WO2017075053A1

WO2017075053A1 - Immune-modifying particles for the treatment of malaria

Info

Publication number: WO2017075053A1
Application number: PCT/US2016/058863
Authority: WO
Inventors: Daniel R. Getts
Original assignee: Cour Pharmaceuticals Development Company Inc.
Priority date: 2015-10-26
Filing date: 2016-10-26
Publication date: 2017-05-04

Abstract

The current invention involves the administration of negatively charged particles, such as polystyrene, PLGA, or diamond particles, to subjects to ameliorate inflammatory immune responses resulting from a malaria. Additionally, the present invention describes methods of inhibiting or treating malaria by administering these same negatively charged particles.

Description

IMMUNE-MODIFYING PARTICLES FOR THE TREATMENT OF MALARIA

RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 62/246,498, filed October 26, 2015, and U.S. Provisional Application No. 62/395,554, filed September 16, 2016, the disclosures of each of which are hereby incorporated by reference in their entirety.

BACKGROUND OF INVENTION

[0002] Malaria is a mosquito-borne infectious disease of humans and other animals caused by parasitic protozoans belonging to the genus Plasmodium. Malaria causes symptoms that typically include fever, fatigue, vomiting, and headaches. In severe cases it can cause yellow skin, seizures, coma or death. The disease is transmitted by the biting of mosquitos, and the symptoms usually begin ten to fifteen days after being bitten. The most severe symptoms of this disease can be neurologically related. In addition to brain pathology, it has become increasingly clear in recent years that infection may cause widespread pathology in other organs. Various reports provide convincing evidence of acute lung pathology and associated respiratory distress in some patients. Metabolic acidosis and liver damage are major features of severe malaria in humans. If not appropriately treated, people may have recurrences of the disease months later.

[0003] Despite intense world-wide effort, malaria is still a major cause of mortality and morbidity, especially in third world countries. According to the World Health Organization's (WHO) "WHO Malaria World Report 2011," there were approximately 216 million episodes of the disease in 2010, which resulted in 655,000 deaths. Approximately 81 % of cases were in the African Region, about 91% being due to P. falciparum. Other groups have estimated the number of cases at between 350 and 550 million for falciparum malaria and deaths in 2010 at 1.24 million up from 1.0 million deaths in 1990. The majority of cases occur in children.

[0004] Malaria is typically treated with antimalarial medications, depending on the type and severity of the disease. Uncomplicated malaria may be treated with oral medications. The most effective treatment for P. falciparum infection is the use of artemisinins in combination with other antimalarials (known as artemisinin-combination therapy, or ACT), which decreases resistance to any single drug component. These additional antimalarials include: amodiaquine, lumefantrine, mefloquine or sulfadoxine/pyrimethamine. ACT is about 90% effective when used to treat uncomplicated malaria.

[0005] While the annual incidences and mortalities of malaria have dropped since 2000, they remain short of the targeted goal of a 50% reduction as proposed in the initial Global Malaria Action Plan of the Roll Back Malaria Partnership. Furthermore, drug resistant strains are emerging in several parts of the world. In Cambodia, Myanmar, Thailand and Vietnam, malarial strains resistant to the primary treatment therapy, artemisinin, are stimulating efforts to contain the spread of these resistant forms. There is currently no vaccine available for prevention.

[0006] While treatments for malaria exist, malaria remains a major cause of mortality and morbidity, and the emergence of drug resistant strains further increase the challenge of treating the disease. Furthermore, no treatment is available to alleviate the immune pathology associated with malaria infection. What is needed in the art are new therapies for the treatment of malaria that can reduce the severity of disease as well augment host anti-parasite immunity, at minimum improving mortality, and as best promoting complete parasite removal.

SUMMARY OF THE I VENTION

[0007] Particular embodiments of the present invention are directed to a method of inhibiting or treating malaria in a subject, said method comprising administering to said subject a pharmaceutical composition comprising negatively charged particles and a pharmaceutically acceptable carrier, wherein said particles are free from attached peptide moieties, antigenic moieties, and bioactive agents. In some embodiments, the malaria is caused by Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, or Plasmodium knowlesi. In particular embodiments, the malaria is caused by Plasmodium falciparum. In certain embodiments, the composition alleviates at least one symptom associated with malaria. In certain embodiments, the at least one symptom is selected from respiratory distress, metabolic acidosis, acute encephalitis/meningitis syndrome, and liver malfunction or failure.

[0008] Particular embodiments of the present invention are directed to a method of inhibiting or treating malaria in a subject, wherein the particles are polystyrene particles, diamond particles,

PLURIONICS stabilized polypropylene sulfide particles, or poly(lactic-co-glycolic acid)

(PLGA) particles. In certain embodiments, the particles are polystyrene particles. In some embodiments, the particles are diamond particles. In some embodiments, the particles are PLGA particles. In particular embodiments, the particles are carboxylated. In certain embodiments, the particles have a zeta potential of less than about -100 mV. By having a zeta potential of less than about -100 mV, it is meant that the zeta potential is between about 0 mV than about -100 mV. In particular embodiments, the particles have a zeta potential between about -100 mV and about -15 mV. In certain embodiments, the particles have a zeta potential between about -100 mV and about -75 mV. In particular embodiments, the particles have a zeta potential between about -50 mV and about -20 mV. In some embodiments, the composition ameliorates an inflammatory immune response.

[0009] Some embodiments of the present invention are directed to a method of inhibiting or treating malaria in a subject, wherein the diameter of said negatively charged particles is between about 0.1 μηι to about 10 μηι. In some embodiments, the diameter of said negatively charged particles is between about 0.3 μηι to about 5 μπι. In particular embodiments, the diameter of said negatively charged particles is between about 0.5 μηι to about 3 μηι. In some embodiments, the diameter of the negatively charged particles is between about 0.5 μηι to about 1 μιη. In particular embodiments, the diameter of the negatively charged particles is about 0.5 μπι.

[0010] Particular embodiments of the present invention are directed to a method of inhibiting or treating malaria in a subject, wherein the composition is administered orally, nasally, intravenously, intramuscularly, ocularly, transdermally, intra-lymphatically, or subcutaneously. In certain embodiments, the subject is a human.

[0011] Certain embodiments of the present invention are directed to a method for removing pro-inflammatory mediators from the inflammatory milieu in a subject with malaria, said method comprising administering to said subject a pharmaceutical composition comprising negatively charged particles and a pharmaceutically acceptable carrier, wherein said particles are free from attached peptide moieties, antigenic moieties, and bioactive agents. In some embodiments, the malaria is caused by Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, or Plasmodium knowlesi. In certain embodiments, the malaria is caused by is Plasmodium falciparum. In some embodiments, the composition alleviates at least one symptom associated with malaria. In some embodiments, the at least one symptom is selected from respiratory distress, metabolic acidosis, acute encephalitis/meningitis syndrome, and liver malfunction or failure. In certain embodiments, the pro-inflammatory mediators produced in the subject bind to the negatively charged particles.

[0012] Particular embodiments of the present invention are directed to a method for removing pro-inflammatory mediators from the inflammatory milieu in a subject with malaria, wherein the particles are polystyrene particles, diamond particles, PLURIONICS stabilized polypropylene sulfide particles, or poly(lactic-co-glycolic acid) (PLGA) particles. In certain embodiments, the particles are polystyrene particles. In some embodiments, the particles are diamond particles. In some embodiments, the particles are PLGA particles. In certain embodiments, the particles are citric acid particles. In particular embodiments, the particles are carboxylated. In certain embodiments, the particles have a zeta potential of less than about -100 mV. In particular embodiments, the particles have a zeta potential between about -100 mV and about -15 mV. In certain embodiments, the particles have a zeta potential between about -100 mV and about -75 mV. In particular embodiments, the particles have a zeta potential between about -50 mV and about -20 mV. In some embodiments, the particles have a zeta potential between about -80 mV and about -30 mV. In certain embodiments, the particles have a zeta potential between about -75 mV and about -25 mV. In some embodiments, the composition ameliorates an inflammatory immune response.

[0013] Certain embodiments of the present invention are directed to a method for removing pro-inflammatory mediators from the inflammatory milieu in a subject with malaria, wherein the diameter of said negatively charged particles is between about 0.1 μηι to about 10 μπι. In some embodiments, the diameter of said negatively charged particles is between about 0.3 μηι to about 5 μηι. In particular embodiments, the diameter of said negatively charged particles is between about 0.5 μηι to about 3 μπι. In some embodiments, the diameter of the negatively charged particles is between about 0.5 μηι to about 1 μιη. In particular embodiments, the diameter of the negatively charged particles is about 0.5 μηι.

[0014] Some embodiments are directed to a method for removing pro-inflammatory mediators from the inflammatory milieu in a subject with malaria, wherein the composition is administered orally, nasally, intravenously, intramuscularly, intra-lymphatically, ocularly, transdermally, or subcutaneously. In certain embodiments, the subject is a human.

[0015] Particular embodiments are directed to a method for inducing regulatory T cells in a subject with malaria, said method comprising administering to said subject a pharmaceutical composition comprising negatively charged particles and a pharmaceutically acceptable carrier, wherein the negatively charged particles are free from attached peptide moieties, antigenic moieties and bioactive agents. In some embodiments, the regulatory T cells comprise CD4+ cells. In some embodiments, the regulatory T cells comprise CD8⁺ T cells. In some embodiments, the regulatory T cells comprise TR1 cells. In some embodiments, the malaria is caused by Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, or Plasmodium knowlesi. In some embodiments, the malaria is caused by Plasmodium falciparum. In some embodiments, the composition alleviates at least one symptom associated with malaria. In certain embodiments, the composition alleviates immune pathology associated with malaria. In particular embodiments, the at least one symptom is selected from respiratory distress, metabolic acidosis, acute encephalitis/meningitis syndrome, and liver malfunction or failure.

[0016] Some embodiments are directed to a method for inducing regulatory T cells in a subject with malaria, wherein the particles are polystyrene particles, diamond particles, PLURIONICS stabilized polypropylene sulfide particles, poly(lactic-co-glycolic acid) (PLGA) particles, or citric acid particles. In certain embodiments, the particles are polystyrene particles. In some embodiments, the particles are diamond particles. In some embodiments, the particles are PLGA particles. In particular embodiments, the particles are carboxylated. In certain embodiments, the particles have a zeta potential of less than about -100 mV. In particular embodiments, the particles have a zeta potential between about -100 mV and about -15 mV. In certain embodiments, the particles have a zeta potential between about -100 mV and about -75 mV. In particular embodiments, the particles have a zeta potential between about -50 mV and about -20 mV. In some embodiments, the composition ameliorates an inflammatory immune response.

[0017] Particular embodiments are directed to a method for inducing regulatory T cells in a subject with malaria, wherein the diameter of said negatively charged particles is between about 0.01 μιη to about 10 μηι. In some embodiments, the diameter of said negatively charged particles is between about 0.1 μηι to about 10 μπι. In some embodiments, the diameter of said negatively charged particles is between about 0.3 μηι to about 5 μπι. In particular embodiments, the diameter of said negatively charged particles is between about 0.5 μηι to about 3 μηι. In some embodiments, the diameter of the negatively charged particles is between about 0.5 μηι to about 1 μπι. In particular embodiments, the diameter of the negatively charged particles is about 0.5 μηι.

[0018] Some embodiments are directed to a method for inducing regulatory T cells in a subject with malaria, wherein the composition is administered orally, nasally, intravenously, intramuscularly, ocularly, transdermally, or subcutaneously. In certain embodiments, the subject is a human. [0019] Some embodiments are directed to a method of vaccinating a subject for malaria comprising administering to the subject a pharmaceutical composition comprising negatively charged particles, wherein the particles comprise an antigen comprising one or more epitopes associated with malaria. In some embodiments, the malaria is caused by Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, or Plasmodium knowlesi. In some embodiments, the malaria is caused by Plasmodium falciparum. In certain embodiments, the one or more epitopes is associated with Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, or Plasmodium knowlesi. In some embodiments, the one or more epitopes is associated with Plasmodium falciparum. In particular embodiments, the one or more epitopes is associated with the same species of Plasmodium. In certain embodiments, the one or more epitopes is associated with different species of Plasmodium. In come embodiments, the pharmaceutical composition further comprises an adjuvant.

[0020] Some embodiments are directed to a method of vaccinating a subject for malaria, wherein the particles are polystyrene particles, diamond particles, PLURIONICS stabilized polypropylene sulfide particles, citric acid particles, amino acid particles, nucleic acid particles, or poly(lactic-co-glycolic acid) (PLGA) particles. In certain embodiments, the particles are polystyrene particles. In some embodiments, the particles are diamond particles. In some embodiments, the particles are PLGA particles. In particular embodiments, the particles are carboxylated. In certain embodiments, the particles have a zeta potential of less than about -100 mV. In particular embodiments, the particles have a zeta potential between about -100 mV and about -15 mV. In certain embodiments, the particles have a zeta potential between about -100 mV and about -75 mV. In particular embodiments, the particles have a zeta potential between about -50 mV and about -20 mV. In some embodiments, the composition ameliorates an inflammatory immune response.

[0021] Particular embodiments are directed to a method of vaccinating a subject for malaria, wherein the diameter of said negatively charged particles is between about 0.1 μηι to about 10 μηι. In some embodiments, the diameter of said negatively charged particles is between about

0.3 μηι to about 5 μπι. In particular embodiments, the diameter of said negatively charged particles is between about 0.5 μηι to about 3 μηι. In some embodiments, the diameter of the negatively charged particles is between about 0.5 μηι to about 1 μιη. In particular embodiments, the diameter of the negatively charged particles is about 0.5 μπι. [0022] Some embodiments are directed to a method of vaccinating a subject for malaria, wherein the composition is administered intravenously, intramuscularly, transdermally, or subcutaneously. In certain embodiments, the subject is a human.

[0023] Some embodiments are directed to a method for treating or preventing cerebral malarial in a subject with malaria comprising administering to the subject a pharmaceutical composition comprising negatively charged particles and a pharmaceutically acceptable carrier, wherein the negatively charged particles are free from attached peptide moieties, antigenic moieties, and bioactive agents. In some embodiments, the malaria is caused by Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, or Plasmodium knowlesi. In particular embodiments, the malaria is caused by Plasmodium falciparum. In some embodiments, the composition alleviates at least one symptom associated with malaria. In some embodiments, the at least one symptom is selected from respiratory distress, metabolic acidosis, acute encephalitis/meningitis syndrome, and liver malfunction or failure.

[0024] Certain embodiments are directed to a method for treating or preventing cerebral malarial in a subject with malaria wherein the particles are polystyrene particles, diamond particles, PLURIONICS stabilized polypropylene sulfide particles, or poly(lactic-co-glycolic acid) (PLGA) particles. In certain embodiments, the particles are polystyrene particles. In some embodiments, the particles are diamond particles. In some embodiments, the particles are PLGA particles. In particular embodiments, the particles are carboxylated. In certain embodiments, the particles have a zeta potential of less than about -100 mV. In particular embodiments, the particles have a zeta potential between about -100 mV and about -15 mV. In certain embodiments, the particles have a zeta potential between about -100 mV and about -75 mV. In particular embodiments, the particles have a zeta potential between about -50 mV and about -20 mV. In some embodiments, the particles have a zeta potential between about -80 mV and about -30 mV. In certain embodiments, the particles have a zeta potential between about -75 mV and about -25 mV. In some embodiments, the composition ameliorates an inflammatory immune response.

[0025] Particular embodiments are directed to a method for treating or preventing cerebral malarial in a subject with malaria wherein the diameter of said negatively charged particles is between about 0.1 μιη to about 10 μηι. In some embodiments, the diameter of said negatively charged particles is between about 0.3 μηι to about 5 μηι. In particular embodiments, the diameter of said negatively charged particles is between about 0.5 μηι to about 3 μπι. In some embodiments, the diameter of the negatively charged particles is between about 0.5 μηι to about 1 μπι. In particular embodiments, the diameter of the negatively charged particles is about 0.5 μιη.

[0026] Some embodiments are directed to a method for treating or preventing cerebral malarial in a subject with malaria wherein the composition is administered orally, nasally, intravenously, intramuscularly, ocularly, transdermally, or subcutaneously. In certain embodiments, the subject is a human.

[0027] Some embodiments are directed to treating cerebral malaria comprising administering a synergistically effective amount of a pharmaceutical composition comprising negatively charged particles and a pharmaceutically acceptable carrier, and a synergistically effective amount of an antimalarial, wherein the negatively charged particles are free from attached peptide moieties, antigenic moieties, and bioactive agents. In some embodiments, the malaria is caused by Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, or Plasmodium knowlesi. In particular embodiments, the malaria is caused by Plasmodium falciparum. In certain embodiments, the cerebral malaria is late-stage cerebral malaria. In particular embodiments, the negatively charged particles and the antimalarial are administered in the same pharmaceutical composition. In some embodiments, the negatively charged particles are administered in a separate pharmaceutical composition than the pharmaceutical composition comprising the negatively charged particles.

[0028] In particular embodiments, the antimalarial is administered to the subject prior to, simultaneously with, and/or after the pharmaceutical composition comprising the negatively charged particles is administered to the subject. In certain embodiments, the antimalarial is administered to the subject prior to the pharmaceutical composition comprising the negatively charged particles is administered to the subject. In some embodiments, the antimalarial is administered to the subject simultaneously with the pharmaceutical composition comprising the negatively charged particles is administered to the subject. In some embodiments, the antimalarial is administered to the subject after the pharmaceutical composition comprising the negatively charged particles is administered to the subject.

[0029] In particular embodiments, administering the synergistically effective amount of the pharmaceutical composition comprising the negatively charged particles and the synergistically effective amount of the antimalarial synergistically increases the probability of survival in the subject. In particular embodiments, administering the synergistically effective amount of the pharmaceutical composition comprising the negatively charged particles and the synergistically effective amount of the antimalarial to the subject prevents or synergistically reduces the probability of a coma. In certain embodiments, administering the synergistically effective amount of the pharmaceutical composition comprising the negatively charged particles and the synergistically effective amount of the antimalarial to the subject synergistically reduces the duration of a coma in the subject. In certain embodiments, administering the synergistically effective amount of the pharmaceutical composition comprising the negatively charged particles and the synergistically effective amount of the antimalarial to the subject synergistically ameliorates at least one symptom associated with cerebral malaria.

[0030] In particular embodiments, negatively charged particles are polystyrene particles, diamond particles, PLURIONICS stabilized polypropylene sulfide particles, citric acid particles, or poly(lactic-co-glycolic acid) (PLGA) particles. In some embodiments, the particles are polystyrene particles. In particular embodiments, the particles are diamond particles. In certain embodiments, the particles are PLGA particles. In particular embodiments, the particles are carboxylated. In some embodiments, the particles have a zeta potential of about 0 mV to about - 100 mV. In certain embodiments, the particles have a zeta potential between about -100 mV and about -15 mV. In some embodiments, the particles have a zeta potential between about -75 mV and about -75 mV. In particular embodiments, the particles have a zeta potential between about - 80 mV and about -30 mV.

[0031] In some embodiments, the diameter of the negatively charged particles is between about 0.01 μηι to about 10 μηι. In certain embodiments, the diameter of the negatively charged particles is between about 0.3 μηι to about 5 μηι. In particular embodiments, the diameter of the negatively charged particles is between about 0.5 μηι to about 3 μηι. In some embodiments, the diameter of the negatively charged particles is between about 0.5 μηι to about 1 μιη. In certain embodiments, the diameter of the negatively charged particles is about 0.5 μηι.

[0032] In particular embodiments, the pharmaceutical composition comprising the negatively charged particles is administered orally, nasally, intravenously, intramuscularly, ocularly, transdermally, or subcutaneously.

[0033] In some embodiments, the antimalarial is at least one of Quinine, Chloroquine, Amodiaquine, Pyrimethamine, Proguanil, Sulfonamides, Mefloquine, Atovaquone, Primaquine, Artemisinin, Halofantrine, Doxycycline, Clindamycin, Artesunate, or a derivative thereof. In particular embodiments, the antimalarial is Artesunate. In certain embodiments, the antimalarial is Chloroquine.

[0034] Certain embodiments are directed to a method for preventing reinfection with malaria in a subject infected with malaria comprising administering a pharmaceutical composition comprising negatively charged particles and a pharmaceutically acceptable carrier, and an antimalarial, wherein the negatively charged particles are free from attached peptide moieties, antigenic moieties, and bioactive agents. In some embodiments, the malaria is caused by Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, or Plasmodium knowlesi. In particular embodiments, the malaria is caused by Plasmodium falciparum. In particular embodiments, the antimalarial is administered to the subject prior to, simultaneously with, and/or after the pharmaceutical composition comprising the negatively charged particles is administered to the subject. In certain embodiments, the antimalarial is administered to the subject prior to the pharmaceutical composition comprising the negatively charged particles is administered to the subject. In some embodiments, the antimalarial is administered to the subject simultaneously with the pharmaceutical composition comprising the negatively charged particles is administered to the subject. In some embodiments, the antimalarial is administered to the subject after the pharmaceutical composition comprising the negatively charged particles is administered to the subject.

[0035] In particular embodiments, negatively charged particles are polystyrene particles, diamond particles, PLURIONICS stabilized polypropylene sulfide particles, citric acid particles, or poly(lactic-co-glycolic acid) (PLGA) particles. In some embodiments, the particles are polystyrene particles. In particular embodiments, the particles are diamond particles. In certain embodiments, the particles are PLGA particles. In particular embodiments, the particles are carboxylated. In some embodiments, the particles have a zeta potential of about 0 mV to about - 100 mV. In certain embodiments, the particles have a zeta potential between about -100 mV and about -15 mV. In some embodiments, the particles have a zeta potential between about -75 mV and about -75 mV. In particular embodiments, the particles have a zeta potential between about - 80 mV and about -30 mV.

[0036] In some embodiments, the diameter of the negatively charged particles is between about

0.01 μηι to about 10 μηι. In certain embodiments, the diameter of the negatively charged particles is between about 0.3 μηι to about 5 μηι. In particular embodiments, the diameter of the negatively charged particles is between about 0.5 μηι to about 3 μπι. In some embodiments, the diameter of the negatively charged particles is between about 0.5 μιη to about 1 μιη. In certain embodiments, the diameter of the negatively charged particles is about 0.5 μηι.

[0037] In particular embodiments, the antimalarial is at least one of Quinine, Chloroquine, Amodiaquine, Pyrimethamine, Proguanil, Sulfonamides, Mefloquine, Atovaquone, Primaquine, Artemisinin, Halofantrine, Doxycycline, Clindamycin, Artesunate, or a derivative thereof. In particular embodiments, the antimalarial is Artesunate. In certain embodiments, the antimalarial is Chloroquine.

BRIEF DESCRIPTION OF THE DRAWINGS

[0038] FIG. 1 is a schematic illustrating the switch from protective to pathogenic immune responses in malaria and the restoration of a balanced immune response with immune modifying nanoparticles.

[0039] FIG. 2 shows a schematic of the Plasmodium berghei ANKA (PbA) infection mouse model of malaria.

[0040] FIGS. 3A and 3B show graphs illustrating clinical score and survival in PbA-infected C57BL/6 mice. FIG. 3A shows a time course of clinical scores observed in PbA-infected C57BL/6 mice with and without treatment with negatively charged carboxylated polystyrene particles (IMP). FIG. 3B shows a survival curve of PbA-infected C57BL/6 mice with and without treatment with negatively charged carboxylated polystyrene particles (IMP). Data represented as mean ± SEM.

[0041] FIG. 4 shows the flow cytometry gating for analysis of PbA-infected tissues.

[0042] FIGS. 5A and 5B show cell counts from central nervous system (CNS) tissue collected from uninfected (Mock), PbA-infected, and PbA-infected with negatively charged carboxylated polystyrene particle treated mice. FIG. 5A shows cell counts of monocyte and macrophage cell populations. FIG. 5B shows cell counts of T cell populations. Data represented as mean ± SEM.

[0043] FIGS. 6A-6E show charts illustrating the effects of negatively charged carboxylated polystyrene particle treatment on total CD45+ cell populations and Ly6Chi+ monocytes in spleen and brain in PbA-infected CBA mice. FIG. 6A and FIG. 6C displays total numbers of

CD45+ cells in the spleen (FIG. 6A) and brain (FIG. 6C) from uninfected (Mock), PbA-infected, and PbA-infected with negatively charged carboxylated polystyrene particle treated mice. FIG

6B and FIG. 6D shows numbers of Ly6Chi monocytes in spleen (FIG. 6B) and brain (FIG. 6D) in mock, PbA-infected, and PbA-infected negatively charged carboxylated polystyrene particle treated mice. FIG. 6E shows FlowJo plots showing Ly6Chi monocytes in the brains of uninfected (Mock), PbA-infected, and PbA-infected with negatively charged carboxylated polystyrene particle treated mice. Data represented as mean ± SEM.

[0044] FIG. 7 shows representative histological images of non-infected (top row), PbA-infected and untreated (middle row), and PbA-infected with negatively charged carboxylated polystyrene particle treated mice (bottom row) of brain (left panels) and lung (right panels).

[0045] FIG. 8 shows a graph displaying parasitaemia of PbA-infected (circles) and PbA-infected with negatively charged carboxylated polystyrene particle (IMP) treatment (squares). Data represented as mean ± SEM.

[0046] FIGS. 9A-9D show graphs comparing the manifestation of cerebral malaria between PbA-infected CBA mice with or without negatively charged carboxylated polystyrene particle (IMP) treatment. FIG. 9A shows a survival curve of PbA infection in IMP treated (n = 28) and untreated (n = 21) mice. FIG. 9B shows clinical evaluation scores allocated to the two groups. FIG. 9C shows changes in weight over the course of the infection. FIG. 9D shows the evolution of parasitaemia. Data represented as mean ± SEM. The Q marking indicates the beginning of quinine treatment.

[0047] FIGS. 10A and 10B shows survival curves of PbA-infected C57BL/6 (FIG. 10A) and CBA (FIG. 10B) mice with or without treatment with negatively charged carboxylated polystyrene particles (IMP). Data points for PbA-infected mice are shown as circles; data points for PbA-infected and IMP treated mice are shown as squares.

[0048] FIGS. 11A-11D show graphs illustrating that treatment with PLGA particles (IMP) increases survival and reduces symptoms in PbA-infected CBA and C57BL/6 mice. FIG. 11A shows a survival curve of PbA-infected CBA mice with and without IMP treatment. FIG. 11B shows clinical scores of PbA-infected CBA mice with and without IMP treatment. FIG. 11 C shows a survival curve of PbA-infected C57BL/6 mice with and without IMP treatment. FIG. 1 ID shows clinical scores of PbA-infected C57BL/6 mice with and without IMP treatment. Data points for PbA-infected mice are shown as circles; data points for PbA-infected and IMP treated mice are shown as squares.

[0049] FIGS. 12A-C show survival and clinical scores in PbA-infected CBA mice that are untreated, treated with PLGA particles (IMP), or treated with IMP and Chloroquine. FIG. 12A shows survival curves of PbA-infected CBA mice that are untreated (circles), treated with IMPS at day 3 (square), day 5 (triangle), day 7 (inverted triangle), or treated with IMP at day 7 and Chloroquine (diamond). FIG. 12B shows a graph displaying parasitaemia of the experimental groups. FIG. 12C shows a graph displaying clinical scores of the experimental groups.

[0050] FIG. 13 shows survival curves for PbA-infected with mice that are untreated (circles) or treated with negatively charged carboxylated PLGA particles (IMP) from days 3-10 (closed squares), artesunate treatment from day 7-10 (triangles), IMP and artesunate from day 7-10 (inverted triangles), or IMP treatment from day 7-10 (open squares).

[0051] FIG. 14 shows survival curves for PbA-infected CBA mice that are untreated or treated with negatively charged carboxylated PLGA particles (IMP) once at day 7, artesunate treatment once at day 7, and IMP and artesunate once at day 7.

[0052] FIG. 15 shows survival curves for PbA-infected CBA mice that are untreated or treated with negatively charged carboxylated PLGA particles (IMP) once at day 7, artesunate treatment once at day 7, and IMP and artesunate once at day 7.

[0053] FIG. 16 shows survival curves for PbA-infected CBA mice that are untreated or treated with negatively charged carboxylated PLGA particles (IMP), artesunate, and IMP and artesunate (left panel) and the survival curves for PbA infected mice that were treated with IMP and artesunate that were reinfected with PbA at day 28, as compared to untreated control mice infected with PbA for the first time (right panel).

DETAILED DESCRIPTION OF THE INVENTION

[0054] The present inventors have surprisingly found that negatively charged particles, such as polystyrene, PLGA, or diamond particles of a certain size and zeta potential, can be administered to subjects to treat, ameliorate, alleviate, and/or reduce the severity of malaria or symptoms associated with malaria. Administering the negatively charged particles can increase survival of subjects suffering from malaria, reduce the severity of symptoms associated with malaria, reduce tissue damage associated with malaria, and/or reduce the inflammation associated with malaria, and such improvements can be observed when the particles are administered in the early or in the late stages of the disease progression. The present inventors have also observed that combined treatment of negatively charged particles with antimalarials, e.g., Quinine, Chloroquine, or artesunate, synergistically improve survival and ameliorate symptoms of malaria. Combined treatment of negatively charged particles and antimalarials induces immunity, as subjects treated with the particles and antimalarials develop sterilizing immunity and cannot be re- infected with malarial parasites.

[0055] Certain embodiments contemplate that the negatively charged particles themselves are the bioactive agent capable of reducing the immune response, inducing monocyte and/or neutrophil apoptosis and improved clearance, inducing toleragenic dendritic cells, suppressor cells, and regulatory T cells, and removing pro-inflammatory T cells, as opposed to merely being a delivery system for a bioactive agent. Thus, in certain embodiments, administration of the negatively charged particles alone are effective in treating subjects infected with malaria, and do not require that the particles attached to any peptides, antigens, pharmaceuticals, drugs, oligonucleotides, phospholipids, or any other bioactive agents. One of skill in the art would understand, however, that the particles themselves are the active agents.

[0056] As used herein, "malaria" is a parasitic disease that involves high fevers, shaking chills, flu-like symptoms, and anemia. Malaria includes but is not limited to Quartan malaria, Falciparum malaria, Biduoterian fever, Blackwater fever, Tertian malaria, Plasmodium, uncomplicated malaria and severe malaria. Malaria is a disease caused by infection by a species of the genus Plasmodium. Most severe cases are associated with Plasmodium falciparum.

[0057] "Severe malaria" as used herein, refers to malaria associated with high mortality. Examples of clinical manifestations of severe malaria are found in Table 1. From a clinical perspective, there is a continuum from asymptomatic malaria to uncomplicated illness through to severe and lethal malaria. Before artemisinin combination treatments (ACT) became widely available, uncomplicated falciparum malaria was associated with a case-specific mortality of approximately 0.1% when there was ready access to effective antimalarial drug treatment. Thus, 1 patient in 1000 who presented with apparently uncomplicated falciparum malaria would deteriorate despite treatment and die (in the United States this is estimated to be 1-2 patients per year). With worsening resistance and/or delays in access to effective drugs mortality approached 1 in 100 (1%). The mortality with ACTs is lower than 0.1% as artemisinins are particularly effective in the group of patients with high ring-stage parasitemia who may appear only mildly ill, but then deteriorate rapidly coincident with extensive parasite red cell sequestration.

[0058] Nearly all deaths from severe malaria result from infections with P. falciparum. Severe malaria is observed in patients with positive parasitemia and one or more of the clinical or laboratory manifestations defined in table 1. Severe malaria encompasses three major malarial syndromes including cerebral malaria, severe anemia, and acute lung injury/severe respiratory distress.

[0059] "Cerebral malaria" as used herein, refers to the most severe neurological complication of infection with malarial parasites. It is a clinical syndrome characterized by coma and asexual forms of the parasite on peripheral blood smears. Mortality is high and some surviving patients sustain brain injury which manifest as long-term neuro-cognitive impairments. Cerebral malaria is a severe complication of Plasmodium infection and is responsible for an estimated 627,000 deaths annually, particularly among children according to the World Health Organization (WHO) Global Malaria Program: World Malaria Report 2013. Cerebral malaria is strongly associated with parasitised red blood cell sequestration in the vasculature of the brain. The resultant obstruction of blood flow has been proposed as a potential pathological mechanism leading to ischemia and hypoxia of the central nervous system.

[0060] "Late stage cerebral/severe malaria" as used herein, refers to the stage of malaria that is classified by the World Health Organization (WHO) as one or more of the following, occurring in the absence of an identified alternative cause, and in the presence of P. falciparum asexual parasitemia:

• Impaired consciousness (Cerebral Malaria): A Glasgow Coma Score <11 in adults or a Blantyre coma score <3 in children

• Acidosis: A base deficit of >8 meq/1 or, if unavailable, a plasma bicarbonate of <15 mM or venous plasma lactate >5 mM. Severe acidosis manifests clinically as respiratory distress - rapid, deep and labored breathing

• Hypoglycaemia: Blood or plasma glucose <2.2 mM (<40 mg/dl)

• Severe malarial anemia: A haemoglobin concentration <5 g/dl or a haematocrit of <15% in children <12 years of age (<7 g/dl and <20%, respectively, in adults) together with a parasite count >10 000/11

• Acute lung injury/acute respiratory distress syndrome: Radiologically confirmed, or

oxygen saturation <92% on room air with a respiratory rate >30/min, often with chest in drawing and crepitation on auscultation

[0061] "Particle" as used herein refers to any non-tissue derived minute composition of matter, it may be a sphere or sphere-like entity or bead. The term "particle" and the term "bead" may be used interchangeably. Additionally, the term "particle" may be used to encompass beads, rods, and spheres.

[0062] "Carboxylated particles" or "carboxylated beads" or "carboxylated spheres" includes any particle that has been modified to contain a carboxyl group on its surface. In some embodiments the addition of the carboxyl group enhances phagocyte/monocyte uptake of the particles from circulation, for instance through the interaction with scavenger receptors such as MARCO. The carboxylation can be achieved using any compound which adds carboxyl groups, including, but not limited to poly(ethylene-maleic anhydride (PEMA).

[0063] "Negatively charged particles" or "negatively charged beads" or "negatively charged spheres" include any particle that inherently possesses, or has been modified to have a negative charge. In some embodiments, particles are made to have a negative charge by carboxylation of the particles.

[0064] "Antigenic moiety" as used herein refers to any moiety, for example a peptide, that is recognized by the host's immune system. Examples of antigenic moieties include, but are not limited to, autoantigens and/or bacterial or viral proteins, peptides or components. Without being bound by theory, while the negatively charged beads themselves may be recognized by the immune system, the negatively charged beads with nothing more attached thereto are not considered an "antigenic moiety" for the purposes of the invention.

[0065] "Naked beads" or "naked particles" or "naked spheres" as used herein refers to beads, particles or spheres that have not been carboxylated or otherwise modified.

[0066] "Pro-inflammatory mediators" or "pro-inflammatory polypeptides" as used herein refers to polypeptides or fragments thereof which induce, maintain, or prolong inflammation in a subject. Examples of pro-inflammatory mediators include, but are not limited to, cytokines and chemokines.

[0067] "Inflammatory milieu" or "inflammatory foci" as used herein refers to the site of inflammation, or increased concentration of pro-inflammatory mediators, in a subject. The inflammatory milieu may encompass a subjects circulation as a whole. For example, when a subject is suffering from a systemic inflammatory disorder, inflammatory mediators may be found throughout the subject's circulation. Thus, in these embodiments, the inflammatory milieu is not contained within a discreet area.

[0068] By "Regulatory T cell" or "Treg" or "Suppressor T cell" is meant a T cell that is capable of modulating the T cell immune response. For example a Treg may be a CD4⁺ or CD8⁺ T cell that is capable of inhibiting the effector function of either CD4⁺ or CD8⁺ T cell response. In another example, a Treg may be a CD4⁺ or CD8⁺ T cell that is capable of inhibiting the effector function of a B cell, mast cell or a monocyte/macrophage.

[0069] By "about" is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0070] "Treatment" or "treating," as used herein, includes any desirable effect on the symptoms or pathology of a disease or condition, and may include even minimal changes or improvements in one or more measurable markers of the disease or condition being treated. "Treatment" or "treating" does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof. The subject receiving this treatment is any subject in need thereof. Exemplary markers of clinical improvement will be apparent to persons skilled in the art. [0071] "Inhibit," as used herein, is meant to prevent or decrease a function or process. "Inhibit," as used herein, can refer to "decrease," "reduce," "curb," "abate," "diminish," "lessen," "lower," or "weaken."

[0072] "Bioactive agent" or "bioactive compound" as used herein, refers to any agent or compound that has an effect on a living organism, tissue, or cell. In accordance with the present invention, bioactive agents include, but are not limited to, nutrients, pharmaceuticals, drugs, peptides, peptide moieties, oligonucleotides, phospholipids, lipids, and antigenic moieties. Thus, by particles "free of attached bioactive agents" it is meant particles that do not have such bioactive agents conjugated to the surface, embedded within the core, or otherwise associated with the particle. As used herein, the terms "bioactive agent" or "bioactive compound" do not include carboxyl groups. Thus, as used herein, a particle free from bioactive agents and/or bioactive compounds can include particles that are carboxylated.

[0073] A "synergistically effective" dose or amount refers to a dose or amount of a therapeutic agent, e.g., a drug or the particles described herein, that results in a synergistic enhancement of a therapeutic effect, e.g. an increased probability of survival, when the same subject is also administered a synergistically effective dose or amount of a second therapeutic agent that is capable of synergistically interacting with the first therapeutic agent. Thus, if two or more therapeutic agents are administered to a subject, the doses are considered synergistically effective if at least one of the therapeutic effects is synergistic, i.e., the effect is greater than what would have been expected from the effects of either therapeutic agent administered alone.

[0074] A "citric acid particle" or "citric acid nanoparticle" as used herein refers to a particle that comprise citric acid based polymers and/or a particle that comprises a core made from citric acid based polymers.

[0075] The particle may have any particle shape or conformation. However, in some embodiments it is preferred to use particles that are less likely to clump in vivo. Examples of particles within these embodiments are those that have a spherical shape. Particles useful in the methods of the current invention are described in U.S. Patent Applications Nos. U.S. 2013/0323319 and U.S. 2015/0010631, and PCT Application No. PCT/US2015/054922, all of which are hereby incorporated by reference in their entireties.

[0076] The negatively charged particles of the present invention (sometimes referred to herein as "immune modified particles" or "IMPs") specifically inhibit inflammatory monocyte immigration into inflammatory foci. By IMP, it is meant a PLGA immune modified particle, but one of skill in the art will understand that particles composed of other materials, e.g. polystyrene particles, diamond particles, or citric acid based polymers. Inflammatory monocytes take up IMPs in a macrophage receptor with collagenous (MARCO) dependent fashion and migrate to the spleen, whereby they undergo caspase 3 -mediated cell death. Importantly IMP therapy is shown to have positive impacts on West Nile Virus (WNV) encephalitis, peritonitis, experimental autoimmune encephalomyelitis, heart function after myocardial infarction, kidney reperfusion injury and colitis. IMPs provide an alternative and highly specific tool for inhibiting inflammatory monocytes in a MARCO-dependent manner. Harnessing a natural leukocyte clearance pathway, IMPs represent a novel and safe inflammatory monocyte specific therapy.

[0077] It is not necessary that each particle be uniform in size, although the particles must generally be of a size sufficient to trigger phagocytosis in an antigen presenting cell or other MPS cell. Preferable, the particles are microscopic or nanoscopic in size, in order to enhance solubility, avoid possible complications caused by aggregation in vivo and to facilitate pinocytosis. Particle size can be a factor for uptake from the interstitial space into areas of lymphocyte maturation. A particle having an average diameter of from about 0.1 μηι to about 10 μιτι, or from about 0.01 μηι to about 10 μιτι, is capable of triggering phagocytosis. Thus in one embodiment, the particle has a diameter within these limits. In another embodiment, the particle has an average diameter of about 0.2μηι to about 2 μπι. In another embodiment, the particle has an average diameter of about 0.3 μηι to about 5 μπι. In still another embodiment, the particle has an average diameter of about 0.5 μηι to about 3 μπι. In a further embodiment the particle has an average size of about 0.1 μηι, or about 0.2 μηι or about 0.3 μηι or about 0.4 μηι or about 0.5 μηι or about 1.0 μιη or about 1.5 μηι or about 2.0 μηι or about 2.5 μηι or about 3.0 μηι or about 3.5 μηι or about 4.0 μηι or about 4.5 μηι or about 5.0 μπι. In a particular embodiment the particle has a size of about 0.5 μηι. The particles in a composition need not be of uniform diameter. By way of example, a pharmaceutical formulation may contain a plurality of particles, some of which are about 0.5 μιτι, while others are about 1.0 μιη. Any mixture of particle sizes within these given ranges will be useful. Particle sizes discussed herein refer to particle size as measured by dynamic light scattering, and one of skill in the art will understand that measurements performed by other techniques can give other values for particle size.

[0078] In some embodiments, the particle is non-metallic. In these embodiments the particle may be formed from a polymer. In a preferred embodiment, the particle is biodegradable in an individual. In this embodiment, the particles can provide in an individual across multiple doses without there being an accumulation of particles in the individual. Examples of suitable particles include polystyrene particles, PLGA particles, PLURIONICS stabilized polypropylene sulfide particles, citric acid based polymers, and diamond particles.

[0079] Preferably the particle surface is composed of a material that minimizes non-specific or unwanted biological interactions. Interactions between the particle surface and the interstitium may be a factor that plays a role in lymphatic uptake. The particle surface may be coated with a material to prevent or decrease non-specific interactions. Steric stabilization by coating particles with hydrophilic layers such as poly(ethylene glycol) (PEG) and its copolymers such as PLURONICS® (including copolymers of poly(ethylene glycol)-bl-poly(propylene glycol)-bl- poly(ethylene glycol)) may reduce the non-specific interactions with proteins of the interstitium as demonstrated by improved lymphatic uptake following subcutaneous, oral, or lymphatic administration/injections. All of these facts point to the significance of the physical properties of the particles in terms of lymphatic uptake. Biodegradable polymers may be used to make all or some of the polymers and/or particles and/or layers. Biodegradable polymers may undergo degradation, for example, by a result of functional groups reacting with the water in the solution. The term "degradation" as used herein refers to becoming soluble, either by reduction of molecular weight or by conversion of hydrophobic groups to hydrophilic groups. Polymers with ester groups are generally subject to spontaneous hydrolysis, e.g., polylactides and polyglycolides.

[0080] Certain embodiments contemplate that the negatively charged particles themselves are the bioactive agents capable of treating or inhibiting malaria in a subject, as opposed to being a delivery system for a bioactive agent that treats or inhibits malaria. In particular embodiments, negatively charged particles are free from any attached bioactive agents. In some embodiments, negatively charged particles are free from bioactive agents, e.g. nutrients, pharmaceuticals, drugs, peptides, oligonucleotides, phospholipids, lipids, and antigenic moieties. Examples of bioactive agents also include, but are not limited to, a natural or chemically modified polypeptide, an antibody or fragment thereof, a natural or chemically modified small oligopeptide, a natural, unnatural, or chemically modified amino acid, a polynucleotide, a natural or chemically modified oligonucleotide, RNAi, shRNA, siRNA, a small nucleotide, a natural or chemically modified mononucleotide, a lipopeptide, a phospholipid, an antimicrobial, a small molecule, and a pharmaceutical molecule. [0081] Particles of the present invention may also contain additional components. For example, carriers may have imaging agents incorporated or conjugated to the carrier. An example of a carrier nanosphere having an imaging agent that is currently commercially available is the Kodak X-sight nanospheres. Inorganic quantum- confined luminescent nanocrystals, known as quantum dots (QDs), have emerged as ideal donors in FRET applications: their high quantum yield and tuneable size-dependent Stokes Shifts permit different sizes to emit from blue to infrared when excited at a single ultraviolet wavelength. (Bruchez, et al, Science, 1998, 281, 2013; Niemeyer, C. M Angew. Chem. Int. Ed. 2003, 42, 5796; Waggoner, A. Methods Enzymol. 1995, 246, 362; Brus, L. E. J. Chem. Phys. 1993, 79, 5566). Quantum dots, such as hybrid organic/inorganic quantum dots based on a class of polymers known as dendrimers, may be used in biological labelling, imaging, and optical biosensing systems. (Lemon, et al, J. Am. Chem. Soc. 2000, 122, 12886). Unlike the traditional synthesis of inorganic quantum dots, the synthesis of these hybrid quantum dot nanoparticles does not require high temperatures or highly toxic, unstable reagents. (Etienne, et al, Appl. Phys. Lett. 87, 181913, 2005).

[0082] Particles can be formed from a wide range of materials. The particle is preferably composed of a material suitable for biological use. For example, particles may be composed of glass, silica, polyesters of hydroxy carboxylic acids, polyanhydrides of dicarboxylic acids, or copolymers of hydroxy carboxylic acids and dicarboxylic acids. More generally, the carrier particles may be composed of polyesters of straight chain or branched, substituted or unsubstituted, saturated or unsaturated, linear or cross-linked, alkanyl, haloalkyl, thioalkyl, aminoalkyl, aryl, aralkyl, alkenyl, aralkenyl, heteroaryl, or alkoxy hydroxy acids, or polyanhydrides of straight chain or branched, substituted or unsubstituted, saturated or unsaturated, linear or cross-linked, alkanyl, haloalkyl, thioalkyl, aminoalkyl, aryl, aralkyl, alkenyl, aralkenyl, heteroaryl, or alkoxy dicarboxylic acids. Additionally, carrier particles can be quantum dots, or composed of quantum dots, such as quantum dot polystyrene particles (Joumaa et al. (2006) Langmuir 22: 1810-6). Carrier particles including mixtures of ester and anhydride bonds (e.g., copolymers of glycolic and sebacic acid) may also be employed. For example, carrier particles may comprise materials including polyglycolic acid polymers (PGA), polylactic acid polymers (PLA), polysebacic acid polymers (PSA), poly(lactic-co-glycolic) acid copolymers (PLGA), [rho]poly(lactic-co-sebacic) acid copolymers (PLSA), poly(glycolic-co- sebacic) acid copolymers (PGSA), polypropylene sulfide polymers, poly(caprolactone), chitosan, etc. Other biocompatible, biodegradable polymers useful in the present invention include citric acid-based polymers, polymers or copolymers of caprolactones, carbonates, amides, amino acids, orthoesters, acetals, cyanoacrylates and degradable urethanes, as well as copolymers of these with straight chain or branched, substituted or unsubstituted, alkanyl, haloalkyl, thioalkyl, aminoalkyl, alkenyl, or aromatic hydroxy- or di-carboxylic acids. In addition, the biologically important amino acids with reactive side chain groups, such as lysine, arginine, aspartic acid, glutamic acid, serine, threonine, tyrosine and cysteine, or their enantiomers, may be included in copolymers with any of the aforementioned materials to provide surface charge and/or reactive groups for conjugating to antigen peptides and proteins or conjugating moieties. Biodegradable materials suitable for the present invention include diamond, PLA, PGA, polypropylene sulfide polymers, and PLGA polymers. Biocompatible but non-biodegradable materials may also be used in the carrier particles of the invention. For example, non-biodegradable polymers of acrylates, ethylene-vinyl acetates, acyl substituted cellulose acetates, non-degradable urethanes, styrenes, vinyl chlorides, vinyl fluorides, vinyl imidazoles, chlorosulphonated olefins, ethylene oxide, vinyl alcohols, TEFLON^® (DuPont, Wilmington, Del), and nylons may be employed.

[0083] In one embodiment, the buffer solution contacting the immune modified particle may have a basic pH. Suitable basic pH for the basic solution include 7.1, 7.5, 8.0, 8.5, 9.5, 10.0 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, and 13.5. The buffer solution may also be made of any suitable base and its conjugate. In some embodiments of the invention, the buffer solution may include, without limitation, sodium bicarbonate, potassium bicarbonate, lithium bicarbonate, potassium dihydrogen phosphate, sodium dihydrogen phosphate, or lithium dihydrogen phosphate and conjugates thereof.

[0084] In one embodiment of the invention, the immune modified particles contain co-polymers. These co-polymers may have varying molar ratio. Suitable co-polymer ratio of present immune modified particles may be 25:75, 30:70, 35:65, 40:60, 45:55, 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 81 : 19, 82: 18, 83: 17, 84: 16, 85: 15, 86: 14, 87: 13, 88: 12, 89: 11, 90: 10, 91 :9, 92:8, 93:7, 94:6, 95:5, 96:4, 97:3, 98:2, 99: 1, or 100:0. In another embodiment, the co-polymer may be periodical, statistical, linear, branched (including star, brush, or comb co-polymers) copolymers. In some embodiments, the co-polymers ratio may be, but not limited to, polystyrene:poly(vinyl carboxylate)/80:20, polystyrene: poly(vinyl carboxylate)/90: 10, poly(vinyl carboxylate): polystyrene/80: 20, poly(vinyl carboxylate):polystyrene/90: 10, polylactic acid: polyglycolic acid/50: 50, poly lactic acid: polygly colic acid/80: 20, or poly lactic acid: polygly colic acid/90: 10.

[0085] The particles of the instant invention can be manufactured by any means commonly known in the art. Exemplary methods of manufacturing particles include, but are not limited to, microemulsion polymerization, interfacial polymerization, precipitation polymerization, emulsion evaporation, emulsion diffusion, solvent displacement, and salting out (Astete and Sabliov, J. Biomater. Sci. Polymer Edn., 17:247-289(2006)). Manipulation of the manufacturing process for PLGA particles can control particle properties (e.g. size, size distribution, zeta potential, morphology, hydrophobicity/hydrophilicity, polypeptide entrapment, etc). The size of the particle is influenced by a number of factors including, but not limited to, the concentration of PLGA, the solvent used in the manufacture of the particle, the nature of the organic phase, the surfactants used in manufacturing, the viscosity of the continuous and discontinuous phase, the nature of the solvent used, the temperature of the water used, sonication, evaporation rate, additives, shear stress, sterilization, and the nature of any encapsulated antigen or polypeptide.

[0086] Particle size is affected by the polymer concentration; higher particles are formed from higher polymer concentrations. For example, an increase in PLGA concentration from 1% to 4% (w/v) can increase mean particle size from about 205 nm to about 290 nm when the solvent propylene carbonate is used. Alternatively, in ethyl acetate and 5% Pluronic F-127, an increase in PLGA concentration from 1% to 5% (w/v) increases the mean particle size from 120 nm to

230 nm.

[0087] The viscosity of the continuous and discontinuous phase is also an important parameter that affects the diffusion process, a key step in forming smaller particles. The size of the particles increases with an increase in viscosity of the dispersed phase, whereas the size of the particles decreases with a more viscous continuous phase. In general, the lower the phase ratio of organic to aqueous solvent, the smaller the particle size.

[0088] Homogenizer speed and agitation also affect particle size; in general, higher speeds and agitation cause a decrease in particle size, although there is a point where further increases in speed and agitation no longer decrease particle size. There is a favorable impact in the size reduction when the emulsion is homogenized with a high pressure homogenizer compared with just high stirring. For example, at a phase ration of 20% in 5% PVA, the mean particle size with stirring is 288 nm and the mean particle size with homogenization (high pressure of 300 bars) is [0089] An important size reduction of the particles can be achieved by varying the temperature of the water added to improve the diffusion of the solvent. The mean particle size decreases with an increase in water temperature.

[0090] Certain embodiments of the present invention are directed to particles described herein that are coupled to a bioactive agent, wherein the bioactive agent is one or more antimalarials. By "coupled to" it is meant that the particles are associated with, encapsulated with, or attached to one or more antimalarial. In particular embodiments, the particles are carboxylated particles with a negative zeta potential that are coupled to one or more antimalarials. Antimalarials suitable for coupling to particles of the present invention include Quinine and related agents, Chloroquine, Amodiaquine, Pyrimethamine, Proguanil, Sulfonamides, Mefloquine, Atovaquone, Primaquine, Artemisinin and its derivatives, Halofantrine, Doxycycline, Clindamycin, or combinations thereof.

[0091] Some embodiments are directed to two separate formulas, such that the particles may be administered before, simultaneously to, or after administration of one or more antimalarials.

[0092] Certain embodiments of the present invention comprise negatively charged particles that are free from attached antimalarials.

[0093] Particular embodiments of the present invention are directed to pharmaceutical compositions that comprise negatively charged particles and antimalarials. In particular embodiments the pharmaceutical composition comprises negatively charged particles that are free from attached bioactive agents and antimalarials. In particular embodiments, the pharmaceutical composition comprises negatively charged particles are coupled to bioactive agents that are antimalarials.

[0094] Particular embodiments of the present invention are directed to particles described herein that are coupled to a malaria vaccine or components of a malaria vaccine. By "coupled to" it is meant that the particles are associated with, encapsulated with, or attached to the malarial vaccine or components thereof. In some embodiments, the particles are carboxylated, have a negative zeta potential, and coupled with a malarial vaccine or components thereof. In some embodiments, the malaria vaccine or components of a malaria vaccine comprise one or more epitopes associated with malaria e.g. epitopes associated with Plasmodium falciparum,

Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, or Plasmodium knowlesi. In certain embodiments, the particles are coupled to one or more epitopes associated with malaria,

In particular embodiments, the particles are coupled to one or more epitopes that are capable of stimulating antibody production against Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, or Plasmodium knowlesi. In some embodiments, the particles are carboxylated PLGA particles with a negative zeta potential that encapsulate one or more epitopes that are capable of stimulating antibody production against Plasmodium falciparum.

[0095] Certain embodiments of the present invention are directed to negatively charged particles that are free from any attached bioactive agents, including malaria vaccine or components of a malaria vaccine. Some embodiments of the present invention are directed to pharmaceutical compositions that comprise a malaria vaccine and negatively charged particles that are free from attached bioactive agents.

[0096] The nature of the polypeptide encapsulated in the particle also affects particle size. In general, encapsulation of hydrophobic polypeptides leads to the formation of smaller particles compared with the encapsulation of more hydrophilic polypeptides. In the double emulsion process, the entrapment of more hydrophilic polypeptides is improved by using high molecular mass PLGA and a high molecular mass of the first surfactant which causes a higher inner phase viscosity. The interaction between the solvent, polymer, and polypeptide affects the efficiency of incorporating the polypeptide into the particle.

[0097] The PLGA molecular mass impacts the final mean particle size. In general, the higher the molecular mass, the higher the mean particle size. For example, as the composition and molecular mass of PLGA varies (e.g. 12 to 48 kDa for 50 : 50 PLGA; 12 to 98 kDa for 75 : 25 PLGA) the mean particle size varies (about 102 nm -154 nm; about 132 nm to 152 nm respectively). Even when particles are the same molecular mass, their composition can affect average particle size; for example, particles with a 50 : 50 ratio generally form particles smaller than those with a 75 : 25 ratio. The end groups on the polymer also affects particle size. For example, particles prepared with ester end-groups form particles with an average size of 740nm (PI=0.394) compared with the mean size for the acid PLGA end-group is 240 nm (PI=0.225).

[0098] The solvent used can also affect particle size; solvents that reduce the surface tension of the solution also reduce particle size.

[0099] The organic solvent is removed by evaporation in a vacuum to avoid polymer and polypeptide damage and to promote final particle size reduction. Evaporation of the organic solvent under vacuum is more efficient in forming smaller particles. For example, evaporation in vacuum produces a mean particle size around 30% smaller than the mean particle size produced under a normal rate of evaporation.

[00100] The amplitude of the sonication wavelength also affects the particle characteristics. The amplitude of the wavelength should be over 20% with 600 to 800 s of sonication to form sable miniemulsions with no more droplet size changes. However, the main draw-back of sonication is the lack of monodispersity of the emulsion formed.

[00101] Organic phases that may be used in the production of the particles of the invention include, but are not limited to, ethyl acetate, methyl ethyl ketone, propylene carbonate, and benzyl alcohol. The continuous phases that may be used, include but are not limited to the surfactant poloxamer 188.

[00102] A variety of surfactants can be used in the manufacturing of the particles of the invention. The surfactant can be anionic, cationic, or nonionic. Surfactants in the poloxamer and poloaxamines family are commonly used in particle synthesis. Surfactants that may be used, include, but are not limited to PEG, Tween-80, gelatin, dextran, pluronic L-63, PVA, methylcellulose, lecithin and DMAB. Additionally, biodegradable and biocompatible surfactants including, but not limited to, vitamin E TPGS (D-a- tocopherol polyethylene glycol 1000 succinate). In certain embodiments, two surfactants are needed (e.g. in the double emulsion evaporation method). These two surfactants can include a hydrophobic surfactant for the first emulsion, and a hydrophobic surfactant for the second emulsion.

[00103] Solvents that may be used in the production of the particles of the invention include, but are not limited to, acetone, Tetrahydrofuran (THF), chloroform, and members of the chlorinate family, methyl chloride. The choice of organic solvents require two selection criteria: the polymer must be soluble in this solvent, and the solvent must be completely immiscible with the aqueous phase.

[00104] Salts that may be used in the production of the particles of the invention include, but are not limited to magnesium chloride hexahydrate, magnesium acetate tetrahydrate.

[00105] Common salting-out agents include, but are not limited to, electrolytes (e.g. sodium chloride, magnesium acetate, magnesium chloride), or non-electrolytes (e.g. sucrose).

[00106] The stability and size of the particles of the invention may be improved by the addition of compounds including, but not limited to, fatty acids or short chains of carbons. The addition of the longer carbon chain of lauric acid is associated with the improvement of particle characteristics. Furthermore, the addition of hydrophobic additives can improve the particle size, incorporation of the polypeptide into the particle, and release profile. Preparations of particles can be stabilized by lyophilization. The addition of a cryoprotectant such as trehalose can decrease aggregation of the particles upon lyophilization.

[00107] Suitable beads which are currently available commercially include polystyrene beads such as FluoSpheres (Molecular Probes, Eugene, Oreg.).

[00108] Physical properties are also related to a nanoparticle's usefulness after uptake and retention in areas having immature lymphocytes. These include mechanical properties such as rigidity or rubberiness. Some embodiments are based on a rubbery core, e.g., a poly(propylene sulfide) (PPS) core with an overlayer, e.g., a hydrophilic overlayer, as in PEG, as in the PPS- PEG system recently developed and characterized for systemic (but not targeted or immune) delivery. The rubbery core is in contrast to a substantially rigid core as in a polystyrene or metal nanoparticle system. The term rubbery refers to certain resilient materials besides natural or synthetic rubbers, with rubbery being a term familiar to those in the polymer arts. For example, cross-linked PPS can be used to form a hydrophobic rubbery core. PPS is a polymer that degrades under oxidative conditions to polysulphoxide and finally polysulphone, transitioning from a hydrophobic rubber to a hydrophilic, water-soluble polymer. Other sulphide polymers may be adapted for use, with the term sulphide polymer referring to a polymer with a sulphur in the backbone of the mer. Other rubbery polymers that may be used are polyesters with glass transition temperature under hydrated conditions that is less than about 37° C. A hydrophobic core can be advantageously used with a hydrophilic overlayer since the core and overlayer will tend not to mingle, so that the overlayer tends to sterically expand away from the core. A core refers to a particle that has a layer on it. A layer refers to a material covering at least a portion of the core. A layer may be adsorbed or covalently bound. A particle or core may be solid or hollow. Rubbery hydrophobic cores are advantageous over rigid hydrophobic cores, such as crystalline or glassy (as in the case of polystyrene) cores, in that higher loadings of hydrophobic drugs can be carried by the particles with the rubbery hydrophobic cores.

[00109] Another physical property is the surface's hydrophilicity. A hydrophilic material may have a solubility in water of at least 1 gram per liter when it is uncrosslinked. Steric stabilization of particles with hydrophilic polymers can improve uptake from the interstitium by reducing non-specific interactions; however, the particles' increased stealth nature can also reduce internalization by phagocytic cells in areas having immature lymphocytes. The challenge of balancing these competing features has been met, however, and this application documents the creation of nanoparticles for effective lymphatic delivery to DCs and other APCs in lymph nodes. Some embodiments include a hydrophilic component, e.g., a layer of hydrophilic material. Examples of suitable hydrophilic materials are one or more of polyalkylene oxides, polyethylene oxides, polysaccharides, polyacrylic acids, and polyethers. The molecular weight of polymers in a layer can be adjusted to provide a useful degree of steric hindrance in vivo, e.g., from about 1,000 to about 100,000 or even more; artisans will immediately appreciate that all the ranges and values within the explicitly stated ranges are contemplated, e.g., between 10,000 and 50,000.

[00110] The composition of the particles has been found to affect the length of time the particles persist in the body and tolerance requires rapid particle uptake and clearance/degradation. Since ratios of over 50:50 lactide:glycolide slow the degradation rate, the particles of the invention have a lactide:glycolide ratio of about 50:50 or below. In one embodiment the particles of the invention have about a 50:50 D,L-lactide:glycolide ratio.

[00111] The particles may incorporate functional groups for further reaction. Functional groups for further reaction include electrophiles or nucleophiles; these are convenient for reacting with other molecules. Examples of nucleophiles are primary amines, thiols, and hydroxyls. Examples of electrophiles are succinimidyl esters, aldehydes, isocyanates, and maleimides.

[00112] The efficacy of colloidal therapeutics, such as the negatively charged particles of the present invention, is closely related to the particles' in vivo distribution. The distribution of a colloidal system can be predicted by determining the zeta potential. The zeta potential is measure of the potential difference between the dispersion medium and the stationary layer of fluid attached to the dispersed particle, and indicates the degree of repulsion between adjacent, similarly charged particles in a dispersion. A high zeta potential predicts stability and good dispersion of the colloidal formulation. In preferred embodiments, the zeta potential of the pharmaceutical formulations of the present invention predicts good dispersion of the formulation in vivo.

[00113] The particles of the current invention can possess a particular zeta potential. In certain embodiments, the zeta potential is negative. In one embodiment, the zeta potential is less than about -100 mV. In one embodiment, the zeta potential is less than about -50 mV. In certain embodiments, the particles possess a zeta potential between -100 mV and 0 mV. In a further embodiment, the particles possess a zeta potential between -75 mV and 0 mV. In a further embodiment, the particles possess a zeta potential between -60 mV and 0 mV. In a further embodiment, the particles possess a zeta potential between -: 50 mV and 0 mV. In still a further embodiment, the particles possess a zeta potential between -40 mV and 0 mV. In a further embodiment, the particles possess a zeta potential between -30 mV and 0 mV. In a further embodiment, the particles possess a zeta potential between -20 mV and 0 mV. In a further embodiment, the particles possess a zeta potential between -10 mV and -0 mV. In a further embodiment, the particles possess a zeta potential between^■ ^■100 mV and -50mV. In a further embodiment, the particles possess a zeta potential between^■ -75 mV and -50 mV. In a further embodiment, the particles possess a zeta potential between^■ -50 mV and -40 mV. In a further embodiment, the particles possess a zeta potential between - 100 mV and -75 mV. In a further embodiment, the particles possess a zeta potential between - 100 mV and -25 mV. In a further embodiment, the particles possess a zeta potential between - 100 mV and -20 mV. In a further embodiment, the particles possess a zeta potential between^■ ■100 mV and -15 mV. In a further embodiment, the particles possess a zeta potential between^■ -50 mV and -25 mV. In a further embodiment, the particles possess a zeta potential between^■ -50 mV and -20 mV. In a further embodiment, the particles possess a zeta potential between^■ -50 mV and -15 mV. In a further embodiment, the particles possess a zeta potential between^■ -80 mV and -30 mV. In a further embodiment, the particles possess a zeta potential between -75 mV and -25 mV.

[00114] In some embodiments, particles have a negative zeta potential. In one embodiment, the zeta potential is more negative than about -100 mV, about -95 mV, about -90 mV, about 85 mV, about -80 mV, about 75 mV, about -70 mV, about -65 mV, about -60 mV, about -55 mV, about -50 mV, about -45 mV, about -40 mV, about -35 mV, about -30 mV, about -25 mV, about

-20 mV, about -15 mV, or about - 10 mV. In certain embodiments, the particles possess a zeta potential between about -100 mV and about 0 mV. In a further embodiment, the particles possess a zeta potential between about -75 mV and about -100 mV. In a further embodiment, the particles possess a zeta potential between -60 mV and 0 mV. In a further embodiment, the particles possess a zeta potential between about -50 mV and about 0 mV. In still a further embodiment, the particles possess a zeta potential between about -40 mV and about 0 mV. In a further embodiment, the particles possess a zeta potential between about -30 mV and about 0 mV. In a further embodiment, the particles possess a zeta potential between about -20 mV and about +0 mV. In a further embodiment, the particles possess a zeta potential between about -10 mV and about -0 mV. In a further embodiment, the particles possess a zeta potential between about -100 mV and about -50mV. In a further embodiment, the particles possess a zeta potential between about -75 mV and about -50 mV. In a further embodiment, the particles possess a zeta potential between about -50 mV and about -40mV. In a further embodiment, the particles possess a zeta potential between about -100 mV and about -75 mV. In a further embodiment, the particles possess a zeta potential between about -100 mV and about -25 mV. In a further embodiment, the particles possess a zeta potential between about -100 mV and about -20 mV. In a further embodiment, the particles possess a zeta potential between about -100 mV and about - 15 mV. In a further embodiment, the particles possess a zeta potential between about -50 mV and about -25 mV. In a further embodiment, the particles possess a zeta potential between about -50 mV and about -20 mV. In a further embodiment, the particles possess a zeta potential between about -50 mV and about -15 mV. As used herein, when referring to zeta potential, "less than" refers to the amplitude (i.e. the absolute value) of the charge. For example, the phrase "less than -100 mV" refers to a range of charges between 0 and -100 mV.

[00115] The particles of the current invention can be given in any dose effective to dampen the inflammatory immune response in a subject in need thereof or to treat a bacterial, viral, or parasitic infection in a subject in need thereof. In particular embodiments, the parasitic invention is malaria. In certain embodiments, about 10² to about 10²⁰ particles are provided to the

3 15

individual. In a further embodiment between about 10 to about 10 particles are provided. In yet a further embodiment between about 10⁶ to about 10¹² particles are provided. In still a further embodiment between about 10⁸ to about 10¹⁰ particles are provided. In a preferred embodiment the preferred dose is 0.1% solids/ml. Therefore, for 0.5 μηι beads, a preferred dose is approximately 4 x 10⁹ beads, for 0.05μηι beads, a preferred dose is approximately 4 x 10¹² beads, for 3μηι beads, a preferred dose is 2 x 10⁷ beads. However, any dose that is effective in treating the particular condition to be treated is encompassed by the current invention.

[00116] As illustrated by Fig. 1, particular embodiments contemplate a model whereby an parasitic infection caused by a species of Plasmodium in a subject progresses in stages. The

Plasmodium species, e.g. Plasmodium falciparum, initially invades cells and multiplies. The first defense is the activation of the intracellular anti-parasitic response, followed by activation of the innate-immune response. At this stage, the response may progress to a well-coordinated adaptive response culminating in the elimination of the parasite and long-lasting immunity.

Alternatively, aberrant activation of the innate and/or adaptive immune response may trigger a hyperactive immune response involving, amongst many other molecules, CCL2, and cells including but not limited to, monocytes and T cells, leading to immune pathology, tissue damage, and death. A timed intervention during the acute phase of immune dysregulation that can reverse the dysregulation will reduce mortality. Certain embodiments contemplate that administration of negatively charged particles described herein to a subject infected with a Plasmodium species will prevent, reduce, or reverse the aberrant immune stimulation and improve probability of survival. In particular embodiments, a subject infected with a Plasmodium species is administered a pharmaceutical composition comprising negatively charged particles described herein and a carrier to prevent, reduce, or reverse an aberrant immune stimulation caused by the infection.

[00117] Particular embodiments contemplate a model whereby administration of a pharmaceutical composition comprising negatively charged particles with no associated bioactive agents to a subject with malaria will reduce the subject's aberrant immune response to malaria, and thereby ameliorate symptoms of malaria. In some embodiments, administration of the particles to the subject with malaria reduces the aberrant immune response and increases or improves the specific immune response of the subject to the parasite associated with the malaria. Particular embodiments contemplate that increasing or improving the specific immune response to the parasite associated with malaria will reduce, inhibit, and/or prevent the severity and/or the duration of the malaria invention.

[00118] As used herein, the term "immune response" includes T cell mediated and/or B cell mediated immune responses. Exemplary immune responses include T cell responses, e.g., cytokine production and cellular cytotoxicity. In addition, the term immune response includes immune responses that are indirectly effected by T cell activation, e.g., antibody production (humoral responses) and activation of cytokine responsive cells, e.g., macrophages. Immune cells involved in the immune response include lymphocytes, such as B cells and T cells (CD4⁺, CD8⁺, Thl and Th2 cells); antigen presenting cells (e.g., professional antigen presenting cells such as dendritic cells, macrophages, B lymphocytes, Langerhans cells, and nonprofessional antigen presenting cells such as keratinocytes, endothelial cells, astrocytes, fibroblasts, oligodendrocytes); natural killer cells; myeloid cells, such as macrophages, eosinophils, mast cells, basophils, and granulocytes. In some embodiments, the modified particles of the present invention are effective to reduce inflammatory cell trafficking to the site of inflammation.

[00119] As used herein, the term "inflammatory monocyte" refers to any myeloid cell expressing any combination of CD14/CD16 and CCR2. As used herein, the term "inhibitory neutrophil" encompasses monocyte derived suppressor cells, and/or neutrophils. As used herein, the term "anergy," "tolerance," or "antigen-specific tolerance" refers to insensitivity of T cells to T cell receptor-mediated stimulation. Such insensitivity is generally antigen- specific and persists after exposure to the antigenic peptide has ceased. For example, anergy in T cells is characterized by lack of cytokine production, e.g., IL-2. T-cell anergy occurs when T cells are exposed to antigen and receive a first signal (a T cell receptor or CD-3 mediated signal) in the absence of a second signal (a costimulatory signal). Under these conditions, re-exposure of the cells to the same antigen (even if re-exposure occurs in the presence of a costimulatory molecule) results in failure to produce cytokines and subsequently failure to proliferate. Thus, a failure to produce cytokines prevents proliferation. Anergic T cells can, however, proliferate if cultured with cytokines (e.g., IL-2). For example, T cell anergy can also be observed by the lack of IL-2 production by T lymphocytes as measured by ELISA or by a proliferation assay using an indicator cell line. Alternatively, a reporter gene construct can be used. For example, anergic T cells fail to initiate DL-2 gene transcription induced by a heterologous promoter under the control of the 5' IL-2 gene enhancer or by a multimer of the API sequence that can be found within the enhancer (Kang et al. 1992 Science. 257: 1134).

[00120] As used herein, the term "immunological tolerance" refers to methods performed on a proportion of treated subjects in comparison with untreated subjects where: a) a decreased level of a specific immunological response (thought to be mediated at least in part by antigen-specific effector T lymphocytes, B lymphocytes, antibody, or their equivalents); b) a delay in the onset or progression of a specific immunological response; or c) a reduced risk of the onset or progression of a specific immunological response. "Specific" immunological tolerance occurs when immunological tolerance is preferentially invoked against certain antigens in comparison with others. "Non- Specific" immunological tolerance occurs when immunological tolerance is invoked indiscriminately against antigens which lead to an inflammatory immune response. "Quasi-Specific" immunological tolerance occurs when immunological tolerance is invoked semi-discriminately against antigens which lead to a pathogenic immune response but not to others which lead to a protective immune response.

[00121] A proxy for tolerogenic activity is the ability of a particle to stimulate the production of an appropriate cytokine at the target site. The immunoregulatory cytokine released by T suppressor cells at the target site is thought to be TGF-β (Miller et al., Proc. Natl. Acad. Sci.

USA 89:421, 1992). Other factors that may be produced during tolerance are the cytokines IL4 and IL-10, and the mediator PGE. In contrast, lymphocytes in tissues undergoing active immune destruction secrete cytokines such as IL-I, IL-2, IL-6, and IFNy. Hence, the efficacy of a modified particle can be evaluated by measuring its ability to stimulate the appropriate type of cytokines.

[00122] With this in mind, a rapid screening test for modified particles, effective mucosal binding components, effective combinations, or effective modes and schedules of mucosal administration can be conducted using animal model systems. Animals are treated at a mucosal surface with the test particle composition, and at some time are challenged with administration of the disease causing antigen or an infectious agent. Spleen cells are isolated, and cultured in vitro in the presence of the disease causing antigen or an antigen derived from the infectious agent at a concentration of about 50 μg/ml. Cytokine secretion into the medium can be quantitated by standard immunoassay.

[00123] The ability of the particles to suppress the activity of cells can be determined using cells isolated from an animal immunized with the modified particles, or by creating a cell line responsive to a disease causing antigen or viral antigen target antigen (Ben-Nun et al, Eur. J. Immunol. 11 : 195, 1981). In one variation of this experiment, the suppressor cell population is mildly irradiated (about 1000 to 1250 rads) to prevent proliferation, the suppressors are co- cultured with the responder cells, and then tritiated thymidine incorporation (or MTT) is used to quantitate the proliferative activity of the responders. In another variation, the suppressor cell population and the responder cell population are cultured in the upper and lower levels of a dual chamber transwell culture system (Costar, Cambridge Mass.), which permits the populations to coincubate within 1 mm of each other, separated by a polycarbonate membrane (WO 93/16724). In this approach, irradiation of the suppressor cell population is unnecessary, since the proliferative activity of the responders can be measured separately.

[00124] The effectiveness of compositions and modes of administration for treatment of specific disease can also be elaborated in a corresponding animal disease model. The ability of the treatment to diminish or delay the symptomatology of the disease is monitored at the level of circulating biochemical and immunological hallmarks of the disease, immunohistology of the affected tissue, and gross clinical features as appropriate for the model being employed. Non- limiting examples of animal models that can be used for testing are included in the following section. [00125] In another aspect of the present invention, negatively charged particles act as sink to mop up pro-inflammatory mediators, pathological proteins and cellular debris from the blood of a subject with an inflammatory response. Alternatively, or in addition to, the negatively charged particles of the present invention may concentrate regulatory proteins by binding to regulatory proteins in the blood of a subject with an inflammatory response and present these regulatory proteins to their cognate receptors to further ameliorate an immune response. The negatively charged particles of the current invention may be used in broad scale diagnostic methods of blood samples where other methods, such as mass spectrometry and other proteomic methods have failed. When inflammatory plasma or serum is incubated with the negatively charged particles described herein, this results in the binding and subsequent purification of proteins not found in the serum/plasma under non-inflammatory or homeostatic conditions.

[00126] In another aspect of the present invention, negatively charged particles encompassing antigens are provided.

[00127] In one embodiment, the negatively charged particles of the invention are coupled to antigens comprising one or more epitopes associated with malaria. The antigens may comprise one or more copies of an epitope. In one embodiment, the antigens comprise a single epitope associated with a malaria. In one embodiment, the epitope is associated with a Plasmodium species. In some embodiments, the epitope is associated with Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, or Plasmodium knowlesi. In a further embodiment, the antigens comprise more than one epitope associated with the same Plasmodium species. In yet a further embodiment, the antigens comprise more than one epitope associated with different Plasmodium species. In a further embodiment, the antigens comprise one or more epitopes associated with one or more infections of Plasmodium species. Particular embodiments of the present invention contemplate that negative charged particles coupled to one or more epitopes associated with malaria are useful as a malaria vaccine. Some embodiments contemplate that administering to a subject negatively charged particles coupled with one or more epitopes associated with malaria will vaccinate the subject to malaria, i.e., result in malaria vaccination of the subject.

[00128] Not all epitopes are linear epitopes; epitopes can also be discontinuous, conformational epitopes.

[00129] The term 'epitope' refers to a portion of a larger protein which defines a sequence that by itself or as part of a larger sequence, an is capable of binding to an antibody or a T cell generated in response to that protein. In some embodiments, an epitope is a sequence of at least about 3 to 5, at least about 5 to 10, at least about 5 to 15, at least about 10 to 20, at least about 10 to 25, or at least about 15 to 30 amino acids or more than 30 amino acids, and typically not more than about 500, or about 1,000 amino acids. In some embodiments, an epitope is a linear epitope comprising an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 98%, or 100% identical to a region of protein found in Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, or Plasmodium knowlesi that is a three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty- two, twenty-three, twenty-four, or twenty five, or greater than twenty five contiguous amino acids in length. In some embodiments, an epitope is not limited to a polypeptide having a sequence identical to the portion of the parent protein from which is derived. Thus the term 'epitope' encompasses sequences identical to the native sequence as well as modifications, such as deletions substitutions and/or insertions into the native sequence. In some embodiments term "epitope" specifically includes 'mimitopes' i.e. sequences that do not identify a continuous linear sequence or do not necessarily occur in a native protein, but functionally mimic an epitope on a native protein. The term 'epitope' specifically includes linear and conformational epitopes. Particular embodiments contemplate that one of skill in the art will be able to recognize or identify a linear and/or a conformational epitope of a given protein found in an organism that is thought to cause or is otherwise thought to be associated with malaria, e.g., Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, or Plasmodium knowlesi.

[00130] In another aspect of the present invention, particles encapsulating antigens are provided. Particles which encapsulate antigens inside the particle can be used to induce immunity and/or antibody production in a subject. Examples of antigens which can be encapsulated within the particles of the invention include, but are not limited to, exogenous antigens, such as parasite antigens. In one embodiment, the antigens comprise one or more epitopes associated with Plasmodium species. In one embodiment, the one or more epitopes is associated with Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, or Plasmodium knowlesi. In a further embodiment, the antigens comprise more than one epitope associated with the same Plasmodium infection. In yet a further embodiment, the antigens comprise more than one epitope associated with different Plasmodium infections. In a further embodiment, the antigens comprise one or more epitopes associated with one or more Plasmodium infections.

[00131] Particular embodiments of the present invention contemplate that treatment with negatively charged particles are effective at treating malaria. Effective treatment of malaria includes, but is not limited to, increasing probability of survival, and/or a decreasing the severity or incidence of respiratory distress, metabolic acidosis, acute encephalitis/meningitis syndrome, and liver malfunction or failure. Effective treatment may also include clearing the parasite from the subject, and/or reducing the number of parasites in the subject.

[00132] In particular embodiments, a pharmaceutical composition is administered to a subject for the treatment of malaria, wherein the pharmaceutical composition contains negatively charged particles free from attached bioactive agents and a pharmaceutically acceptable carrier. In particular embodiments, the pharmaceutical composition treats at least one symptom in the subject. In some embodiments, the pharmaceutical composition is administered to the subject to increase the probability of survival. In certain embodiments, the pharmaceutical composition is administered to the subject to decrease the severity or incidence of respiratory distress. In particular embodiments, the pharmaceutical composition is administered to the subject to decrease the severity or incidence of metabolic acidosis. In some embodiments, the pharmaceutical composition is administered to the subject to decrease the severity or incidence of acute encephalitis/meningitis syndrome. In some embodiments, the pharmaceutical composition is administered to the subject to decrease the severity or incidence of liver malfunction or failure. In particular embodiments, the pharmaceutical composition comprising particles and a separate antimalarial is administered to the subject to clear the parasite from the subject, and/or reduce the number of parasites in the subject.

[00133] Monocytes and macrophages play central roles in the initiation and resolution of inflammation, principally through phagocytosis, the release of inflammatory cytokines, reactive oxygen species and the activation of the acquired immune system (Auffray et al, 2009 Annu

Rev Immunol 27:669-692). Typically, monocytes circulate in the bloodstream for a very short time before undergoing apoptosis, however, stimulatory signals can trigger monocyte survival by inhibiting the apoptotic pathway, and thus contribute to the maintenance of the inflammatory response. Anti-apoptotic proteins work by inhibiting caspases or the activation of the apoptotic program. Phosphatidyl inositol 3 -kinase (PI-3K)/Akt, ERK, Fas, TNF, heat shock proteins and anti-apoptotic molecules, among others, play key roles in determining monocyte life span. During the inflammatory response, inflammatory cells, such as monocytes and macrophages, are recruited to the sites of inflammation. This recruitment is essential for effective control and clearance of infection, but recruited monocytes also contribute to the pathogenesis of inflammatory and degenerative diseases. The accumulation of monocytes can be harmful and aggravate disease such as atherosclerosis, arthritis, and multiple sclerosis. Resolution of inflammation requires the reduction and/or inhibition of inflammatory cells to the inflammatory foci, and apoptosis of the inflammatory cells already present. Apoptotic caspases play a fundamental role by proteolytically dismantling cells by degrading proteins with diverse biological functions. For instance, caspase-3 activation is essential for CD14⁺ monocyte apoptosis (Fahy et al., 1999 J. Immunol. 163: 1755-1762).

[00134] In one aspect, the methods of the current invention include inducing apoptosis in monocytes, granulocytes and/or neutrophils in a subject to reduce the severity or duration of an inflammatory response. In one embodiment, administering the negatively charged particles of the invention induces monocyte, granulocyte and/or neutrophil apoptosis and clearance, thereby aiding in the resolution of inflammation.

[00135] Certain embodiments are directed to administering a pharmaceutical composition to a subject with Plasmodium infection, the pharmaceutical composition comprising negatively charged particles and a carrier, wherein the negatively charged particles are free from attached peptide moieties, antigenic moieties, and/or bioactive agents. In some embodiments, the pharmaceutical composition is administered to treat or inhibit Plasmodium infection. In certain embodiments, the pharmaceutical composition is administered to remove pro-inflammatory mediators from the inflammatory milieu in a subject with Plasmodium infection. In various embodiments, the pharmaceutical composition is administered to a subject to induce regulatory T cells in a subject with a Plasmodium infection. In some embodiments, the pharmaceutical composition is administered to control a pathologic and/or unwanted inflammatory immune response in a subject with a Plasmodium infection. Particular aspects of the present invention contemplate that negatively charged particles described herein are sufficient to treat or inhibit infection, remove pro-inflammatory mediators from the inflammatory milieu, induce regulatory T cells , and/or control a pathologic and/or an unwanted inflammatory response in a subject with a Plasmodium infection, without attached or added bioactive agents, peptide moieties, and/or antigenic moieties. [00136] Particular embodiments are directed to administering to a subject a pharmaceutical composition to a subject with Plasmodium infection, the pharmaceutical composition comprising carboxylated PLGA particles and a carrier, wherein the particles are free from attached peptide moieties, antigenic moieties, and bioactive agents, and wherein the carboxylated PLGA particles have a negative zeta potential. Particular aspects of the present invention contemplate that carboxylated PLGA particles with negative zeta potential are sufficient to treat or inhibit infection, remove pro-inflammatory mediators from the inflammatory milieu, induce regulatory T cells, and/or control a pathologic and/or an unwanted inflammatory response in a subject Wit Plasmodium infection, without attached or additional bioactive agents, peptide moieties, and/or antigenic moieties. Such embodiments contemplate that the carboxylated PLGA particles with negative zeta potential are themselves the bioactive agent.

[00137] Particular embodiments of the present invention contemplate that treatment with negatively charged particles and an antimalarial are more effective at treating malaria than treatment with either the particles or the antimalarial alone. More effective treatment may be manifested by an increased probability of survival and reduced mortality, and/or a decrease in the severity or incidence of a respiratory distress, metabolic acidosis, acute encephalitis/meningitis syndrome, and liver malfunction or failure. More effective treatment may also include clearing the parasite from the subject, and/or reducing the number of parasites in the subject. Certain embodiments contemplate that the negatively charged particles and the antimalarial are administered separately as part of the same treatment regimen, or are administered together in the same pharmaceutical composition. The pharmaceutical compositions may contain negatively charged particles that are free from attached bioactive agents and the antimalarial, or the composition may contain negatively charged particles that are coupled to the particles.

[00138] Some embodiments are directed to methods of treating a subject with malaria by administering to the subject a pharmaceutical composition that contains negatively charged particles and an antimalarial. In certain embodiments, the pharmaceutical composition comprises negatively charged particles that are free from attached bioactive agents and an antimalarial, wherein the antimalarial and the particles are separate from each other. In some embodiments, the negatively charged particles are coupled to the antimalarial. In some embodiments, the pharmaceutical composition comprises an antimalarial selected from Quinine and related agents, Chloroquine, Amodiaquine, Pyrimethamine, Proguanil, Sulfonamides, Mefloquine, Atovaquone, Primaquine, Artemisinin and its derivatives, Halofantrine, Doxycycline, Clindamycin, or combinations thereof.

[00139] In some embodiments, an antimalarial is administered to a subject having, suspected of having, or at risk of having malaria, after the subject has been treated with any of the negatively charged particles described herein. In particular embodiments, an antimalarial is administered to the subject while the subject is treated with any of the negatively charged particles described herein. In certain embodiments, the antimalarial is administered to the subject before the subject is treated with any of the negatively charged particles described herein. In particular embodiments, subjects treated with both negatively charged particles and an antimalarial, either simultaneously or as separate treatments, are not only treated efficiently but also develop immunity to malaria. In particular embodiments, subjects treated with both negatively charged carboxylated PLGA particles and an antimalarial, e.g. Chloroquine, Quinine, or artesunate, develop immunity to malaria. In certain embodiments, subjects treated with both negatively charged carboxylated polystyrene particles and an antimalarial, e.g. Chloroquine, Quinine, or artesunate, develop immunity to malaria. In some embodiments, subjects treated with both negatively charged carboxylated citric acid particles and an antimalarial, e.g. Chloroquine, Quinine, or artesunate, develop immunity to malaria. In certain embodiments, immunity to malaria refers to a reduced probability of developing malaria, developing at least one symptom associated with malaria, and/or an increased probability of survival following exposure to parasite associated with malaria, e.g. Plasmodium falciparum.

[00140] In a particular embodiment, a subject having, suspected of having, or at risk of having malaria is treated with negatively charged carboxylated PLGA particles with a zeta potential of -80 mV to -30 mV and a diameter of 0.3 μηι to 5 μηι and is also treated with an antimalarial either before, during, or after the treatment with the PLGA particles, which results in effective treatment of the malaria and/or results in the subject having immunity to malaria. In particular embodiments, the negatively charged carboxylated PLGA particles have a zeta potential of -80 mV to -30 mV and a diameter of 0.5 μηι to 1 μιη. In some embodiments, the antimalarial is artesunate. In particular embodiments, the antimalarial is Chloroquine.

[00141] In a certain embodiment, a subject having, suspected of having, or at risk of having malaria is treated with negatively charged carboxylated PLGA particles with a zeta potential of -75 mV to -25 mV and a diameter of 0.3 μιη to 5 μηι and is also treated with an antimalarial either before, during, or after the treatment with the PLGA particles, which results in effective treatment of the malaria and/or results in the subject having immunity to malaria. In particular embodiments, the negatively charged carboxylated PLGA particles have a zeta potential of -75 mV to -25 mV and a diameter of 0.5 μηι to 1 μιη. In some embodiments, the antimalarial is artesunate. In particular embodiments, the antimalarial is Chloroquine.

[00142] In a certain embodiment, a subject having, suspected of having, or at risk of having malaria is treated with negatively charged carboxylated polystyrene or citric acid particles with a zeta potential of -80 mV to -30 mV and a diameter of 0.3 μηι to 5 μηι and is also treated with an antimalarial either before, during, or after the treatment with the polystyrene or citric acid particles, which results in effective treatment of the malaria and/or results in the subject having immunity to malaria. In particular embodiments, the negatively charged carboxylated polystyrene or citric acid particles have a zeta potential of -80 mV to -30 mV and a diameter of 0.5 μηι to 1 μιη. In some embodiments, the antimalarial is artesunate. In particular embodiments, the antimalarial is Chloroquine.

[00143] In a certain embodiment, a subject having, suspected of having, or at risk of having malaria is treated with negatively charged carboxylated polystyrene or citric acid particles with a zeta potential of -75 mV to -25 mV and a diameter of 0.3 μηι to 5 μηι and is also treated with an antimalarial either before, during, or after the treatment with the polystyrene or citric acid particles, which results in effective treatment of the malaria and/or results in the subject having immunity to malaria. In particular embodiments, the negatively charged carboxylated polystyrene or citric acid particles have a zeta potential of -75 mV to -25 mV and a diameter of 0.5 μηι to 1 μιη. In some embodiments, the antimalarial is artesunate. In particular embodiments, the antimalarial is Chloroquine.

[00144] In particular embodiments, an antimalarial is administered to a subject having, suspected of having, or at risk of having malaria, before, during, and/or after the subject has been treated with any of the negatively charged particles described herein. In particular embodiments, subjects treated with both negatively charged particles and an antimalarial, either simultaneously or as separate treatments, have an enhanced probability of survival as compared to untreated subjects and/or as compared to subjects that are treated with particles or an antimalarial alone. In particular embodiments, subjects treated with both negatively charged carboxylated PLGA particles and an antimalarial, e.g. Chloroquine, Quinine, or artesunate, have an increased probability of surviving malaria compared to untreated subjects and/or compared to subjects that are treated with PLGA particles or the antimalarial alone. In certain embodiments, subjects treated with both negatively charged carboxylated polystyrene particles and an antimalarial, e.g. Chloroquine, Quinine, or artesunate, have an increased probability of surviving malaria compared to untreated subjects and/or compared to subjects that are treated with polystyrene particles or the antimalarial alone. In some embodiments, subjects treated with both negatively charged carboxylated citric acid particles and an antimalarial, e.g. Chloroquine, Quinine, or artesunate, have an increased probability of surviving malaria compared to untreated subjects and/or compared to subjects that are treated with citric acid particles or the antimalarial alone. In particular embodiments, the combined treatment of the negatively charged particles and the antimalarial results in a synergistic increase on the probability of survival, i.e. an increased probability of survival that is greater than what would have been expected based on the probability of survival resulting from either treatment alone. In certain embodiments, the antimalarial and the negatively charged particles are administered to the subject in synergistically effective amounts.

[00145] In particular embodiments, a pharmaceutical composition is administered to a subject for the treatment of malaria, wherein the pharmaceutical composition contains negatively charged particles free from attached bioactive agents and an antimalarial which are separate from each other. In particular embodiments, the pharmaceutical composition treats at least one symptom in the subject. In certain embodiments, the pharmaceutical composition is more effective at treating the at least one symptom than the antimalarial alone. In particular embodiments, the pharmaceutical composition is more effective at treating the at least one symptom than the particles alone. In some embodiments, the pharmaceutical composition comprising particles and a separate antimalarial is administered to the subject to increase survival. In certain embodiments, the pharmaceutical composition comprising particles and a separate antimalarial is administered to the subject to decrease the severity or incidence of respiratory distress. In particular embodiments, the pharmaceutical composition comprising particles and a separate antimalarial is administered to the subject to decrease the severity or incidence of metabolic acidosis. In some embodiments, the pharmaceutical composition comprising particles and a separate antimalarial is administered to the subject to decrease the severity or incidence of acute encephalitis/meningitis syndrome. In some embodiments, the pharmaceutical composition comprising particles and a separate antimalarial is administered to the subject to decrease the severity or incidence of liver malfunction or failure. In particular embodiments, the pharmaceutical composition comprising particles and a separate antimalarial is administered to the subject to clear the parasite from the subject, and/or reduce the number of parasites in the subject.

[00146] In particular embodiments, a pharmaceutical composition is administered to a subject for the treatment of malaria, wherein the pharmaceutical composition contains negatively charged particles coupled to an antimalarial. In particular embodiments, the pharmaceutical composition treats at least one symptom in the subject. In certain embodiments, the particles coupled to an antimalarial are more effective at treating the at least one symptom than the antimalarial alone. In particular embodiments, the particles coupled to an antimalarial are more effective at treating the at least one symptom than the particles alone. In some embodiments, the pharmaceutical composition comprising particles coupled to an antimalarial is administered to the subject to increase survival. In certain embodiments, the pharmaceutical composition particles coupled to an antimalarial is administered to the subject to decrease the severity or incidence of respiratory distress. In particular embodiments, the pharmaceutical composition comprising particles coupled to an antimalarial is administered to the subject to decrease the severity or incidence of metabolic acidosis. In some embodiments, the pharmaceutical composition comprising particles coupled to an antimalarial is administered to the subject to decrease the severity or incidence of acute encephalitis/meningitis syndrome. In some embodiments, the pharmaceutical composition comprising particles coupled to an antimalarial is administered to the subject to decrease the severity or incidence of liver malfunction or failure. In particular embodiments, the pharmaceutical composition comprising particles coupled to an antimalarial is administered to the subject to clear the parasite from the subject, and/or reduce the number of parasites in the subject.

[00147] Particular aspects contemplate that administration of negatively charged particles and a malaria vaccine to a subject will improve the subject's response to the vaccine. An improved response may be manifested by increased immunity against malaria by the subject, longer lasting immunity to malaria by the subject, and/or reduced negative side effects associated with vaccines. Certain embodiments contemplate that the vaccine and the particles are administered in the same pharmaceutical compositions. Some embodiments contemplate that administering negatively charged particles with the vaccine will improve the subject's response to the vaccine if the particles are coupled to the vaccine, e.g. the vaccine is imbedded within the particles, or if the vaccine is physically separate from the negatively charged particles. In certain embodiments, the vaccine is coupled to the particles. In some embodiments, the vaccine is embedded within the particles. In particular embodiments, the vaccine is surface-coupled to the particles.

[00148] Certain aspects of the present invention are directed to methods of vaccinating a subject by administering to the subject a pharmaceutical composition that contains negatively charged particles and a malaria vaccine or components thereof. In certain embodiments, the pharmaceutical composition comprises negatively charged particles that are free from attached bioactive agents and a malaria vaccine, wherein the malaria vaccine and the particles are separate from each other. In some embodiments, the negatively charged particles are coupled to the vaccine or components thereof. In some embodiments, the vaccine is surface-coupled to the particles. In certain embodiments, the vaccine is embedded in the particles. In some embodiments, the vaccine is monovalent. In certain embodiments, the vaccine is divalent or polyvalent. In particular embodiments, the malaria vaccine or components thereof comprise one or more epitopes associated with malaria or with a parasite that causes malaria, e.g. one or more Plasmodium species.

[00149] In some embodiments, a subject is administered with a pharmaceutical composition that contains negatively charged particles free from attached bioactive agents and a malaria vaccine that contains one or more epitopes associated with malaria, wherein the particles and the vaccine are separate from each other, that results in the subject's immunity to Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and/or Plasmodium knowlesi. In particular embodiments, a subject is administered with a pharmaceutical composition that contains negatively charged particles free from bioactive agents and a vaccine that contains one or more epitopes associated with malaria that results in production of antibodies by the subject against Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and/or Plasmodium knowlesi. In certain embodiments, the malaria vaccine is embedded in the particles.

[00150] In some embodiments, a pharmaceutical composition comprising negatively charged particles coupled to a malaria vaccine comprises an additional bioactive agent. In certain embodiments, the additional bioactive agent is separate from the vaccine-coupled negatively charged particles. In a particular embodiment, the additional bioactive agent is coupled to the negatively charged particles. In some embodiments, the additional bioactive agent is surface-coupled to the negatively charged particles. In certain embodiments, the additional bioactive agent is embedded in the negatively charged particles. In a certain embodiment, the additional bioactive agent is a small molecule inhibitor of the Target of Rapamycin Complex I (TORCI) and or the Target of Rapamycin Complex II (TORCH). In some embodiments, the additional bioactive agent is rapamycin or a derivative thereof. Examples of rapamycin derivatives (rapalogs) include, but are not limited to, RAD001, everolimus, CCI-779, temsiroliumus, AP23573, deforolimus, and ridaforolimus,

[00151] In particular embodiments, a subject is administered with a pharmaceutical composition that contains negatively charged particles coupled to one or more epitopes associated with malaria that results in the subject's immunity to Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and/or Plasmodium knowlesi. In some embodiments, a subject is administered with a pharmaceutical composition that contains negatively charged particles coupled to one or more epitopes associated with malaria that results in production of antibodies by the subject against Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and/ 'or Plasmodium knowlesi.

[00152] In one aspect, the methods of the current invention contemplate using the particles of the invention as "molecular sinks" that bind to inflammatory molecules and polypeptides produced by the cell, thereby preventing them from exerting their activity. When inflammation happens, pro-inflammatory mediators such as cytokines and chemokines are released by cells, such as macrophages and monocytes, into the surrounding pro- inflammatory milieu. Examples of pro-inflammatory mediators include, but are not limited to interleukins, members of the TNF family, interferons, and colony stimulating factors. These mediators potentiate the inflammatory response, thereby exacerbating the inflammatory pathology. As described herein, the particles of the invention bind to inflammatory mediators in the serum of animals experiencing an inflammatory immune response. The inflammatory mediators to which the particles of the invention bind include, but are not limited to, heat shock protein beta -1, protein S100-A7, protein S100-A8, protein S100-A9, fatty acid-binding protein, annexin Al and ubiquitin cross- reactive protein precursor. Administration of uncoated particles of the invention to animals results in a decrease of inflammatory monocytes present in the inflammatory foci, a decrease in inflammatory symptoms, and an increase in survival of infected animals.

[00153] In another aspect, the methods of the current invention contemplate using particles to bind to DNA and/or RNA.

[00154] In another aspect, the methods of the current invention contemplate using the particles as "molecular sinks" that bind virions, viral proteins, viral RNA and/or DNA. [00155] As discussed above, this invention provides novel compounds that have biological properties useful for the treatment of immune mediated disorders.

[00156] Accordingly, in another aspect of the present invention, pharmaceutical compositions are provided, which comprise the particles and optionally comprise a pharmaceutically acceptable carrier. In certain embodiments, these compositions optionally further comprise one or more additional therapeutic agents. The particles of the current invention may be administered to a patient in need thereof in combination with the administration of one or more other therapeutic agents. For example, additional therapeutic agents for conjoint administration or inclusion in a pharmaceutical composition with a compound of this invention may be an approved anti-inflammatory agent, or it may be any one of a number of agents undergoing approval in the Food and Drug Administration that ultimately obtain approval for the treatment of any disorder characterized by an uncontrolled inflammatory immune response or a bacterial or viral infection. It will also be appreciated that certain of the modified particles of present invention can exist in free form for treatment, or where appropriate, as a pharmaceutically acceptable derivative thereof.

[00157] The pharmaceutical compositions of the present invention additionally comprise a pharmaceutically acceptable carrier, which, as used herein, includes any and all solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's Pharmaceutical Sciences, Sixteenth

Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof.

Except insofar as any conventional carrier medium is incompatible with the compounds of the invention, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this invention. Some examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatine; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil, sesame oil; olive oil; corn oil and soybean oil; glycols; such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogenfree water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.

[00158] Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[00159] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S. P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

[00160] The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[00161] In order to prolong the effect of a drug, it is often desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension or crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution that, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include (poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues. In certain embodiments, drugs and therapeutics may be encapsulated in the particles of the invention for administration to the subject.

[00162] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the modified particles are mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.

[00163] Solid compositions of a similar type may also be employed as fillers in soft and hard- filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner.

Examples of embedding compositions that can be used include polymeric substances and waxes. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

[00164] The modified particles can also be in micro-encapsulated form with one or more excipients as noted above. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms the active compound may be admixed with at least one inert diluent such as sucrose, lactose and starch. Such dosage forms may also comprise, as in normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. They may optionally contain opacifying agents and can also be of a composition that they release the modified particles only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes.

[00165] The present invention encompasses pharmaceutically acceptable topical formulations of the inventive modified particles. The term "pharmaceutically acceptable topical formulation", as used herein, means any formulation which is pharmaceutically acceptable for intradermal administration of modified particles of the invention by application of the formulation to the epidermis. In certain embodiments of the invention, the topical formulation comprises a carrier system. Pharmaceutically effective carriers include, but are not limited to, solvents (e.g., alcohols, poly alcohols, water), creams, lotions, ointments, oils, plasters, liposomes, powders, emulsions, microemulsions, and buffered solutions (e.g., hypotonic or buffered saline) or any other carrier known in the art for topically administering pharmaceuticals. A more complete listing of art-known carriers is provided by reference texts that are standard in the art, for example, Remington's Pharmaceutical Sciences, 16th Edition, 1980 and 17th Edition, 1985, both published by Mack Publishing Company, Easton, Pa., the disclosures of which are incorporated herein by reference in their entireties. In certain other embodiments, the topical formulations of the invention may comprise excipients. Any pharmaceutically acceptable excipient known in the art may be used to prepare the inventive pharmaceutically acceptable topical formulations.

Examples of excipients that can be included in the topical formulations of the invention include, but are not limited to, preservatives, antioxidants, moisturizers, emollients, buffering agents, solubilizing agents, other penetration agents, skin protectants, surfactants, and propellants, and/or additional therapeutic agents used in combination to the modified particles. Suitable preservatives include, but are not limited to, alcohols, quaternary amines, organic acids, parabens, and phenols. Suitable antioxidants include, but are not limited to, ascorbic acid and its esters, sodium bisulfite, butylated hydroxytoluene, butylated hydroxyanisole, tocopherols, and chelating agents like EDTA and citric acid. Suitable moisturizers include, but are not limited to, glycerine, sorbitol, polyethylene glycols, urea, and propylene glycol. Suitable buffering agents for use with the invention include, but are not limited to, citric, hydrochloric, and lactic acid buffers. Suitable solubilizing agents include, but are not limited to, quaternary ammonium chlorides, cyclodextrins, benzyl benzoate, lecithin, and polysorbates. Suitable skin protectants that can be used in the topical formulations of the invention include, but are not limited to, vitamin E oil, allatoin, dimethicone, glycerin, petrolatum, and zinc oxide.

[00166] In certain embodiments, the pharmaceutically acceptable topical formulations of the invention comprise at least the modified particles of the invention and a penetration enhancing agent. The choice of topical formulation will depend or several factors, including the condition to be treated, the physicochemical characteristics of the inventive compound and other excipients present, their stability in the formulation, available manufacturing equipment, and costs constraints. As used herein the term "penetration enhancing agent" means an agent capable of transporting a pharmacologically active compound through the stratum corneum and into the epidermis or dermis, preferably, with little or no systemic absorption. A wide variety of compounds have been evaluated as to their effectiveness in enhancing the rate of penetration of drugs through the skin. See, for example, Percutaneous Penetration Enhancers, Maibach H. I. and Smith H. E. (eds.), CRC Press, Inc., Boca Raton, Fla. (1995), which surveys the use and testing of various skin penetration enhancers, and Buyuktimkin et al, Chemical Means of Transdermal Drug Permeation Enhancement in Transdermal and Topical Drug Delivery Systems, Gosh T. K., Pfister W. R, Yum S. I. (Eds.), Interpharm Press Inc., Buffalo Grove, 111. (1997). In certain exemplary embodiments, penetration agents for use with the invention include, but are not limited to, triglycerides (e.g., soybean oil), aloe compositions (e.g., aloe-vera gel), ethyl alcohol, isopropyl alcohol, octolyphenylpolyethylene glycol, oleic acid, polyethylene glycol 400, propylene glycol, N-decylmethylsulfoxide, fatty acid esters (e.g., isopropyl myristate, methyl laurate, glycerol monooleate, and propylene glycol monooleate) and N- methylpyrrolidone.

[00167] In certain embodiments, the compositions may be in the form of ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. In certain exemplary embodiments, formulations of the compositions according to the invention are creams, which may further contain saturated or unsaturated fatty acids such as stearic acid, palmitic acid, oleic acid, palmito-oleic acid, cetyl or oleyl alcohols, stearic acid being particularly preferred. Creams of the invention may also contain a non-ionic surfactant, for example, polyoxy-40-stearate. In certain embodiments, the active component is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required. Ophthalmic formulation, eardrops, and eye drops are also contemplated as being within the scope of this invention. Additionally, the present invention contemplates the use of transdermal patches, which have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms are made by dissolving or dispensing the compound in the proper medium. As discussed above, penetration enhancing agents can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.

[00168] The modified particles can be administered by aerosol. This is accomplished by preparing an aqueous aerosol, liposomal preparation or solid particles containing the modified particles. A nonaqueous (e.g., fluorocarbon propellant) suspension could be used.

[00169] Ordinarily, an aqueous aerosol is made by formulating an aqueous solution or suspension of the agent together with conventional pharmaceutically acceptable carriers and stabilizers. The carriers and stabilizers vary with the requirements of the particular compound, but typically include nonionic surfactants (Tweens, Pluronics®, or polyethylene glycol), innocuous proteins like serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars or sugar alcohols. Aerosols generally are prepared from isotonic solutions.

[00170] It will also be appreciated that the modified particles and pharmaceutical compositions of the present invention can be formulated and employed in combination therapies, that is, the compounds and pharmaceutical compositions can be formulated with or administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures for the treatment of malaria. Other treatments for malaria, i.e. antimalarial medications or antimalarials, include but are not limited to, Quinine and related agents, Chloroquine, Amodiaquine, Pyrimethamine, Proguanil, Sulfonamides, Mefloquine, Atovaquone, Primaquine, Artemisinin and its derivatives, Halofantrine, Doxycycline, Clindamycin, or combinations thereof. The particular combination of therapies (therapeutics or procedures) to employ in a combination regimen will take into account compatibility of the desired therapeutics and/or procedures and the desired therapeutic effect to be achieved. It will also be appreciated that the therapies employed may achieve a desired effect for the same disorder (for example, an inventive compound may be administered concurrently with another anti-inflammatory agent), or they may achieve different effects (e.g., control of any adverse effects).

[00171] In certain embodiments, the pharmaceutical compositions containing the modified particles of the present invention further comprise one or more additional therapeutically active ingredients (e.g., anti-inflammatory and/or palliative). For purposes of the invention, the term "Palliative" refers to treatment that is focused on the relief of symptoms of a disease and/or side effects of a therapeutic regimen, but is not curative. For example, palliative treatment encompasses painkillers, antinausea medications and anti-sickness drugs.

[00172] The modified particles are administered in an amount sufficient to regulate an immune response. As described herein, regulation of an immune response may be humoral and/or cellular, and is measured using standard techniques in the art and as described herein.

[00173] Subjects treated by the particles of the present invention are preferably human, however, the particles are useful in treating non-human animal species. Non-human animal species which may be treated by the particles of the present invention include, but are not limited to, dogs, cats, chickens, geese, ducks, sheep, cows, goats, pigs, non-human primates, monkey, rabbits, mice, rats, guinea pigs, hamsters, gerbils, and horses.

[00174] Animal models for the study of the pathology of malaria infections are known in the art. For example, mice, and non-human primates have been used to study the pathogenesis of malaria or to test vaccines.

[00175] In some embodiments, the invention relates to uses of compositions of this invention prior to the onset of disease. In other embodiments, the invention relates to uses of the compositions of this invention to inhibit ongoing disease. In some embodiments, the invention relates to ameliorating disease in a subject. By ameliorating disease in a subject is meant to include treating, preventing or suppressing the disease in the subject. [00176] In some embodiments, the invention relates to preventing the relapse of disease. For example, an unwanted immune response can occur at one region of a peptide (such as an antigenic determinant). Relapse of a disease associated with an unwanted immune response can occur by having an immune response attack at a different region of the peptide. Since the negatively charged particles of the current invention are free from attached peptides or antigenic moieties, the particles will be effective against multiple epitopes.

EXAMPLES MATERIALS AND METHODS

Plasmodium infection

[00177] Mice were infected with the rodent-infective plasmodium species P. berghei species of the P. berghei ANKA (PbA) strain. The PbA strain was maintained and used as previously reported (see de Oca et ah, (2013) Methods Mol. Biol. 1031 :203-13; hereby incorporated by reference in its entirety). Unless otherwise stated, All PbA-infected mice were infected with a single intraperitoneal (i.p.) injection with 10⁶ parasitized red blood cells (pRBC). The day the PbA infection was performed was considered to be day 0.

[00178] More specifically, animals were older than 6-weeks-of-age females, that were given food and water ad libitum. Severe Malaria was induced in mice via intraperitoneal injection of diluted erythrocytes (200 μΐ) containing the malaria parasite Plasmodium berghei ANKA. The 200-μ1 aliquot of blood contained a PRBC count of approximately 1 x 10⁶.

Plasmodium Re-Challenge

[00179] Immunity was tested in infected animals that had cleared the primary inoculation

(as determined by blood smear). Animals were re-inoculated via intraperitoneal injection with diluted erythrocytes (200 μΐ) containing the malaria parasite Plasmodium berghei ANKA. The 200-μ1 aliquot of blood contained a PRBC count of approximately 1 x lO⁶ This was conducted on day 28 after initial infection, which is approximately 20 days after the completion of treatment regimens of charged particles and/or antimalarial were completed.

Polystyrene Particles

[00180] Negatively charged carboxylated polystyrene particles were custom made by Bangs laboratories (Fishers, Indiana). [00181] For treatment with polystyrene particles, mice were injected with approximately

4.4 xlO⁹ of negatively charged carboxylated polystyrene particles in 300 μΐ PBS i.v. Untreated control mice were injected with a 300 μ 1 PBS vehicle, i.v.

PLGA Particles

[00182] Particles were prepared as described in PCT Application Pub. NO. WO 2014/089160, hereby incorporated by reference in its entirety. Briefly, PLGA (Lactel B6013, inherent viscosity 0.15-0.25 dL/g) was dissolved in 10 ml methylene chloride. The PLGA solution was mixed with 100 ml 1% polyvinyl alcohol solution and homogenized at 18,400 rpm for 45 seconds with an IKA T25_digital_ULTRA-TURRAX Homogenizer. The resulting emulsion was poured to a glass container and stirred magnetically at 400 rpm for 4 hours to allow the evaporation of the solvent. The nanoparticles were washed three times with distilled water before they were lyophilized. Particle size and zeta potential were determined with a Beckman-Coulter LS320 Laser Diffraction Particle Size Analyzer. The average particle size was found to be 680 nm.

Preparation of Carboxylated PLGA Nanoparticles via Precipitation Process with a Short- Chain PLGA Polymer

[00183] 0.42 g PLGA polymer (Lakeshore DLG 5050 1A, inherent viscosity 0.05-0.15 dL/g) was dissolved in 10 ml acetone. This PLGA/acetone solution was added using a syringe pump at an addition rate of about 25 mL/hour to 60 mL 1 mM NaOH solution. The resulting nanoparticle suspension was mixed with 1 liter of distilled water and concentrated to approximately 20 mL with a tangential flow filtration device and a 500 kDa molecular weight cut-off module. The concentrated nanoparticle suspension was lyophilized. Particle size and zeta potential were determined with a Malvern particle size analyzer (Worcestershire, UK). The average particle size was found to be 230.4 nm and zeta potential was -31.1 mV.

Preparation of Carboxylated PLGA Nanoparticles via Emulsification Process with a Short- Chain PLGA Polymer Containing Two Terminal Carboxyl Groups

[00184] PLGA (Lactel B6013, initiated by gly colic acid) containing terminal COOH groups on both chain ends was dissolved in 10 ml methylene chloride. The PLGA solution was mixed with 150 ml 1% polyvinyl alcohol in 1 mM NaOH solution and homogenized at 18,000 rpm for 60 seconds on a NISSEN Homogenizer. The resulting emulsion was poured to a glass container and stirred magnetically at 500 rpm for 4 hours to allow the evaporation of the solvent. The nanoparticles were washed three times with distilled water before lyophilization.

[00185] Zeta potential of resulting particles ranged from -25mv to -45mv depending on batch. Z average size was 400-1200nm.

Treatment with PLGA Particles

[00186] For treatment with PLGA particles, mice were injected with approximately

4.4* 10⁹ (approx. 0.3 to 2.5mg of polymer) of negatively charged carboxylated PLGA particles in 300 μΐ PBS i.v. Untreated control mice were injected with a 200-300 μΐ PBS vehicle, i.v.

Antimalarials

[00187] Antimalarials were obtained commercially from Sigma Aldrich (St. Louis, MO) and were administered as summarized in Table El .

Table El: Antimalarials

Clinical Score

[00188] A five point score from 0 to 4 was used to score the severity of symptoms relating to the PbA infection. Higher numbers reflected the presence of more severe symptoms. Symptoms were evaluated as shown in Table E2. Table E2: Clinical Evaluation Scores

[00189] Experiments examining survival were performed according to procedures approved by the local animal ethics committee where the experiments took place. In these experiments, the point at which euthanasia is used as an endpoint is determined based on time, combined with clinical score. If mice remain at a score of 3 or greater, with continued treatment, over a period of several days (i.e. 2-3 days), with no improvement, then these mice are euthanized. A typical time-course consists of mice developing cerebral malaria on day 7-9 post infection, and unsuccessfully treated mice being euthanized on day 10, at which point it could be clearly determined that the treatment would not succeed in rescuing them from the syndrome.

Parasitaemia

[00190] The percentage of parasitized red blood cells was used as a measurement for parasitaemia. A thin blood smear was prepared on a glass slide using a drop of blood taken from the tail of the mouse. The smear was then stained using the Geimsa protocol, with eosin and azure B (ProSciTech, ARS 1-500 and ARS 1 1-500) after fixation in methanol. This slide was then enumerated under the microscope to count the number of parasitised vs. normal red blood cells and expressed as a percentage of the number of parasitised cells divided by number of total red blood cells.

EXAMPLE 1

TREATMENT WITH NEGATIVELY CHARGED POLYSTYRENE PARTICLES IN A MOUSE MODEL OF CEREBRAL MALARIA. [00191] Various mouse models have been used to model lethal malaria, usually involving infection of inbred mouse strains with rodent-infective Plasmodium species such as P. chabaudi, P. yoelii, P. vinckei, and P. berghei. Within the P. berghei species there are various strains that have been used experimentally, most commonly used is P. berghei ANKA (PbA). The most severe symptoms of this disease in mice appear to be neurologically related. In addition to brain pathology, it has become increasingly clear in recent years that PbA infection causes widespread pathology in other organs. Various reports provide convincing evidence of acute lung pathology and associated respiratory distress in PbA infection in various inbred mouse strains. Metabolic acidosis is a major feature of severe malaria in humans, and this too is observed in PbA-infected mice. Liver damage is also a feature of severe malaria in humans, which can be reversed by clearance of the parasite. This observation has been mirrored in PbA infection of C57BL/6J mice.

[00192] Thirteen week-old C57BL/6 mice were split into three experimental groups: non- infected controls (n=7), PbA-infected (n=7), and PbA-infected coupled with polystyrene IMP treatment (n=7). Parasitemia was determined by thin blood smears prepared from tail bleeds. Smears were stained using the Diff-Quick kit (Lab Aids, Narrabeen, NSW, Australia).

[00193] Cerebral malaria was modeled with the PbA-infected mouse model which has been previously described (see Pai et al, PLOS Pathogens: 10(7) el 004236; hereby incorporated by reference in its entirety). PbA infection occurred at Day 0. On day 3, the PbA-infected mice were injected with either approximately 4.4 xlO⁹ (approximately 0.3-2.5 mg) of negatively charged carboxylated polystyrene particles in 300 μΐ PBS or PBS alone, i.v. These injections continued once daily from day 3 to day 10. At the time of the occurrence of clinical signs, 3 mice from each group were sacrificed for flow cytometry and histology analysis, and clinical score and survival were assessed in the remaining 4 mice per group.

[00194] Clinical score was assessed in PbA infected mice with and without negatively charged carboxylated polystyrene particle treatment. As shown in FIG. 3, injection of negatively charged carboxylated polystyrene particles reduced the severity of the PbA-infection. Treatment with negatively changed carboxylated polystyrene particles significantly reduced the clinical score of PbA-infected mice (FIG. 3A). Furthermore, treatment with negatively changed carboxylated polystyrene particles resulted with 75% increase in survival (FIG. 3B). The experiment ceased on day 27 post infection. This reflects a >200% increase in survival time. [00195] As noted above, 3 mice per group were sacrificed at the onset of symptoms. The mice were slowly perfused, and tissue from brain, spleen, bone marrow, and cervical lymph nodes were taken for analysis with flow cytometry. In addition, tissue from brain, lung, and spleen were collected for histological analysis.

[00196] Flow cytometry was performed as illustrated in FIG. 4. Inflammatory monocytes and macrophages and T cells populations were examined in the central nervous systems (CNS) of uninfected controls, PbA-infected mice, and PbA-infected mice treated with negatively charged polystyrene particles. The total amounts of CD45+ cells and cells with high levels of Ly6C expression were significantly increased in CNS tissues of PbA-infected mice as compared to CNS obtained from uninfected mice, while no significant differences were observed between CNS tissues of non-infected mice and PbA-infected mice treated with negatively charged carboxylated polystyrene particles (FIG. 6A and B). Specific populations of monocytes and macrophages and T cells present in CNS tissues were examined. PbA infection significantly increased levels of CNS CD45+ monocytes and macrophages that express Ly6Chi as compared to uninfected controls, and a trend of increased amounts Ly6C-int cells, Ly6C-microglia, and neutrophils in CNS tissue obtained from PbA-infected mice as compared to uninfected mice was observed. These increases were not observed in PbA-infected CNS tissues harvested from mice treated with negatively charged carboxylated polystyrene particles (FIG. 5A). Similarly, a trend of increased amounts of CD45+ T cells, CD3+ T cells, and CD8+ T cells in CNS tissues harvested from PbA-infected mice was observed as compared to non-infected controls, and these amounts were lower in CNS tissues taken from PbA-infected mice treated with negatively charged polystyrene particle-treated particles (FIG. 5B).

[00197] Inflammatory monocytes and macrophages and T cells populations were examined in the spleens of uninfected controls, PbA-infected mice, and PbA-infected mice treated with negatively charged polystyrene particles. Spleens from PbA-infected mice had significantly higher levels of CD45+ cells and cells that express high levels of LyC6 than uninfected mice and untreated PbA-infected mice (FIG. 6).

[00198] Histopathology of brain and lung tissues were examined in uninfected control mice, untreated PbA-infected mice, and PbA-infected mice treated with negatively charged carboxylated polystyrene particles. Brains from untreated PbA infected mice had increased sequestration, hemorrhages, and enlarged perivascular spaces as compared to uninfected controls. Lungs from untreated PbA-infected mice has increased cellularity, thickening of alveolar septa, and haemorrhages as compared to uninfected control mice. Brains and lungs from PbA-infected mice treated with negatively changed carboxylated polystyrene particles had less severe pathology than untreated PbA-infected mice and more closely resembled brain and lung tissues from the uninfected control mice (FIG. 7).

[00199] Parasitaemia was measured in PbA-infected mice that were untreated or treated with negatively charged carboxylated polystyrene particles. No difference was observed in the percentage of parasitized red blood cells (PRBC) between treated and untreated mice (FIG. 8).

[00200] These data illustrate that negatively changed carboxylated particles are sufficient to reduce the severity of the disease in a mouse model of malaria. Mortality associated with PbA infection in this model is usually 100% between days 7-12 post infection. Treatment with negatively charged carboxylated polystyrene particles reduced the severity of the malaria and increased survival without reducing the parasite load in PbA-infected mice. Furthermore, the particles are effective in reducing the observed clinical scores and increasing survival in a mouse model of malaria without any additional drugs or bioactive agents that were attached to the particles themselves or administered along with the particles. The particles are therefore considered the active agent that reduces mortality in the PbA-infected mouse model of malaria.

EXAMPLE 2

TREATMENT WITH POLYSTYRENE PARTICLES AND AN ANTIMALARIAL IN A MOUSE MODEL OF CEREBRAL MALARIA.

[00201] Administration of negatively changed carboxylated polystyrene particles alone were sufficient to improve mortality and reduce the disease severity in a mouse model of malaria infection. To examine the effects of combined treatment with polystyrene particles and an antimalarial, eight to nine week old CBA mice were infected with PbA as described as above. Mice were divided into three experimental groups. Uninfected controls, untreated PbA-infected mice, and PbA-infected mice treated with negatively charged carboxylated polystyrene particles and Quinine (n=7). Treatment with polystyrene particles was performed once daily from day 3 to day 10 post PbA infection, and untreated PbA-infected mice were administered a PBS vehicle. After the treatment for severe malaria with the polystyrene particles was completed, Quinine was administered once daily (50 mg/kg LP.) for seven days starting at 21 days post PbA infection. [00202] The combined treatment with polystyrene particles and Quinine significantly enhanced survival of PbA-infected mice. While no untreated PbA-infected mice (n = 21) survived to day 12 post infection, more than half the mice receiving the combined treatment (n = 28) survived past 30 days post infection (FIG. 9A). Clinical evaluation scores were measured in treated and untreated PbA-infected mice between day 4 and day 15 post infection. As observed in Example 1, treatment with negatively charged carboxylated particles reduced the clinical scores as compared untreated PbA-infected mice (FIG. 9B). While treatment with polystyrene particles did not reduce the parasite load in PbA-infected mice, subsequent treatment with Quinine starting at day 21 did reduce parasite load.

[00203] These data demonstrate that PbA-infected mice can be treated with an antimalarial to increase survival and reduce parasite load after successful treatment with negatively charged carboxylated polystyrene particles.

EXAMPLE 3

TREATMENT WITH POLYSTYRENE PARTICLES IN A MOUSE MODEL OF CEREBRAL MALARIA IN TWO DIFFERENT MOUSE STRAINS

[00204] To confirm that administration of negatively changed carboxylated polystyrene particles were sufficient to increase survival in a mouse model of cerebral malaria, two mouse strains, C57BL/6 and CBA, were tested. Mice were divided into 5 experimental groups: uninfected control C57BL/6 mice (n = 4), untreated PbA-infected C57BL/6 mice (n = 7), PbA- infected C57BL/6 mice treated with negatively changed carboxylated polystyrene particles (n = 7), untreated PbA-infected CBA mice (n = 5), and PbA-infected CBA mice treated with negatively changed carboxylated polystyrene particles (n = 11). For both C57BL/6 and CBA particle treated mice, the negatively charged carboxylated polystyrene particles were administered once daily beginning at day 3 and ending at day 10 post infection. As shown in FIG 10, treatment with the negatively charged carboxylated polystyrene particles increased survival in PbA-infected mice in both C57BL/6 and CBA strains.

EXAMPLE 4

TREATMENT WITH PLGA PARTICLES IN A MOUSE MODEL OF CEREBRAL

MALARIA IN TWO DIFFERENT MOUSE STRAINS [00205] Negatively charged carboxylated poly(lactic-co-gly colic acid) (PLGA) particles administered to PbA-infected mice to determine the efficacy of PLGA particles for the treatment of cerebral malaria. PLGA particle treatment was tested in two mouse strains, C57BL/6 and CBA. Mice were divided into five experimental groups: untreated PbA-infected C57BL/6 mice (n=5), PbA-infected C57BL/6 mice treated with negatively changed carboxylated PLGA particles (n=l l), uninfected CBA control mice (n = 4), untreated PbA-infected CBA mice (n = 7), and PbA-infected CBA mice treated with negatively changed carboxylated PLGA particles (n = 7).

[00206] Treatment with PLGA particles was performed by injection of approximately

4.4*10⁹ (Approx 0.3-2.5mg) of negatively charged carboxylated polystyrene particles in 300 μΐ PBS, i.v., once daily from day 3 to day 10 post PbA infection. Untreated PbA-infected mice were injected with 300 μΐ PBS, i.v. once daily from day 3 to day 10 post PbA infection. As shown in FIG. 11, treatment with particles increased survival and reduced symptoms in PbA- infected mice in both C57BL/6 and CBA mouse strains.

EXAMPLE 5

TREATMENT WITH PLGA PARTICLES IN A MOUSE MODEL OF CEREBRAL MALARIA AT DIFFERENT ΉΜΕ POINTS FOLLOWING INFECTION

[00207] The effectiveness of administering negatively charged carboxylated PLGA particles at different times during the course of PbA infection was tested. Treatments with the antimalarial Chloroquine were also tested. CBA mice aged approximately 13 weeks were divided into 6 experimental groups: untreated PbA-infected mice (n = 5), PbA-infected mice treated with Chloroquine from day 7 to day 10 post infection (n = 7), PbA-infected mice treated with PLGA particles from day 3 to day 10 post infection (n = 7), PbA-infected mice treated with PLGA particles from day 5 to day 10 post infection (n = 7), PbA-infected mice treated with PLGA particles from day 7 to day 10 post infection (n = 7), and PbA-infected mice treated with PLGA particles and Chloroquine from day 7 to day 10 post infection (n = 7).

[00208] As shown in FIG. 12, the combined treatment of PLGA particles and Chloroquine were effective when administered beginning at a time point (day 7 post infection) when the disease was severe. Chloroquine treatment alone resulted in only 1 of 7 animals surviving to day 10. While the treatments of PLGA particles alone improved survival, with 1 of 7 animals surviving to day 10 after treatment at day 7, the combined treatment of Chloroquine and PLGA particles greatly enhanced survival to a degree (6/7 mice surviving to day 10) that would not have been expected from treatment of either agent alone (FIG. 12A). Parasite load was reduced in PbA-infected mice that were treated with both PLGA particles and Chloroquine (FIG. 12b), and the combined treatment of PLGA particles and Chloroquine resulted in greater reduction in clinical score than any of the treatments of PLGA particles alone. These data illustrate that chloroquine and PLGA particles act synergistically to improve survival, reduce parasite burden, and reduce clinical symptoms in the PbA-infected mouse model of cerebral malaria.

EXAMPLE 6

TREATMENT WITH PLGA PARTICLES IN A MOUSE MODEL OF CEREBRAL MALARIA AT DIFFERENT TIME POINTS FOLLOWING PbA INFECTION

[00209] The effectiveness of administering negatively charged carboxylated PLGA particles at different times during the course of PbA infection with or without an antimalarial agent (artesunate). CBA mice aged approximately 13 weeks were divided into 5 experimental groups: untreated PbA-infected mice (n = 5), PbA-infected mice treated with artesunate from day 7 to day 10 post infection (n = 10), PbA-infected mice treated with PLGA particles from day 3 to day 10 post infection (n = 7), PbA-infected mice treated with PLGA particles from day 7 to day 10 post infection (n = 7), and PbA-infected mice treated with PLGA particles and artesunate from day 7 to day 10 post infection (n = 7).

[00210] As shown in FIG. 13, the combined treatment of PLGA particles and artesunate resulted in greater survival in PbA-infected mice as compared to treatments with PLGA particles or artesunate alone.

[00211] The effectiveness of administering a single dose of PLGA particles, a single dose of an anti-malarial, or a single dose of a combined treatment of PLGA particles and an antimalarial was also tested. Treatments were administered at day 7 post injection, a time point when the severe symptoms begin to develop. CBA mice aged approximately 13 weeks were divided into 4 experimental groups: untreated PbA-infected mice (n = 5), PbA-infected mice treated with a single dose of artesunate at day 7 post infection (n = 7), PbA-infected mice treated with a single dose of PLGA particles at day 7 post infection (n = 7), PbA-infected mice treated with a single combined treatment of PLGA particles and artesunate at day 7 post infection (n =

7). As shown in FIG. 14, the single treatment of PLGA particles and artesunate resulted in maximum survival. The combined treatment of PLGA particles and artesunate resulted in greater survival of PbA-infected mice than single treatment of PLGA particles or artesunate alone.

[00212] The ability of a single treatment of PLGA particles, artesunate, or a combined treatment of PLGA treatment and artesunate when administered at the late stages of severe malaria were tested. Treatments were administered when animals displayed a clinical score of 2- 3, which is considered to be at the late stages of the severe malaria of the PbA-infected mouse model of cerebral malaria. CBA mice aged approximately 13 weeks were divided into 4 experimental groups: untreated PbA-infected mice (n = 5), PbA-infected mice treated with a single dose of artesunate at day 8 post infection (n = 7), PbA-infected mice treated with a single dose of PLGA particles at day 8 post infection (n = 7), PbA-infected mice treated with a single combined treatment of PLGA particles and artesunate at day 8 post infection (n = 7).

[00213] As shown in FIG. 15, only the combined treatment of PLGA particles and artesunate resulted in increased survival as compared to untreated PbA-infected mice. This represents a significant step forward in malaria treatment as there are currently no options for patents in the severe stages of malaria.

[00214] Furthermore, the combined treatment of PLGA particles and the antimalarial induces immunity, with all animals surviving re-inoculation with PbA. The surviving mice develop sterilizing immunity to the parasites and they cannot be re-infected with PbA. PbA- infected mice were untreated, or treated with a single dose of PLGA particles, artesunate, or a combination PLGA particles and artesunate that were administered at the late stages of severe malaria (when the mice displayed a clinical score of at least 2). In agreement with the experiments described above, mice receiving the combination of the PLGA particles and artesunate displayed increased survival (FIG. 16, left panel). At day 28 post infection, the surviving mice that had received the combination treatment at the onset of late-stage severe malaria were rechallenged with a second lethal dose of malaria. At the same time a set of naive animals (that had not been previously infected or treated) were infected as a control sentinel group. While all of the sentinel control animals developed severe malaria and did not survive past 8 days after the PbA infection, mice that had received the combined treatment during the primary infection still demonstrated 100% survival 15 days after the PbA reinfection (FIG. 16, right panel). These data demonstrate that the mice treated with PLGA particles and artesunate developed immunity to malaria. Of note, there is currently no vaccine or treatment option capable of inducing immunity to malaria. [00215] While specific embodiments of the invention have been described and illustrated, such embodiments should be considered illustrative of the invention only and not as limiting the invention as construed in accordance with the accompanying claims.

[00216] All patents, applications, and other references cited herein are incorporated by reference in their entireties.

Claims

WHAT IS CLAIMED IS:

1. A method of inhibiting or treating malaria in a subject, said method comprising administering to said subject a pharmaceutical composition comprising negatively charged particles and a pharmaceutically acceptable carrier, wherein said particles are free from attached peptide moieties, antigenic moieties, and bioactive agents.

2. The method of claim 1, wherein the malaria is caused by Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, or Plasmodium knowlesi.

3. The method of claim 2, wherein the malaria is caused by Plasmodium falciparum.

4. The method of claim any of claims 1-3, wherein said composition alleviates at least one symptom associated with malaria.

5. The method of claim 4, wherein the at least one symptom is selected from respiratory distress, metabolic acidosis, acute encephalitis/meningitis syndrome, and liver malfunction or failure.

6. The method of any of claims 1-5, wherein said negatively charged particles are polystyrene particles, diamond particles, PLURIONICS stabilized polypropylene sulfide particles, citric acid particles, or poly(lactic-co-glycolic acid) (PLGA) particles.

7. The method of claim 6, wherein said particles are polystyrene particles.

8. The method of claim 6, wherein said particles are diamond particles.

9. The method of claim 6, wherein said particles are PLGA particles.

10. The method of any of claims 1-9, wherein the particles are carboxylated.

11. The method of any of claims 1-10, wherein the particles have a zeta potential of less than about -100 mV.

12. The method of claim 11, wherein the particles have a zeta potential between about -100 mV and about -15 mV.

13. The method of claim 12, wherein the particles have a zeta potential between about -100 mV and about -75 mV.

14. The method of claim 12, wherein the particles have a zeta potential between about -50 mV and about -20 mV.

15. The method of any of claims 1-14, wherein said composition ameliorates an inflammatory immune response.

16. The method of any of claims 1-15, wherein the diameter of said negatively charged particles is between about 0.01 μηι to about 10 μπι.

17. The method of claim 16, wherein the diameter of said negatively charged particles is between about 0.3 μηι to about 5 μηι.

18. The method of claim 16, wherein the diameter of said negatively charged particles is between about 0.5 μηι to about 3 μηι.

19. The method of claim 16, wherein the diameter of said negatively charged particles is between about 0.5 μηι to about 1 μιη.

20. The method of claim 16, wherein the diameter of said negatively charged particles is about 0.5 μηι.

21. The method of any of claims 1-20, wherein said composition is administered orally, nasally, intravenously, intramuscularly, ocularly, transdermally, or subcutaneously.

22. The method of any of claims 1-21, wherein said subject is a human.

23. A method for removing pro-inflammatory mediators from the inflammatory milieu in a subject with malaria, said method comprising administering to said subject a pharmaceutical composition comprising negatively charged particles and a pharmaceutically acceptable carrier, wherein said particles are free from attached peptide moieties, antigenic moieties, and bioactive agents.

24. The method of claim 23, wherein the malaria is caused by Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, or Plasmodium knowlesi.

25. The method of claim 24, wherein the malaria is caused by is Plasmodium falciparum.

26. The method of any of claims 22-25, wherein said composition alleviates at least one symptom associated with malaria.

27. The method of claim 26, wherein the at least one symptom is selected from respiratory distress, metabolic acidosis, acute encephalitis/meningitis syndrome, and liver malfunction or failure.

28. The method of any of claims 23-27, wherein said pro-inflammatory mediators produced in the subject bind to the negatively charged particles.

29. The method of any of claims 23-28, wherein said negatively charged particles are polystyrene particles, diamond particles, PLURIONICS stabilized polypropylene sulfide particles, citric acid particles, or poly(lactic-co-glycolic acid) (PLGA) particles.

30. The method of claim 29, wherein said particles are polystyrene particles.

31. The method of claim 29, wherein said particles are diamond particles.

32. The method of claim 29, wherein said particles are PLGA particles.

33. The method of any of claims 23-32, wherein said particles are carboxylated.

34. The method of any of claims 23-33, wherein the particles have a zeta potential of less than about -100 mV.

35. The method of claim 34, wherein the particles have a zeta potential between about -100 mV and about -15 mV.

36. The method of claim 34, wherein the particles have a zeta potential between about -100 mV and about -75 mV.

37. The method of claim 34, wherein the particles have a zeta potential between about -50 mV and about -20 mV.

38. The method of any of claims 23-37, wherein said composition ameliorates an inflammatory immune response.

39. The method of any of claims 23-38, wherein the diameter of said negatively charged particles is between about 0.01 μηι to about 10 μπι.

40. The method of claim 39, wherein the diameter of said negatively charged particles is between about 0.3 μηι to about 5 μηι.

41. The method of claim 39, wherein the diameter of said negatively charged particles is between about 0.5 μηι to about 3 μηι.

42. The method of claim 39, wherein the diameter of said negatively charged particles is between about 0.5 μηι to about 1 μιη.

43. The method of claim 39, wherein the diameter of said negatively charged particles is about 0.5 μπι.

44. The method of any of claims 23-43, wherein said subject is a human.

45. The method of any of claims 23-44, wherein said composition is administered orally, nasally, intravenously, intramuscularly, ocularly, transdermally, or subcutaneously.

46. A method for inducing regulatory T cells in a subject with malaria, said method comprising administering to said subject a pharmaceutical composition comprising negatively charged particles and a pharmaceutically acceptable carrier, wherein the negatively charged particles are free from attached peptide moieties, antigenic moieties and bioactive agents.

47. The method of claim 46 wherein the regulatory T cells comprise CD4+ cells.

48. The method of claim 46 or 47 wherein the regulatory T cells comprise CD8⁺ T cells.

49. The method any of claims 46-48, wherein the malaria is caused by Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, or Plasmodium knowlesi.

50. The method of claim 49, wherein the malaria is caused by Plasmodium falciparum.

51. The method of claim 50, wherein said composition alleviates at least one symptom associated with malaria.

52. The method of claim 51, wherein the at least one symptom is selected from respiratory distress, metabolic acidosis, acute encephalitis/meningitis syndrome, and liver malfunction or failure.

53. The method of claim 52, wherein said negatively charged particles are polystyrene particles, diamond particles, PLU IONICS stabilized polypropylene sulfide particles, citric acid particles, or poly(lactic-co-glycolic acid) (PLGA) particles.

54. The method of claim 53, wherein said particles are polystyrene particles.

55. The method of claim 53, wherein said particles are diamond particles.

56. The method of claim 53, wherein said particles are PLGA particles.

57. The method of claim 53, wherein the particles are carboxylated.

58. The method of any of claims 46-57, wherein the particles have a zeta potential of less than about -100 mV.

59. The method of claim 58, wherein the particles have a zeta potential between about -100 mV and about -15 mV.

60. The method of claim 58, wherein the particles have a zeta potential between about -100 mV and about -75 mV.

61. The method of claim 58, wherein the particles have a zeta potential between about -50 mV and about -20 mV.

62. The method of any of claims 46-61, wherein said composition ameliorates an inflammatory immune response.

63. The method of any of claims 46-62, wherein the diameter of said negatively charged particles is between about 0.01 μηι to about 10 μπι.

64. The method of claim 63, wherein the diameter of said negatively charged particles is between about 0.3 μηι to about 5 μηι.

65. The method of claim 63, wherein the diameter of said negatively charged particles is between about 0.5 μηι to about 3 μηι.

66. The method of claim 63, wherein the diameter of said negatively charged particles is between about 0.5 μηι to about 1 μιη.

67. The method of claim 63, wherein the diameter of said negatively charged particles is about 0.5 μπι.

68. The method of any of claims 46-67, wherein said subject is a human.

69. The method of any of claims 46-67, wherein said composition is administered orally, nasally, intravenously, intramuscularly, ocularly, transdermally, or subcutaneously.

70. A method for controlling a pathologic and/or unwanted inflammatory immune response in a subject with malaria comprising administering to the subject a pharmaceutical composition comprising negatively charged particles and a pharmaceutically acceptable carrier, wherein the negatively charged particles are free from attached peptide moieties, antigenic moieties, and bioactive agents.

71. The method of claim 70, wherein the malaria is caused by Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, or Plasmodium knowlesi.

72. The method of claim 70, wherein the malaria is caused by Plasmodium falciparum.

73. The method of any of claims 70-72, wherein said composition alleviates at least one symptom associated with malaria.

74. The method of claim 73, wherein the at least one symptom is selected from respiratory distress, metabolic acidosis, acute encephalitis/meningitis syndrome, and liver malfunction or failure.

75. The method of any of claims 70-74, wherein said negatively charged particles are polystyrene particles, diamond particles, PLURIONICS stabilized polypropylene sulfide particles, citric acid particles, or poly(lactic-co-glycolic acid) (PLGA) particles.

76. The method of claim 75, wherein said particles are polystyrene particles.

77. The method of claim 75, wherein said particles are diamond particles.

78. The method of claim 75, wherein said particles are PLGA particles.

79. The method of claim 75, wherein said particles are carboxylated.

80. The method of any of claims 70-79, wherein the particles have a zeta potential of less than about -100 mV.

81. The method of claim 80, wherein the particles have a zeta potential between about -100 mV and about -25 mV.

82. The method of claim 80, wherein the particles have a zeta potential between about -100 mV and about -25 mV.

83. The method of claim 80, wherein the particles have a zeta potential between about -50 mV and about -20 mV.

84. The method of any of claims 70-83, wherein said composition ameliorates an inflammatory immune response.

85. The method of any of claims 70-83, wherein the diameter of said negatively charged particles is between about 0.01 μηι to about 10 μπι.

86. The method of claim 85, wherein the diameter of said negatively charged particles is between about 0.3 μηι to about 5 μηι.

87. The method of claim 85, wherein the diameter of said negatively charged particles is between about 0.5 μηι to about 3 μηι.

88. The method of claim 85, wherein the diameter of said negatively charged particles is between about 0.5 μηι to about 1 μιη.

89. The method of claim 85, wherein the diameter of said negatively charged particles is about 0.5 μπι.

90. The method of any of claims 70-89, wherein said subject is a human.

91. The method of any of claims 70-90, wherein said composition is administered orally, nasally, intravenously, intramuscularly, ocularly, transdermally, or subcutaneously.

92. A method of vaccinating a subject for malaria comprising administering to the subject a pharmaceutical composition comprising negatively charged particles, wherein the particles comprise an antigen comprising one or more epitopes associated with malaria.

93. The method of claim 92, wherein the malaria is caused by Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, or Plasmodium knowlesi.

94. The method of claim 93, wherein the malaria is caused by Plasmodium falciparum.

95. The method of any of claims 92-94, wherein the one or more epitopes is associated with Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, or Plasmodium knowlesi.

96. The method of claim 95, wherein the one or more epitopes is associated with Plasmodium falciparum.

97. The method of claim 92-96, wherein the one or more epitopes is associated with the same species of Plasmodium.

98. The method of any of claims 92-96, wherein the one or more epitopes is associated with different species of Plasmodium.

99. The method of any of claims 92-98, wherein the pharmaceutical composition further comprises an adjuvant.

100. The method of any of claims 92-99, wherein said particles are polystyrene particles, diamond particles, PLURIONICS stabilized polypropylene sulfide particles, citric acid particles, or poly(lactic-co-glycolic acid) (PLGA) particles.

101. The method of claim 100, wherein said particles are polystyrene particles.

102. The method of claim 100, wherein said particles are diamond particles.

103. The method of claim 100, wherein said particles are PLGA particles.

104. The method of any of claims 92-103, wherein said particles are carboxylated.

105. The method of any of claims 92-104, wherein the particles have a zeta potential of less than about -100 mV.

106. The method of claim 105, wherein the particles have a zeta potential between about -100 mV and about -25 mV.

107. The method of claim 105, wherein the particles have a zeta potential between about -100 mV and about -75 mV.

108. The method of claim 105, wherein the particles have a zeta potential between about -50 mV and about -20 mV.

109. The method of any of claims 92-108, wherein the diameter of said negatively charged particles is between about 0.01 μηι to about 10 μπι.

110. The method of claim 109, wherein the diameter of said negatively charged particles is between about 0.3 μηι to about 5 μηι.

111. The method of claim 109, wherein the diameter of said negatively charged particles is between about 0.5 μηι to about 3 μηι.

112. The method of claim 109, wherein the diameter of said negatively charged particles is between about 0.5 μηι to about 1 μιη.

113. The method of claim 109, wherein the diameter of said negatively charged particles is about 0.5 μπι.

114. The method of any of claims 92-113, wherein said subject is a human.

115. The method of any of claims 92-114, wherein said composition is administered intravenously, intramuscularly, transdermally, or subcutaneously.

116. A method for treating or preventing cerebral malaria in a subject with malaria comprising administering to the subject a pharmaceutical composition comprising negatively charged particles and a pharmaceutically acceptable carrier, wherein the negatively charged particles are free from attached peptide moieties, antigenic moieties, and bioactive agents.

117. The method of claim 116, wherein the malaria is caused by Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, or Plasmodium knowlesi.

118. The method of claim 117, wherein the malaria is caused by Plasmodium falciparum.

119. The method of any of claims 116-118, wherein said composition alleviates at least one symptom associated with malaria.

120. The method of claim 119, wherein the at least one symptom is selected from respiratory distress, metabolic acidosis, acute encephalitis/meningitis syndrome, and liver malfunction or failure.

121. The method of any of claims 116-120, wherein said negatively charged particles are polystyrene particles, diamond particles, PLURIONICS stabilized polypropylene sulfide particles, citric acid particles, or poly(lactic-co-glycolic acid) (PLGA) particles.

122. The method of claim 121, wherein said particles are polystyrene particles.

123. The method of claim 121, wherein said particles are diamond particles.

124. The method of claim 121, wherein said particles are PLGA particles.

125. The method of claim 121, wherein said particles are carboxylated.

126. The method of any of claims 116-125, wherein the particles have a zeta potential of less than about -100 mV.

127. The method of claim 126, wherein the particles have a zeta potential between about -100 mV and about -25 mV.

128. The method of claim 126, wherein the particles have a zeta potential between about -100 mV and about -25 mV.

129. The method of claim 126, wherein the particles have a zeta potential between about -50 mV and about -20 mV.

130. The method of any of claims 116-129, wherein said composition ameliorates an inflammatory immune response.

131. The method of any of claims 116-130, wherein the diameter of said negatively charged particles is between about 0.01 μηι to about 10 μπι.

132. The method of claim 131, wherein the diameter of said negatively charged particles is between about 0.3 μηι to about 5 μηι.

133. The method of claim 131, wherein the diameter of said negatively charged particles is between about 0.5 μηι to about 3 μηι.

134. The method of claim 131, wherein the diameter of said negatively charged particles is between about 0.5 μηι to about 1 μιη.

135. The method of claim 131, wherein the diameter of said negatively charged particles is about 0.5 μπι.

136. The method of any of claims 116-135, wherein said subject is a human.

137. The method of any of claims 116-136, wherein said composition is administered orally, nasally, intravenously, intramuscularly, ocularly, transdermally, or subcutaneously.

138. A method for treating cerebral malaria comprising administering a synergistically effective amount of a pharmaceutical composition comprising negatively charged particles and a pharmaceutically acceptable carrier, and a synergistically effective amount of an antimalarial, wherein the negatively charged particles are free from attached peptide moieties, antigenic moieties, and bioactive agents.

139. The method of claim 138, wherein the malaria is caused by Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, or Plasmodium knowlesi.

140. The method of claim 139, wherein the malaria is caused by Plasmodium falciparum.

141. The method of any of claims 138-140, wherein the cerebral malaria is late-stage cerebral malaria.

142. The method of any of claims 138-141, wherein the negatively charged particles and the antimalarial are administered in the same pharmaceutical composition.

143. The method of any of claims 138-141, wherein the negatively charged particles are administered in a separate pharmaceutical composition than the pharmaceutical composition comprising the negatively charged particles.

144. The method of claim 143, wherein the antimalarial is administered to the subject prior to, simultaneously with, and/or after the pharmaceutical composition comprising the negatively charged particles is administered to the subject.

145. The method of claim 144, wherein the antimalarial is administered to the subject prior to the pharmaceutical composition comprising the negatively charged particles is administered to the subject.

146. The method of claim 144, wherein the antimalarial is administered to the subject simultaneously with the pharmaceutical composition comprising the negatively charged particles is administered to the subject.

147. The method of claim 144, wherein the antimalarial is administered to the subject after the pharmaceutical composition comprising the negatively charged particles is administered to the subject.

148. The method of any of claims 138-147, wherein administering the synergistically effective amount of the pharmaceutical composition comprising the negatively charged particles and the synergistically effective amount of the antimalarial synergistically increases the probability of survival in the subject.

149. The method of claim any of claims 138-148, wherein administering the synergistically effective amount of the pharmaceutical composition comprising the negatively charged particles and the synergistically effective amount of the antimalarial to the subject prevents or synergistically reduces the probability of a coma.

150. The method of claim any of claims 138-148, wherein administering the synergistically effective amount of the pharmaceutical composition comprising the negatively charged particles and the synergistically effective amount of the antimalarial to the subject synergistically reduces the duration of a coma in the subject.

151. The method of claim any of claims 138-148, wherein administering the synergistically effective amount of the pharmaceutical composition comprising the negatively charged particles and the synergistically effective amount of the antimalarial to the subject synergistically ameliorates at least one symptom associated with cerebral malaria.

152. The method of any of claims 138-151, wherein said negatively charged particles are polystyrene particles, diamond particles, PLURIONICS stabilized polypropylene sulfide particles, citric acid particles, or poly(lactic-co-glycolic acid) (PLGA) particles.

153. The method of claim 152, wherein said particles are polystyrene particles.

154. The method of claim 152, wherein said particles are diamond particles.

155. The method of claim 152, wherein said particles are PLGA particles.

156. The method of any of claims 138-155, wherein the particles are carboxylated.

157. The method of any of claims 138-156, wherein the particles have a zeta potential of about O mV to about -100 mV.

158. The method of claim 157, wherein the particles have a zeta potential between about -100 mV and about -15 mV.

159. The method of claim 157, wherein the particles have a zeta potential between about -75 mV and about -75 mV.

160. The method of claim 157, wherein the particles have a zeta potential between about -80 mV and about -30 mV.

161. The method of any of claims 138-160, wherein the diameter of said negatively charged particles is between about 0.01 μηι to about 10 μπι.

162. The method of claim 161, wherein the diameter of said negatively charged particles is between about 0.3 μηι to about 5 μηι.

163. The method of claim 161, wherein the diameter of said negatively charged particles is between about 0.5 μηι to about 3 μηι.

164. The method of claim 161, wherein the diameter of said negatively charged particles is between about 0.5 μηι to about 1 μιη.

165. The method of claim 161, wherein the diameter of said negatively charged particles is about 0.5 μηι.

166. The method of any of claims 1-20, wherein the pharmaceutical composition comprising the negatively charged particles is administered orally, nasally, intravenously, intramuscularly, ocularly, transdermally, or subcutaneously.

167. The method of any of claims 138-166, wherein the antimalarial is at least one of Quinine, Chloroquine, Amodiaquine, Pyrimethamine, Proguanil, Sulfonamides, Mefloquine, Atovaquone, Primaquine, Artemisinin, Halofantrine, Doxycycline, Clindamycin, Artesunate, or a derivative thereof.

168. The method of claim 167, wherein the antimalarial is Artesunate.

169. The method of claim 167, wherein the antimalarial is Chloroquine.

170. A method for preventing reinfection with malaria in a subject infected with malaria comprising administering a pharmaceutical composition comprising negatively charged particles and a pharmaceutically acceptable carrier, and an antimalarial, wherein the negatively charged particles are free from attached peptide moieties, antigenic moieties, and bioactive agents.

The method of claim 170, wherein the malaria is caused by Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, or Plasmodium knowlesi.

172. The method of claim 171, wherein the malaria is caused by Plasmodium falciparum.

173. The method of any of claims 170-172, wherein the negatively charged particles and the antimalarial are administered in the same pharmaceutical composition.

The method of any of claims 170-172, wherein the negatively charged particles are administered in a separate pharmaceutical composition than the pharmaceutical composition comprising the negatively charged particles.

The method of claim 174, wherein the antimalarial is administered to the subject prior to, simultaneously with, and/or after the pharmaceutical composition comprising the negatively charged particles is administered to the subject.

The method of claim 174, wherein the antimalarial is administered to the subject prior to the pharmaceutical composition comprising the negatively charged particles is administered to the subject.

The method of claim 174, wherein the antimalarial is administered to the subject simultaneously with the pharmaceutical composition comprising the negatively charged particles is administered to the subject.

The method of claim 174, wherein the antimalarial is administered to the subject after the pharmaceutical composition comprising the negatively charged particles is administered to the subject.

179. The method of any of claims 170-178, wherein said negatively charged particles are polystyrene particles, diamond particles, PLURIONICS stabilized polypropylene sulfide particles, citric acid particles, or poly(lactic-co-glycolic acid) (PLGA) particles.

180. The method of claim 179, wherein said particles are polystyrene particles.

181. The method of claim 179, wherein said particles are diamond particles.

182. The method of claim 179, wherein said particles are PLGA particles.

183. The method of any of claims 170-182, wherein the particles are carboxylated.

184. The method of any of claims 170-183 wherein the particles have a zeta potential of about O mV to about -100 mV.

185. The method of claim 184, wherein the particles have a zeta potential between about -100 mV and about -15 mV.

186. The method of claim 184, wherein the particles have a zeta potential between about -75 mV and about -75 mV.

187. The method of claim 184, wherein the particles have a zeta potential between about -80 mV and about -30 mV.

188. The method of any of claims 170-187, wherein the diameter of said negatively charged particles is between about 0.01 μηι to about 10 μπι.

189. The method of claim 188, wherein the diameter of said negatively charged particles is between about 0.3 μηι to about 5 μηι.

190. The method of claim 188, wherein the diameter of said negatively charged particles is between about 0.5 μηι to about 3 μηι.

191. The method of claim 188, wherein the diameter of said negatively charged particles is between about 0.5 μηι to about 1 μιη.

192. The method of any of claims 170-191, wherein the antimalarial is at least one of Quinine, Chloroquine, Amodiaquine, Pyrimethamine, Proguanil, Sulfonamides, Mefloquine, Atovaquone, Primaquine, Artemisinin, Halofantrine, Doxycycline, Clindamycin, Artesunate, or a derivative thereof.

193. The method of claim 192, wherein the antimalarial is Artesunate.

194. The method of claim 192, wherein the antimalarial is Chloroquine.