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

MARIA ALICIA DELFINO; TRINITARIO, SEBASTIÁN N.; CARDOSO LADANBURU ALEJANDRO; MELISSA RUSSO; NATACHA CERNY; AUGUSTO E BIVONA; EMILIO L MALCHIODI; SANCHEZ ALBERTI ANDRES

Congreso; Reunión Anual de Sociedades de Biociencias 2020; 2020

SAI-SAIC-SAFIS

DNA vaccines areefficient Th1 and CD8 inducers and have shown efficacy to control intracellularpathogens such as Trypanosoma cruzi. Live attenuated vectors, like rareserotype Adenovirus, used as vaccine DNA-delivery system, improveimmunogenicity and guarantee a strong and long-lasting response.Considering thesefacts, we generated a vaccine based on rare serotype human adenovirus (Ad48)carrying Traspain gene, a novel T. cruzi chimeric antigen developed in ourlaboratory. With the aim of studying innate immune activation by this Adserotype and the spatiotemporal tracking of the antigen we developed an Ad48carrying Traspain gene fused with the monomeric red fluorescent proteinmScarlet and analyzed its performance. mScarlet taggedTraspain was constructed by traditional cloning. Ad48-Traspain-mScarlet viruswas obtained by homologous recombination in HEK-293 cells, 15 dayspost-transfection. Seven clones wereisolated by agarose plaque assay and further analyzed. Traspain-mScarlet genewas detected by PCR,  in vitro expressiondemonstrated by Western-Blot and Fluorescent Microscopy in infected cellsshowed full cytopathic effect. Three brighter cloneswere compared employing a high-throughput imaging system (IN-Cell Analyzer2200, GE). Clone 2 was selected because it showed a signal/noise ratio of 100and 2-fold mScarlet MFI compared to other ones. Purification of this clone by sucrose density gradientultracentrifugation, resulted in titers higher than 2.108 TCID50/ml. Low rateof impurities were found by SDS-PAGE and A280/A260 ratio = 1.40-1.60. Traspain specificimmune response was assessed by flow cytometry after immunization of C57BL6mice with two subcutaneous doses of the virus. A strong antigen-specific CTLresponse was detected by tetramer staining of whole blood from immunized mice. In conclusion, therecombinant viral vector Ad48 carrying Traspain-mScarlet was generated and its in vitro and in vivo performance confirmed the feasibility of the vaccine approach.