WO2016057909A1

WO2016057909A1 - Immune-modifying particles for the treatment of ebola virus

Info

Publication number: WO2016057909A1
Application number: PCT/US2015/054922
Authority: WO
Inventors: Daniel R. Getts
Original assignee: COUR PHARMACEUTICALS DEVELOPMENT Co
Current assignee: COUR PHARMACEUTICALS DEVELOPMENT Co
Priority date: 2014-10-10
Filing date: 2015-10-09
Publication date: 2016-04-14
Anticipated expiration: 2017-04-10

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 filovirus infection. Additionally, the present invention describes methods of inhibiting or treating filovirus infections by administering these same negatively charged particles.

Description

IMMUNE-MODIFYING PARTICLES FOR THE TREATMENT OF EBOLA VIRUS

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 62/062,694, filed on October 10, 2014, which is incorporated by reference herein in its entirety.

BACKGROUND OF INVENTION

[0002] Ebola virus disease (EVD), formerly known as Ebola hemorrhagic fever, is a severe hemorrhagic fever syndrome of humans and nonhuman primates caused by ebolaviruses, which are enveloped negative-strand RNA viruses of the Filoviridae family. The family Filoviridae includes three genera: Cuevavirus, Marburgvirus and Ebolavirus. The five known species in the genus Ebolavirus are Bundibugyo ebolavirus, Reston ebolavirus, Sudan ebolavirus, Tai Forest ebolavirus (originally Cote d'lvoire ebolavirus) and Zaire ebolavirus. Ebola virus (EBOV) was first discovered in Zaire (now the Democratic Republic of the Congo) and Zaire ebolavirus (ZEBOV) is the reference species for the genus Ebolavirus. The Zaire species was involved in the first documented outbreak of the disease in 1976 and is currently thought to be responsible for the outbreak with the most deaths, although there are about 115 mutations in the latest Zaire strain relative to the last Zaire strain outbreak.

[0003] Transmission of Ebola virus from infected animals to humans occurs via contact with bodily fluids, especially sweat, of the ill or dead animals through broken skin or unprotected mucous membranes. The animals can be, but are not limited to, fruit bats, chimpanzees, gorillas, monkeys, forest antelope and porcupines. Similarly, human-to-human transmission is through direct contact with the blood, secretions, organs or other bodily fluids of infected people and/or items contaminated with these fluids. The virus can still be transmitted even up to 7 weeks through semen of male survivors.

[0004] A person becomes infectious after the onset of symptoms, which can exhibit anywhere between 2-21 days after infection with the virus. The pathogenesis of disease results in a biphasic pattern of symptom development, with the first symptoms including fever fatigue, muscle pain, headache and sore throat. Disease may appear to be getting better before the second wave of symptoms which can include vomiting, diarrhea, rash, impaired kidney and liver function, and in some cases, both internal and external bleeding are experienced. Low white blood cell and platelet counts and elevated liver enzymes can be found in infected individuals. [0005] It is sometimes difficult to distinguish EVD from other infectious diseases such as malaria, typhoid fever and meningitis. Diagnosis of EVD can be confirmed by antibody- capture enzyme-linked immunosorbent assay (ELISA), antigen-capture detection tests, serum neutralization test, reverse transcriptase polymerase chain reaction (RT-PCR) assay, electron microscopy and/or virus isolation by cell culture.

[0006] The mortality rate for EVD is 50-90%. Prevention and control of EVD relies on awareness of risk factors for transmission, good hygiene, infrastructure for prompt burial of the dead, identification of infected individuals and safe practices for health-care and laboratory workers. Rehydration with oral or intravenous fluids and treatment of specific symptoms improves survival, but currently there is no treatment for EVD. Though safety issues are still of concern for use of live, attenuated Ebola virus for prevention, recombinant

Ebola virus vaccine vectors are being developed with less virulent viral systems such as adenovirus, vesicular stomatitis virus and human parainfluenza virus. Ebola virus-like particles (VLPs) based upon coexpression of the viral matrix protein VP40, nucleoprotein and glycoprotein have been generated but efficacy issues still exist for non-human primates.

[0007] Ebola virus plays a crucial role in dysregulation of the innate immune response

(Wauqier et al. 2010). Studies in nonhuman primates and guinea pigs suggest that monocytes, macrophages and dendritic cells (DCs) are the preferred sites of viral replication. Upon activation of these cells, upregulation of specific receptors on their surfaces render them amenable to infection by EBOV around 7 days after infection. As part of the innate immune response, monocytes and DCs secrete proinflammatory cytokines, chemokines and growth factors as a first line of defense against pathogens. In lethal cases of ZEBOV, a cytokine storm is unleashed, where levels of cytokines (IL-lb, IL-1RA, IL-6, IL-8, IL-15 and IL-16), chemokines and growth factors (ΜΙΡ-Ι , ΜΙΡ-Ιβ, MCP-1, M-CSF, MIF, IP- 10, GRO-a and eotaxin) rise very rapidly after symptom onset and reach very high levels two days before death. This so-called cytokine storm may in turn recruit more monocytes and macrophages and possibly neutrophils to sites of infection. The innate immune response is also dysregulated through the disruption of the anti-viral interferon (IFN) Type I response pathway. Interferon production is blocked in virus-infected macrophages, peripheral blood mononuclear cells and DCs in vitro and in vivo. The viral VP35 protein is an interferon antagonist that blocks the activation of interferon regulatory factors 3 and 7, which are transcription factors important for IFN-α/β synthesis. The viral VP24 protein causes infected cells to become resistant to IFN-α/β and is linked to lethal disesase in mice and guinea pigs.

Additionally, the Ebola soluble envelope glycoprotein (sGP) inhibits neutrophil activation. [0008] Ebola virus infection is also associated with a suppression of the adaptive immune response (Wauqier et al. 2010). The adaptive immune response is affected through the decrease in mature and activated myeloid DCs, massive apoptosis of T CD4 and CD8 lymphocytes and low amounts of circulating cytokines produced by T lymphocytes. Infection of immature DCs by Ebola virus prevents their maturation and function. Infected DCs are unable to secrete pro-inflammatory cytokines and costimulatory molecules, thus inhibiting upregulation of major histocompatibility (MHC) molecules and ultimately activation of T cells. The soluble apoptotic factor nitric oxide synthesized by infected macrophages, apoptosis-inducing ligands FasL and TRAIL and the viral glycoprotein are several players that have been implicated in lymphocyte apoptosis.

[0009] Patients who survived Ebola virus infection had prominent CD8+ T cell activation, above -normal numbers of T cells, lower viral genome copies/mL serum, detectable antibodies in blood at onset of symptoms and low levels of the pro-apoptotic soluble factor nitric oxide compared to patients who succumbed to the infection (Sanchez 2004). It is becoming clear that both an early, robust innate immune response and later activation of the adaptive immune response is important for survival. Mortality is estimated to be 50-90% when the immune pathological switch is engaged.

[0010] The pathogenesis of an Ebola infection involves both damage caused by the proinflammatory mediators secreted by infected macrophages and monocytes and direct damage to host tissues and organs from the virus itself. It is thought that the virus is carried by monocytes, macrophages and DCs through the lymphatic system, possibly via binding to cell surface lectin binding proteins on macrophages and monocytes or to DC-expressed cell- surface receptors. Viral replication directly causes endothelial cell damage and lysis and necrosis of cells in organs such as the liver, spleen, kidneys and gonads. Coagulation abnormalities may arise from decreased production of clotting factors due to liver damage. Vascular permeability appears to be caused by the viral envelope glycoprotein (GP), as studies have shown both in vitro and ex vivo that endothelial cell rounding and detachment occurs in the presence of GP. Viral GP is essential for the viral life cycle, and GPi,₂ mediates attachment and fusion of the viral and host membranes, leading to endothelial cell disruption and cytotoxicity. The adrenal cortex controls blood pressure homeostasis, and its disturbance by the virus leads to hypotension and sodium loss, possibly contributing to the shock that sometimes arises from this viral infection.

[0011] Current drugs in development to combat the Ebola virus include monoclonal antibodies, RNAi, small molecules, DNA vaccines and viral vaccines. Treatment of coagulation abnormalities with anticoagulant modulators could be promising, as this therapeutic focus has provided some protection in rhesus monkeys and is correlated with human survival. The monoclonal antibodies and some of the DNA and viral vaccines target the viral envelope glycoprotein (GP). Survival in infected mice, guinea pigs and nonhuman primates has been found to correlate with levels of total immunoglobulin G (IgG) specific to the Zaire ebolavirus glycoprotein (Wong et al. 2012). Small molecules have been developed to target the viral RNA-dependent RNA polymerase. Additionally, antisense oligonucleotides have been created against Ebola VP24 and VP35 transcripts. The effectiveness of passive immunization of infected patients with human convalescent blood or serum is currently unclear, with consistent success only seen so far in rodents treated with neutralizing monoclonal antibodies specific for the glycoprotein of Ebola virus generated from different species. A study with Favipiravir, a small molecule drug being tested against Ebola virus and already approved in Japan for influenza, suggested that survival of viral infection may only be correlated with viral load when the drug was given at early stages after infection. When Favipiravir was administered late to infected subjects, the viral load was still decreased by the small molecule, but survival remained poor, suggesting that at later stages of infection, the immune pathology is already developing and may contribute to lethality. None of the current drugs being developed, however, target the immunopathology of the virus.

[0012] Considering the role of inflammatory monocytes, macrophages and dendritic cells in filovirus infections, their therapeutic accessibility in the blood stream, and their inherent propensity to interact with particles, particle-based therapeutics are potentially better equipped to specifically target these cells than antibodies or small molecules. Natural leukocyte clearance and the ability to induce apoptosis remains a primary goal for therapies that aim to reduce pathology associated with specific cell subsets including inflammatory derived macrophages and dendritic cells. It has been surprisingly found that immune- modified particles (IMPs) derived from polystyrene, nanodiamonds, or biodegradable poly(lactic-co-glycolic) acid, when infused are taken up by inflammatory monocytes through the macrophage receptor with collagenous structure (MARCO), triggering the migration and sequestration of monocytes in the spleen, where they undergo Caspase 3-mediated apoptosis. Perhaps more surprisingly, targeted administration of Immune-Modifying Particles (IMPs) in acute models of inflammation not only reduced inflammatory monocyte accumulation at the primary site of inflammation, but reduced pathology and disease severity in all models. IMPs are a versatile, readily translatable therapeutic option for diseases caused or potentiated by inflammatory monocytes. IMPs represent a novel and safe inflammatory monocyte specific therapy to reduce the pathology of an Ebola virus infection and promote survival.

SUMMARY OF THE INVENTION

[0013] The current invention provides a method of inhibiting or treating filo virus infection in a subject, said method comprising administering to said subject a pharmaceutical composition comprising negatively charged particles and a pharmaceutical acceptable carrier, wherein said particles are free from attached peptide moieties, antigenic moieties, and bioactive agents. In some embodiments, the filovirus is Cuevavirus, Marburgvirus or Ebolavirus. In some embodiments, the Ebolavirus is Bundibugyo ebolavirus, Reston ebolavirus, Sudan ebolavirus, Tai Forest ebolavirus, Zaire ebolavirus or a new strain or species of Ebolavirus. In some embodiments, the Ebola virus is Zaire ebolavirus. In some embodiments, the negatively charged particles are polystyrene particles. In other embodiments, the negatively charged particles are diamond particles. In still other embodiments, the negatively charged particles are poly(lactic-co-glycolic acid) (PLGA) particles. In some embodiments, the particles are PLURONICS® stabilized polypropylene sulfide particles. In still other embodiments, the negatively charged particles are carboxylated.

[0014] In one embodiment, the current invention provides a method for removing proinflammatory mediators from the inflammatory milieu in a subject with a filovirus infection, said method comprising administering to said subject a pharmaceutical composition comprising negatively charged particles and a pharmaceutical acceptable carrier, wherein said particles are free from attached peptide moieties, antigenic moieties, and bioactive agents. In some embodiments, the filovirus is Cuevavirus, Marburgvirus or Ebolavirus. In some embodiments, the Ebolavirus is Bundibugyo ebolavirus, Reston ebolavirus, Sudan ebolavirus, Tai Forest ebolavirus, Zaire ebolavirus, or a new strain or species of Ebolavirus. In some embodiments, the Ebola virus is Zaire ebolavirus. In some embodiments, said proinflammatory mediators produced in the subject bind to the negatively charged particles. In some embodiments, the negatively charged particles are polystyrene particles. In other embodiments, the negatively charged particles are diamond particles. In still other embodiments, the negatively charged particles are poly(lactic-co-glycolic acid) (PLGA) particles. In some embodiments, the particles are PLURONICS® stabilized polypropylene sulfide particles. In still other embodiments, the negatively charged particles are carboxylated.

[0015] In one embodiment, the current invention provides a method for inducing regulatory T cells in a subject with a filo virus infection, said method comprising administering said subject a pharmaceutical composition comprising negatively charged particles and a pharmaceutical acceptable carrier, wherein the negatively charged particles are free from attached peptide moieties, antigenic moieties, and bioactive agents. In a further embodiment, the regulatory T cells comprise CD4⁺ T cells. In a further embodiment, the regulatory T cells comprise CD8⁺ T cells. In some embodiments, the filovirus is Cuevavirus, Marburgvirus, or Ebolavirus. In some embodiments, the Ebolavirus comprises Bundibugyo ebolavirus, Reston ebolavirus, Sudan ebolavirus, Tai Forest ebolavirus, Zaire ebolavirus, or a new strain or species of Ebolavirus. In some embodiments, the Ebola virus is Zaire ebolavirus. In some embodiments, said pro-inflammatory mediators produced in the subject bind to the negatively charged particles. In some embodiments, the negatively charged particles are polystyrene particles. In other embodiments, the negatively charged particles are diamond particles. In still other embodiments, the negatively charged particles are poly(lactic-co-glycolic acid) (PLGA) particles. In some embodiments, the particles are PLURONICS® stabilized polypropylene sulfide particles. In still other embodiments, the negatively charged particles are carboxylated.

[0016] In one embodiment, the current invention provides a method for decreasing a viral load in a subject with a filovirus infection comprising administering to said subject a pharmaceutical composition comprising negatively charged particles and a pharmaceutical acceptable carrier, wherein the negatively charged particles are free from attached peptide moieties, antigenic moieties, and bioactive agents. In some embodiments, said viral load is measured by the number of viral genome copies present per milliliter of blood serum. In some embodiments, said viral load decreases to less than about 10⁷ viral genome copies per milliliter of blood serum. In some embodiments, said viral load is measured by the number of plaque forming units present per milliliter of blood serum. In some embodiments, said viral load decreases to less than about 10⁵ plaque forming units present per milliliter of blood serum. In some embodiments, said viral load is measured by viral envelope glycoprotein expression. In some embodiments, the viral envelope glycoprotein expression of said subject is compared to the viral envelope glycoprotein expression of a subject with a filovirus infection not administered said composition. In some embodiments, the viral envelope glycoprotein expression of the administered subject is 10- to 100-fold lower compared to the viral envelope glycoprotein expression of a subject with a filo virus infection not administered said composition. In some embodiments, the filovirus is Cuevavirus, Marburgvirus or Ebolavirus. In some embodiments, the Ebolavirus is Bundibugyo ebolavirus, Reston ebolavirus, Sudan ebolavirus, Tai Forest ebolavirus, Zaire ebolavirus, or a new strain or species of Ebolavirus. In some embodiments, the Ebola virus is Zaire ebolavirus. In some embodiments, said pro-inflammatory mediators produced in the subject bind to the negatively charged particles. In some embodiments, the negatively charged particles are polystyrene particles. In other embodiments, the negatively charged particles are diamond particles. In still other embodiments, the negatively charged particles are poly(lactic-co-glycolic acid) (PLGA) particles. In some embodiments, the particles are PLURONICS® stabilized polypropylene sulfide particles. In still other embodiments, the negatively charged particles are carboxylated.

[0017] In still a further embodiment, the current invention provides a method for controlling a pathologic and/or unwanted inflammatory immune response in a subject with a filovirus infection comprising administering to the subject a pharmaceutical composition comprising negatively charged particles and a pharmaceutical acceptable carrier, wherein the negatively charged particles are free from attached peptide moieties, antigenic moieties, and bioactive agents. In some embodiments, the filovirus is Cuevavirus, Marburgvirus or

Ebolavirus. In some embodiments, the Ebolavirus is Bundibugyo ebolavirus, Reston ebolavirus, Sudan ebolavirus, Tai Forest ebolavirus, Zaire ebolavirus, or a new strain or species of Ebolavirus. In some embodiments, the Ebola virus is Zaire ebolavirus. In some embodiments, the negatively charged particles are polystyrene particles. In other embodiments, the negatively charged particles are diamond particles. In still other embodiments, the negatively charged particles are poly(lactic-co-glycolic acid) (PLGA) particles. In some embodiments, the particles are PLURONICS® stabilized polypropylene sulfide particles. In a further embodiment, the negatively charged particles are carboxylated.

[0018] In still a further embodiment, particles encapsulating antigens are provided. In one embodiment, the antigens comprise one or more epitopes associated with a filovirus infection. In one embodiment, the one or more epitopes is associated with Cuevavirus,

Marburgvirus, or Ebolavirus. In some embodiments, the one or more epitopes is associated with Bundibugyo ebolavirus, Reston ebolavirus, Sudan ebolavirus, Tai Forest ebolavirus,

Zaire ebolavirus, or a new strain or species of Ebolavirus. In one embodiment, the one or more epitopes is associated with Zaire ebolavirus. In a further embodiment, the antigens comprise more than one epitope associated with the same filovirus infection. In yet a further embodiment, the antigens comprise more than one epitope associated with different filovirus infections. In a further embodiment, the antigens comprise one or more epitopes associated with one or more filovirus infections.

[0019] In one embodiment, the negatively charged particles have a zeta potential of less than about -100 mV. In one embodiment, the negatively charged particles have a zeta potential between about -100 mV and -25 mV. In some embodiments, the negatively charged particles with a zeta potential between -100 mV and -25 mV are carboxylated. In another embodiment, the negatively charged particles have a zeta potential between -100 mV and -75 mV. In another embodiment, the negatively charged particles have a zeta potential between - 50 mV and -20 mV. In a particular embodiment, the negatively charged particles have a zeta potential between -50 mV and -40 mV. In another embodiment, the charged particles have a zeta potential between -100 mV and -50 mV. In one embodiment, the charged particles have a zeta potential between -75 mV and -50 mV.

[0020] In one embodiment, the pharmaceutical formulations of the current invention decrease and/or inhibit the infiltration of inflammatory monocytes to inflammatory foci. In another embodiment, the pharmaceutical formulations of the current invention ameliorate an inflammatory immune response.

[0021] In one embodiment, the pharmaceutical formulations of the current invention increase the number of regulatory T cells. In another embodiment, the pharmaceutical formulations of the current invention ameliorate an inflammatory immune response.

[0022] In one embodiment, the pharmaceutical formulations of the current invention comprise negatively charged particles with an average diameter of about 0.1 μιη to about 10 μιη. In another embodiment the negatively charged particles have an average diameter of about 0.2 μιη to about 2 μιη. In a further embodiment, the negatively charged particles have an average diameter of about 0.3 μιη to about 5 μιη. In yet a further embodiment the negatively charged particles have an average diameter of about 0.5 μιη to about 3 μιη. In still a further embodiment, the negatively charged particles have an average diameter of about 0.5 μιη.

[0023] In one embodiment, the subject has a filovirus infection. In a further embodiment, the subject is human. In one embodiment, the composition alleviates at least one symptom associated with filovirus infection. In a further embodiment, said at least one symptom is hemorrhagic fever.

[0024] In one embodiment, the method includes administering the negatively charged particles by any suitable means. In one embodiment, the composition is administered orally, nasally, intravenously, intramuscularly, ocularly, transdermally, or subcutaneously. In a particular embodiment, the carboxylated particles are administered nasally. In still another embodiment, the negatively charged particles are administered intravenously. In still another embodiment, the negatively charged particles are administered subcutaneously.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Fig. 1 is a schematic illustrating the switch from protective to pathogenic immune responses in viral disease and the restoration of a balanced immune response with immune modifying nanoparticles.

DETAILED DESCRIPTION OF THE INVENTION

[0026] The present inventors have surprisingly found that when negatively charged particles, such as polystyrene, PLGA, or diamond particles of a certain size and zeta potential, are administered to subjects, inflammatory immune responses are ameliorated. Additionally, the present inventors have also surprisingly found that these same negatively charged particles, when administered to subjects, induce monocyte and/or neutrophil apoptosis and improved clearance in the subject. It was also surprising that these same negatively charged particles, when administered to subjects, induce toleragenic dendritic cells, suppressor cells, and regulatory T cells. These negatively charged particles have also been found to remove pro-inflammatory mediators from the inflammatory milieu. Similarly, the negatively charged particles may also concentrate and present regulatory mediators to further induce a regulatory T cell response or otherwise ameliorate the inflammatory immune response. Negatively charged particles, therefore, may be useful in the treatment of any disease or condition characterized by an excessive inflammatory immune response, such as viral infections.

[0027] 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 Ebola virus, and do not require that the particles attached to any peptides, antigens, pharmaceuticals, drugs, oligonucleotides, phospholipids, or any other bioactive agents. [0028] "Filovirus" as used herein refers to a virus belonging to the family Filoviridae in the order of Mononegavirales. The filo viruses are enveloped, non-segmented, negative- stranded R A viruses of varying morphology, and the virus particles are filamentous in nature. The family Filoviridae includes three genera: Cuevavirus, Marburgvirus and Ebolavirus. The five known species in the genus Ebolavirus are Bundibugyo ebolavirus, Reston ebolavirus, Sudan ebolavirus, Tai Forest ebolavirus (originally Cote d'lvoire ebolavirus) and Zaire ebolavirus. Ebola viruses have a uniform diameter of 80 nm but can vary greatly in length, with particle length being as long as 14000 nm.

[0029] "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 and spheres.

[0030] "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).

[0031] "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.

[0032] "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.

[0033] "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.

[0034] "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. [0035] "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.

[0036] 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.

[0037] 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.

[0038] "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.

[0039] "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."

[0040] "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.

[0041] 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 PCT Applications, PCT/US2011/060537 and PCT/US2014/026719, both of which are hereby incorporated by reference in their entireties.

[0042] 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 μιη to about 10 μιη 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 0.2μιη to about 2 μιη. In another embodiment, the particle has an average diameter of about 0.3 μιη to about 5 μιη. In still another embodiment, the particle has an average diameter of about 0.5 μιη to about 3 μιη. In a further embodiment the particle has an average size of about 0.1 μιη, or about 0.2 μιη or about 0.3 μιη or about 0.4 μιη or about 0.5 μιη or about 1.0 μιη or about 1.5 μιη or about 2.0 μιη or about 2.5 μιη or about 3.0 μιη or about 3.5 μιη or about 4.0 μιη or about 4.5 μιη or about 5.0 μιη In a particular embodiment the particle has a size of about 0.5 μιη. 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 μιη, while others are about 1.0 μιη. Any mixture of particle sizes within these given ranges will be useful.

[0043] 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, and diamond particles.

[0044] Preferably the particle surface is composed of a material that minimizes nonspecific 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 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.

[0045] Certain embodiments contemplate that the negatively changed particles themselves are the bioactive agents capable of treating or inhibiting filo virus infection in a subject, as opposed to being a delivery system for a bioactive agent that treats or inhibits filovirus infection. 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, R Ai, shR A, siRNA, a small nucleotide, a natural or chemically modified mononucleotide, a lipopeptide, a phospholipid, an antimicrobial, a small molecule, and a pharmaceutical molecule.

[0046] 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 tunable 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 used in biological labeling, 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).

[0047] 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]oly(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 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 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 nonbiodegradable 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.

[0048] 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.

[0049] In one embodiment of the invention, the immune modified particles contain copolymers. 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) co-polymers. 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, polylactic acid: polyglycolic acid/80:20, or polylactic acid: polyglycolic acid/90:10.

[0050] 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. [0051] 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.

[0052] 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.

[0053] 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 231 nm.

[0054] 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.

[0055] 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.

[0056] 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).

[0057] The solvent used can also affect particle size; solvents that reduce the surface tension of the solution also reduce particle size.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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-tocopheryl 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.

[0062] 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. [0063] 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.

[0064] 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).

[0065] 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.

[0066] Suitable beads which are currently available commercially include polystyrene beads such as FluoSpheres (Molecular Probes, Eugene, Oreg.).

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] 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 or viral infection in a subject in need thereof. In particular embodiments, the viral invention is an filovirus infection. In certain embodiments, about 10² to about 10²⁰ particles are provided to the individual. In a further embodiment between about 10³ to about 10¹⁵ 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 μιη beads, a preferred dose is approximately 4 x 10⁹ beads, for 0.05μιη beads, a preferred dose is approximately 4 x 10¹² beads, for 3μιη 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.

[0075] The invention is useful for treatment of immune related disorders such as autoimmune disease, transplant rejection, inflammatory diseases and/or disorders, ischemia reperfusion, stroke, myocardial infarction and allergic reactions. Substitution of a synthetic, biocompatible particle system to induce immune tolerance could lead to ease of manufacturing, broad availability of therapeutic agents, increase uniformity between samples, increase the number of potential treatment sites and dramatically reduce the potential for allergic responses to a carrier cell. [0076] As illustrated by Fig. 1, particular embodiments contemplate a model whereby a viral infection in a subject progresses in stages. The virus initially invades tissues and multiplies. The first defense is the activation of the intracellular anti-viral 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 virus and life-long 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 not limited to , monocytes, and T cells, leading to immune pathology, tissue damage, and death. In the case of infection with Ebola virus, as few as 10% of subjects and up to 100%, depending on the outbreak and strain, experience an aberrant immune response, resulting in a mortality of 50-90%. 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 Ebola virus will prevent, reduce, or reverse the aberrant immune stimulation and improve probability of survival. In particular embodiments, a subject infected with Ebola virus 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.

[0077] 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.

[0078] 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).

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] In another aspect of the present invention, negatively charged particles encompassing antigens are provided. Nanoparticles carrying antigen on their surface have been successfully used to induce T-cell tolerance (Getts et al, 2012 Nature Biotechnology 30: 1217-1223). Tolerance induced by peptide-coupled particles depends on both the induction of T-cell anergy and the activity of regulatory T cells and may represent an alternative way to treat autoimmune disorders by inducing T-cell tolerance. This T-cell tolerance was observed when using peptides couple to biodegradable (PLG) particles.

[0086] In one embodiment, the negatively charged particles of the invention are coupled to antigens comprising one or more epitopes associated with a filovirus infection. The antigens may comprise one or more copies of an epitope. In one embodiment, the antigens comprise a single epitope associated with a filovirus infection. In one embodiment, the epitope is associated with Cuevavirus, Marburgvirus or Ebolavirus. In some embodiments, the epitope is associated with Bundibugyo ebolavirus, Reston ebolavirus, Sudan ebolavirus, Tai Forest ebolavirus, Zaire ebolavirus or a new strain or species of Ebolavirus. In a further embodiment, the antigens comprise more than one epitope associated with the same filovirus infection. In yet a further embodiment, the antigens comprise more than one epitope associated with different filovirus infections. In a further embodiment, the antigens comprise one or more epitopes associated with one or more filovirus infections.

[0087] Not all epitopes are linear epitopes; epitopes can also be discontinuous, conformational epitopes.

[0088] In another aspect of the present invention, particles encapsulating antigens are provided. Particles which encapsulate antigens inside the particle can be used to induce T- cell tolerance 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 viral and bacterial antigens, endogenous antigens, autoantigens, tumor antigens, and/or native antigens.

In one embodiment, the antigens comprise one or more epitopes associated with a filovirus infection. In one embodiment, the one or more epitopes is associated with Cuevavirus, Marburgvirus or Ebolavirus. In some embodiments, the one or more epitopes is associated with Bundibugyo ebolavirus, Reston ebolavirus, Sudan ebolavirus, Tai Forest ebolavirus, Zaire ebolavirus or a new strain or species of Ebolavirus. In a further embodiment, the antigens comprise more than one epitope associated with the same filovirus infection. In yet a further embodiment, the antigens comprise more than one epitope associated with different filovirus infections. In a further embodiment, the antigens comprise one or more epitopes associated with one or more filovirus infections.

[0089] 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 proteo lyrically 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).

[0090] 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. 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.

[0091] 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.

[0092] Certain embodiments are directed to administering a pharmaceutical composition to a subject with filovirus infection, the pharmaceutical composition comprising negatively charged particles and a carrier, wherein the negatively charged particles are free from attached are free from attached peptide moieties, antigenic moieties, and/or bioactive agents. In some embodiments, the pharmaceutical composition is administered to treat or inhibit filovirus infection. In certain embodiments, the pharmaceutical composition is administered to remove pro-inflammatory mediators from the inflammatory milieu in a subject with filovirus infection. In various embodiments, the pharmaceutical composition is administered to a subject to induce regulatory T cells in a subject with a filovirus infection. In some embodiments, the pharmaceutical composition is administered to reduce a viral load in a subject with a filovirus infection. In some embodiments, the pharmaceutical composition is administered to control a pathologic and/or unwanted inflammatory immune response in a subject with a filovirus 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, reduce a viral load, and/or control a pathologic and/or an unwanted inflammatory response in a subject with filovirus infection, without attached or added bioactive agents, peptide moieties, and/or antigenic moieties.

[0093] Particular embodiments are directed to administering to a subject a pharmaceutical composition to a subject with filovirus 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, reduce a viral load, and/or control a pathologic and/or an unwanted inflammatory response in a subject with filo virus 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.

[0094] 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 proinflammatory 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.

[0095] In another aspect, the methods of the current invention contemplate using particles to bind to DNA and/or R A.

[0096] 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.

[0097] As discussed above, this invention provides novel compounds that have biological properties useful for the treatment of immune mediated disorders.

[0098] 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.

[0099] 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.

[00100] 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.

[00101] 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.

[00102] 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.

[00103] 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.

[00104] 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.

[00105] 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.

[00106] 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.

[00107] 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.

[00108] 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.

[00109] 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.

[00110] 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.

[00111] 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 (T weens, 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.

[00112] 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. 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).

[00113] 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.

[00114] The invention provides methods of regulating an immune response in an individual, preferably a mammal, more preferably a human, comprising administering to the individual the modified particles described herein. Methods of immunoregulation provided by the invention include those that suppress and/or inhibit an innate immune response or an adaptive immune response, including, but not limited to, an immune response stimulated by immunostimulatory polypeptides or viral or bacterial components.

[00115] 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.

[00116] 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.

[00117] Animal models for the study of the pathology of Ebola virus infections are known in the art. For example, mice, guinea pigs and non-human primates have all been used to study the pathogenesis of the virus or to test vaccines.

[00118] In certain embodiments, the individual suffers from a viral infection. An individual having a viral infection is an individual with a recognizable symptom of an existing viral infection.

[00119] A non- limiting list of viral infections treatable with the modified particles of the current invention includes filovirus infections, herpes virus infections, hepatitis virus infections, west nile virus infections, flavivrus infections, influenza virus infections, rhinovirus infections, papillomavirus infections, paramyxovirus infections, parainfluenza virus infections, and retrovirus infections. Preferred viruses are those viruses that cause manifestations such as systemic (prostration), gastrointestinal (anorexia, nausea, vomiting, abdominal pain, diarrhea), respiratory (chest pain, shortness of breath, cough, nasal discharge), vascular (conjunctival injection, postural hypotension, edema), and neurological (headache, confusion, coma). Preferred viruses also include those that cause hemorrhagic fever, multi-organ failure and/or septic shock.

[00120] 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.

[00121] 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.

[00122] 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.

[00123] 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 filo virus infection 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 filo virus is Cuevavirus, Marburgvirus, or Ebolavirus.

3. The method of claim 2, wherein the Ebolavirus is Bundibugyo ebolavirus, Reston ebolavirus, Sudan ebolavirus, Tai Forest ebolavirus, Zaire ebolavirus or a new strain or species of Ebolavirus.

4. The method of claim 3, wherein the Ebolavirus is Zaire ebolavirus.

5. The method of any of claims 1-4, wherein said composition alleviates at least one symptom associated with filovirus infection.

6. The method of claim 5, wherein said at least one symptom is hemorrhagic fever.

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

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

9. The method of claim 7, wherein said particles are diamond particles.

10. The method of claim 7, wherein said particles are PLGA particles.

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

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

13. The method of any of claims 1-1 1, wherein the particles have a zeta potential between about -100 mV and about -15 mV.

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

15. The method of claim 13, wherein the particles have a zeta potential between about -50 mV and about -20 mV.

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

17. The method of any of claims 1-16, wherein the diameter of said negatively charged particles is between about 0.1 μιη to about 10 μιη.

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

19. The method of claim 17, wherein the diameter of said negatively charged particles is between about 0.5 μιη to about 3 μιη.

20. The method of claim 17, wherein the diameter of said negatively charged particles is between about 0.5 μιη to about 1 μιη.

21. The method of claim 17, wherein the diameter of the said negatively charged particles is about 0.5 μιη.

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

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

24. A method for removing pro-inflammatory mediators from the inflammatory milieu in a subject with a filo virus infection, 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.

25. The method of claim 24, wherein the filovirus is Cuevavirus, Marburgvirus, or Ebolavirus.

26. The method of claim 25, wherein the Ebolavirus is Bundibugyo ebolavirus, Reston ebolavirus, Sudan ebolavirus, Tai Forest ebolavirus, Zaire ebolavirus or a new strain or species of Ebolavirus.

27. The method of claim 26, wherein the Ebolavirus is Zaire ebolavirus.

28. The method of any of claims 24-27, wherein said composition alleviates at least one symptom associated with filovirus infection.

29. The method of claim 28, wherein said at least one symptom is hemorrhagic fever.

30. The method of any of clams 24-29, wherein said pro-inflammatory mediators produced in the subject bind to the negatively charged particles.

31. The method of any of claims 24-30, wherein said negatively charged particles are polystyrene particles, diamond particles, PLURIONICS stabilized polypropylene sulfide particles, or poly(lactic-co-glycolic acid) (PLGA) particles.

32. The method of claim 31 , wherein said particles are polystyrene particles.

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

34. The method of claim 31, wherein said particles are PLGA particles.

35. The method of claim 24, wherein said particles are carboxylated.

36. The method of any of claims 24-35, wherein the particles have a zeta potential of less than about -100 mV.

37. The method of any of claims 24-35, wherein the particles have a zeta potential between about -100 mV and about -15 mV.

38. The method of claim 37, wherein the particles have a zeta potential between about - 100 mV and about -75 mV.

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

40. The method of any of claims 24-39, wherein said composition ameliorates an inflammatory immune response.

41. The method of any of claims 24-40, wherein the diameter of said negatively charged particles is between about 0.1 μιη to about 10 μιη.

42. The method of claim 41, wherein the diameter of said negatively charged particles is between about 0.3 μιη to about 5 μιη.

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

44. The method of claim 41, wherein the diameter of said negatively charged particles is between about 0.5 μιη to about 1 μιη.

45. The method of claim 41, wherein the diameter of the said negatively charged particles is about 0.5 μιη.

46. The method of any of claims 24-45, wherein said subject is a human.

47. The method of any of claims 24-46, wherein said composition is administered orally, nasally, intravenously, intramuscularly, ocularly, transdermally, or subcutaneously.

48. A method for inducing regulatory T cells in a subject with a filovirus infection, 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.

49. The method of claim 48 wherein the regulatory T cells comprise CD4+ cells.

50. The method of claim 48 or 49 wherein the regulatory T cells comprise CD8⁺ T cells.

51. The method of any of claims 48-50, wherein the filovirus is Cuevavirus, Marburgvirus or Ebolavirus.

52. The method of claim 51, wherein the Ebolavirus is Bundibugyo ebolavirus, Reston ebolavirus, Sudan ebolavirus, Tai Forest ebolavirus, Zaire ebolavirus or a new strain or species of Ebolavirus.

53. The method of claim 52, wherein the Ebolavirus is Zaire ebolavirus.

54. The method of any of claims 48-53, wherein said composition alleviates at least one symptom associated with filovirus infection.

55. The method of claim 54, wherein said at least one symptom is hemorrhagic fever.

56. The method of any of claims 48-55, wherein said negatively charged particles are polystyrene particles, diamond particles, PLURIONICS stabilized polypropylene sulfide particles, or poly(lactic-co-glycolic acid) (PLGA) particles.

57. The method of claim 56, wherein said particles are polystyrene particles.

58. The method of claim 56, wherein said particles are diamond particles.

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

60. The method of any of claims 48-59, wherein the particles are carboxylated.

61. The method of any of claims 48-60, wherein the particles have a zeta potential of less than about -100 mV.

62. The method of any of claims 48-60, wherein the particles have a zeta potential between about -100 mV and about -15 mV.

63. The method of claim 62, wherein the particles have a zeta potential between about 100 mV and about -75 mV.

64. The method of claim 62, wherein the particles have a zeta potential between about -50 mV and about -20 mV.

65. The method of any of claims 48-64, wherein said composition ameliorates an inflammatory immune response.

66. The method of any of claims 48-65, wherein the diameter of said negatively charged particles is between about 0.1 μιη to about 10 μιη.

67. The method of claim 66, wherein the diameter of said negatively charged particles is between about 0.3 μιη to about 5 μιη.

68. The method of claim 66, wherein the diameter of said negatively charged particles is between about 0.5 μιη to about 3 μιη.

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

70. The method of claim 66, wherein the diameter of the said negatively charged particles is about 0.5 μιη.

71. The method of any of claims 48-70, wherein said subject is a human.

72. The method of any of claims 48-71, wherein said composition is administered orally, nasally, intravenously, intramuscularly, ocularly, transdermally, or subcutaneously.

73. A method for decreasing a viral load in a subject with a filo virus infection 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.

74. The method of claim 73, wherein said viral load is measured by the number of viral genome copies present per milliliter of blood serum.

75. The method of claim 74, wherein said viral load decreases to less than about 10⁷ viral genome copies per milliliter of blood serum.

76. The method of claim 73, wherein said viral load is measured by the number of plaque forming units present per milliliter of blood serum.

77. The method of claim 76, wherein said viral load decreases to less than about 10⁵ plaque forming units present per milliliter of blood serum.

78. The method of claim 73, wherein said viral load is measured by viral envelope glycoprotein expression.

79. The method of claim 78, wherein the viral envelope glycoprotein expression of said subject is compared to the viral envelope glycoprotein expression of a subject with a filovirus infection not administered said composition.

80. The method of claim 79, wherein the viral envelope glycoprotein expression of said subject is 10- to 100-fold lower compared to the viral envelope glycoprotein expression of a subject with a filovirus infection not administered said composition.

81. The method of any of claims 73-80, wherein the filovirus is Cuevavirus, Marburgvirus, or Ebolavirus.

82. The method of any of claims 73-80, wherein the Ebolavirus is Bundibugyo ebolavirus, Reston ebolavirus, Sudan ebolavirus, Tai Forest ebolavirus, Zaire ebolavirus, or a new strain or species of Ebolavirus.

83. The method of claim 82, wherein the Ebolavirus is Zaire ebolavirus.

84. The method of any of claims 73-83, wherein said composition alleviates at least one symptom associated with filovirus infection.

85. The method of claim 84, wherein said at least one symptom is hemorrhagic fever.

86. The method of any of claims 73-85, wherein said negatively charged particles are polystyrene particles, diamond particles, PLURIONICS stabilized polypropylene sulfide particles, or poly(lactic-co-glycolic acid) (PLGA) particles.

87. The method of claim 86, wherein said particles are polystyrene particles.

88. The method of claim 86, wherein said particles are diamond particles.

89. The method of claim 86, wherein said particles are PLGA particles.

90. The method of any of claims 73-89, wherein said particles are carboxylated.

91. The method of any of claims 73-90, wherein the particles have a zeta potential of less than about -100 mV.

92. The method of claim 91, wherein the particles have a zeta potential between about - 100 mV and about -15 mV.

93. The method of claim 91, wherein the particles have a zeta potential between about - 100 mV and about -75 mV.

94. The method of claim 91, wherein the particles have a zeta potential between about -50 mV and about -20 mV.

95. The method of any of claims 73-94, wherein said composition ameliorates an inflammatory immune response.

96. The method of any of claims 73-95, wherein the diameter of said negatively charged particles is between about 0.1 μιη to about 10 μιη.

97. The method of claim 96, wherein the diameter of said negatively charged particles is between about 0.3 μιη to about 5 μιη.

98. The method of claim 96, wherein the diameter of said negatively charged particles is between about 0.5 μιη to about 3 μιη.

99. The method of claim 96, wherein the diameter of said negatively charged particles is between about 0.5 μιη to about 1 μιη.

100. The method of claim 96, wherein the diameter of the said negatively charged particles is about 0.5 μιη.

101. The method of any of claims 73-100, wherein said subject is a human.

102. The method of any of claims 73-101, wherein said composition is administered orally, nasally, intravenously, intramuscularly, ocularly, transdermally, or subcutaneously.

103. A method for controlling a pathologic and/or unwanted inflammatory immune response in a subject with a filo virus infection 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.

104. The method of claim 103, wherein the filovirus is Cuevavirus, Marburgvirus, or Ebolavirus.

105. The method of claim 104, wherein the Ebolavirus is Bundibugyo ebolavirus, Reston ebolavirus, Sudan ebolavirus, Tai Forest ebolavirus, Zaire ebolavirus, or a new strain or species of Ebolavirus.

106. The method of claim 105, wherein the Ebolavirus is Zaire ebolavirus.

107. The method of any of claims 103-106, wherein said composition alleviates at least one symptom associated with filovirus infection.

108. The method of claim 107, wherein said at least one symptom is hemorrhagic fever.

109. The method of any of claims 103-108, wherein said negatively charged particles are polystyrene particles, diamond particles, PLURIONICS stabilized polypropylene sulfide particles, or poly(lactic-co-glycolic acid) (PLGA) particles.

110. The method of claim 109, wherein said particles are polystyrene particles.

111. The method of claim 109, wherein said particles are diamond particles.

112. The method of claim 109, wherein said particles are PLGA particles.

113. The method of any of claims 103-112, wherein said particles are carboxylated.

114. The method of any of claims 103-113, wherein the particles have a zeta potential of less than about -100 mV.

115. The method of claim 114, wherein the particles have a zeta potential between -about 100 mV and about -25 mV.

116. The method of claim 114, wherein the particles have a zeta potential between about - 100 mV and about -25 mV.

117. The method of claim 114, wherein the particles have a zeta potential between about - 50 mV and about -20 mV.

118. The method of any of claims 103-117, wherein said composition ameliorates an inflammatory immune response.

119. The method of any of claims 103-118, wherein the diameter of said negatively charged particles is between about 0.1 μιη to about 10 μιη.

120. The method of claim 119, wherein the diameter of said negatively charged particles is between about 0.3 μιη to about 5 μιη.

121. The method of claim 119, wherein the diameter of said negatively charged particles is between about 0.5 μιη to about 3 μιη.

122. The method of claim 119, wherein the diameter of said negatively charged particles is between about 0.5 μιη to about 1 μιη.

123. The method of claim 119, wherein the diameter of the said negatively charged

particles is about 0.5 μιη.

124. The method of any of claims 103-123, wherein said subject is a human.

125. The method of any of claims 103-124, wherein said composition is administered orally, nasally, intravenously, intramuscularly, ocularly, transdermally, or subcutaneously.

126. A method of inhibiting or treating filo virus infection in a subject, said method

comprising administering to said subject a pharmaceutical composition comprising negatively charged particles, wherein said particles comprise an antigen comprising one or more epitopes associated with said filovirus infection.

127. The method of claim 126, wherein the filovirus is Cuevavirus, Marburgvirus, or Ebolavirus. The method of claim 127, wherein the Ebolavirus is Bundibugyo ebolavirus, Reston ebolavirus, Sudan ebolavirus, Tai Forest ebolavirus, Zaire ebolavirus, or a new strain or species of Ebolavirus.

The method of claim 128, wherein the Ebolavirus is Zaire ebolavirus.

The method of any of claims 126-129, wherein the one or more epitopes is associated with Cuevavirus, Marburgvirus, or Ebolavirus.

1. The method of any of claims 126-129, wherein the one or more epitopes is associated with Bundibugyo ebolavirus, Reston ebolavirus, Sudan ebolavirus, Tai Forest ebolavirus, Zaire ebolavirus, or a new strain or species of Ebolavirus.

The method of claim 131, wherein the one or more epitopes is associated with Zaire ebolavirus.

The method of any of claims 126-132, wherein the one or more epitopes is associated with the same filovirus infection.

The method of any of claims 126-132, wherein the one or more epitopes is associated with different filovirus infections.

The method of any of claims 126-134, wherein said composition alleviates at least one symptom associated with filovirus infection.

The method of claim 135, wherein said at least one symptom is hemorrhagic fever.

The method of any of claims 126-136, wherein said negatively charged particles are polystyrene particles, diamond particles, PLURIONICS stabilized polypropylene sulfide particles, or poly(lactic-co-glycolic acid) (PLGA) particles.

The method of claim 137, wherein said particles are polystyrene particles.

The method of claim 137, wherein said particles are diamond particles.

The method of claim 137, wherein said particles are PLGA particles. The method of any of claims 126-140, wherein said particles are carboxylated.

The method of any of claims 126-141, wherein the particles have a zeta potential of less than about -100 mV.

The method of any of claims 126-141, wherein the particles have a zeta potential between about -100 mV and about -25 mV.

The method of claim 143, wherein the particles have a zeta potential between about - 100 mV and about -75 mV.

The method of claim 143, wherein the particles have a zeta potential between about - 50 mV and about -20 mV.

The method of any of claims 126-145, wherein said composition ameliorates an inflammatory immune response.

The method of any of claims 126-146, wherein the diameter of said negatively charged particles is between about 0.1 μιη to about 10 μιη.

The method of claim 147, wherein the diameter of said negatively charged particles is between about 0.3 μιη to about 5 μιη.

The method of claim 147, wherein the diameter of said negatively charged particles is between about 0.5 μιη to about 3 μιη.

The method of claim 147, wherein the diameter of said negatively charged particles is between about 0.5 μιη to about 1 μιη.

1. The method of claim 147, wherein the diameter of the said negatively charged

particles is about 0.5 μιη.

The method of any of claims 126-151, wherein said subject is a human. The method of any of claims 126-152, wherein said composition is administered orally, nasally, intravenously, intramuscularly, ocularly, transdermally, or subcutaneously.