METHODS AND PHARMACEUTICAL COMPOSITIONS FOR INDUCING IMMUNE TOLERANCE BY MUCOSAL VACCINATION WITH FC-COUPLED ANTIGENS

Abstract

The present invention relates to methods and pharmaceutical compositions of inducing immune tolerance by mucosal vaccination with Fc-coupled antigens. In particular, the present invention relates to a method for inducing tolerance to one antigen of interest in a subject in need thereof, comprising the mucosal administration to the subject of a therapeutically effective amount of a recombinant chimeric construct comprising a FcRn targeting moiety and an antigen-containing moiety.

Claims

1. A method for inducing tolerance to one antigen of interest in a subject in need thereof, comprising the mucosal administration to the subject of a therapeutically effective amount of a recombinant chimeric construct comprising a FcRn targeting moiety and an antigen-containing moiety.

2. The method of claim 1 wherein the antigen is an auto-antigen, the antigen is an allergen or the antigen is a molecule that is exogenously administered for therapeutic purposes.

3. The method of claim 1 wherein the subject is an adult, a pregnant woman or a child.

4. The method of claim 1 wherein the subject is a newborn or a neonate.

5. The method of claim 1 wherein the subject is predisposed or believed to be predisposed to developing, or has already developed or is developing an autoimmune disease.

6. The method of claim 1 wherein the subject is predisposed or believed to be predisposed to developing, or has already developed or is developing an allergy.

7. The method of claim 1 wherein the subject is predisposed or believed to be predisposed to developing, or has already developed or is developing an immune reaction against molecules that are exogenously administered for therapeutic or other purposes.

8. The method of claim 1 wherein the subject is predisposed or believed to be predisposed to developing, or has already developed or is developing an immune reaction against a grafted tissue or grafted hematopoietic cells or grafted blood cells.

9. The method of claim 1 wherein the FcRn targeting moiety is an Fc of an IgG antibody, preferably of an IgG1 or IgG4 antibody, even more preferably of an IgG1 antibody, or a portion of the Fc.

10. The method of claim 1 wherein the recombinant chimeric construct of the present invention is a fusion protein that comprises an amino acid sequence consisting of a portion of an Fc region and an amino acid sequence that comprises the antigenic portion of the antigen.

11. The method of claim 1 wherein the recombinant chimeric construct is administered to the subject with the means of recombinant bacteria that express the construct.

12. The method of claim 1 wherein the recombinant chimeric construct is delivered via the oral cavity.

13. The method of claim 1 wherein the recombinant chimeric construct is delivered via the respiratory tract.

14. The method of claim 13, wherein the recombinant chimeric construct is delivered via the nasal cavity.

Description

FIGURES

[0045] FIG. 1. Schematic of the delivery strategy. The physiological pathway that transfers maternal breastmilk IgG to the newborn is exploited. This pathway is FcRn-dependent, and fusion of preproinsulin (PPI) with an IgG Fc fragment grants it access to the bloodstream through the intestinal epithelium.

[0046] FIG. 2. Orally administered PPI-Fc remains confined to the gut in the absence of FcRn. One-day-old C56BL/6 wild-type (FcRn wt) and FcRn−/− mice were force-fed with 50 μg of Alexa-labeled PPI-Fc and imaged after 72 h. PPI-Fc fluorescence remains detectable in the gut of FcRn−/− but not FcRn wt mice.

[0047] FIG. 3. Systemic and thymic PPI-Fc delivery upon oral administration. One-day-old PPI T-cell receptor (TCR)-transgenic G9C8 NOD newborn mice were force-fed with 50 μg of Alexa-labeled PPI-Fc or PPI and imaged after 72 h. PPI-Fc but not PPI accumulation is detected at the whole body level (top) and in the thymus (bottom). Results refer to a representative experiment out of two performed.

[0048] FIG. 4. Serum concentrations of PPI-Fc and PPI upon oral administration. ELISA quantification on serum samples collected at the indicated time points following force-feeding at day 1 as above. Data are depicted as mean+SEM.

[0049] FIG. 5A-B. Oral PPI-Fc administration induces immune tolerance. A. Percent spleen CD8.sup.+CD3.sup.+ and CD4.sup.+CD3.sup.+ T cells in 4-week-old G9C8 mice treated at 1 day of life as above. B. Percent spleen CD4.sup.+ T-cell subsets in the same mice. Bars represent median and interquartile ranges. ***, p<0.001; **, p<0.01; *, p<0.05.

[0050] FIG. 6. Gating strategy for the analysis of antigen-presenting cells. Relevant gates are numbered as follows: 1, macrophages; 2, B cells; 3, plasmacytoid dendritic cells (DCs); 4, migratory CD103.sup.+CD11b.sup.− cDCs; 5, resident CD103.sup.−CD11b.sup.− SIRPα.sup.− cDCs; 6, resident CD103.sup.−CD11b.sup.−SIRPα.sup.+ cDCs; 7, migratory CD103.sup.+CD11b.sup.+ cDCs; 8, resident CD103.sup.−CD11b.sup.+ cDCs. Representative results from the spleen of a 2-week-old G9C8 mouse are shown.

[0051] FIG. 7. Gating strategy for the analysis of T-cell subsets. After gating on viable CD3+ cells (not shown), CD8+ (panel A) and CD4+ T cells (panel F) were analysed for the expression of different markers. Relevant gates are numbered as follows: 1, CD8.sup.+ T cells; 2, activated/memory CD8.sup.+ T cells; 3, naïve CD8.sup.+ T cells; 4, CCR9+ activated CD8.sup.+ T cells; 5, α4β7.sup.+ activated CD8.sup.+ T cells; 6, CCR9.sup.+ naïve CD8+ T cells; 7, α4β7.sup.+ naïve CD8.sup.+ T cells; 8, CD4.sup.+ T cells; 9, activated/memory CD4.sup.+ T cells; 10, naïve CD4.sup.+ T cells; 11, CCR9.sup.+ activated CD4.sup.+ T cells; 12, α4β7.sup.+ activated CD4.sup.+ T cells; 13, CCR9.sup.+ naïve CD4.sup.+ T cells; 14, α4β7.sup.+ naïve CD4.sup.+ T cells; 15, thymic-derived Tregs; 16, peripheral Tregs; 17, Th3 cells; 18, Tr1 cells. Representative results from the spleen of a 2-week-old G9C8 mouse are shown.

[0052] FIG. 8. CCR9.sup.hi CD8.sup.+ T cells in PPI-Fc vs. IgG1-treated G9C8 mice. A-B. Percent (A) and counts (B) of activated (CD44.sup.hiCD62L.sup.−) CCR9.sup.hiCD8.sup.+ T cells in 2-week-old G9C8 mice treated at 1 day of life as above. C-D. Percent (C) and counts (D) of naïve (CD44.sup.−CD62L.sup.+) CCR9.sup.hiCD8.sup.+ T cells in the same mice. Bars represent medians. *, p<0.05.

[0053] FIG. 9. α4β7.sup.+ CD4.sup.+ T cells in PPI-Fc vs. IgG1-treated G9C8 mice. A-B. Percent (A) and counts (B) of activated (CD44.sup.hiCD62L.sup.−) α4β7.sup.+CD4.sup.+ T cells in 2-week-old G9C8 mice treated at 1 day of life as above. C-D. Percent (C) and counts (D) of naïve (CD44.sup.−CD62L.sup.+) α4β7.sup.+CD4.sup.+ T cells in the same mice. Bars represent medians. *, p<0.05.

[0054] FIG. 10. Peripheral and thymic Tregs in PPI-Fc vs. IgG1-treated G9C8 mice. A-B. Percent (A) and counts (B) of peripheral (NRP1.sup.−) Foxp3.sup.+CD4.sup.+ Tregs in 2-week-old G9C8 mice treated at 1 day of life as above. C-D. Percent (C) and counts (D) of thymic (NRP1.sup.+) Foxp3.sup.+CD4.sup.+ Tregs in the same mice. Bars represent medians. *, p<0.05.

[0055] FIG. 11. Orally delivered PPI-Fc protects from diabetes. Diabetes incidence in G9C8 mice force-fed at day 1 with 50 μg PPI-Fc (solid line), equimolar amounts of PPI (dashed line) or IgG1 (dotted line). Diabetes was subsequently induced by immunization with PPIB15-23 and CpG at 4 and 6 weeks of age. **P<0.01 by Mann Whitney U test.

EXAMPLE

Material & Methods

[0056] Generation of Mouse PPI1-Fc and PPI2-Fc Fusion Proteins

[0057] Sequences encoding PPI1 and PPI2 were PCR-amplified from pancreatic and thymic cDNA, respectively, and inserted into pCR4-TOPO plasmids (Invitrogen).sup.35. Following digestion with the appropriate restriction enzymes, PPI1/2 sequences were inserted at EcoRV/BglII sites by cohesive end ligation into pFUSE-hIgG1-Fc2 expression vector (InvivoGen), downstream of an IL-2 signal peptide and upstream of the human Fcγ1 sequence. PPI1-Fc and PPI2-Fc sequences were then re-amplified by PCR and ligated at XbaI/XhoI sites into the pFastBac1 expression vector (Invitrogen). These constructs were inserted into the Bac-to-Bac Baculovirus Expression System (Invitrogen), expressed in Hi5 insect cells and protein products purified on Sepharose-coupled protein G (GE Healthcare). Protein identity was confirmed by reducing SDS-PAGE and Western blot using rabbit anti-insulin polyclonal antibody (H-86, Santa Cruz) and mouse anti-human Fc monoclonal Ab (Southern Biotech). PPI1 and PPI2 were purified from Hi5 insect cell pellets as previously described.sup.56. PPI1-Fc (hereinafter referred to as PPI-Fc) was used in the experiments depicted.

[0058] Mice

[0059] C56BL/6 wild-type and C56BL/6 FcRn−/− mice were obtained from the Janvier Labs and the Jackson Laboratory, respectively. G9C8 Cα.sup.−/− NOD mice are transgenic for a PPI.sub.B15-23 TCR and have been previously described and characterized.sup.35,37.

[0060] In Vivo PPI-Fc Imaging

[0061] PPI-Fc and PPI proteins were conjugated with Alexa Fluor (AF)680 using SAIVI Rapid Antibody/Protein labeling kit (Invitrogen). One-day-old newborn mice were force-fed with 50 μg PPI-Fc or equimolar amounts of PPI. Fluorescence was detected using the Fluobeam imaging system (Fluoptics) at a 690 nm excitation and >700 nm emission wavelengths, with 50-100 ms exposures.

[0062] ELISA Quantification of Serum PPI-Fc and PPI Concentrations

[0063] Following force-feeding as above, blood was collected at the indicated time points for ELISA quantification, with standard curves obtained by sequential dilutions of PPI-Fc and PPI proteins. Both PPI-Fc and PPI were captured with plate-coated H-86 anti-insulin Ab (Santa Cruz). PPI-Fc was detected with a horseradish peroxidase-labeled goat anti-human Fc antibody (Southern Biotech). PPI was revealed with an anti-proinsulin monoclonal Ab (KL-1; kindly provided by Dr. L. Harrison, Walter and Eliza Hall Institute, Parkville, Australia).

[0064] Spleen T-Cell Phenotyping

[0065] The following monoclonal antobodies were used on splenocytes retrieved from treated mice: PE-labeled anti-Foxp3, APC-eFluor780-labeled anti-CD3ε (eBioscience); APC-labeled anti-neuropilin-1 (NRP1; R&D); Brilliant Violet (BV)605-labeled anti-CD4 and BV711-labeled anti-CD8a (BioLegend). Cells were additionally stained with Live/Dead Red (Invitrogen).

[0066] K.sup.d Multimer Preparation

[0067] Monomers composed of the mouse MHC class I heavy chain H-2Kd, human β2-microglobulin and the PPI B15-23 peptide (LYLVCGERL) were synthesized as described.sup.35 and incubated with BV650-coupled streptavidin (mole:mole ratio 4:1) for 1 h. D-biotin and bovine serum albumin were then added at 25 μM and 0.5% concentrations, respectively. The obtained multimers (MMrs) were stored at 4° C. protected from light and used the same day.

[0068] Phenotyping of Subsets of Antigen presenting Cells and T Cells in the Spleen and Lymph Nodes.

[0069] All cells were washed and resuspended in PBS 1x prior to transfer into 96-well V-bottom plated (200 μl/well) for FACS staining BD CompBeads (10 μl/well, 1 drop in 500 μl PBS) were added to each well prior to staining to normalize cell counts. Two antibody panels were used, as follows.

[0070] T-cell panel. Dasatinib (50 μM, 100 μl/well) was added to each cell pellet for 30 min at 37° C. After centrifugation, cells were resuspended in 18 μl of MMr solution containing a 20% dilution of 50 μM dasatinib for 20 min at room temperature, followed by incubation for 20 min at 4° C. with 18 μL of the following antibody premix: Live/DEAD Aqua (Invitrogen, 1/1,000), CD3-APC-eFluor780 (clone 145-2C11, eBioscience, 1/100), CD4-BV711 (clone RM4-5, BD Biosciences, 1/200), CD8-AF700 (clone 53-6.7, BD Biosciences, 1/150), CCR9-PE-Cy7 (clone CW-1.2, BioLegend, 1/100); NRP1-APC (clone 3E12, BioLegend, 1/50), LPAM-1 (α4β7)-PE-CF594 (clone ATK32, BD Biosciences, 1/100), latency-associated peptide (LAP)-BV421 (clone TW7-16B4, BioLegend, 1/200), CD62L-BV605 (clone MEL-14, BD Biosciences, 1/200), CD44-BV786 (clone IM7, BD Biosciences, 1/100). A second 10 μL antibody mix was then added for 15 min at 37° C.: CD49b-FITC (clone HMα2, BioLegend, 1/200), LAG3-PerCP-Cy5.5 (clone C9B7W, BD Biosciences, 1/200). Cells were then washed in PBS, fixed and permeabilised with the Foxp3 Fix/Perm Buffer Set (BioLegend), and incubated with 30 μl anti-Foxp3 antibody (Foxp3-PE, clone FJK-16s, eBioscience, 1/50 in FoxP3/Perm buffer). After a final wash, cells were resuspended in 200 μl PBS 1x and kept at 4° C. prior to flow cytometry acquisition.

[0071] Antigen-presenting cell (APC) panel. An identical staining protocol was applied without fixation and permeabilisation, with an incubation for 20 min at 4° C. with 30 μl of the following antibody mix: Live/DEAD Aqua (Invitrogen, 1/1,000), NK1.1-B V510 (clone PK136, BD Biosciences, 1/100), CD3-BV510 (clone 145-2C11, BD Biosciences, 1/100), F4/80-BV711

[0072] (clone BM8, BioLegend, 1/50), CD8-AF700 (clone 53-6.7, BD Biosciences, 1/150), B220-PE-Cy7 (clone RA3-6B2, BD Biosciences, 1/150), SIRPα-PerCP-eF1710 (clone P84, eBioscience, 1/50), PDCA-1-Pacific Blue (clone 927, BioLegend, 1/200), CD11b-BV650 (clone M1/70, BD Biosciences, 1/150), CD11c-BV605 (clone N418, BioLegend, 1/50), CD103-BV786 (clone M290, BD Biosciences, 1/100).

[0073] PPI-Fc Treatment, Diabetes Induction and Follow-Up

[0074] G9Cα−/−.NOD mice were force-fed on day 1 after birth with 50 μg PPI-Fc or control proteins, namely equimolar quantities of Fc-devoid PPI or Herceptin IgG1. For diabetes induction, 4-week-old mice were primed with 50 μg PPI.sub.B15-23 peptide and 100 μg CpG 4 days after weaning, followed by a second identical immunization 15 days later. Diabetes development was monitored by testing glycosuria and confirmed by glycaemia when positive. Diabetic mice were sacrificed by cervical dislocation.

Results

[0075] The Intestinal Transfer of Orally Administered PPI-Fc is FcRn- and Fc-Dependent

[0076] A schematic of the strategy used is depicted in FIG. 1. We exploited the intestinal FcRn pathway that physiologically delivers breastmilk IgG to the newborn. To this end, we fused the PPI1 or PPI2 protein with the N-terminus of the CH2-CH3 Fc domain from human IgG1 to obtain PPI1-Fc and PPI2-Fc fusion proteins. While Fc-devoid PPI1/2 is not able to cross the intestinal epithelium, addition of the Fc moiety should favor this transfer. We explored this strategy using a PPI1-Fc construct (hereinafter referred to as PPI-Fc).

[0077] We first performed ex vivo imaging on 1-day-old FcRn−/− and wild-type C56BL/6 mice force-fed with fluorescently labeled PPI1-Fc and sacrificed after 72 h (FIG. 2). The PPI-Fc fluorescence was still detectable in the intestines of FcRn−/− but not of wild-type mice, suggesting lack of transfer in the absence of FcRn.

[0078] To verify the occurrence of such transfer, 1-day-old G9C8 newborn mice were force-fed with fluorescently labeled PPI-Fc or Fc-devoid PPI (FIG. 3). After 72 h, systemic PPI-Fc accumulation was promptly visualized, which was not the case for PPI. PPI-Fc, but not PPI, was also visualized in the thymus.

[0079] Serum PPI1-Fc concentrations (FIG. 4) were of ˜1 μg/ml at 24 h after administration (day 2) and remained relatively stable up to 72 h after (day 3; ˜0.75 μg/ml). PPI1 was not detected at any of these time points.

[0080] Collectively, these results show that oral administration of a single 50 μg dose of PPI-Fc, but not of PPI, results in intestinal transfer, systemic antigen bioavailability and delivery to the thymus, which is FcRn- and Fc-dependent.

[0081] Oral PPI-Fc Vaccination induces Tolerogenic T-Cell Modifications.

[0082] We next assessed whether thymic PPI-Fc delivery induced T-cell modifications compatible with immune tolerance (FIG. 5A). Splenocytes obtained at 4 weeks of age following treatment at day 1 of life as above displayed significantly decreased numbers of CD8.sup.+ effector T cells in PPI-Fc-treated newborns compared with PBS-treated ones. No significant difference was highlighted in the number of total CD4.sup.+ T cells between PPI-Fc- and PBS-treated mice. However, when analyzing CD4.sup.+ T cell subsets, significant differences were detected (FIG. 5B). Compared with PBS-treated mice, PPI1-Fc-treated animals displayed decreased numbers of CD4.sup.+ effector T cells (Foxp3.sup.−) and increased numbers of Foxp3.sup.+ regulatory T cells (Tregs), both thymic-derived (NRP1/CD304.sup.−) and peripherally induced (NRP1/CD304.sup.−).

[0083] A second set of experiments was performed by comparing G9C8 mice force fed with either PPI-Fc or IgG1, in order to differentiate the effects induced by the Fc moiety by those relying on the PPI antigenic portion. These mice were analyzed at 2 weeks of age, i.e. closer to the first day of life at which they were orally vaccinated. Both APC and T-cell subsets were analyzed in the spleen, mesenteric lymph nodes (MLNs) and pancreatic lymph nodes (PLNs).

[0084] The APC gating strategy is depicted in FIG. 6. After gating on forward and side scatter and live cells (not shown), CD3.sup.−NK1.1.sup.− (lineage-negative) cells were selected to exclude T and NK cells, respectively (FIG. 6A). Three gates were then selected according to two markers: F4/80.sup.−B220.sup.−, F4/80.sup.−B220.sup.+ and F4/80.sup.−B220.sup.− (FIG. 6B). F4/80.sup.+B220.sup.− cells were then plotted for CD11c and CD11b expression to identify CD11c.sup.−CD11b.sup.+ macrophages (FIG. 6C, gate 1). Similarly, F4/80.sup.−B220.sup.+ cells were plotted for CD11c and CD11b markers to gate CD11c.sup.−CD11b.sup.− B cells (FIG. 6D, gate 2) and CD11b.sup.−CD11c.sup.+ cells, which reveal the plasmacytoid dendritic cell (pDC) population upon further gating for PDCA-1 marker (FIG. 6E, gate 3) and for the migration marker CD103. Among F4/80.sup.−B220.sup.− cells, two gates were selected according to CD11c and CD11b markers (FIG. 6F). Collectively, these CD11c.sup.+ fractions correspond to conventional dendritic cells (cDCs), which are further divided in CD11b.sup.− cDCs, either migratory (CD103.sup.+, gate 4) or resident (CD103.sup.−, gate 5, FIG. 6G); and CD11b.sup.+ cDCs, again either migratory (CD103.sup.+, gate 7) or resident (CD103.sup.−, gate 8, FIG. 6H). An additional population of CD103.sup.−SIRPα.sup.+ CD11b.sup.− cDCs not detailed in the literature was also visualized (gate 6, FIG. 6G). When comparing PPI-Fc- and IgG1-fed 2-week-old mice, no differences were highlighted in the frequency or counts of these different APC populations (not shown). Collectively, these results show that APC composition is not influenced by prior oral vaccination, at least at the 2-week time point analyzed.

[0085] The T-cell gating strategy is depicted in FIG. 7. Among lineage.sup.−CD3.sup.+ cells, CD8.sup.+ T cells were gated (FIG. 7A, gate 1). Among CD8.sup.+ cells, MMr.sup.+ cells were selected to verify their specificity for the PPI.sub.B15-23 peptide (FIG. 7B). CD8.sup.+ T cells were further gated for CD44 and CD62L. While the CD44.sup.hiCD62L.sup.− subset corresponds to activated/memory CD8.sup.+ T cells (gate 2), CD44.sup.−CD62L.sup.+ are naïve cells (gate 3, FIG. 7C). Finally, both activated and naïve CD8.sup.+ T cells were analysed for the expression of the gut-homing markers CCR9 and α4β7 (gates 4-5, FIG. 7D; and gates 6-7, FIG. 7E). For CD4.sup.+ T cells (FIG. 7F), the same phenotypic markers (CD44 and CD62L, FIG. 7G; and CCR9 and α4β7, FIG. 7H-I) were used. In addition, three Treg populations were analysed: classical Tregs, either thymic-derived (Foxp3.sup.+NPR1.sup.+; gate 15) or peripherally induced (Foxp3.sup.+NRP1.sup.−; gate 16, FIG. 7J); T helper (Th)3 cells (LAP.sup.+FoxP3.sup.−; gate 17, FIG. 7K) and T regulatory 1 (Tr1) cells (Foxp3.sup.−CD49b.sup.+LAG3.sup.+; gate 18, FIG. 7L). Using this gating strategy, few differences between PPI-Fc- and IgG1-fed animals were already visible at 2 weeks of age. First, the proportion of splenic CCR9.sup.hiCD8.sup.+ T cells, which are likely to originate in the gut, was increased in PPI-Fc-fed mice (FIG. 8). Such increase was confined to activated CCR9.sup.hiCD8.sup.+ T cells (FIG. 8A-B) and was not observed in the naïve CD8.sup.+ subset (FIG. 8C-D), further suggesting that T cells migrate to the spleen from the gut upon encounter with their PPI B15-23 cognate antigen that is recognized by most T cells in this G9C8 TCR -transgenic model. Second, a similar increase in the PPI-Fc group was observed for splenic α4β7±CD4.sup.+ T cells (FIG. 9), another population reported to originate in the gut. Also in this case, this increase was observed only in the activated (FIG. 9A-B) but not in the naïve subset of these cells (FIG. 9C-D), likely reflecting prior cognate PPI-Fc priming in the gut. Third, a minor increase in splenic peripheral Tregs was observed with the PPI-Fc treatment (FIG. 10A-B), while splenic thymus-derived Tregs were not increased at this time point (FIG. 10C-D).

[0086] Collectively, these results show that oral PPI-Fc vaccination induces T-cell modifications characteristic of oral tolerance, namely an increase in gut-derived activated CD8.sup.+ and CD4.sup.+ T cells and in peripheral Tregs at 2 weeks of age; and modifications suggestive of deletional and regulatory tolerance mechanisms, namely decreased CD8.sup.+ and CD4.sup.+ effector T cells and increased CD4.sup.+ Tregs at 4 weeks. Of further note, the proposed mechanism of action for oral PPI-Fc vaccination is different than for classical oral tolerance with Fc-devoid antigens. Systemic and thymic antigen-Fc bioavailability is here achieved, boosting both peripheral and central tolerance mechanisms, as evidenced by the increased numbers of both thymic- and peripheral-derived Tregs.

[0087] Neonatal Oral PPI-Fc Vaccination Protects G9C8 Mice from Diabetes Development

[0088] Finally, we verified whether PPI-Fc oral vaccination and the associated T-cell modifications resulted in diabetes protection later in life. To this end, 1-day-old newborn G9C8 mice were orally vaccinated with 50 μg PPI-Fc. At 4 and 6 weeks of age, they were then immunized with PPI B15-23 peptide and CpG to induce diabetes and prospectively followed. For controls, equimolar amounts of recombinant IgG1 (i.e., irrelevant protein with preserved FcRn binding) and PPI (i.e., cognate antigen with no FcRn binding) were administered. In IgG1-fed mice, diabetes development was rapid and synchronous with prime-boost immunizations, affecting 93% of mice. Conversely, only 44% of mice developed diabetes when fed with PPI-Fc (p<0.01). As expected, PPI gave an intermediate protection, with 72% of mice ultimately developing diabetes. Collectively, these results demonstrate that oral vaccination with PPI-Fc protects G9C8 mice from diabetes more efficiently than Fc-devoid PPI.

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