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Plant-based vaccines for oral delivery of type 1 diabetes-related autoantigens: Evaluating oral tolerance mechanisms and disease prevention in NOD mice

View Article: PubMed Central - PubMed

ABSTRACT

Autoantigen-specific immunological tolerance represents a central objective for prevention of type 1 diabetes (T1D). Previous studies demonstrated mucosal antigen administration results in expansion of Foxp3+ and LAP+ regulatory T cells (Tregs), suggesting oral delivery of self-antigens might represent an effective means for modulating autoimmune disease. Early preclinical experiments using the non-obese diabetic (NOD) mouse model reported mucosal administration of T1D-related autoantigens [proinsulin or glutamic acid decarboxylase 65 (GAD)] delayed T1D onset, but published data are conflicting regarding dose, treatment duration, requirement for combinatorial agents, and extent of efficacy. Recently, dogma was challenged in a report demonstrating oral insulin does not prevent T1D in NOD mice, possibly due to antigen digestion prior to mucosal immune exposure. We used transplastomic plants expressing proinsulin and GAD to protect the autoantigens from degradation in an oral vaccine and tested the optimal combination, dose, and treatment duration for the prevention of T1D in NOD mice. Our data suggest oral autoantigen therapy alone does not effectively influence disease incidence or result in antigen-specific tolerance assessed by IL-10 measurement and Treg frequency. A more aggressive approach involving tolerogenic cytokine administration and/or lymphocyte depletion prior to oral antigen-specific immunotherapy will likely be required to impart durable therapeutic efficacy.

No MeSH data available.


Related in: MedlinePlus

Creation of transplastomic tobacco lines expressing human GAD65 and evaluation of expression.(A) Schematic diagram of chloroplast vectors containing the hGAD65 gene constructed into the chloroplast transformation vector. (Prrn = chloroplast rRNA operon promoter; aadA = aminoglycoside 3′-adenylytransferase gene; PpsbA = psbA gene promoter; hGAD65 = coding sequence of human GAD; TpsbA = 3′ UTR of psbA gene; trnI = isoleucyl-tRNA; trnA = alanyl-tRNA). (B) Southern blot analysis of ApaI digested total DNA from untransformed control (UT) and GAD transplastomic tobacco lines (#1, #2, and #3) probed with the radioisotope-labeled flanking region fragment as shown in (A). (C) Western blot probed with GAD antibody; 15 μg of total leaf soluble protein was loaded in each lane; UT: untransformed plant; young, mature, and old transplastomic leaves were harvested at 6 PM. GST-GAD: 100 ng, positive control. ELISA quantitation using goat polyclonal anti-GAD65 (diluted 1:1500), is shown as a percentage of the total soluble proteins (TSP). Data expressed as mean ± SD of three independent experiments.
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f1: Creation of transplastomic tobacco lines expressing human GAD65 and evaluation of expression.(A) Schematic diagram of chloroplast vectors containing the hGAD65 gene constructed into the chloroplast transformation vector. (Prrn = chloroplast rRNA operon promoter; aadA = aminoglycoside 3′-adenylytransferase gene; PpsbA = psbA gene promoter; hGAD65 = coding sequence of human GAD; TpsbA = 3′ UTR of psbA gene; trnI = isoleucyl-tRNA; trnA = alanyl-tRNA). (B) Southern blot analysis of ApaI digested total DNA from untransformed control (UT) and GAD transplastomic tobacco lines (#1, #2, and #3) probed with the radioisotope-labeled flanking region fragment as shown in (A). (C) Western blot probed with GAD antibody; 15 μg of total leaf soluble protein was loaded in each lane; UT: untransformed plant; young, mature, and old transplastomic leaves were harvested at 6 PM. GST-GAD: 100 ng, positive control. ELISA quantitation using goat polyclonal anti-GAD65 (diluted 1:1500), is shown as a percentage of the total soluble proteins (TSP). Data expressed as mean ± SD of three independent experiments.

Mentions: Transplastomic tobacco plants expressing CTB-hpINS were created as published previously19. Plants expressing human GAD were created to test the potential for synergistic benefit in combining two autoantigens to induce oral tolerance and prevent TID in NOD mice. Antigen fusion to CTB has been shown to lower the dose required for oral tolerance induction1737. However, attempts to express CTB-GAD in plant chloroplasts were unsuccessful. Therefore, this study was performed only with GAD expressed in chloroplasts and not CTB-GAD. The human GAD (hGAD65) gene was inserted into the chloroplast transformation vector – pLDutr. The constructed transformation vector was designed to allow for the specific integration of hGAD65 expression cassette into the intergenic space between isoleucyl-tRNA synthetase (trnI) and alanyl-tRNA synthetase (trnA) of the wild type chloroplast genome by double homologous recombination (Fig. 1A). The hGAD65 gene expression in chloroplasts was driven by the light regulated psbA promoter and 5′ untranslated region (UTR) to increase expression, and the expressed transcripts were stabilized by the psbA 3′ UTR (Fig. 1A). Transplastomic plants were subject to Southern blot assay to evaluate homoplasmy (transformation of all chloroplast genomes). Three independent lines showed transformed fragments of 7.3 kb while the untransformed wild type control fragment migrated to 4.0 kb (Fig. 1B) when probed with the flanking sequence (Fig. 1A). Absence of the native untransformed chloroplast genome 4.0 kb fragment in transplastomic lines (Fig. 1B) confirmed homoplasmy.


Plant-based vaccines for oral delivery of type 1 diabetes-related autoantigens: Evaluating oral tolerance mechanisms and disease prevention in NOD mice
Creation of transplastomic tobacco lines expressing human GAD65 and evaluation of expression.(A) Schematic diagram of chloroplast vectors containing the hGAD65 gene constructed into the chloroplast transformation vector. (Prrn = chloroplast rRNA operon promoter; aadA = aminoglycoside 3′-adenylytransferase gene; PpsbA = psbA gene promoter; hGAD65 = coding sequence of human GAD; TpsbA = 3′ UTR of psbA gene; trnI = isoleucyl-tRNA; trnA = alanyl-tRNA). (B) Southern blot analysis of ApaI digested total DNA from untransformed control (UT) and GAD transplastomic tobacco lines (#1, #2, and #3) probed with the radioisotope-labeled flanking region fragment as shown in (A). (C) Western blot probed with GAD antibody; 15 μg of total leaf soluble protein was loaded in each lane; UT: untransformed plant; young, mature, and old transplastomic leaves were harvested at 6 PM. GST-GAD: 100 ng, positive control. ELISA quantitation using goat polyclonal anti-GAD65 (diluted 1:1500), is shown as a percentage of the total soluble proteins (TSP). Data expressed as mean ± SD of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5304332&req=5

f1: Creation of transplastomic tobacco lines expressing human GAD65 and evaluation of expression.(A) Schematic diagram of chloroplast vectors containing the hGAD65 gene constructed into the chloroplast transformation vector. (Prrn = chloroplast rRNA operon promoter; aadA = aminoglycoside 3′-adenylytransferase gene; PpsbA = psbA gene promoter; hGAD65 = coding sequence of human GAD; TpsbA = 3′ UTR of psbA gene; trnI = isoleucyl-tRNA; trnA = alanyl-tRNA). (B) Southern blot analysis of ApaI digested total DNA from untransformed control (UT) and GAD transplastomic tobacco lines (#1, #2, and #3) probed with the radioisotope-labeled flanking region fragment as shown in (A). (C) Western blot probed with GAD antibody; 15 μg of total leaf soluble protein was loaded in each lane; UT: untransformed plant; young, mature, and old transplastomic leaves were harvested at 6 PM. GST-GAD: 100 ng, positive control. ELISA quantitation using goat polyclonal anti-GAD65 (diluted 1:1500), is shown as a percentage of the total soluble proteins (TSP). Data expressed as mean ± SD of three independent experiments.
Mentions: Transplastomic tobacco plants expressing CTB-hpINS were created as published previously19. Plants expressing human GAD were created to test the potential for synergistic benefit in combining two autoantigens to induce oral tolerance and prevent TID in NOD mice. Antigen fusion to CTB has been shown to lower the dose required for oral tolerance induction1737. However, attempts to express CTB-GAD in plant chloroplasts were unsuccessful. Therefore, this study was performed only with GAD expressed in chloroplasts and not CTB-GAD. The human GAD (hGAD65) gene was inserted into the chloroplast transformation vector – pLDutr. The constructed transformation vector was designed to allow for the specific integration of hGAD65 expression cassette into the intergenic space between isoleucyl-tRNA synthetase (trnI) and alanyl-tRNA synthetase (trnA) of the wild type chloroplast genome by double homologous recombination (Fig. 1A). The hGAD65 gene expression in chloroplasts was driven by the light regulated psbA promoter and 5′ untranslated region (UTR) to increase expression, and the expressed transcripts were stabilized by the psbA 3′ UTR (Fig. 1A). Transplastomic plants were subject to Southern blot assay to evaluate homoplasmy (transformation of all chloroplast genomes). Three independent lines showed transformed fragments of 7.3 kb while the untransformed wild type control fragment migrated to 4.0 kb (Fig. 1B) when probed with the flanking sequence (Fig. 1A). Absence of the native untransformed chloroplast genome 4.0 kb fragment in transplastomic lines (Fig. 1B) confirmed homoplasmy.

View Article: PubMed Central - PubMed

ABSTRACT

Autoantigen-specific immunological tolerance represents a central objective for prevention of type 1 diabetes (T1D). Previous studies demonstrated mucosal antigen administration results in expansion of Foxp3+ and LAP+ regulatory T cells (Tregs), suggesting oral delivery of self-antigens might represent an effective means for modulating autoimmune disease. Early preclinical experiments using the non-obese diabetic (NOD) mouse model reported mucosal administration of T1D-related autoantigens [proinsulin or glutamic acid decarboxylase 65 (GAD)] delayed T1D onset, but published data are conflicting regarding dose, treatment duration, requirement for combinatorial agents, and extent of efficacy. Recently, dogma was challenged in a report demonstrating oral insulin does not prevent T1D in NOD mice, possibly due to antigen digestion prior to mucosal immune exposure. We used transplastomic plants expressing proinsulin and GAD to protect the autoantigens from degradation in an oral vaccine and tested the optimal combination, dose, and treatment duration for the prevention of T1D in NOD mice. Our data suggest oral autoantigen therapy alone does not effectively influence disease incidence or result in antigen-specific tolerance assessed by IL-10 measurement and Treg frequency. A more aggressive approach involving tolerogenic cytokine administration and/or lymphocyte depletion prior to oral antigen-specific immunotherapy will likely be required to impart durable therapeutic efficacy.

No MeSH data available.


Related in: MedlinePlus