Construction of fluorescent-tagged adenoviral vaccine candidate as a tool for studying immune responses upon vaccination

Abstract

Efficacy to control intracellular pathogens such as Trypanosoma cruzi. Live attenuated vectors, like rare serotype Adenovirus, used as vaccine DNA-delivery system, improve immunogenicity andguarantee a strong and long-lasting response. Considering these facts, we generated a vaccine based on rare serotype human adenovirus (Ad48) carrying Traspain gene, a novel T. cruzi chimeric antigen developed in our laboratory. With the aim of studying immune activation by this Ad serotype and the spatiotemporal tracking of the antigen we developed an Ad48 carrying Traspain gene fused with the monomeric red fluorescent protein mScarlet and analyzed its performance. mScarlet tagged Traspain was constructed by traditional cloning. Ad48-Traspain-mScarlet virus was obtained by homologous recombination in HEK-293 cells, 15 days post-transfection. Seven clones were isolated by agarose plaque assay and further analyzed. Traspain-mScarlet gene was detected by PCR, in vitro expression demonstrated by Western-Blot and Fluorescent Microscopy in infected cells showed full cytopathic effect. Three brighter clones were compared employing a high-throughputimaging system (IN-Cell Analyzer 2200, GE). Clone 2 was selected because it showed a signal/noise ratio of 100 and 2-fold mScarlet MFI compared to other ones. Purification of this clone by sucrosedensity gradient ultracentrifugation, resulted in titers higher than 2.108 TCID50/ml. Low rate of impurities were found by SDS-PAGE and A280/A260 ratio = 1.40-1.60. Traspain specific immune response was assessed by flow cytometry after immunization of C57BL6 mice with two subcutaneous doses of the virus. A strong antigen-specific CTL response was detected bytetramer staining of whole blood from immunized mice. In conclusion, the recombinant viral vector Ad48 carrying Traspain- mScarlet was generated and its in vitro and in vivo performance confirmed the feasibility of the vaccine approach.