FIELD OF INVENTION The invention relates to a method of treating or preventing multiple sclerosis in an animal subject using one or more neural regeneration peptides (NRPs), In particular, the invention relates to the use of NRP 2945 and closely related analogues for treating or preventing multiple sclerosis or seizures in an animal subject. More particularly, the invention relates to the use of NRP 2945 and closely related analogues to prevent axonal damage or demyelination of central nervous system cells.

BACKGROUND ART

Neural Regeneration Peptides (NRPs) are small peptides that promote proliferation, migration, differentiation and survival of neural precursor and committed neural stem cells under conditions of central nervous system disease or injury (Gorba et al., 2006; Singh et al., 2010). The discovery of NRPs dates back to experimentation with a developed in vitro model of traumatic brain injury (Sieg et al., 1999). Administration of biochemically fractionated cell culture supernatant derived from hippocampal organotypic tissue cultures (OTCs) led to a neuronal bridge formation in thalamocortical OTC co-cultures within 3-4 days (Landgraf et al., 2005). The first completely identified gene that encodes for bioactive peptides displaying femtomolar potency for neuronal survival-promoting activity was experimentally shown to be encoded on chromosome 12 in mice and is coding for a 135 amino acid long protein (Gorba et al., 2006). NRP2945 derives from the human NRP gene located at position 7q31.35 also known as calcium dependent activator of protein secretion 2 (CAPS2; Sadakata et al., 2004) and represents amino acid position 40-50 within the CAPS2 sequence (Speidel et al., 2003).

NRPs have both neurodegenerative and anti-inflammatory effects (Landgraf et al. 2005; Gorba et al., 2006 and Sieg & Anionic, 2007). Several studies using human primary brain cells, human embryonic neural stem cells and animal cells subsequently confirmed that NRPs at sub-nanomolar concentrations have both neuroprotective and anti-inflammatory efficacy suitable for the treatment of neuroinflammatory diseases (Gorba et al., 2006).

Experimental autoimmune encephalomyelitis (EAE) is a T cell mediated autoimmune disease characterized by perivascular infiltration of CD4+ T cells and mononuclear cells of the central nervous system (CNS). The disease is described by a progressive loss of the myelin sheath as well as damage to axons, leading to progressive hind-limb paralysis. This model is considered relevant for the human disease multiple sclerosis (MS). The experimental disease is induced by the injection of a self-peptide derived from myelin oligodendrocyte glycoprotein (MOG), a CNS specific antigen, together with pertussis toxin and potent mycobacterial antigens used as adjuvants. In the mouse strain C57BL/6, the disease follows a monophasic chronic-progressive clinical course, with no spontaneous remission. The pathogenesis is characterized by an initial migration of activated myelin- specific T cells to the CNS, which then secrete chemokines and cytokines. These effector molecules attract peripheral mononuclear phagocytes into the CNS parenchyma, which are then, together with brain-resident microglial cells, activated by the T cell-derived cytokines. Demyelination and axonal damage ensues, most likely as the result of phagocytic activity of monocytes/macrophages, the inflammatory process and the cytotoxic effects of cytokines.

It is an object of this invention to provide a method for treating or preventing multiple sclerosis or preventing demyelination in an animal subject using a neural regeneration peptide or to at least to provide a useful choice.

STATEMENT OF INVENTION

In a first aspect the invention provides a method of treating or preventing multiple sclerosis in a subject comprising administering an effective amount of a neural regenerative peptide to a subject in need thereof. In one embodiment the invention provides a method of treating or preventing multiple sclerosis in a subject comprising administering an effective amount of a neural regeneration peptide selected from one or more of the sequences:

GlyArgArgAlaAlaProGlyArgAibGlyGly (SEQ ID NO:1) or

GlyArgArgAlaAlaProGlyArgAibGlyGly-NH ₂ (SEQ ID NO:2); (NRP2945) or GlyArgArgAlaAlaProGlyArg β-alanine GlyGly-NH2 (SEQ ID NO:3); (NRP2983) to a subject in need thereof.

In one embodiment the peptide consists of the 11 amino acid residue sequence:

GlyArgArgAlaAlaProGlyArgAibGlyGly and functionally acceptable derivatives thereof. One such acceptable derivative includes the sequence wherein the C-terminus of the peptide is amidated to give:

GlyArgArgAlaAlaProGIyArgAibGlyGly-NH ₂ (SEQ ID NO:2). In another embodiment the peptide consists of the 11 amino acid residue sequence:

GlyArgArgAlaAlaProGlyArg beta-alanine GlyGly and functionally acceptable derivatives thereof. One such acceptable derivative includes the sequence wherein the C-terminus of the peptide is amidated to give:

GlyArgArgAlaAlaProGlyArg β-alanine GlyGly-NH ₂ (SEQ ID NO:3).

In one embodiment the administration step to the subject is by way of injection, including but not limited to intra peritoneal, intravenous, intramuscular, intra arterial and intra spinal injection. In another embodiment the administration step to the subject is by way of oral administration. In another embodiment the effective amount of the NRP is at least 1 pg/kg administered to the subject. In another embodiment the effective amount of the NRP is between about 10-200^g/kg of the NRP administered to the subject.

In a second aspect the invention provides a method of preventing axonal damage or demyelination of central nervous system cells in a subject comprising administering an effective amount of a neural regeneration peptide to a subject in need thereof.

In one embodiment the invention provides a method of preventing (i) axonal damage or (ii) demyelination of central nervous system cells in a subject comprising administering an effective amount of a neural regeneration peptide selected from one or more of the sequences: GlyArgArgAlaAlaProGlyArgAibGlyGly (SEQ ID NO:1) or GlyArgArgAlaAiaProGlyArgAibGlyGly-NH ₂ (SEQ ID NO:2); GlyArgArgAlaAlaProGlyArg β-alanine GlyGly-NH2 (SEQ ID NO:3) and to a subject in need thereof.

In one embodiment the peptide consists of the 1 amino acid residue sequence: GlyArgArgAlaAlaProGlyArgAibGiyGly and acceptable derivatives thereof. One such acceptable derivative includes the sequence wherein the C-terminus of the peptide is amidated to give:

GlyArgArgAlaAlaProGlyArgAibGlyGly-NH ₂ (SEQ ID NO:2).

In another embodiment the peptide consists of the 11 amino acid residue sequence: GlyArgArgA!aAlaProGlyArg β-alanine GlyGly

In one embodiment the administration step to the subject is by way of injection, including but not limited to intra peritoneal, intravenous, intramuscular, intra arterial and intra spinal injection. In another embodiment the administration step to the subject is by way of oral administration.

In another embodiment the effective amount of the NRP is at least 1 pg/kg administered to the subject. In another embodiment the effective amount of the NRP is between about 1- 200pg/kg of the NRP administered to the subject. In one embodiment the methods defined above include the step of administering an effective amount of a peptide selected from one or more of the sequences:

GlyArgArgAlaAlaProGlyArgAibGlyGly (SEQ ID NO:1) or

GiyArgArgAlaAlaProGlyArgAibGlyGIy-NH ₂ (SEQ ID NO:2);

GlyArgArgAlaAlaProGlyArg β-alanine GlyGly-NH2 (SEQ ID NO:3) and to a subject in need thereof on an at least once a day basis.

In one embodiment the method includes the step of administering an effective amount of a peptide comprising the sequence:

GlyArgArgAlaAlaProGlyArgAibGlyGly (SEQ ID NO:1) to a subject in need thereof on an at least once a day basis. In one embodiment the methods defined above include the step of administering the peptide on an at least twice a day basis.

In one embodiment the subject is selected from the group consisting of: humans and companion animals.

In one embodiment the administration step to the subject is by way of injection, including but not limited to intra peritoneal, intravenous, intramuscular, intra-arterial and intra spinal injection. In another embodiment the administration step to the subject is by way of oral administration. In another embodiment the effective amount of the NRP is at least 5Mg/kg administered to the subject. In another embodiment the effective amount of the NRP is between about 10-200pg/kg of the NRP administered to the subject.

In a third aspect the invention provides the use in the manufacture of a medicament of an effective amount of neural regenerative peptide for treating or preventing

(i) multiple sclerosis; or

(ii) demylination of central nervous system cells; or

(iii) axonal damage in a subject in need thereof. In one embodiment the invention provides the use in the manufacture of a medicament of an effective amount of at least one of the peptides selected from:

GlyArgArgAlaAlaProGlyArgAibGlyGly (SEQ ID NO:1)or

GlyArgArgAlaAlaProGlyArgAibGlyGly-NH ₂ (SEQ ID NO:2);

GlyArgArgAlaAlaProGlyArg β-alanine GlyGly-NH2 (SEQ ID NO:3) or for treating or preventing

(iv) multiple sclerosis; or

(v) demyelination of central nervous system cells; or

(vi) axonal damage in a subject in need thereof. In one embodiment the administration step to the subject is by way of injection, including but not limited to intra peritoneal, intravenous, intramuscular, intra-arterial and intra spinal injection. In another embodiment the administration step to the subject is by way of oral administration.

In one embodiment the medicament is adapted for at least once daily administration. In one embodiment the medicament is adapted for at least twice daily administration.

In one embodiment the subject is selected from the group consisting of: humans and companion animals. It is to be understood from the following description that the effective amount of the peptide of SEQ ID NO:1 or SEQ ID NO: 2 or SEQ ID NO:3 is indicated to be in the range of 1-20pg/kg dose amounts when administered in rodent models and in the range of 5-200 pg/kg dose amounts when administered to larger subjects such as dogs or humans. In the description and claims of this specification the following acronyms, terms and phrases have the meaning provided:

"Effective amount" means an amount effective to treat or prevent demyelination of a central nervous system cell in a given subject or an amount effective to treat or prevent axonal damage. "Functionally acceptable derivatives" means derivatives of the peptide defined in SEQ ID NO:1 , 2 and 3 obtained by amidation, acylation, alkylation, carboxylation, glycosylation, phosphorylation, prenylation, salification, sulfation, or a combination thereof, that are suitable for inclusion in a composition for administration to the eye.

The invention will now be described with reference to embodiments or examples and the figures of the accompanying drawings pages.

BRIEF DESCRIPTION OF DRAWINGS

Figure 1. shows the prophylactic impact of NRP 2945 (SEQ ID No:1) and NRP 2983 (SEQ ID NO: 3) on the development of EAE in C57BL/6 mice. EAE was induced by

immunization with 200pg of pMOG ₃ 5-55. Four days later, animals were treated with one of the NRP peptides and thereafter were scored daily for clinical signs of EAE. Data represent the mean ± sem. Note that the mean clinical score of mice treated with 10 pg/kg NRP 2945 IP was significantly different from that of the EAE (1way ANOVA; pO.0001) and Saline groups (p<0.001). Likewise, the mean clinical score in mice treated with 4 pg/kg NRP 2945 IP at the experimental end point was similar to that of the 10 pg/kg NRP 2945 IP group and was significantly different from the EAE and Saline groups (p<0.001 and p<0.05 respectively). Subcutaneous injection of NRP 2945 also lead to a significant reduction of the clinical signs of EAE, compared to EAE group (p<0.01). No significant effect was observed on the clinical score with the peptidomimetic NRP 2983.

Figure 2: shows a summary of the histological analysis of the spinal cords of mice treated with the NRP peptides: prophylactic study. Sections were stained with hematoxylin and eosin (HE) for inflammatory infiltrates, luxol fast blue (LFB) for demyelination,

Bielschowski silver stain (BSS) for axonal loss and damage. Data are presented as mean score of inflammation, demyelination and axonal damage or loss + sem. *p<0.05, **p<0.01 compare to the EAE control group. Figure 3. shows representative histological sections of spinal cords from mice treated with the NRP peptides of the invention. Sections were stained with hematoxylin and eosin (HE) for inflammatory infiltrates, luxol fast blue (LFB) for demyelination Bielschowski silver stain (BSS) for axonal loss and damage. Black arrows indicate areas of inflammatory cell accumulation, corresponding to areas of demyelination and axon damage. Original magnification: x4. See appendix for higher magnification.

Figure 4: shows the results of a splenocyte proliferation assay. Proliferative response of splenic T cells to MOG35-55 and the non-specific mitogens [PMA/lonomycin (P A/lono) and anti-CD3/CD28 antibodies (CD3/CD28)] from EAE C57BL/6 mice treated with the NRP peptides. Data are expressed as the mean ± sem of H3 incorporation (CPM) obtained at 72hours in culture. No statistically significant differences were observed between groups.

Figure 5. shows the detection of pro- and anti-inflammatory cytokines in splenocytes of EAE-C57BL/6 mice treated with the NRP peptides. Supernatants of spleen cells stimulated with MOG35-55 (20pg/ml) and/or with the non-specific mitogen (CD3/CD28) were collected after 72 hours and cytokines were quantified by cytometric bead array (CBA). Histograms show TH1 /17 (IFNy, IL-2, TNF, IL-17), TH2 (IL-4, IL-6, IL-10) cytokines secretion for all groups of animals. Data are expressed as the mean ± sem of secretion in pg/ml. Statistically significant at *p<0.05; **p< 0.01 ; *** p<0.001 ; **** p<0.0001 using 2 way ANOVA.

Figure 6: shows anti-NRP 2945 antibody responses: Sera of mice treated with NRP 2945 were tested 40 days post EAE induction and 36 days after the start of the treatment. Data are expressed as the mean of specific absorbance at OD 492 nm ± sem. As indicated by the specific absorbance (OD of the sample tested minus OD from the reactivity against an irrelevant scramble peptide), no difference in the level of anti-NRP antibodies could be found between the EAE and Saline control group, implying a total absence of antibody response against NRP 2945.

Figure 7. shows the therapeutic impact of NRP 2945 on the development of EAE in C57BL/6 mice. EAE was induced by immunization with 200pg of pMOG35-55. Fourteen days later, animals were treated IP with 40 or 20 g/kg NRP 2945 peptide. They were scored daily for clinical signs of EAE. Data are expressed as the mean ± sem. Note that at day 37, (end point) the mean clinical score (1.44± 0.42) of mice treated with 20 pg/kg NRP 2945 (panels A, B and C) was significantly different (pO.0001 ; 2 way ANOVA) from that of the EAE (3.38±0.34). Panel A also shows that when EAE mice, with the skin ulcerations were removed from the group treated with 40 pg/kg NRP 2945, the mean clinical score at the experimental end point (2.0+ 0.7) was almost similar to that of the 20 pg/kg NRP 2945 group and significantly different from the EAE group (p<0.05 to p<0.01 ; 2 way ANOVA). Panels B and panel C show the mean clinical scores of EAE observed in all the mice treated with 40pg/kg NRP 2945 with that observed in the group treated with 20pg/kg NRP 2945 when none of the mice (n=10) were excluded from the analysis (panel C) or when the 2 severely affected EAE mice (n=8) were removed from the analysis (panel B). In this later group, the difference in clinical score was significantly different (## p< 0.01) when compared with that of mice treated with 40pg/kg NRP 2945.

Figure 8: shows the histological analysis of the spinal cords of mice therapeutically treated with the NRP peptides. Sections were stained with hematoxylin and eosin (HE) for inflammatory infiltrates, luxol fast blue (LFB) for demyelination Bielschowski silver stain (BSS) for axonal loss and damage. Data are presented as mean score of inflammation , demyelination and axonal damage or loss ± sem. *p<0.05, **p<0.01 compare to the EAE control group. Figure 9. shows representative histological sections of spinal cords from mice

therapeutically treated with the NRP peptides. Sections were stained with hematoxylin and eosin (HE) for inflammatory infiltrates, luxol fast blue (LFB) for demyelination

Bielschowski silver stain (BSS) for axonal loss and damage. Black arrows indicate areas of inflammatory cell accumulation, corresponding to areas of demyelination and axon damage. Please note that in contrast to the EAE control mice, which had numerous and diffused inflammatory lesions, associated with extensive areas of demyelination and axonal damage and loss, mice treated with NRP 2945, particularly at the dosage of 20 pg/kg, only displayed few localized areas of inflammation, demyelination and axonal damage. Original magnification: x4. See Appendix 3 for higher magnification. Figure 10: shows the results of the splenocyte proliferation assay. Proliferative response of splenic T cells to incremental concentration of MOG35-55 and to the non-specific mitogens PMA/lonomycin (PMA/lono) and to anti-CD3/CD28 antibodies (CD3/CD28)] from EAE C57BL/6 mice treated with the 2 concentration of NRP peptides. Data are expressed as the mean ± sem of H3 incorporation (CPM) obtained at 72hours in culture. * p<0.05; ** p<0.01 ; **** p<0.001.

Figure 11. shows the detection of pro- and anti-inflammatory cytokines in splenocytes of EAE-C57BL/6 mice treated therapeutically with the NRP 2945. Supernatants of spleen cells stimulated with MOG35-55 (20pg/ml) and/or with the non-specific mitogen

(CD3/CD28) were collected after 72 hours and cytokines were quantified by cytometric bead array (CBA). Histograms show TH1/17 (IFNy, IL-2, TNF, IL-17), TH2 (IL-4, IL-6, IL- 10) cytokines secretion for all groups of animals. Data are expressed as the mean ± sem of secretion in pg/ml. Statistically significant at *p<0.05 ; **p< 0.01 ; *** p<0.001 ; **** p<0.0001 using 2 way ANOVA.

Figure 12.shows the anti-MOG antibodies response in mice treated with NRP 2945. Sera were collected at day 39, the experimental end point and tested by ELISA against MOG. Data are expressed as the mean of specific absorbance at OD 492 nm ± sem. Statistically significant at *p<0.05 compare to Not grafted group using Unpaired t test.

Figure 13: shows the anti-NRP 2945 antibodies responses in mice treated therapeutically with NRP 2945: Sera of mice treated with NRP 2945 were tested 39 days post EAE induction and 25 days after the start of the treatment. Data are expressed as the mean of specific absorbance at OD 492 nm ± sem. As indicated by the specific absorbance, no difference in the level of anti-NRP antibodies could be found between the EAE and NRP- treated mice group, implying NRP 2945 is not immunogenic.

Further aspects of the invention will become apparent with reference to the accompanying Figures and Example described below:

Example 1 : Efficacy of NRP 2945 (SEQ ID NO 1) and 2983 (SEQ ID NO 3) in an experimental model of multiple sclerosis (MS) in mice by assessment of clinical signs, immunological responses and pathological examination:

Materials and Methods

The animal experiments were performed in the laboratories of Prof. Claude Bernard at the Monash University in Melbourne, Australia.

Animals Female C57BL/6 mice weighing between 20 to 22 g were obtained from Monash Animal Services, Australia. Mice were group housed during the acclimatization period (7-10 days) and during the actual experiments received appropriate environmental enrichment (card board, tissues and seeds) in the cages. All animals were maintained under identical conditions and had free access to drinking water and normal pelleted food (Ridley Agriproducts, Australia). All animals were housed in climate-controlled and specific pathogen-free conditions with a 12-h light/dark cycle. All studies were performed in accordance with the Australian code of practice for the care and use of animals for scientific purposes (NHMRC, 1997), after the approval by Monash University Animal Ethics committee (Clayton/Melbourne, Australia). In order to enable the recognition of individual mice of a given group, each animal was marked at the tip of the tail.

Preparation of test compounds NRP 2945 and NRP 2983, The test compounds, NRP 2945 and NRP 2983 (mimetic of NRP2945), were prepared and supplied by CuroNZ Ltd, New Zealand and stored at -80°C protected from light before being used.

1.1. Identity:

SEQ ID NO; 2 also known as NRP2945 is an amidated peptidomimetic (H- GGRRAAPGRAIBGG-NH2) where AIB stands for alpha-aminoisobutyric acid. The provided batches were synthesized by GlycoSyn in February 2013. NRP2945 derives from the human CAPS-2 gene (position 40-50), There is more than 90% identity (and 100% homology) to rodent CAPS-2 regarding this particular region.

SEQ ID NO: 3 or NRP2983 is: GRRAAPGR β-alanine GG-NH2. 1.2. Formulation:

NRP2945 after cleavage from its resin (solid phase synthesis) is formulated in excess of 170 x times D (+)-trehalose and lyophilised. Further storage is done under an argon atmosphere. NRP2983 is also formulated like NRP2945 in 170xtimes of D (+)-trehalose. 1 vial contains 500 ig of NRP2983 + 84.5mg trehalose (just alike NRP2945 vials). The same formulation was prepared for NRP2983.

1.3. Storage:

Lyophilised contents of the vials of NRP2945/2983 were stored at -80°C. Each vial has been resuspended at a concentration of 1 mg/ml of peptide in 0.9% saline / PBS (pH 7.2), then aliquoted and stored at -800C until use. None of the vials were freezed-thawed. Twenty vials of 1 mg/ml NRP2945/2983 in saline (500 μΙ of saline per vial) were prepared. For every experimental day working solutions from freshly thawed peptide aliquots were prepared for each of the different groups (intraperitoneal (IP) and subcutaneous (SC) injections). After injection, all remaining working solutions were discarded.

Experimental protocol for the prophylactic im act of NRP2945 and NRP 2983 on OG-EAE, EAE mice 1-10: control, no treatment (n=10).

Saline mice 11-20: vehicle, IP daily; from day 4 post-immunization (p.i.) to day 18 p.i. (n=10).

NRP2945 mice 21-30: 4pg/kg, IP daily; from day 4 p.i. to day 18 p.i. (n=10). NRP2945 mice 31-40: 10pg/kg, IP daily; from day 4 p.i. to day 18 p.i, (n=10).

NRP2945 mice 41-50: 10pg/kg, SC daily; from day 4 p.i. to day 18 p.i. (n=10).

NRP2983 mice 51-60: 10pg/kg, IP daily; from day 4 p.i. to day 18 p.i. (n=10).

Mice were immunized with MOG at day 0, injected with peptide during 15 days from day 4 to day 18. At day 4, 30 and 40, mice were bled retro-orbitally for further anti-NRP2945 antibodies detection by EL1SA. Mice were monitored daily for 40 days for clinical signs of EAE. At the end of the experiment, the mice were sacrificed for immunological analysis, which included splenocytes proliferation and cytokines production. In addition, central nervous system (CNS) along with optic nerves were also dissected and prepared for the pathological analysis, which included the blind assessment of inflammation, demyelination as well as axonal damage and loss in the spinal cord. Optic nerves were sent to the inventor for subsequent analysis.

Experimental protocol for the therapeutic impact of N P2945 on MOG-EAE.

NRP2945 mice 1-10: 20pg/kg, IP every second day; from day 14 p.i. to day 35 p.i. (n=10).

NRP2945 mice 1-20: 40pg/kg, IP every second day; from day 14 p.i. to day 35 p.i. (n=10).

Saline mice 21-30: vehicle, IP every second day; from day 14 post-immunization (p.i.) to day 35 p.i. (n=10).

EAE mice 31-40: control, no treatment (n=10).

Mice were immunized with MOG at day 0, injected with peptide every second day from day 14 to day 35. Animals were monitored daily for 37 days for clinical signs of EAE. At the end of the experiment, the mice were sacrificed for immunological analysis, which included splenocytes proliferation and cytokines production. In this protocol, serum anti- MOG antibodies responses were also assessed. Central nervous system (CNS) along with optical nerves were dissected and prepared for the pathological analysis, which included the blind assessment of inflammation, demyelination as well as axonal damage and loss in the spinal cord. Optic nerves were provided to CuroNZ for subsequent analysis.

Induction of EAE: Female C57BL/6 mice were immunized with 200μ9 of the

encephalitogenic peptide MOG35-55 (MEVGWYRSPFSRVVHLYRNGK, from GL

Biochem, Shanghai, China), emulsified 1 :1 (0.1 ml: 0.1 ml) in complete Freund's adjuvant (CFA) (Difco, Detroit, USA) containing 4mg/ml_ Mycobacterium tuberculosis (BD, Australia). On Day 0, the encephalitogenic mixture was injected subcutaneously into each flank of the abdomen. Immediately thereafter and again 48h later, each mouse was injected IP with 350 ng of pertussis vaccine (List Biological Laboratories, Campbell, USA) in 0.2 ml of PBS.

Assessment of clinical score:

Mice were monitored daily and neurological impairments were quantified on an arbitrary scale: 0, no detectable impairment; 1 , flaccid tail; 2, hind limb weakness; 3, hind limb paralysis; 4, hind limb paralysis and ascending paralysis; 5, moribund or deceased. Following the recommendations of the animal ethics committee, mice were euthanized after reaching a clinical score of 4 and were given an arbitrary score of 5. Likewise, any mice that exhibited unexpected signs such as skin ulceration at a site of injection or ascites, deemed to cause pain and/or suffering were humanely killed immediately and given a score of 5, as per above. .

Histology:

At the completion of the experiments, mice were anesthetized, their blood and spleen collected (for subsequent antibody determination, T cell proliferation assays and cytokine profiles), and brain and spinal cord carefully removed, prior to immersion in 10% buffered neutral formalin (Sigma, Cat. No HT501128, USA) for 7 days. Fixed tissues were dehydrated in ethanol gradient (60%, 75%, 90%, 100% ) prior to their immersion in xylene (Grale, Cat.No 5001 , Australia) and being embedded in paraffin wax. Sections of 5μιπ thickness were cut on a microtome (Leica, Model RM2135) from various regions of the spinal cord. Sections were stained with haemotoxylin-eosin (HE), Luxol Fast Blue (LFB) and Bielshowsky for evidence of inflammation, demyelination and axonal damage, respectively.

Staining details: For HE staining, sections were deparaffinized in xylene (Grale, 5001 , Australia) and 100% ethanol before rinsed in running tap water. Nuclei was stained with hematoxylin (Sigma, H-3136, USA) for 5 minutes, followed by differentiation with acid/alcohol solution

(hydrochloric acid (Ajax Finechem, A256, Australia) 1 mL and 70% ethanol, 100mL) for 2-3 sec, and washing with Scott's tap water (ammonium hydroxide (Ajax Finechem, A43, Australia) 2mL and distilled water, lOOOmL) for 8- 0 sec. Brief rinse was conducted 3 times with running tap water between each step. Then sections were counterstained with eosin (Amber Scientific, E0A1 , Australia) for 5-7 min. Finally, sections were dehydrated and cleared through 95% ethanol, 100% ethanol, and xylene (Grale, 5001 , Australia) before mounted with DPX mounting solution (Merck, 101979, Australia).

For LFB staining, sections were deparaffinized in xylene and gradient ethanol (100%, 95%, 80%) before rinsed in running tap water; and subsequently left in 0.1% LFB solution (Amber Scientific, LFB-25g, Australia, 0.1mg and 95% ethanol, 100 ml, glacial acetic acid, Lab-Scan, A8401 , Thailand, 0.5mL) for 2 hours at 60°C, before excess stain was rinsed off in 70% ethanol. After being rinsed in tap water; sections were differentiated twice firstly in lithium carbonate solution (saturated lithium carbonate, Merck, 05680, Australia, 4mL and distilled water, 96mL) for 5 sec, and then in the 70% ethanol for 10 sec; with sections rinsed with tap water in between. After this step, sections were checked under microscope to see if gray matter was clear and white matter sharply defined; otherwise differentiation steps would be repeated if necessary. At last, sections were counterstained with HE as described above; before mounted with DPX mounting solution (Merck, 101979, Australia).

For Bielschowsky staining, sections were deparaffinized and placed in pre-warmed (37°C) 10% silver nitrate (Ajax Finechem, 631 , Australia) solution for 15 min at 37°C. The silver nitrate solution was removed and kept for following steps, and sections were washed 3 times in distilled water. Concentrated ammonium hydroxide (Ajax Finechem, A43, Australia) was added drop-wise into the silver nitrate solution from last step until precipitation was formed and cleared again. Sections were transferred back to this ammoniacal silver solution and stained in a 37°C oven for 10 min; prior to be incubated in 1%) ammonium hydroxide (Ajax Finechem, A43, Australia) solution for 2 min. Sections were developed in developer working solution (10% formalin, Sigma, HT501128, USA 20mL, nitric acid, concentrated, Merck, 101799, Australia, 1 drop, citric acid, Merck, 100244, Australia, 0.5mg and distilled water, 100mL) for 3-5 min; and terminated by dipping sections into 1% aqueous ammonium hydroxide solution (Ajax Finechem, A43, Australia). Then sections were washed in distilled water 3 times of 1 min each; and incubated in 5% sodium thiosulfate (Merck, 106516, Australia) solution for 5 min. After being washed again in 3 changes of distilled water for 5 min each; sections were dehydrated and cleared through 95% ethanol, 100% ethanol and xylene (Grale, 5001 , Australia); before final mounting with DPX mounting solution (Merck, 101979, Australia).

Evaluation of histological lesions:

Semi-quantitative histological evaluation for inflammation, demyelination, and axonal damage/loss was performed and scored in a blinded fashion. Between 12 to 20 sections, cut at different locations along the spinal cord were examined for each mouse.

Inflammation scores were defined as follows: 0, no inflammation; 1 , small and localized cellular infiltrates; 2, mild cellular infiltrates in parenchyma; 3, moderate cellular infiltrates in parenchyma; 4, severe and diffuse cellular infiltrates in parenchyma. Myelin breakdown was scored as follows: 0, no demyelination; 1 , mild demyelination; 2, moderate demyelination; 3, severe demyelination. Axonal damage was assessed as follows: 0, axonal damage; 1 , mild axonal damage; 2, moderate axonal damage; 3, severe axonal damage.

Measurement of serum anti-NRP 2945 antibodies:

The anti-NRP2945 antibody response was assessed at day 40 PI for the prophylactic experiment and at day 39 days PI for the therapeutic experiments. These time point were chosen to allow maximum time for the generation of putative antibodies. Maxisorp 96-well C-96 plates (Nunc) were coated overnight at 4°C with 100ul of NRP 2945 at 10ug/mL in carbonate buffer ( 5mM Na2C03, 35mM NaHC03, pH 9.6). Plates were rinsed twice with 150uL of PBS (136.9mM NaCI, 2.68mM KCl, 10.1 mM Na2HP04, 1.76mM KH2P04, pH 7.4), then blocked for 3 hours with 1.5% BSA. Plates were washed three times with 150uL of PBS/0.05% Tween-20 before adding 100uL of mouse serum for 1 hour at room temperature. Each mouse serum was diluted in PBS at 1/50. Plates were washed four times before adding 100uL of anti-mouse IgG, A, M HRP-conjugated antibody (diluted 1/2000 in PBS) (Invitrogen) for 1 hour at room temperature. Plates were washed five times and then 100ul of substrate was added (0.4mg/mL OPD substrate (Sigma), 0.05M phosphate citrate pH5 (Sigma), 0.024% hydrogen peroxide). Plates were left in the dark for 15min before the reaction was stopped with 30uL of 3M HCI, and the OD was measured at 492nm using the Benchmark Plus microplate spectrophotometer (BioRad).

Data are expressed as specific absorbance. To that effect, an irrelevant scramble peptide was used as negative control. OD obtained with this scramble peptide was then subtracted from the OD obtained with the NRP peptide, to determine the specific reactivity of the sera against NRP 2945. Given that no positive anti-NRP antibodies were available, an anti-MOG antibody was reacted against MOG 35-55 as a quality control for the ELISA assay.

Measurement of anti-MOG antibodies:

To assess the anti-MOG antibody response in mice, blood was collected by cardiac puncture at the experimental end point and sera were isolated by centrifugation.

Maxisorp 96-well C-96 plates (Nunc) were coated overnight at 4°C with 100ul of MOG35- 55 peptide (GL-Biochem, Shanghai) at 5ug/mL in carbonate buffer (15mM Na2C03, 35mM NaHC03, pH 9.6). Plates were rinsed twice with 150uL of PBS (136.9mM NaCI, 2.68mM KCI, 10.1mM Na2HP04, 1.76mM KH2P04, pH 7.4), then blocked for 3 hours with 1.5% BSA. Plates were washed three times with 150uL of PBS/0.05% Tween-20 before adding 100uL of mouse serum for 1 hour at room temperature (each mouse serum was serially diluted in PBS at 1/50 or 1/250 and 1/1000). Plates were washed four times before adding 100uL of anti-mouse IgG, A, M HRP-conjugated antibody (diluted 1/2000 in PBS) (Invitrogen) for 1 hour at room tsmperature. Plates were washed five times and then 100ul of substrate was added (0.4mg/mL OPD substrate (Sigma), 0.05M phosphate citrate pH5 (Sigma), 0.024% hydrogen peroxide). Plates were left in the dark for 15min before the reaction was stopped with 30uL of 3M HCI, and the OD was measured at 492nm using the Benchmark Plus microplate spectrophotometer (BioRad). All samples were tested in triplicate. Proliferation of splenocytes:

For anti-CD38 and anti-CD28 stimulation, a fiat bottom 96-well plate (Nunc, USA, cat#167008) was coated with 50ul of an anti-CD3 (BD), anti-CD28 (BD) cocktail (each at 10ug/mL), overnight at 4°C. The contents of these wells were aspirated immediately prior to the addition of the splenocyte preparations. For MOG35-55 stimulation (specific stimulation), splenocytes were cultured in presence of soluble MOG35-55 (Gl_ Biochem, Shanghai, China) at a final concentration of 20, 10, and 5ug/mL. For the non-specific response, splenocytes were cultured in presence of the non-specific mitogens, phorbol 12-myristate 13-acetate and ionomycin (PMA/lono) at a final concentration of 20 and 800ng/ml respectively. Spleens were removed ascepticaily and placed in media (RPMI (Invitrogen), 10% FCS (Gibco, Australia), 1 mM sodium pyruvate (Sigma), 50uM 2-mercaptoethanol (Sigma), 100units/mL Penicillin and 100ug/mL Streptomycin (Gibco), 200mM L-Glutamine (Gibco)) on ice. In a laminar flow hood they were cut then physically homogenized through a 70um filter (BD), then spun at 300xg (1300 rpm) for 5min before RBC lysis (155mM NH4CI, 10mM KHC03, 0.0892mM EDTA, pH 7.3); after 3 min lysis was stopped by the addition of media. Splenocytes were filtered a second time through a 70um membrane, then washed twice by pelleting and re-suspension in 14mL of media. Splenocytes were finally re- suspended in 1 mL of media and counted with a Coulter counter (Z2 Coulter Particle Count and Size Analyzer, Beckman Coulter). Splenocytes were then adjusted to equal densities and 2.5x105 cells were added to each well of the 96-well plate in a final volume of 200ul. Each activation was performed in triplicate. Splenocytes were kept in media on ice during all incubations. Cells were incubated at 37°C and 5% C02 for 48 hours then 10ul of media containing 1 uCi of tritiated thymidine was added to each well. After a further 18 hours of incubation, the plates were harvested with Filtermate Harvester (Packard) onto a glass fiber filter (Perkin Elmer). After these were dried, a drop of Microscint-0 Scintillant (Perkin Elmer) was added to each well and counts were measured using Top Count NXT microplate scintillation and luminescence counter (Perkin Elmer). Data are expressed as stimulation index, whereby spontaneous incorporation of 3H-thymidine in wells containing medium alone was arbitrarily set to 1 and proliferation in presence of MOG peptide as multiple thereof.

Detection of pro- and anti-inflammatory cytokines

For anti-CD3e and anti-CD28 stimulation, a flat bottom 24-well plate (Nunc, USA) was coated with 300ul of an anti-CD3 (BD), anti-CD28 (BD) cocktail (each at lOug/mL), overnight at 4°C. The contents of these wells were aspirated immediately prior to the addition of the splenocyte preparation. For OG35-55 stimulation, splenocytes were cultured in presence of soluble MOG35-55 (GL Biochem, Shanghai, China) at a final concentration of 20ug/mL.

Spleens were removed asceptically and placed in media (RP I (Invitrogen), 10% FCS (Gibco, Australia), 1 mM sodium pyruvate (Sigma), 50uM 2-mercaptoethanol (Sigma), 100units/ml_ Penicillin and 100ug/mL Streptomycin (Gibco), 200mM L-Glutamine (Gibco)) on ice. In a laminar flow hood they were cut then physically homogenised through a 70um filter (BD), then spun at 300xg (1300 rpm) for 5min before RBC lysis (155m NH4CI, 10mM KHC03, 0.0892mM EDTA, pH 7.3); after 3 min lysis was stopped by the addition of media. Splenocytes were filtered a second time through a 70um membrane, then washed twice by pelleting and re-suspension in 14mL of media. Splenocytes were finally re- suspended in 1 mL of media and counted with a Coulter counter (Z2 Coulter Particle Count and Size Analyzer, Beckman Coulter). Splenocytes were then adjusted to equal densities and 2.5x106 cells were added to each well of the 24-well plate in a final volume of 1 ml. Splenocytes were kept in media on ice during all incubations. Cells were incubated at

37°C and 5% C02 for 72 hours then supernatant of culture were collected for cytokines detection analysis by flow cytometry using BD Cytometric Bead Array (CBA) mouse TH1/2/17 kit. Briefly, 15μΙ of supernatant of culture are incubated 2 hours at room temperature in the dark with 15μΙ of antibody-conjugated beads to capture the analytes and 15μΙ of a second antibody with a phycoerythrin (PE) label to detect the analytes bound to a bead. After one wash with FACS Buffer (1X PBS, 1 % FBS, 5 mM EDTA, 0.02% NaN3) the quantity of cytokines bound to beads are analysed using BD FACS Canto II. Each bead has a unique fluorescence intensity that resolves as a unique population on the flow cytometer. The PE signal is proportional to the amount of bound analyte. The concentration of cytokines in every sample was calculated using the analysis software FCAP Array™.

Results.

3.1 : Prophylactic impact of NRP peptides on the development of EAE in C57BL/6 mice.

In this study, the potential of NRP 2945 and NRP 2983 to prevent as well as to influence disease progression in the EAE model was investigated. Both NRP peptides were administrated daily for a period of 15 days, starting from day 4 post EAE induction (PI). NRP 2983 was administrated IP at a concentration of 10pg/kg while NRP2945 was injected either IP (A^g/kg or ^g/kg) or SC (10pg/kg). Mice were followed for EAE signs until day 39, PI. a) Clinical signs of EAE in mice treated with NRP peptides. As shown in Figure 1 , treatment with NRP2945, particularly at the dosage of 10pg/kg injected IP, prevented the development of clinical signs of EAE in 50% of the mice, as compared to the 90% incidence, observed in the EAE and saline control groups. As evidenced by the reduction in clinical scores (mean clinical scores + sem of 0.95±0.32, 0.95±0.30 and 1.45+0.36 for NRP1945-1 Opg/kg: IP, NRP2945-4Mg/kg:IP and NRP2945- 10pg/kg: SC, respectively), the maximum score (0.95+0.32, 0.95±0.30 and 1 ,45±0.36 for NRP2945-10pg/kg: IP, NRP1945-4pg/kg:IP and NRP1945-10pg/kg:SC, respectively) and the cumulative score (26.25±9.16, 30.64+7.96 and 35.25±9.18 for NRP1945-1 Opg/kg: IP, NRP2945-4pg/kg:IP and NRP2945- 0pg/kg:SC, respectively), observed up to day 39 post EAE induction, this effect was long lasting. By contrast severe clinical signs of EAE were observed in the 2 control groups (mean clinical scores at day 39 being; maximum scores

53.72±8.67 and 43.60±7.34, for EAE and saline controls, respectively). The

peptidomimetic NRP2983 only showed a mid effect on the signs EAE, which was not statistically significant as compared to the control groups (Figure 1). Thus, the mean clinical score of mice treated with 10 pg/kg NRP 2945 IP was significantly different from that of the EAE (1way ANOVA; p<0.0001) and Saline groups (p<0.001 ). Likewise, the mean clinical score in mice treated with 4 ug/kg NRP 2945 IP at the experimental end point was similar to that of the 10 μg/kg NRP 2945 IP group and was significantly different from the EAE and Saline groups (pO.001 and p<0.05 respectively). Subcutaneous injection of NRP 2945 also lead to a significant reduction of the clinical signs of EAE, compared to EAE group (p<0.01). b) Histological analysis of EAE.

In addition to the impact of the NRP peptides on the clinical signs of EAE, their influence on inflammation, demyelination and axonal damage and loss in the CNS was examined by histological studies on fixed CNS tissues using haemotoxylin-eosin, Luxol fast blue and Bielshowsky staining. As illustrated in Figures 2 and 3 and Table 1 , evaluation of stained spinal cord sections of control EAE and saline-treated mice showed extensive inflammatory lesions, demyelination and axonal damage with mean inflammatory scores of 2.80+0.2 and 2.45+0.25, mean demyelinating scores of 1.80+1.00 and 1.40±0.17, mean axonal damage scores of 1.92±0.10 and 1.55±0.30, respectively. This contrast with the pathology observed in spinal cord of mice treated with NRP 2945 where inflammatory lesions were less extensive and numerous (mean scoreisem; 1.62±0.40, 1.50+0.69 and 1.4010.53 for NRP2945-4Mg/kg: IP, NRP1945-10pg/kg:IP and NRP2945-10Mg/kg: SC, respectively). This was associated with a decrease in the amount and extent of demyelination (0.92±0.24, 0.95±0.45 and 1.00+0.35 for NRP2945-4 g/kg: IP, NRP2945- 10pg/kg:IP and NRP2945-10pg/kg: SC, respectively) and a decrease axonal damage or loss (0.92+0.24, 1.00+0.48 and 1.20±0.40 for NRP1945-4Mg/kg: IP, NRP2945-10Mg/kg:IP and NRP2945-10Mg/kg: SC, respectively).

Histological assessment of the spinal cord of animal treated with NRP2983, similarly showed a reduction in inflammation, demyelination and axonal damage loss as compared to control mice (Figures 2, 3; Table 1 and Appendix 1 and 2, below).

Table 1 : Summary of the mean histological scores of EAE for the four groups of mice treated with the NRP peptides using the prophylactic model. Data represent the mean ± sem. *p<0.05, **p<0.01 compare to EAE control group (unpaired t test). c) Peripheral immune responses to MOG.

1) T cell proliferation assay.

Prevention of EAE in this model is generally thought to be a consequence of reduced antigen presentation or antigen recognition of the MOG peptide by antigen-presenting cells and T cells, respectively. This would lead to a reduced expansion of antigen-specific T cells in the periphery. In order to investigate this possibility, the presence of MOG peptide-recognizing T cells was assessed in an antigen-specific T cell proliferation assay. To this end, splenocytes from animals of different treatment groups were incubated with MOG 33-55 and T cell proliferation was determined using radioactive thymidine incorporation. Given the reduction of EAE signs and lesions in NRP-treated mice, we thus ask whether the T cell immune responses to MOG in such treated mice was affected, particularly because auto-reactivity to MOG is the driving force behind the pathogenesis of EAE. To that effect, we performed re-call proliferation assays to assess the ex vivo proliferative response of splenic T cells to MOG and to non-specific mitogens. As indicated in Figure 4, reactivity to MOG35-55 (20 pg/ml), was detected in each of the groups tested, regardless of their treatment. No differences in the capacity of splenic T cells to proliferate in response to MOG or to the two non-specific mitogens,

(PMA/lonomycin and the anti-CD3/anti-CD28 antibodies) were observed between groups. As judged by the strong T cell proliferation obtained, it would appear that none of the NRP peptides interfere with the T cell-mediated autoimmune response to MOG. Clearly, the continued presence of NRP during the induction phase of EAE (from day 4 onward) did not prevent the formation of MOG peptide specific T cell clones.

2) Cytokine production

Given that the treatment with NRP peptides had little impact on the T cell reactivity to MOG, we next assessed whether in such animals the phenotype of T cells was altered, by determining the cytokines profiles of cultured splenocytes in presence of MOG and the non-specific mitogens, PMA/lonomycin and the anti-CD3/anti-CD28 antibodies. Culture supernatants were collected after 72h in culture and were analyzed by FACS for the production of pro-inflammatory TH1/17 cytokines (IL-2, IFNy, TNF and IL-17), and anti- inflammatory TH2 cytokines (IL-4, IL-6 and IL-10). Upon MOG stimulation, the overall amounts of Th1/17 cytokines produced by the splenocytes of NRP-treated mice were similar, although slightly reduced as compared to the control groups (Figure 5). A small, albeit significant reduction (p<0.05; 2-way ANOVA) in the IFNy level was observed in the group treated with NRP 2945, administrated SC. Figure 5 also shows that when

NRP2945 or NRP2983 were administrated to the mice, the level of the TH2 cytokines, IL- 4 produced in response to MOG, was significantly lower (p<0.05 to p<0.001) than that of the EAE group (Figure 5). No significant differences could be found in the production of the 2 other TH2 cytokines, namely IL10 and IL-4, even though their levels were reduced as compared to controls (Figure 5). Upon CD3/CD28 stimulation, splenocytes from mice treated with the higher concentration of NRP 2945 or NRP 2983 significantly produced reduced levels of TNF-alpha and IL-17A (p<0.001 to p<0.0001 ; 2 way ANOVA) as compared to splenocytes from the EAE control group. Significant differences were observed in levels of IL-4, IL-6 and IL-10 (p<0.01 and p<0.0001 ; 2 way ANOVA) (Figure 5). On the basis of these results, it would appear that the modulation of EAE, following treatment with NRP peptides might be the result of a shift to a TH2 response that occurs through bystander suppression, rather than an effect on MOG specific T lymphocytes.

3) Antibody responses to NRP 2945.

The potential of NRP 2945 to be immunogenic and thus produce an unwanted antibody response, was investigated by ELISA in sera collected 40 post-immunization. Each serum was tested at 1/50 dilution against 10 g/ml of NRP 2945. As shown in Figure 6, no significant level of anti-NRP 2945 antibody could be found in any of the animals treated with NRP 2945 or in the 2 control groups. As indicated by the reactivity obtained with a positive control antibody directed against MOG, the ELISA technique employed here worked appropriately. These results are therefore taken to indicate that under the experimental condition used, NRP 2945 is not immunogenic.

Therapeutic impact of NRP 2945 on progression of EAE in C57BL/6 mice. a) Clinical signs of EAE in mice treated with NRP 2945 peptide.

In this study, the potential of NRP 2945 to influence disease progression in the EAE model was investigated. To that effect, NRP 2945 was injected IP 14 days after the induction of EAE, at a time when all the mice presented with an EAE clinical score of 2 or slightly above. NRP 2495 was administrated either 40 pg/kg or 20 pg/kg every second day up to day 35 post-EAE induction. As for the prophylactic experiment, EAE was induced using 200 pg of MOG35-55. Given that no significant differences were observed between the EAE and Saline control groups for all the experimental paradigms tested (see above) and in order to minimize the number of animals, (a constraint imposed by the Monash

Animal Ethic Committee), only the EAE control was included. At the end of the experiment, the mice were sacrificed for immunological analysis, which included splenocytes proliferation, cytokines production, serum anti-MOG and anti-NRP 2945 antibodies responses. CNS was dissected and prepared for the pathological analysis, which included the blind assessment of inflammation, demyelination as well as axonal damage and loss in the spinal cord. As for the prophylactic experiment, optic nerves were dissected, sectioned and sent to CuroNZ for subsequent analysis. After randomizing mice in each treatment group 9 days post-EAE induction, two mice in the 20pg/kg NRP group developed a few days later, unusually severe and progressive signs of paralysis (EAE score 3 at day 14), and therefore needed to be excluded from that group. We were also required to euthanize four paralyzed mice in the 4( g/kg NRP group and one in the EAE group as these presented with skin ulceration in their tails. In both cases, mice were given a score of 5 (see Assessment of clinical score section, above). According to the Monash Veterinarian, this occurred as the consequence of some bacterial infections, likely associated with loss of sensation and over grooming. In order to take into account each of these events, the results obtained on the therapeutic effects of NRP 2945 are presented separately in Figure 7 A, B C and in Tables 2 A, B, C.

As clearly indicated in Figure 7, treatment with the 20pg/kg NRP 2945 (Figure 7 A, B, C) significantly reduced the progression of EAE and also led to functional recovery, regardless of whether animals that progressed rapidly to severe signs of EAE or suffered skin ulcerations were removed from the analysis (Figure 7 B and C; Table 2 B, C). As illustrated in Table 2 A, B, the mean (± sem) end clinical and cumulative scores

(1.44±0.42 and 47.94± 5.16 respectively and 1.55+0.34 and 52.55±5.10 for Table 2 C) were significantly different (p<0.05) from that observed in the EAE control group (3.38± 0.34 and 75.39±6.12, respectively for Table 2 A). Recovery of mice treated with 20pg/kg NRP 2945, became evident some 8 days after the start of the treatment and by 25 onward, was significantly different from the control EAE group (p<0.05 to p<0.0001 ; 2 way ANOVA), Figure 7 A, also shows that when EAE mice, with skin ulcerations were removed from the analysis, animals treated with 40 g/kg display a significant reduction in their mean clinical scores: from 2.6 at day 5 post EAE induction to a mean of 2.0 at termination end point (p<0.05 to p<0.01 ; 2 way ANOVA). These data are summarized in Table 2 below.

Cumulative score 76.72 ± 6.97 56.00 + 12.02 47.94 ± 5.17*

B

C

Table 3 summarizes the data pertaining to the clinical signs of EAE for the 2 groups of mice treated with NRP 2945. Data represent the mean ± sem, statistically significant at * p<0.05 and **p<0.01 compare to the EAE group using Unpaired t test. b) Histological analysis of EAE.

The influence of the therapeutic treatment with NRP 2945 on inflammation, demyelination and axonal damage and loss in the spinal cord of the animals was assessed. Histological assessment was performed on fixed CNS tissues using haemotoxylin-eosin, Luxol Fast Blue and Bielshowsky staining. As illustrated in Figure 8 and summarized Table 4, evaluation of stained spinal cord sections of control EAE mice (n=10) showed extensive inflammatory lesions, demyelination and axonal damage with mean inflammatory scores (± sem) of 3.18+0,19, mean demyelinating scores of 2.30+0.11 and mean axonal damage scores of 2.35+0.08, respectively, This contrasts with the pathology observed in spinal cord of mice treated with 20pg/kg NRP 2945 (n=8), where inflammatory lesions, signs of demyelination and axonal damage and loss, were significantly fewer and less extensive (mean score±sem; 1.53+0.22, 0.97 ± 0.2 and 1.25 + 0.23 respectively), than the EAE control (p<0.01 to p<0.0001 ; one-way ANOVA). The mean score of inflammation for this group was also significantly different (p<0.05) from that of the 40yg/kg NRP 2945-treated group (Table 4). Histological analysis of the spinal cord of animal treated with 40yg/kg NRP 2945 (n=10), similarly showed a reduction in inflammation (2.48 ± 0.27), a decrease in the extent of demyelination (1.58 + 0.22) and a decrease in axonal damage/loss (1.48 + 0.24**) as compared to control EAE mice. These differences were statistically significant at p<0.05 for the inflammation and demyelination and at p<0.01 , for axonal damage and loss using one-way ANOVA.

Table 4 summarizes the mean histological scores of EAE for the 3 groups of mice studied in the therapeutic model. Data represent the mean + sem. Histogram representation of these data are outlined in Figure 8.

* p<0.05, ** p<0.05, *** p<0.05, **** p<0.05 compared with EAE Δ p<0.05 compared with NRP 40ug/kg C) Peripheral immune responses to MOG. 1) T cell proliferation assay.

Given the differences observed in the severity of clinical signs and histological lesions of EAE, between the animals treated with the NRP peptides and the EAE control mice, we then ask whether the former groups of mice had reduced T cell responses to the encephalitogen MOG as compared to the control EAE mice. Thus, we performed re-call proliferation assays to assess the ex vivo proliferative response to MOG and to the nonspecific mitogens, PMA/lonomycin and anti-CD3/CD28 antibodies. To that effect, splenocytes from animals of different treatment groups were incubated with incremental amount of MOG 33-55 as well as the 2 non-specific mitogens, PMA/lonomycin and anti- CD3/CD28 antibodies. T cell proliferation was determined using radioactive thymidine incorporation. In vitro proliferation assay shows that overall, splenocytes from C57BL/6 mice treated with NRP 2945 proliferated upon stimulation with each concentration of MOG35-55 and to the non-specific mitogens, regardless of the NRP dosage (Figure 10). There was a small, but nonetheless significant reduction (p<0.05 to p<0.001) in the capacity of splenic T cells from the NRP 2945-treated mice to proliferate in response to MOG35-55. Under non-specific stimulation via the protein kinase C pathway (PMA/lono), the 20ug/kg NRP 2945 leads to 30% down regulation of the proliferative response. Under full TCR response stimulation (CD3/CD28), this effect becomes dramatic for the 20ug/kg NRP2945 group: the proliferation rate is down regulated by more than 75% when compared to the EAE group (Figure 10). Given that good T cell proliferation was observed in each group of mice tested, it would appear that the T cell-mediated autoimmune response to MOG was not greatly affected by the NRP therapeutic treatment. Clearly, and as observed in the prophylactic experiment, the putative presence of NRP during the effectors' phase of EAE (from day 14 onward) did not, at least in vitro, affect the ability of T cells to recognize as well as to be stimulated by MOG.

2) Cytokine production

Given that the treatment with NRP peptides had some impact on the T cell reactivity to MOG, we next assessed whether in such animals the phenotype of T cells was altered, by determining the cytokines profiles of cultured splenocytes in presence of MOG and the non-specific mitogen, anti-CD3/anti-CD28 antibodies. Culture supernatants were collected after 72h in culture and were analyzed by FACS for the production of pro-inflammatory TH1/17 cytokines (IL-2, IFNy, TNF and IL-17), and anti-inflammatory TH2 cytokines (IL-4, IL-6 and IL-10). In line with the observation made with the prophylactic treatment, the overall amounts of Th1/17 cytokines produced by the splenocytes of NRP-treated mice upon MOG stimulation, were similar to that observed in the EAE control group (Figure 1 1). Upon stimulation with anti-CD3/anti-CD28 antibodies, IL-2 secretion in the 20ug/kg NRP 2945-treated mice was enhanced by more than 100% compared to the negative control (p<0.01 ; 2-way ANOVA), while in the group treated with the higher dose of NRP 2945 (40ug/kg), TNF-alpha secretion only increased 30%, when compared to the EAE control group (p<0.05; 2-way ANOVA). Figure 11 also shows that when NRP 2945 was administrated therapeutically to the mice, the level of the TH2 cytokines, IL-4 was increased by a massive 700%, as compared to the EAE group (p<0.001 ; 2-way ANOVA). Although not as dramatic, the levels of IL-6 produced in response to the CD3/CD28 stimulation (but not to MOG) were also significantly higher (p<0.01 to pO.0001 ; 2-way ANOVA)) than that of the EAE group (Figure 11). No significant differences could be found in the production of the other TH2 cytokine, namely IL10. Collectively, these data clearly indicate that under the influence of NRP 2945, the differentiation profile has shifted to a Th2 phenotype. In this context and given the very high of II-2 produced in response to NRP 2945, it should be noted that while CD4+CD25+ regulatory T cells do not produce IL-2, this cytokine is essential for their regulatory function, mediated by anti-inflammatory cytokines, such as IL-4 and IL-6.

3) Anti-MOG antibody responses.

Sera were collected from all mice at the end of the experiment (day 39) and analyzed by ELISA for their anti-MOG antibody responses at 1/50, 1/250 and 1/1000 dilutions. As shown in Figure 12, no significant differences, in the level of specific anti-MOG antibodies (as determined by the specific absorbance) was observed between sera from mice treated with NRP 2945 and the EAE control group.

4) Antibody responses to NRP 2945.

The potential of NRP 2945 to be immunogenic was once again investigated here by ELISA in sera collected 39 post EAE induction. Each serum was tested at 1/50 dilution against 10 pg/ml of NRP 2945. As shown in Figure 13, no significant level of anti-NRP 2945 antibody could be found in any of the animals treated with NRP 2945. As indicated by the reactivity obtained with a positive control antibody directed against MOG, the ELISA was used was appropriate. Given that the level of reactivity to NRP2945 was identical to that of the untreated control EAE mice, these results are taken to indicate that under the experimental condition used here, NRP 2945 is not immunogenic.

This study was undertaken to assess the effects of NRP 2945 and NRP 2983 on prevention and treatment of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS) that mimic many features of this human disease. EAE was induced in G57BL/6 mice by immunization with a peptide derived from myelin oligodendrocyte glycoprotein (MOG), a specific central nervous system autoantigen that is also a target in MS.

In the prophylactic intervention, NRP 2945 was dosed at 4 and 10 pg/kg and injected intraperitonealiy (IP) starting on Day 4 after the immunization for 5 days (up to day 18 post EAE induction). A separate group of mice also received 10 pg/kg of NRP 2945 subcutaneously (SC), starting on Day 4 after the immunization for 15 days. Finally, the peptidomimetic NRP2983 dosed at 10 pg/kg and injected intraperitonealiy (IP) was administrated on Day 4 for 15 days. In the therapeutic treatment, NRP 2945 was dosed at 20 and 40 pg/kg and injected intraperitoneal^ (IP) every second day, starting on Day 14 after the immunization up to day 35 post-EAE induction.

Under the Prophylactic protocol, both concentrations of NRP 2495 either administrated IP or SC were found to significantly reduce the severity of clinical signs as well as the progression of EAE, as compared to controls mice. Mice treated with NRP 2945 injected IP showed an increased efficacy as compared to the group of mice treated with NRP 2495, administrated SC. Notably, this effect was long lasting. NRP 2983 had no significant effect on the clinical course of the disease. The protective effects of NRP 2945 in this EAE model were further emphasized by the observation that cellular infiltrates in the CNS as well as demyelination and axonal damage were markedly diminished as compared to control EAE mice. Despite its lack of impact on clinical signs of EAE, NRP 2983 administration significantly reduced the histological features of this MS-like disease. Since no differences were observed between NRP-treated and controls mice in the ability of their T ceils to proliferate to MOG and the non-specific mitogens, it is unlikely that protection against EAE is the result of an inhibition of the adaptive immune response. Quantification of the pro-inflammatory cytokines obtained under non-specific stimulation reveals that mice treated with the higher dose of NRP produced lower levels of pro- inflammatory cytokines. This included the TNF alpha and IL17A, both of which have been shown to activate T cells and play a crucial role in the development of EAE. Finally, as shown by the fact that no antibodies were produce after 15 injections, NRP 2945 is unlikely to be immunogenic.

Under the Therapeutic protocol, NRP 2495 administrated IP at a concentration of 20 pg/kg, significantly reduced the progression of EAE and also led to functional recovery. This was associated with a significant decrease in cellular infiltrates as well as demyelination and axonal damage in the spinal cord. These effects were also observed when NRP 2945 was administrated at a dosage of 40 pg/kg, albeit to a lesser extent. While T cells from both groups proliferated upon stimulation with MOG and the non- specific mitogen, anti-CD3e and anti~CD28, a significant reduction in T cell proliferation was observed as compared to the control untreated EAE mice. There were no differences in the level of pro-and anti-inflammatory cytokines upon stimulation to MOG. However, quantification of the pro-and anti-inflammatory cytokines obtained under non-specific stimulation reveals that mice treated with NRP produced greater levels of pro-and anti- inflammatory cytokines, namely, IL-2, TNF alpha, IL-4 and IL-6. NRP 2945 treatment had no effect on the production of anti-MOG antibodies and animal injected with NRP 2945 did not produce anti-NRP 2945 antibodies.

In summary, the data presented in this report shows that under defined EAE experimental conditions, NRP 2945 (and to a far lesser extent, NRP 2983) can prevent as well as suppress the severity of EAE clinical signs as well as the pathology associated with this disease. From the immunological and phenotypic assessments of the peripheral T cells of EAE-protected mice, it is unlikely (although not totally excluded) that mechanism(s) by which NRP peptides produced their effects, involve specific T cell anergy, peripheral immunoregulation or clonal deletion. In view of the differential expression of both the TH1/17 pro-inflammatory and TH2 anti-inflammatory cytokines, such as IL-2, IL-6, IL-4 and IL-10 and the purported role that NRP 2945 has on proliferation, migration, differentiation and survival of neural precursor cells, we would suggest that perhaps NRP peptides interfere with the secretion of these cytokines within the CNS, by either T cells and/or brain cells such astrocytes and microglia, which are known to be the main source of these cytokines. The examination of the optic nerve for potential regeneration may further help in clarify this important issue. In this context it is noteworthy that while stem cells (know to have important regenerative properties) can interfere with the induction phase EAE, these have at least in our hand, have had little effect when transplanted at the time when EAE is well established (see publications at the end of the report). More over and in contrast to the potential regenerative potential of NRP2945, our work as well as that of others, have indicated that the prevention of EAE by stem cell transplantation, is likely to involve a direct effect on pathogenic T cells via the modulation of antigen presenting cell function. One of the approved relapsing-remitting drugs, Fingolimod (Gilenya(r)), is showing efficacy as low as 1 mg/kg in the therapeutic mouse MOG-EAE model (see but toxicity issues have restricted the FDA-approved human dosage to 0.5mg per human patient (which is a factor >100 below the effective dosage in the respective mouse model) which makes the drug unlikely to work in the current progressive MS clinical phase 3 trials. NRP2945 has already been shown effective therapeutic data in dogs displaying chronic spinal cord injury at a dosage of 10-15ug/kg. This

pharmacodynamic efficacy of NRP2945 renders it potentially to be effective in the future when trialled in human progressive MS patients.

Although the invention has been described with reference to an embodiment or example it should be appreciated that variations and modifications may be made to this embodiment or example without departing from the scope of the invention. Where known equivalents exist to specific features, such equivalents are incorporated as if specifically referred to in this specification. In particular, it is anticipated that functionally similar peptide sequences may be obtained by substitution of one or more amino acids of the biosequence with a functionally similar amino acid. It is suggested that the functionality of similar peptide sequences may be confirmed without undue additional experimentation by use of the method disclosed in this specification.

REFERENCES:

Sara A Litwak, Natalie Payne, Naomi Campanale, Ezgi Ozturk, Jae Young Lee, Steven Petratos, Christopher Siatskas, Maha Bakhuraysah and Claude C.A. Bernard. Nogo- Receptor 1 deficiency has no influence on immune repertoire or function during experimental autoimmune encephalomyelitis. Plos One. (2013) 8:e82101

Nicolas Molnarfi, Ulf Schulze-Topphoff, Martin S. Weber, Juan C. Patarroyo, Thomas Prod'homme, Michel Varrin-Doyer, Aparna Shetty, Christopher Linington, Anthony J. Slavin, Juan Hidalgo, Dieter E. Jenne, Hartmut Wekerle, Raymond A. Sobel, Claude C.A. Bernard, Mark J. Shlomchik, and Scott S. Zamvil. MHC class ll-dependent B cell APC function is required for induction of CNS autoimmunity independent of myelin- specific antibodies. Journal Experimental Medicine. (2013) 210: 2921-2937.

Natalie Payne, Guizhi Sun, Courtney McDonald, Leon Moussa, Ashley Emerson-Weber, Severine Loisel-Meyer, Jeffrey A. Medin, Christopher Siatskas and Claude C.A. Bernard. Human adipose-derived mesemchymal stem cells engineered to secrete IL-10 inhibit APC function and limit CNS autoimmunity. Brain, Behavior and Immunity. (2013) 30:103-114.

Natalie L Payne, Guizhi Sun, Daniella Herszfeld, Pollyanna A Tat, Paul J Verma, Helena C Parkington, Harold A Coleman, Mary A Tonta, Christopher Siatskas, Claude C.A. Bernard.. Comparative study on the therapeutic potential of neurally differentiated stem cells in a mouse model of multiple sclerosis. PloS One (2012) 7; e35093. Christopher Siatskas, Natalie Seach, Guizhi Sun, Ashley Emerson-Webber, Aude Silvain, Ban-Hock Toh, Frank Alderuccio, B Thomas Backstrom, Richard L Boyd, and Claude C. A, Bernard. Thymic gene transfer of myelin oligodendrocyte glycoprotein ameliorates the onset but not the progression of autoimmune demyelination. Molecular Therapy (2012) 20, 1349-1359 Steven Petratos, Ezgi Ozturk, Michael F Azari, Courtney McDonald, Leon Moussa, Sara Litwak, Kasra Taghian, Stephen M Strittmatter and Claude C. A. Bernard. Limiting Nogo-A receptor 1 -dependent phosphorylation of CRMP-2 reduces axonal degeneration in EAE, Brain. (2012). 135:1794-1818. Jianmei Ma, Kenji F. Tanaka, Takahiro Shimizu, Claude C.A. Bernard, Akiyoshi Kakita, Hitoshi Takahashi, Steven E. Pfeiffer, Kazuhiro Ikenaka, Microglial cystatin F expression is a sensitive indicator for ongoing demyelination and active remyelination. Journal of Neuroscience Research. (2011) 89: 639-349. Chan, J., Ban, E.J., Chun, K.H., Wang, S., McQualter, J., Bernard, C.C.A., Toh, B.H. and Alderuccio, F. Methylprednisolone induces reversible clinical and pathological remission and loss of lymphocyte reactivity to myelin oligodendrocyte glycoprotein in experimental autoimmune encephalomyelitis. Autoimmunity, 2008; 41 : 405-413.

Jonathan L. McQualter and Claude C.A. Bernard. Multiple Sclerosis: A battle between degeneration and repair. J. Neurochem. 2006; 100:295-306.

Wang, D.W., Ayers, M.M., Catmull. D.V., Hazelwood, L.J., Bernard, CCA. and J.M.Orian. Astrocyte associated axonal damage in pre-onset stages of experimental autoimmune encephalomyelitis. Glia. 2005; 51 :235-240.

Tara Karnezis, Wim Mandemakers, Jonathan L. McQualter, Binhai Zheng, Peggy P. Ho, Kelly A. Jordan, Belinda M. Murray, Ben Barres, Marc Tessier-Lavigne and Claude C.A. Bernard. The neurite outgrowth inhibitor Nogo A is involved in autoimmune-mediated demyelination. Nature Neuroscience. 2004. 7: 736-744.

Ayers, M. M., Hazelwood, L. J., Catmull, D. V., Wang, D., McKormack, Q., Bernard, C. C. A., and Orion, J. M. Early Glial Responses in Murine Models of Multiple Sclerosis.

Neurochem. Int. 2004, 45:409-419.

Strle K, Zhou JH, Shen WH, Broussard SR, Johnson RW, Freund GG, Dantzer R, Kelley KW. lnterleukin-10 in the brain. Crit Rev Immunol. 2001 ; 21(5):427-49.

Maurus de la Rosa, Sascha Rutz, Heike Dorninger and Alexander Scheffold. lnterleukin-2 is essential for CD4+CD25+ regulatory T cell function. Eur. J. Immunol. 2004:34; 2480 - 2488.

Yang, JF, Tao.HQ, Liu, YM, Zhan XX, Liu, XY, Wang, JH, et al. Characterization of the interaction between astrocytes and encephalitogenic lymphocytes during the development of experimental autoimmune encephalomyelitis (EAE) in mice. Clin Exp Immunol. 2012; 170: 254-65. Zhou, X, Spittau, B, Krieglstein, K. TGFp signalling plays an important role in IL4-induced alternative activation of microglia. J Neuroinflammation. 2012; 4; 9:210. Choi, JW, Gardell, SE, Herr, DR, Rivera, R, Lee, CW, Noguchi, K, Teo, ST, , Yung, YC, Lu, M, Kennedy, G, Chun, J. FTY720 (fingoiimod) efficacy in an animal model of multiple sclerosis requires astrocyte sphingosine 1-phosphate receptor 1 (S1 P1) modulation. Proc Natl Acad Sci U S A. 2011 ;108 :751-6.