Abstract
The present disclosure relates to a vaccine composition for treating multiple sclerosis. The vaccine composition of the present disclosure induces immune tolerance and suppresses autoimmune response itself, thus can be usefully applied to the treatment of multiple sclerosis.
Claims
1. A vaccine composition for treating multiple sclerosis, the vaccine composition comprising: biocompatible porous nanoparticles; and a myelin-derived self-antigen loaded in the nanoparticles.
2. The vaccine composition of claim 1, wherein the biocompatible porous nanoparticles have mesopores with a diameter of 5 nm to 40 nm.
3. The vaccine composition of claim 1, wherein the biocompatible porous nanoparticles comprise three-dimensional radial pores.
4. The vaccine composition of claim 1, wherein the biocompatible porous nanoparticles are inorganic nanoparticles.
5. The vaccine composition of claim 4, wherein the inorganic nanoparticles are at least one selected from the group consisting of silica nanoparticles, iron oxide nanoparticles, cerium oxide nanoparticles, manganese oxide nanoparticles, platinum nanoparticles, selenium nanoparticles, and carbon nanoparticles.
6. The vaccine composition of claim 1, wherein the biocompatible porous nanoparticles are organic nanoparticles.
7. The vaccine composition of claim 1, wherein the myelin-derived self-antigen is at least one peptide selected from the group consisting of myelin oligodendrocyte glycoprotein peptide, myelin proteolipid protein peptide, myelin basic protein peptide, and αB-crystalline peptide (CRYAB), or a fragment thereof.
8. The vaccine composition of claim 7, wherein the fragment of the at least one peptide selected from the group consisting of myelin oligodendrocyte glycoprotein peptide, myelin proteolipid protein peptide, myelin basic protein peptide, and αB-crystalline peptide (CRYAB) is 5 to 25 amino acids in length.
9. The vaccine composition of claim 1, further comprising ceria nanoparticles bound to the surface of the porous nanoparticles.
10. A method for preparing a vaccine composition for treating multiple sclerosis, the method comprising loading a myelin-derived self-antigen in biocompatible porous nanoparticles.
11. The method of claim 10, wherein the biocompatible porous nanoparticles have mesopores with a diameter of 5 nm to 40 nm.
12. The method of claim 10, wherein the biocompatible porous nanoparticles comprise three-dimensional radial pores.
13. The method of claim 10, wherein the myelin-derived self-antigen is at least one peptide selected from the group consisting of myelin oligodendrocyte glycoprotein peptide, myelin proteolipid protein peptide, myelin basic protein peptide, and αB-crystalline peptide (CRYAB), or a fragment thereof.
14. The method of claim 13, wherein the fragment of the at least one peptide selected from the group consisting of myelin oligodendrocyte glycoprotein peptide, myelin proteolipid protein peptide, myelin basic protein peptide, and αB-crystalline peptide (CRYAB) is 5 to 25 amino acids in length.
15. The method of claim 10, further comprising binding ceria nanoparticles to the biocompatible porous nanoparticles.
16. A pharmaceutical composition for inducing immune tolerance, the pharmaceutical composition comprising: biocompatible porous nanoparticles; a myelin-derived self-antigen loaded in the nanoparticles; and ceria nanoparticles bonded to the surface of the porous nanoparticles.
17. The pharmaceutical composition of claim 16, wherein the biocompatible porous nanoparticles have mesopores with a diameter of 5 nm to 40 nm.
18. The pharmaceutical composition of claim 16, wherein the myelin-derived self-antigen is a fragment of at least one peptide selected from the group consisting of myelin oligodendrocyte glycoprotein peptide, myelin proteolipid protein peptide, myelin basic protein peptide, and αB-crystalline peptide (CRYAB), the fragment being 5 to 25 amino acids in length.
19. The pharmaceutical composition of claim 16, wherein the biocompatible porous nanoparticles comprise three-dimensional radial pores.
20. The pharmaceutical composition of claim 16, wherein the inducing immune tolerance is an auto-immunosuppression on a multiple sclerosis patient.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0087] FIG. 1 shows proposed schematics of the immunosuppressive therapeutic nanoparticle vaccine to treat EAE (experimental autoimmune encephalomyelitis) via re-establishing antigen-specific immune tolerance. The accumulation of CeNPs-decorated, MOG-loaded MSNs in the APCs resulted in the reduction of intracellular ROS and expression of costimulatory molecules (CD86, CD40) on APCs, leading to the suppression of APCs activation, thus making APCs more tolerogenic. The interaction between the MOG peptide presented by MHC-II on semi-mature, tolerogenic APCs and T-cell receptor on naive CD4.sup.+ T cells enables the induction of Foxp3.sup.+ Tregs. The peripherally induced Tregs subsequently inhibit the infiltration of MOG-specific autoreactive CD4.sup.+ T cells to the CNS. Consequently, the reduction of infiltrated CD4.sup.+ T cells in CNS hampers neuronal self-destruction and prevents epitope spreading within CNS. Thus, neuro-immune homeostasis can be achieved for the treatment of late, chronic-stage multiple sclerosis.
[0088] FIGS. 2a, 2b, 2c, 2d, 2e, 2f, and 2g show experimental data of intravenous injection of myelin oligodendrocyte glycoprotein (MOG)-loaded MSNs which suppressed EAE development. FIG. 2a shows transmission electron microscopy (TEM) image of MSNs, scale bar: 200 nm; the experiment was repeated independently at least three times. FIG. 2b. shows representative histograms of rhodamine B isothiocyanate (RITC) signal from splenocytes of C57BL/6 mice that were left untreated (no injection) or intravenously injected with RITC-MSNs 24 h before flow cytometry analysis. FIG. 2c shows the percentage of immune cells that engulfed RITC-MSNs in spleens, n=4 biologically independent animals. FIG. 2d shows the schematics for MOG loading in MSNs. FIG. 2e shows that EAE was induced in C57BL/6 mice before being injected intravenously with bare MSNs (MSN), MOG.sub.35-55 peptide-loaded MSNs (MSN-MOG), OVA.sub.323-339 peptide-loaded MSNs (MSN-OVA), soluble MOG (MOG), or left untreated on days 4, 7, and 10 (n=6). FIG. 2f shows EAE clinical score for different groups of mice. FIG. 2g shows Kaplan-Meier curves demonstrating the percentage of EAE-free mice over time, P values were calculated using log-rank (Mantel-Cox) test. Data in FIG. 2c are represented as mean±standard deviation (SD). Data in FIG. 2f are represented as mean±standard error (SE). Data in FIG. 2f were subjected to one-way analysis of variance (ANOVA) with Dunnett's multiple comparisons test. P<0.05 was considered significant.
[0089] FIGS. 3a, 3b, 3c, 3d, 3e, 3f, 3g, 3h, 3i, 3j, 3k, 31, 3m, 3n, 3o, 3p, 3q, 3r, 3s, 3t, and 3u show experimental data indicating that MSN-MOG induced peripheral tolerance in the spleen and MSN-MOG vaccine suppressed CNS-infiltrating APCs and CD4.sup.+ cells. FIG. 3a shows that EAE-induced mice were intravenously injected with MSN-OVA, MSN-MOG, or left untreated on days 4, 7, and 10 after EAE induction. Splenocytes were isolated and analyzed by flow cytometry on day 20 after EAE induction. FIG. 3b and FIG. 3c shows CD86 and MHC (major histocompatibility complex)-II expression on CD11c.sup.+DCs, F4/80.sup.+ macrophages, and B220.sup.+ B cells, respectively. FIG. 3d shows the frequency of CD4.sup.+ T cells. FIG. 3e and FIG. 3f show percentage of forkhead box P3.sup.+ (Foxp3.sup.+) among CD4.sup.+ T cells and their representative pseudocolor plots, respectively. FIG. 3g shows the number of Foxp3.sup.+ regulatory T cells (Treg). FIG. 3h, FIG. 3i, FIG. 3j and FIG. 3k shows the levels of IL-17A, TNF-α, GM-CSF, and IL-10 secreted by splenocytes stimulated ex vivo with MOG.sub.35-55, respectively. In FIG. 3b-3e and FIG. 3g-3k, n=4 (untreated and MSN-OVA) or 5 (MSN-MOG) biologically independent animals. FIG. 3l shows the percentage of CD11c.sup.+ DC, F4/80.sup.+ macrophages, and B220.sup.+ B cells in the spinal cords and FIG. 3m shows their representative pseudocolor plots, respectively. FIG. 3n shows the number of CD11c.sup.+ DCs, F4/80.sup.+ macrophages, and B220.sup.+ B cells in spinal cords. FIG. 3o shows the percentage of APCs expressing MHC-II in spinal cords. FIG. 3p shows the number of CD4.sup.+ T-cells infiltrating spinal cords. FIG. 3q and FIG. 3r show the percentage of Iba1 expressed in spinal cord cells and representative pseudocolor plots, respectively. FIG. 3j shows EAE mean clinical score of EAE mice that were injected with MSN-MOG, MSN-MOG and control antibody (MSN-MOG+Ctrl Ab), MSN-MOG and anti-CD25 antibody (MSN-MOG+αCD25), or left untreated (n=4). FIG. 3t and FIG. 3u show the percentage of APCs and CD4.sup.+ T cells in the spinal cord on day 20 after disease induction, respectively. Data in FIG. 3b-3e, 3g-31, 3n-3q, 3t and 3u, are represented as mean±SD and were subjected to a one-way ANOVA with Dunnett's multiple comparisons tests. P<0.05 was considered significant, ns=not significant.
[0090] FIGS. 4a, 4b, 4c, 4d, 4e, 4f, 4g, and 4h show MSN-MOG vaccine therapeutically suppressing the developed EAE. FIG. 4a shows the experiment schedule; EAE was induced in mice before administering three MSN-MOG injections starting from day 12 (early therapeutics), day 15 (late therapeutics), or left untreated. FIG. 4b, FIG. 4c, and FIG. 4d show the clinical score, body weight of the mice, and incidence of complete paralysis, respectively, measured over time. n=5 (untreated) or 6 (early therapeutics, late therapeutics) biologically independent animals. FIG. 4e is a photograph of an EAE-induced mouse from the late therapeutic group before receiving an injection on day 15 (left) and after 3 MSN-MOG injections on day 22. FIG. 4f and FIG. 4g shows the EAE clinical score and body weight of mice that received additional MSN-MOG injections on days 28 and 35, respectively; n=5 (untreated) or 6 (early therapeutics, late therapeutics) biologically independent animals. FIG. 4h is images of Hematoxylin and eosin (H&E)-stained thoracic vertebrae cross sections of EAE-induced mice on day 50, the experiment was repeated independently at least twice. The discontinuous lines indicate the border between the gray matter (on the lower part) and the ventral white matter of the spinal cord (scale bar: 100 μm). The data in FIG. 4b, FIG. 4c, FIG. 4f, and FIG. 4g are represented as mean±SE and were analyzed by one-way ANOVA. Dunnett's multiple comparisons tests were performed in FIG. 4b, FIG. 4c, and FIG. 4f. Tukey's multiple comparisons test was performed in FIG. 4g. P<0.05 was considered significant, ns=not significant.
[0091] FIGS. 5a, 5b, 5c, 5d, 5e, 5f, 5g, 5h, 5i, 5j, 5k, 5l, 5m, 5n, 5o and 5p show ROS-scavenging CeNPs inducing tolerogenic APCs. FIG. 5a shows a scheme demonstrating the catalytic property of CeNPs to scavenge intracellular ROS for the suppression of APCs activation. FIG. 5b TEM images of CeNPs (scale bar: 20 nm), the experiment was repeated independently at least three times. FIG. 5c Hydrodynamic size distribution of CeNPs and pegylated CeNPs in complete RPMI 1640 medium. FIG. 5d Apoptosis of BMDCs after 48 h incubation with various cerium concentrations. BMDCs were incubated with pegylated CeNPs (Ce, 50 μM cerium), OVA.sub.323-339 (OVA, 1 μg/mL), pegylated CeNPs plus OVA.sub.323-339 (Ce+OVA), or left untreated for 24 h; following by LPS treatment (1 μg/mL) for the next 24 h; control is the bare BMDCs; n=3 biologically independent samples. FIG. 5e, FIG. 5f and FIG. 5g show expressions of CD86, CD40, and MHC-II on BMDCs, respectively; n=4 biologically independent samples. FIG. 5h BMDCs were incubated with pegylated CeNPs (Ce, 50 μM cerium), OVA.sub.323-339 (OVA, 1 μg/mL), pegylated CeNPs plus OVA.sub.323-339 (Ce+OVA), or left untreated for 24 h; following by LPS treatment (1 μg/mL) for the next 24 h before being co-cultured with OT-II CD4.sup.+ T cells for 72 h. FIG. 5i, FIG. 5j, FIG. 5k, and FIG. 5l show the percentage of CD25.sup.highFoxp3, IL-10, IFN-γ, and IL-17A expression in the gate of Vα2.sup.+ CD4.sup.+ T cells, respectively. FIG. 5m, FIG. 5n, FIG. 5o and FIG. 5p show ratios of CD25.sup.highFoxp3.sup.+ to IL-17A.sup.+ T cells, of IL-10.sup.+ to IL-17A.sup.+ T cells, of CD25.sup.highFoxp3.sup.+ to IFN-γ.sup.+ T cells, and of IL-10.sup.+ to IFN-γ.sup.+ T cells, respectively. In FIG. 5i-5p, n=5 biologically independent samples. The data in FIG. 5d-5g, FIG. 5i-5p are represented as mean±SD and were analyzed by one-way ANOVA with Tukey's multiple comparisons test. P<0.05 was considered significant, ns=not significant.
[0092] FIGS. 6a, 6b, 6c, 6d, 6e, 6f, 6g, 6h, 6i, 6j, 6k, 6l, 6m, 6n and 6o show CeNPs-decorated MSN-MOG enhancing efficacy of the therapeutic vaccine against EAE at chronic phase. FIG. 6a shows the TEM image of MSN-MOG-Ce vaccine, scale bar: 50 nm; the experiment was repeated independently at least twice. BMDCs were treated with MSN-MOG, MSN-MOG-Ce, or left untreated for 24 h, followed by stimulation with LPS (100 ng/mL) for the next 12 h. FIG. 6b shows intracellular ROS levels in BMDCs, as measured by H.sub.2DCFDA assay, n=4 biologically independent samples. FIG. 6c shows the percentage of CD11c+ BMDCs expressing CD86, n=4 biologically independent samples. FIG. 6d shows EAE clinical scores of mice that were intravenously injected with MSN-MOG, MSN-MOG-Ce, or left untreated on days 15, 18, and 21 after disease induction; n=5 biologically independent animals. FIG. 6e and FIG. 6f show the expression levels of CD86 and CD40 on APCs in spleens on day 31, respectively. FIG. 6g and FIG. 6h show the percentage of Foxp3.sup.+ cells among CD3.sup.+ CD4.sup.+ T cells in spleens on day 31 and their representative contour plots, respectively. FIG. 6i and FIG. 6j show the number and frequency of CD3.sup.+ CD4.sup.+ T cells infiltrating the spinal cords on day 31. FIG. 6k shows the representative pseudocolor plots showing the frequency of CD3.sup.+ CD4.sup.+ T cells in the spinal cord of the indicated groups. FIG. 6l shows the number of MOG-specific CD4.sup.+ T cells infiltrating to the spinal cord on day 31. FIG. 6m and FIG. 6n show the percentage of APCs in the spinal cord and MHC-II expression on these cells on day 31, respectively. FIG. 6o shows the frequency of CD3.sup.+ CD4.sup.+ T cells in cervical lymph nodes on day 31. In FIG. 6e-6g, 6i, 6k, 6m-6p, n=5 biologically independent animals. The data in FIG. 6b, 6c, 6e-6g, 6i, 6j, 6l-6o are represented as mean±SD and were analyzed by one-way ANOVA. Tukey's multiple comparisons tests were performed in FIG. 6b and FIG. 6c. Dunnett's multiple comparisons tests were performed in FIG. 6e-6g, 6i, 6j, 6l-6o. The data in FIG. 6d are represented as mean±SE and were subjected to a one-way ANOVA with Dunnett's multiple comparisons test. P<0.05 was considered significant, ns=not significant.
[0093] FIGS. 7a, 7b and 7c show the results of MSN characterization. FIG. 7a shows the scanning electron microscope (SEM) image of MSNs, scale bar: 200 nm; the experiment was repeated independently at least three times. FIG. 7b shows Nitrogen adsorption/desorption isotherms of MSNs, P/Po indicates the ratio between equilibrium (P) and saturation (Po) pressure of nitrogen at the adsorption temperature. The graph inside shows the distribution of desorption pore-size of MSNs. FIG. 7c shows Cell Counting Kit (CCK)-8 viability assay of RAW 264.7 cells incubated for 24 h with various concentrations of MSNs (n=6 biologically independent samples). Data in FIG. 7c are represented as mean±standard deviation (SD).
[0094] FIG. 8 shows in vitro degradation of MSNs, and shows TEM images demonstrating the degradation of MSNs in phosphate-buffered saline at 37° C. over time.
[0095] FIG. 9 shows the amounts of loaded MOG peptide and OVA peptide per 1 mg MSNs and the administration doses of each peptide and MSNs used in the present disclosure.
[0096] FIGS. 10a, 10b, and 10c show the effect of MSN amount on semi-therapeutic efficacy. FIG. 10a shows that EAE was induced in C57BL/6 mice before intravenous injection with different amounts of MSNs on day 7. The amount of MOG.sub.35-55 loaded was unchanged. FIG. 10b and FIG. 10c show EAE clinical scores and body weights, of mice treated with normal (1×MSN-MOG) and double amount of MSNs (2×MSN-MOG), while maintaining the same dose of the MOG peptide. n=4 (untreated) or 5 (1×MSN-MOG and 2×MSN-MOG) biologically independent animals. The data in FIG. 10b and FIG. 10c are represented as mean±standard error (SE) and were subjected to one-way ANOVA. Dunnett's multiple comparisons tests were performed in FIG. 10b and FIG. 10c, ns=not significant.
[0097] FIGS. 11a, 11b, and 11c show the analysis of APCs in spleens after administering semi-therapeutics. FIG. 11a shows that EAE was induced in C57BL/6 mice on days 0 and 1, which was followed by intravenous injection of MSN-OVA, MSN-MOG, or no treatment; n=4 for untreated and MSN-OVA, n=5 for MSN-MOG. Splenocytes were isolated on day 20 after EAE induction for flow cytometry analysis. FIG. 11b and FIG. 11c show the percentages and numbers of APCs, namely, CD11c.sup.+, F4/80.sup.+, and B220.sup.+ cells, respectively. The data in FIG. 11b and FIG. 11c have been represented as mean±SD and were subjected to a two-way ANOVA. Dunnett's post-hoc test was performed in FIG. 11b and FIG. 11c. P<0.05 was considered significant, ns=not significant.
[0098] FIGS. 12a and 12b show the analysis of APCs in the CNS after administering semi-therapeutics. FIG. 12a shows that EAE was induced in C57BL/6 mice on days 0 and 1, which was followed by intravenous injection of MSN-OVA, MSN-MOG, or no treatment; n=4 for untreated and MSN-OVA, n=5 for MSN-MOG. Cells were isolated from the spinal cord on day 20 after EAE induction for flow cytometry analysis. FIG. 12b shows the numbers of MHC-II molecules expressed by APCs, namely, CD11c.sup.+, F4/80.sup.+, and B220.sup.+ cells. The data in FIG. 12b have been represented as mean±SD and were subjected to a two-way ANOVA. Dunnett's post-hoc test was performed in FIG. 12b. P<0.05 was considered significant, ns=not significant.
[0099] FIGS. 13a, 13b, 13c, and 13d show the analysis of immune tolerance when Treg are depleted. FIG. 13a. shows the experiment schedule; EAE mice were injected with MSN-MOG, MSN-MOG and control antibody (MSN-MOG+Ctrl Ab), MSN-MOG and anti-CD25 antibody (MSN-MOG+αCD25), or left untreated. FIG. 13b shows the number of APCs in the spinal cord on day 20, n=4 biologically independent animals. FIG. 13c and FIG. 13d show representative plots showing the percentage of APCs (CD11c.sup.+ cells and F4/80.sup.+ cells) and CD4.sup.+ T-cells in the spinal cord on day 20, respectively. Data in FIG. 13b are represented as mean±SD and were subjected to a one-way ANOVA with Dunnett's multiple comparisons tests. P<0.05 was considered significant, ns=not significant.
[0100] FIGS. 14a and 14b shows the results of characterization of cerium oxide nanoparticles. FIG. 14a shows the cell viability of RAW264.7 cells that were incubated with various concentration of cerium for 24 h. FIG. 14b shows the TEM image of the cerium oxide nanoparticles. scale bar: 20 nm.
[0101] FIG. 15 shows CeNPs inducing Tregs in vitro. BMDCs were incubated with pegylated CeNPs (Ce, 50 μM cerium), OVA.sub.323-339 (OVA, 1 μg/mL), pegylated CeNPs plus OVA.sub.323-339 (Ce+OVA), or left untreated for 24 h; following by LPS treatment (1 μg/mL) for the next 24 h before being co-cultured with OT-II CD4.sup.+ T-cells for 72 h. FIG. 15 shows the representative pseudocolor plots of the expression of CD25.sup.highFoxp3.sup.+, IL-10, IFN-γ, and IL-17A in the gate of OT-II CD4.sup.+ T-cells.
[0102] FIG. 16 shows the zeta potential of MSN, CeNPs, MSN-MOG and MSN-MOG-Ce.
[0103] FIG. 17 shows in vitro BMDCs suppression. BMDCs were treated with MSN-MOG, MSN-MOG-Ce, or left untreated for 24 h, followed by stimulation with 100 ng/mL LPS for the next 12 h; n=4 biologically independent samples. Figure shows the percentage of CD11c.sup.+ BMDCs expressing MHC-II. The data are represented as mean±SD and were analyzed by one-way ANOVA with Tukey's multiple comparisons test, ns=not significant.
[0104] FIGS. 18a and 18b show the body weight changes during late therapeutic treatment. FIG. 18a shows the experiment schedule; EAE was induced in C57BL/6 mice, followed by thrice intravenous injection of MSN-MOG or MSN-MOG-Ce, or left untreated, starting from day 15 (late therapeutics); n=5 biologically independent animals. FIG. 18b shows the body weights of mice during the study; the arrows indicate the injection time points. The data in FIG. 18b are represented as mean±SE and were subjected to a one-way ANOVA with Tukey's multiple comparisons test. P<0.05 was considered significant, ns=not significant.
[0105] FIGS. 19a, 19b, 19c, 19d, 19e, 19f and 19g show the analysis of APCs in spleens after administering late therapeutics. FIG. 19a shows the experiment schedule; EAE was induced in C57BL/6 mice, followed by thrice intravenous injection of MSN-MOG or MSN-MOG-Ce, or left untreated, on days 15, 18, and 21 prior to flow cytometry analysis of splenocytes on day 31; n=5 biologically independent animals. FIG. 19b, FIG. 19c and FIG. 19d show the frequencies of CD11c.sup.+, F4/80.sup.+, and B220.sup.+ cells, respectively. FIG. 19e, FIG. 19f and FIG. 19g show the numbers of CD11c.sup.+, F4/80.sup.+, and B220.sup.+ cells, respectively. The data in FIG. 19b-19g are represented as mean±SD and were subjected to a one-way ANOVA with Dunnett's multiple comparisons tests. P<0.05 was considered significant, ns=not significant.
[0106] FIGS. 20a, 20b, 20c and 20d show the analysis of MHC-II expression on APCs in spleens after administering late therapeutics. FIG. 20a shows the experiment schedule; EAE was induced in C57BL/6 mice, followed by intravenous injection of MSN-MOG, MSN-MOG-Ce, or left untreated, on days 15, 18, and 21 prior to flow cytometry analysis of splenocytes on day 31; n=5 biologically independent animals. FIG. 20b, FIG. 20c and FIG. 20d shows the expression of MHC-II on CD11c.sup.+ DCs, F4/80.sup.+ macrophages, and B220.sup.+ cells, respectively. Data in FIG. 20b-20d are represented as mean±SD and were subjected to a one-way ANOVA with Dunnett's multiple comparisons tests, ns=not significant.
[0107] FIGS. 21a, 21b and 21c show the analysis of T-cells in the spleens after treatment with late therapeutics. FIG. 21a shows the experiment schedule; EAE was induced in C57BL/6 mice, followed by intravenous injection of MSNMOG, MSN-MOG-Ce, or left untreated, on days 15, 18, and 21 prior to flow cytometry analysis of splenocytes on day 31; n=5 biologically independent animals. FIG. 21b and FIG. 21c show the frequency and number of CD4.sup.+ T-cells, respectively, in the spleens. Data in FIG. 21b and FIG. 21c are represented as mean±SD and were subjected to a one-way ANOVA with Dunnett's multiple comparisons tests, ns=not significant.
[0108] FIG. 22 shows MSN-MOG-Ce vaccine suppressing infiltration of APCs into CNS and their antigen presentation capacity in late therapeutics. FIG. 22 is representative pseudocolor plots showing the percentages of CD11c.sup.+ cells, F4/80.sup.+ cells, B220.sup.+ cells, and MHC-II expression on the cells in the CNS of EAE mice on day 31 after late therapeutics study with MSN-MOG and MSN-MOG-Ce. The numbers in the plots depict the percentage.
[0109] FIG. 23 shows MSN-MOG-Ce vaccine inhibiting CD4.sup.+ T-cell in the CNS-draining lymph node in late therapeutics. FIG. 23 is representative plots of frequency of CD4.sup.+ T-cell in the cervical lymph node in EAE mice after late therapeutics with MSN-MOG and MSN-MOG-Ce.
DETAILED DESCRIPTION OF THE EXEMPLARY EMBODIMENTS
[0110] Hereinafter, the present invention will be described in more detail with reference to examples. It would be obvious to those skilled in the art that these examples are intended to be more concretely illustrative, and the scope of the present disclosure as set forth in the appended claims is not limited to or by the examples.
EXAMPLE
Example 1: Materials
[0111] Hexadecyltrimethylammonium bromide (CTAB), ammonium hydroxide solution, phorbol 12-myristate 13-acetate (PMA), ionomycin, 6-aminohexanoic acid, formic acid, tetraethyl orthosilicate (TEOS), lipopolysaccharide from Escherichia coli, rhodamine B isothiocyanate (RITC), and RPMI 1640 were purchased from Sigma-Aldrich (St. Louis, Mo., USA). Dulbecco's modified Eagle's medium (DMEM) was purchased from Lonza (Basel, Switzerland). 2′,7-Dichlorofluorescein diacetate (H2DCFDA) was purchased from Invitrogen (Carlsbad, Calif., USA). Cerium (III) nitrate hexahydrate was purchased from Alfa Aesar (Tewksbury, Mass., USA). Ethanol, methanol, hydrochloric acid, and ethyl acetate were purchased from Samchun (Seoul, South Korea). Endotoxin free ultra-pure water was purchased from EMD Millipore (MA, USA). MOG35-55 peptide (sequence: MEVGWYRSPFSRWHLYRNGK) and OVA323-339 peptide (sequence: ISQAVHAAHAEINEAGR) were synthesized by Anygen (Gwangju, South Korea). Methoxy poly(ethylene glycol) succinimidyl glutarate (Mw=5000) was purchased from SunBio (Gyeonggi, South Korea). Fluorescein isothiocyanate (FITC)-conjugated anti-CD3, eFluor 450 and R-phycoerythrin (PE)-conjugated anti-F4/80, PE-Cyanine7 (PE-Cy7)-conjugated anti-CD4, APC-conjugated anti-CD11c, eFluor 450-conjugated anti-CD86, FITC-conjugated anti-CD40, PE-Cy7-conjugated anti-B220, FITC-conjugated anti-MHC-II, and APC-conjugated anti-Foxp3 monoclonal antibodies were purchased from eBioscience (CA, USA). PE-conjugated I-Ab MOG.sub.35-55 tetramer was purchased from MBL (Japan). FcR blocking reagent, APC-conjugated anti-Foxp3, PE-conjugated anti-CD25, FITC-conjugated anti-IFN-γ, PE-Vio770-conjugated anti-CD4, APC-conjugated anti-IL-10, PE-conjugated anti-IL-17A monoclonal antibodies, CD4.sup.+ T-cell isolation kit, LS column, and MidiMACS separator were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). Pacific blue-conjugated anti-mouse TCR Vα2 antibody was purchased from BioLegend (CA, USA). Alexa Fluor 647-conjugated anti-lba1/AIF-1 monoclonal antibody was purchased from Cell Signaling Technology, Inc.
Example 2: Synthesis of Large-Pore Mesoporous Silica Nanoparticles
[0112] MSNs were synthesized according to a previous study (Nguyen, T. L., Cha, B. G., Choi, Y., Im, J. & Kim, J. Injectable dual-scale mesoporous silica cancer vaccine enabling efficient delivery of antigen/adjuvant-loaded nanoparticles to dendritic cells recruited in local macroporous scaffold. Biomaterials 239, 119859 (2020)). First, 500 μL of Fe3O4 (6 mg/mL) nanocrystals, which were prepared from an iron-oleate complex by a heat-up reaction, was mixed with 10 mL of a 0.055 M CTAB aqueous solution with vigorous stirring for 30 min. The mixture was then heated at 60° C. for 15 min before being poured into 95 mL of deionized (DI) water. Subsequently, 3 mL of ammonium hydroxide solution, 5 mL of methanol, 20 mL of ethyl acetate, and 500 μL of TEOS were added to the mixture and allowed to react overnight. The resulting nanoparticles were washed thrice with ethanol. The CTAB template and Fe3O4 nanocrystal core were removed by stirring the MSNs in ethanol containing HCl for 3 h at 60° C. Finally, the MSNs were washed thrice with ethanol and stored in ethanol until use. To track MSN accumulation in immune cells in vivo, RITC-MSN was prepared by mixing RITC with MSNs in methanol for 48 h under dark conditions. An intensive wash was applied to completely remove the unbound RITC.
Example 3: Synthesis of Cerium Oxide Nanoparticles
[0113] 6-Aminohexanoic acid (6-AHA) (10 mmol) and cerium (III) nitrate hexahydrate (2.5 mmol) were dissolved in 60 and 50 mL of DI, respectively. The 6-AHA solution was then heated. When the temperature of the solution reached 95° C. under continuous stirring, 70 μL of HCl was added. Thereafter, the cerium (III) salt solution was immediately poured into the heated 6-AHA solution under vigorous stirring. To produce CeNPs with a 3 nm diameter, the mixture was allowed to react for 1 min before washing thrice with excess acetone. The CeNPs were collected under vacuum pressure and re-dispersed in sterile DI water. To pegylate CeNPs, 10 mg CeNPs were allowed to react with 250 mg methoxy poly(ethylene glycol) succinimidyl glutarate in 20 mL ethanol at pH 8. The resulted nanoparticles were washed thrice with excess acetone and collected after being dried under vacuum pressure.
Example 4: MSN and CeNP Characterization
[0114] The porous properties of MSNs were measured using the Brunauer-Emmett-Teller (BET) method. The nanoparticle size and morphology were analyzed using transmission electron microscopy (JEM-2100F, JEOL, Akishima, Japan) and scanning electron microscopy (JSM-7000F, JEOL), respectively. Energy dispersive X-ray spectroscopy elemental mapping was conducted using a JEM-2100F field emission electron microscope (JEOL, Akishima, Japan). The concentration of cerium was measured using inductively coupled plasma-optical emission spectrometry (ICP-OES, Varian, CA, USA).
Example 5: Cell Counting Kit (CCK)-8 Cytotoxicity Assay
[0115] 10.sup.4 RAW264.7 cells (ATCC) were seeded per well in a 96-well plate and incubated for 24 h at 37° C. Then, various concentrations of MSNs (25, 50, 100, and 200 μg/mL) and cerium (5, 10, 20, 50, and 100 μg/mL) were incubated with the cells for the next 24 h. Finally, 10 μL of CCK-8 solution (Dojindo, Japan) was added to each well and incubated for 2 h at 37° C. Absorbance was measured at 450 and 600 nm using a microplate reader (Thermo Fisher Scientific, MA, USA). The cell viability was calculated according to the manufacturer's instructions.
Example 6: Preparation of Peptide-Loaded MSNs
[0116] Five hundred microliters of MOG.sub.35-55 peptide solution (1 μg/μL in DI water) and five hundred microliters OVA.sub.323-339 peptide solution (1 μg/μL in DI water) were separately mixed with 1 mg MSNs each, and incubated for 3 h at 25° C. Subsequently, the nanoparticles were washed in DI water three times under sterilized conditions, and the loading efficiency was measured using the Pierce BCA Protein Assay Kit (Thermo Fisher, MA, USA). The peptide-loaded nanoparticles were finally re-dispersed in saline buffer prior to retro-orbital injection using a BD insulin syringe (BD, NJ, USA). To prepare MSN-MOG-Ce, MOG.sub.35-55-loaded MSNs were mixed with 1 mg CeNPs (2 mg/mL) in DI water and gently shaken for 5 min. The mixture was then centrifuged (10,000×g, 5 min) and washed twice with DI water to remove unbound CeNPs. The loaded CeNPs were measured using the ultraviolet-visible method at 310 nm. Finally, the nano-composition was re-dispersed in saline buffer prior to retro-orbital injection using a BD Insulin Syringe.
Example 7: Animals
[0117] Female C57BL/6 mice aged 9 weeks were purchased from OrientBio (Seongnam, South Korea). The experimental and control animals were co-housed under specific pathogen-free condition during the study. Female OT-II (C57BL/6-Tg (TcraTcrb)425Cbn/Crl) mice of 7-9-week age were used for in vitro generation of Tregs. OT-II mice were a kind gift from Prof. Suk-Jo Kang from Korea Advanced Institute of Science and Technology (KAIST). Animals were acclimatized for at least 1 week before immunization. At the end of each study, mice were euthanized by carbon dioxide (flow rate: 3 L/min). All experiments were approved by the Institutional Animal Care and Use Committee of Sungkyunkwan University (SKKUIACUC, No. 2020-01-15-1).
Example 8: In Vivo Cellular Uptake in Spleen
[0118] Twenty-four hours prior to intravenous administration of RITC-MSN into C57BL/6 mice, splenocytes were isolated. The cells were stained with FcR blocking reagent for 10 min at 4° C. and washed. Subsequently, cells were stained with antibodies against CD11c, F4/80, B220, and CD3 for 20 min at 4° C. Positive signals from stained surface markers were gated among the RITC.sup.+ cells to determine the cell types that engulfed RITC-MSN.
Example 9: EAE Induction
[0119] EAE was induced in female C57BL/6 mice aged 10-11 weeks using a kit (EK-2110) from Hooke Laboratory (Lawrence, Mass., USA). Briefly, on day 0, mice were subcutaneously injected with an emulsion containing MOG.sub.35-55 peptide and complete Freund's adjuvant (CFA), in the lower and upper back. After 2 and 24 h, the animals were intraperitoneally injected with pertussis toxin (PTX) according to the manufacturer's instructions. EAE clinical score was evaluated after day 8, post-EAE induction based on the standard protocol in a blinded manner (0, no obvious symptoms; 0.5, tip of tail was limp; 1, limp tail; 1.5, limp tail and hind leg inhibition; 2, limp tail and weakness of hind legs; 2.5, limp tail and dragging of hind legs; 3, complete paralysis of hind legs; 3.5, complete paralysis of hind legs and hind legs together on one side of the body; 4, full hind leg and partial front leg paralysis; 4.5 full hind leg and partial front leg paralysis, no movement). Paralyzed mice were given easier access to water and food. Mice were euthanized if any of the following conditions were observed unable to eat, unresponsive when scored as 4, when scored as 4 for two consecutive days, both hind limbs and forelimbs were completely paralyzed.
Example 10: Therapeutic Vaccine Studies
[0120] For semi-therapeutic treatment (vaccination after disease establishment but before the onset of symptoms), the mice were injected with different formulations of material components (MSN, MOG, MSNMOG, MSN-OVA) on days 4, 7, and 10 after EAE induction or left untreated. FIG. 9 shows the dose for each formulation in detail. To examine the immune responses after semi-therapeutic treatment of MSN-OVA, MSN-MOG, or no treatment in EAE-induced mice, mice from each group were euthanized for spleen and spinal cord collection on day 10 after the final injection (the respective treatments were administered on days 4, 7, and 10 post-EAE induction). For the early therapeutic study, EAE-induced mice were injected with MSN-MOG or left untreated on days 12, 15, and 18. For the late therapeutic study, EAE-induced mice were injected with MSN-MOG or left untreated on days 15, 18, and 21.
Example 11: Histology
[0121] The vertebral columns were collected on day 50 post-EAE induction and fixed in 4% buffered formaldehyde solution for 48 h. The tissues were then washed in DI water before decalcification in an aqueous solution containing 4% formic acid and 4% hydrochloric acid for 72 h. The acid solution was replaced daily. Subsequently, the mouse spines were neutralized in an ammonia solution, washed in DI water, and embedded in paraffin. The tissues were cut into 4-μm thick sections. Finally, the spinal cord sections were stained with H&E, and an optical microscope (ECLIPSE Ti-U, Nikon, Japan) was used to visualize them.
Example 12: Tissue Processing
[0122] Spleens were excised and processed by mechanical disruption using a 70 μm cell strainer. Cells were then centrifuged for 5 min, 400×g, 4° C. and treated with ammonium-chloride-potassium (ACK) lysing buffer (Lonza) for 4 min to remove red blood cells. Splenocytes were then filtered through a 40 μm cell strainer and washed in cold PBS. Spinal cords were dissociated in PBS containing 1 mg/mL collagenase type IV and 20% EDTA/trypsin and incubated at 37° C. for 20 min. RPMI 1640 containing 10% fetal bovine serum was added to each sample to inhibit enzymatic activity, and the cells were filtered through a 40 μm cell strainer. The cells were collected by centrifugation according to standard protocol.
Example 13: Flow Cytometry
[0123] Immediately after stimulation or obtaining single-cell suspensions from tissues, cells were incubated with the FcR blocking reagent for 10 min at 4° C. to prevent non-specific binding. Then, antibodies against surface markers CD11c, B220, F4/80, CD86, CD40, MHC-II were used to stain for APC analysis. For T-cell analysis, I-Ab MOG.sub.35-55 tetramer was used to stain the cells for 40 min at 4° C., then antibodies against CD3 and CD4 were used to stain the cells for 20 min at 4° C. before being washed in FACS buffer. After that, the cells were either analyzed immediately or fixed and permeabilized for transcription factor staining. Foxp3/Transcription Factor Staining buffer set (eBioscience 00-5523-00, CA, USA) was used to fix and permeabilize the cells prior to staining with antibody against Foxp3. Suitable isotype control antibodies were used as the negative controls. For the analysis of lba1 expression in CNS cells, the cells were first fixed and permeabilized by Intracellular Fixation & Permeabilization buffer set (eBioscience 88-8824-00, CA, USA) prior to lba1 staining for 1 h. The stained cells were analyzed using a MACSQuant VYB flow cytometer (Miltenyi Biotec, Bergisch Gladbach, Germany).
[0124] All cells were gated based on forward-scatter and side-scatter characteristics to exclude dead cells and debris. Thereafter, the forward-scatter height (FSC-H) and forward-scatter area (FSC-A) parameters were used to determine the single-cell population. Finally, the frequency of positively stained cells for each marker was recorded based on the isotype control antibodies. Examples of the gating strategies are shown in FIG. 16. Data were analyzed using FlowJo X 10.0 (Becton, Dickinson and Company).
Example 14: Cytokine Recall Study
[0125] Half the mice from each group were euthanized for splenocyte collection and on day 3, and other half were euthanized for splenocyte collection on day 10 after the final injection. Then, splenocytes (1×10.sup.6) isolated from each mouse were restimulated with 20 μg/mL of MOG.sub.35-55 peptide for 72 h. The culture supernatant was collected and stored at −80° C. until use. The secreted cytokines IL-10, GM-CSF, TNF-α, and IL-17A were quantified by enzyme-linked immunosorbent assay (ELISA, R&D Systems, Minneapolis, Minn., USA) according to the manufacturer's instructions.
Example 15: BMDC Culture
[0126] Bone marrow cells from the femurs of C57BL/6 mice were isolated and filtered using a 70 μm cell strainer. The cells were then cultured in complete RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, 1% penicillin/streptomycin (Sigma-Aldrich), 50 μM β-mercaptoethanol (Sigma-Aldrich), and 20 ng/mL GM-CSF (PeproTech, NJ, USA). The culture medium was refreshed on days 3 and 6. Differentiated cells from days 7 to 9 were used for the cell activation study.
Example 16: CD4.SUP.+ .T Cell Isolation and Culture
[0127] Spleen and lymph nodes of OT-II mice were first processed to obtain single-cell suspensions. Purified CD4.sup.+ T cells were isolated by magnetic activated cell sorting (MACS) using CD4.sup.+ T cell isolation kit (Miltenyi Biotec, 130-104-454). OT-II CD4.sup.+ T cells were then washed and cultured with BMDCs in complete RMPI 1640 supplemented with 10% heat-inactivated fetal bovine serum (Gibco), 1% penicillin/streptomycin (Sigma-Aldrich), 50 μM β-mercaptoethanol (Sigma-Aldrich) at 37° C. in 5% CO.sub.2.
Example 17: Apoptosis Assay
[0128] BMDCs were seeded in 6-well culture plates (1×10.sup.6 cells/well). The cells were then co-incubated for 48 h with pegylated CeNPs at different cerium concentrations. Subsequently, the cells were washed with FACS buffer and stained using the FITC Annexin V Apoptosis Kit with PI (BioLegend, CA, USA) according to the manufacturer's instructions before performing flow cytometry analysis.
Example 18: BMDC Activation Study
[0129] BMDCs were seeded in 6-well culture plates (1×10.sup.6 cells/well). The cells were then treated with either pegylated CeNPs (Ce, 50 μM cerium), OVA.sub.323-339 peptide (OVA, 1 μg/m L), pegylated CeNPs and OVA.sub.323-339 peptide (Ce+OVA), MSN-MOG, MSN-MOG-Ce, or left untreated for 24 h, followed by stimulation with 1 μg/mL or 100 ng/mL LPS for the next 24 or 12 h. Finally, the cells were washed and stained with H.sub.2DCFDA and antibodies against CD11c, CD86, CD40, and MHC-II before flow cytometry analysis.
Example 19: T-Cell Differentiation In Vitro
[0130] Day 7 BMDCs were treated with PBS (control), pegylated CeNPs (Ce, 50 μM cerium), OVA.sub.323-339 peptide (OVA, 1 μg/mL), or pegylated CeNPs plus OVA (Ce+OVA) in 24 h, following by LPS treatment (1 μg/mL) in the next 24 h. Then BMDCs were washed twice and co-cultured with OT-II CD4.sup.+ T cells at 1:10 (30,000:300,000) BMDCs to T-cell ratio for 72 h. Subsequently, the cells were stimulated in a culture medium containing PMA (100 ng/mL), ionomycin (1 μg/mL) for 5 h and protein transport inhibitor (GolgiStop, BD Bioscience) in the last 3 h. Finally, cells were fixed, permeabilized, and stained with antibodies against Vα2, CD4, CD25, Foxp3, IL-10, IFN-γ, IL-17A before flow cytometry analysis.
Example 20: ROS-Scavenging Study
[0131] BMDCs were seeded in 6-well culture plates (1×10.sup.6 cells/well). The cells were then treated with either MSN-MOG or MSN-MOG-Ce, or left untreated for 24 h, followed by stimulation with 100 ng/mL LPS to induce excessive intracellular ROS for the next 12 h. Subsequently, the cells were stained with 5 μM H.sub.2DCFDA and CD11c antibody for 20 min at 4° C. before being washed and analyzed by flow cytometry.
Example 21: Statistics and Reproducibility
[0132] EAE clinical score values were expressed as the mean±standard error (SE). All other values are expressed as mean±standard deviation (SD) unless indicated otherwise. An unpaired two-tailed t-test was performed to compare the statistical significance between the two groups. For multiple comparisons, one-way ANOVA was performed using GraphPad Prism 7.00.
[0133] Results
[0134] 1. Myelin Oligodendrocyte Glycoprotein (MOG)-Loaded MSNs Suppresses EAE Development in Semi-Therapeutic Study
[0135] First, MSNs with 10-30-nm large mesopores along with 3-nm conventional mesopores were synthesized according to previous reports (Kwon, D. et al. Extra-large pore mesoporous silica nanoparticles for directing in vivo M2 macrophage polarization by delivering IL-4. Nano Lett. 17,2747-2756 (2017)., Nguyen, T. L., Cha, B. G., Choi, Y., Im, J. & Kim, J. Injectable dual-scale mesoporous silica cancer vaccine enabling efficient delivery of antigen/adjuvant-loaded nanoparticles to dendritic cells recruited in local macroporous scaffold. Biomaterials 239, 119859 (2020).) (FIG. 2a, FIG. 7). Before the in vivo tests, the cytotoxicity of various concentrations of MSNs was examined, showing that the MSNs were highly biocompatible (FIG. 1d). The biodegradability of MSNs has been confirmed in phosphate-buffered saline (PBS) at physiological pH (FIG. 8) and under physiological conditions of lysosomes (Croissant, J. G., Fatieiev, Y. & Khashab, N. M. Degradability and clearance of silicon, organosilica, silsesquioxane, silica mixed oxide, and mesoporous silica nanoparticles. Adv. Mater. 29, 1604634 (2017).). In line with previous studies (Kwon, D. et al. Extra-large pore mesoporous silica nanoparticles for directing in vivo M2 macrophage polarization by delivering IL-4. Nano Lett. 17,2747-2756 (2017)., Bindini, E. et al. Following in situ the degradation of mesoporous silica in biorelevant conditions: at last, a good comprehension of the structure influence. ACS Appl. Mater. Interfaces 12, 13598-13612 (2020).), the present inventors demonstrated that the systemic administration of MSNs led to the accumulation of nanoparticles in the spleen (FIG. 2b), which may be attributed to the intrinsic capacity of the mononuclear phagocytic system to massively capture foreign nanoparticles. APCs, including CD11c.sup.+ dendritic cells (DCs), F4/80.sup.+ macrophages, and B220.sup.+ B cells were the major cell populations that engulfed MSNs in the spleen (FIG. 2c). Owing to their large pore size, MSNs used in this study exhibited high antigen-loading capacity; thus, large-pore MSNs could carry antigen (MOG.sub.35-55 peptide), equivalent to that used in previous studies (Hunter, Z. et al. A biodegradable nanoparticle platform for the induction of antigen-specific immune tolerance for treatment of autoimmune disease. ACS Nano 8, 2148-2160 (2014)., Cho, J. J. et al. An antigen-specific semi-therapeutic treatment with local delivery of tolerogenic factors through a dual-sized microparticle system blocks experimental autoimmune encephalomyelitis. Biomaterials, 79-92 (2017). and Saito, E. et al. Design of biodegradable nanoparticles to modulate phenotypes of antigen-presenting cells for antigen-specific treatment of autoimmune disease. Biomaterials, 119432 (2019).) at a substantially lower dose (FIG. 2d and FIG. 9).
[0136] The present inventors tested the hypothesis of the present disclosure in EAE-induced C57BL/6 mice (FIG. 2e). Intravenous injection of bare MSNs into EAE-induced mice prior to disease onset (days 4, 7, and 10; semi-therapeutics) was unable to prevent typical EAE progression (FIG. 2f). Injecting soluble MOG slightly delayed EAE progression, whereas MOG-loaded MSNs (MSNMOG) markedly impeded EAE development, resulting in a high percentage of healthy animals (FIG. 2g). These results indicate that loading antigens on MSNs is critical for suppressing EAE development. Conversely, the OVA.sub.323-339 peptide (unrelated peptide antigen from ovalbumin)-loaded MSNs (MSN-OVA) did not suppress EAE development, indicating that the reduction in disease severity induced by MSN-MOG was antigen-specific. Meanwhile, the increase in nanoparticle amount when maintaining MOG peptide dose was unable to show comparable benefits (FIG. 10).
[0137] 2. MOG-Loaded MSN Vaccine Generates Induced Tregs and Antigen-Specific Tolerogenic Immune Responses
[0138] The present inventors evaluated cellular responses in EAE-induced mice after MSN-MOG injection to gain a better insight into the mechanism underlying immune tolerance. Phenotypic alterations in CD11c.sup.+ DCs, F4/80.sup.+ macrophages, and B220.sup.+ B cells in the spleen, were examined by flow cytometry after vaccination (FIG. 3a). CD86 expressions on F4/80.sup.+ macrophages were significantly lower in the MSN-MOG group than those in the untreated group (FIG. 3b). No significant difference in the activation of MHC class II (MHC-II) on APCs between the three groups was observed (FIG. 3c); indicating that antigen presentation by MHC-II was not affected by immunization. The number of APCs increased (FIG. 11), while the expression of activation markers on APCs did not increase significantly. These data suggest that systemic administration of peptide antigen (MOG)-loaded MSNs did not promote APC maturation in the lymphoid organ in EAE mice, which is necessary for the polarization of naive T cells into Tregs and/or anergic T cells.
[0139] Given the inability of MSN-MOG to activate splenic APCs in EAE mice, the present inventors further examined whether MSN-MOG administration could induce Foxp3.sup.+ Tregs. Following treatment with peptide-loaded MSNs, the present inventors observed a decrease in the percentage of splenic CD4.sup.+ T cells on day 20 (FIG. 3d). In contrast, there were noticeable increases of both frequencies and numbers of Foxp3.sup.+ Tregs in mice vaccinated with MSN-MOG (FIG. 3e, FIG. 3f and FIG. 3g). These observations suggest that the systemic delivery of self-antigen (MOG)-loaded MSNs modified the cellular composition of the spleen into more tolerogenic T cell population in the EAE mice, which is a desirable change to treat EAE.
[0140] The present inventors next evaluated the responsiveness of immune cells retrieved from spleen upon re-stimulation with EAE-associated antigens (MOG) at different time points (days 13 and 20 after EAE induction, corresponding to days 3 and 10 after the last semi-therapeutic intervention) to confirm whether an antigen-specific tolerogenic environment was established. Interleukin 17A (IL-17A) and tumor necrosis factor-α (TNF-α), the central mediators of EAE and MS progression, were significantly lower in the MSN-MOG-treated group than in the untreated group (FIG. 3h and FIG. 3i). Similarly, granulocyte-macrophage colony-stimulating factor (GM-CSF) production, a key recruitment factor that attracts APCs and pathogenic monocyte-derived cells to the CNS31, was significantly inhibited in the MSN-MOG-treated group (FIG. 3j). The present inventors detected IL-10 in the MSN-MOG-injected group, but not in the untreated or MSN-OVA groups (FIG. 3k). IL-10 is a potent anti-inflammatory cytokine that plays a vital role in hampering immune responses against self-antigens.
[0141] To assess immune tolerance at the disease site, the present inventors characterized the disease-associated immune cells in the CNS of EAE-induced mice intravenously injected with MSN-MOG, MSN-OVA, or left untreated. Frequencies (FIG. 3l and FIG. 3m) and numbers (FIG. 3n) of CNS-infiltrating APCs decreased in the MSN-MOG-treated group. The percentage (FIG. 3o) and the number (FIG. 12) of CD11c.sup.+MHC-II.sup.+, F4/80.sup.+MHC-II.sup.+, and B220.sup.+MHC-II.sup.+ cells in the CNS decreased in MSN-MOG-injected mice. CD11c.sup.+ DCs expressing MHC-II could reactivate primed T cells and initiate EAE as DCs within inflamed CNS encounter myelin epitopes and present them to the infiltrated T cells. The resulting self-reactive T cells contribute to epitope spreading in the CNS. A decrease in the frequency and number of APCs in the CNS after MSN-MOG treatment might reduce T-cell responses in the CNS as there would be less antigen processing by APCs. Since the induced Tregs in secondary lymphoid organs (FIG. 3e, FIG. 3f and FIG. 3g) suppress a generation of autoreactive T cells, the number of autoreactive T cells migrating into the CNS may be decreased. Consistently, CD4.sup.+ T-cell infiltration in the CNS was significantly inhibited in the MSN-MOG group (FIG. 3p).
[0142] Moreover, the expression of ionized calcium-binding adapter molecule 1 (lba1), a marker of macrophage and microglia, in the cells retrieved from CNS was also notably suppressed by MSN-MOG vaccination (FIG. 3q and FIG. 14), presenting that the MSN-MOG vaccination could suppress the pathological cellular responses of multiple sclerosis.
[0143] As the present inventors have observed the increase of Tregs after vaccination in EAE mice (FIG. 3e-3g), the present inventors further investigated whether the depletion of Tregs via anti-Treg antibody administration over the vaccination process would inhibit the therapeutic efficacy of MSN-MOG vaccine. The results showed that the depletion of Tregs in MSN-MOG-vaccinated mice diminished the therapeutic effect of MSN-MOG vaccine, and thus could not prevent EAE development (FIG. 3s). In addition, the cellular analysis revealed that depletion of Tregs led to the infiltration of CD11c.sup.+ cells, F4/80.sup.+ cells, and CD4.sup.+ T cells into the CNS (FIG. 3t, FIG. 3u and FIG. 13).
[0144] Taken together, these data (FIG. 3a-31) demonstrate the potency of self-antigen-loaded MSNs in inducing systemic antigen-specific tolerance via Treg generation and reduction of Th1 and Th17-biased inflammatory cytokine secretion.
[0145] 4. MSN-MOG Vaccine Reduces Disease Severity Following Therapeutic Intervention at the Late Stage of the Disease
[0146] Although the present inventors have shown that MSN-MOG could suppress the development of EAE in semi-therapeutic study (FIG. 2f and FIG. 2g), MS in human is initiated before clinical symptoms appear, leading to the high demand for therapeutics to treat MS after the clinical diagnosis. Next, the present inventors investigated whether MSN-MOG suppressed EAE during the onset and chronic phases. EAE-induced mice received three intravenous MSNMOG injections at 3 days intervals, starting on day 12 (the point of intermediate disease severity, early therapeutics) or day 15 (the peak of disease severity, late therapeutics) (FIG. 4a). The results showed that for both treatment regimens, progression of clinical episodes was strongly hampered immediately after the first injection (FIG. 4b), leading to body weight recovery of the mice (FIG. 4c). In both treated groups, complete paralysis disappeared three days after the first injection (FIG. 4d). For example, mice completely paralyzed on day 15 after EAE induction were able to walk on day 22 after vaccinations on days 15, 18, and 21 (FIG. 4e). These data reveal that MSN-MOG mediated functional recovery from neuroinflammation in EAE.
[0147] As the clinical scores remained stable after three shots in both early and late therapeutic studies (FIG. 4b), the present inventors examined whether motor impairment could be further ameliorated by providing additional vaccinations on days 28 and 35, to all mice except the untreated group. EAE severity in the early and late therapeutic groups reduced strongly after two more shots (FIG. 4f). Especially, in late therapeutics group, the clinical score became significantly lower than in the untreated group, indicating the significance of therapeutic efficacy of nanovaccine to treat the late chronic stage of EAE. At the end of the study, the body weights of mice had recovered completely, and there were no differences between the early and late therapeutic groups (FIG. 4g). Representative hematoxylin and eosin (H&E)-stained histological images revealed fewer inflammatory cells in the ventral area of the white matter of MSN-MOG-treated mice in both early and late therapeutics groups than that of the untreated mice, indicating that the vaccination induced an improvement of neuro-immune homeostasis comparable to the healthy mice (naive mice) (FIG. 4h).
[0148] 5. ROS-Scavenging CeNPs Suppresses Activation of APCs and Induces Tolerogenic APCs
[0149] Since oxidative stress derived from high intracellular ROS is known to activate APC, especially in MS, the present inventors hypothesized that scavenging intracellular ROS in APCs would further suppress their activation and potentially enhance the tolerogenic phenotype of APCs (FIG. 5a). The ceria nanoparticles (CeNPs) were recently engineered for the treatment of ROS-associated diseases, due to their ROS-scavenging catalytic properties that mimic catalase and superoxide dismutase; however, the immunological impact of these nanoparticles has not been investigated intensively. Water-dispersible, positively charged CeNPs (4 nm) were synthesized according to our previous reports (Jeong, H. G. et al. Ceria Nanoparticles Fabricated with 6-Aminohexanoic Acid that Overcome Systemic Inflammatory Response Syndrome. Adv. Healthc. Mater. 8, 1-10 (2019)) (FIG. 5b and FIG. 14). To improve the dispersion of CeNPs in the cell culture medium, polyethylene glycol (PEG) was coated on the CeNPs (FIG. 5c and FIG. 14). The resulting CeNPs were highly biocompatible (FIG. 5d) and were directly used to examine the capacity to suppress the activation of bone marrow-derived dendritic cells (BMDCs). BMDCs were cultured with CeNPs and treated with lipopolysaccharides (LPS), a TLR4 agonist known to induce excessive intracellular ROS production and enhance activation of APCs, resulting in a substantially decreased expression levels of costimulatory molecules (CD86 and CD40) and MHC-II (FIG. 5e, FIG. 5f and FIG. 5g). These data demonstrate the ROS-scavenging CeNPs could prevent activation of DCs and maintain the tolerogenic phenotype of DCs even under the presence of immune-activating TLR4 agonist.
[0150] The present inventors next evaluated whether the OVA.sub.323-339 peptide-experienced semi-mature DCs induced by CeNP could trigger the CD4.sup.+ T-cell differentiation to Tregs by co-culturing these DCs with CD4.sup.+ T cells retrieved from OT-II transgenic mice (FIG. 5h). The CD4.sup.+ T cells in OT-II mice have T-cell receptors that specifically recognize OVA.sub.323-339 peptide. A significantly higher percentage of CD25.sup.+Foxp3.sup.+ Tregs appeared in the CeNP+OVA-treated group than the others (FIG. 5i and FIG. 15). Interestingly, the elevated population of IL-10 secreting CD4.sup.+ T cells, known as type 1 regulatory T cells (Tr1) that inhibit DC maturation and establish T-cell tolerance, was observed in CeNP-treated group and the CeNP+OVA-treated group (FIG. 5j and FIG. 15). In contrast, the IFN-γ secreting CD4.sup.+ T cells (activated Th1 cells) was significantly suppressed in CeNP+OVA-treated group compared to OVA-treated one (FIG. 5k and FIG. 15). Although there was no difference in IL-17A secretion within CD4.sup.+ T cells between the OVA-treated group and the CeNP+OVA-treated group (FIG. 5l and FIG. 15), the ratios of CD25.sup.+Foxp3/IL-17A.sup.+ and IL-10/IL-17A.sup.+ in CeNP+OVA-treated group were highest among groups (FIG. 5m and FIG. 5n). In addition, the bias of naive CD4.sup.+ T cells into Tregs and Tr1 rather than Th1 by CeNPs was demonstrated by the greater ratios of CD25.sup.+Foxp3.sup.+/IFN-γ.sup.+ and IL-10.sup.+/IFN-γ.sup.+ in the CeNP+OVA-treated group, respectively (FIG. 5o and FIG. 5p). Taken together, these data show that CeNPs could induce tolerogenic DCs which can skew naive CD4.sup.+ T cells toward Tregs and Tr1 in vitro, representing that CeNPs could be used as an immunosuppressive agent to enhance immune tolerance.
[0151] 6. ROS-Scavenging MSN-MOG Vaccine Enhances the Therapeutic Efficacy in the Late Chronic Phase of EAE
[0152] To enhance the therapeutic efficacy of autoimmune disease nanovaccine, CeNPs were additionally attached on MSN-MOG via electrostatic interactions between the negatively charged MSN-MOG and positively charged CeNPs (FIG. 16), resulting in the ROS-scavenging MSN-MOG-Ce nanovaccine (FIG. 6a). The ability of MSN-MOG-Ce to scavenge intracellular ROS in BMDCs was examined in the presence of LPS. The level of intracellular ROS was significantly lower in MSN-MOG-Ce-treated BMDCs than in LPS- and MSNMOG-treated BMDCs (FIG. 6b), revealing that MSN-MOG-Ce attenuated oxidative stress in BMDCs under inflammatory conditions. Consistently, BMDCs treated with MSN-MOG-Ce expressed lower levels of CD86 and MHC-II than LPS-treated cells; MSN-MOG had no impact on the BMDC activation markers (FIG. 6c and FIG. 17). MSN-MOG-Ce could prevent oxidative stress in BMDCs and maintain their semi-maturity under an inflammatory environment, representing its potential to further aid DCs in enhancing peripheral tolerance in vivo.
[0153] Next, to investigate the therapeutic efficacy of ROS-scavenging vaccine, EAE-induced mice in the late chronic phase were intravenously injected with MSN-MOG, MSN-MOG-Ce, or left untreated. The present inventors observed an additional reduction in the clinical score for MSN-MOG-Ce-injected EAE mice compared to that for MSN-MOG-injected EAE mice (FIG. 6d, see FIG. 18 for weight change). After the late therapeutic treatment (day 22), MSN-MOG-Ce-treated EAE mice could take complete coordinated strides, unlike the visibly irregular/wobble walk of MSN-MOG-treated mice.
[0154] To further investigate the immunosuppressive role of ROS-scavenging CeNPs in the therapeutic autoimmune disease nanovaccine, the present inventors compared the cellular phenotypes and responses in mice with late EAE therapeutics after MSN-MOG and MSN-MOG-Ce administration. The frequency and number of splenic APCs of the treated mice showed no significant change compared to the untreated group, except for an increase in the frequency of B220.sup.+ cells in the MSN-MOG-treated group (FIG. 19). CD86 expressions on CD11c.sup.+ DCs, F4/80.sup.+ macrophages, and B220.sup.+ cells were significantly lower in the MSN-MOG-Ce-treated group than in the MSN-MOG-treated group (FIG. 6e). Similarly, CD40 expression on CD11c.sup.+ DCs and F4/80.sup.+ macrophages was significantly reduced in the MSN-MOG-Ce-treated group but not in the MSN-MOG-treated group (FIG. 6f). These results are consistent with the suppressive effects of CeNPs in vitro. There was no difference in MHC-II expression on APCs between groups (Supplementary FIG. 20), indicating that loading CeNPs on the nanovaccine only inhibited the expression of costimulatory molecules (CD86 and CD40) without affecting MHC-II, thus making the APCs more tolerogenic. Consequently, the MSN-MOG-Ce-treated group exhibited higher Foxp3.sup.+ Tregs in the spleens of EAE-induced mice (FIG. 6g and FIG. 6h). The percentage of Foxp3.sup.+ T cells among the CD4.sup.+ T cells was twice as high in MSN-MOG-Ce-treated mice (day 31) compared to that in untreated mice. The frequency and number of CD4.sup.+ T cells were similar among the three groups (FIG. 21). These results indicate that coating MSN-MOG with CeNPs did not elicit APC and helper T-cell proliferation in the spleen but drove the induction of Foxp3.sup.+ Tregs via the immunosuppressive effects on APCs.
[0155] The infiltrated CD4.sup.+ T cells in CNS is one of the indications to show severity of autoimmune response in EAE. The present inventors further examined the infiltration of autoreactive CD4.sup.+ T cells into the CNS after vaccination. The numbers and percentages of CD4.sup.+ T cells in the CNS in both MSN-MOG-treated mice and MSN-MOG-Ce-treated mice were significantly lower compared to the untreated group (FIG. 6i, FIG. 6j, FIG. 6k). Importantly, the number of MOG-specific CD4.sup.+ T cells in CNS, which is the main cause of demyelination, was strongly reduced in MSNMOG-Ce-vaccinated mice (FIG. 6l). The enhanced peripheral tolerance induced by MSN-MOG-Ce resulted in the inhibition of CNS-infiltrating MOG-specific CD4.sup.+ T cells and ameliorated disease severity in the late stage (FIG. 6d). Interestingly, the frequencies of CD11c.sup.+ and B220.sup.+ cells in the CNS were greatly suppressed by vaccination (FIG. 6m and FIG. 22). A diminishment in MHC-II expression on APCs was more significant in the MSN-MOG-Ce-treated group than in MSN-MOG counterpart (FIG. 6n and FIG. 22), probably owing to the inhibition of CNS-infiltrated autoreactive CD4.sup.+ T cells. Consequently, the present inventors observed a reduction in CD4.sup.+ T-cell frequencies in the cervical lymph nodes of the MSN-MOG-Ce-treated group at the late phase of MS (FIG. 6o and FIG. 23). Taken together, the introduction of ROS-scavenging nanovaccine did not affect the proliferation of splenic APCs but suppressed their costimulatory signals, which prevents activation of APCs and induces antigen-presenting tolerogenic APCs.
[0156] As a result, a higher frequency of peripheral Tregs could be generated in peripheral lymphoid organ and sequentially inhibit the infiltration of autoreactive CD4.sup.+ T cells into CNS, which led to suppression of ongoing chronic phase MS (FIG. 1).
