaccines against Foot-and-Mouth Disease Virus

Figures

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  Figure 1. Plasmid map of a pJJPKan VLP expression plasmid containing the FMDV P1-2A and 3C(L127P) sequences in a single open reading frame. Restriction sites BamHI and ApaI are utilized to insert the P1-2A sequence from any desired serotype or strain into the plasmid.

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  Figure 2. Virus-like particle (VLP) production process using transiently transfected mammalian cell cultures. (1) DNA plasmids encoding the FMDV P1 and 3C protease sequences were transfected into cell cultures using polyethylenimine. Transfected cells were incubated for 24 h to allow expression and processing of the P1 polypeptide resulting in the formation of cellular VLP arrays. Enlargement of individual arrays are presented inside of color-coded boxes; blue bar represents 500 nm. (2) VLPs were harvested by lysis of cells using LB9 and (3) concentrated by a centrifugal concentrator column. (4) The retained high molecular weight fraction was aliquoted into doses and stored frozen until (5) administered to swine or cattle.

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  Figure 3. VNTs at the time of challenge for treatment groups and individuals. The mean VNTs for swine treatment groups, 12 μg, 16 μg, and 20 μg, are presented in blue, while the mean VNTs for the 16 μg cattle group are presented in green. Protected swine are represented by blue circles, protected cattle are represented by green circles, and unprotected swine represented are represented by red circles, all grouped by treatment. Baseline VNTs observed in unvaccinated animals, both swine and cattle, are 0.6 and represented by a red line.

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  Figure 4. (A) Cellular VLP array images captured in cells transfected with constructs expressing FMDV O1M, Asia1, and SAT2 containing constructs. The light blue bar represents 100 nm. (B) The visual bands present in the 1.38 g/cm3 fraction, localized between tube markings, of cesium chloride gradients demonstrated an antigen of the correct size for VLPs in serotypes A24, Asia1, C3I, SAT1, SAT2, and SAT3. (C) Western blot of the VLP band from cesium chloride gradients utilizing the F1412SA monoclonal antibody, reactive with VP2 and its progenitor VP0, confirmed the presence of both VP2 and VP0 in VLPs.