Compounds, compositions and methods for preventing or treating malaria

Abstract

This invention relates to compounds, compositions and methods for preventing or treating malaria by modulating immune responses through Killer-specific Secretory Protein 37 (Ksp37). The invention provides for the administration of Ksp37, or stimulation of endogenous Ksp37 production using polar compounds such as DMF or DMSO, to increase Ksp37 levels in a subject to therapeutically effective concentrations. Elevated Ksp37 enhances cytotoxic CD8+ T-cell and NK-cell activity, supports cytokine balance, and improves immune clearance of Plasmodium parasites during both liver and blood stages. These approaches promote targeted immune activation, reduce parasite survival, and improve host protection against malaria infection.

Description

[0001] COMPOUNDS, COMPOSITIONS AND METHODS FOR PREVENTING OR

[0002] TREATING MALARIA

[0003] FIELD OF THE INVENTION

[0004] This invention relates to compounds, compositions and methods for preventing or treating malaria.

[0005] BACKGROUND TO THE INVENTION

[0006] Malaria is an infectious parasitic disease caused by Plasmodium species and transmitted to humans through the bites of infected female Anopheles mosquitoes. It remains a major global health concern. In many affected regions, malaria continues to account for significant levels of illness and mortality, particularly in sub-Saharan Africa. Infants, young children, pregnant women and individuals with limited immunity are at an increased risk of developing severe disease.

[0007] Current malaria prevention and control strategies rely heavily on insecticide-treated nets (ITNs), indoor residual spraying (IRS), preventive chemotherapy and antimalarial medicines. However, the growing resistance of Anopheles mosquitoes to insecticides and the increasing resistance of Plasmodium parasites to antimalarial drugs are undermining the long-term effectiveness of these tools. Although malaria vaccines have recently become available, they offer only partial and time-limited protection. The immune response to malaria involves a coordinated interaction between innate and adaptive immune pathways. Natural killer (NK) cells and gamma-delta (y5) T cells play important roles during early infection, while CD8+ cytotoxic T lymphocytes eliminate infected hepatocytes during the liver stage. CD4+ T helper cells support antibody production and cytokine coordination throughout infection. Regulatory T cells and cytokines such as interleukin-10 (IL-10) help prevent excessive inflammation, whereas high levels of tumour necrosis factor-alpha (TNF-a) and interferon-gamma (IFN-y) are associated with severe disease.

[0008] The Plasmodium parasite follows a complex lifecycle in humans. After transmission, sporozoites enter the bloodstream and migrate to the liver, where they infect hepatocytes and multiply during the pre-erythrocytic stage. Merozoites released from the liver subsequently infect red blood cells, initiating the erythrocytic stage responsible for most clinical symptoms, including fever, chills, headache, anaemia and fatigue. In severe cases, excessive inflammatory responses can lead to complications such as cerebral malaria.

[0009] Published studies indicate that healthy individuals typically exhibit Ksp37 plasma levels of approximately 250-350 ng / mL, which are insufficient to provide the therapeutic effects described in this invention.

[0010] Despite advances in understanding malaria immunology, there is no therapeutic approach that safely enhances or regulates these immune pathways in a targeted manner. In particular, no method has been disclosed for stimulating or elevating the CD4+, CD8+, NK and yb T-cell responses required for effective control of Plasmodium infection.

[0011] There remains a clear need for targeted therapeutic strategies capable of strengthening protective immunity, assisting early parasite clearance, regulating inflammatory responses and reducing the risk of severe malaria complications.

[0012] OBJECT OF THE INVENTION

[0013] It is an object of this invention to provide compounds, compositions and methods for preventing or treating malaria, which at least partially, alleviates some of the abovementioned difficulties.

[0014] SUMMARY OF THE INVENTION

[0015] In accordance with this invention, there are provided compounds, compositions, and methods for preventing or treating malaria.

[0016] The invention provides compounds including, but not limited to, Killer-specific Secretory Protein 37 (Ksp37) and clinically modified or genetically engineered forms thereof, formulated to be carried by one or more polar compounds such as dimethylformamide (DMF) (C3H7NO) or dimethylsulfoxide (DMSO) (C2H6OS). These polar compounds may act as carriers for Ksp37 or may facilitate the delivery, stability, absorption, or endogenous production of Ksp37 within a subject. In this invention, administration of Ksp37, or stimulation of endogenous Ksp37 production via polar compounds or vectors encoding Ksp37 may modulate immune pathways. This modulation may include increased activation of CD4+T helper cells and CD8+cytotoxic T lymphocytes, enhanced natural killer (NK)-cell and gamma-delta (yd) T-cell activity, and changes in cytokine profiles such as reduced tumour necrosis factor-alpha (TNF-a) and interferon-gamma (IFN-y) levels. This modulation may also contribute to reduced inflammatory damage, improved clearance of infected hepatocytes and red blood cells, and the promotion of regulatory cytokines such as interleukin-10 (IL-10) associated with balanced immune responses.

[0017] The method may include the step of raising the level of Ksp37 in the blood plasma of a subject to a therapeutically effective concentration. A concentration of 400 ng / mL or higher may be used as such a therapeutically effective level.

[0018] The amount of Ksp37 which is released or produced may be influenced by the polar compound and / or associated transport mechanisms.

[0019] The levels of Ksp37 in a subject may be increased by one or more of the following routes: a. by intravenously administering a medicament comprising a therapeutically effective amount of a clinically modified or genetically engineered Ksp37 protein to the subject; and / or b. by stimulating the production of Ksp37 in the subject to a therapeutically effective level by administering a vector encoded with a Ksp37 gene, which will translate to a Ksp37 protein; and / or c. by stimulating the production of Ksp37 in the subject to a therapeutically effective level by chemically treating the subject with a polar compound capable of activating increased Ksp37 production; and / or d. by orally administering a medicament comprising a therapeutically effective amount of a clinically modified or genetically engineered Ksp37 protein to the subject, optionally by encapsulating Ksp37 with nanoparticles to avoid degradation in the digestive system.

[0020] Appropriate vectors for gene therapy may include, but are not limited to, pGEM-T vectors, pCMV3-C-GFPSpark vectors, pcDNA3.1 , AAV vectors or AAV8 vectors. Ksp37 proteins used in the invention may be produced through bacterial expression systems (e.g., Escherichia coli) or plant-based expression systems to obtain therapeutic quantities.

[0021] The invention further provides for the use of a clinically modified or genetically engineered Ksp37 protein, and / or a vector encoded with a Ksp37 gene which will translate to a Ksp37 protein, in the manufacture of a medicament for preventing or treating malaria, wherein a polar compound, such as dimethylformamide (DMF) or dimethylsulfoxide (DMSO) is used as a carrier of Ksp37 to increase the level of Ksp37 in the blood plasma of a subject to a minimum concentration level of 400 ng / mL.

[0022] The pharmaceutical composition or medicament may additionally include one or more pharmaceutically acceptable excipients. These may include aqueous vehicles such as saline, phosphate-buffered solution, Ringer’s solution, dextrose solution or Hank’s solution, as well as non-aqueous vehicles such as fixed oils, sesame seed oil, ethyl oleate or triglycerides. The pharmaceutical composition or medicament may also be formulated using nanoparticle-based delivery systems or other encapsulation techniques, and may include controlled-release compositions capable of slowly releasing Ksp37 into a subject.

[0023] The compounds, compositions or medicaments may be administered to a subject via acceptable administration routes, including nasal, oral, topical, inhalation, transdermal, rectal or parenteral administration.

[0024] In certain embodiments, transdermal administration may include the use of a patch containing a polar compound such as DMF for maintaining or supporting increased Ksp37 levels over time.

[0025] In certain embodiments, long-term transdermal administration of a polar compound such as DMF may be employed to support or maintain increased Ksp37 levels over an extended period of time.

[0026] Further provided is a method of preventing or treating malaria in a subject in need thereof, said method comprising administering to said subject a clinically modified or genetically engineered Ksp37 protein and / or proteins, and / or a vector encoded with a Ksp37 gene which will translate to a Ksp37 protein and / or proteins, wherein a polar compound is used as a carrier of the Ksp37 to increase the level of Ksp37 in the blood plasma of a subject to a therapeutic concentration level, which is a minimum concentration of 400 ng / mL.

[0027] One or more pharmaceutically acceptable excipients may be administered to the subject, selected from aqueous vehicles such as saline, phosphate buffered solution, Ringer’s solution, dextrose solution or Hank’s solution, or non-aqueous vehicles such as fixed oils, sesame seed oil, ethylene oleate, or triglycerides.

[0028] A nanoparticle-based delivery system or other encapsulation techniques may be employed using this method.

[0029] Furthermore, a controlled release substance may be used that is capable of slowly releasing Ksp37 into a subject.

[0030] The method may also include the step of administering the clinically modified or genetically engineered Ksp37 protein and / or proteins, and / or a vector encoded with a Ksp37 gene which will translate to a Ksp37 protein and / or proteins to the subject via acceptable administration routes, including nasal, oral, topical, inhalation, transdermal, rectal or parenteral administration.

[0031] The transdermal administration may include the use of a patch containing the polar compound for maintaining or supporting increased Ksp37 levels over time.

[0032] Long-term transdermal administration of the polar compound may be achieved via the subject wearing the transdermal patch for at least two years prior to or during treatment, to aid the increase of Ksp37 and to keep it at a maintained level. The Ksp37 used in the method may be produced using bacterial expression systems including Escherichia coli, or plant-based expression systems.

[0033] These and other features of the invention are described in more detail below.

[0034] DETAILED DESCRIPTION OF THE INVENTION

[0035] The following description is provided to illustrate the principles of the invention and is not intended to limit the scope thereof.

[0036] For consistency in the specification and claims, the term “compound” is used in a broad pharmaceutical sense and includes both protein molecules such as Ksp37 and smallmolecule polar compounds such as dimethylformamide (DMF) (C3H7NO) and dimethylsulfoxide (DMSO) (C2H6OS).

[0037] According to one aspect of the invention, provided are compounds, compositions and methods for preventing or treating malaria. Malaria is caused by Plasmodium parasites transmitted to humans through infected Anopheles mosquitoes. The invention provides therapeutic strategies aimed at elevating immune responses by activating and expanding cytotoxic immune cells at the defined therapeutic concentrations, responsible for clearing infected liver cells and red blood cells, regulating inflammatory pathways, and preventing severe malaria outcomes. Killer-specific Secretory Protein 37 (Ksp37) plays an important role in activating the immune system. Ksp37 is naturally expressed in cytotoxic lymphocytes, including CD4+T helper cells, CD8+cytotoxic T lymphocytes, natural killer (NK) cells and gamma-delta (yd) T cells. These immune cells are central to the control of Plasmodium at both the liver and blood stages. By enhancing the activation, proliferation and cytotoxic behaviour of these cells, Ksp37 may improve early parasite clearance, promote regulated cytokine responses and reduce the risk of excessive inflammation associated with complications such as cerebral malaria.

[0038] Compounds including, but not limited to, polar compounds such as DMF and DMSO may act as carriers or facilitators of Ksp37 delivery or production in a subject. These polar compounds, being aprotic solvents, may enhance the stability, absorption and biological effect of Ksp37 and may stimulate endogenous production of Ksp37 within immune cells. Compounds of the general formula RI-CO-NR2R3may similarly support Ksp37 delivery or activation.

[0039] Increasing or elevating the production of Ksp37 in a subject to define the therapeutic concentrations may modulate several immune mechanisms involved in controlling malaria infection. Ksp37 at these concentrations may enhance CD4+T helper-cell activity, improving cytokine coordination and supporting antibody production required for binding and neutralising free parasites. CD4+T-cell activation may further support macrophage responses and assist in regulating inflammatory cascades during early and late stages of infection. Ksp37 at defined therapeutic concentrations may strengthen CD8+cytotoxic T-cell responses. CD8+cells are responsible for identifying and eliminating Plasmodium- infected hepatocytes during the pre-erythrocytic stage. By activating and expanding CD8+T-cell activation, cytotoxic activity and recognition of infected cells, Ksp37 may reduce liver-stage parasite load and limit the release of merozoites into the bloodstream.

[0040] Ksp37 may further influence innate immune mechanisms. NK cells play an essential role in early malaria control by recognising and destroying infected red blood cells. Elevating Ksp37 to defined therapeutic concentrations may improve NK-cell activation, enhance their cytotoxic efficiency, and extend the duration of their interactions with Plasmodium-infected erythrocytes. Similarly, gamma-delta (yb) T cells, which are known to expand rapidly during early Plasmodium infection, may be elevated by increased Ksp37 availability, improving early innate protection and contributing to the development of effective adaptive responses.

[0041] Ksp37 may modulate cytokine pathways associated with malaria pathology. Excessive production of tumour necrosis factor-alpha (TNF-a) and interferon-gamma (IFN-y) contributes to severe malaria, endothelial dysfunction and cerebral involvement. By regulating these cytokines and supporting the production of anti-inflammatory cytokines such as interleukin-10 (IL-10), Ksp37 may reduce inflammatory tissue damage and support more balanced immune responses. IL-10-mediated pathways may also support regulatory T-cell activity (CD25+ / FoxP3+), which helps limit harmful inflammation during acute infection. By modulating cytokine release, activating and expanding cytotoxic immune cells at defined concentration levels, and regulating the inflammatory pathways associated with Plasmodium infection, Ksp37 may offer a novel therapeutic approach for preventing or treating malaria by decreasing harmful inflammation, supporting efficient clearance of infected cells, and maintaining immune homeostasis. These effects arise from targeted immune activation rather than general immune support.

[0042] Additional experimental observations indicate that Ksp37 exerts a direct, concentration-dependent influence on cytotoxic and regulatory immune cell populations. In vitro exposure of human PBMCs to Ksp37 at approximately 1 pg / mL has been associated with measurable expansion of CD8+T cells and NK cells, while exposure at around 2 pg / mL has been found to support increased activation of CD4+T cells expressing CD69 and increased representation of regulatory CD25+FoxP3+subsets.

[0043] These concentration-dependent effects are consistent with the immune-modulating activity described above, including support for immune balance, reduced inflammatory damage and improved clearance of Plasmodium-infected hepatocytes and erythrocytes. These effects are in line with the therapeutic mechanisms proposed for preventing or treating malaria and for reducing progression to severe disease.

[0044] The compounds of the invention, including Ksp37 carried by polar compounds, may increase or elevate the production of Ksp37 in the subject to defined therapeutic concentrations and thereby modulate immune pathways that contribute to parasite clearance. Polar compounds such as DMF and DMSO may additionally possess immunomodulatory properties that support therapeutic effects in malaria, including influencing cytokine behaviour and aiding immune-cell activation.

[0045] Ksp37 used in this invention may be produced using bacterial expression systems such as Escherichia coli or plant-based expression systems capable of generating therapeutic quantities. The Ksp37 protein may be modified, purified or formulated to enhance stability and bioavailability. In certain embodiments, encapsulation techniques such as nanoparticle-based systems may be employed to protect orally administered Ksp37 from digestive degradation.

[0046] Appropriate vectors for gene therapy may include pGEM-T vectors, pCMV3-C- GFPSpark vectors, AAV vectors or AAV8 vectors, which may be used to increase endogenous Ksp37 production in a subject. Such vectors may be formulated with polar compounds or excipients that enhance cellular uptake and expression of the Ksp37 gene.

[0047] The compounds, compositions or medicaments may be administered to a subject via acceptable administration routes including nasal, oral, topical, inhalation, transdermal, rectal or parenteral administration. In certain embodiments, transdermal administration may include the use of a patch containing a polar compound such as DMF for maintaining or elevating increased Ksp37 levels over time. Long-term transdermal administration may be employed to support or maintain increased Ksp37 levels during treatment.

[0048] These and other embodiments of the invention are described in further detail below. EXPERIMENTAL RESULTS

[0049] Experiment 1 : The Role of NK Cells and Macrophages in Early Malaria Infection

[0050] 1 . Introduction / Backqround

[0051] Natural killer (NK) cells are a critical component of the innate immune system, known for their ability to identify and kill infected cells without prior sensitisation. In malaria, caused by Plasmodium parasites, the early immune response is vital to controlling parasite levels before adaptive immunity is fully activated. This experiment aimed to investigate the roles of NK cells and macrophages during the initial stages of malaria infection and to explore the mechanisms by which NK cells interact with infected red blood cells (RBCs).

[0052] 2. Materials and Methods

[0053] Animal Studies:

[0054] Mice were used as the experimental model. Groups of mice were treated to selectively deplete either NK cells or macrophages using targeted interventions. Malaria infection was induced, and parasite levels were measured during the first two days of infection.

[0055] Human NK Cell Assays:

[0056] Human NK cells were isolated and placed in samples containing both infected and uninfected RBCs. The interaction duration between NK cells and both types of RBCs was observed and recorded.

[0057] The ability of NK cells to kill infected RBCs was evaluated.

[0058] 3. Results

[0059] Impact of Cell Depletion:

[0060] Macrophage depletion had minimal effect on controlling parasite levels during the first two days of infection.

[0061] In mice lacking NK cells, parasite levels increased sevenfold, indicating a critical role for NK cells in early infection control.

[0062] NK Cell Interactions:

[0063] Human NK cells randomly interacted with both infected and uninfected RBCs but showed significantly longer interaction times with infected RBCs.

[0064] NK cells successfully killed infected RBCs during these extended interactions.

[0065] 4. Discussion

[0066] The findings demonstrate that NK cells are essential for controlling malaria infection in its early stages. Their absence leads to a dramatic increase in parasite levels, while macrophages play a less significant role during this period. The prolonged interaction of NK cells with infected RBCs, leading to their destruction, suggests a targeted immune defence mechanism. These results highlight the potential for exploiting NK cell function as a therapeutic strategy against malaria. By expanding NK cell proliferation and activation, it may be possible to improve early parasite control, reduce disease severity, and complement other treatments.

[0067] EXPERIMENTAL RESULTS RELATING TO CONCENTRATION-DEPENDENT IMMUNE MODULATION BY KSP37

[0068] Experiment 2: In vitro concentration-dependent immune modulation by Ksp37

[0069] Human peripheral blood mononuclear cells (PBMCs) were cultured in vitro and exposed to increasing concentrations of Ksp37 protein (0-2 pg / mL, with a repeat series up to 8 pg / mL). After incubation, flow cytometry was used to evaluate major immune cell subsets, including CD4+T cells, CD8+T cells, NK cells and FoxP3+regulatory cells, as well as activation (CD69) and proliferation (Ki67) markers. The purpose of the assay was to determine whether defined concentrations of Ksp37 produce measurable activation, expansion or regulatory shifts in these immune populations.

[0070] Across multiple donors, Ksp37 showed a clear concentration-dependent effect on cytotoxic immune cells. At approximately 1 pg / mL, CD8+T cells increased by about 27%, NK cells by about 41 %, and monocytes by about 19%. At around 2 pg / mL, Ksp37 supported increased activation of CD4+T cells (CD69+) and an increase in CD8+CD25+FoxP3+regulatory subsets. Ki67 expression decreased in both CD4+and CD8+T cells, consistent with controlled activation rather than excessive proliferation. In a higher-dose repeat series (2-8 pg / mL), FoxP3+regulatory subsets increased in a consistent dose-dependent manner. These observations indicate that Ksp37 can directly influence cytotoxic and regulatory immune cell behaviour at defined concentrations. This concentration-dependent activity supports the proposed therapeutic role of Ksp37 in preventing or treating malaria by enhancing cytotoxic clearance mechanisms, supporting immune balance and reducing inflammatory damage associated with severe Plasmodium infection.

Claims

CLAIMS1. A compound including Killer-specific Secretory Protein 37 (Ksp37) or clinically modified or genetically engineered forms thereof, for use in preventing or treating malaria in a subject in need thereof, wherein one or more polar compounds are used as a carrier of the Ksp37 to increase the level of Ksp37 in blood plasma of the subject to a therapeutic quantity, which is a minimum concentration level of 400 ng / mL.

2. The compound for use of claim 1 , wherein the one or more polar compounds are selected from the group including, but not limited to, dimethylformamide (DMF) (C3H7NO) or dimethylsulfoxide (DMSO) (C2H6OS).

3. The compound for use of claim 1 , wherein the one or more polar compounds facilitate the delivery, stability, absorption, or production of Ksp37 within the subject.

4. The compound for use of claim 1 , wherein the levels of Ksp37 in the subject are increased by one or more of the following routes, including:- via intravenous administration of the compound including the Ksp37 or clinically modified or genetically engineered forms thereof;- via administration of a vector encoded with a Ksp37 gene, which will translate to a Ksp37 protein and / or proteins;- via treatment of the subject with the polar compound or compounds, to activate increased Ksp37 production; and / or- via oral administration of the compound including the Ksp37 or clinically modified or genetically engineered forms thereof, wherein the clinically modified Ksp37 is achieved by encapsulating the Ksp37 with nanoparticles to avoid degradation in the digestive system of the subject.

5. The compound for use of claim 4, wherein appropriate vectors include, but are not limited to, pGEM-T vectors, pCMV3-C-GFPSpark, pcDNA3.1 vectors, AAV vectors orAAV8 vectors.

6. The compound for use of claim 1 , wherein Ksp37 proteins are produced through bacterial expression systems (including Escherichia coli) or through plant-based expression systems to obtain the therapeutic quantities of at least 400 ng / mL.

7. Use of a clinically modified or genetically engineered Ksp37 protein and / or proteins, and / or a vector encoded with a Ksp37 gene which will translate to a Ksp37 protein and / or proteins, in the manufacture of a medicament for preventing or treating malaria in a subject in need thereof, wherein a polar compound is used as a carrier of the Ksp37 to increase the level of Ksp37 in the blood plasma of a subject to a therapeutic concentration level, which is a minimum concentration of 400 ng / mL.

8. The use of claim 7, wherein the medicament includes one or more pharmaceutically acceptable excipients, selected from aqueous vehicles such as saline, phosphate buffered solution, Ringer’s solution, dextrose solution orHank’s solution, or non-aqueous vehicles such as fixed oils, sesame seed oil, ethylene oleate, or triglycerides.

9. The use of claim 8, wherein the medicament further includes a nanoparticlebased delivery systems or other encapsulation techniques.

10. The use of claim 9, wherein the medicament includes a controlled release substance that is capable of slowly releasing Ksp37 into a subject.11 . The use of claim 7, wherein the medicament is formulated for administration to the subject via acceptable administration routes, including nasal, oral, topical, inhalation, transdermal, rectal or parenteral administration.

12. The use of claim 11 , wherein the transdermal administration includes the use of a patch containing the polar compound for maintaining or supporting increased Ksp37 levels over time.

13. The use of claim 12, wherein long-term transdermal administration of the polar compound is achieved via the subject wearing the transdermal patch for at least two years prior to or during treatment, to aid the increase of Ksp37 and to keep it at a maintained level.

14. The use of claim 7, wherein the Ksp37 is produced using bacterial expression systems including Escherichia coli, or plant-based expression systems.

15. A method of preventing or treating malaria in a subject in need thereof, said method comprising administering to said subject a clinically modified or genetically engineered Ksp37 protein and / or proteins, and / or a vector encoded with a Ksp37 gene which will translate to a Ksp37 protein and / or proteins, wherein a polar compound is used as a carrier of the Ksp37 to increase the level of Ksp37 in the blood plasma of a subject to a therapeutic concentration level, which is a minimum concentration of 400 ng / mL.

16. The method of claim 15, wherein one or more pharmaceutically acceptable excipients are administered to the subject, selected from aqueous vehicles such as saline, phosphate buffered solution, Ringer’s solution, dextrose solution or Hank’s solution, or non-aqueous vehicles such as fixed oils, sesame seed oil, ethylene oleate, or triglycerides.

17. The method of claim 15, wherein a nanoparticle-based delivery system or other encapsulation techniques are employed.

18. The method of claim 15, wherein a controlled release substance is used that is capable of slowly releasing Ksp37 into a subject.

19. The method of claim 15, wherein the clinically modified or genetically engineered Ksp37 protein and / or proteins, and / or a vector encoded with a Ksp37 gene which will translate to a Ksp37 protein and / or proteins is administered to the subject via acceptable administration routes, including nasal, oral, topical, inhalation, transdermal, rectal or parenteral administration.

20. The method of claim 19, wherein the transdermal administration includes the use of a patch containing the polar compound for maintaining or supporting increased Ksp37 levels over time.

21. The method of claim 20, wherein long-term transdermal administration of the polar compound is achieved via the subject wearing the transdermal patch for at least two years prior to or during treatment, to aid the increase of Ksp37 and to keep it at a maintained level.

22. The method of claim 15, wherein the Ksp37 is produced using bacterial expression systems including Escherichia coli, or plant-based expression systems.