Production of recombinant GAD65 by insect larvae and its evaluation as antigen-diabetogenic splenocytes proliferation inductor

MARFIA, JI; BOMBICINO, SS; FUERTES, F; SABLJIC, AV; MIRANDA MV; PERONE, MJ; VALDEZ, SN; TRABUCCHI, A

Congreso; Reunión de Sociedades de Biociencias 2020; LXV SAIC, LXVIII SAI, SAFIS; 2020

(529)PRODUCTION OF RECOMBINANT GAD65 BY INSECT LARVAE AND ITS EVALUATION ASANTIGEN-DIABETOGENIC SPLENOCYTES PROLIFERATION INDUCTORMarfíaJI1;2, Bombicino SS1;2, Fuertes F5, Sabljic AV1;2, Miranda MV3;4, Perone MJ5,Valdez SN1;2, Trabucchi A1;2. 1. Cátedra de Inmunología, Facultad deFarmacia y Bioquímica, Universidad de Buenos Aires (UBA) 2.Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni(IDEHU, CONICET-UBA) 3. Cátedra de Biotecnología, Facultad de Farmacia yBioquímica, Universidad de Buenos Aires (UBA) 4. Instituto de Nanobiotecnología(Nanobiotec, CONICET- UBA) 5. Instituto de Investigaciones en MedicinaTraslacional (CONICET), Universidad Austral, Facultad de CienciasBiomédicas. The65kDa isoform of glutamate decarboxylase (GAD65) is one of the main autoantigensin Autoimmune Diabetes Mellitus. The aim of this work was to expressrecombinant GAD65 (rGAD65) in insect larvae and assay its capacity asantigen-driven proliferation of NOD mice-derived splenocytes. GAD65 wasexpressed in Spodoptera frugiperda larvae using the baculovirusexpression system with 97% purity yielding 5.7 mg/g of larvae. rGAD65immunoreactivity was corroborated by radiometric assay using sera from diabeticpatients with antibodies against GAD65. Proliferation assays were performed toevaluate the capability of splenocytes to recognize rGAD65. Splenocytes frompre-diabetic and diabetic NOD mice were cultured in triplicates in 96-well U-bottomplates with RPMI (basal proliferation) or with different concentration of thefollowing diabetogenic antigens: 0.01ug/mL to 1 ug/mL of rGAD65, 0.1 ug/mL to 4ug/mL of insulin and pancreatic islet lysate and 10 ug/mL of ovoalbumin asnegative control. A positive unspecific control was carried out with ConA 10ug/ml. The cells were cultured for 5 days, [3H]TdR was added in the last 18h ofthe assay. Cells were harvested and the radioactivity incorporated was determinedby liquid scintillation counter. Cell proliferation was expressed asStimulation Index (SI = antigen-proliferation/basal proliferation). SI obtainedfor the different doses of each treatment were not significant. Besides, allantigen tested induced proliferation of NOD splenocytes compared to de basalcondition (p˂0.01). SI of pre-diabetic NOD splenocytes ranged from 0.28±0.06 to2.45±0.25 for rGAD65 at 1 ug/mL to 0.01ug/mL, from 5.34±1.38 to 4.06±0.44 forinsulin at 4 ug/mL to 0.1ug/mL and from 5.15±0.03 to 3.58±0.48 for isletlysates at 4 ug/mL to 0.1ug/mL. SI of overt-diabetic NOD splenocytes rangedfrom 2.06±0.32 to 2.35±0.11, from 3.07±0.19 to 2.95±0.42 and from 2.83±0.28 to2.01±0.44, respectively. SI for OVA was 0.44±0.6 and 0.57±0.02, and ConA56.32±5.84 and 15.16±2.52 for pre-diabetic and diabetic, respectively. In sum,rGAD65 was successfully produced in S. frugiperda. Moreover, rGAD65stimulated diabetogenic splenocytes proliferation obtained from NOD mice,fortunately, similarly to insulin and islets lysate. The dose of 1μg/ml ofrGAD65 seems to be toxic for cells. Our preliminary results suggested thatrGAD65 can be a potential candidate to generate immunotolerance to preventexperimental autoimmune diabetes.<!-- /* Font Definitions */@font-face{font-family:Arial;panose-1:2 11 6 4 2 2 2 2 2 4;mso-font-charset:0;mso-generic-font-family:auto;mso-font-pitch:variable;mso-font-signature:-536859905 -1073711037 9 0 511 0;}@font-face{font-family:"ＭＳ 明朝";panose-1:0 0 0 0 0 0 0 0 0 0;mso-font-charset:128;mso-generic-font-family:roman;mso-font-format:other;mso-font-pitch:fixed;mso-font-signature:1 134676480 16 0 131072 0;}@font-face{font-family:"ＭＳ 明朝";panose-1:0 0 0 0 0 0 0 0 0 0;mso-font-charset:128;mso-generic-font-family:roman;mso-font-format:other;mso-font-pitch:fixed;mso-font-signature:1 134676480 16 0 131072 0;}@font-face{font-family:Cambria;panose-1:2 4 5 3 5 4 6 3 2 4;mso-font-charset:0;mso-generic-font-family:auto;mso-font-pitch:variable;mso-font-signature:-536870145 1073743103 0 0 415 0;} /* Style Definitions */p.MsoNormal, li.MsoNormal, div.MsoNormal{mso-style-unhide:no;mso-style-qformat:yes;mso-style-parent:"";margin:0cm;margin-bottom:.0001pt;mso-pagination:widow-orphan;font-size:12.0pt;font-family:Cambria;mso-ascii-font-family:Cambria;mso-ascii-theme-font:minor-latin;mso-fareast-font-family:"ＭＳ 明朝";mso-fareast-theme-font:minor-fareast;mso-hansi-font-family:Cambria;mso-hansi-theme-font:minor-latin;mso-bidi-font-family:"Times New Roman";mso-bidi-theme-font:minor-bidi;mso-ansi-language:ES-TRAD;}.MsoChpDefault{mso-style-type:export-only;mso-default-props:yes;font-family:Cambria;mso-ascii-font-family:Cambria;mso-ascii-theme-font:minor-latin;mso-fareast-font-family:"ＭＳ 明朝";mso-fareast-theme-font:minor-fareast;mso-hansi-font-family:Cambria;mso-hansi-theme-font:minor-latin;mso-bidi-font-family:"Times New Roman";mso-bidi-theme-font:minor-bidi;mso-ansi-language:ES-TRAD;}size:595.0pt 842.0pt;margin:72.0pt 90.0pt 72.0pt 90.0pt;mso-header-margin:35.4pt;mso-footer-margin:35.4pt;mso-paper-source:0;}div.WordSection1{page:WordSection1;}