title: Trimming of two major type 1 diabetes driving antigens, GAD65 and IA-2, allows for successful expression in Lactococcus lactis.
abstract: Type 1 diabetes (T1D) is a chronic autoimmune disease characterised by excessive immune reactions against auto-antigens of pancreatic β-cells. Restoring auto-antigen tolerance remains the superior therapeutic strategy. Oral auto-antigen administration uses the tolerogenic nature of the gut-associated immune system to induce antigen-specific tolerance. However, due to gastric degradation, proper mucosal product delivery often imposes a challenge. Recombinant Lactococcus lactis have proven to be effective and safe carriers for gastrointestinal delivery of therapeutic products: L. lactis secreting diabetes-associated auto-antigens in combination with interleukin (IL)-10 have demonstrated therapeutic efficacy in a well-defined mouse model for T1D. Here, we describe the construction of recombinant L. lactis secreting the 65 kDa isoform of glutamic acid decarboxylase (GAD65) and tyrosine phosphatase-like protein ICA512 (IA-2), two major T1D-related auto-antigens. Attempts to secrete full size human GAD65 and IA-2 protein by L. lactis were unsuccessful. Trimming of GAD65 and IA-2 was investigated to optimise antigen secretion while maintaining sufficient bacterial growth. GAD65370-575 and IA-2635-979 showed to be efficiently secreted by recombinant L. lactis. Antigen secretion was verified by immunoblotting. Plasmid-derived GAD65 and IA-2 expression was combined in single strains with human IL-10 expression, a desired combination to allow tolerance induction. This study reports the generation of recombinant L. lactis secreting two major diabetes-related auto-antigens: human GAD65 and IA-2, by themselves or combined with the anti-inflammatory cytokine human IL-10. Prohibitive sequence obstacles hampering antigen secretion were resolved by trimming the full size proteins.
authors.affiliation: 1 Clinical and Experimental Endocrinology (CEE), KU Leuven, Herestraat 49 bus 902, 3000 Leuven, Belgium.
authors.forename: S
authors.initials: S
authors.lastname: Robert
authors.forename: K
authors.initials: K
authors.lastname: Van Huynegem
authors.forename: C
authors.initials: C
authors.lastname: Gysemans
authors.forename: C
authors.initials: C
authors.lastname: Mathieu
authors.forename: P
authors.initials: P
authors.lastname: Rottiers
authors.forename: L
authors.initials: L
authors.lastname: Steidler
mesh_terms: D001324:Autoantigens / Q000235:genetics / Q000378:metabolism; D003922:Diabetes Mellitus, Type 1 / Q000628:therapy; D015870:Gene Expression; D005968:Glutamate Decarboxylase / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D016753:Interleukin-10 / Q000235:genetics / Q000378:metabolism; D013294:Lactococcus lactis / Q000235:genetics* / Q000378:metabolism*; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8 / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion
chemical_list: D001324:Autoantigens; C508609:IL10 protein, human; D050505:Mutant Proteins; D011994:Recombinant Proteins; D016753:Interleukin-10; C516862:PTPRN protein, human; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8; D005968:Glutamate Decarboxylase; C401141:glutamate decarboxylase 2
keywords: oral tolerance; recombinant bacteria; secretion efficiency