key: cord-0712944-2j2h0btf
authors: Matsoukas, John M.; Ligielli, Irene; Chasapis, Christos T.; Kelaidonis, Konstantinos; Apostolopoulos, Vasso; Mavromoustakos, Thomas
title: Novel Approaches in the Immunotherapy of Multiple Sclerosis: Cyclization of Myelin Epitope Peptides and Conjugation with Mannan
date: 2021-11-29
journal: Brain Sci
DOI: 10.3390/brainsci11121583
sha: e78aa55907dd3303ef4b04ebcd31fbbc2035740b
doc_id: 712944
cord_uid: 2j2h0btf

Multiple Sclerosis (MS) is a serious autoimmune disease. The patient in an advanced state of the disease has restrained mobility and remains handicapped. It is therefore understandable that there is a great need for novel drugs and vaccines for the treatment of MS. Herein we summarise two major approaches applied for the treatment of the disease using peptide molecules alone or conjugated with mannan. The first approach focuses on selective myelin epitope peptide or peptide mimetic therapy alone or conjugated with mannan, and the second on immune-therapy by preventing or controlling disease through the release of appropriate cytokines. In both approaches the use of cyclic peptides offers the advantage of increased stability from proteolytic enzymes. In these approaches, the synthesis of myelin epitope peptides conjugated to mannan is of particular interest as this was found to protect mice against experimental autoimmune encephalomyelitis, an animal model of MS, in prophylactic and therapeutic protocols. Protection was peptide-specific and associated with reduced antigen-specific T cell proliferation. The aim of the studies of these peptide epitope analogs is to understand their molecular basis of interactions with human autoimmune T-cell receptor and a MS-associated human leucocyte antigen (HLA)-DR2b. This knowledge will lead the rational design to new beneficial non-peptide mimetic analogs for the treatment of MS. Some issues of the use of nanotechnology will also be addressed as a future trend to tackle the disease. We highlight novel immunomodulation and vaccine-based research against MS based on myelin epitope peptides and strategies developed in our laboratories.

Multiple Sclerosis (MS) is a serious systemic demyelinating disease that leads to the partial paralysis of the patient who needs the assistance of medicare in order to survive with low quality of life. The need for an effective treatment in the form of medication or vaccine is more urgent than ever before [1] [2] [3] [4] [5] . This chronic inflammatory and neurodegenerative disease is initiated by autoreactive T helper (Th) cells and affects approximately 2.5 million people worldwide. Thus, there is an urgent need to develop effective treatments. Advances in the immunotherapy of MS have been recently reported in excellent reviews [6] [7] [8] [9] . The pathogenesis of MS has been extensively studied over the last years, which shows a complex immunological involvement. Myelin epitopes have been identified as a target for autoimmune CD4+ T cells and antibodies, and much focus has been around the modulation of Th1 pro-inflammatory autoreactive CD4+ T cells against myelin epitopes, namely myelin basic protein (MBP), proteolipid protein (PLP), and myelin oligodendrocyte glycoprotein (MOG) [10] [11] [12] [13] [14] [15] [16] [17] [18] .

Herein, we summarize the efforts utilized to develop novel approaches to manage MS, which are primarily efforts of immune modulation via the synthesis of selective peptide epitopes of MBP, PLP, and MOG with or without conjugation to mannan. In addition, we provide the efforts applied for rational design to synthesize non-peptide mimetic analogues, as well as some nanotechnology approaches. We review evidence on how the knowledge of the molecular basis of interactions of the studied peptide analogues in complex with the major histocompatibility (MHC) class II molecule (HLA-DR2b) and its interaction with the T-cell receptor (TCR) can lead to the rational design of new therapeutic non-peptide mimetic analogs for the treatment of MS. The utilized strategies to approach the disease are ( Figure 1 ):

(a) Use linear MBP, PLP, and MOG epitopes with or without amino acid mutations to gain insights into the molecular basis of the disease. This information is aided by nuclear magnetic resonance (NMR) studies and structural information of the epitope ligand bound to the MHC [19] ; (b) Perform cyclization of these epitopes to increase their stability [20] [21] [22] [23] [24] [25] [26] ; (c) Conjugate selective epitopes with mannan [27] [28] [29] [30] ; (d) Synthesis of non-peptide mimetic analogs to decrease their flexibility and increase their therapeutic index; (e) Apply nanotechnology as a means of antigen delivery [31] [32] [33] [34] [35] [36] [37] [38] [39] Brain Sci. 2021, 11, x FOR PEER REVIEW 2 of 15 degenerative disease is initiated by autoreactive T helper (Th) cells and affects approximately 2.5 million people worldwide. Thus, there is an urgent need to develop effective treatments. Advances in the immunotherapy of MS have been recently reported in excellent reviews [6] [7] [8] [9] . The pathogenesis of MS has been extensively studied over the last years, which shows a complex immunological involvement. Myelin epitopes have been identified as a target for autoimmune CD4+ T cells and antibodies, and much focus has been around the modulation of Th1 pro-inflammatory autoreactive CD4+ T cells against myelin epitopes, namely myelin basic protein (MBP), proteolipid protein (PLP), and myelin oligodendrocyte glycoprotein (MOG) [10] [11] [12] [13] [14] [15] [16] [17] [18] .

Herein, we summarize the efforts utilized to develop novel approaches to manage MS, which are primarily efforts of immune modulation via the synthesis of selective peptide epitopes of MBP, PLP, and MOG with or without conjugation to mannan. In addition, we provide the efforts applied for rational design to synthesize non-peptide mimetic analogues, as well as some nanotechnology approaches. We review evidence on how the knowledge of the molecular basis of interactions of the studied peptide analogues in complex with the major histocompatibility (MHC) class II molecule (HLA-DR2b) and its interaction with the T-cell receptor (TCR) can lead to the rational design of new therapeutic non-peptide mimetic analogs for the treatment of MS. The utilized strategies to approach the disease are ( Figure 1 ): a) Use linear MBP, PLP, and MOG epitopes with or without amino acid mutations to gain insights into the molecular basis of the disease. This information is aided by nuclear magnetic resonance (NMR) studies and structural information of the epitope ligand bound to the MHC [19] ; b) Perform cyclization of these epitopes to increase their stability [20] [21] [22] [23] [24] [25] [26] ; c) Conjugate selective epitopes with mannan [27] [28] [29] [30] ; d) Synthesis of non-peptide mimetic analogs to decrease their flexibility and increase their therapeutic index; e) Apply nanotechnology as a means of antigen delivery [31] [32] [33] [34] [35] [36] [37] [38] [39] 

Linear myelin epitopes MBP83-99, MBP82-98, MBP85-99, MBP87-99 (Figures 2 and 3) , MOG35-55, and PLP139-151 have been identified as agonist peptides inducing disease in humans and in animal models of MS. These peptides bind to MHC class II alleles, however, peptides binding to MHC class I have also been identified, primarily HLA-A*0301 (HLA-A3) in complex with a PLP45-53 peptide and the crystal structure known (Figure 4 ). Mutant analogs of linear agonist MHC class II peptides have been used to obtain information on the molecular basis of the disease. Data points out that mutant analogs of Figure 1 . Immune modulation strategies used to tackle MS.

Linear myelin epitopes MBP [83] [84] [85] [86] [87] [88] [89] [90] [91] [92] [93] [94] [95] [96] [97] [98] [99] , MBP 82-98 , MBP [85] [86] [87] [88] [89] [90] [91] [92] [93] [94] [95] [96] [97] [98] [99] , MBP [87] [88] [89] [90] [91] [92] [93] [94] [95] [96] [97] [98] [99] (Figures 2 and 3) , MOG , and PLP 139-151 have been identified as agonist peptides inducing disease in humans and in animal models of MS. These peptides bind to MHC class II alleles, however, peptides binding to MHC class I have also been identified, primarily HLA-A*0301 (HLA-A3) in complex with a PLP [45] [46] [47] [48] [49] [50] [51] [52] [53] peptide and the crystal structure known (Figure 4 ). Mutant analogs of linear agonist MHC class II peptides have been used to obtain information on the molecular basis of the disease. Data points out that mutant analogs of diseaseassociated epitopes can inhibit disease through two distinct mechanisms, one via the activation of antigen-specific regulatory T cells, or two, by activation and secretion of appropriate cytokines. The application of MBP 83-99 -based altered peptide ligands inhibits MBP-reactive T cell proliferation in vitro [30] ; this is attributed to anti-inflammatory Th2 cytokine secretion by T cells, primarily IL-4 and IL-10. These obtained results point out that cytokine regulation is the major mechanism through which T-cell receptor (TCR)-specific CD4+ T cells regulate encephalitogenic and potentially other bystander Th1 cells. Thus, the modulation of cytokine secretion by auto-reactive T cells through peptide or non-peptide mimetics, even in longstanding autoimmune disease through cytokine therapy, might be beneficial therapeutically. This beneficial response is achieved by switching the function of myelin reactive T cells.

disease-associated epitopes can inhibit disease through two distinct mechanisms, one via the activation of antigen-specific regulatory T cells, or two, by activation and secretion of appropriate cytokines. The application of MBP83-99-based altered peptide ligands inhibits MBP-reactive T cell proliferation in vitro [30] ; this is attributed to anti-inflammatory Th2 cytokine secretion by T cells, primarily IL-4 and IL-10. These obtained results point out that cytokine regulation is the major mechanism through which T-cell receptor (TCR)-specific CD4 + T cells regulate encephalitogenic and potentially other bystander Th1 cells. Thus, the modulation of cytokine secretion by auto-reactive T cells through peptide or non-peptide mimetics, even in longstanding autoimmune disease through cytokine therapy, might be beneficial therapeutically. This beneficial response is achieved by switching the function of myelin reactive T cells. One of the first human clinical studies for patients with secondary progressive MS (MAESTRO-01) used the agonist MBP82-98 peptide (dirucotide). Intravenous injection of MBP82-98 peptide delayed disease progression. However, this effort was suspended at phase III stage since it lacked efficacy and did not meet primary endpoints [40, 41] .

Two mutant peptides namely [R 91 , A 96 ]MBP87-99 and [A 91 , A 96 ]MBP87-99 derived from the immunodominant agonist identified in MS (MBP87-99) were synthesized. The chosen mutations occurred at amino acids K 91 and P 96 as they are critical TCR contact sites. Immunization of mice with these altered peptide ligands (ALPs) emulsified in complete One of the first human clinical studies for patients with secondary progressive MS (MAESTRO-01) used the agonist MBP 82-98 peptide (dirucotide). Intravenous injection of MBP 82-98 peptide delayed disease progression. However, this effort was suspended at phase III stage since it lacked efficacy and did not meet primary endpoints [40, 41] .

Two [87] [88] [89] [90] [91] [92] [93] [94] [95] [96] [97] [98] [99] ) were synthesized. The chosen mutations occurred at amino acids K 91 and P 96 as they are critical TCR contact sites. Immunization of mice with these altered peptide ligands (ALPs) emulsified in complete Freund's adjuvant induced both IFNγ and interleukin-4 (IL-4) responses while the native MBP 87-99 peptide induced only IFNγ responses. The linear MBP 72-85 peptide (EKSERSEDENPV) is well known to induce experimental autoimmune encephalitis (EAE), and D 79 mutation with A 79 resulted in an analog that suppressed the induction of EAE [22] . In addition, the immunodominant peptide from PLP, HSLGKWLGHPDKF, is a naturally processed epitope. A double mutation of the agonist PLP 139-151 , in which both of the TCR binding sites are replaced with Leu or Arg ([L 144 , R 147 ]PLP 139-151 ), is able to antagonize PLP-specific T cell clones in vitro [42] . The mutated analog inhibited EAE and prevented clinical disease progression when administrated in the early stage of EAE induction [42] . Antibodies against the minor protein MOG have been noted in inflammation areas of MS. This proves that antibodies do play a role in MS and cooperate with antigen-presenting cells in myelin destruction. Blocking the effects of these MOG antibodies with secondary antibodies or non-peptide mimetics might be an important avenue for future therapy.

Linear peptide are known to be sensitive to proteolytic enzymes, which results in their degradation. Cyclization of linear peptides increases their stability in vitro and in vivo [43] .

In an attempt to develop non-peptide mimetics, cyclic peptides are an important intermediate step towards this. As such, cyclic counterparts of linear peptides have been synthesized in an effort to improve their biological properties and structural stability [20] [21] [22] [23] [24] [25] [26] . Cyclic MBP 82-98 exerts strong binding to the HLA-DR2 allele but has lower affinity binding to the HLA-DR4 allele. Cyclic analogues of dirucotide proved to be promising leads, and it is Brain Sci. 2021, 11, 1583 5 of 14 proposed that they be evaluated for their ability to alter T cell responses for therapeutic benefit against MS [40, 41] .

Spectroscopic data combined with Molecular Dynamics (MD) calculations showed that the linear MBP 72-85 peptide adopts a pseudo-cyclic conformation. Based on this information, the cyclic analogue QKSQRSQDQNPV-NH 2 was rationally designed. The cyclization of this molecule was achieved by connecting the side-chain amino and carboxyl groups of Lys and Glu at positions 2 and 9. This cyclic analogue exerted similar biological activity to the linear peptide; however, in EAE experiments, the cyclic analogue completely suppressed EAE by co-injection with the agonist peptide in Lewis rats. The similar potencies propose that cyclization does not substantially affect the conformational properties of its linear analogue and provides support to its proposed pseudo-cyclic conformation. In addition, this study proposes that a pseudo cyclic conformation for the MBP 72-85 epitope allows D 81 and K 78 binding to the trimolecular complex MHC-peptide-TCR, and as a consequence, it inhibits EAE [23] .

A cyclic analogue, cyclo(87-99)MBP 87-99 ( Figure 5 ), of the human immunodominant MBP 87-99 epitope, was synthesized based on the same rational as MBP 72-85 epitope. This cyclic analogue in the same manner was shown to mimic the effects of the linear MBP 87 [87] [88] [89] [90] [91] [92] [93] [94] [95] [96] [97] [98] [99] , suppressed, to a varying degree, EAE, and possessed the following immunomodulatory properties: (i) they suppressed the proliferation of a CD4 T-cell line raised from a MS patient; (ii) they scored the best in vitro Th2/Th1 cytokine ratio in peripheral blood mononuclear cell (PBMC) cultures, inducing IL-10 selectively; (iii) they bound to HLA-DR4, first to be reported for cyclic MBP peptides; and (iv) they were found to be more stable to lysosomal enzymes and Cathepsin B, D, and H, compared to their linear counterparts. Such beneficial properties establish these synthetic peptides as putative immunotherapeutics for treating MS and potentially other Th1-mediated autoimmune diseases [23] . The mutations have been chosen as they identified the major TCR contact sites by X-ray crystallographic studies of human MHC and Molecular Dynamics (MD) studies using murine MHC.

The cyclic-MOG35-55 peptide, cyclized at the C-and N-terminal amino acids (cyclic-MOG35-55), altered the 3D conformation of the linear MOG35-55 peptide ( Figure 5 ). Following the injection of cyclic-MOG35-55 during disease induction, EAE, demyelination, and chronic axonopathy in acute and chronic phases of disease were reduced. Molecular docking and spectroscopic data revealed milder interactions between the cycic-MOG35-55 and mouse or human MHC class II alleles (H2-IA b and HLA-DR2) [44] . Likewise, synthesis of cyclic PLP139-151 peptide and injection in SJL/J mice showed that cylic-PLP139-151 analog is minimally encephalitogenic when administered to induce EAE ( Figure 5 ). In particular, cyclic-PLP139-151 analog showed low disease burden and minimal inflammatory, demyelinating, and axonopathic pathology compared to its linear counterpart. Proliferation assays confirmed the low stimulatory potential of the cyclic-PLP139-151 compared to linear PLP139-151 as well as the induction of lower antibody responses. Comparative molecular modeling studies between the two molecules may explain the biological data, as it was shown that different amino acids are involved in the TCR recognition [42] . It is clear that cyclic modification of linear peptide counterparts may provide novel approaches for future, immunomodulative treatments against MS. 

Mannan, a poly-mannose isolated from the cell wall of yeasts, has been shown to exert immunomodulatory effects in the cancer settings in vitro [45] [46] [47] [48] [49] [50] , in vivo (inbred mice, transgenic mice, rats, rabbits, and chickens) [51] [52] [53] [54] [55] [56] [57] [58] [59] [60] [61] [62] , in rhesus macaques [63] [64] [65] , and in human clinical trials [66] [67] [68] [69] [70] [71] [72] [73] [74] [75] . Mannan targets antigens to the mannose receptor, antigens endocytosed for MHC class I or II presentation, and modulation of appropriate T cells [45, [53] [54] [55] [76] [77] [78] [79] [80] [81] . In relation to autoimmune disorders, mannan conjugates (i) represent a new class of immunoregulators that directly and selectively target a population of immune cells that are implicated in the pathogenesis and progression of disease; (ii) provide first line treatment that selectively tolerates or inactivates disease-inducing cells in patients and also prevents progression of disease by stopping diversification of the autoimmune response to additional epitopes; (iii) allows easier formulation of newly discovered molecules within the mannan matrix platform; and (iv) can achieve block-buster status as a global vaccine drug for efficient treatment of MS [27] [28] [29] [30] . The cyclic-MOG peptide, cyclized at the C-and N-terminal amino acids (cyclic-MOG , altered the 3D conformation of the linear MOG 35-55 peptide ( Figure 5 ). Following the injection of cyclic-MOG 35-55 during disease induction, EAE, demyelination, and chronic axonopathy in acute and chronic phases of disease were reduced. Molecular docking and spectroscopic data revealed milder interactions between the cycic-MOG and mouse or human MHC class II alleles (H2-IA b and HLA-DR2) [44] . Likewise, synthesis of cyclic PLP 139-151 peptide and injection in SJL/J mice showed that cylic-PLP 139-151 analog is minimally encephalitogenic when administered to induce EAE ( Figure 5 ). In particular, cyclic-PLP 139-151 analog showed low disease burden and minimal inflammatory, demyelinating, and axonopathic pathology compared to its linear counterpart. Proliferation assays confirmed the low stimulatory potential of the cyclic-PLP 139-151 compared to linear PLP 139-151 as well as the induction of lower antibody responses. Comparative molecular modeling studies between the two molecules may explain the biological data, as it was shown that different amino acids are involved in the TCR recognition [42] . It is clear that cyclic modification of linear peptide counterparts may provide novel approaches for future, immunomodulative treatments against MS.

Mannan, a poly-mannose isolated from the cell wall of yeasts, has been shown to exert immunomodulatory effects in the cancer settings in vitro [45] [46] [47] [48] [49] [50] , in vivo (inbred mice, transgenic mice, rats, rabbits, and chickens) [51] [52] [53] [54] [55] [56] [57] [58] [59] [60] [61] [62] , in rhesus macaques [63] [64] [65] , and in human clinical trials [66] [67] [68] [69] [70] [71] [72] [73] [74] [75] . Mannan targets antigens to the mannose receptor, antigens endocytosed for MHC class I or II presentation, and modulation of appropriate T cells [45, [53] [54] [55] [76] [77] [78] [79] [80] [81] . In relation to autoimmune disorders, mannan conjugates (i) represent a new class of immunoregulators that directly and selectively target a population of immune cells that are implicated in the pathogenesis and progression of disease; (ii) provide first line treatment that selectively tolerates or inactivates disease-inducing cells in patients and also prevents progression of disease by stopping diversification of the autoimmune response to additional epitopes; (iii) allows easier formulation of newly discovered molecules within the mannan matrix platform; and (iv) can achieve block-buster status as a global vaccine drug for efficient treatment of MS [27] [28] [29] [30] .

Altered peptide ligands, where one to two amino acid mutations are made to those interacting with the TCR are able to alter an agonist peptide into an antagonist peptide by reduction of hydrogen bond interactions [82] . Cyclization of peptides allows for their stronger stability and protection against enzymatic and proteolytic degradation [43] . As such, a cyclic APL, cyclo(87-99)[A 91 ,A 96 ]MBP [87] [88] [89] [90] [91] [92] [93] [94] [95] [96] [97] [98] [99] ] reduced Th1 responses, but when conjugated to reduced mannan, an additional significant reduction of Th1 responses and moderate Th2 responses was induced (Table 1) [27, 30] . Likewise, APL of linear and cyclic MBP 83-99 analogs, MBP 83-99 (A 91 ,A 96 ), conjugated to reduced mannan, resulted in diversion of Th1 response to Th2 [27] . The use of reduced mannan to further divert immune responses to Th2 when conjugated to MBP peptides constitutes a novel strategy for immunotherapy of the disease. The main advantages of mannan conjugates is their stability and non-toxicity. In addition, linear and cyclic peptide analogs based on MBP 83-99 immunodominant epitope conjugated to reduced mannan via (KG) 5 or keyhole limpet hemocyanin (KLH) linkers, were evaluated for their biological/immunological profiles in SJL/J mice. Of all the peptide analogs tested, linear MBP 83-99 (F 91 ) and MBP 83-99 (Y 91 ) conjugated to reduced mannan and cyclic MBP 83-99 (F 91 ) conjugated to reduced mannan yielded the best immunological profile and constitute novel candidates for further immunotherapeutic studies against MS for translation into human clinical trials. Immune responses were diverted from Th1 to Th2 in SJL/J mice and generated antibodies that did not cross-react with native MBP protein. [21] . Furthermore, MBP 87-99 (R 91 , A 96 ) conjugated to reduced mannan induced 70% less IFNγ compared with the native MBP 87-99 peptide. However, MBP 87-99 (A 91 ,A 96 ) conjugated to reduced mannan did not induce IFNγ-secreting T cells, elicited very high levels of IL-4, and antibodies generated did not cross-react with the native MBP 87-99 peptide ( Table 1 ). It is clear that this double-mutant peptide analog conjugated to reduced mannan is able to divert immune responses from Th1 to Th2 and is a promising mutant peptide analogue for use in studies exploring potential treatments for MS [21] . Table 1 . Biological effects of myelin-derived peptides (linear, cyclic, mannan conjugated).

Major Effects MBP [83] [84] [85] [86] [87] [88] [89] [90] [91] [92] [93] [94] [95] [96] [97] [98] [99] and PLP 139-151 [10] [11] [12] [13] [14] [15] [16] [17] [18] These agonist peptides are involved in the pathophysiology of MS and also induce EAE in animal models.

MBP [82] [83] [84] [85] [86] [87] [88] [89] [90] [91] [92] [93] [94] [95] [96] [97] [98] [40, 41] Dirucotide in animal models inhibits disease and in early human clinical trials showed efficacy; however, the peptide did not meet primary endpoints in phase III-trials. cyclic (87) (88) (89) (90) (91) (92) (93) (94) (95) (96) (97) (98) (99) [MBP [87] [88] [89] [90] [91] [92] [93] [94] [95] [96] [97] [98] [99] ] [23] Stimulates Th2 cytokines and inhibits EAE in mice. MBP [87] [88] [89] [90] [91] [92] [93] [94] [95] [96] [97] [98] [99] (R 91 ,A 96 ), MBP 87-99 (A 91 ,A 96 ) [21] Induces IL-4 and antagonizes IFNγ responses in mice. MBP [72] [73] [74] [75] [76] [77] [78] [79] [80] [81] [82] [83] [84] [85] [22] These agonist peptides induce EAE in mice and Th1 responses in humans. MBP [72] [73] [74] [75] [76] [77] [78] [79] [80] [81] [82] [83] [84] [85] (A 79 ) [22] Suppresses EAE in mice. PLP 139-151 (L 144 , R 147 ) [42] Antagonizes PLP-specific T-clones in vitro. Induces T-cell proliferation and IFNγ secretion in mice.

In studies by Tseveleki et al. [28] , MOG peptide conjugated to mannan in oxidized or reduced forms protected mice against EAE in prophylactic and therapeutic protocols, with oxidized-conjugated peptides giving the best results. Protection was peptide-specific and associated with reduced antigen-specific T cell proliferation. Mannan-MOG peptide pulsed bone marrow-derived dendritic cells (DC) showed up-regulated expression of co-stimulatory molecules and induced active T cell tolerance, suppressing ongoing EAE; whilst mannan-MOG 35-55 peptide vaccinated mice did not reduce the proliferation of transferred MOG-specific T cells. MOG 35-55 -specific T cells cultured with mannan-MOG 35-55loaded DCs showed reduced proliferation and equal Th1 and Th17 cell differentiation as those with MOG 35-55 -loaded DC. Results show that mannan-conjugated myelin peptides protect mice against EAE through the expansion of antigen-specific Th1 and Th17 cells with impaired proliferation responses and DC-induced co-stimulatory signals that are required for licensing them to become fully pathogenic T cells [28] . In another study by Dagkonaki et al. [29] , CNS autoantigens conjugated to oxidized mannan were shown to induce antigen-specific T cell tolerance and protection against EAE in mice ( Table 1 ). The results showed that peptides conjugated with mannan induce peripheral type 2 myeloid cell responses and T cell anergy, and suggests that mannan-peptide conjugates may be useful for suppressing antigen-specific CD4+ T-helper cell responses in the context of human autoimmune CNS demyelination [29] .

Further, the immunogenicity of linear (Cit 91 ,A96,Cit 97 )MBP 87-99 and its cyclic analogcyclo(87-99)(Cit 91 ,A96,Cit 97 )MBP 87-99 when conjugated to mannan were studied in SJL/J mice. It was found that mannosylated cyclic citrullinated APL induced stronger T cell proliferative responses and IFNγ cytokine secretion in respect to its linear counterpart [25] .

Citrullination is a post-translational modification of peptidyl-arginine that plays a role in normal functioning of the immune system and also suspected to play a pathophysiological role in MS [83] [84] [85] [86] [87] [88] [89] . Citrullinated proteins of MBP are present in white matter lesions in the central nervous system in MS ( [25, [83] [84] [85] [86] [87] [88] [89] [87] [88] [89] [90] [91] [92] [93] [94] [95] [96] [97] [98] [99] shows similar properties to the linear counterpart [25] . Thus, citrullination of self-antigens may potentially trigger disease in susceptible individuals. This result triggers future research to new substances that inhibit citrullination and arrest epitope spreading and worsening of MS [25] .

Different molecules such as the fumaric acid and its esters are reported to be promising for MS treatment, exerting an antioxidative mechanism of action [31] . FTY720 (Gilenya TM ) is derived from the natural product myriocin and constitutes the first oral treatment for MS [32] (Figure 6 ). Recently, monomethyl fumarate (MMF, Bafiertam) was reported to be well tolerated, safe, and effective [33] (Figure 7 ). Searching the literature, there is only one publication that claims the discovery of non-peptide mimetic molecules derived directly from peptides. The example is referred to the conformational analysis of MBP83-96 epitope. The authors searched for molecules that inhibit the trimolecular complex formation and consequently the proliferation of activated T-cells. They generated a structure-based pharmacophore and used ZINC as a chemical database to extract candidates. They used semi-empirical and density functional theory methods to predict the binding energy between the proposed non-peptide mimetics and the TCR. They decided, using this analysis, to synthesize six molecules. From these molecules, the following two were the most promising as they inhibited the stimulation of T-cells by the immunodominant MBP83-99 peptide from immunized mice (Figure 8 ) [34] . Searching the literature, there is only one publication that claims the discovery of non-peptide mimetic molecules derived directly from peptides. The example is referred to the conformational analysis of MBP83-96 epitope. The authors searched for molecules that inhibit the trimolecular complex formation and consequently the proliferation of activated T-cells. They generated a structure-based pharmacophore and used ZINC as a chemical database to extract candidates. They used semi-empirical and density functional theory methods to predict the binding energy between the proposed non-peptide mimetics and the TCR. They decided, using this analysis, to synthesize six molecules. From these molecules, the following two were the most promising as they inhibited the stimulation of T-cells by the immunodominant MBP83-99 peptide from immunized mice (Figure 8 ) [34] . Searching the literature, there is only one publication that claims the discovery of non-peptide mimetic molecules derived directly from peptides. The example is referred to the conformational analysis of MBP 83-96 epitope. The authors searched for molecules that inhibit the trimolecular complex formation and consequently the proliferation of activated T-cells. They generated a structure-based pharmacophore and used ZINC as a chemical database to extract candidates. They used semi-empirical and density functional theory methods to predict the binding energy between the proposed non-peptide mimetics and the TCR. They decided, using this analysis, to synthesize six molecules. From these molecules, the following two were the most promising as they inhibited the stimulation of T-cells by the immunodominant MBP 83-99 peptide from immunized mice (Figure 8 ) [34] .

tivated T-cells. They generated a structure-based pharmacophore and used ZINC as a chemical database to extract candidates. They used semi-empirical and density functional theory methods to predict the binding energy between the proposed non-peptide mimetics and the TCR. They decided, using this analysis, to synthesize six molecules. From these molecules, the following two were the most promising as they inhibited the stimulation of T-cells by the immunodominant MBP83-99 peptide from immunized mice ( Figure 8 ) [34] . 

In the light of nanotechnology development, the approaches of discovering peptides linear or cyclic in structure or conjugated with oxidized or reduced mannans can be improved. Drug delivery vehicles can be developed that can selectively drive these simple or conjugated peptides to their site of action. Examples are mentioned in the literature for such application. A bifunctional peptide (small and soluble) and PLGA NPs (large and insoluble) resulted in relative greater EAE suppression [31] [32] [33] [34] [35] [36] [37] [38] [39] .

In a recent review article, the recent advances in nanomedicines for MS therapy were outlined [35] . Nanomaterials have the potential to carry therapeutic agents through 

In the light of nanotechnology development, the approaches of discovering peptides linear or cyclic in structure or conjugated with oxidized or reduced mannans can be improved. Drug delivery vehicles can be developed that can selectively drive these simple or conjugated peptides to their site of action. Examples are mentioned in the literature for such application. A bifunctional peptide (small and soluble) and PLGA NPs (large and insoluble) resulted in relative greater EAE suppression [31] [32] [33] [34] [35] [36] [37] [38] [39] .

In a recent review article, the recent advances in nanomedicines for MS therapy were outlined [35] . Nanomaterials have the potential to carry therapeutic agents through the blood brain barrier towards the lesion sites and can promote the remyelination process, achieving neuroprotection and neurodegeneration. In addition, they can act as effective ingredients for stem cell therapies. There is an intense effort to develop nanomedicines for clinical trials by reducing their toxicity and adverse effects [90] .

In another recent article entitled "Nanocariers as potential drug delivery candidates for overcoming the blood-brain barrier: challenges and possibilities" [39] the different nanosystems and their potential as drug carriers are outlined. An optimistic view is presented for the future of nanocarriers in the field of treatment of CNS-related disorders, as their ability to pass the blood brain barrier to deliver therapeutic doses of the drug, is very promising [90] .

The development of an efficient treatment against MS has a long way to go. The existing therapies are not satisfactory. The multifactorial disease is difficult to be tackled. The different approaches mentioned herein will certainly aid in new and more efficient treatments. More efforts must be made for developing non-peptide analogs using rational design and the knowledge derived from the spectroscopic and computational chemistry studies on peptides. The cyclization of myelin epitope peptides and the conjugation of linear or cyclic peptides with mannan is a promising field that must be strengthened. Furthermore, nanotechnology has emerged to aid in this effort by providing vehicles that will lead potential drug leads specifically to the right target. The steps in the discovering of new leads to treat MS are illustrated below (Figure 9 ).

Brain Sci. 2021, 11, x FOR PEER REVIEW 10 of 15 the blood brain barrier towards the lesion sites and can promote the remyelination process, achieving neuroprotection and neurodegeneration. In addition, they can act as effective ingredients for stem cell therapies. There is an intense effort to develop nanomedicines for clinical trials by reducing their toxicity and adverse effects [90] . In another recent article entitled "Nanocariers as potential drug delivery candidates for overcoming the blood-brain barrier: challenges and possibilities" [39] the different nanosystems and their potential as drug carriers are outlined. An optimistic view is presented for the future of nanocarriers in the field of treatment of CNS-related disorders, as their ability to pass the blood brain barrier to deliver therapeutic doses of the drug, is very promising [90] .

The development of an efficient treatment against MS has a long way to go. The existing therapies are not satisfactory. The multifactorial disease is difficult to be tackled. The different approaches mentioned herein will certainly aid in new and more efficient treatments. More efforts must be made for developing non-peptide analogs using rational design and the knowledge derived from the spectroscopic and computational chemistry studies on peptides. The cyclization of myelin epitope peptides and the conjugation of linear or cyclic peptides with mannan is a promising field that must be strengthened. Furthermore, nanotechnology has emerged to aid in this effort by providing vehicles that will lead potential drug leads specifically to the right target. The steps in the discovering of new leads to treat MS are illustrated below (Figure 9 ). 

Linear MBP, PLP, and MOG epitopes were used as tools to study the molecular basis of MS. To increase their stability and enhance their pharmacological profile, linear peptides were cyclized. As such, cyclic-MOG and cyclic-PLP altered the conformation of the 

Linear MBP, PLP, and MOG epitopes were used as tools to study the molecular basis of MS. To increase their stability and enhance their pharmacological profile, linear peptides were cyclized. As such, cyclic-MOG and cyclic-PLP altered the conformation of the peptide, resulting in reduced EAE in animal models. In addition, one to two amino acid mutations were made to TCR contact residues (altered peptide ligands), which were shown to enhance Th2 anti-inflammatory responses over the predominant Th1 pro-inflammatory responses. However, this enhancement is not adequate, and as such, conjugation to mannan is required, which further shifts Th1 to Th2 responses. The future holds promise for novel immune modulators including cyclic peptides, altered peptide ligands, and the use of mannan to deliver the peptides to appropriate antigen-presenting cells. Funding: The preparation of this review did not receive any external funding.

Informed Consent Statement: Not applicable.

The data presented in this study are available on request from the corresponding author.

Multiple sclerosis

Multiple Sclerosis: A Coordinated Immunological Attack against Myelin in the Central Nervous System

Time trends in the incidence and prevalence of multiple sclerosis in Norway during eight decades

Diagnostic criteria for multiple sclerosis: 2010 Revisions to the McDonald criteria

Currently approved and emerging oral therapies in multiple sclerosis: An update for the ophthalmologist

Multiple Sclerosis: Immunopathology and Treatment Update

Anti-CD20 Agents for Multiple Sclerosis: Spotlight on Ocrelizumab and Ofatumumab

Recent Advances in Antigen-Specific Immunotherapies for the Treatment of Multiple Sclerosis

Molecular Interventions towards Multiple Sclerosis Treatment

Interleukin-10 plays a crucial role in suppression of experimental autoimmune encephalomyelitis by Bowman-Birk inhibitor

Peptide 53-78 of myelin P2 protein is a T cell epitope for the induction of experimental autoimmune neuritis

Induction of severe experimental autoimmune neuritis with a synthetic peptide corresponding to the 53-78 amino acid sequence of the myelin P2 protein

Immunological Aspects of Demyelinating Diseases

Immunologic Mechanisms and Therapy in Multiple Sclerosis

Induction of a non-encephalitogenic type 2 T helper-cell autoimmune response in multiple sclerosis after administration of an altered peptide ligand in a placebo-controlled, randomized phase II trial

Encephalitogenic potential of the myelin basic protein peptide (amino acids 83-99) in multiple sclerosis: Results of a phase II clinical trial with an altered peptide ligand

HLA-DPB1*03 as Risk Allele and HLA-DPB1*04 as Protective Allele for Both Early-and Adult-Onset Multiple Sclerosis in a Hellenic Cohort

Differential activation of human autoreactive T cell clones by altered peptide ligands derived from myelin basic protein peptide (87-99)

Structural Requirements for Binding of Myelin Basic Protein (MBP) Peptides to MHC II: Effects on Immune Regulation

Design and Synthesis of a Cyclic Double Mutant Peptide (cyclo(87−99)[A91,A96]MBP87−99) Induces Altered Responses in Mice after Conjugation to Mannan: Implications in the Immunotherapy of Multiple Sclerosis

A double mutation of MBP83-99 peptide induces IL-4 responses and antagonizes IFN-γ responses

Design and Synthesis of a Potent Cyclic Analogue of the Myelin Basic Protein Epitope MBP72-85: Importance of the Ala81 Carboxyl Group and of a Cyclic Conformation for Induction of Experimental Allergic Encephalomyelitis

Design And Synthesis of a Novel Potent Myelin Basic Protein Epitope 87−99 Cyclic Analogue: Enhanced Stability and Biological Properties of Mimics Render Them a Potentially New Class of Immunomodulators

Properties of myelin altered peptide ligand cyclo(87-99)(Ala91,Ala96)MBP87-99 render it a promising drug lead for immunotherapy of multiple sclerosis

Epitope Elicits a Th1 Polarized Response by T Cells Isolated from Multiple Sclerosis Patients: Implications in Triggering Disease

Design of Linear and Cyclic Mutant Analogues of Dirucotide Peptide (MBP82-98) against Multiple Sclerosis: Conformational and Binding Studies to MHC Class II

Mannosylation of mutated MBP83-99 peptides diverts immune responses from Th1 to Th2

Mannan-conjugated myelin peptides prime non-pathogenic Th1 and Th17 cells and ameliorate experimental autoimmune encephalomyelitis

Mannan-MOG35-55 Reverses Experimental Autoimmune Encephalomyelitis, Inducing a Peripheral Type 2 Myeloid Response, Reducing CNS Inflammation, and Preserving Axons in Spinal Cord Lesions

Design of Novel Cyclic Altered Peptide Ligands of Myelin Basic Protein MBP83−99That Modulate Immune Responses in SJL/J Mice

Fumaric Acid and its Esters: An Emerging Treatment for Multiple Sclerosis

From Natural Product to the First Oral Treatment for Multiple Sclerosis: The Discovery of FTY720 (Gilenya™)?

Urits, I. Monomethyl Fumarate (MMF, Bafiertam) for the Treatment of Relapsing Forms of Multiple Sclerosis (MS)

Design and Synthesis of Non-Peptide Mimetics Mapping the Immunodominant Myelin Basic Protein (MBP83-96) Epitope to Function as T-Cell Receptor Antagonists

Targeting the brain lesions using peptides: A review focused on the possibility of targeted drug delivery to multiple sclerosis lesions

Structure, Size, and Solubility of Antigen Arrays Determines Efficacy in Experimental Autoimmune Encephalomyelitis

Nanoparticle-mediated codelivery of myelin antigen and a tolerogenic small molecule suppresses experimental autoimmune encephalomyelitis

Recent Advances in Nanomedicines for Multiple Sclerosis Therapy

Nanocarriers as Potential Drug Delivery Candidates for Overcoming the Blood-Brain Barrier: Challenges and Possibilities

Intravenous synthetic peptide MBP8298 delayed disease progression in an HLA Class II-defined cohort of patients with progressive multiple sclerosis: Results of a 24-month double-blind placebo-controlled clinical trial and 5 years of follow-up treatment

A phase III study evaluating the efficacy and safety of MBP8298 in secondary progressive MS

Cyclization of PLP139-151 peptide reduces its encephalitogenic potential in experimental autoimmune encephalomyelitis

Round and round we go: Cyclic peptides in disease

Apostolopoulos, V.; Matsoukas, J. Cyclic MOG35-55 ameliorates clinical and neuropathological features of experimental autoimmune encephalomyelitis

Ex vivo targeting of the macrophage mannose receptor generates anti-tumor CTL responses

Delivery of antigen using a novel mannosylated dendrimer potentiates immunogenicity in vitro and in vivo

The adjuvanticity of a mannosylated antigen reveals TLR4 functionality essential for subset specialization and functional maturation of mouse dendritic cells

Mannan derivatives induce phenotypic and functional maturation of mouse dendritic cells

Protein/peptide and DNA vaccine delivery by targeting C-type lectin receptors

Oxidized and reduced mannan mediated MUC1 DNA immunization induce effective anti-tumor responses

MUC1-specific immune responses in human MUC1 transgenic mice immunized with various human MUC1 vaccines

Peptide mimics of a tumor antigen induce functional cytotoxic T cells

Role of the mannose receptor in the immune response

Aldehyde-mannan antigen complexes target the MHC class I antigen-presentation pathway

Oxidative/reductive conjugation of mannan to antigen selects for T1 or T2 immune responses

Cell-mediated immune responses to MUC1 fusion protein coupled to mannan

Use of the mannan receptor to selectively target vaccine antigens for processing and antigen presentation through the MHC class I and class II pathways

The effect of T1 and T2 cytokines on the cytotoxic T cell response to mannan-MUC1

Immunotherapy with mannan-MUC1 and IL-12 in MUC1 transgenic mice

Cytokine production from murine CD4 and CD8 cells after mannan-MUC1 immunization

Induction of T1 (cytotoxic lymphocyte) and/or T2 (antibody) responses to a mucin-1 tumour antigen

Mannan-mediated gene delivery for cancer immunotherapy

Natural human anti-Gal alpha(1,3)Gal antibodies react with human mucin peptides

The immune response of mice and cynomolgus monkeys to macaque mucin 1-mannan. Vaccine

Induction of humoral and cellular responses in cynomolgus monkeys immunised with mannan-human MUC1 conjugates

MUC1 cross-reactive Gal alpha(1,3)Gal antibodies in humans switch immune responses from cellular to humoral

Pilot phase III immunotherapy study in early-stage breast cancer patients using oxidized mannan-MUC1

Dendritic cell immunotherapy: Clinical outcomes

Antibody and T cell responses of patients with adenocarcinoma immunized with mannan-MUC1 fusion protein

Flow cytometric measurement of intracellular cytokines detects immune responses in MUC1 immunotherapy

Mannan Mucin-1 Peptide Immunization: Influence of Cyclophosphamide and the Route of Injection

Mannan-MUC1-pulsed dendritic cell immunotherapy: A phase I trial in patients with adenocarcinoma

A phase 2, single-arm study of an autologous dendritic cell treatment against mucin 1 in patients with advanced epithelial ovarian cancer

In vivo tracking of dendritic cells in patients with multiple myeloma

Up to 15-year clinical follow-up of a pilot Phase III immunotherapy study in stage II breast cancer patients using oxidized mannan-MUC1

Strongly increased efficiency of altered peptide ligands by mannosylation

Combining mannose receptor mediated nanovaccines and gene regulated PD-L1 blockade for boosting cancer immunotherapy

Human epidermal Langerhans cells lack functional mannose receptors and a fully developed endosomal/lysosomal compartment for loading of HLA class II molecules

Potential Use of Thioalkylated Mannose-Modified Dendrimer (G3)/alpha-Cyclodextrin Conjugate as an NF-kappaB siRNA Carrier for the Treatment of Fulminant Hepatitis

Design and evaluation of thioalkylated mannose-modified dendrimer (G3)/alpha-cyclodextrin conjugates as antigen-presenting cell-selective siRNA carriers

Mannose receptor mediated uptake of antigens strongly enhances HLA-class II restricted antigen presentation by cultured dendritic cells

A functional hot spot for antigen recognition in a superagonist TCR/MHC complex

Citrullination under physiological and pathological conditions

Localisation of citrullinated proteins in normal appearing white matter and lesions in the central nervous system in multiple sclerosis

Role for peptidylarginine deiminase enzymes in disease and female reproduction

Potential role of peptidylarginine deiminase enzymes and protein citrullination in cancer pathogenesis

Deimination of myelin basic protein. 1. Effect of deimination of arginyl residues of myelin basic protein on its structure and susceptibility to digestion by cathepsin D

Deimination of myelin basic protein. 2. Effect of methylation of MBP on its deimination by peptidylarginine deiminase

An immunochemical comparison of human myelin basic protein and its modified, citrullinated form, C8

Biotechnological agents for the immunotherapy of multiple sclerosis. Principles, problems and perspectives

The authors declare no conflict of interest.