Treatment methods for autoimmune diseases

Abstract

The present disclosure generally relates to autoimmune diseases, disorders and conditions associated with a Group A Streptococcus infections and methods of treating or preventing such diseases, disorders or conditions, or more particularly rheumatic fever, rheumatic heart disease and Sydenham's chorea.

Description

[0001] Treatment methods for autoimmune diseases

[0002] Cross-reference to related applications

[0003] The present application claims priority from Australian Provisional Patent Application No. 2024902462 filed on 8 August 2024, the contents of which are incorporated herein by reference in their entirety.

[0004] Technical Field

[0005] The present disclosure generally relates to autoimmune diseases, disorders and conditions associated with a Group A Streptococcus infections and methods of treating or preventing such diseases, disorders or conditions, or more particularly rheumatic fever, rheumatic heart disease and Sydenham’s chorea.

[0006] Background

[0007] Rheumatic Heart Disease (RHD) is a neglected disease of poverty that affects 40 million people worldwide and annually leads to more than 350,000 deaths. It accounts for nearly 2% of all deaths from cardiovascular diseases and is the commonest cause of pediatric acquired heart disease (Watkins et al., 2017). In susceptible individuals, infections with Group A Streptococcus (GAS) initiate an autoimmune process that leads to Acute Rheumatic Fever (ARF). Approximately 60% of patients who experience at least one episode of ARF will develop irreversible damage of the heart valves, which characterizes RHD (Good, 2020). Up to 30% of patients with ARF will also develop Sydenham’s Chorea (SC), a neurob ehavi oral condition characterized by involuntary choreiform movements and neuropsychiatric impairment.

[0008] Currently, there is no specific treatment to arrest the progression of ARF to RHD and other associated sequelae. Additionally, there is no vaccine available to prevent repetitive Strep A infection, which can exacerbate ARF and drive the pathological process. At present, patients diagnosed with ARF are prescribed with regular (4 weekly) and long-term (up to 10 years or more) administration of penicillin or the like to prevent recurrent Strep A infections (Carapetis et al., 2016; Stollerman et al., 1955). Accordingly, there remains a clinical need for therapeutic agents and methods that are effective in treating autoimmune diseases associated with GAS infections and related sequelae.

[0009] Summary

[0010] The present disclosure is based, in part, on the surprising discovery that low dose IL-2 demonstrates efficacy in preventing the development of cardiac inflammation, conduction abnormalities and valvular disease in a well-established rodent model of RHD by reducing the production of cross-reactive antibodies against cardiac tissue. Moreover, low dose IL-2 was demonstrated to significantly decrease the production of autoantibodies that recognise connective tissue and neuronal proteins. Accordingly, low dose IL-2 may be effective in preventing or treating the autoimmune sequelae that can result from a Group A Streptococcus (GAS) infection.

[0011] In a first aspect, the present disclosure provides a method of treating or preventing an autoimmune disease, disorder or condition associated with a Group A Streptococcus (GAS) infection in a subject, said method including the step of administering a therapeutically effective amount of an IL-2 protein or a nucleic acid molecule encoding the IL-2 protein to the subject to thereby treat or prevent the autoimmune disease, disorder or condition.

[0012] In a second aspect, the present disclosure provides a method of preventing or inhibiting the production of autoantibodies in a subject with or at risk of developing a GAS infection, said method including the step of administering a therapeutically effective amount of an IL-2 protein or a nucleic acid molecule encoding the IL-2 protein to the subject to thereby prevent or inhibit the production of autoantibodies therein.

[0013] In a third aspect, the present disclosure relates to a method of increasing a level and / or activity of regulatory T cells in a subject with or at risk of developing a GAS infection, said method including the step of administering a therapeutically effective amount of an IL-2 protein or a nucleic acid molecule encoding the IL-2 protein to the subject to thereby increase the level and / or activity of regulatory T cells therein.

[0014] For the methods of the second and third aspects, the subject suitably has or is at risk of developing an autoimmune disease, disorder or condition associated with the GAS infection.

[0015] Suitably, the autoimmune disease, disorder or condition of the above aspects is selected from the group consisting of rheumatic fever, rheumatic heart disease (RHD) and Sydenham’s chorea. In some examples, the autoimmune disease, disorder or condition is rheumatic fever. In other examples, the autoimmune disease, disorder or condition is rheumatic heart disease. In various examples, the autoimmune disease, disorder or condition is Sydenham’s chorea.

[0016] According to particular examples of the above aspects, the therapeutically effective amount of the IL-2 protein or the encoding nucleic acid molecule is a low dose thereof or is equivalent to a low dose thereof. In certain examples, the IL-2 protein or the encoding nucleic acid molecule is administered at a dose of or a dose equivalent to less than about 3.5 MIU / day. For other examples, the IL-2 protein or the encoding nucleic acid molecule is administered at a dose of or a dose equivalent to between about 0.3 MIU / day to about 3.0 MIU / day.

[0017] In some examples, the aforementioned methods comprise administering at least a first course of the IL-2 protein or the encoding nucleic acid molecule to the subject, wherein the first course comprises administering the IL-2 protein or the encoding nucleic acid molecule once per day for at least 3 consecutive days to the subject.

[0018] Suitably, the IL-2 protein of the above aspects is or comprises a functional variant, fragment or derivative of a native or wildtype IL-2 protein. To this end, the IL-2 protein or the encoding nucleic acid molecule can be selected from the group consisting of aldesleukin, rezpegaldesleukin, NKTR-214, RO7049665, CC-92252, XmAb27564, MK-6194, AMG592, CUG252, mRNA-6231, BNT151, BNT153, AVB-001, salspera, TILT-123 and any combination thereof. In certain examples, the IL-2 protein or the encoding nucleic acid molecule is or comprises aldesleukin.

[0019] Referring to some examples of the above aspects, administration of the IL-2 protein or the encoding nucleic acid molecule increases a level and / or activity of regulatory T cells in the subject.

[0020] In other examples of the above aspects, administration of the IL-2 protein or the encoding nucleic acid molecule does not substantially modulate or alter, such as substantially increase, a level and / or activity of effector T cells in the subject. In certain examples of the above aspects, administration of the IL-2 protein or the encoding nucleic acid molecule decreases a level and / or activity of effector T cells in the subject.

[0021] According to certain examples of the above aspects, administration of the IL-2 protein or the encoding nucleic acid molecule prevents or inhibits production of autoantibodies to a cardiac protein, a connective tissue protein and / or a neural protein in the subject. The cardiac protein can be one or more of cardiac myosin and tropomyosin. The connective tissue protein can be one or more of laminin and keratin. The neural protein can be one or more of dopamine receptor 1, dopamine receptor 2, lysoganglioside and tubulin.

[0022] Suitably, the subject of the aforementioned aspects is receiving an antimicrobial agent for treatment or prevention of the GAS infection. To this end, the above methods may include the further step of administering an antimicrobial agent for treatment or prevention of the GAS infection to the subject. The antimicrobial agent can be selected from the group consisting of a penicillin antibiotic, a lincosamide antibiotic, a macrolide antibiotic, a glycopeptide antibiotic, a cephalosporin antibiotic and any combination thereof. In particular examples, the antimicrobial agent is selected from the group consisting of penicillin, amoxicillin, benzathine penicillin, clindamycin, erythromycin, clarithromycin, azithromycin, vancomycin, cephalosporin C, cefadroxil, cephalexin and any combination thereof.

[0023] In a third aspect, the present disclosure provides a kit for use in the method of any one of the aforementioned aspects, said kit comprising a low dose of an IL-2 protein or a nucleic acid molecule encoding the IL-2 protein, optionally an antimicrobial agent and optionally instructions for use. In a fourth aspect, the present disclosure provides a method of treating or preventing an autoimmune disease, disorder or condition associated with a GAS infection in a subject, said method including the step of administering a therapeutically effective amount of regulatory T cells to the subject to thereby treat or prevent the autoimmune disease, disorder or condition.

[0024] Brief description of the drawings

[0025] The following figures form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these figures in combination with the detailed description of specific embodiments presented herein.

[0026] Figure 1. Experimental timeline summarizing development of low-dose IL-2 (LD-IL-2) therapeutics for ARF / RHD using the Rat Autoimmune Valvulitis (RAV) model. Rats received 3 boosters with Strep A rM5 at weekly intervals. LD-IL-2 treatment began either on Day 8 or Day 21 post-primary injection with Strep A rM5. Rats were euthanized at Day 35 for assessment of carditis and immune responses.

[0027] Figure 2. Low-dose IL-2 induces functional improvement of the heart and reduces carditis. (A) Functional assessment of the heart before (•) and after (A) injection of PBS or rM5 by ECG. Antigen -injected rats (n=5-6 females / grp) received LD-IL-2 therapy at Day 8 (D8) or Day 21 (D21) post-primary injection or were left untreated. Statistical analysis performed by two-way ANOVA. n.s. non-significant, ** p<0.01. (B) Inflammatory changes in the myocardium and valvular tissue, characterized by mononuclear cell infiltration, were scored in rats treated with PBS, rM5, and LD-IL-2 therapy started at Day 8 or Day 21 post-primary rM5 injection. Box-and- whisker plot show min to max carditis scores within each treatment group. Statistical analysis for carditis score were obtained by one-way ANOVA. n.s. non-significant, *p<0.05, *** p<0.005, ***p<0.001. (C) Representative histological images of myocardium and valvular tissues. Arrows indicate inflammatory cell infiltration in the myocardium (blue arrows) and valves (red arrows). Scale bar = 50pm.

[0028] Figure 3. LD-IL-2 therapy reduces serum IgG cross-reactivity to host cardiac and neuronal tissue proteins. Serum IgG levels against cardiac proteins (tropomyosin and cardiac myosin), connective tissues (laminin and keratin) and neuronal proteins (dopamine receptors 1 and 2, lysoganglioside and tubulin). Absorbance values of Day-35 rat sera at 1 :400 dilution is shown (n=5-6 females / grp). Statistical analysis performed by one-way ANOVA with Dunnett multiple comparison test. n.s. non-significant, *p < 0.05, **p < 0.01, ***p<0.005, ****p< 0.001. Bars represent standard deviation (SD). Figure 4. LD-IL-2 therapy efficacy is associated with increase of Treg. Flow cytometry analysis of Treg cells in mediastinal lymph nodes. (A) Violin plot is used to compare the proportion on CD4+Treg (CD3+CD4+CD25+FoxP3+) between rats treated with PBS, rM5 only and rM5+LD- IL-2 (n=5-6). Each symbol represents an individual rat analyzed. (B) Representative gating strategy of CD4+ Treg. (C) Proportion on CD8+ Treg (CD3+CD8+CD25+FoxP3+) between treatment groups. Each symbol represents an individual rat analyzed (n=5-6 females / grp). *P < 0.05, **P < 0.01, ***P < 0.001. Non-parametric one-way ANOVA test with Bonferroni correction. Figure 5. LD-IL-2 therapy does not alter the frequency of conventional T-cells. Flow cytometry analysis of conventional T-cells in mediastinal lymph nodes. (A) Violin plot is used to compare the proportion of (A) total T-cells, (B) CD4+ T-cells and (C) CD8+ T-cells between rats treated with PBS, rM5 only and rM5+LD-IL-2. Each symbol represents an individual donor analysed (n=6 females / grp). Non-parametric one-way ANOVA test with Bonferroni correction, ns = non-significant.

[0029] Figure 6. LD-IL-2 therapy does not impair IgG antibody responses to a foreign antigen. Analysis of serum antibodies levels using ELISA. All rats (n=5-6) were given 2 doses of pertussis toxin (on days 3 and 5), regardless of the treatment group. Serum IgG antibody levels against pertussis toxin were measured at a 1 :400 dilution in healthy rats (PBS-injected), rM5-injected rats treated with low-dose IL-2 (LD-IL-2), and rM5-injected rats left untreated. Statistical analysis was performed by one-way ANOVA with Dunnett multiple comparison test. No significant difference was observed between the treatment groups.

[0030] Figure 7. LD-IL-2 restores germinal center (GC) B cell and regulatory T cell (Treg) balance in the spleen. Representative gating strategy for defining GC B cells and Treg is shown. Violin plot is used to show the proportion of (A) GC B cells (CD19+Bcll-6+CD38+) and (B) Tregs (CD3+CD4+CD25+FoxP3+) in rats injected with PBS or rM5, or given LD-IL-2 following rM5 boosters (n=5). Each symbol represents an individual rat analysed. Statistical analysis was performed by one-way ANOVA with Dunnett multiple comparison test. n.s. non-significant, *P<0.05 and **P<0.01.

[0031] Figure 8. LD-IL-2 therapy re-establishes immune homeostasis within the spleen. Analysis of spleen transcriptomes using bulk RNA sequencing. (A) Principal component analysis illustrates the relationship of mRNA from five biological replicates across PBS, rM5 and rM5+LD-IL-2 groups. (B) Venn diagrams show the overlap between quantified mRNA obtained from the different treatment groups. (C) Heatmaps display the expression patterns of top 50 mRNA transcripts differentially expressed across the treatment groups. Yellow: upregulated, blue: downregulated. (D) Box-plots present differentially expressed genes in rM5 versus rM5+LD-IL-2 groups (n=5). Genes were selected based on their involvement in inflammatory and autoimmune pathways associated with the activation of effector CD4+T cells. Each symbol represents an individual rat analysed. Statistical analysis was performed by one-way ANOVA with Dunnett multiple comparison test. n.s. non-significant, *P<0.05, **P<0.01 and ***0<0.005.

[0032] Figure 9. Adoptive transfer of CD4+ T cells isolated from LD-IL-2 treated rats reverse rM5- induced cardiac dysfunction. (A) Schematic showing isolation of CD4+ T cells from donor rats and treatment regimen of recipient rats. (B) Functional assessment of the heart before (•, Day 0) and after (A, Day 27) injection of PBS or rM5 by ECG. Antigen -injected rats (n=5 female s / group) received intravenous injection of 10 million LD-IL-2-expanded CD4+ T cells or were left untreated. Statistical analysis performed by two-way ANOVA. n.s. non-significant, ** p<0.01. (C) Inflammatory changes in the myocardium and valvular tissue, characterized by mononuclear cell infiltration, were scored in rats injected with PBS or rM5, which were either treated with CD4+ T cells or left untreated, i. Box-and-whisker plot shows min to max carditis scores within each treatment group. Statistical analysis for carditis score was performed by one-way ANOVA. n.s. non-significant, ****p < 0.001. ii. Representative histological images of myocardium and valvular tissues. Arrows indicate inflammatory cell infiltration in the myocardium and valves. Scale bar = 50 pm. (D) Serum IgG antibody levels were measured against i. cardiac myosin and ii. laminin. Statistical analysis was performed by one-way ANOVA with Dunnett multiple comparison test, n.s. non-significant, **P<0.01 and ****0<0.001.

[0033] Key to the Sequence Listing

[0034] SEQ ID NO: 1 Amino acid sequence of human IL-2 protein with signal sequence

[0035] SEQ ID NO: 2 Amino acid sequence of human IL-2 protein without signal sequence SEQ ID NO: 3 Nucleic acid sequence of mRNA sequence encoding human IL-2 protein SEQ ID NO: 4 Nucleic acid sequence of cDNA sequence encoding human IL-2 protein SEQ ID NO: 5 Amino acid sequence of aldesleukin

[0036] Detailed Description

[0037] General

[0038] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, feature, composition of matter, group of steps or group of features or compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, features, compositions of matter, groups of steps or groups of features or compositions of matter.

[0039] Those skilled in the art will appreciate that the present disclosure is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

[0040] The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the present disclosure.

[0041] Any example of the present disclosure herein shall be taken to apply mutatis mutandis to any other example of the disclosure unless specifically stated otherwise.

[0042] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as comm only understood by one of ordinary skill in the art (for example, in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0043] Unless otherwise indicated, the recombinant DNA, recombinant protein, cell culture, and immunological techniques utilized in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991 ), D M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988), and J.E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).

[0044] Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", is understood to imply the inclusion of a stated step or element or integer or group of steps or elements or integers but not the exclusion of any other step or element or integer or group of elements or integers.

[0045] By “consisting essentially of’ in the context of an amino acid sequence, such as an IL-2 protein, is meant the recited amino acid sequence together with an additional one, two or three amino acids at the N- or C-terminus thereof. By “consisting essentially of’ in the context of a nucleotide sequence is meant the recited nucleotide sequence together with an additional one, two or three amino nucleic acids at the 5’ or 3’ end thereof.

[0046] The term “and / or”, e.g., “X and / or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning. Those skilled in the art will appreciate that the present disclosure is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features. The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally equivalent products, compositions and methods are clearly within the scope of the disclosure, as described herein.

[0047] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e., one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.

[0048] As used herein, the singular forms of “a”, “and” and “the” include plural forms of these words, unless the context clearly dictates otherwise. For example, a reference to “a bacterium” includes a plurality of such bacteria, and a reference to “an allergen” is a reference to one or more allergens.

[0049] Throughout the present specification, various aspects and components of the disclosure can be presented in a range format. The range format is included for convenience and should not be interpreted as an inflexible limitation on the scope of the present disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range, unless specifically indicated. For example, description of a range such as from 1 to 5 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 5, from 3 to 5 etc., as well as individual and partial numbers within the recited range, for example, 1, 2, 3, 4, 5, 5. 5 and 6, unless where integers are required or implicit from context. This applies regardless of the breadth of the disclosed range. Where specific values are required, these will be indicated in the specification.

[0050] The term “about” in relation to a numerical value x is optional and means, for example, any number within (i.e., plus or minus) 1%, 5% or 10% of the referenced number. In certain examples, the term “about” encompasses the exact number recited.

[0051] The term “substantially” does not exclude “completely” (e.g., a composition which is “substantially free” from Y may be completely free from Y).

[0052] All computer programs, algorithms, gene and protein accession numbers and their associated sequences, patents, patent applications and scientific literature referred to herein is incorporated herein by reference. Methods of treating autoimmune diseases

[0053] The inventors have surprisingly shown for the first time that low dose IL-2 therapy demonstrates efficacy in preventing rheumatic heart disease in the Rat Autoimmune Valvulitis Model (RAV) model, which has been extensively characterized to evaluate the early events of rheumatic fever and its associated complications and sequelae. In this model, low dose IL-2 therapy was demonstrated to prevent the development of cardiac tissue inflammation and conduction abnormalities, whilst effectively reducing the production of cross-reactive antibodies against cardiac tissue, connective tissue and neural proteins. Administration of low dose IL-2 also induced a significant increase in classical (e.g., CD4+) regulatory T-cells (Tregs) and CD8+ Tregs in the mediastinal (heart-draining) lymph nodes. Such immunotherapeutic therapy therefore offers promise as a treatment for the autoimmune sequelae that may develop subsequent to a GAS infection, such as rheumatic fever, rheumatic heart disease and Sydenham’s chorea.

[0054] In one broad form, the present disclosure relates to a method of preventing or treating an autoimmune disease, disorder or condition associated with a Group A Streptococcus infection, said method including the step of administering a therapeutically effective amount of an IL-2 protein or a nucleic acid molecule encoding the IL-2 protein to the subject to thereby treat or prevent the autoimmune disease, disorder or condition.

[0055] In a related form, the present disclosure relates to the use of a therapeutically effective amount of an IL-2 protein or a nucleic acid molecule encoding the IL-2 protein in the manufacture of a medicament for treating or preventing an autoimmune disease, disorder or condition associated a Group A Streptococcus infection in a subject.

[0056] In yet another form, the present disclosure provides a therapeutically effective amount of an IL-2 protein or a nucleic acid molecule encoding the IL-2 protein for use in a method of treating or preventing an autoimmune disease, disorder or condition associated a Group A Streptococcus infection in a subject.

[0057] As used herein, the terms “Group A Streptococcus”, “Group A Streptococci”, “Group A Streptococcal”, “Group A Strep” and the abbreviation “GAS” refer to streptococcal bacteria of Lancefield serogroup A which are gram positive P-haemolytic bacteria of the species Streptococcus pyogenes. An important virulence factor of GAS infections, and also associated autoimmune disease, disorders or conditions, is the M protein, which is strongly anti -phagocytic and binds to serum factor H, destroying C3-convertase and preventing opsonization by C3b. Suitably, the GAS strain in question expresses an M protein. These also include virulent “mutants” such as CovR / S or CovRS mutants (see, e.g., Graham et al., 2002, PNAS USA 99 13855). Diseases and conditions caused by GAS infections include cellulitis, erysipelas, impetigo, scarlet fever, throat infections such as acute pharyngitis (“strep throat”), bacteraemia, toxic shock syndrome, necrotizing fasciitis, acute rheumatic fever and acute glomerulonephritis, although without limitation thereto. In various examples, the GAS infection is selected from the group consisting of cellulitis, erysipelas, impetigo, scarlet fever and acute pharyngitis. More particularly, the GAS infection can be scarlet fever (i.e., a skin infection) or acute pharyngitis (i.e., a throat infection). For certain examples, the GAS infection is scarlet fever. Referring to other examples, the GAS infection is acute pharyngitis.

[0058] As used herein, the term “autoimmune disease, disorder or condition” refers to a disease, disorder or condition in which a subject's immune system has an aberrant immune response against a substance, such as a self-protein or native protein, that does not normally elicit an immune response in a healthy subject. Similarly, the term “autoimmune disease, disorder or condition associated with a Group A Streptococcus infection” refers to an autoimmune disease, disorder or condition in which a subject's immune system has an aberrant immune response against a protein or other antigenic substance derived from a Group A Streptococcus bacteria that leads to the activation and / or induction of immune system components or elements, such as B cells and / or T cells, that recognise a self-protein or native protein. To this end, the autoimmune disease, disorder or condition may be characterized by or associated with the production of autoantibodies that cross-react with a self-protein of the subject, such as a cardiac protein, a connective tissue protein and a neural protein, and a GAS protein, such as an M protein. In this regard, the autoimmune disease, disorder or condition may be characterized or associated with molecular mimicry, when similarities between a foreign protein or antigen and a self-protein favours an activation of autoreactive T or B cells by the foreign protein in a susceptible individual.

[0059] As such, the autoimmune disease, disorder or condition may be diagnosed or detected in the subject by detecting the presence of autoantibodies or cross-reactive B cells or T cells therein. Furthermore, the autoimmune disease, disorder or condition may be characterized or associated with the induction or activation of effector immune cells, such as effector T cells (e.g., Thl and Thl7 cells) and / or effector B cells (e.g., cross-reactive B cells), in the subject. Moreover, the autoimmune disease, disorder or condition may be characterized or associated with the suppression or reduced levels of regulatory T cells, such as CD4+ Tregs and / or CD8+ Tregs, in the subject. Similarly, the autoimmune disease, disorder or condition may be diagnosed or detected in the subject by determining a level and / or activity of one or both of an effector T cell and a regulatory T cell, such as those described herein, in the subject.

[0060] Suitably, the IL-2 protein or the encoding nucleic acid molecule are administered after detection or diagnosis of the autoimmune disease, disorder or condition in the subject. The methods described herein may therefore include the earlier or initial step of diagnosing or detecting the autoimmune disease, disorder or condition in the subject. Such diagnosis or detecting may be performed by any means or method known in the art and inclusive of those provided herein.

[0061] Further to the above, the IL-2 protein or the encoding nucleic acid molecule can be administered to a subject determined to be at risk of developing the autoimmune disease, disorder or condition. The methods described herein may therefore include the earlier or initial step of determining a risk or likelihood of the subject developing the autoimmune disease, disorder or condition. Such determination may be based, for example, on the level and / or activity of effector T cells, effector B cells, regulatory T cells and autoantibodies in the subject.

[0062] In particular examples, the autoimmune disease, disorder or condition disclosed herein is rheumatic fever, rheumatic heart disease (RHD) or Sydenham’s chorea. More particularly, the autoimmune disease, disorder or condition may be rheumatic fever. More particularly, the autoimmune disease, disorder or condition may be rheumatic heart disease (RHD). More particularly, the autoimmune disease, disorder or condition may be Sydenham’s chorea.

[0063] Suitably, the autoimmune disease, disorder or condition is rheumatic fever. Accordingly, there is provided herein a method of treating or preventing rheumatic fever or a disease, disorder or condition associated therewith in a subject, said method including the step of administering a therapeutically effective amount of an IL-2 protein or a nucleic acid molecule encoding the IL-2 protein to the subject to thereby treat or prevent rheumatic fever or the disease, disorder or condition associated therewith.

[0064] In a related form, the present disclosure relates to the use of a therapeutically effective amount of an IL-2 protein or a nucleic acid molecule encoding the IL-2 protein in the manufacture of a medicament for treating or preventing rheumatic fever or a disease, disorder or condition associated therewith in a subject.

[0065] In yet another form, the present disclosure provides a therapeutically effective amount of an IL-2 protein or a nucleic acid molecule encoding the IL-2 protein for use in a method of treating or preventing rheumatic fever or a disease, disorder or condition associated therewith in a subject.

[0066] The terms “acute rheumatic fever”, “ARF” and “rheumatic fever” are used interchangeably herein and refer to a multifocal autoimmune disease that may affect the heartjoints, skin, kidneys and brain. To this end, rheumatic fever or the disease, disorder or condition associated can encompass, for example, rheumatic heart disease and Sydenham’s chorea. Accordingly, in some examples, the disease, disorder or condition associated with rheumatic fever is rheumatic heart disease and / or Sydenham’s chorea. Rheumatic fever typically develops two to four weeks after an untreated GAS infection, such as strep throat or scarlet fever, with clinical symptoms including fever, multiple painful joints, involuntary muscle movements, and occasionally a characteristic non-itchy rash known as erythema marginatum. Referring to some examples, the autoimmune disease, disorder or condition, or more particularly the disease, disorder or condition associated with rheumatic fever, is rheumatic heart disease. As such, in one form, the present disclosure provides a method of treating or preventing rheumatic heart disease in a subject, said method including the step of administering a therapeutically effective amount, such as a low dose, of an IL-2 protein or a nucleic acid molecule encoding the IL-2 protein to the subject to thereby treat or prevent rheumatic heart disease therein.

[0067] In a related form, the present disclosure relates to the use of a therapeutically effective amount, such as a low dose, of an IL-2 protein or a nucleic acid molecule encoding the IL-2 protein in the manufacture of a medicament for treating or preventing rheumatic heart disease in a subject.

[0068] In yet another form, the present disclosure provides a therapeutically effective amount, such as a low dose, of an IL-2 protein or a nucleic acid molecule encoding the IL-2 protein for use in a method of treating or preventing rheumatic heart disease in a subject. To this end, administration of the IL-2 protein or the encoding to the subject may at least partly treat or prevent the development of, for example, cardiac tissue inflammation, conduction abnormalities (e.g., arrhythmias) and / or valvular disease associated with the rheumatic heart disease in the subject.

[0069] The terms “rheumatic heart disease” and “RHD” are intended to encompass conditions affecting the heart following acute rheumatic fever including damage to the mitral valve and / or the aortic valve, myocarditis and pericarditis. To this end, RHD is typically considered to be a sequela of GAS infections in which antibodies and T-cells raised against epitopes in the structural M-protein cross-react with host heart proteins resulting in heart damage. This process, generally known as molecular mimicry, is a normal part of the host’s immune response to GAS infections (Cunningham, 2019). Previous studies have identified cross-reactive antibodies and T helper cells - producers of IFN-y and IL-17 (Thl and Thl7, respectively) - as key mediators of RHD heart lesions (Cunningham, 2000; Sikder et al., 2018). For initiation of disease, evidence suggests that a two-hit hypothesis may play a role: (i) autoantibodies binding and activating the valvular endothelium and (ii) subsequent extravasation of T-cells through activated endothelium into the valve. This is believed to lead to recognition of autoantigens and potentiation of the inflammatory process followed by tissue destruction (Carapetis et al., 2016). A deficiency of regulatory T-cells (Treg) has been reported in patients with RHD as well as a high Thl7 / Treg ratio, a phenotype exacerbated in patients with multivalvular compared to univalvular involvement (Bas et al., 2014; Mukhopadhyay et al., 2013), although a functional role for Tregs in the pathogenesis of RHD was not established in these studies.

[0070] In certain examples, the autoimmune disease, disorder or condition, or more particularly the disease, disorder or condition associated with rheumatic fever, is Sydenham chorea. As such, in one form, the present disclosure provides a method of treating or preventing Sydenham chorea in a subject, said method including the step of administering a therapeutically effective amount, such as a low dose, of an IL-2 protein or a nucleic acid molecule encoding the IL-2 protein to the subject to thereby treat or prevent Sydenham chorea therein.

[0071] In a related form, the present disclosure relates to the use of a therapeutically effective amount, such as a low dose, of an IL-2 protein or a nucleic acid molecule encoding the IL-2 protein in the manufacture of a medicament for treating or preventing Sydenham chorea in a subject.

[0072] In yet another form, the present disclosure provides a therapeutically effective amount, such as a low dose, of an IL-2 protein or a nucleic acid molecule encoding the IL-2 protein for use in a method of treating or preventing Sydenham chorea in a subject.

[0073] The terms “Sydenham chorea” and “Sydenham’s chorea” refer to an autoimmune sequelae or complication of rheumatic fever that is characterized by muscular weakness and chorea. Sydenham chorea (also referred to as St. Vitus dance) is a major manifestation of rheumatic fever occurring in up to 40% of patients with rheumatic fever. The resultant muscle weakness and involuntary movements may lead to a clumsy gait, slurred speech, and the inability to hold a grip. Sydenham chorea is thought to result from an autoimmune process mediated by antineuronal antibodies. The self-reactive antibodies appear to arise in response to GAS infections and then cross-react with antigens on neuronal cells within the basal ganglia and other brain regions.

[0074] In another broad form, the present disclosure provides a method of treating or preventing an autoimmune disease, disorder or condition associated with a GAS infection in a subject, said method including the step of administering a therapeutically effective amount of regulatory T cells, such as CD4+ regulatory T cells, to the subject to thereby treat or prevent the autoimmune disease, disorder or condition.

[0075] In various examples, the regulatory T cells may be derived from or contained within a population of T cells, such as a population of CD4+ T cells. As such, the method may include the step of administering a population of T cells to the subject, wherein the therapeutically effective amount of regulatory T cells is contained or present within the population of T cells. The regulatory T cells may comprise at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%,

[0076] 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%,

[0077] 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,

[0078] 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,

[0079] 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,

[0080] 98%, 99%, or about 100% of the population of T cells, or any range therein.

[0081] Alternatively, the regulatory T cells may be isolated from the population of T cells prior to administration to the subject. Accordingly, the present method may include the earlier or initial step of isolating a population of regulatory T cells from the population of T cells, such as a population of CD4+ T cells.

[0082] In some examples, the regulatory T cells may be isolated from the population of T cells and treated with IL2 prior to administration of the regulatory T cells to the subject. As such, the present method may include the earlier or initial steps of isolating a population of regulatory T cells from the population of T cells and treating the regulatory T cells with an IL-2 protein prior to their administration to the subject.

[0083] In other examples, the regulatory T cells may be derived from or contained within a population of T cells, such as a population of CD4+ T cells, that has been treated with an IL-2 protein. As such, the present method may include the earlier or initial step of treating the population of T cells with an IL-2 protein prior to the administration of the population of T cells, or of the isolated regulatory T cells, to the subject.

[0084] In some examples, the regulaettory T cells or T cell population may be isolated from a subject that has been treated with an IL-2 protein.

[0085] In other examples, the regulatory T cells and the population of T cells may be derived or obtained from a single subject or from a combination of multiple subjects (e.g., a plurality or two or more subjects). Suitably, regulatory T cells and / or the population of T cells are autologous and / or for use as an autologous therapy. Alternatively, the regulatory T cells and / or the population of T cells are allogeneic and / or for use as an allogeneic therapy.

[0086] It is envisaged that in some examples the regulatory T cells are modified regulatory T cells, such as chimeric antigen receptor (CAR)-regulatory T cells (CAR-Tregs). CAR-Treg cells are described, for example, in Arjomandnejad et al., 2022, Biomedicines (DOI 10.3390 / biomedicinesl0020287) and Doglio et al., 2024, Nature Communications (DOI 10.1038 / s41467-024-46448-9), herein incorporated by reference in their entirety.

[0087] The term “autologous”, as used herein, generally refers to cells or tissues, such as regulatory T cells, derived from the same individual or involving one individual as both donor and recipient. “Autologous therapy” refers to therapy that involves harvesting, isolating and / or generating cells or tissues, such as regulatory T cells, from an individual and using those cells or tissues as therapies for the same individual.

[0088] The term “allogeneic”, as used herein, generally refers to cells or tissues, such as regulatory T cells, derived from, or being from individuals of the same species that are not genetically identical to the intended recipient. “Allogeneic therapy” refers to therapy that involves harvesting, isolating or generating cells or tissues, such as regulatory T cells, from an individual or individuals and using those cells or tissues as therapies for a different individual or individuals. As used herein, the terms “treating”, “treat” or “treatment” and variations thereof, refer to clinical intervention designed to alter the natural course of the individual or cell being treated during the course of clinical pathology. Desirable effects of treatment include decreasing the rate of disease progression, ameliorating or palliating the disease state, and remission or improved prognosis. Accordingly, the therapeutically effective amount of the IL-2 protein or the encoding nucleic acid molecule described herein can be administered to the subject after development or diagnosis of the autoimmune disease, disorder or condition therein. Moreover, the therapeutically effective amount of the IL-2 protein or the encoding nucleic acid molecule described herein can be administered to the subject after determining the subject is at risk of developing the autoimmune disease, disorder or condition therein, such as when diagnosed as having the GAS infection.

[0089] As used herein, the terms “prevent”, “prevented”, or “preventing”, refer to a prophylactic treatment which increases the resistance of a subject to developing the disease or condition or, in other words, decreases the likelihood that the subject will develop the disease or condition as well as a treatment after the disease or condition has begun in order to reduce or eliminate it altogether or prevent it from becoming worse. These terms also include within their scope preventing the disease or condition from occurring in a subject which may be predisposed to the disease or condition but has not yet been diagnosed as having it. Accordingly, the therapeutically effective amount of the IL-2 protein, the encoding nucleic acid molecule, or the regulatory T cells described herein can be administered to the subject prior to development or diagnosis of the autoimmune disease, disorder or condition therein. Instead, the subject may have been diagnosed with the GAS infection and be determined to be at risk of developing the autoimmune disease, disorder or condition associated therewith.

[0090] As used herein, the term “subject” refers to any animal, for example, a mammalian animal, including, but not limited to humans, non-human primates, livestock (e.g., sheep, horses, cattle, pigs, donkeys), companion animals (e.g., pets such as dogs and cats), laboratory test animals (e.g., mice, rabbits, rats, guinea pigs), performance animals (e.g., racehorses, camels, greyhounds) or captive wild animals. In various examples, the “subject” is a human, such as a male human or a female human. Typically, the terms “subject” and “patient” are used interchangeably, particularly in reference to a human subject.

[0091] The term “therapeutically effective amount” describes a quantity of a specified agent, such as an IL-2 protein, an encoding nucleic acid molecule thereof, or regulatory T cells, sufficient to achieve a desired effect in a subject being treated with that agent or composition. For example, this can be the amount of the agent and optionally one or more further therapeutic agents (e.g., one or more antimicrobial agents), necessary to reduce, alleviate and / or prevent an autoimmune disease, disorder or condition associated with a GAS infection. Suitably, a “therapeutically effective amount” is sufficient to reduce or eliminate a symptom of the autoimmune disease, disorder or condition, inclusive of rheumatic fever, rheumatic heart disease and Sydenham chorea. More particularly, a “therapeutically effective amount” may be an amount sufficient to achieve a desired biological effect, for example an amount that is effective to decrease or prevent disease progression, such as the production of cross-reactive autoantibodies and the induction of effector T cells and / or effector B cells in the subject.

[0092] Ideally, a therapeutically effective amount of an agent is an amount sufficient to induce the desired result without causing a substantial cytotoxic effect in the subject. The therapeutically effective amount of an agent useful for reducing, alleviating and / or preventing the diseases, disorders and conditions described herein will be dependent on the subject being treated, the type and severity of any associated disease, disorder and / or condition (e.g., disease progression), and the manner of administration of the therapeutic composition. Suitably, a therapeutically effective amount of the agent is administered parenterally, or more particularly subcutaneously, to the subject.

[0093] Suitably, the therapeutically effective amount of the IL-2 protein or the encoding nucleic acid molecule represents or contains a low or reduced dose or is equivalent to a low or reduced dose of such agents. The term “low dose” or “reduced dose” as used herein refers to a therapeutically effective dose of a treatment, such as an IL-2 protein or an encoding nucleic acid molecule thereof provided herein, whose dose is significantly or substantially less (e.g., at least about 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% etc less) than the usual or the conventional dose required to produce a therapeutic effect, such as in a different indication (e.g., a dose of an IL-2 protein or an encoding nucleic acid molecule required to produce an anti-cancer effect). In particular examples, a low dose of the IL-2 protein or the encoding nucleic acid molecule is sufficient to promote induction or activation of regulatory T cells in the subject without promoting or substantially promoting the induction or activation of effector T cells and / or effector B cells. Such a low dose may also be sufficient to prevent or inhibit the production of autoantibodies in the subject.

[0094] Suitably, the present methods further including the earlier or initial step of identifying whether the subject’s autoimmune disease, disorder or condition, such as from a sample (e.g., a biopsy sample or a biological sample, such as a blood, plasma, serum, urine, CSF etc.) obtained from the subject is associated with one or more of: an elevated level and / or activity of effector T cells; a decreased level and / or activity of regulatory T cells; and an elevated level or presence of autoantibodies. In such instances, the present method may include the step of determining one or more of: a level of effector T cell expression and / or activity; a level of regulatory T cell expression and / or activity; and a level (or presence or absence) of autoantibodies in the subject. Well known assays, such as those described herein, can be used for determining such levels in the subject to be treated if such prior determination is desired.

[0095] Accordingly, the methods disclosed herein may further include the step of determining the level and / or activity of regulatory T cells, effector T cells and / or autoantibodies before and / or after administration of the IL-2 protein or the encoding nucleic acid molecule to the subject. In some examples, the level and / or activity of regulatory T cells, effector T cells and / or autoantibodies may be determined before administration of the IL-2 protein or the encoding nucleic acid molecule to the subject. For other examples, the level and / or activity of regulatory T cells, effector T cells and / or autoantibodies may be determined after administration of the IL-2 protein or the encoding nucleic acid molecule to the subject. In particular examples, the level and / or activity of regulatory T cells, effector T cells and / or autoantibodies may be determined before and after administration of the IL-2 protein or the encoding nucleic acid molecule to the subject.

[0096] Suitable methods to determine the level and / or activity of regulatory T cells, effector T cells and / or autoantibodies are known in the art, such as those provided herein. For example, flow cytometry, microscopy, western blot, spectrophotometry and / or ELISA methods may be utilised in this regard.

[0097] In various examples, the level and / or activity of effector T cells in the subject are elevated prior to administering the IL-2 protein or the encoding nucleic acid molecule to the subject. For some examples, the level of autoantibodies in the subject are elevated prior to administering the IL-2 protein or the encoding nucleic acid molecule to the subject. Additionally, or alternatively, the level and / or activity of regulatory T cells in the subject are decreased prior to administering the low dose of the IL-2 protein or the encoding nucleic acid molecule to the subject.

[0098] As used herein, the level and / or activity of regulatory T cells, effector T cells and / or autoantibodies is considered “elevated” or “increased” when it is higher (including relatively or absolutely higher) than a predetermined reference, control or threshold level thereof . With respect to the level of autoantibodies, this may be considered to be elevated when relevant autoantibodies are detected as being present in the subject. Moreover, the level and / or activity of regulatory T cells, effector T cells and / or autoantibodies is considered “reduced” or “decreased” when it is lower (including relatively or absolutely lower) than a predetermined reference, control or threshold level thereof. Thus, any of the methods disclosed herein may comprise a step of establishing a reference, control or threshold level and / or activity of regulatory T cells, effector T cells and / or autoantibodies. Suitable threshold levels can then be determined according to the particular methodology used to measure the levels of regulatory T cells, effector T cells and / or autoantibodies, which threshold levels can establish a significant difference in the levels and / or activity of regulatory T cells, effector T cells and / or autoantibodies and a healthy control (e.g., a mean or median level in a healthy population of subjects), such a difference then being indicative of an increase or decrease in the level and / or activity of regulatory T cells, effector T cells and / or autoantibodies in the subject. It will be appreciated that the precise threshold levels will vary depending on the samples used to establish those threshold levels of regulatory T cells, effector T cells and / or autoantibodies and according to the particular analytical methodology used in each instance.

[0099] The term “control sample”, “control level”, “reference sample” or “reference level” typically refers to a biological sample or a respective level from a (healthy) non-diseased individual not having a GAS infection or an autoimmune disease, disorder or condition associated with such an infection. Alternatively, the control sample or level may be from a subject in remission from an autoimmune disease, disorder or condition associated with a GAS infection. The control sample may be a pooled, average or an individual sample. An internal control is a marker from the same biological sample being tested.

[0100] In some examples, a reference level or amount is determined from measurements, such as respective levels of immune cells, inclusive of those described herein, and / or autoantibodies, taken from a population of healthy individuals. The term “healthy individual” as used herein refers to a person or populations of persons who are known not to have a GAS infection or an autoimmune disease, disorder or condition associated with such an infection. In some examples, the control or reference level is determined from measurements in a “typical population”. Preferably, a "typical population" will exhibit a spectrum of GAS infection at different stages of disease progression. It is particularly preferred that a “typical population” exhibits the expression characteristics of a cohort of subjects as described herein.

[0101] In another example, a reference level or amount may be derived from an established data set including one or more of:

[0102] 1. a data set comprising measurements for a population of subjects known to have a GAS infection;

[0103] 2. a data set comprising measurements for a population of subjects known to have an autoimmune disease, disorder or condition associated with a GAS infection;

[0104] 3. a data set comprising measurements for the subject being tested wherein said measurements have been made previously, such as, for example, when the subject was known to be healthy or, in the case of a subject having a GAS infection, when the subject was diagnosed or at an earlier stage in disease progression (e.g., prior to development of an autoimmune disease, disorder or condition); and / or

[0105] 4. a data set comprising measurements for a healthy individual or a population of healthy individuals. The “normal” level and / or activity of effector T cells, regulatory T cells and autoantibodies may be determined by selecting any suitable biological sample or samples from which to derive the levels thereof in a non-disease state (e.g., a mean or median level). The presence of a modulated level and / or activity of effector T cells, regulatory T cells and / or autoantibodies may be determined by selecting any suitable biological sample from which to derive the level and / or activity thereof in a disease state. The level and / or activity of effector T cells, regulatory T cells and / or autoantibodies may be measured in any one or more tissues (e.g., mediastinal lymph node levels, spleen levels), organs or biological samples (e.g., circulating levels in a blood, plasma or serum sample of the subject). The biological sample may be or may comprise a bodily fluid. The bodily fluid may blood, serum, plasma, CSF or urine. Alternatively, the biological sample may be or comprise a tissue sample, such as a biopsy sample of a lymph node. The biological sample may be subject to any suitable pre-treatment steps before the measurement is performed, in order to improve the accuracy and / or efficiency of the measurement. Thus, any of the methods disclosed herein may comprise a step of taking a biological sample from a subject and determining the level and / or activity of effector T cells, regulatory T cells and / or autoantibodies in the sample. Alternatively, any of the methods disclosed herein may not comprise a step of taking a biological sample from the subject and determining the level and / or activity of effector T cells, regulatory T cells and / or autoantibodies in the sample. Instead, the level and / or activity of effector T cells, regulatory T cells and / or autoantibodies in the sample may have been determined previously.

[0106] In view of the foregoing, the present method may include the initial step of selecting a subject as being suitable for treatment with the IL-2 protein or the encoding nucleic acid molecule by determining a level and / or activity of regulatory T cells, effector T cells and / or autoantibodies in the subject. Suitably, an elevated level and / or activity of effector T cells and / or autoantibodies is indicative of the subject being suitable for administration of the low dose of the IL-2 protein or the encoding nucleic acid molecule. In other examples, a decreased level and / or activity of regulatory T cells is indicative of the subject being suitable for administration of the low dose of the IL-2 protein or the encoding nucleic acid molecule.

[0107] As such, in another form, the present disclosure provides a method of treating a subject with an autoimmune disease, disorder or condition associated with a GAS infection, including the steps: (a) determining a level and / or activity of effector T cells, regulatory T cells and / or autoantibodies in the subject; and (b) based on the determination in (a) initiating, continuing, modifying or discontinuing administration of a therapeutically effective amount of an IL-2 protein or a nucleic acid molecule encoding the IL-2 protein to the subject.

[0108] In a related form, the present disclosure provides a method of selecting a subject with a GAS infection for treatment with a therapeutically effective amount of an IL-2 protein or a nucleic acid molecule encoding the IL-2 protein, said method including the steps of: (a) determining a level and / or activity of effector T cells, regulatory T cells and / or autoantibodies in the subject; and (b) based on the determination in (a) determining whether the subject has or is at risk of developing an autoimmune disease, disorder or condition associated with the GAS infection.

[0109] In certain examples, the present method includes administering the IL-2 protein or the encoding nucleic acid molecule to the subject if they are determined to have or be at risk of developing the autoimmune disease, disorder or condition.

[0110] Interleukin-2

[0111] For the methods described herein, the subject is suitably administered a therapeutically effective amount of, and more particularly a low dose of, an IL-2 protein or a nucleic acid molecule encoding the IL-2 protein.

[0112] The term “IL-2” is intended to encompass any synthetic, recombinant, naturally occurring, native or wildtype form of IL-2 protein, whether monomeric or multimeric, including dimers, trimers, etc., which may be derived from any suitable organism. Further, the term encompasses unprocessed IL-2 as well as any form of IL-2 that results from processing in the cell. In particular examples, IL-2 refers to a mammalian IL-2, such as human, rat or mouse IL-2, as well as nonhuman primate, bovine, ovine, or porcine IL-2. For some examples, the IL-2 protein is human (see, e.g., SEQ ID NOs: 1 and 2) or “hIL-2”. It is also envisaged that the term “IL-2” encompasses fragments, portions, variants (inclusive of splice variants and allelic variants), isoforms, analogues, derivatives, fusion proteins and other homologs of such IL-2 proteins. Such fragment, variant or derivative IL-2 proteins will generally be characterized by having the same type of activity or function as naturally occurring IL-2, such as the ability to bind an IL-2 receptor, and the ability to induce receptor-mediated activity and downstream signalling (e.g., phosphorylation of STAT5). In this regard, the IL-2 fragments, variants or derivatives described herein are suitably active or functional fragments, variants or derivatives. Determining whether a IL-2 protein fragment, variant or derivative is active or functional may be assessed by any method or means known in the art.

[0113] IL-2 has been used in the clinic for boosting effector immune responses in cancers and infectious diseases. In this context, IL-2 has demonstrated limited clinical efficacy, which may be at least partly explained by IL-2’s role in the peripheral survival and suppressive function of regulatory T cells (Tregs), which are known to supress antitumour effector responses. In fact, IL- 2 / IL-2 receptor (IL-2R) signalling is important during immune responses of both effector T cells (Teffs) and Tregs. On the one hand, extensive IL-2R signalling is necessary for the development of terminally differential short-lived effector T cells that exhibit enhanced functional activity, and for eliciting proper T cell memory. On the other hand, IL-2 / IL-2R signalling is essential for Treg development and homeostasis as shown by the fact IL-2 knock-out mice lack Tregs. The therapeutic efficacy of low dose interleukin-2 therapy has previously been described for a number of other autoimmune conditions, such as type 1 diabetes (Hartemann et al., 2013), systemic lupus erythematosus (He et al., 2016), and vasculitis associated with chronic hepatitis C virus. Unlike conventional immunosuppressive treatments, LD-IL-2 therapy generally promotes immune tolerance without inducing generalized immunosuppression (Churlaud et al., 2014; Zhou et al., 2021). In this regard, low doses of IL-2 tend to preferentially induce regulatory T cells over effector T cells and NK cells (Maelk and Castro, 2010).

[0114] By “protein” is meant an amino acid polymer. The amino acids may be natural or nonnatural amino acids, D- or L-amino acids as are well understood in the art.

[0115] The term “protein” includes and encompasses “peptide”, which is typically used to describe a protein having no more than fifty (50) amino acids (e.g., no more than 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 amino acids and any range therein) and “polypeptide”, which is typically used to describe a protein having more than fifty (50) amino acids.

[0116] As used herein, a protein, polypeptide or peptide “variant” shares a definable amino acid sequence relationship with a reference amino acid sequence. In particular examples, the reference amino acid sequence is that of IL-2 protein sequence. As such, the reference amino acid sequence may be the amino acid sequence of SEQ ID NOs: 1, 2 or 5. The “variant” protein, polypeptide or peptide may have one or a plurality of amino acids of the reference amino acid sequence deleted, inserted / added or substituted by different amino acids. It is well understood in the art that some amino acids may be substituted, inserted / added or deleted without changing the activity of the IL- 2 protein (i.e., conservative substitutions). Accordingly, one or more (e.g., 1, 2, 3, 4, 5 etc) of the residues of an IL-2 protein, such as that of SEQ ID NOs: 1 or 2, may be conservatively modified e.g., by amino acid substitution or deletion) without altering the biological activity, function, or other desired property thereof, such as its affinity or its specificity for an IL-2 receptor.

[0117] Suitably, protein variants share at least 60% or 65%, 66%, 67%, 68%, 69%, preferably at least 70%, 71%, 72%, 73%, 74% or 75%, more particularly at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% or 89%, and even more particularly at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleotide sequence identity with an isolated protein of the present disclosure (e.g., SEQ ID NOs: 1, 2 or 5). Percent sequence identity may be determined by any method known in the art, such as that described herein.

[0118] Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Vai, Leu, and He; interchange of the hydroxyl residues Ser and Thr; exchange of the acidic residues Asp and Glu; substitution between the amide residues Asn and Gin; exchange of the basic residues Lys and Arg; and replacements among the aromatic residues Phe and Tyr. Guidance concerning which amino acid changes are likely to be phenotypically silent can be found in, for example, Bowie et al., Science 247: 1306-1310 (1990).

[0119] Mutant or variant forms of the IL-2 protein are well known in the art. Exemplary variants of IL-2 are provided in SEQ ID NO: 5 and PCT / US2004 / 023317, PCT / EP2012 / 051991, PCT / US2019 / 032321 and PCT / US2020 / 036454 which are incorporated by reference herein. Variant IL-2 proteins may include the sequence of SEQ ID NO: 1 or 2 with one or more, two or more, three or more, four or more or five or more amino acid substitutions relative to SEQ ID NO: 1 or 2. For some examples, the mutant or variant IL-2 proteins disclosed herein include: a substitution of the cysteine residue at position 125 with another residue (e.g., serine); and / or a deletion of the alanine residue at position 1 of SEQ ID NO: 2.

[0120] The present disclosure also provides for fragments of the IL-2 proteins described herein. As used herein, a “fragment” is a segment, domain, portion or region of a protein or peptide (such as those set forth in SEQ ID NOs: 1 and 2) which constitutes less than 100% of the amino acid sequence of the protein or peptide.

[0121] In general, fragments may comprise, consist essentially of or consist of up to 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151 or 152 (inclusive of any range therein) contiguous amino acids of an IL-2 protein (such as one of SEQ ID NOs: 1 or 2).

[0122] As used herein, “derivatives” refers to proteins or peptides, inclusive of fragments or variants thereof, that have been altered, for example by conjugation or complexing with other chemical moieties, such as by post-translational modification (e.g., phosphorylation, acetylation, ubiquitination, glycosylation and the like), modification of glycosylation (e.g., adding, removing or altering glycosylation), lipidation and / or inclusion of additional amino acid sequences as would be understood in the art. Derivatives contemplated by the disclosure include, but are not limited to, modification to side chains, incorporation of unnatural amino acids and / or their derivatives during peptide, or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the IL-2 proteins of the disclosure. In this regard, the skilled person is referred to Chapter 15 of CURRENT PROTOCOLS IN PROTEIN SCIENCE, Eds. Coligan et al. (John Wiley & Sons NY 1995-2008) for more extensive methodology relating to chemical modification of proteins.

[0123] Further derivatives may include conjugates of the IL-2 protein, such as those described herein. The term “conjugated” can be used in the context of the present disclosure to describe the IL-2 protein conjugated to another compound or structure, such as a label or carrier molecule or protein. Accordingly, in one example, the IL-2 protein is “conjugated”. The IL-2 protein may be modified via conjugation or complexing with other chemical moieties, such as chemical modification (e.g., cross-linking, acetylation, biotinylation, oxidation or reduction) and / or conjugation with labels (e.g., fluorophores, enzymes, radioactive isotopes) and / or other functional elements (e.g., a half-life extender, such as PEG, albumin, an Fc region or an albumin binding domain, a CNS targeting moiety), as are known in the art.

[0124] The term “nucleic acid molecule encoding the IL-2 protein” or the like means that nucleic acid, if present in the appropriate environment, such as within a cell of the subject, can be expressed to produce an IL-2 protein it encodes. The nucleic acids described herein may be recombinant and / or isolated molecules. Such nucleic acids may be administered to the subject by any appropriate delivery system known in the art, inclusive of viral and non-viral systems, such as virus-like particles, lipid nanoparticles (LNPs), polymeric nanoparticles, inorganic nanoparticles, liposomes and exosomes.

[0125] For the purposes of the present disclosure, by “isolated” is meant material that has been removed from its natural state or otherwise been subjected to human manipulation. Isolated material (e.g., IL-2 proteins or encoding nucleic acids) may be substantially or essentially free from components that normally accompany it in its natural state, or may be manipulated so as to be in an artificial state together with components that normally accompany it in its natural state.

[0126] Referring to the methods described herein, the IL-2 protein or the encoding nucleic acid molecule may be administered in any form known in the art, such as a recombinant IL-2 protein, inclusive of fragments, variants and derivatives thereof, a cell line expressing the IL-2 protein (e.g., cells transformed with the nucleic acid encoding the IL-2 protein) and an mRNA molecule encoding the IL-2 protein. In some examples, the IL-2 protein or the encoding nucleic acid molecule is suitably selected from the group consisting of aldesleukin (a recombinant form of IL- 2), rezpegaldesleukin (a PEGylated IL-2), interking (a recombinant IL-2 with a serine at residue 125), AVB-001 (polymer encapsulated cells that produce IL-2), salspera (an attenuated Salmonella typhimurium strain secreting unmodified IL-2), NKTR-214 (a PEGylated IL-2 with approximately six PEG moieties added at lysine residues), RO7049665 (a CD25-biased IL-2 harbouring a N88D mutation and fused to IgGl), CC-92252 (a CD25-biased IL-2 mutein-Fc fusion protein), XmAb27564 (a CD25-biased IL-2 mutein-Fc fusion protein), MK-6194 (a CD25-biased IL-2-Fc fusion protein), AMG592 (a mutated IL-2-Fc fusion protein with increased Treg selectivity), CUG252 (a mutated IL-2-Fc fusion protein with increased Treg selectivity), mRNA-6231 (an mRNA coding for human serum albumin fused to IL-2 mutein with increased Treg selectivity), BNT151 (a liposome or LNP encapsulated mRNA encoding a modified IL-2 fused with albumin for half-life prolongation), BNT153 (a liposome or LNP encapsulated IL-2 encoding mRNA), TILT- 123 (a virus, such as an adenovirus, engineered to encode IL-2) and any combination thereof. In certain examples, the IL-2 protein is or comprises aldesleukin (Proleukin®). Aldesleukin is a recombinant unglycosylated variant of mature human IL-2 comprising two amino acid modifications as compared to the sequence of mature human IL-2 (i.e., SEQ ID NO: 2): the deletion of the first amino acid (alanine) and the substitution of cysteine at position 125 by serine (i.e., SEQ ID NO: 5). Accordingly, in some examples, the IL-2 protein is or comprises the amino acid sequence set forth in SEQ ID NO: 5.

[0127] Aldesleukin (SEQ ID NO: 5)

[0128] PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLE EELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRW ITFSQSIISTLT

[0129] In view of the above, and in one particular form, there is provided herein a method of treating or preventing rheumatic fever or a disease, disorder or condition associated therewith in a subject, said method including the step of administering a therapeutically effective amount of a nucleic acid molecule encoding an IL-2 protein to the subject to thereby treat or prevent rheumatic fever or the disease, disorder or condition associated therewith. Suitably, the therapeutically effective amount of the nucleic acid molecule encoding the IL-2 protein is equivalent to a low dose of the IL-2 protein.

[0130] In some examples, the nucleic acid molecule encoding the IL-2 protein is in the form of a genetic construct suitable for administration to a mammal, such as a human. More particularly, the genetic construct may be suitable for DNA delivery of the IL-2 protein to a mammal, such as a human. A useful reference describing DNA delivery of peptides is DNA Vaccines, Methods and Protocols, Second Edition (Volume 127 of Methods in Molecular Medicine series, Humana Press, 2006).

[0131] According to certain examples, the nucleic acid molecule encoding the IL-2 protein is in the form of RNA, such as mRNA, suitable for administration to a mammal, such as a human. In various examples, the nucleic acid is or comprises an mRNA having an open reading frame encoding an IL-2 protein provided herein. mRNA vaccines are described, for example, in International Patent Application Nos. PCT / US2015 / 027400 and PCT / US2016 / 044918, herein incorporated by reference in their entirety. The mRNA delivery of functional proteins, such as antibodies, is further described in PCT / US2018 / 037918, which is also incorporated by reference herein.

[0132] It will be appreciated that nucleic acid, and more particularly mRNA, delivery of the IL-2 protein provides a unique therapeutic alternative to protein-based or DNA-based methods of administering such agents. When the mRNA is delivered to a cell, the mRNA will be processed into a polypeptide or peptide by the intracellular machinery which can then process the polypeptide or peptide into the IL-2 protein capable of binding and activating an IL-2 receptor, such as expressed by immune cells of the subject.

[0133] Compositions comprising isolated nucleic acids or polynucleotides that encode the IL-2 protein described herein, inclusive of fragments, variants or derivatives thereof that may be used for such methods, are also contemplated by the present disclosure. For such examples, the composition suitably comprises a delivery agent, such as a nanoparticle, as are known in the art. In various examples, the nanoparticle has a mean diameter of 50-200 nm. In some examples, the composition comprising the mRNA polynucleotide (e.g., an mRNA polynucleotide having an open reading frame that encodes a polypeptide conjugate described herein) is formulated in a lipid nanoparticle (LNP). Exemplary encoding nucleic acid molecules formulated in this manner include BNT151, BNT153 and mRNA-6231.

[0134] Protein sequences of IL-2 are publicly available (e.g., P60568) and an exemplary amino acid sequences is set forth in SEQ ID NO: 1 below. Thus, IL-2 protein may have an amino acid sequence which is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 1 or 2, or a fragment or derivative thereof. The sequence in SEQ ID NO: 1 includes a signal peptide at the beginning of the protein (i.e., amino acid residues 1 to 20 of SEQ ID NO: 1), with the sequence for the mature IL-2 protein beginning at Ala 21 of SEQ ID NO: 1, which is absent in the mature IL-2 protein of SEQ ID NO: 2. It will further be understood by the skilled person that the IL-2 protein generally demonstrates a high level of conservation in terms of sequence, structure and function thereof across different mammalian species.

[0135] Human IL-2 protein with signal peptide (SEQ ID NO: 1)

[0136] MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRM LTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSE TTFMCEYADETATIVEFLNRWITFCQSIISTLT

[0137] Mature Human IL-2 protein without signal peptide (SEQ ID NO: 2)

[0138] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCL EEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFCQSIISTLT Encoding nucleic acid sequences of IL-2 are also publicly available (e.g., NM_000586.4) and an exemplary nucleotide sequence is set forth in SEQ ID NO: 3 and SEQ ID NO: 4 below. Variants of such encoding nucleic acids are contemplated herein. As used herein, a nucleic acid “variant” shares a definable nucleotide sequence relationship with a reference nucleic acid sequence (e.g., SEQ ID NO:3 or 4). The “variant” nucleic acid may have one or a plurality of nucleic acids of the reference nucleic acid sequence deleted or substituted by different nucleic acids. Suitably, nucleic acid variants share at least 60% or 65%, 66%, 67%, 68%, 69%, preferably at least 70%, 71%, 72%, 73%, 74% or 75%, more particularly at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% or 89%, and even more particularly at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleotide sequence identity with an isolated nucleic acid of the present disclosure (e.g., SEQ ID NOs: 3 or 4). Percent sequence identity may be determined by any method known in the art, such as that described herein. Thus, the nucleic acid molecule encoding the IL-2 protein may have an nucleotide sequence which is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical to the nucleotide sequence set forth in SEQ ID NO: 3 or 4, or a fragment or derivative thereof.

[0139] Human IL-2 encoding mRNA (SEQ ID NO: 3)

[0140] CTATCACCTAAGTGTGGGCTAATGTAACAAAGAGGGATTTCACCTACATCCATTCAG

[0141] TCAGTCTTTGGGGGTTTAAAGAAATTCCAAAGAGTCATCAGAAGAGGAAAAATGAA

[0142] GGTAATGTTTTTTCAGACAGGTAAAGTCTTTGAAAATATGTGTAATATGTAAAACAT

[0143] TTTGACACCCCCATAATATTTTTCCAGAATTAACAGTATAAATTGCATCTCTTGTTCA

[0144] AGAGTTCCCTATCACTCTCTTTAATCACTACTCACAGTAACCTCAACTCCTGCCACAA

[0145] TGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACAAACA

[0146] GTGCACCTACTTCAAGTTCTACAAAGAAAACACAGCTACAACTGGAGCATTTACTGC

[0147] TGGATTTACAGATGATTTTGAATGGAATTAATAATTACAAGAATCCCAAACTCACCA

[0148] GGATGCTCACATTTAAGTTTTACATGCCCAAGAAGGCCACAGAACTGAAACATCTTC

[0149] AGTGTCTAGAAGAAGAACTCAAACCTCTGGAGGAAGTGCTAAATTTAGCTCAAAGC

[0150] AAAAACTTTCACTTAAGACCCAGGGACTTAATCAGCAATATCAACGTAATAGTTCTG

[0151] GAACTAAAGGGATCTGAAACAACATTCATGTGTGAATATGCTGATGAGACAGCAAC

[0152] CATTGTAGAATTTCTGAACAGATGGATTACCTTTTGTCAAAGCATCATCTCAACACT

[0153] GACTTGATAATTAAGTGCTTCCCACTTAAAACATATCAGGCCTTCTATTTATTTAAAT

[0154] ATTTAAATTTTATATTTATTGTTGAATGTATGGTTTGCTACCTATTGTAACTATTATTC

[0155] TTAATCTTAAAACTATAAATATGGATCTTTTATGATTCTTTTTGTAAGCCCTAGGGGC

[0156] TCTAAAATGGTTTCACTTATTTATCCCAAAATATTTATTATTATGTTGAATGTTAAAT

[0157] ATAGTATCTATGTAGATTGGTTAGTAAAACTATTTAATAAATTTGATAAATATAAA Human IL-2 encoding cDNA (SEQ ID NO: 4)

[0158] ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACAAAC AGTGCACCTACTTCAAGTTCTACAAAGAAAACACAGCTACAACTGGAGCATTTACTG CTGGATTTACAGATGATTTTGAATGGAATTAATAATTACAAGAATCCCAAACTCACC AGGATGCTCACATTTAAGTTTTACATGCCCAAGAAGGCCACAGAACTGAAACATCTT CAGTGTCTAGAAGAAGAACTCAAACCTCTGGAGGAAGTGCTAAATTTAGCTCAAAG CAAAAACTTTCACTTAAGACCCAGGGACTTAATCAGCAATATCAACGTAATAGTTCT GGAACTAAAGGGATCTGAAACAACATTCATGTGTGAATATGCTGATGAGACAGCAA CCATTGTAGAATTTCTGAACAGATGGATTACCTTTTGTCAAAGCATCATCTCAACAC TGACTTGA

[0159] Terms used generally herein to describe sequence relationships between respective nucleotides or polypeptides include “sequence identity”, “percentage of sequence identity” and “substantial identity”. Because respective nucleotides or polypeptides may each comprise (i) only one or more portions of a complete nucleotide or polypeptide sequence that are shared by the nucleotides or amino acids, and (ii) one or more portions which are divergent between the nucleotides or amino acids, sequence comparisons are typically performed by comparing sequences over a “comparison window” to identify and compare local regions of sequence similarity. A “comparison window” refers to a conceptual segment of typically 6, 9 or 12 contiguous residues that is compared to a reference sequence. The comparison window may comprise additions or deletions (i.e., gaps) of about 20% or less as compared to the reference sequence for optimal alignment of the respective sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerised implementations of algorithms (Geneworks program by Intelligenetics; GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA, incorporated herein by reference) or by inspection and the best alignment (i.e. resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et al., 1997, Nucl. Acids Res. 25 3389, which is incorporated herein by reference. A detailed discussion of sequence analysis can be found in Unit 19.3 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al. (John Wiley & Sons Inc NY, 1995-1999).

[0160] The term “sequence identity” is used herein in its broadest sense to include the number of exact nucleotide or amino acid matches having regard to an appropriate alignment using a standard algorithm, having regard to the extent that sequences are identical over a window of comparison. Thus, a “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U) or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For example, “sequence identity” may be understood to mean the “match percentage” calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA).

[0161] T cells and autoantibodies

[0162] Suitably, administration of the IL-2 protein or the encoding nucleic acid molecule modulates a level and / or an activity of an immune cell, such as effector immune cells like effector T and / or B cells or regulatory T cells, in the subject.

[0163] Referring to various examples, administration of the IL-2 protein or the encoding nucleic acid molecule increases a level and / or activity of regulatory T cells in the subject. Put another way, administration of the IL-2 protein or the encoding nucleic acid molecule can promote induction or activation of regulatory T cells in the subject. Such induction or activation of regulatory T cells may be selective, in that induction or activation of regulatory T cells can occur essentially without concomitant activation or induction of other T cell subsets (e.g. effector T cells, such as CD4+ T helper cells, CD8+ cytotoxic T cells) or natural killer (NK) cells.

[0164] Accordingly, in one form, the present disclosure relates to a method of increasing a level and / or activity of (e.g., promoting the induction of) regulatory T cells in a subject with or at risk of developing an autoimmune disease, disorder or condition associated with a GAS infection, said method including the step of administering a therapeutically effective amount of an IL-2 protein or a nucleic acid molecule encoding the IL-2 protein to the subject to thereby increase the level and / or activity of regulatory T cells therein. Suitably, the subject has or is at risk of developing an autoimmune disease, disorder or condition associated with the GAS infection, such as rheumatic fever.

[0165] The terms “regulatory T cell” and “Treg” are used interchangeably herein and are intended to encompass specialized type of T cell that can suppress the responses of other T cells, and more particularly effector T cells. Tregs are typically characterized by expression of CD4 or less commonly CD8, the a-subunit of the IL-2 receptor (CD25), and the transcription factor forkhead box P3 (FOXP3) (Sakaguchi, Annu Rev Immunol 22, 531-62 (2004)) and can play a role in the induction and maintenance of peripheral self-tolerance to antigens. In some examples, the regulatory T cells include CD4+ Tregs. In certain examples, the regulatory T cells include CD8+ Tregs. In particular examples, the regulatory T cells include CD4+ Tregs and CD8+ Tregs.

[0166] Accordingly, in some examples, administration of the IL-2 protein or the encoding nucleic acid molecule increases a level and / or an activity of CD4+ Tregs and CD8+ Tregs in the subject. In other examples, administration of the IL-2 protein or the encoding nucleic acid molecule increases a level and / or an activity of CD4+ Tregs in the subject. For certain examples, administration of the IL-2 protein or the encoding nucleic acid molecule increases a level and / or an activity of CD8+ Tregs in the subject. Methods for identifying and determining the level of such regulatory T cells are well known in the art and also described herein. The assessment of activity levels may include induction of IL-2 receptor signaling (e.g., detection of phosphorylated STAT5), induction of proliferation (e.g., detection of Ki-67), and / or up-regulation of expression of activation markers (e.g., CD25, FOXP3), and expansion of cell numbers.

[0167] Suitably, administration of the IL-2 protein or the encoding nucleic acid molecule does not increase or does not substantially increase a level and / or activity of effector T cells in the subject. In other words, administration of the IL-2 protein or the encoding nucleic acid molecule does not promote or substantially promote the induction or activation of effector T cells in the subject (e.g., a population of effector T cells has increased by no more than 1%, 5% or 10% in said subject as a result of treatment with the IL-2 protein or the encoding nucleic acid molecule).

[0168] Referring to other examples, the IL-2 protein or the encoding nucleic acid molecule decreases a level and / or activity of effector T cells in the subject. In other words, administration of the IL-2 protein or the encoding nucleic acid molecule inhibits, prevents or reverses the induction or activation of effector T cells in the subject.

[0169] As such, in another form, the present disclosure relates to a method of decreasing a level and / or activity of (e.g., inhibiting the induction of) effector T cells in a subject with or at risk of developing an autoimmune disease, disorder or condition associated with a GAS infection, said method including the step of administering a therapeutically effective amount of an IL-2 protein or a nucleic acid molecule encoding the IL-2 protein to the subject to thereby decrease the level and / or activity of effector T cells therein.

[0170] For the above examples, the effector T cells may be CD4+ T helper cells and / or CD8+ cytotoxic T cells. CD4+ effector T cells can contribute to the development of several immunologic processes, including maturation of B cells into plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages. CD8+ effector T cells destroy virus-infected cells and tumour cells. More particularly, the effector T cell can be a T helper 17 cell or a Thl7 cell (i.e., a subset of pro-inflammatory T helper cells typically defined by their production of interleukin 17). In other examples, the effector T cell can be a T helper 1 cell or a Thl cell (i.e., a subset of pro- inflammatory T helper cells typically defined by their production of IFN-y, IL-2 and TNF-a). Suitably, the effector T cell or population thereof in question recognize a self-protein, such as a cardiac protein, a connective tissue protein and / or a neural protein, inclusive of those disclosed herein. Again, methods for determining the levels and / or activity of effector T cells are well known in the art and may include those described herein.

[0171] The induction of regulatory T cells and the absence of substantial induction of effector T cells by the methods disclosed herein may be assessed by a measure of the ratio or the balance Treg / Teff in the treated subject. This balance may be calculated, for example, on the number of Tregs and the number of Teffs in a biological sample from the subject. In particular examples, the methods disclosed herein result in an increase by about 20%, 30%, 40%, 50%, 75%, 100% or more (or any range therein) of the Treg / Teff ratio in the subject.

[0172] The level and / or activity of regulatory T cells and / or effector T cells may be assessed in a biological sample obtained from the subject. Suitably, the biological sample is or comprises a lymphoid tissue, such as thymus, bone marrow, lymph node and spleen, or a blood sample (e.g., a circulating level thereof). Accordingly, the methods described herein may include the step of obtaining a biological sample from the subject in question.

[0173] Suitably, administration of the IL-2 protein or the encoding nucleic acid molecule modulates a level of autoantibodies in the subject. In particular examples, administration of the IL-2 protein or the encoding nucleic acid molecule prevents or inhibits production of autoantibodies in the subject. Accordingly, administration of the IL-2 protein or the encoding nucleic acid molecule may decrease or reduce a level of autoantibodies in the subject. By extension, administration of the IL-2 protein or the encoding nucleic acid molecule may prevent or inhibit induction or activation of effector B cells in the subject, such as those that produce autoantibodies.

[0174] Therefore, in another form, the present disclosure provides a method of preventing or inhibiting the production of (e.g., decreasing a level of) autoantibodies in a subject with or at risk of developing an autoimmune disease, disorder or condition associated with a GAS infection, said method including the step of administering a therapeutically effective amount of an IL-2 protein or a nucleic acid molecule encoding the IL-2 protein to the subject to thereby prevent or inhibit the production of autoantibodies therein.

[0175] Such methods may result in a reduction in a level and / or activity of B cells, such as those that produce those autoantibodies described herein, in the subject. This level may be calculated, for example, on the number of B cells, such germinal center B cells, in a biological sample, such as a lymphoid tissue like the spleen, from the subject. Additionally, the present methods can result in a modulation, such as an increase or a decrease, in a level of one or more cytokines and / or cytokine receptors in the subject. In particular examples, the present methods result in a decreased level of one or more cytokines and / or cytokine receptors in the subject, such as one or more of IL- 17, IFN-y, IL- 10 and IL-13. In other examples, the present methods result in an increased level of one or more cytokines and / or cytokine receptors in the subject, such as one or more of IL2RA (CD25) and IL5RA. Again, this level of the one or more cytokines and / or cytokine receptors may be calculated in a biological sample, such as a lymphoid tissue like the spleen, from the subject.

[0176] By the terms “autoantibody” and “autoantibodies” is meant an antibody that is directed against one or more of the subjects own proteins (i.e., a self-protein). Referring to particular examples, the autoantibodies bind or are directed against a cardiac protein, a connective tissue protein and / or a neural protein in the subject. Suitably, the autoantibodies also bind or are directed against a GAS protein, such as an M protein, as are known in the art.

[0177] It is contemplated that the autoantibodies described herein may bind the self-protein and / or the GAS protein with high affinity. The terms “high affinity” and “relatively high affinity” are used interchangeably herein and refer to a binding affinity between a binding agent, such as an autoantibody, and the target molecule of interest with a KD of at least about 10'6M, more particularly at least about 10'7M, even more particularly at least about 10'7M and still even more particularly between about 10'8M to about IO'10M. The determination of such affinity may be conducted under standard competitive binding immunoassay procedures, such as equilibrium dialysis, ELISA, isothermal titration calorimetry (ITC), surface plasmon resonance (SPR; e.g., Biacore assays), fluorescent-activated cell sorting (FACS) binding assays and the like.

[0178] Suitably, the autoantibodies are directed to or bind, such as with high affinity, a cardiac protein, a connective tissue protein and / or a neural protein in the subject. In particular examples, the autoantibodies are directed or bind to a cardiac protein. For such examples, the cardiac protein can be one or more of cardiac myosin and tropomyosin. In other examples, the autoantibodies are directed or bind to a connective tissue protein, such as a valvular connective tissue protein. For such examples, the connective tissue protein can be one or more of laminin and keratin. In various examples, the autoantibodies are directed or bind to a neural protein, or more particularly a neuronal protein. For such examples, the neural protein can be one or more of dopamine receptor 1, dopamine receptor 2, lysoganglioside and tubulin.

[0179] Terms such as “higher”, “increased” and “greater” as used herein refer to an elevated level or activity of an immune cell, such as a regulatory T cell, or autoantibodies when compared to a control, threshold or reference level thereof. The level or activity of the immune cell or autoantibodies may be relative or absolute (i.e., relatively or absolutely higher, increased or greater). In some examples, the level or activity of the immune cell or autoantibodies is higher, increased or greater if its level or activity is more than about 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 300%, 400% or at least about 500% above the level or activity of a control, threshold or reference level thereof, such as that in the absence of the IL-2 protein or the encoding nucleic acid molecule (e.g., prior to the administration of the IL-2 protein).

[0180] The terms, “lower”, “reduced” and “decreased”, as used herein refer to a lower level or activity of an immune cell, such as an effector T cell, an effector B cell or autoantibodies, when compared to a control, threshold or reference level thereof. The level or activity of the immune cell or autoantibodies may be relative or absolute (i.e., relatively or absolutely lower, reduced or decreased). In some examples, the level or activity of the immune cell or autoantibodies is lower, reduced or decreased if its level or activity is less than about 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10%, or even less than about 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01%, 0.001% or 0.0001% of the level or activity of a control, threshold or reference level thereof, such as that in the absence of the IL-2 protein or the encoding nucleic acid molecule.

[0181] Antimicrobial agents

[0182] In any of the methods disclosed herein, the subject may be receiving an antimicrobial agent, and more particularly an antibiotic agent or antibiotic, in addition to the low dose of the IL-2 protein or the encoding nucleic acid molecule. Moreover, any of the present methods may include the step of administering to the subject an antimicrobial agent for treatment of the GAS infection or an autoimmune disease, disorder or condition associated therewith.

[0183] The term “antimicrobial agent” refers to a substance that kills microorganisms or inhibits their growth. A microorganism in this context can be any microbe or microscopic organism, including for example, a bacterium, a fungus, or a virus. In particular examples, the antimicrobial agent is an antibiotic or an antibacterial agent. Such terms are used interchangeably herein and refer to any agent capable of having bactericidal or bacteriostatic effects on bacterial growth.

[0184] Suitably, the strain or type of GAS bacteria, and more particularly Streptococcus pyogenes, which is at least partly causative of or responsible for the subject’s autoimmune disease, disorder or condition, such as rheumatic fever, demonstrates sensitivity to the antimicrobial agent described herein. To this end, the methods described herein may include the earlier or initial step of determining a level of bacterial sensitivity to the antimicrobial agent prior to its administration to the subject. Determining bacterial sensitivity to the antimicrobial agents described herein may be assessed by any means known in the art, such as a disc diffusion test, antimicrobial gradient method, broth microdilution and genetic testing to detect the presence or absence of antibiotic resistance genes or markers.

[0185] Exemplary antibiotic agents for use in the treatment of an autoimmune disease, disorder or condition associated with GAS, such as rheumatic fever or a disease, disorder or condition associated therewith, include a penicillin antibiotic (e.g., penicillin, amoxicillin, benzathine penicillin (benzylpenicillin or penicillin G), cefixime, cefpodoxine, cefotaxime, ceftriaxone, oxacillin), a lincosamide antibiotic (e.g., clindamycin), a macrolide antibiotic (e.g., erythromycin, clarithromycin, azithromycin, spiramycin), a glycopeptide antibiotic (e.g., vancomycin, teicoplanin), a cephalosporin antibiotic (e.g., cephalosporin C, cefadroxil, cephalexin) a streptogramine antibiotic (e.g., pristinamycin), a ketolide antibiotic (e.g., telithromycin) a phenicol antibiotic (e.g., chloramphenicol), a fluoroquinolone antibiotic (e.g., levofloxacin) and a tetracycline antibiotic (e.g., tetracycline).

[0186] Suitably, the antibiotic is a beta-lactam antibiotic, such as a penicillin derivative (penams), a cephalosporin, a cephamycin (cephems), a monobactam, a carbapenem and a carbacephem. According to particular examples, the antibiotic agent is a penicillin antibiotic. For such examples, the antibiotic can be penicillin. In other examples, the antibiotic is amoxicillin. For various examples, the antibiotic is benzathine penicillin.

[0187] In various examples, the antibiotic agent is selected from the group consisting of penicillin, amoxicillin, oxacillin, erythromycin, azithromycin, clarithromycin, cephalothin, cefoxitin, cefixime, cefuroxime, cefotaxime, ceftriaxone, vancomycin, clindamycin, ifampicin, ciprofloxacin, tetracycline, cotrimoxazole, chloramphenicol and any combination thereof.

[0188] The antimicrobial agent may be administered sequentially in any order, at the same time (i.e., simultaneously) or at different times to the IL-2 protein or the encoding nucleic acid molecule. For example, the subject may have received an antimicrobial agent prior to receiving the IL-2 protein or the encoding nucleic acid molecule described herein. In another example, the subject may receive an antimicrobial agent after receiving the IL-2 protein or the encoding nucleic acid molecule described herein. In yet another example, the subject may receive an antimicrobial agent at the same time as the IL-2 protein or the encoding nucleic acid molecule described herein.

[0189] In any of the methods disclosed herein, the method may include the further step of administering an antimicrobial agent, such as an antibiotic agent, to the subject. The antimicrobial agent may be administered simultaneously with, before or after treatment with the IL-2 protein or the encoding nucleic acid. In various examples, the antimicrobial agent is administered to the subject simultaneously with the IL-2 protein or the encoding nucleic acid. In another example, the antimicrobial agent is administered to the subject before the IL-2 protein or the encoding nucleic acid. In another example, the antimicrobial agent is administered to the subject after the IL-2 protein or the encoding nucleic acid.

[0190] It will be appreciated that the therapeutic effect of the various agents described herein (e.g., the IL-2 protein, the encoding nucleic acid molecule and / or the antimicrobial agent) need not be absolute to elicit a biological effect, such as treatment of the subject’s rheumatic fever or a disease, disorder or condition associate therewith. Accordingly, therapeutic efficacy provided by an agent described herein can be partial or incomplete (e.g., a readout, indicator or biomarker of disease progression in the subject is reduced by about 20%, 30%, 40%, 50%, 60% or 70%, 80%, 90%, 95%, 96%, 97%, 98% and 99%, including any intermediate value therebetween with administration of an agent as described herein).

[0191] Dosage and administration

[0192] The IL-2 protein, the encoding nucleic acid molecule, the antimicrobial agent, and the compositions disclosed herein can be administered to a patient suffering from or at risk of developing the autoimmune disease, disorder or condition, such as rheumatic fever, in an amount sufficient to cure, at least partially arrest the autoimmune disease, disorder or condition and one or more of its complications or sequelae or at least partially prevent development of the autoimmune disease, disorder or condition in the subject as a result of the GAS infection.

[0193] In other examples, the methods, compositions and uses disclosed herein also include or relate to prophylactic or preventative application of a suitable effective dose of the IL-2 protein or the encoding nucleic acid molecule described herein. In some embodiments, the IL-2 protein or the encoding nucleic acid molecule provided herein, in an appropriate effective dose, is used as a maintenance therapy.

[0194] The therapeutically effective dose level for any particular patient or subject, will depend upon a variety of factors familiar to one skilled in the art, including, for example: the causative GAS bacteria of the autoimmune disease, disorder or condition, such as rheumatic fever, being treated and / or the severity of the disease, the age, body weight, general health, sex and diet of the patient, the time of administration, the route of administration, the duration of the treatment, drugs used in combination or coincidental with the treatment, together with other related factors well known in medicine. It will be understood, however, that the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, gender, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, a patient's body surface area, whether the patient is undergoing therapy, and any specific contraindications. As hereinbefore described, the therapeutically effective dose of the IL-2 protein or the encoding nucleic acid molecule is suitably a low dose thereof, as is known in the art.

[0195] In one example, the IL-2 protein or the encoding nucleic acid molecule described herein is administered at a dose of or, in the case of the encoding nucleic acid, a dose equivalent to about 0.05 MIU / day to about 10 MIU / day (e.g., about 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10 MIU of IL-2 / day or any range therein). In some examples, the IL-2 protein or the encoding nucleic acid molecule is administered at a dose of or a dose equivalent to about 0.1 MIU / day to about 8 MIU / day. In other examples, the IL-2 protein or the encoding nucleic acid molecule is administered at a dose of or a dose equivalent to about 0.2 MIU / day to about 5 MIU / day. In further examples, the IL-2 protein or the encoding nucleic acid molecule is administered at a dose of or a dose equivalent to about 0.3 MIU / day to about 3.5 MIU / day. In related examples, the IL-2 protein or the encoding nucleic acid molecule is administered at a dose of or a dose equivalent to about 0.5 MIU / day to about 5 MIU / day. In specific examples, the IL-2 protein or the encoding nucleic acid molecule is administered at a dose of or a dose equivalent to about 0.4 MIU / day to about 4 MIU / day. In other examples, the IL-2 protein or the encoding nucleic acid molecule is administered at a dose of or a dose equivalent to about 1 MIU / day to about 3 MIU / day. In some examples, the IL-2 protein or the encoding nucleic acid molecule is administered at a dose of or a dose equivalent to about 1 MIU / day to about 2 MIU / day. In other examples, the IL-2 protein or the encoding nucleic acid molecule is administered at a dose of or a dose equivalent to about 0.5 MIU / day to about 2.5 MIU / day.

[0196] As used herein, the term “IU” means International Unit and the term “MIU” means mega or million International Units. One microgram of vitamin D is about 40 international units. The conversion between International Units (IU) and other units for IL-2 can vary depending on the specific context and reference standard used. However, generally, IU is a measure of biological activity, not a fixed mass, and is calibrated against a reference standard like the WHO international standard of IL-2 (NIBSC code: 86 / 500). Accordingly, the MIU values provided herein suitably refer to or are defined by such a standard (e.g., an MIU of a wildtype human IL-2 protein).

[0197] According to various examples, the IL-2 protein or the encoding nucleic acid molecule is administered, such as parenterally administered, at a dose, such as a daily dose, of or a dose equivalent to about 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10 MIU of IL- 2 or any range therein. In some examples, the IL-2 protein or the encoding nucleic acid molecule is administered at a dose of or a dose equivalent to less than about 10 MIU / day (e.g., less than about 10, 9, 8, 7, 6, 5, 4, 3.5, 3, 2.5, 2, 1.5, 1 or 0.5 MIU of IL-2 / day or any range therein). For other examples, the IL-2 protein or the encoding nucleic acid molecule is administered at a dose of or a dose equivalent to less than about 5 MIU / day. More particularly, the IL-2 protein or the encoding nucleic acid molecule can be administered at a dose of or a dose equivalent to less than about 3 MIU / day. Even more particularly, the IL-2 protein or the encoding nucleic acid molecule can be administered at a dose of or a dose equivalent to less than about 2.5 MIU / day.

[0198] It is further contemplated that the IL-2 protein or the encoding nucleic acid molecule may be administered daily or alternatively every 2nd, 3rd, 4th, 5th, 6thor 7thday, such as with those dosages hereinbefore provided. In particular examples, the IL-2 protein or the encoding nucleic acid molecule is administered once, twice, three times, four times or five times per week over the course of the treatment regime. For some examples, the IL-2 protein or the encoding nucleic acid molecule is administered at a dosage of or a dose equivalent to about 0.05 MIU / day to about 10 MIU / day, about 0.1 MIU / day to about 8 MIU / day, about 0.2 MIU / day to about 5 MIU / day, about 0.3 MIU / day to about 3.5 MIU / day, about 0.5 MIU / day to about 5 MIU / day, about 0.4 MIU / day to about 4 MIU / day, or about 1 MIU / day to about 3 MIU / day, at least about two, three, four or five times per week.

[0199] Further, it will be apparent to one of ordinary skill in the art that the optimal quantity and spacing of dosing may depend on the autoimmune disease, disorder or condition being treated, the form, route and site of administration, and the nature of individual dosages will be determined by the nature and extent the particular individual being treated. Also, such optimum conditions can be determined by conventional techniques.

[0200] Suitably, administration of the IL-2 protein or the encoding nucleic acid molecule is continued until cessation of one or more clinical symptoms of the autoimmune disease, disorder or condition. In any of the methods disclosed herein, the IL-2 protein or the encoding nucleic acid molecule may be administered daily for a period of at least about 1 day (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60 days or any range therein), or at least about 2 days, or at least about 3 days, or at least about 4 days, or at least about 5 days, or at least about 6 days, or at least about 7 days, or at least about 8 days, or at least about 9 days, or at least about 10 days, or at least about 11 days, or at least about 12 days, or at least about 13 days or at least about 2 weeks or any range therein. In particular examples, the IL-2 protein or the encoding nucleic acid molecule is administered daily for a period of at least about 5 days. In other examples, the IL-2 protein or the encoding nucleic acid molecule is administered daily for a period of between about 3 days and about 7 days. For certain examples, the IL-2 protein or the encoding nucleic acid molecule is administered daily for a period of between about 4 days and about 6 days. In various examples, the IL-2 protein or the encoding nucleic acid molecule is administered daily for a period of about 5 days. Moreover, the IL-2 protein or the encoding nucleic acid molecule may be administered daily for a period of at least about 2 weeks (e.g., at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 weeks or any range therein), or at least about 3 weeks, or at least about 4 weeks, or at least about 5 weeks, or at least about 6 weeks. In certain examples, the IL-2 protein or the encoding nucleic acid molecule is administered, such as parenterally administered, daily for a period of at least about 2 weeks. In another example, the IL-2 protein or the encoding nucleic acid molecule is administered, such as parenterally administered, daily for a period of at least about 1 week.

[0201] Referring to some examples, the methods described herein may include administering a first course or a loading dose of the IL-2 protein or the encoding nucleic acid molecule. For such examples, the first course suitably comprises administering the IL-2 protein or the encoding nucleic acid molecule once per day for at least 3 consecutive days, more particularly for at least 4 consecutive days, even more particularly for at least 5 consecutive days, still more particularly for at least 6 consecutive days, or still even more particularly for at least 7 consecutive days. Suitably, the first course is continued until cessation of one or more clinical symptoms of the autoimmune disease, disorder or condition.

[0202] It is further envisaged that the first course may be followed by a suitable maintenance dose, such as at an appropriate time interval or period, or more particularly a regular time interval or period, after administration of the first course. Accordingly, the methods described herein may include administering the IL-2 protein or the encoding nucleic acid molecule at one or more time intervals or periods, such as once, twice, three times four times etc every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks (or any range therein) after the first course or loading dose of the IL-2 protein or the encoding nucleic acid molecule. It is envisaged that the first course and the maintenance dose of the IL-2 protein or the encoding nucleic acid molecule, inclusive of the timing, dose and duration, may be altered according to the subject’s response, such as based on the effects of treatment on the levels and / or activity of regulatory T cells and / or effector T cells and / or levels of autoantibodies in the subject.

[0203] Those skilled in the art will appreciate that in accordance with the presently disclosed methods and uses, the IL-2 protein, the encoding nucleic acid molecule or the compositions disclosed herein may be administered alone or in conjunction with one or more additional agents, such as an antimicrobial agent described herein, as part of a combination therapy.

[0204] Any safe route of administration may be employed for providing a patient with the composition of the present disclosure. For example, oral, rectal, parenteral, sublingual, buccal, intravenous, intra-articular, intra-muscular, intra-dermal, subcutaneous, inhalational, intraocular, intraperitoneal, intracerebroventricular, transdermal and the like may be employed. Suitably, the IL-2 protein or the encoding nucleic acid molecule is administered or formulated to be administered parenterally. Referring to various examples, the IL-2 protein or the encoding nucleic acid molecule is administered subcutaneously, intravenously or intramuscularly. In some examples, the IL-2 protein or the encoding nucleic acid molecule is administered subcutaneously. In certain examples, the agents described herein, such as the antimicrobial agent are administered orally. The terms “oral administration”, “orally administering” and the like represent any method of administration in which an active agent can be administered by swallowing, chewing, sucking or drinking an oral dosage form of said active agent. Such solid or liquid oral dosage forms are traditionally intended to substantially release and or deliver the active agent in the gastrointestinal tract beyond the mouth and / or buccal cavity. Examples of solid dosage forms include conventional tablets, multi-layer tablets capsules, caplets, etc., which do not substantially release the drug in the mouth or in the oral cavity.

[0205] Compositions

[0206] Compositions comprising an IL-2 protein or an encoding nucleic acid molecule, together with an acceptable carrier or diluent, can be useful in the methods disclosed herein. Therapeutic compositions can be prepared by mixing the desired compounds having the appropriate degree of purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers (see, e.g., Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)), in the form of lyophilized formulations, aqueous solutions or aqueous suspensions. Acceptable carriers, excipients, or stabilizers are preferably nontoxic to recipients at the dosages and concentrations employed, and include buffers such as Tris, HEPES, PIPES, phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; and / or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

[0207] Additional examples of such carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts, or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, and cellulose-based substances.

[0208] Therapeutic compositions to be used for in vivo administration should be sterile. This is readily accomplished by filtration through sterile filtration membranes, prior to or following lyophilization and reconstitution. The composition may be stored in a lyophilized form or in solution if to be administered systemically. If in a lyophilized form, it is typically formulated in combination with other ingredients for reconstitution with an appropriate diluent at the time for use. An example of a liquid formulation is a sterile, clear, colourless unpreserved solution filled in a single-dose vial for subcutaneous injection.

[0209] Single or multiple administrations of the compositions are administered depending on the dosage and frequency as required and tolerated by the patient. The dosage and frequency will typically vary according to factors specific for each patient depending on the specific therapeutic or prophylactic agents administered, the severity and type of disease or condition, the route of administration, as well as age, body weight, response, and the past medical history of the patient. Suitable regimens can be selected by one skilled in the art by considering such factors and by following, for example, dosages reported in the literature and recommended in the Physician's Desk Reference (56th ed., 2002). Generally, the dose is sufficient to treat or ameliorate symptoms or signs of disease without producing unacceptable toxicity to the patient.

[0210] EXAMPLES

[0211] Example 1. Immunotherapeutic strategy to prevent progression and complications of Acute Rheumatic Fever

[0212] The present Example utilized the Rat Autoimmune Valvulitis (RAV) model (Rafeek et al., 2022; Reynolds et al., 2023; Sikder et al., 2019) to investigate whether LD-IL-2 therapy can be repurposed to treat active ARF and prevent RHD.

[0213] Material and methods

[0214] Animals

[0215] All experimental protocols involving animals were approved by the Animal Ethics Committee of University of New England (UNE) (ARA23-009). Female Lewis rats (LEW / SsN; Albino^h^RT1) aged 4 to 6 weeks were purchased from the Centre for Animal Research and Teaching at UNE and acclimatised for 5 days prior to experiments. Preparation of recombinant Strep A M5 proteins

[0216] Recombinant M5 protein of Strep A (rM5) was cloned and purified as described previously (Gorton, et al., 2009). Briefly, Strep A emm5 was cloned into pREP4 vector and expressed in E.coli BL21. Recombinant M proteins were purified using Ni-NTA resin. Lipopolysaccharide (LPS) contamination in recombinant protein preparations was removed by Triton X-114 assisted LPS extraction as previously described (Teodorowicz et al., 2017). The rM5 protein preparations was determined to be free of LPS using Pierce Chromogenic Endotoxin Quantification Kit (ThermoFisher, USA) as per manufacturer’s instructions.

[0217] Induction of autoimmune carditis and valvulitis

[0218] For induction of carditis, rats were injected with 0.5 mg / 100 pL of recombinant Strep A M5 protein emulsified in Complete Freund's Adjuvant (CFA) or PBS emulsified in CFA. Baseline Electrocardiography (ECG) was performed prior to injection. All priming injections were performed under isoflurane inhalation anaesthesia (5% induction and 2% maintenance) in the hock as described previously (Gorton et al., 2010). On day 1 and 3 all rats were intraperitoneally (i.p.) injected with 300 ng of Bordetella pertussis (BPTx) toxin (Gibco, USA) in 200 pL PBS. Rats were boosted with respective antigens in Incomplete Freund’s Adjuvants (IF A) on the flank on days 7, 14, and 21. The rats were euthanized on day 35 from the day of primary injection with the overdose of sodium pentobarbital (260mg / kg, i.p), blood was collected by cardiac puncture and heart tissue was collected and fixed with 4% paraformaldehyde for histopathological analysis. Heart draining mediastinal lymph nodes were collected for flow cytometry analysis.

[0219] Low dose interleukin-2 (LD-IL-2) treatment

[0220] Twenty-three age matched female Lewis rats were divided into four treatment groups (n=5 / 6); (i) PBS, (ii) Strep A rM5, (iii) Strep A rM5 + IL-2 (D8) and (iv) Strep A rM5 + IL-2 (D21). Recombinant Human IL-2 (#200-02, Peprotech, USA) was reconstituted in lOOmM acetic acid and further diluted in PBS containing 0.1% Bovine Serum Albumin (BSA) to achieve 20,000 IU concentration. Rats in group iii received s.c injection of LD-IL-2 from Day 8 of primary injection to Day 24 on every other day. Rats in group iv received s.c injection of LD-IL-2 from Day 21 of primary injection to Day 34 on every other day (Figure 1).

[0221] Electrocardiography (ECG)

[0222] ECG traces were recorded for 1-2 min using Bio Amp with PowerLab data acquisition system (ADInstruments, USA) in all rats prior to injection and a day before euthanasia to assess the conduction abnormalities in the heart. Peak values of P and R points at three different segments of ECG from each rat was individually extracted and analysed (Rafeek et al., 2021).

[0223] Histopathological analysis of cardiac tissue

[0224] To examine the extent of inflammation, formalin-fixed paraffin-embedded (FFPE) cardiac tissue was processed, embedded in paraffin, sectioned, and stained with Harris H&E using standard procedures as previously described (Gorton, et al., 2009). Slides were examined microscopically for infiltration of inflammatory cells as evidence of myocarditis or valvulitis. The extent of inflammation was expressed as a “carditis score” based on the number of inflammatory cells and focal lesions from each rat (Reynolds et al., 2023). The scoring system assesses and scores valvular and myocardial sections (5 randomly selected areas per animal) from the different groups. These sections were blindly scored using a validated semi -quantitative scoring system.

[0225] Detection of tissue-reactive serum antibodies

[0226] Serum IgG antibody against purified host proteins including cardiac myosin, tropomyosin, laminin, keratin, dopamine receptors 1&2, lysogangliosideGMi and tubulin (Sigma, USA) were determined using indirect ELISA as described previously (Rafeek et al., 2022). Briefly purified proteins were coated onto Maxisorp 96-well plates (Nunc, USA) in carbonate-bicarbonate buffer. Plates were blocked and incubated with individual rat sera in duplicate at 1 :400 dilution. Following repeated washing of the plates, each well was incubated with 100 pL of goat anti -rat IgG HRP conjugated secondary antibody (1 :5000; Jackson Immunoresearch, USA) for an hour at room temperature. Each well was washed and incubated for 20 min with 100 pL of SIGMAFAST™ OPD (Sigma, USA) . Absorbance was measured at 450 nm using SpectraMax M2 / M2e (Molecular Devices, USA).

[0227] Multiparametric flow cytometry for analysis of circulating Treg

[0228] Mediastinal lymph nodes were dissociated into single-cells suspension were obtained from mediastinal lymph nodes using a plunger from a sterile syringe. Red blood cells were lysed with ACK lysis buffer and washed with RPMI (Thermo Fisher Scientific, AUS). The cell populations were determined by flow cytometry. The cells were surface-stained with a master mix containing dead cell exclusion dye (Zombie violet), CD4-APC-Cy7, CD3-PerCP, CD8-PE-Cy7 and CD25- APC. Cells were stained on ice in the dark for 40 min. Intranuclear staining for FoxP3-PE was achieved using FoxP3 Fix / Perm Buffer Set for nuclear staining (BioLegend, San Diego, California, USA). Samples were washed in PBS and acquired Samples were acquired by a BD Fortessa multiparametric flow cytometer (BD Biosciences, Franklin Lakes, New Jersey, USA).

[0229] Data were analyzed using FlowJo V.10.7 (BD).

[0230] Adoptive T cell transfer

[0231] To investigate the immunomodulatory effects of CD4+T cells, an adoptive transfer model was employed in Lewis rats. Donor rats were injected with recombinant M5 (rM5) protein and treated with nine injections of low-dose IL-2 to expand CD4+regulatory T cells (Tregs), using the same method previously described. On day 35, spleens were harvested, and CD4+T cells were isolated via magnetic separation. Flow cytometry confirmed enrichment of a pure CD4+T cell population, comprising approximately 12% Tregs. Recipient rats received an intravenous injection of 10* 106CD4+T cells from rM5 + LD-IL-2— treated donors one day after their third rM5 booster immunisation. The effects of adoptive transfer were evaluated on day 27 by electrocardiography (ECG), histopathological scoring of heart tissue, and ELISA-based detection of cardiac autoantibodies.

[0232] Statistical analysis

[0233] Descriptive statistics of results were made using GraphPad Prism version 8.0. Antibody titre data are presented as geometric mean. One-way ANOVA with Dunnett post hoc method for multiple comparisons was used for pairwise comparisons. Results were taken as significant with P -values <0.05 or <0.01. Two-way ANOVA was used to assess ECG differences between rats. All data with p-values <0.05 were considered significant.

[0234] Results and Discussion

[0235] The rat autoimmune valvulitis (RAV) model has been previously developed and characterized to determine the early events that lead to the clinical syndromes of ARF complications. In this model, multiple injections of rats with recombinant M protein type 5 (rM5, Strep A M5 is a classical “rheumatogenic” strain) emulsified in Freund's adjuvant induces antibodies and T-cells that cross-react with the cardiac tissue (Gorton et al., 2009; Lymbury et al., 2003; Sikder et al., 2019). The histological, immunological, and functional changes in the hearts resembles that of RHD, including Aschoffs nodules. To investigate whether LD-IL-2 therapy can be repurposed to treat carditis, PBS-injected control rats were compared with rats injected with rM5, which were either treated with LD-IL-2 or left untreated. Treatment regimen consisted of repeated subcutaneous injections of LD-IL-2 after rats have received the first (Day 8) or last (Day 21) booster with Strep A rM5 (Fig. 1). For both regimens, LD-IL-2 was administered daily for 5 days, followed by 4 injections with a 2-day interval between each injection. Euthanasia was performed on Day 35, either 11 days following the conclusion of LD-IL-2 treatment (Day 8) or 2 days following the end of treatment (Day 21). 5 -day course of LD-IL-2 therapy with daily injections was shown to harness IL-2's immunomodulatory properties and mitigate autoimmune responses in humans and in preclinical models (Churlaud et al., 2015; Grasshoff et al., 2021).

[0236] LD-IL-2 therapy prevents cardiac functional and histological changes associated with RHD.

[0237] An important characteristic of the RAV model is its ability to induce observable functional and histological changes to the heart, mirroring those clinically observed in patients with ARF / RHD. To measure conduction abnormalities in the heart and assess cardiac function, all rats underwent electrocardiography (ECG) before culled on Day 35. Baseline heart function prior to the first Strep A rM5 injection (Day 0) was used to compare functional changes. Peak values of P and R points at three different segments of ECG from each rat was individually extracted and analyzed. A prolongation of the P-R interval was found in all rM5 -treated rats, indicating a conduction abnormality of the heart (Fig. 2A). In contrast, rats demonstrated no change to P-R interval following both LD-IL-2 therapeutic regimen.

[0238] To investigate the effect of LD-IL-2 therapy on inflammatory changes associated with ARF / RHD, infiltrating mononuclear cells were assessed by hematoxylin and eosin (H&E) staining. The extent of inflammation in myocardium and valves was expressed as a “carditis score” based on the number of mononuclear cell infiltrates and focal lesions in both the myocardium and mitral valve. Carditis scoring was undertaken blinded using a scoring matrix previously developed (Reynolds et al., 2023). An increased mononuclear cell infiltration was observed in rats injected with rM5 compared to PBS control group. In contrast, minimal mononuclear cell infiltration was observed in rats injected with rM5 that were treated with LD-IL-2 from either Day 8 or Day 21, which presented scores similar to control rats injected with PBS only. Both LD-IL-2 treatment regimens resulted in a significant reduction in carditis, with minimal difference observed between the two groups (Fig. 2B and 2C).

[0239] It is possible that the timing of LD-IL-2 treatment in ARF can impact the effectiveness of LD-IL-2 therapy, due to the stage and chronicity of the autoimmune process. Notwithstanding this, the present Example demonstrates that commencing IL-2 immunotherapy either early or later in the time course of M protein exposure and hence disease onset, were both effective in treating any existing disease and preventing the development of autoantibodies and any associated sequelae of ARF in this rodent model. In the RAV model, starting LD-IL-2 treatment at Day 21 may allow for better resolution of the acute inflammatory response triggered by rM5 in comparison with treatment starting at Day 8. This could be because LD-IL-2 treatment during active disease phases or when specific T-cell subsets are dysregulated could help modulate the ongoing response and prevent further exacerbation of heart damage. While the idea of monitoring immune profiles to adjust LD-IL-2 therapy holds theoretical promise for enhancing efficacy and safety in autoimmune diseases, ongoing research and clinical trials are crucial to establish definitive guidelines and protocols for implementation in clinical practice.

[0240] Reduction of cross-reactive M-protein antibodies following LD-IL-2 therapy

[0241] Tissue cross-reactive antibodies against streptococcal antigens play a significant role in initiating the autoimmune pathology in ARF / RHD. A key aspect of the RAV model is its ability to generate antibodies that cross-react with cardiac and neuronal tissue. To investigate whether LD-IL-2 therapy can inhibit the induction of cross-reactive antibodies, we conducted enzyme- linked immunosorbent assay (ELISA) against cardiac myosin and tropomyosin (heart muscle), laminin (heart valve extracellular matrix), and dopamine receptors type 1 and 2, tubulin and lysoganglioside (brain). Although the rM5-injected group exhibited significant cross-reactivity with all tissue proteins compared to the negative control PBS group, no cross-reactivity to cardiac tissues was observed for the antisera from both LD-IL-2 treatment groups (Fig. 3 A). LD-IL-2 also resulted in significant reduction in the levels of IgG recognizing connective and neuronal proteins (Fig. 3B and 3C). No significant difference was observed between both treatment groups, and treatment started at Day 21 resulted in antibody levels similar to the control group (Fig. 3B and 3C).

[0242] In ARF / RHD, tissue cross-reactive antibodies activate the valvular endothelium and trigger carditis. In SC, cross reactive antibodies that transverse the compromised blood brain barrier and bind to neuronal proteins in the basal ganglia are considered to be the cause for the onset of neurob ehavi oral symptoms (Carapetis et al., 2016; Seller et al., 2023). Particularly, autoantibodies against dopamine receptors may lead to a receptor imbalance and induce greater sensitivity to dopamine signaling potentially leading to neuropsychiatric symptoms in SC (Ben-Pazi et al., 2013).

[0243] LD-IL-2 can indirectly affect B cell differentiation and antibody production. By promoting a regulatory environment through Tregs, LD-IL-2 helps to control the differentiation of B cells into antibody-secreting plasma cells that are enriched for a regulatory B cell signature (Inaba et al., 2023), thereby reducing the production of self-reactive antibodies. Besides, LD-IL-2 suppress the activation and differentiation of T follicular helper (Tfh) cells (Ballesteros-Tato, 2014; Liang et al., 2021). This modulation helps to limit excessive germinal center responses that contribute to autoantibody production and promotes immune tolerance, highlighting LD-IL-2 as a potential therapeutic strategy for managing ARF / RHD. LD-IL-2 therapy selectively expands regulatory T-cells (Treg) in mediastinal lymph nodes

[0244] Tregs are a specialized T-cell subset that regulates the immune system, maintaining homeostasis and self-tolerance (Singh et al., 2023; Wong et al., 2021). Decreased numbers or defective function of Tregs has been implicated in the pathogenesis of various autoimmune diseases (Dominguez- Villar and Hafler, 2018; Weerakoon et al., 2021). Although higher doses of IL-2 therapy has been traditionally approved for cancer therapy, the concept of using LD-IL-2 for treating autoimmune and inflammatory diseases emerged due to its potential to induce Treg cells compared to conventional T-cells and NK cells (Malek and Castro, 2010).

[0245] The present Example investigated the impact of LD-IL-2 therapy on Treg and conventional T-cells from mediastinal (heart-draining) lymph nodes using flow cytometry. Strikingly, LD-IL-2 therapy resulted in 50% increase of classical Treg (CD4+CD25+FoxP3+) in lymph nodes of rats injected with rM5 (Fig. 4A). This is consistent with the expansion achieved in other rodent models assessing efficacy of LD-IL-2 therapy for treating autoimmune disorders (Zhou et al., 2021). Noteworthy, an increase of rare CD8+Treg (CD8+CD25+FoxP3+) was observed when rats were treated with LD-IL-2 from Day 21, but not when they were treated from Day 8 (Fig. 4B). In contrast to classical Tregs, the origin and roles of CD8+Treg in the pathogenesis of autoimmune diseases are less understood. While naturally occurring CD8+Treg are rare, they can be induced in vivo (Flippe et al., 2019) and are generally considered to maintain less stable expression of FoxP3 than classical Tregs (lamsawat et al., 2018; Wang et al., 2021). Nonetheless, CD8+Tregs show heightened sensitivity to IL-2 for proliferation compared to T effector cells and have been induced by LD-IL-2 therapy in both mice and humans, leading to benefits in alleviating autoimmune disorders (Aoyama et al., 2012; Churlaud et al., 2015; Dinesh et al., 2010). In the present Example, there was no induction of CD8+Tregs when rats were treated with LD-IL-2 therapy starting from Day 8. This lack of induction is likely due to the instability of FoxP3 expression in induced Tregs, which could not be sustained until the experiment endpoint, at Day 35. Similar to their CD4+counterpart, CD8+Tregs have been shown to play important roles in immune regulation suppressing immune responses through a variety of mechanisms that include secretion of cytokines, cell-to-cell contact, induction of a tolerogenic phenotype in APCs that can then induce classical Tregs, and cytotoxic activity (Mishra et al., 2021; Vieyra-Lobato et al., 2018).

[0246] Although administrating LD-IL-2 expanded Tregs and reduced disease severity in several autoimmune diseases (Hartemann et al., 2013; He et al., 2016; Zheng et al., 2022), IL-2 is also a growth factor for potentially pathogenic conventional T-cells due to the widespread expression of IL-2R subunits. Thus, IL-2's dual role in promoting both tolerance and activation makes predicting its therapeutic effects challenging. Moreover, low dose IL-2 has been demonstrated to exhibit limited or no therapeutic efficacy, as well as heterogeneity in clinical responsiveness, in various autoimmune and alloimmune conditions (Lim et al., J Hepatol. 2023 Jan;78(l): 153-164; GraBhoff et al., Front Immunol. 2021; 12: 648408). Furthermore, a therapeutic role for low dose IL-2 in autoimmune diseases mediated by bacterial antigens and subsequent molecular mimicry has not been previously described.

[0247] To determine if the LD-IL-2 therapeutic regimen used in this study could also expand conventional T-cells, the frequencies of total T-cells and the CD4+and CD8+T-cell subsets were analyzed. Notably, LD-IL-2 therapy did not alter the frequencies of conventional T-cells but specifically expanded Tregs (Fig. 5) suggesting that LD-IL-2 is a safe and specific approach for treating ARF and preventing RHD.

[0248] LD-IL2 therapy does not impair IgG antibody responses to a foreign antigen

[0249] To determine whether the LD-IL-2 therapeutic regimen used in this study impaired IgG antibody responses to a foreign antigen, rats were given 2 doses of pertussis toxin and serum IgG antibody levels against pertussis toxin were measured. Although LD-IL-2 decreased serum IgG recognition of cardiac, connective tissue and brain proteins (Rafeek et al., 2025), it did not change responses to pertussis toxin, suggesting it specifically decreases responses to autoantigens but not to foreign antigens (Fig. 6).

[0250] LD-IL-2 restores germinal center (GC) B cells and regulatory T cells (Tregs) balance in the spleen

[0251] The present Example also investigated the effect of LD-IL-2 treatment on GC B cells and Tregs in the spleen. Increased autoantibody levels following injections with rM5 (Rafeek et al. ,2025) were associated with an increase in GC B cells and decrease of Tregs in the spleen (Fig. 7). LD-IL-2 therapy decreased the frequency of germinal center B cells and reestablished regulatory T cells towards the levels observed in healthy rats (PBS-injected).

[0252] LD-IL-2 therapy re-establishes immune homeostasis within the spleen

[0253] In order to assess the effect of LD-IL-2 therapy on gene expression within the spleen, the present Example analysed the splenic transcriptome with bulk RNA sequencing. LD-IL-2 therapy modulated the expression of multiple genes associated with immune responses and restored the transcriptome landscape of rM5-injected rats towards healthy levels (Fig 8). This was partly attributed to reduced expression of pro-inflammatory cytokine receptors and ligands, including IL- 17, IFN-y, IL- 10, and IL-13, which were upregulated following rM5 injections. In contrast, I12ra (CD25, the receptor for IL-2) and I15ra (the receptor for IL-5, both implicated in protective roles in certain autoimmune diseases, were enriched following LD-IL-2 therapy. All of these cytokines are known to be secreted by CD4+T cells.

[0254] Adoptive transfer of CD4+ T cells isolated from LD-IL-2 treated rats reverse rM5-induced cardiac dysfunction.

[0255] In the present Example, it was experimentally demonstrated that repeated injection of LD- IL-2 following rM5 injection resulted in expansion of both CD4+and CD8+Tregs and thereby reduced the associated cardiac inflammation. However, the short half-life of IL-2 necessitates a course of repetitive injections to maintain therapeutic levels of Tregs for treatment of autoimmune diseases (Lotze et al., 1985).

[0256] The present Example therefore also investigated whether CD4+ T cells isolated from the spleens of rats treated with LD-IL-2 could treat carditis and prevent cardiac dysfunction following adoptive transfer into rM5 injected rats. Adoptive transfer of LD-IL-2-expanded CD4+ T cells (containing approximately 12% of Tregs) prevented cardiac inflammation and dysfunction following injections with rM5 (Fig 9). It is known that when rats were injected with CD4+ T cells, at day 21, carditis was already in place. Importantly, this data shows that their therapeutic effect occurs even when pathogenic autoantibodies are present, implying that the protection is not due to suppression of antibody production, but rather due to modulation of tissue inflammation — likely through regulatory mechanisms mediated by the transferred T cells, and particularly Tregs. This highlights a potential tissue-protective or anti-inflammatory role of LD-IL-2-expanded CD4+T cells, independent of antibody-mediated pathology.

[0257] Treg cells constitutively express high levels of the heterotrimeric high-affinity IL-2 receptor (IL-2R) complex and the highest expression of the high-affinity a chain (CD25) of the IL-2R complex, making them particularly sensitive to even small amounts of IL-2 in the body. However, a genetic deficiency in the IL-2 / IL-2R pathway may leads to systemic autoimmunity (Sadlack et al., 1993). Due to this unique property LD-IL-2 therapy has become an emerging immunotherapeutic approach for treating certain autoimmune conditions. The present Example suggests that LD-IL-2 therapy holds promise for managing ARF by effectively reducing autoimmune responses and potentially preventing carditis. By targeting immune system dysregulation, LD-IL-2 could represent a valuable therapeutic approach. This approach may lead to improved clinical outcomes and reduced incidence of cardiac complications in individuals affected by ARF. References

[0258] Aoyama, A., D. Klarin, Y. Yamada, S. Boskovic, O. Nadazdin, K. Kawai, D. Schoenfeld, J.C. Madsen, A.B. Cosimi, G. Benichou, and T. Kawai. 2012. Low-dose IL-2 for In vivo expansion of CD4+ and CD8+ regulatory T cells in nonhuman primates. Am J Transplant 12:2532-2537.

[0259] Ballesteros-Tato, A. 2014. Beyond regulatory T cells: the potential role for IL-2 to deplete T- follicular helper cells and treat autoimmune diseases. Immunotherapy 6:1207-1220.

[0260] Bas, H.D., K. Baser, E. Yavuz, H.A. Bolayir, B. Yaman, S. Unlu, A. Cengel, E.U. Bagriacik, and R. Yalcin. 2014. A shift in the balance of regulatory T and T helper 17 cells in rheumatic heart disease. J Investig Med 62:78-83.

[0261] Ben-Pazi, H., J. A. Stoner, and M.W. Cunningham. 2013. Dopamine receptor autoantibodies correlate with symptoms in Sydenham's chorea. PLoS One 8:e73516.

[0262] Carapetis, J.R., A. Beaton, M.W. Cunningham, L. Guilherme, G. Karthikeyan, B.M. Mayosi, C. Sable, A. Steer, N. Wilson, R. Wyber, and L. Zuhlke. 2016. Acute rheumatic fever and rheumatic heart disease. Nat Rev Dis Primers 2: 15084.

[0263] Churlaud, G., V. Jimenez, J. Ruberte, M. Amadoudji Zin, G. Fourcade, G. Gottrand, E. Casana, B. Lambrecht, B. Bellier, E. Piaggio, F. Bosch, and D. Klatzmann. 2014. Sustained stimulation and expansion of Tregs by IL2 control autoimmunity without impairing immune responses to infection, vaccination and cancer. Clin Immunol 151 : 114-126.

[0264] Churlaud, G., F. Pitoiset, F. Jebbawi, R. Lorenzon, B. Bellier, M. Rosenzwajg, and D. Klatzmann. 2015. Human and Mouse CD8(+)CD25(+)FOXP3(+) Regulatory T Cells at Steady State and during Interleukin-2 Therapy. Front Immunol 6: 171.

[0265] Cunningham, M.W. 2000. Pathogenesis of group A streptococcal infections. Clin Microbiol Rev 13:470-511.

[0266] Cunningham, M.W. 2019. Molecular Mimicry, Autoimmunity, and Infection: The Cross-Reactive Antigens of Group A Streptococci and their Sequelae. Microbiol Spectr 7:

[0267] Dinesh, R.K., B.J. Skaggs, A. La Cava, B.H. Hahn, and R.P. Singh. 2010. CD8+ Tregs in lupus, autoimmunity, and beyond. Autoimmun Rev 9:560-568.

[0268] Flippe, L., S. Bezie, I. Anegon, and C. Guillonneau. 2019. Future prospects for CD8(+) regulatory T cells in immune tolerance. Immunol Rev 292:209-224.

[0269] Good, M.F. 2020. Streptococcus: An organism causing diseases beyond neglect. PLoSNegl Prop Dis 14:e0008095.

[0270] Gorton, D., S. Blyth, J.G. Gorton, B. Govan, and N. Ketheesan. 2010. An alternative technique for the induction of autoimmune valvulitis in a rat model of rheumatic heart disease. J Immunol Methods 355:80-85. Gorton, D., B. Govan, C. Olive, and N. Ketheesan. 2009. B- and T-cell responses in group a streptococcus M-protein- or Peptide-induced experimental carditis. Infect Immun 77:2177-2183.

[0271] Gorton., B. Govan, C. Olive, and N. Ketheesan. 2009. B- and T-cell responses in group a streptococcus M-protein- or Peptide-induced experimental carditis. Infect Immun 77:2177-2183. Grasshoff, H., S. Comduhr, L.R. Monne, A. Muller, P. Lamprecht, G. Riemekasten, and J.Y. Humrich. 2021. Low-Dose IL-2 Therapy in Autoimmune and Rheumatic Diseases. Front Immunol 12:648408.

[0272] Hartemann, A., G. Bensimon, C.A. Payan, S. Jacqueminet, O. Bourron, N. Nicolas, M. Fonfrede, M. Rosenzwajg, C. Bernard, and D. Klatzmann. 2013. Low-dose interleukin 2 in patients with type 1 diabetes: a phase 1 / 2 randomised, double-blind, placebo-controlled trial. Lancet Diabetes Endocrinol 1 :295-305.

[0273] He, J., X. Zhang, Y. Wei, X. Sun, Y. Chen, J. Deng, Y. Jin, Y. Gan, X. Hu, R. Jia, C. Xu, Z. Hou, Y.A. Leong, L. Zhu, J. Feng, Y. An, Y. Jia, C. Li, X. Liu, H. Ye, L. Ren, R. Li, H. Yao, Y. Li, S. Chen, X. Zhang, Y. Su, J. Guo, N. Shen, E.F. Morand, D. Yu, and Z. Li. 2016. Low-dose interleukin-2 treatment selectively modulates CD4(+) T cell subsets in patients with systemic lupus erythematosus. Nat Med 22:991-993. lamsawat, S., A. Daenthanasanmak, J.H. Voss, H. Nguyen, D. Bastian, C. Liu, and X.Z. Yu. 2018. Stabilization of Foxp3 by Targeting JAK2 Enhances Efficacy of CD8 Induced Regulatory T Cells in the Prevention of Graft-versus-Host Disease. J Immunol 201 :2812-2823.

[0274] Inaba, A., Z.K. Tuong, T.X. Zhao, A.P. Stewart, R. Mathews, L. Truman, R. Sriranjan, J. Kennet, K. Saeb-Parsy, L. Wicker, F. Waldron-Lynch, J. Cheriyan, J. A. Todd, Z. Mallat, and M.R. Clatworthy. 2023. Low-dose IL-2 enhances the generation of IL-10-producing immunoregulatory B cells. Nat Commun 14:2071.

[0275] Liang, K., J. He, Y. Wei, Q. Zeng, D. Gong, J. Qin, H. Ding, Z. Chen, P. Zhou, P. Niu, Q. Chen, C. Ding, L. Lu, X.X. Chen, Z. Li, N. Shen, D. Yu, and J. Deng. 2021. Sustained low-dose interleukin-2 therapy alleviates pathogenic humoral immunity via elevating the Tfr / Tfh ratio in lupus. Clin Transl Immunology 10:el293.

[0276] Lymbury, R.S., C. Olive, K.A. Powell, M.F. Good, R.G. Hirst, J.T. LaBrooy, and N. Ketheesan. 2003. Induction of autoimmune valvulitis in Lewis rats following immunization with peptides from the conserved region of group A streptococcal M protein. J Autoimmun 20:211-217.

[0277] Malek, T.R., and I. Castro. 2010. Interleukin-2 receptor signaling: at the interface between tolerance and immunity. Immunity 33: 153-165.

[0278] Mishra, S., W. Liao, Y. Liu, M. Yang, C. Ma, H. Wu, M. Zhao, X. Zhang, Y. Qiu, Q. Lu, and N. Zhang. 2021. TGF-beta and Eomes control the homeostasis of CD8+ regulatory T cells. J Exp Med 218: Mukhopadhyay, S., S. Varma, S. Gade, J. Yusuf, V. Trehan, and S. Tyagi. 2013. Regulatory T- cell deficiency in rheumatic heart disease: a preliminary observational study. J Heart Valve Dis 22: 118-125.

[0279] Rafeek, R.A., A.S. Hamlin, N.M. Andronicos, C.S. Lawlor, D.J. McMillan, K.S. Sriprakash, and N. Ketheesan. 2022. Characterization of an experimental model to determine streptococcal M protein-induced autoimmune cardiac and neurob ehavi oral abnormalities. Immunol Cell Biol 100:653-666.

[0280] Rafeek, R.A.M., C.M. Lobbe, E.C. Wilkinson, A.S. Hamlin, N.M. Andronicos, D.J. McMillan, K.S. Sriprakash, and N. Ketheesan. 2021. Group A streptococcal antigen exposed rat model to investigate neurob ehavi oral and cardiac complications associated with post-streptococcal autoimmune sequelae. Animal Model Exp Med 4: 151-161.

[0281] Reynolds, S., R. A.M. Rafeek, A. Hamlin, A. Lepletier, M. Pandey, N. Ketheesan, and M.F. Good. 2023. Streptococcus pyogenes vaccine candidates do not induce autoimmune responses in a rheumatic heart disease model. NPJ Vaccines 8:9.

[0282] Sikder, S., G. Price, M.A. Alim, A. Gautam, R. Scott Simpson, C. Margaret Rush, B. Lee Govan, and N. Ketheesan. 2019. Group A streptococcal M-protein specific antibodies and T-cells drive the pathology observed in the rat autoimmune valvulitis model. Autoimmunity 52:78-87.

[0283] Sikder, S., N.L. Williams, A.E. Sorenson, M.A. Alim, M.E. Vidgen, N.J. Moreland, C.M. Rush, R.S. Simpson, B.L. Govan, R.E. Norton, M.W. Cunningham, D.J. McMillan, K.S. Sriprakash, and N. Ketheesan. 2018. Group G Streptococcus Induces an Autoimmune Carditis Mediated by Interleukin 17A and Interferon gamma in the Lewis Rat Model of Rheumatic Heart Disease. J Infect Dis 218:324-335.

[0284] Singh, T.P., C. Farias Amorim, V.M. Lovins, C.W. Bradley, L.P. Carvalho, E.M. Carvalho, E.A. Grice, and P. Scott. 2023. Regulatory T cells control Staphylococcus aureus and disease severity of cutaneous leishmaniasis. J Exp Med 220:

[0285] Seller, T., K.V. Roberts, B.F. Middleton, and A.P. Ralph. 2023. Sydenham chorea in the top end of Australia's Northern Territory: A 20-year retrospective case series. J Paediatr Child Health 59: 1210-1216.

[0286] Stollerman, G.H., J.H. Rusoff, and I. Hirschfeld. 1955. Prophylaxis against group A streptococci in rheumatic fever; the use of single monthly injections of benzathine penicillin G. N Engl J Med 252:787-792.

[0287] Teodorowicz, M., O. Perdijk, I. Verhoek, C. Govers, H.F. Savelkoul, Y. Tang, H. Wichers, and K. Broersen. 2017. Optimized Triton X-114 assisted lipopolysaccharide (LPS) removal method reveals the immunomodulatory effect of food proteins. PLoS One 12:e0173778. Vieyra-Lobato, M.R., J. Vela-Ojeda, L. Montiel-Cervantes, R. Lopez-Santiago, andM.C. Moreno- Lafont. 2018. Description of CD8(+) Regulatory T Lymphocytes and Their Specific Intervention in Graft-versus-Host and Infectious Diseases, Autoimmunity, and Cancer. J Immunol Res 2018:3758713.

[0288] Wang, W., T. Hong, X. Wang, R. Wang, Y. Du, Q. Gao, S. Yang, and X. Zhang. 2021. Newly Found Peacekeeper: Potential of CD8+ Tregs for Graft-Versus-Host Disease. Front Immunol 12:764786.

[0289] Watkins, D.A., C.O. Johnson, S.M. Colquhoun, G. Karthikeyan, A. Beaton, G. Bukhman, M.H. Forouzanfar, C.T. Longenecker, B.M. Mayosi, G.A. Mensah, B.R. Nascimento, A.L.P. Ribeiro,

[0290] C.A. Sable, A.C. Steer, M. Naghavi, A.H. Mokdad, C.J.L. Murray, T. Vos, J.R. Carapetis, and G.A. Roth. 2017. Global, Regional, and National Burden of Rheumatic Heart Disease, 1990-2015. N Engl J Med 377 : 713 -722.

[0291] Welfare, A.I.o.H.a. 2022. Acute rheumatic fever and rheumatic heart disease in Australia 2016- 2020. Australian Government catalogue number CVD 95, AIHW,:

[0292] Wong, H.S., K. Park, A. Gola, A.P. Baptista, C.H. Miller, D. Deep, M. Lou, L.F. Boyd, A Y. Rudensky, P.A. Savage, G. Altan-Bonnet, J.S. Tsang, and R.N. Germain. 2021. A local regulatory T cell feedback circuit maintains immune homeostasis by pruning self-activated T cells. Cell 184:3981-3997 e3922.

[0293] Zheng, X., R. Su, F. Hu, Y. Liu, X. Li, C. Gao, and C. Wang. 2022. Low-dose IL-2 therapy restores imbalance between Thl7 and regulatory T cells in patients with the dermatomyositis combined with EBV / CMV viremia. Autoimmun Rev 21 : 103186.

[0294] Zhou, P., J. Chen, J. He, T. Zheng, J. Yunis, V. Makota, Y.O. Alexandre, F. Gong, X. Zhang, W. Xie, Y. Li, M. Shao, Y. Zhu, J.E. Sinclair, M. Miao, Y. Chen, K.R. Short, S.N. Mueller, X. Sun,

[0295] D. Yu, and Z. Li. 2021. Low-dose IL-2 therapy invigorates CD8+ T cells for viral control in systemic lupus erythematosus. PLoS Pathog 17:el009858.

[0296] Rukshan Ahamed Mohamed Rafeek, Natkunam Ketheesan, Michael F. Good, Manisha Pandey, Ailin Lepletier. Low-dose interleukin 2 therapy halts the progression of post-streptococcal autoimmune complications in a rat model of rheumatic heart disease. mBio. April 2025, Volume 16, Issue 4, e03823-24.

Claims

CLAIMS:

1. A method of treating or preventing an autoimmune disease, disorder or condition associated with a Group A Streptococcus (GAS) infection in a subject, said method including the step of administering a therapeutically effective amount of an IL-2 protein or a nucleic acid molecule encoding the IL-2 protein to the subject to thereby treat or prevent the autoimmune disease, disorder or condition.

2. The method of claim 1, wherein the autoimmune disease, disorder or condition is selected from the group consisting of rheumatic fever, rheumatic heart disease (RHD) and Sydenham’s Chorea.

3. The method of claim 1 or claim 2, wherein the autoimmune disease, disorder or condition is RHD.

4. The method of any one of the preceding claims, wherein the therapeutically effective amount of the IL-2 protein or the encoding nucleic acid molecule is a low dose thereof or is equivalent to a low dose thereof.

5. The method of any one of the preceding claims, wherein the IL-2 protein or the encoding nucleic acid molecule are administered at a dose of or a dose equivalent to less than about 3.5 MIU / day.

6. The method of any one of the preceding claims, wherein the IL-2 protein or the encoding nucleic acid molecule are administered at a dose of or a dose equivalent to between about 0.3 MIU / day to about 3.0 MIU / day.

7. The method of any one of the preceding claims, wherein the method comprises administering at least a first course of the IL-2 protein or the encoding nucleic acid molecule, wherein the first course comprises administering the IL-2 protein or the encoding nucleic acid molecule once per day for at least 3 consecutive days.

8. The method of any one of the preceding claims, wherein the IL-2 protein is or comprises a functional variant, fragment or derivative of a native or wildtype IL-2 protein.

9. The method of any one of the preceding claims, wherein the IL-2 protein or the encoding nucleic acid molecule is selected from the group consisting of aldesleukin, rezpegaldesleukin, NKTR-214, RO7049665, CC-92252, XmAb27564, MK-6194, AMG592, CUG252, mRNA-6231, BNT151, BNT153, AVB-001, salspera, TILT-123 and any combination thereof.

10. The method of any one of the preceding claims, wherein administration of the IL-2 protein or the encoding nucleic acid molecule increases a level and / or activity of regulatory T cells in the subject.

11. The method of any one of the preceding claims, wherein administration of the IL-2 protein or the encoding nucleic acid molecule does not substantially increase a level and / or activity of effector T cells in the subject.

12. The method of any one of the preceding claims, wherein administration of the IL-2 protein or the encoding nucleic acid molecule prevents or inhibits production of autoantibodies to a cardiac protein, a connective tissue protein and / or a neural protein in the subject.

13. The method of claim 12, wherein the cardiac protein is one or more of cardiac myosin and tropomyosin.

14. The method of claim 12, wherein the connective tissue protein is one or more of laminin and keratin.

15. The method of claim 12, wherein the neural protein is one or more of dopamine receptor 1, dopamine receptor 2, lysoganglioside and tubulin.

16. The method of any one of the preceding claims, wherein the subject is receiving an antimicrobial agent for treatment or prevention of the GAS infection.

17. The method of any one of the preceding claims, including the further step of administering an antimicrobial agent for treatment or prevention of the GAS infection to the subject.

18. The method of claim 16 or claim 17, wherein the antimicrobial agent is selected from the group consisting of a penicillin antibiotic, a lincosamide antibiotic, a macrolide antibiotic, a glycopeptide antibiotic, a cephalosporin antibiotic and any combination thereof.

19. The method of any one of claims 16 to 18, wherein the antimicrobial agent is selected from the group consisting of penicillin, amoxicillin, benzathine penicillin, clindamycin, erythromycin, clarithromycin, azithromycin, vancomycin, cephalosporin C, cefadroxil, cephalexin and any combination thereof.

20. A method of preventing or inhibiting the production of autoantibodies in a subject with or at risk of developing a GAS infection, said method including the step of administering a therapeutically effective amount of an IL-2 protein or a nucleic acid molecule encoding the IL-2 protein to the subject to thereby prevent or inhibit the production of autoantibodies therein.

21. A method of increasing a level and / or activity of regulatory T cells in a subject with or at risk of developing a GAS infection, said method including the step of administering a therapeutically effective amount of an IL-2 protein or a nucleic acid molecule encoding the IL-2 protein to the subject to thereby increase the level and / or activity of regulatory T cells therein.

22. The method of claim 20 or claim 21, wherein the subject has or is at risk of developing an autoimmune disease, disorder or condition associated with the GAS infection.

23. The method of claim 22, wherein the autoimmune disease, disorder or condition is rheumatic fever or rheumatic heart disease.

24. A kit for use in the method of any one of claims 1 to 23, said kit comprising a low dose of an IL-2 protein or a nucleic acid molecule encoding the IL-2 protein, optionally an antimicrobial agent and optionally instructions for use.

25. A method of treating or preventing an autoimmune disease, disorder or condition associated with a GAS infection in a subject, said method including the step of administering a therapeutically effective amount of regulatory T cells to the subject to thereby treat or prevent the autoimmune disease, disorder or condition.

Images