CONSTRUCTION OF FLUORESCENT-TAGGED ADENOVIRAL VACCINE CANDIDATE AS A TOOL FOR STUDYING IMMUNE RESPONSES UPON VACCINATION

DELFINO MA; TRINITARIO SN; CARDOSO A; RUSSO M; CERNY N; BIVONA A; MALCHIODI EL; SÁNCHEZ ALBERTI A

Congreso; REUNIÓN DE SOCIEDADES DE BIOCIENCIAS 2020; 2020

DNA vaccines are efficient Th1 and CD8 inducers and have shownefficacy to control intracellular pathogens such as Trypanosomacruzi. Live attenuated vectors, like rare serotype Adenovirus, usedas vaccine DNA-delivery system, improve immunogenicity andguarantee a strong and long-lasting response.Considering these facts, we generated a vaccine based on rare serotypehuman adenovirus (Ad48) carrying Traspain gene, a novel T.cruzi chimeric antigen developed in our laboratory. With the aim ofstudying immune activation by this Ad serotype and the spatiotemporaltracking of the antigen we developed an Ad48 carrying Traspaingene fused with the monomeric red fluorescent protein mScarletand analyzed its performance.mScarlet tagged Traspain was constructed by traditional cloning.Ad48-Traspain-mScarlet virus was obtained by homologous recombinationin HEK-293 cells, 15 days post-transfection.Seven clones were isolated by agarose plaque assay and furtheranalyzed. Traspain-mScarlet gene was detected by PCR, in vitro expressiondemonstrated by Western-Blot and Fluorescent Microscopyin infected cells showed full cytopathic effect.Three brighter clones were compared employing a high-throughputimaging system (IN-Cell Analyzer 2200, GE). Clone 2 was selectedbecause it showed a signal/noise ratio of 100 and 2-fold mScarletMFI compared to other ones. Purification of this clone by sucrosedensity gradient ultracentrifugation, resulted in titers higher than2.108 TCID50/ml. Low rate of impurities were found by SDS-PAGEand A280/A260 ratio = 1.40-1.60.Traspain specific immune response was assessed by flow cytometryafter immunization of C57BL6 mice with two subcutaneous doses ofthe 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 performanceconfirmed the feasibility of the vaccine approach.