ABSTRACT
With the recent outbreaks of Zika and Dengue virus infections in various countries worldwide, production of vaccines or diagnostic kits is an urgent public health demand. Production of a monoclonal antibody (mAb) that specifically binds to a common antigen shared by the Flavivirus genus will be necessary for new diagnostic kits or characterization and viral identity tests during vaccine development. This study aimed to cultivate, in serum-free conditions, the 4G2 hybridoma that produces an mAb, which recognizes a shared epitope from the Flavivirus genus. We compared 4G2 hybridoma growth and biochemical profiles between cells cultivated in batch mode over 10 days in roller bottles containing Dulbecco's modified Eagle's medium high glucose containing 10% fetal bovine serum medium or hybridomas directly adapted to Ex-Cell serum-free medium. Cellular parameters such as specific growth rate (µ), maximum cell concentration, specific l-lactate, and glucose and IgG rates were evaluated. Thereafter, we also compared total mAb volumetric productivity, purification yield, and mAb staining of Vero cells infected with Zika and Dengue-2 virus. Direct adaptation to serum-free conditions did not change hybridoma growth rate and mAb production under the conditions tested. Instead, serum-free mAb purification showed a higher yield with no alterations on mAb structure or mAb staining of Zika and Dengue Vero-infected cells.
Subject(s)
Antibodies, Monoclonal/immunology , Hybridomas/cytology , Zika Virus/immunology , Animals , Antibodies, Monoclonal/isolation & purification , Antibodies, Monoclonal/metabolism , Batch Cell Culture Techniques/instrumentation , Batch Cell Culture Techniques/methods , Chlorocebus aethiops , Culture Media, Serum-Free , Electrophoresis, Polyacrylamide Gel , Epitopes , Flavivirus/immunology , Mice, Inbred BALB C , Vero CellsABSTRACT
Os linfócitos T γδ desempenham funções importantes na resposta imune contra infecções, na resposta alérgica e na imunidade anti-tumoral. Durante processos inflamatórios, é observado um acúmulo destas células nos tecidos, um fenômeno que acontece em função da migração induzida pela liberação de mediadores quimiotáticos, incluindo a quimiocina CCL2. [...] Porém, o envolvimento de LTB4 na migração de linfócitos T γδ ainda não havia sido descrito. Desta forma, o objetivo deste trabalho foi investigar o papel do LTB4 na migração e na ativação de linfócitosT δ de forma direta, ou de forma indireta, via produção de CCL2. Nossos resultados demonstraram que o LTB4 induziu a migração de linfócitos T γδ in vivo e in vitro. Demonstramos que linfócitos T δexpressam o receptor de alta afinidade para o LTB4, o BLT1, e que o LTB4 induziu reorganização nocitoesqueleto de linfócitos T γδ isolados. Demonstramos ainda que a resposta inflamatória pleuralinduzida por LPS levou ao aumento na produção de LTB4 (e de CCL2), o que ocorreu em paralelo aoacúmulo de linfócitos T γδ. A inibição da síntese de LTB4, assim como o bloqueio de BLT1, foi capazde inibir a migração de linfócitos T γδ in vitro e in vivo em diferentes modelos de pleurisia (induzidapor LPS, alérgeno e Mycobacterium bovis BCG)...
γδ T lymphocytes play important roles during immune responses to infections, allergy and contribute to tumorimmunosurveillence. During inflammatory process, these cells accumulate in the tissues, a phenomena orchestratedby chemotatic mediators, including the chemokine CCL2. [...] In addition, it had beenshown that LTB4 induces CCL2 production, and vice versa. Therefore, the aim of this work was to investigate therole of LTB4 in δ T cell migration and activation, directly or indirectly, via CCL2 production. Our resultsdemonstrate that LTB4 induces γδ T cell migration in vivo and in vitro. We also demonstrated that δ Tlymphocytes express the high affinity receptor for LTB4, BLT1, and LTB4 increased polimerazed f-actin levels inisolated γδ T lymphocytes. In addition, we demonstrated that the pleural inflammatory response induced by LPStriggered an increase in LTB4 (and CCL2) production in vivo, what happened in parallel with the accumulation ofγδ T lymphocytes. The inhibition of LTB4 synthesis or the BLT1 blockade inhibited γδ T cell migration in vivo inresponse to different inflammatory stimuli (LPS, allergen and Mycobacterium Bovis BCG), and also in vitro, in achemotatic assay. Interestingly, only the Vγ4 subtype of γδ T lymphocyte was sensitive to BLT1 blockade in vivo,but the Vδ6.3 and Vγ4 subtypes were not. In vitro, chemoattraction of Vγ4 cells was observed in response to bothLTB4 and CCL2. In accordance, Vγ4 lymphocytes express BLT1 and CCR2, a CCL2 receptor. LTB4 intrapleuralinjection in CCR2 knockout mice caused γδ T cell accumulation in pleural cavities, but not of the Vγ4 subtype...
