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

MARFÍA, JUAN IGNACIO; BOMBICINO, SILVINA S.; FUERTES, FLORENCIA; SABLJIC, ADRIANA V; MIRANDA, MA. VICTORIA; PERONE, MARCELO J.; VALDEZ, SILVINA N.; TRABUCCHI, ALDANA

Buenos Aires

Congreso; Reunión Anual de Sociedades de Biociencia; 2020

Sociedad Argentina de Inmunología

The 65kDa isoform of glutamate decarboxylase (GAD65) is one of the main autoantigens in Autoimmune Diabetes Mellitus. The aim of this work was to express recombinant GAD65 (rGAD65) in insect larvae and assay its capacity as antigen-driven proliferation of NOD mice-derived splenocytes.GAD65 was expressed in Spodoptera frugiperda larvae using the baculovirus expression system with 97% purity yielding 5.7 mg/g of larvae. rGAD65 immunoreactivity was corroborated by radiometric assay using sera from diabetic patients with antibodies against GAD65. Proliferation assays were performed to evaluate the capability of splenocytes to recognize rGAD65. Splenocytes from pre-diabetic and diabetic NOD mice were cultured in triplicates in 96-well U-bottom plates with RPMI (basal proliferation) or with different concentration of the following diabetogenic antigens: 0.01ug/mL to 1 ug/mL of rGAD65, 0.1 ug/mL to 4 ug/mL of insulin and pancreatic islet lysate and 10 ug/mL of ovoalbumin as negative control. A positive unspecific control was carried out with ConA 10 ug/ml. The cells were cultured for 5 days, [3H]TdR was added in the last 18h of the assay. Cells were harvested and the radioactivity incorporated was determined by liquid scintillation counter. Cell proliferation was expressed as Stimulation Index (SI = antigen-proliferation/basal proliferation). SI obtained for the different doses of each treatment were not significant. Besides, all antigen tested induced proliferation of NOD splenocytes compared to de basal condition (p˂0.01). SI of pre-diabetic NOD splenocytes ranged from 0.28±0.06 to 2.45±0.25 for rGAD65 at 1 ug/mL to 0.01ug/mL, from 5.34±1.38 to 4.06±0.44 for insulin at 4 ug/mL to 0.1ug/mL and from 5.15±0.03 to 3.58±0.48 for islet lysates at 4 ug/mL to 0.1ug/mL. SI of overt-diabetic NOD splenocytes ranged from 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 to 2.01±0.44, respectively. SI for OVA was 0.44±0.6 and 0.57±0.02, and ConA 56.32±5.84 and 15.16±2.52 for pre-diabetic and diabetic, respectively.In sum, rGAD65 was successfully produced in S. frugiperda. Moreover, rGAD65 stimulated diabetogenic splenocytes proliferation obtained from NOD mice, fortunately, similarly to insulin and islets lysate. The dose of 1µg/ml of rGAD65 seems to be toxic for cells. Our preliminary results suggested that rGAD65 can be a potential candidate to generate immunotolerance to prevent experimental autoimmune diabetes.