Development of rare serotype Adenovirus codifying a novel chimeric antigen fussed to a fluorescent tag as a tool for vaccination studies

DELFINO, MARÍA ALICIA; TRINITARIO, SEBASTIÁN NICOLÁS; CARDOSO LANDABURU, ALEJANDRO; RUSSO, MELISSA; BIVONA, AUGUSTO ERNESTO; MALCHIODI, EMILIO LUIS; SANCHEZ-ALBERTI, ANDRÉS

Buenos Aires

Congreso; REUNIÓN ANUAL DE SOCIEDADES DE BIOCIENCIAS SAIC - SAI - SAFIS; 2020

Sociedad Argentina de Inmunología (SAI)

DNA vaccines are efficient Th1 and CD8 inducers and have shown efficacy tocontrol intracellular pathogens such as Trypanosoma cruzi. Live attenuatedvectors, like rare serotype Adenovirus, used as vaccine DNA-delivery system,improve immunogenicity and guarantee a strong and long-lasting response.Considering these facts, we generated a vaccine based on rare serotype humanadenovirus (Ad48) carrying Traspain gene, a novel T. cruzi chimeric antigendeveloped in our laboratory. With the aim of studying immune activation by thisAd serotype and the spatiotemporal tracking of the antigen we developed anAd48 carrying Traspain gene fused with the monomeric red fluorescent proteinmScarlet and analyzed its performance.mScarlet tagged Traspain was constructed by traditional cloning. Ad48-TraspainmScarlet virus was obtained by homologous recombination in HEK-293 cells, 15days post-transfection.Seven clones were isolated by agarose plaque assay and further analyzed.Traspain-mScarlet gene was detected by PCR, in vitro expression demonstratedby Western-Blot and Fluorescent Microscopy in infected cells showed fullcytopathic effect.Three brighter clones were compared employing a high-throughput imagingsystem (IN-Cell Analyzer 2200, GE). Clone 2 was selected because it showed asignal/noise ratio of 100 and 2-fold mScarlet MFI compared to other ones.Purification of this clone by sucrose density gradient ultracentrifugation, resultedin titers higher than 2.108 TCID50/ml. Low rate of impurities were found by SDSPAGE and A280/A260 ratio = 1.40-1.60.Traspain specific immune response was assessed by flow cytometry afterimmunization of C57BL6 mice with two subcutaneous doses of the virus. A strongantigen-specific CTL response was detected by tetramer staining of whole bloodfrom immunized mice.