Journal: Journal of Virology
Article Title: Development of Virus-Like-Particle Vaccine and Reporter Assay for Zika Virus
Figure Lengend Snippet: Expression of ZIKV prM-E alone releases VLPs into the supernatant, while C-prM-E requires the NS2B-3 protease for efficient VLP release. (A) 293T cells were transfected with the pCDNA3.1 vector expressing ZIKV prM-E or C-prM-E along with the indicated expression vectors. Cells were radiolabeled with [ 35 S]Met/Cys, and culture supernatants were harvested and ultracentrifuged. Cell and virion samples were lysed and immunoprecipitated with MAB10216-coated protein A beads, resolved by SDS-PAGE, and visualized by PhosphorImager analysis. (B) Culture supernatants were harvested from cells expressing ZIKV C-prM-E or prM-E as indicated in Materials and Methods. Twenty-five to 30 ml of supernatant was transferred to ultracentrifuge tubes and carefully underlaid with 5 ml of 25% glycerol in TNE buffer. VLPs were pelleted by centrifugation at 110,500 × g for 3 h at 4°C. Thereafter, the supernatant was carefully removed and the VLP pellet resuspended in TNE buffer. An aliquot of concentrated VLPs was subjected to lysis with 10× RIPA buffer, and E protein in the preparations was detected by Western blotting. (C) VLPs were concentrated as described above, and images were acquired after negative staining, using a JEOL1010 transmission electron microscope with a Hamamatsu digital camera. Bars, ∼30 nm. (D and E) Immunization studies in mice. (D) BALB/c mice were divided into groups of six mice each. Mice received the primary immunization on day 0, followed by 2 boosters, at days 14 and 28, and were finally sacrificed at day 63 post-primary immunization. (E) Mice were divided into 5 groups and received immunizations with either C-prM-E/prM-E DNA or VLPs. For DNA immunization, a total of 50 μg of DNA in a volume of 100 μl PBS was injected intramuscularly (i.m.). For VLPs, the first immunization consisted of a VLP preparation mixed with TiterMax Gold adjuvant in a total volume of 100 μl injected intramuscularly. For subsequent boosters, mice received VLPs alone, without adjuvant. Control mice were injected with PBS.
Article Snippet: The prM-E construct was generated by PCR amplification of the prM-E region spanning amino acids 105 to 795, using a Phusion high-fidelity PCR kit (New England BioLabs), and was cloned into the pcDNA3.1+ vector (Invitrogen).
Techniques: Expressing, Transfection, Plasmid Preparation, Immunoprecipitation, SDS Page, Centrifugation, Lysis, Western Blot, Negative Staining, Transmission Assay, Microscopy, Mouse Assay, Injection