Patent Grant
8278083
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 25, 2012, is Sequence_Listing_CRF_NIHC6029 and is 1,930 bytes in size.
The invention is related to a method for universal inactivation of viruses, parasites and tumor cells. These inactivated agents can be used as vaccines against the diseases caused by such viruses, parasites and tumor cells. The inventive inactivation method preserves the integrity of structural and conformational features of the agent. Hence, the immunogenicity of the agent as a whole is maintained and can be safely used for vaccination without the threat of infection.
Vaccination against pathogens has been one of the major accomplishments of medicine over the past century. While effective vaccines have been developed for a large number of diseases, development of safe and effective vaccines for a number of other diseases remains problematic. For example, the most commonly used approach for inactivation of enveloped viruses is cross-linking of surface glycoproteins by formaldehyde. While this approach results in inactivation of the enveloped viruses, covalent cross-linking of the surface proteins can severely distort the structure of immunogenic epitopes (Koch et al., Apmis 104:115-25 (1996)). The use of inactivated or killed microbial agents as a vaccine, although generally safe, will not always be effective if the immunogenic characteristics of the agent are altered. Indeed, the preferential degradation of certain antigens on the inactivated microorganisms might produce a weak or poorly targeted immune response that permits a pathological response when the host is later challenged with the live microorganism. On the other hand, while the preparation of live attenuated microbial agents as vaccines will often provide improved immunologic reactivity, use of such live attenuated microbial agents has an increased risk that the vaccine itself will be infectious. Such live attenuated vaccines can be infectious, for example, as a result of reversion, or the organism may be able to propagate and provide a reservoir for future infection.
Thus, one must generally choose between improved effectiveness and greater degree of safety when selecting between the viral inactivation and viral attenuation techniques for vaccine preparation. The choice is particularly difficult when the virus is resistant to inactivation and requires highly rigorous inactivation conditions that are likely to degrade the antigenic characteristics.
It is therefore desirable to provide improved methods for inactivating agents such as viruses, bacteria, cancer cells and other cell types, where the methods are capable of inactivating these agents without causing substantial degradation of the antigenic structure of the agents. In particular, the inactivated agents should be useful as vaccines and free from adverse side effects at the time of administration as well as upon subsequent challenge with the live agent.
The invention provides methods for inactivating an infective agent or cancer cell that involve exposing the agent or cell to a hydrophobic photoactivatable compound, for example, 1,5-iodonaphthylazide (INA). These photoactivatable compounds are non-toxic, hydrophobic compounds that penetrate into the innermost regions of biological membrane bilayers and selectively accumulate in such inner membrane regions. Upon irradiation with light, a reactive derivative of the compound is generated that binds to membrane proteins deep in the lipid bilayer. This process specifically inactivates integral membrane proteins embedded in the membrane while maintaining the structural integrity and activity of the proteins that protrude from the extracellular surface of the membrane. Such inactivation is so successful that the inactivated virus, microbe, infective agent, cancer cell or other agent of interest, is non-infective and can be used as a vaccine. In some embodiments, the agent is an inactivated virus, for example, influenza virus.
One aspect of the invention is a composition comprising a pharmaceutically acceptable carrier and an inactivated influenza virus, wherein the inactivated influenza virus is made by (a) contacting an influenza virus with an effective amount of a photoactivatable hydrophobic compound to form a mixture of the influenza virus with the photoactivatable hydrophobic compound, and (b) exposing the mixture to light for a time sufficient to generate the inactivated influenza virus.
The photoactivatable hydrophobic compound employed in the present compositions and methods is a compound of formula (I):
X—Ar—Y I
wherein:
The Ar group is a moiety that preferentially partitions out of an aqueous environment and into a cellular or viral membrane. In another embodiment, the Ar group is a linear, branched, cyclic or acyclic hydrocarbon or a combination thereof. In another embodiment, the Ar group is a fatty acid, alkyl, adamantane, phenyl, naphthyl, anthracene, pyrene, or phenanthracene group.
The X and Y reactive groups separately are azido, halo, halo lower alkyl, diazirene, azidocarbonyl)oxy, haloacetamide, amine, maleimide, isocyanato, isothiocyanato, acyl halide, succinimidyl ester, or sulfosuccinimidyl ester.
Examples of photoactivatable hydrophobic compounds that can be used in the invention include azidobenzene, 1-azidonaphthalene, 4-azido-2-nitro-1-(phenylthio)benzene, 1-azido-4-iodobenzene, 1-azido-5-iodonaphthalene, 3-phenyl-3H-diazirene, 3-phenyl-3-(trifluoromethyl)-3H-diazirene, 3-(3-iodophenyl)-3-(trifluoromethyl)-3H-diazirene, 1-azidopyrene, adamantanediazirene, 12-(4-azido-2-nitrophenoxy)-stearic acid, w-(m-diazirinophenoxy)fatty acid, 12-[(azidocarbonyl)oxy]stearic acid, 12-azidostearic acid, 11-(3-azidophenoxy)undecanoic acid or w-(m-diazirinophenoxy)undecanoic acid. In some embodiments, the photoactivatable hydrophobic compound is 1,5-iodonaphthyl azide.
The light employed can be ultraviolet light. In other embodiments, the light is visible light and an effective amount of a photosensitizer chromophore is included in the mixture. Examples of photosensitizer chromophores that can be used include porphyrin, chlorin, bacteriochlorin, purpurin, phthalocyanine, naphthalocyanine, merocyanines, carbocyanine, texaphyrin, or non-tetrapyrrole chromphores. Further examples include the photosensitizer chromophore is fluorescein, eosin, bodipy, nitro-benzo-diazol (NBD), erythrosine, acridine orange, doxorubicin, rhodamine 123, or picoerythrin.
A variety of viruses can be inactivated and used in the compositions of the invention. One example of a virus that can be inactivated and used in the compositions and methods of the invention is an influenza virus. The influenza virus can be an influenza virus type A, influenza virus type B or any other type of influenza virus. Further examples of viruses that can be inactivated by use of the inventive methods include a hepatitis A virus, hepatitis B virus, hepatitis C virus, simian immunodeficiency virus, human immunodeficiency virus, Ebola virus, poxvirus, herpes virus, adenovirus, papovavirus, parvovirus, reovirus, orbivirus, picornavirus, rotavirus, alphavirus, rubivirus, influenza virus type A, influenza virus type B, flavivirus, coronavirus, paramyxovirus, morbillivirus, pneumovirus, rhabdovirus, lyssavirus, orthmyxovirus, bunyavirus, phlebovirus, nairovirus, hepadnavirus, arenavirus, retrovirus, enterovirus, rhinovirus, filovirus, hemorrhagic fever virus, Chikungunya virus, Japanese encephalitis virus, Monkey pox virus, variola virus, Congo-Crimean hemorrhagic fever virus, Junin virus, Omsk hemorrhagic fever virus, Venezuelan equine encephalitis virus, Dengue fever virus, Lassa fever virus, Rift valley fever virus, Western equine encephalitis virus, Eastern equine encephalitis virus, Lymphocytic choriomeningitis virus, Russian Spring-Summer encephalitis virus, White pox, Machupo virus, Smallpox virus, Yellow fever virus, Hantaan virus, Marburg virus, or Tick-borne encephalitis virus. In some embodiments, the virus is an enveloped virus.
Another aspect of the invention is a vaccine comprising the any of the inactivated viral, bacterial, fungal, cellular or parasite compositions of the invention. In one embodiment the vaccine comprises an inactivated influenza virus.
Another aspect of the invention is a method of inactivating a virus, for example, an influenza virus, comprising contacting the virus with an effective amount of a photoactivatable hydrophobic compound to form a mixture of the virus and the photoactivatable hydrophobic compound, and exposing the mixture to light for a time sufficient to inactivate the virus. In some embodiments the light is ultraviolet light. In other embodiments, the light is visible light and an effective amount of a photosensitizer chromophore is included in the mixture.
Another aspect of the invention is a method of inactivating an influenza virus comprising contacting the influenza virus with an effective amount of 1,5-iodonaphthyl azide to form a mixture of the influenza virus and the 1,5-iodonaphthyl azide, and exposing the mixture to ultraviolet light for a time sufficient to inactivate the influenza virus.
As described herein, while treatment with the hydrophobic photoactivatable compound (e.g., 1,5-iodonaphthylazide (INA)) produces non-infective virus compositions, the structural elements needed for generating a strong immune response against the virus are preserved. Thus, for example, influenza virus treated with INA still exhibits hemagglutinin and/or neuraminidase activity. Moreover, while INA-treated influenza viruses can still bind to mammalian cells, these viruses cannot fuse with the cells and therefore cannot initiate an infective cycle.
According to the invention, treatment of influenza virus with a photoactivatable hydrophobic compound of the invention blocks viral infectivity and fusion of influenza virus with mammalian cells. Moreover, administration of influenza virus treated with such a photoactivatable hydrophobic compound protects mammals against the effects of subsequent influenza viral infections, even if the mammal becomes infected or exposed to a different strain or type of influenza virus. Thus, the invention provides immunological compositions and vaccines that contain influenza virus with a photoactivatable hydrophobic compound of the invention.
Similarly, treatment of tumor cells with a photoactivatable hydrophobic compound of the invention blocks tumor cell division and tumor cell colony formation with substantially no detectable damage to the structural integrity of the tumor cells. Moreover, when live HIV, SIV and Ebola viral particles are treated with appropriate concentration of such photoactivatable hydrophobic compounds, substantially no infectivity is observed. Minor, generally insubstantial changes in the structural integrity of virus particles were observed. These modified viral particles reacted with monoclonal antibodies directed against selected viral proteins and the inactivated viruses bound to their target cells. However, viral fusion with the membrane was impaired by use of the present inventive methods.
Hence, the invention provides new methods for inactivating viruses, bacteria, parasites and tumor cells. These inactivated agents can be used in compositions to stimulate an immune response against active viruses, bacteria, parasites and tumor cells. In another embodiment, the invention provides vaccines to prevent the diseases caused by such viruses, bacteria, parasites and tumor cells.
Photoactivatable Hydrophobic Compounds
Accordingly, as provided herein, a photoactivatable hydrophobic compound of the following formula (I) can be used to inactivate viruses, parasites and tumor cells.
X—Ar—Y I
wherein:
Ar is a hydrophobic moiety; and
X and Y are each independently hydrogen or a reactive group, provided that at least one of X or Y is a reactive group.
The Ar hydrophobic moiety can be any moiety that preferentially partitions out of an aqueous environment and into a cellular or viral membrane. Examples of Ar hydrophobic moieties include linear, branched, cyclic and acyclic hydrocarbons and combinations thereof. The cyclic groups employed can be non-aromatic or aromatic ring moieties. For example, the Ar hydrophobic moiety can be a fatty acid, alkyl, adamantane, phenyl, naphthyl, anthracene, pyrene, phenanthracene or similar moiety.
The X and Y reactive groups are functional groups that are chemically reactive (or that can be made or activated to be chemically reactive) with functional groups typically found in biological materials, or with functional groups that can be readily converted to chemically reactive groups using methods well known in the art. In one embodiment of the invention, the X and/or Y reactive groups are separately azido (—N3), halo (Cl, Br or I), halo lower alkyl (e.g. CF3), diazirene, azidocarbonyl)oxy (—O—CO—N3), haloacetamide (—NH—(C═O)—CH2—Z), where Z is Cl, Br or I. Alternatively, the reactive groups are separately amine, maleimide, isocyanato (—N═C═O), isothiocyanato (—N═C═S), acyl halide, succinimidyl ester, or sulfosuccinimidyl ester. In another embodiment, the reactive groups are carboxylic acid (COOH), or derivatives of a carboxylic acid. An appropriate derivative of a carboxylic acid includes an alkali or alkaline earth metal salt of carboxylic acid. Alternatively, the reactive groups are reactive derivatives of a carboxylic acid (—COOR), where the reactive group R is one that activates the carbonyl group of —COOR toward nucleophilic displacement. In particular, R is any group that activates the carbonyl towards nucleophilic displacement without being incorporated into the final displacement product. Examples of COOR groups include esters of phenol or naphthol that are further substituted by at least one strong electron withdrawing group, or a carboxylic acid activated by carbodiimide. In some embodiments the X and Y reactive groups are acyl chlorides, or azido, succinimidyl or sulfosuccinimidyl esters. Additional charged groups include, among others, sulfonyl halides, sulfonyl azides, alcohols, thiols, semicarbazides, hydrazines or hydroxylamines.
Examples of photoactivatable hydrophobic compounds that can be used in the invention include the following compounds:
11-(3-azidophenoxy)undecanoic acid w-(m-diazirinophenoxy)undecanoic acid
In one embodiment, 1,5-iodonaphthyl azide (INA) is employed as a photoactivatable hydrophobic compound. INA is a non toxic hydrophobic compound. The structure for 1,5-iodonaphthyl azide (INA) is provided below. See also, Bercovici and Gitler 1978, Biochemistry, 17: 1484-89.
Upon exposure to cells, photoactivatable hydrophobic compounds of the invention will penetrate into the innermost regions of biological membrane bilayers and will accumulate selectively in these regions. Photoactivatable hydrophobic compounds of the invention are also light sensitive. Upon irradiation with ultraviolet light (e.g. 320 to 400 nm) a reactive derivative is generated that binds to membrane proteins deep in the lipid bilayer. This process specifically inactivates integral membrane proteins embedded in the membrane while maintaining the integrity and activity of the proteins that protrude from the extracellular surface of the membrane.
In another embodiment, the photoactivatable hydrophobic compounds of the invention can be used for inactivation of viruses, bacteria, parasites and tumor cells using visible light. However, when visible light is used a photosensitizer chromophore is needed. This photosensitizer chromophore has an absorption maximum in the visible light range and can photosensitize the photoactivatable hydrophobic compounds of the invention. In general, the photosensitizer chromophore have absorption maxima in the range of about 450 to about 525 nm or about 600 to about 700 nm. The photosensitizer chromophore can be a porphyrin, chlorin, bacteriochlorin, purpurin, phthalocyanine, naphthalocyanine, merocyanines, carbocyanine, texaphyrin, non-tetrapyrrole, or other photosensitizer known to one of skill in the art. Specific examples of photosensitizer chromophores include fluorescein, eosin, bodipy, nitro-benzo-diazol (NBD), erythrosine, acridine orange, doxorubicin, rhodamine 123, picoerythrin and the like.
Treatment with Photoactivatable Hydrophobic Compounds
As provided herein, viruses, bacteria, parasites and tumor cells can be inactivated by exposure to photoactivatable hydrophobic compounds. In some embodiments the photoactivatable hydrophobic compound is 1,5-iodonaphthyl azide (INA) or a related compound. After contacting the photoactivatable hydrophobic compound with the virus, parasite or tumor cell to form a mixture thereof, the mixture is exposed to light. If the virus, parasite or tumor cell is contacted with just the photoactivatable hydrophobic compound, ultraviolet light is used. If the virus, parasite or tumor cell is contacted with both the photoactivatable hydrophobic compound and a photosensitizer chromophore that absorbs visible light, then visible light can be used instead. Exposure to ultraviolet light directly photoactivates the photoactivatable hydrophobic compound within viral and cellular membranes. Exposure to visible light first photoactivates the photosensitizer chromophore, which then activates or photosensitizes the photoactivatable hydrophobic compound within viral or cellular membranes. In either case, a reactive derivative of the photoactivatable hydrophobic compound is generated that binds to membrane proteins deep within the lipid bilayer. This process causes specific inactivation of integral membrane proteins embedded in the membrane, while maintaining the integrity and activity of proteins that protrude outside of the membrane.
Prior to exposure to a photoactivatable hydrophobic compound, the viruses, parasites or tumor cells can be washed to remove media, waste and other materials that might reduce partitioning of the photoactivatable hydrophobic compound into viral or cellular membranes. For example, the viruses, parasites or tumor cells can be washed in serum-free media, phosphate-buffered saline or other solutions selected by one of skill in the art.
The amount of photoactivatable hydrophobic compound used to inactivate a virus, bacteria, parasite or tumor cell can vary and may depend upon the type of virus, bacteria, parasite or tumor cell as well as the conditions under which the photoactivatable hydrophobic compound is reacted with the virus, bacteria, parasite or tumor cell. For example, if competing hydrophobic molecules are present in the media, then larger amounts of the photoactivatable hydrophobic compound may be needed.
In some embodiments, the concentration of photoactivatable hydrophobic compound employed in a mixture with a virus, parasite or tumor can vary from about 0.1 micromolar to about 1 millimolar, or from about 1 micromolar to about 700 micromolar, or from about 10 micromolar to about 500 micromolar, or from about 20 micromolar to about 400 micromolar, or from about 50 micromolar to about 300 micromolar, or from about 100 micromolar to about 250 micromolar.
When expressed as a ratio of the amount of photoactivatable hydrophobic compound employed per amount of viral, parasite or tumor protein, this ratio can vary from about 0.1 micrograms photoactivatable hydrophobic compound per milligram of viral, parasite or tumor protein to about 500 micrograms photoactivatable hydrophobic compound per milligram of viral, parasite or tumor protein. In other embodiments, the amount of photoactivatable hydrophobic compound used can vary from about 0.5 to about 200, or about 1 to about 150, or about 2 to about 125, or about 3 to about 100 micrograms photoactivatable hydrophobic compound per milligram of viral, parasite or tumor protein.
The amount of photosensitizer chromophore used to activate the photoactivatable hydrophobic compound can also vary and depends to some extent on the photosensitizer chromophore used, the photoactivatable hydrophobic compound employed and the type of virus, bacteria, parasite or tumor cell. For example, about 0.01 mg/ml to about 50 mg/ml photosensitizer chromophore can be used, or about 0.1 mg/ml to about 5 mg/ml photosensitizer chromophore can be used, or about 0.3 mg/ml to about 1 mg/ml photosensitizer chromophore can be used.
After forming a mixture of the virus, bacteria, parasite or tumor cell with a photoactivatable hydrophobic compound, the mixture is exposed to light for a time and under conditions sufficient for generating a reactive hydrophobic derivative that can bind to membrane proteins within the lipid bilayer. The ultraviolet light employed when only the photoactivatable hydrophobic compound is present has a wavelength that is generally above that absorbed by proteins and nucleic acids. Such a wavelength of ultraviolet light does not cause substantial damage to such proteins and nucleic acids. Thus, for example, the wavelength can be about 320 nm to about 400 nm. In some embodiments, the wavelength is about 330 nm to about 380 nm. In other embodiments, the wavelength is about 340 nm to about 360 nm.
Visible light of an appropriate wavelength can be used when a photosensitizer chromophore is employed that is incubated with or is localized in the vicinity of the hydrophobic photoactivatable compound. In general, the photosensitizer chromophores have absorption maxima in the range of about 450 to about 525 nm or about 600 to about 700 nm.
Light for photoactivation of the photosensitizer chromophore or the hydrophobic derivative can be from various light sources. For example, suitable light sources include broadband conventional light sources, broad arrays of LEDs, laser beams, defocused laser beams, optical fiber devices and transillumination. The light can be filtered to eliminate certain types or wavelengths of light. Hence, the light can be filtered to provide ultraviolet light (e.g. 320 to 400 nm), or visible light of selected wavelengths (e.g., 450 to 525 nm or 600 to 700 nm). The light can also be filtered to reduce heat production, for example, by passing the light through water.
Different light sources of different powers can be used: An incandescent light source like tungsten or halogen lamps will have a power range from 100-200 Watt. Mercury or Xenon light sources have a power range of about 100-1000 Watt. A laser source will have the power range of 1-10 Watts. When visible light is used in the presence of a photosensitizer chromophore, the tungsten, halogen, Mercury and Xenon light sources should be equipped with optical filters or a monochromator that will filter out all wavelengths below 400 nm. When a laser is used, the appropriate wavelength line of 400 nm or higher should be used depending on the photosensitizer chromophore employed. Regardless of the light source the intensities of light on the target sample should be in the range of 1-50 milliwatt/cm2/min depending on the nature of the sample and the area irradiated.
Light exposure times can vary. For example, one of skill in the art may choose to expose a mixture of a photosensitizer chromophore and/or a photoactivatable hydrophobic compound with a virus, bacteria, parasite or tumor cell to a light source for about 1 second to about 20 minutes, or about 3 seconds to about 15 minutes, or about 5 seconds to about 10 minutes, or about 7 seconds to about 7 minutes, or about 30 seconds to about 5 minutes. A series of short (e.g. about 1 to about 60 seconds) or longer (e.g. about 20 to about 60 seconds) light exposures can also be employed. When a laser is used, substantially shorter exposure times are typically used, for example, about 0.1 second to about 5 seconds, or about 0.5 seconds to about 3 seconds.
As is appreciated by one of skill in the art, the exposure time can vary depending on the wattage of the light employed. Either cultures or plates of viruses, bacteria, parasites or tumor cells can be treated with a selected photoactivatable hydrophobic compound and/or a photosensitizer chromophore and then exposed to light. The exposure time and wattage of the light employed may be different if a culture or plate of viruses/cells is employed. For example, less exposure may be needed for plated viruses/cells than for viruses/cells cultured in suspension because the depth of the culture may influence the degree to which the light penetrates the culture. Hence, some variation and deviation from the ranges provided herein is possible without deviating from the scope of the invention.
As described in more detail herein, INA has been shown by the inventors to penetrate into the inner most segments of membrane bilayers and accumulate selectively in this domain. As shown herein, upon irradiation of the organism or cell with ultraviolet light (e.g., 320-400 nm), INA is photoactivated in the membrane to generate a reactive derivative that binds to membrane proteins deep within the lipid bilayer. This process causes specific inactivation of integral membrane proteins embedded in the membrane, while maintaining the integrity and activity of proteins that protrude outside the membrane (Raviv et al, 1984 Biochemistry, 23, 503-508).
Methods of Using the Inactivated Microbes, Parasites and Tumor Cells
The invention provides a method that can universally inactivate viruses, bacteria, parasites and tumor cells in a way that they can be safely used as immunological compositions or vaccines to inhibit the disease they cause. The inactivation kills the organism or cell in a specific manner that maintains it's structural and conformational. Hence, the structure of the inactivated virus/cell is similar to that of the live virus/cell. In this way, the immunogenicity of the organism or cell as a whole is maintained and can be safely used to stimulate the immune system of a subject animal or patient. Similarly, the inactivated viruses, bacteria, cancer cells or parasites of the invention can be used for vaccination without causing disease or other negative side effects.
A study conducted by the inventors showed that INA treatment of tumor cells blocked their ability to divide and form colonies, with no detectable damage to the structural integrity of the cells.
Studies by the inventors show that INA can also be used to inactivate live HIV, SIV and Ebola viruses. In particular, INA treatment produced inactive viruses with no detectable infectivity (Table 1 and
Hence, the INA treatment procedures of the invention generate inactive viruses that can be used in a manner similar to aldrithiol inactivated HIV (developed by the AIDS vaccine program SAIC). Alternatively, the INA-inactivation procedures of the invention can be used in conjunction with aldrithiol inactivation procedures to generate inactive HIV that comply with the requirements of the FDA. Thus, two mechanistically independent methods of inactivation can be used to provide a prophylactic AIDS or HIV vaccine.
The present invention is therefore directed to methods of treating or preventing or otherwise ameliorating microbial or parasitic infections in a mammal, as well as other animals, such as farm animals and birds. In another embodiment, the invention provides to methods of treating or preventing or otherwise ameliorating cancer in a mammal, as well as other animals, such as farm animals and birds. These methods include administering to the animal an effective amount, for example, a therapeutically effective amount of an inactivated agent of the present invention, wherein the agent may cause an infection or cancer when not inactivated as described herein.
Prevention or treatment of microbial infections, parasitic infections or cancer is intended to include the alleviation of or diminishment of at least one symptom typically associated with the infection or cancer. Prevention or treatment also includes alleviation or diminishment of more than one symptom. Ideally, treatment with the inactivated agents of the invention generates immunity in the animal towards the agent while prevention by the inactivated agents of the invention substantially eliminates the symptoms associated with the infection or cancer.
Microbial infections that can be treated by the present inactivated agents include infections by any target microbial organisms that can infect a mammal or other animal. Such target microbial organisms include essentially any virus, bacterium, fungus, single cell organism or parasite that can infect an animal, including mammals. For example, target microbial organisms include viruses, bacteria, fungi, yeast strains and other single cell organisms. In another embodiment, the inactivated agents of the invention can give rise to immunity against both gram-negative and gram-positive bacteria.
Treatment of, or prevention of, viral, bacterial, fungal, microbial or parasitic infections is intended to include the alleviation of or diminishment of at least one symptom typically associated with the infection. The treatment also includes alleviation or diminishment of more than one symptom. The treatment may cure the infection, e.g., it may substantially prevent the infection and/or eliminate the symptoms associated with the infection.
Exemplary viral infections that can be treated by the present inactivated agents include infections by any virus that can infect animals (including but not limited to mammals), including enveloped and non-enveloped viruses, DNA and RNA viruses, viroids, and prions. Hence, for example, infections or unwanted levels of the following viruses and viral types can be treated, prevented or addressed by the present inactivated agents: human immunodeficiency viruses (HIV), simian immunodeficiency viruses (SIV), Ebola viruses, hemorrhagic fever viruses, hepatitis A viruses, hepatitis B viruses, hepatitis C viruses, influenza viruses, poxviruses, herpes viruses, adenoviruses, papovaviruses, parvoviruses, reoviruses, orbiviruses, picornaviruses, rotaviruses, alphaviruses, rubiviruses, influenza virus type A and B, flaviviruses, coronaviruses, paramyxoviruses, morbilliviruses, pneumoviruses, rhabdoviruses, lyssaviruses, orthmyxoviruses, bunyaviruses, phleboviruses, nairoviruses, hepadnaviruses, arenaviruses, retroviruses, enteroviruses, rhinoviruses and the filoviruses.
In some embodiments, the viruses are influenza or filoviruses. Filoviruses are viruses belonging to the family Filoviridae, which is in the order Mononegavirales. These viruses are single stranded negative sense RNA viruses that target primates. There are two general viruses, the Ebola virus (Ebolavirus, with four species) and the Marburg virus (Marburgvirus). These viruses cause horrific viral hemorrhagic fevers, characterized by massive bleeding from every orifice of the body. Ebola destroys the immune system in an explosive manner. Marburg virus typically has a mortality rate of at least 25%, while Ebola virus, depending on the species, has a mortality rate of anywhere from 50% to 90%. The virus is spread through bodily fluids. They are classified by the Centers for Disease Control and Prevention as Biosafety Level 4. This means that they are among the most lethal and destructive viruses known to man. The filovirus viral particles are characteristically shaped as long, cylindrical, filamentous particles which may be straight, curved, coiled, or found in a “6” or “U” shaped configuration. They are occasionally branched and the particles vary greatly in length but the diameter (about 80 nm) is consistently observed. The filovirus virions are produced by budding from an infected cell, and consist of the viral RNA strand and proteins encapsulated in a lipid membrane formed from the host cell's plasma membrane.
Infections or unwanted levels of the following target viruses and viral types that are believed to have potential as biological weapons can be treated, prevented or addressed by the present inactivated agents: hemorrhagic fever viruses (HFVs), Filoviruses, Chikungunya virus, Japanese encephalitis virus, Monkey pox virus, variola virus, Congo-Crimean hemorrhagic fever virus, Junin virus, Omsk hemorrhagic fever virus, Venezuelan equine encephalitis virus, Dengue fever virus, Lassa fever virus, Rift valley fever virus, Western equine encephalitis virus, Eastern equine encephalitis virus, Lymphocytic choriomeningitis virus, Russian Spring-Summer encephalitis virus, White pox, Ebola virus, Machupo virus, Smallpox virus, Yellow fever virus, Hantaan virus, Marburg virus, and Tick-borne encephalitis virus.
Similarly, infections or unwanted levels of the following examples of target microbial organisms can be treated, prevented or addressed by the present inactivated agents: Aeromonas spp. (including, for example, Aeromonas hydrophila, Aeromonas caviae and Aeromonas sobria), Bacillus spp. (including, for example, Bacillus cereus, Bacillus anthracis and Bacillus thuringiensis), Bacteroides spp. (including, for example, B. fragilis, B. thetaiotaomicron, B. vulgatus, B. ovatus, B. distasonis, B. uniformis, B. stercoris, B. eggerthii, B. merdae, and B. caccae), Campylobacter spp. (including, for example, Campylobacter jejuni, Campylobacter laridis, and Campylobacter hyointestinalis), Clostridium spp. (such as the pathogenic clostridia including all types of Clostridium botulinum (including those in Groups I, II, III and IV, and including those that produce botulism A, B, C, D, E, F and G), all types of Clostridium tetani, all types of Clostridium difficile, and all types of Clostridium perfringens), Ebola spp. (e.g. EBOV Zaire), Enterobacter spp. (including, for example, Enterobacter aerogenes (also sometimes referred to as Klebsiella mobilis), Enterobacter agglomerans (also sometimes referred to as Pantoea agglomerans), Enterobacter amnigenus, Enterobacter asburiae, Enterobacter cancerogenus (also sometimes referred to as Enterobacter taylorae and/or Erwinia cancerogena), Enterobacter cloacae, Enterobacter cowanii, Enterobacter dissolvens (also sometimes referred to as Erwinia dissolvens), Enterobacter gergoviae, Enterobacter hormaechei, Enterobacter intermedium, Enterobacter intermedius (also sometimes referred to as Enterobacter intermedium), Enterobacter kobei, Enterobacter nimipressuralis (also sometimes referred to as Erwinia nimipressuralis), Enterobacter sakazakii, and Enterobacter taylorae (also sometimes referred to as Enterobacter cancerogenus)), Enterococcus spp. (including, for example, Vancomycin Resistant Enterococcus (VRE), Enterococcus faecalis, Enterococcus faecium, Enterococcus durans, Enterococcus gallinarum, and Enterococcus casseliflavus), Escherichia spp. (including the enterotoxigenic (ETEC) strains, the enteropathogenic (EPEC) strains, the enterohemorrhagic (EHEC) strain designated E. coli O157:H7, and the enteroinvasive (EIEC) strains), Gastrospirillum spp. (including, for example, Gastrospirillum hominis (also sometimes now referred to as Helicobacter heilmannii), Helicobacter spp. (including, for example, Helicobacter pylori and Helicobacter hepaticus), Klebsiella spp. (including, for example, Klebsiella pneumoniae, Klebsiella ozaenae, Klebsiella rhinoscleromatis, Klebsiella oxytoca, Klebsiella planticola, Klebsiella terrigena, and Klebsiella ornithinolytica), Salmonella spp. (including, for example, S. typhi and S. paratyphi A, B, and C, S. enteritidis, and S. dublin), Shigella spp. (including, for example, Shigella sonnei, Shigella boydii, Shigella flexneri, and Shigella dysenteriae), Staphylococcus spp. (including, for example, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus saprophyticus and Staphylococcus epidermis), Streptococcus ssp. (including Groups A (one species with 40 antigenic types, Streptococcus pyogenes), B, C, D (five species (Streptococcus faecalis, Streptococcus faecium, Streptococcus durans, Streptococcus avium, and Streptococcus bovis)), F, and G, including Streptococcus pneumoniae), Pseudomonas spp. (including, for example, Pseudomonas aeriginosa, Pseudomonas maltophilia, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas cepacia, Pseudomonas stutzeri, Pseudomonas mallei, Pseudomonas pseudomallei and Pseudomonas putrefaciens), Vibrio spp. (including, for example, Vibrio cholera Serogroup O1 and Vibrio cholera Serogroup Non-O1, Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio furnissii, Vibrio carchariae, Vibrio hollisae, Vibrio cincinnatiensis, Vibrio metschnikovii, Vibrio damsela, Vibrio mimicus, Vibrio vulnificus, and Vibrio fluvialis), Yersinia spp. (including, for example, Yersinia pestis, Yersinia enterocolitica and Yersinia pseudotuberculosis), Neisseria, Proteus, Citrobacter, Aerobacter, Providencia, Serratia, Brucella, Francisella tularensis (also sometimes referred to as Pasteurella tularensis, Bacillus tularensis, Brucella tularensis, tularemia, rabbit fever, deerfly fever, Ohara's disease, and/or Francis disease), and the like. Thus, for example, various bacterial infections or unwanted levels of bacteria that can be treated, prevented or addressed by the present inactivated agents include but are not limited to those associated with anthrax (Bacillus anthracis), staph infections (Staphylococcus aureus), typhus (Salmonella typhi), food poisoning (Escherichia coli, such as O157:H7), bascillary dysentery (Shigella dysenteria), pneumonia (Psuedomonas aerugenosa and/or Pseudomonas cepacia), cholera (Vibrio cholerae), ulcers (Helicobacter pylori), Bacillus cereus, Salmonella, Clostridium perfringens, Campylobacter, Listeria monocytogenes, Vibrio parahaemolyticus, botulism (Clostridium botulinum), smallpox (variola major), listeriosis (Listeria monocytogenes), tularemia (Francisella tularensis), plague (Yersinia pestis; also sometimes referred to as bubonic plague, pneumonic plague, and/or black death) and others. E. coli serotype 0157:H7 has been implicated in the pathogenesis of diarrhea, hemorrhagic colitis, hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP). As indicated herein, the inactivated agents of the invention are also active against drug-resistant and multiply-drug resistant strains of bacteria, for example, multiply-resistant strains of Staphylococcus aureus and vancomycin-resistant strains of Enterococcus faecium and Enterococcus faecalis.
Fungal infections that can be treated or prevented by the present inactivated agents include infections by fungi that infect a mammal, including Histoplasma capsulatum, Coccidioides immitis, Cryptococcus neoformans, Candida ssp. including Candida albicans, Aspergilli ssp. including Aspergillus fumigatus, Sporothrix, Trichophyton ssp., Fusarium ssp., Tricosporon ssp., Pneumocystis carinii, and Trichophyton mentagrophytes. Hence, for example, infections or unwanted levels of target fungi can be treated, prevented or addressed by the present inactivated agents. Such fungi also include fungal pathogens that may have potential for use biological weapons, including Coccidioides immitis and Histoplasma capsulatum.
Anti-microbial activity can be evaluated against these varieties of microbes (viruses, bacteria, fungi and parasites) using methods available to one of skill in the art. In one embodiment, anti-microbial activity is the amount of the inactivated agent that stimulates an immune response against the microbe. In another embodiment, anti-microbial activity is the amount of the inactivated agent that effectively immunizes a mammal against the microbe.
Treatment of, or treating, cancer is intended to include the alleviation of or diminishment of at least one symptom typically associated with the disease. The treatment also includes alleviation or diminishment of more than one symptom. The treatment may cure the cancer, e.g., it may reduce the number of cancer cells and/or arrest the growth of the cancerous tumor.
Cancers that can be treated by the present inactivated agents include solid mammalian tumors as well as hematological malignancies. Solid mammalian tumors include cancers of the head and neck, lung, mesothelioma, mediastinum, esophagus, stomach, pancreas, hepatobiliary system, small intestine, colon, colorectal, rectum, anus, kidney, urethra, bladder, prostate, urethra, penis, testis, gynecological organs, ovaries, breast, endocrine system, skin central nervous system; sarcomas of the soft tissue and bone; and melanoma of cutaneous and intraocular origin. Hematological malignancies include childhood leukemia and lymphomas, Hodgkin's disease, lymphomas of lymphocytic and cutaneous origin, acute and chronic leukemia, plasma cell neoplasm and cancers associated with AIDS. In addition, a cancer at any stage of progression can be treated, such as primary, metastatic, and recurrent cancers. Information regarding numerous types of cancer can be found, e.g., from the American Cancer Society (www.cancer.org), or from, e.g., Wilson et al. (1991) Harrison's Principles of Internal Medicine, 12.sup.th Edition, McGraw-Hill, Inc. Both human and veterinary uses are contemplated.
Anti-cancer activity can be evaluated against varieties of cancers using methods available to one of skill in the art. Anti-cancer activity, for example, is determined by identifying the LD100 or ED50 of an inactivated tumor or cancer cell of the present invention that prevents the growth of a cancer. In one embodiment, anti-cancer activity is the amount of the inactivated agent that effectively immunizes a mammal against that cancer type.
According to the present invention, the inactivated agents provided herein do not have substantial or undesired toxicity or infectivity within the mammalian organism to be treated.
Administration of the Inactivated Agents
The inactivated agents (e.g., inactivated viruses) of the invention are administered so as to achieve a reduction in at least one symptom associated with an infection, cancer, tumor or other disease, or a decrease in the amount of antibody associated with the infection, cancer, tumor or other disease.
To achieve the desired effect(s), the inactivated agent, or a combination of inactivated agents, may be administered as single or divided dosages, for example, of at least about 0.01 mg/kg to about 500 to 750 mg/kg, of at least about 0.01 mg/kg to about 300 to 500 mg/kg, at least about 0.1 mg/kg to about 100 to 300 mg/kg or at least about 1 mg/kg to about 50 to 100 mg/kg of body weight, although other dosages may provide beneficial results.
In some embodiments, the dosage is measured by the number of viral particles or the number of cells. Thus, for example, about 20 to about 50,000 inactivated viral particles or inactivated cells can be administered, or about 50 to about 25,000 inactivated viral particles or inactivated cells can be administered, or about 100 to about 10,000 inactivated viral particles or inactivated cells can be administered.
The amount administered will vary depending on various factors including, but not limited to, the inactivated agent chosen, the disease, the weight, the physical condition, the health, the age of the mammal, or whether prevention or treatment is to be achieved. Such factors can be readily determined by the clinician employing animal models or other test systems that are available in the art.
Administration of the therapeutic agents in accordance with the present invention may be in a single dose, in multiple doses, in a continuous or intermittent manner, depending, for example, upon the recipient's physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners. The administration of the inactivated agents of the invention is generally intermittent over a preselected period of time, for example, in a series of spaced doses. Both local and systemic administration is contemplated.
To prepare the composition, inactivated agents are prepared according to the methods described herein, and purified as necessary or desired. In some embodiments the inactivated agents can be lyophilized and/or stabilized. The inactivated agent can then be adjusted to the appropriate concentration, and optionally combined with other agents.
The absolute weight of a given inactivated agent included in a unit dose can vary widely. For example, about 0.01 to about 2 g, or about 0.1 to about 500 mg, of at least one inactivated agent of the invention, or a plurality of inactivated agents, can be administered. Alternatively, the unit dosage can vary from about 0.01 g to about 5 g, from about 0.01 g to about 3.5 g, from about 0.01 g to about 2.5 g, from about 0.1 g to about 1 g, from about 0.1 g to about 0.8 g, from about 0.1 g to about 0.4 g, or from about 0.1 g to about 0.2 g.
One or more suitable unit dosage forms comprising the therapeutic inactivated agents of the invention can be administered by a variety of routes including oral, parenteral (including subcutaneous, intravenous, intramuscular and intraperitoneal), rectal, dermal, transdermal, intrathoracic, intrapulmonary, mucosal and intranasal (respiratory) routes. In some embodiments, the agents of the invention are administered by mucosal, intranasal, or subcutaneous routes.
In some embodiments, the agents of the invention are administered by injection. The therapeutic inactivated agents may also be formulated for sustained release (for example, using microencapsulation, see WO 94/07529, and U.S. Pat. No. 4,962,091). The formulations may, where appropriate, be conveniently presented in discrete unit dosage forms and may be prepared by any of the methods well known to the pharmaceutical arts. Such methods may include the step of mixing the therapeutic agent with liquid carriers, solid matrices, semi-solid carriers, finely divided solid carriers or combinations thereof, and then, if necessary, introducing or shaping the product into the desired delivery system.
When the therapeutic inactivated agents of the invention are prepared for oral administration, they are generally combined with a pharmaceutically acceptable carrier, diluent or excipient to form a pharmaceutical formulation, or unit dosage form. For oral administration, the inactivated agents may be present as a powder, a granular formulation, a solution, a suspension, an emulsion or in a natural or synthetic polymer or resin for ingestion of the agents from a chewing gum. The inactivated agents may also be presented as a bolus, electuary or paste. Orally administered therapeutic inactivated agents of the invention can also be formulated for sustained release, e.g., the inactivated agents can be coated, micro-encapsulated, or otherwise placed within a sustained delivery device. The total active ingredients in such formulations comprise from 0.1 to 99.9% by weight of the formulation.
By “pharmaceutically acceptable” it is meant a carrier, diluent, excipient, and/or salt that is compatible with the other ingredients of the formulation, and not deleterious to the recipient thereof.
Pharmaceutical formulations containing the therapeutic inactivated agents of the invention can be prepared by procedures known in the art using well-known and readily available ingredients. For example, the inactivated agent can be formulated with common excipients, diluents, or carriers, and formed into tablets, capsules, solutions, suspensions, powders, aerosols and the like. Examples of excipients, diluents, and carriers that are suitable for such formulations include buffers, as well as fillers and extenders such as starch, cellulose, sugars, mannitol, and silicic derivatives. Binding agents can also be included such as carboxymethyl cellulose, hydroxymethylcellulose, hydroxypropyl methylcellulose and other cellulose derivatives, alginates, gelatin, and polyvinyl-pyrrolidone. Moisturizing agents can be included such as glycerol, disintegrating agents such as calcium carbonate and sodium bicarbonate. Agents for retarding dissolution can also be included such as paraffin. Resorption accelerators such as quaternary ammonium compounds can also be included. Surface active agents such as cetyl alcohol and glycerol monostearate can be included. Adsorptive carriers such as kaolin and bentonite can be added. Lubricants such as talc, calcium and magnesium stearate, and solid polyethyl glycols can also be included. Preservatives may also be added. The compositions of the invention can also contain thickening agents such as cellulose and/or cellulose derivatives. They may also contain gums such as xanthan, guar or carbo gum or gum arabic, or alternatively polyethylene glycols, bentones and montmorillonites, and the like.
For example, tablets or caplets containing the inactivated agents of the invention can include buffering agents such as calcium carbonate, magnesium oxide and magnesium carbonate. Caplets and tablets can also include inactive ingredients such as cellulose, pre-gelatinized starch, silicon dioxide, hydroxy propyl methyl cellulose, magnesium stearate, microcrystalline cellulose, starch, talc, titanium dioxide, benzoic acid, citric acid, corn starch, mineral oil, polypropylene glycol, sodium phosphate, zinc stearate, and the like. Hard or soft gelatin capsules containing at least one inactivated agent of the invention can contain inactive ingredients such as gelatin, microcrystalline cellulose, sodium lauryl sulfate, starch, talc, and titanium dioxide, and the like, as well as liquid vehicles such as polyethylene glycols (PEGs) and vegetable oil. Moreover, enteric-coated caplets or tablets containing one or more inactivated agents of the invention are designed to resist disintegration in the stomach and dissolve in the more neutral to alkaline environment of the duodenum.
The inactivated agents of the invention can also be formulated as elixirs or solutions for convenient oral administration or as solutions appropriate for parenteral administration, for instance by intramuscular, subcutaneous, intraperitoneal or intravenous routes. The pharmaceutical formulations of the therapeutic inactivated agents of the invention can also take the form of an aqueous or anhydrous solution or dispersion, or alternatively the form of an emulsion or suspension or salve.
Thus, the therapeutic inactivated agents may be formulated for parenteral administration (e.g., by injection, for example, bolus injection or continuous infusion) and may be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion containers or in multi-dose containers. As noted above, preservatives can be added to help maintain the shelve life of the dosage form. The inactivated agents and other ingredients may form suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the inactivated agents and other ingredients may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution, for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.
These formulations can contain pharmaceutically acceptable carriers, vehicles and adjuvants that are well known in the art. It is possible, for example, to prepare solutions using one or more organic solvent(s) that is/are acceptable from the physiological standpoint, chosen, in addition to water, from solvents such as acetone, ethanol, isopropyl alcohol, glycol ethers such as the products sold under the name “Dowanol,” polyglycols and polyethylene glycols, C1-C4 alkyl esters of short-chain acids, ethyl or isopropyl lactate, fatty acid triglycerides such as the products marketed under the name “Miglyol,” isopropyl myristate, animal, mineral and vegetable oils and polysiloxanes.
It is possible to add, if desired, an adjuvant chosen from antioxidants, surfactants, other preservatives, film-forming, keratolytic or comedolytic agents, perfumes, flavorings and colorings. Antioxidants such as t-butylhydroquinone, butylated hydroxyanisole, butylated hydroxytoluene and α-tocopherol and its derivatives can be added.
Also contemplated are combination products that include one or more inactivated agents of the present invention and one or more other anti-microbial agents. For example, a variety of antibiotics can be included in the pharmaceutical compositions of the invention, such as aminoglycosides (e.g., streptomycin, gentamicin, sisomicin, tobramycin and amicacin), ansamycins (e.g. rifamycin), antimycotics (e.g. polyenes and benzofuran derivatives), β-lactams (e.g. penicillins and cephalosporins), chloramphenical (including thiamphenol and azidamphenicol), linosamides (lincomycin, clindamycin), macrolides (erythromycin, oleandomycin, spiramycin), polymyxins, bacitracins, tyrothycin, capreomycin, vancomycin, tetracyclines (including oxytetracycline, minocycline, doxycycline), phosphomycin and fusidic acid.
Additionally, the inactivated agents are well suited to formulation as sustained release dosage forms and the like. The formulations can be so constituted that they release the inactivated agent, for example, in a particular part of the intestinal or respiratory tract, possibly over a period of time. Coatings, envelopes, and protective matrices may be made, for example, from polymeric substances, such as polylactide-glycolates, liposomes, microemulsions, microparticles, nanoparticles, or waxes. These coatings, envelopes, and protective matrices are useful to coat indwelling devices, e.g., stents, catheters, peritoneal dialysis tubing, draining devices and the like.
For topical administration, the inactivated agents may be formulated as is known in the art for direct application to a target area. Forms chiefly conditioned for topical application take the form, for example, of creams, milks, gels, dispersion or microemulsions, lotions thickened to a greater or lesser extent, impregnated pads, ointments or sticks, aerosol formulations (e.g., sprays or foams), soaps, detergents, lotions or cakes of soap. Other conventional forms for this purpose include wound dressings, coated bandages or other polymer coverings, ointments, creams, lotions, pastes, jellies, sprays, and aerosols. Thus, the therapeutic inactivated agents of the invention can be delivered via patches or bandages for dermal administration. Alternatively, the inactivated agent can be formulated to be part of an adhesive polymer, such as polyacrylate or acrylate/vinyl acetate copolymer. For long-term applications it might be desirable to use microporous and/or breathable backing laminates, so hydration or maceration of the skin can be minimized. The backing layer can be any appropriate thickness that will provide the desired protective and support functions. A suitable thickness will generally be from about 10 to about 200 microns.
Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents. Lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, or coloring agents. The inactivated agents can also be delivered via iontophoresis, e.g., as disclosed in U.S. Pat. Nos. 4,140,122; 4,383,529; or 4,051,842. The percent by weight of a therapeutic agent of the invention present in a topical formulation will depend on various factors, but generally will be from 0.01% to 95% of the total weight of the formulation, and typically 0.1-85% by weight.
Drops, such as eye drops or nose drops, may be formulated with one or more of the inactivated agents in an aqueous or non-aqueous base also comprising one or more dispersing agents, solubilizing agents or suspending agents. Liquid sprays are conveniently delivered from pressurized packs. Drops can be delivered via a simple eye dropper-capped bottle, or via a plastic bottle adapted to deliver liquid contents dropwise, via a specially shaped closure.
The therapeutic inactivated agent may further be formulated for topical administration in the nose, mouth or throat. For example, the active ingredients may be formulated as a lozenge further comprising a flavored base, for example, sucrose and acacia or tragacanth; pastilles comprising the composition in an inert base such as gelatin and glycerin or sucrose and acacia; and mouthwashes comprising the composition of the present invention in a suitable liquid carrier.
The pharmaceutical formulations of the present invention may include, as optional ingredients, pharmaceutically acceptable carriers, diluents, solubilizing or emulsifying agents, and salts of the type that are available in the art. Examples of such substances include normal saline solutions such as physiologically buffered saline solutions and water. Specific non-limiting examples of the carriers and/or diluents that are useful in the pharmaceutical formulations of the present invention include water and physiologically acceptable buffered saline solutions such as phosphate buffered saline solutions pH 7.0-8.0.
The inactivated agents of the invention can also be administered to the respiratory tract. Thus, the present invention also provides aerosol pharmaceutical formulations and dosage forms for use in the methods of the invention. In general, such dosage forms comprise an amount of at least one of the agents of the invention effective to treat or prevent the clinical symptoms of a specific infection, cancer, tumor or disease. Any statistically significant attenuation of one or more symptoms of an infection, cancer, tumor or disease that has been treated pursuant to the methods of the present invention is considered to be a treatment or prevention of such infection, cancer, tumor or disease within the scope of the invention.
Alternatively, for administration by inhalation or insufflation, the composition may take the form of a solution or a dry powder, for example, a powder mix of the therapeutic agent and a suitable powder base such as lactose or starch. The powder composition may be presented in unit dosage form in, for example, capsules or cartridges, or, e.g., gelatin or blister packs from which the powder may be administered with the aid of an inhalator, insufflator, or a metered-dose inhaler (see, for example, the pressurized metered dose inhaler (MDI) and the dry powder inhaler disclosed in Newman, S. P. in A
Therapeutic inactivated agents of the present invention can also be administered in an aqueous solution when administered in an aerosol or inhaled form. Thus, other aerosol pharmaceutical formulations may comprise, for example, a physiologically acceptable buffered saline solution containing between about 0.1 mg/ml and about 100 mg/ml of one or more of the inactivated agents of the present invention specific for the indication or disease to be treated or prevented. Dry aerosol in the form of finely divided solid inactivated agent that are not dissolved or suspended in a liquid are also useful in the practice of the present invention.
Inactivated agents of the present invention may be formulated as dusting powders and comprise finely divided particles having an average particle size of between about 1 and 5 μm, alternatively between 2 and 3 μm. Finely divided particles may be prepared by pulverization and screen filtration using techniques well known in the art. The particles may be administered by inhaling a predetermined quantity of the finely divided material, which can be in the form of a powder. It will be appreciated that the unit content of active ingredient or ingredients contained in an individual aerosol dose of each dosage form need not in itself constitute an effective amount for treating or preventing the particular infection, indication or disease since the necessary effective amount can be reached by administration of a plurality of dosage units. Moreover, the effective amount may be achieved using less than the dose in the dosage form, either individually, or in a series of administrations.
For administration to the upper (nasal) or lower respiratory tract by inhalation, the therapeutic inactivated agents of the invention are conveniently delivered from a nebulizer or a pressurized pack or other convenient means of delivering an aerosol spray. Pressurized packs may comprise a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Nebulizers include, but are not limited to, those described in U.S. Pat. Nos. 4,624,251; 3,703,173; 3,561,444; and 4,635,627. Aerosol delivery systems of the type disclosed herein are available from numerous commercial sources including Fisons Corporation (Bedford, Mass.), Schering Corp. (Kenilworth, N.J.) and American Pharmoseal Co., (Valencia, Calif.). For intra-nasal administration, the therapeutic agent may also be administered via nose drops, a liquid spray, such as via a plastic bottle atomizer or metered-dose inhaler. Typical of atomizers are the Mistometer (Wintrop) and the Medihaler (Riker).
Furthermore, the active ingredients may also be used in combination with other therapeutic agents, for example, pain relievers, anti-inflammatory agents, antihistamines, bronchodilators and the like, whether for the conditions described or some other condition.
The present invention further pertains to a packaged pharmaceutical composition for controlling microbial infections or cancer such as a kit or other container. The kit or container holds a therapeutically effective amount of a pharmaceutical composition for controlling microbial infections, or cancer or tumor growth and instructions for using the pharmaceutical composition for control of the microbial infection or for control of the cancer or tumor. The pharmaceutical composition includes at least one inactivated agent of the present invention, in a therapeutically effective amount such that microbial infection, cancer or tumor is controlled.
The invention is further illustrated by the following non-limiting Examples.
This Example provides many of the reagents and procedures employed for several experiments with SIV and/or HIV.
Materials and Methods
Antibodies and their sources were as follows: anti-HLA-DR IgG L243 (mAb from Elena Chertova), anti-HLA-DR IgG DA6-147 (mAb from Paul Roche), and anti-Gp32 IgG (rabbit polyclonal Ab from Raoul Benveniste). [125]INA (300 mCi/mmol) was purchased from Lofstrand Laboratories (Gaithersburg, Md.). All other biochemical reagents used were of the highest purity available and were obtained from regular commercial sources.
Viruses. HIV-1MN/H9 clone 4 was propagated in H9 cells, as described previously (Ott at al. 1995). SIVmne was obtained from supernatants of the cloned E11 S cell lines derived from a culture of HuT-78 cells infected with SIVmne (Benveniste at al. 1990). Concentrated virus preparations were produced by sucrose gradient banding in a continuous-flow centrifuge (Bess at al. 1997). Inactivation of SIV by treatment with aldrithiol-2 was performed as described (Rossio at al. 1998).
Cell cultures. Ghost-345 cells (derived from human osteosarcoma cells) that stably express CD4, as well as CXCR4 and CCR5, and NIH3T3 CD4/X4 were obtained from Dan Littman and Vineet KewalRamani. TF228 cells derived from the BJAB human B cell line and that stably express the HIV-1LAI envelope glycoprotein (Jonak at al. 1993) were from Zdenka L. Jonak (Smith-Kline & Beecham, King of Prussia, Pa.). SupT1 (human CD4-expressing T-Lymphoblastic cell line) and TF228 were grown in RPMI supplemented with 10% fetal bovine serum (FBS) (Life Technologies, Inc., Rockville). NIH3T3 CD4 cells were grown in Dulbecco's modified Eagle's medium+10% FBS (D10). NIH3T3 CD4/X4 cells were grown in D10+3 mg/ml puromycin. Ghost 345 cells were grown in D10+500 mg/ml G418+100 mg/ml hygromycin+1 mg/ml puromycin. All the cells were grown in the presence of penicillin and streptomycin.
Treatment with INA. Viruses or cells were suspended in Phosphate Buffered saline (PBS) at a concentration of 0.5-1.0 mg/ml. A stock solution of 30 mM INA in DMSO was prepared. INA was added to the cell or viral suspension under dim light to a final concentration of 1-200 μM. The INA was added so that the total DMSO will not exceed 1% of the total sample volume. Addition of INA was done in installments of 3-4 aliquots while mixing vigorously after each aliquot. The sample was incubated at room temperature for 30 minutes and washed once in PBS.
The suspension was then irradiated with an ozone free 100 W mercury arc lamp and through a water filter to eliminate heat and a 320 nm cut-off filter. Time of irradiation vary with the size of the sample. For a 1 ml sample and a cross-area of 1 cm2 the irradiation time was 2 minutes. For a 20 ml sample and a cross area of 10 cm2 the irradiation time was 5 minutes.
Labeling of the target cells. The fluorescent lipid DiO (Molecular Probes, Eugene, Oreg.) was diluted in 50% Diluent C (Sigma-Aldrich, St. Louis, Mo.) and 50% serum-free RPMI (RPMI) to a final concentration of 50 mM. After two washes in RPMI the cells were incubated in the DiO solution for 30 min at room temperature. They were then washed once with clear RPMI and further incubated 30 min in medium at room temperature. They were then washed three times with PBS, in which they were finally resuspended. At this point [125I]INA (1 Ci/mmol) was added in the amount of 10 mCi for each experimental group. Upon 20 min incubation in the dark, the cells were washed with PBS and subsequently used for the photolabeling experiment.
Virus infectivity assay. The infectivity of HIV-1(MN) and SIV Mne samples was determined by using AA2 clone 1 and clone 5 cells, respectively, as described by Raviv et al., Virology 293:243-251 (2002). Cells were cultured in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 0.1 μg/ml gentamicin, and 20 mM HEPES buffer. To determine infectious titers of untreated virus, serial dilutions of the virus were prepared in cell culture medium. One hundred microliters of virus from each dilution was added to 2.5×106 cells in 0.9 ml of medium (resulting in an additional 1:10 dilution of the virus) and incubated at 37° C. in a 15-ml tube for 18 h while being slowly rocked. After the addition of 9 ml of incomplete medium, the cells were washed to remove unbound virus. The cells were washed twice more, and the pellet was resuspended in 5 ml of complete medium. One milliliter (5×105 cells) was placed in each of 4 wells of a 24-well plate (Costar no. 3524). Mock-infected cells were included as negative controls for each set of titrations. Plates were incubated at 37° C. in a humidified, 5% CO2 incubator. On day 3, 1 ml of complete medium was added to each well. These cultures were passaged by removing 50% of the culture and adding fresh medium on days 3, 7, 10, 14, 17, and 21 to maintain actively dividing cell populations. On days 7, 14, and 21, samples were analyzed for viral capsid protein concentration to determine if progeny virions could be detected. These samples were clarified from cells by centrifugation at 600×g for 5 min, and the supernatant was lysed by the addition of a 1/10 volume of 10% Triton X-100. Capsid protein concentrations were determined using either an SIV p28 or HIV-1 p24 antigen capture assay (house assay kits; AIDS Vaccine Program, SAIC). Samples were considered positive if they produced capsid protein concentrations above the antigen capture assay cutoff value and these concentrations increased over time. Infectious titers were determined by the Reed and Muench method (Roos et al., Virology 273:307-315 (2000)) using capsid concentration results from day 21.
Infectivity assay of INA-treated virus. To determine if INA-treated virus preparations were free of detectable infectious virus, samples were analyzed at the lowest possible dilution and against a greater number of target cells: 0.5 ml of INA-treated virus was added to 1.25×107 cells in 2.5 ml of medium and incubated at 37° C. in a 15-ml tube for 18 h while being slowly rocked. Due to the high concentration of virus in the inoculum, these samples were washed four times by centrifugation to remove unbound virus. The resulting cell pellets were resuspended in 25 ml of medium (final cell concentration was 5.0×105/ml), planted in T75 flasks, and incubated at 37° C. in 5% CO2. These cultures were passaged and analyzed for capsid antigen concentration. All the other details were as described above for the untreated virus.
Measurement of fusion by photo-sensitized labeling. The HLA-DR+ virions are incubated with the HLA-DR− target cells labeled with the fluorescent lipid analog 3,3′-dioctadecyloxacarbocyanine (DiO) and [125I]INA for binding at room temperature. Plasma membranes of target cells bearing CD4 and coreceptors are labeled with the fluorescent lipid analog 3-dioctadecyloxa-carbocyanine (DiO). [125I]INA spontaneously partitions from the medium into viral and other target membranes. In the bound state only integral membrane proteins of the DiO-labeled target membranes react with [125I]INA following photoactivation by visible light. Upon incubation of virus-cell complexes at 37° C., DiO becomes part of the viral membrane as a result of fusion and therefore photoactivation using visible light results in covalent attachment of [125I]INA to viral membrane-resident proteins. At different times following incubation at 37° C., samples are irradiated with visible light, the cells are lysed, and the HIV or SIV Env, as well other viral envelope-resident proteins such as HLA-DR, is isolated from other radioactively labeled proteins by immunoprecipitation. The extent of radioactivity incorporated into these proteins is then a quantitative measure of viral fusion at the plasma membrane level.
In the case of HIV-1, 1 ml virus (0.79 mg/ml capsid) was added to 3×108 SupT1 cells in 3 ml. In case of SIVmne, 0.2 ml of virus (0.084 mg/ml capsid) was added to 3 ml medium overlaid on attached Ghost-345 cells. The unbound virions were then removed and the samples subjected to fusion at the desired temperature. At defined times cells were irradiated with an argon laser (Lexel Laser, Inc., Freemont, Calif.) in the multiline mode of 488/514 nm. Suspension cells were irradiated horizontally for two consecutive 10-s periods with a beam of 400 mW that was passed through a UV cut-off filter and focused on an area of 1 cm−2 (133 mW/cm2/min). Plated cells were irradiated for 60 s vertically using a 5-W beam focused on an area of 144 cm2 (11 mW/cm2/min).
The cells were then collected and lysed (2% Triton X-100 in Tris-buffered saline (TBS; 50 mM Tris, 138 mM NaCl, 2. mM KCl, pH 8) containing protease inhibitors) for 2 h at 4° C. The insoluble material was spun down at 15,000 rpm for 15 min in an Eppendorf microcentrifuge. The supernatant was then diluted twice in TBS and total protein was measured using the BCA protein determination reagent (Pierce, Rockford, Ill.). Samples were subjected to immunoprecipitation using L243 (for HLA-DR) or anti-SIV gp32 for the SIV Env. Upon overnight incubation with the respective antibody, protein G-agarose was added for 2 h and washed five times with TBS containing 1% Triton X-100. Proteins were separated by 14% SDS-PAGE and transferred to nitrocellulose membranes. Blots were incubated for 1 h in PBST (phosphate-buffered saline, 0.2% Tween 20) containing 5% powdered skim milk. Membranes were incubated for 2 h with the primary antibody in a 3% BSA solution containing 0.2% Tween 20 and for 1 h 30 min with a peroxidase-conjugated secondary antibody in PBST. Immunoreactivity was detected by using an ECL kit (Amersham, Piscataway, N.J.) and an imaging system with high dynamic range (Bio-Rad GS 505 Molecular Imager System, Hercules, Calif.). The blots were then exposed to Phosphorimager screens; bands were quantified using a Storm system (Molecular Dynamics Sunnyvale, Calif.) and the Image Quant software (Molecular Dynamics).
HIV-1 envelope glycoprotein-mediated cell-cell fusion. For the photo-sensitized labeling experiments HLA-DR+ TF228.1.16 effector cells and DiO-labeled HLA-DR target cells were loaded with [125I]INA and incubated for various times at 37° C. The plates were irradiated for 60 s with a 5-W laser beam over an area of 144 cm2 (11 mW/cm2/min) and incorporation of [125I]INA into HLA-DR was measured as described above. For the dye redistribution experiments target cells were labeled with the cytoplasmic dye 5- and 6-([(4-chloromethyl)benzoyl]amino)tetramethylrhodamine (CMTMR) at a concentration of 1.5 mM for 1 h at 37° C. Envelope-expressing cells were labeled with calcein AM at a concentration of 1 mM for 1 h at 37° C. Calcein-labeled effector cells were co-cultured with CMTMR-labeled target cells for 2 h at 37° C., and dye redistribution was monitored microscopically as described previously (Munoz-Barroso et al. 1998). The extent of fusion was calculated as:
This Example describes the results of experiments showing that INA treatment inactivates viruses but leaves them substantially intact. However, such treatment inhibits viral fusion with host cells and prevents viral infection.
However, as shown by reverse phase HPLC analysis of viral proteins under reducing conditions (
When INA was used to treat SIV, viral infectivity was reduced to zero (
Thus, the infectivity of SIV was 100% blocked by treatment with appropriate levels of INA. Table 1 illustrates that INA treatment completely blocks infection of SIV and HIV as measured by the expression of the viral protein P-28 for SIV and P-24 for HIV at different times after the introduction of the virus. In particular, at 200 μM INA infectivity was blocked by 100%.
1Average of determined capsid protein concentrations from quadruplicate cultures.
2Culture result as assessed on day 21. “Pos” indicates the number of the quadruplicate cultures that became productively infected with virus. “Neg” indicates the number of cultures that remained uninfected.
3Reciprocal Tissue Culture Infectious Dose 50 was determined from the culture result data using the Method of Reed and Muench (Roos et al., Virology 273: 307-315 (2000)).
4Negative control cultures were maintained and remained uninfected though out the 21 day test period.
Even though INA-treated virions can bind to host cells, they exhibit substantially reduced fusion with those host cells. As shown by
These data indicate that INA treatment gives rise to viral particles that have minor but significant structural changes. The structural changes do not affect the ability of the viral particles to be recognized by antibodies (
This Example describes the results of experiments showing that INA treatment inactivates human immunodeficiency viral transcription, thereby illustrating by another procedure that INA treatment inactivates HIV.
Infectivity assay was carried out using the luciferase reporter gene assay, essentially as described in Spenlehauer, C., Gordon, C., Trkola, A. and Moore, J. (2001) Virology 280, 292-300; and Wei, X., Decker, J., Liu, Z., Zhang, Z., Arani, R., Kilby, M., Saag, M., Wu, X., Shaw, G., and Kappes, J. (2002) Antimicrobial Agents and Chemotherapy, 46, 1896-1905.
Briefly, JC53BL cells were used that express the luciferase enzyme under the transcriptional control of HIV long terminal repeat (LTR). Upon HIV infection the TAT protein from the virus binds to the LTR to induce the expression of Luciferase. The level of Luciferase expression can be assessed by incubation of the sample with a luciferase substrate which triggers a chemiluminescent signal that can be easily quantified by a luminometer.
As shown in
These results further demonstrate the effectiveness of INA for inactivating HIV. No effective vaccines are currently available for preventing HIV infection. However, the results provided herein indicate that the present compositions involving INA-inactivated HIV may be useful as vaccines.
This Example describes the results of experiments showing that INA treatment does not destroy the antigenicity of HIV. Instead, INA-treated HIV readily binds to available anti-HIV neutralizing antibodies.
The antibodies employed were the 2G12 and B12 antibodies that target Gp120 and the 4E10 antibody that targets gp41. Each of these antibody preparations is broadly neutralizing of HIV infectivity.
Antibody binding to HIV virions was measured by an immunocapture procedure essentially as described in Nyambi, P., Burda, S., Bastani, L., and Williams, C. (2001) Journal of Immunological Methods, 253, 253-262. Briefly, 10 microgram of each antibody was coated onto 96 well ELISA plates and non-specific binding was blocked with BSA. HIV was then added and incubated for binding for one hour at 37° C. using different amounts of virus as indicated in
The results are provided in
As illustrated herein, each of these antibody preparations recognizes and binds to INA-inactivated HIV, demonstrating that the epitopes recognized by the antibodies are substantially unaffected by INA treatment.
This Example illustrates that a single immunization of mice with INA-inactivated Ebola virus in the absence of adjuvant resulted in protection against a lethal challenge with 1000 pfu of mouse adapted Ebola virus. INA treatment resulted in complete loss of Ebola viral infectivity of cultured mammalian cells. Electron microscopy combined with immunogold staining as well as virus capture assays indicated that conformational epitopes were preserved on the surface of the virus. Mice injected with 50,000 pfu of INA-inactivated mouse-adapted Zaire-EBOV survived with no sign of disease. Interestingly, INA-inactivated virus, but not irradiated virus, was capable of inducing a short term protective response in 100% of mice when administered 3 days before challenge. These data indicate that INA has significant potential for development of filovirus vaccine and therapeutic agents useful against viral infection.
Materials and Methods
Reagents and cells. Vero E6 cells were cultured in DMEM supplemented with glutamine and 10% fetal calf serum at 37° C. in a humidified CO2 incubator. Anti-Ebola GP monoclonal antibodies were kindly provided by Dr. Mary-Kate Hart (USAMRIID).
Filoviruses. The EBOV Zaire strain of Ebola virus was used for many of these studies. A recombinant Zaire Ebola virus expressing the gene for green fluorescence protein (GFP-EBOVZ) was kindly provided by Dr. Jason Paragas (USAMRIID). EBOV-Zaire virus was propagated and enumerated by standard plaque assay on Vero or Vero E6 cells (Moe et al. J Clin Microbiol 13:791-3 (1981)). The virus was band purified over a sucrose gradient as described by Warfield et al., Proc. Natl. Acad. Sci. USA 100:15889-94 (2003); and Hevey et al. Virology 239: 206-16 (1997). EBOV-infected cells and animals were handled by qualified personnel under maximum containment in a biosafety level (BSL)-4 laboratory at the United States Army Medical Research Institute of Infectious Diseases.
Treatment of virus with INA. Mouse-adapted EBOV-Zaire or GFP-EBOVZ were diluted in phosphate-buffered saline (PBS) to a final concentration of 2×106 pfu/ml. INA from a stock of 30 mM in dimethyl sulfoxide (DMSO) was added to the virus suspension under dim light in three to four installments (for uniform mixing) to final concentrations as indicated in the individual experiments. The suspension was incubated for 30 min at room temperature. Glutathione, reduced form, was added to the suspension to final concentrations between 20 to 30 mM to neutralize any residual INA in the aqueous phase, and the sample was irradiated with UV light. The light source was a 100-W ozone-free mercury arc lamp placed in a lamphouse with a collector lens (Olympus). Samples were irradiated through a 310-nm cutoff filter placed in front of the lens (to allow transmission of the 313-, 334-, and 365-nm mercury emission bands) and through a water filter (to prevent sample heating) at a distance of 5 cm from the light source. At that point, the light dose was 10 mW/cm2 s. Irradiation times were 5 min for sample volumes of up to 1 ml in a clear microcentrifuge tube.
Detection of Virus infectivity using GFP-EBOVZ: Vero cells were infected with GFP-EBOVZ at an multiplicity of infection (MOI) of 5. Infected cells were detected after various time point post infection by flow cytometry.
Real time polymerase chain reaction for detection of EBOV genome: RNA was extracted from test samples by the TRIzol LS reagent (Invitrogen, Carlsbad, Calif.) according to the manufacturer's directions. RNA pellets were dissolved in 100 uL of RNase free water and stored at −80° C. until used. The primers were synthesized using phosphoramidite chemistry on an ABI 394 DNA/RNA synthesizer (ABI Biosystems, Foster City, Calif.). The TaqMan® probes were synthesized with the fluorescent dyes FAM at the 5′ terminus and TAMRA at the 3′ terminus (ABI Biosystems). Real time One-Step RT-PCR assay combining Superscript reverse transcriptase with Platinum Taq polymerase (Invitrogen) and TaqMan probe assay were used. The reactions were carried out in at total volume of 20 uL. Each reaction contained 0.2 mM dNTP, 0.4 uL of RT/Platinum Taq mix (Invitrogen), 0.4 uM of each primer, 100 nM of TaqMan® probe, 100 ng BSA, 4 units of RnaseOUT recombinant inhibitor (Invitrogen) 5.7 uL of water and 2 uL of RNA sample. The reverse transcription and PCR amplification were performed in a single tube on the Light Cycler (Roche) as follows: one cycle of 50° C. for 15 min, one cycle of 95° C. for 2 min followed by 45 cycles of 95° C. for 10 sec, 60° C. for 30 sec. To determine the viral load in each sample, the PCR reactions were monitored by recording the amount of fluorescence emission at each PCR cycle. PCR threshold cycle (Ct) method was used for sample comparison. Standard curves showing plots of Ct values versus Log PFU or genome copy number were obtained by using plasmid DNA or spiked samples with titered virus at a pre-determined PFU.
Vaccinations. Six to eight week old female C57B1/6 mice (National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, Md.) were vaccinated intramuscularly with indicated doses of INA-inactivated, mouse-adapted EBOV diluted in endotoxin-free PBS once or twice at 3-week intervals. Control mice were vaccinated on the same schedule with PBS. Serum samples were obtained from each mouse on days 0 and 28 post vaccination. Mice were challenged 4 weeks after the second vaccination by intraperitoneal injection with 1000 pfu (˜30,000 LD50) of mouse-adapted EBOV diluted in phosphate buffered saline (PBS) (Bray et al., J. Infect. Dis. 178:651-61 (1998)). After challenge, mice were observed at least twice daily for illness. Throughout the experiment, mice were housed in microisolator cages and provided autoclaved water and chow ad libitum.
Research was conducted in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals and adhered to principles stated in the Guide for the Care and Use of Laboratory Animals, National Research Council, 1996. The facility where this research was conducted is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International.
Antibody titers. Levels of EBOV-specific antibodies were determined, as described by Swenson et al., Vaccine 23:3033-42 (2005). Briefly, the wells were coated with sucrose-purified and γ-irradiated/inactivated EBOV-Zaire. Endpoint titers were established as the inverse of the last dilution where the optical density of the sample was ≧0.2 greater than the corresponding control wells (irrelevant heterologous antigen).
Determination of T cell response to INA-inactivated EBOV by intracellular IFN-γ staining. Splenocytes were isolated and cultured at 37° C. for 5 h in the presence of 1-5 μg of peptide(s) or PMA (25 ng/ml) and ionomycin (1.25 μg/ml) in 100 μl of RPMI-EHAA medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM glutamine, 10 μg of gentamicin per ml, 5 mM HEPES, human recombinant interleukin (IL)-2 (10 U/ml, National Cancer Institute), and 0.05 mM β-mercaptoethanol medium and containing 10 μg/ml of brefeldin A (Epicentre Technologies, Madison, Wis.). Cells were blocked with Mab to FcRIII/II receptor and stained with anti-CD44 FITC and either anti-CD8 or anti-CD4 Cy-Chrome (Pharmingen, San Diego, Calif.) in staining wash buffer (PBS, 2% fetal bovine serum 0.01% sodium azide, Sigma, St. Louis, Mo.), with brefeldin A (10 μg/ml). The cells were fixed in 1% formaldehyde (Ted Pella, Redding, Calif.), made permeable with staining wash buffer containing 0.5% saponin (Sigma, St. Louis, Mo.), and stained with anti-IFN-γ phycoerythrin (PE) (Pharmingen, San Diego, Calif.). The data were acquired by a FACSCalibur flow cytometer and analyzed with CELLQuest software (Becton-Dickinson Immunocytometry systems, San Jose, Calif.). Samples were considered positive if the percentage of CD8+, CD44+, INF-γ-positive cells was greater than two fold above background. Background was determined by adding an irrelevant peptide from Lassa N (RPLSAGVYMGNLSSQ, SEQ ID NO: 1) or no peptide in a solution that contained equivalent amounts of the dimethylsulfoxide used to dilute peptides.
Electron Microscopy: Live or INA-inactivated EBOV were applied to 300-mesh, nickel electron microscopy grids pre-coated with formvar and carbon, treated with 1% glutaraldehyde in PBS for 10 min, rinsed in distilled water, and negatively stained with 1% uranyl acetate. For immunoelectron microscopy, viruses were processed as described for fluid specimens (Geisbert et al., Virus Res. 39:129-50 (1995)). Briefly, fractions were applied to grids and immersed for 45 min in dilutions of monoclonal antibodies against EBOV GP. Normal mouse ascetic fluid was tested in parallel. Grids were washed with the TRIS buffer and incubated for 45 min with goat anti-mouse IgG labeled with 10 nm gold spheres (Ted Pella Inc. Redding, Calif.). Grids were washed in PBS, and fixed in 1% glutaraldehyde. After fixation, grids were rinsed in drops of distilled water and negatively stained with 1% uranyl acetate. For pre-embedment staining, cells were stained with anti-Ebola GP mAb followed by gold-anti-mouse Ab, fixed with 2% glutaraldehyde in Millonig's buffer (pH7.4) for 1 h and post-fixed in 1% uranylacetate, dehydrated and embedded in POLY/BED 812 resin (Polysciences, Warrington, Pa.). Resin was allowed to polymerize for 16 h at 60° C., Ultrathin sections (˜80 nm) were cut, placed on 200-mesh copper electron microscopy grids and negatively stained. Stained grids were examined with a JEOL 1200 EX transmission electron microscope at 80 kV.
Results
Treatment with INA followed by UV irradiation inactivates EBOV. To examine if EBOV can be inactivated by INA, a recombinant EBOV engineered to express green fluorescent protein (GFP) (Towner et al., Virology 332:20-7 (2005)) was used for initial experiments. EBOV-GFP was treated with 100 μM INA for 30 min at 4° C. Residual INA in the aqueous phase was then inactivated by 20 mM glutathione (reduced form). INA-treated virus was then exposed to far UV light for 10 minutes using a 1 inch water filter to avoid heating of the sample. As control, virus was UV irradiated in the absence of INA. Vero E6 cells were infected with untreated virus, UV treated virus and INA+UV treated virus. The virus infection was visualized by flow cytometry at 24 h, 48 h, 72 h, and 96 h time points. While UV alone had no effect on infectivity of the virus, UV irradiation of virus pretreated with INA resulted in complete loss of infectivity.
To ensure that INA treatment can also inactivate the authentic (non-recombinant) virus, Zaire EBOV was treated with UV alone or INA+UV and used to infect Vero E6 cells at an MOI of 10. After infection, cells were incubated in full medium at 37° C. Supernatants were harvested after 24 h, 48 h, or 72 h and viral RNA was purified from the supernatants. Genome copy numbers in the culture supernatants were quantified using a real time PCR (RT-PCR) assay. As shown in
The lack of infectivity of INA-inactivated EBOV was then tested in a mouse infection model. When mice were infected with a 1000 Pfu of mouse adapted EBOV-Z pretreated with INA+UV all the mice survived the challenge and there was no sign of disease in the mice (
INA-inactivated virus retains its normal morphology and the antibody reactivity of the glycoprotein: Electron microscopy studies were performed to determine if treatment with INA+UV had any effect on the morphology of the virus or the conformational epitopes of GP. As shown in
To further examine the question of preservation of conformational epitopes, the binding of live and inactivated EBOV to a panel of anti-GP antibodies was examined using a virus capture assay. Different Monoclonal antibodies against Ebola GP or control antibodies AE11 (anti Ebola VP40) and 9E10 (anti-Myc) were immobilized on 96 well plates. After blocking, 5×105 pfu of live or INA-inactivated EBOV were added to each well and incubated for 1 h. After extensive washing in PBS, the bound viruses were lysed in TRIazol reagent. RNA was prepared and the viral genome was quantified using real time PCR. As shown in
Immunization with INA-inactivated EBOV induces antibody response. To investigate if immunization with INA-inactivated EBOV can confer protective immunity against lethal challenge, groups of mice were immunized with one or two intraperitoneal injections (three weeks apart) of a dose of INA-inactivated EBOV equivalent to 5×104 Pfu. Two weeks after each vaccination, sera were collected from the mice to measure the antibody response. Anti-EBOV titer was measured by an ELISA using irradiated whole virus as immobilized antigen. As shown in
Induction of CD8+ T cell responses specific for Ebola epitopes by vaccination with INA-Treated Ebola. As previously described, protective cellular responses can be detected in both C57B1/6 mice and BALB/c vaccinated mice (Olinger et al., J. Virol. 79:14189-96 (2005)). To investigate if INA-inactivated EBOV can induce specific T cell responses against a range of peptides derived from EBOV proteins were examined using IFNγ intracellular staining as described above in the Materials and Methods section. As shown in Table 2, INA-EBOV vaccinated C57B1/6 (H-2b) mice displayed CD8+ T-cell responses against ZEBOV GP: WIPYFGPAAEGIYTE (GP531; SEQ ID NO:2), NP epitopes VYQVNNLEEIC (NP44; SEQ ID NO:3) and DAVLYYHMM (NP663; SEQ ID NO:4).
aMice vaccinated with INA-inactivated EBOV were assessed ex vivo for cellular responses to previously described ZEBOV epitopes in GP, NP, VP24, VP30, VP35, and VP40 (Olinger et al., J. Virol. 79:14189-96 (2005)). Mice received a booster vaccination on day 14. Splenocytes were collected seven days later.
bSplenocytes were used ex vivo for identification of peptides that induced IFN-γ expressing CD8+ T cells. Data shown represent intracellular IFN-γ data after a 5-h re-stimulation with peptide. The data are expressed as the peptide-induced % positive/background % positive.
The strongest response detected was to the VP35 epitope RNIMYDHL (VP35225; SEQ ID NO:5). Lastly a response to the VP40 epitope LRIGNQAFLQEFVLPP (VP40150; SEQ ID NO:6) was detected in vaccinated mice.
BALB/c mice had detectable CD8+ T-cell responses to two of the ZEBOV peptides derived from GP and VP24 (Table 2). A CD8+ response to the NP peptide sequence, SFKAALSSL (NP279; SEQ ID NO:7) was detected as well as the VP24 epitope PGPAKFSLL (SEQ ID NO:8)(Table 2). Combined, these data indicate that vaccination with INA-inactivated induces cellular immune responses.
Vaccination with INA-inactivated EBOV protects against lethal Ebola infection. INA-inactivated virus was then used in a vaccination study to evaluate its vaccine potential. Groups of mice were immunized either once or twice at a 2 week interval with 50000 Pfu of INA-inactivated mouse adapted EBOV-Z. Three weeks after the last immunization mice were challenged with 1000 pfu of mouse adapted EBOV-Z. As shown in
We had previously demonstrated that inoculation of mice with Ebola virus-like particles conveys short term protection against a lethal challenge, an effect primarily conveyed by activation of natural killer cells (Warfield et al., J Exp Med 200:169-79 (2004)). In contrast, irradiated virus is unable to provide such short term protection (id.). To examine if INA-inactivated EBOV can induce a protective innate immune response, mice were injected intraperitoneally with 50000 Pfu of INA-inactivated mouse adapted EBOV-Z or PBS as control and challenged with 1000 pfu of mouse adapted EBOV-Z three days later. All the mice treated with INA-inactivated virus survived the infection while all the control mice succumbed to death (
Antibody titers in these animals were also determined after challenge by ELISA using inactivated EBOV as antigen. As shown in
Taken together, these data indicate that INA is an effective inactivation agent for use in preparing immune system-stimulating compositions of hemorrhagic fever viruses such as Ebola virus.
This Example illustrates that INA-inactivated influenza viruses can be used to immunize animals against death by the disease it causes.
Materials and Methods
Purified influenza virus strain H3N2 was prepared by and obtained from Charles River Laboratories (N. Franklin Conn.). 1,5 Iodonaphthylazide (INA) was synthesized and supplied by “Combinix” Inc. (San Mateo Calif.). 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO), chloromethylfluorescein diacetate (CMFDA) and Amplex Red Neuraminidase assay kit were from Invitrogen-Molecular Probes (Carlsbad, Calif.). PKH-67 and PKH-26 were from Sigma. 3-1 KB Carcinoma cell line was generously supplied by Suresh Ambudkar from the laboratory of Cell Biology, NCI/NIH. CD4-GFP construct was a generous gift from W. Popik from the Oncology Center, The Johns Hopkins University School of Medicine Baltimore, Md.
Inactivation of Influenza Virus: Inactivation of Influenza virus by INA was carried out essentially as described for retroviruses (Example 1). In short, band purified H3N2 influenza virus was suspended in PBS at a concentration of 1 mg/ml protein. INA from a 40 mM stock solution in DMSO was added to the virus suspension in several installments and thoroughly mixed. The virus was incubated for 15 minutes in the dark and irradiated with UV light using a 100 W mercury lamp source for 2 minutes as described above.
In vitro infectivity and neuraminidase activity: Purified influenza virus strain H3N2 (X-31) at a protein concentration of 1 mg/ml was treated with different concentrations of INA and then inactivated by UV irradiation using the protocol described above and in Raviv, Y. et al. (J. Virol 79, pp. 12394-1 2400 (2005)) for the inactivation of HIV. The INA-treated viral preparation was then divided into two experimental groups. One INA-treated viral group was subjected to an infectivity assay and the second INA-treated viral group was tested for neuraminidase activity.
To monitor infection the binding of red blood cells to influenza virus-infected cells as assessed as described by Puri et al., J. Virol. 64: 3824-3832 (1990). Duplicate 25 pg aliquots of the virus in DMEM medium+2% bovine serum was added to KB carcinoma cells grown to confluence in 6 well plates. The virus-cell mixture was incubated for 2-3 hours at 37° C. and then the cells were washed and further incubated with fresh DMEM+10% bovine serum over night at the same temperature. The cells were washed again and labeled with the cytosolic fluorescent probe CMFDA (5-chloromethylfluorescein diacetate; Excitation wavelength 485 nm; Emission wavelength 525 nm) by following the manufacturer protocol (Molecular Probes). The cells were then washed with PBS and incubated with a suspension of 0.5% human erythrocytes pre-labeled with the fluorescent membrane probe PKH-26 (Excitation wavelength 530 nm; Emission wavelength 580 nm) (Sigma). After 15 minutes incubation at room temperature the cells were washed with PBS five times and lysed with a solution of 1% Triton X-100. The fluorescence values of PKH26/CMFDA at 580/525 nm were measured using a “Cytofluor” fluorescent plate reader.
Neuraminidase activity was determined by measuring the hydrogen peroxide induced fluorescence of a fluorescent probe using the “Amplex Red” neuraminidase assay kit (Molecular Probes) following the manufacturer's protocol.
As a control, non-infected cells were incubated with PKH labeled erythrocytes. Background levels of fluorescence were determined from the signal obtained by incubating labeled erythrocytes in wells without cells. The signal from the non-infected cells was not significantly different from background.
Influenza viral-cell fusion assay by photosensitized labeling: Fusion by photosensitized labeling was measured as described by Pak et al. J. Biol. Chem. 269: 14614-14619 (1994) and Raviv et al., Virology, 293: 243-251 (2002), using human erythrocyte resealed ghosts (1 ml) that were labeled with the fluorescent membrane probe PKH67 (Sigma) and incubated with 20 μCi 125INA for 15 minutes on ice. The ghosts were washed and divided into 5 experimental groups. Influenza virus at 1 mg/ml protein was treated with different concentration of INA and irradiated as described above. The virus was mixed with the resealed ghosts for 30 minutes at room temperature, washed, and then the appropriate amount of citric acid was added to the mixture to lower the pH to pH 5.0. The tubes were further incubated at 37° C. for 15 minutes, the viral/erythrocyte ghost mixture was then irradiated at 488 nm for 30 seconds using an argon laser at the intensity of 0.5 W. The ghosts were lysed and the hemagglutinin (HA) was isolated by immunoprecipitation, then the immunoprecipitated proteins were separated by SDS-PAGE. The radioactivity incorporated in the HA molecules was measured by autoradiography using a phosphorimmager.
The controls employed were untreated virus (100%) and untreated virus that was bound to the ghosts and kept at neutral pH 7.4 (signal=0) so that fusion was not triggered by adjustment of the pH to 5.0.
Measurement of lipid mixing by fluorescence dequenching: Labeling of virus with octadecyl rhodamine (R18) and fusion with RBC ghosts were performed as described by Claque et al., Biochemistry. 30: 5491-5497 (1991); Pak et al., J. Biol. Chem. 269: 14614-14619 (1994); Puri et al., J. Virol. 64: 3824-3832 (1990). Influenza virus was inactivated with 100 μM INA as described and labeled with R18. The R18-labeled virus was prebound to RBC resealed ghosts at room temperature for 20 min and washed. The sample was transferred to a cuvette in the fluorimeter at 37° C. and the pH was lowered to 5.0 by adding citric acid. Fluorescence was monitored before and after the addition of the citric acid. Percent fluorescence dequenching was determined as 100×[(F−Fo)/(Ft−Fo), where Fo and F are fluorescence intensities at time zero and at a given time point, respectively, and Ft is the total fluorescence obtained after disruption of cells by Triton X-100. As a control for non-specific dequenching the virus was inactivated by pre-exposure to pH 5.0 followed by incubation with cells at pH 7.4 (Korte et al., J. Virol. 73: 4567-4574 (1999).
Fluorescence recovery after photobleaching (FRAP) acquisition and analysis: FRAP was performed using a Zeiss LSM 510 (Carl Zeiss, Jena, Germany) confocal laser scanning microscope. HeLa cells were plated on 35 mm glass bottom dishes (MatTek, Ashland, Mass.) and either transfected with CD4-GFP 24 hours prior to confocal analysis as described previously (24) or labeled with DiO one hour prior to the FRAP analysis. INA was added to the cells from stock of 30 mM in dimethyl sulfoxide (DMSO) to a final concentration of 20 or 100 μM. After 10 minutes incubation at room temperature, the sample was irradiated with UV light at 10 mW/cm2 for two minutes. The cells were then submitted to FRAP while kept at physiological conditions of 37° C. and 5% CO2 in a stage incubation system (Incubator S; PeCon GmbH, Erbach, Germany). A 488 nm Ar+ laser line was used for excitation and emission light was collected with a 500-550 bandpass filter. A 40X/1.3 NA oil immersion objective lens was used with a zoom factor of 4. The detector pinhole was opened slightly to acquire an optical section of 2 μm thickness. This allowed more light to be collected for better quantification. Three pre-bleach images were acquired to determine the rate of non-purposeful photobleaching. Photobleaching was performed by increasing the transmission of the laser to 100% for 20-50 iterations to optimize the extent of bleaching. Following photobleaching 8-10 images were acquired at one second intervals and then the acquisition rate was changed to ten second intervals to follow the recovery to completion. A total of 20-40 images were acquired. FRAP analysis was performed using the MIPAV (CIT/NIH, Bethesda, Md.) software package using a 1D diffusion FRAP model to retrieve the mobile fraction (Lippincott-Schwartz et al., Nat Rev Mol Cell Biol. 6: 444-456 (2001)). Data were automatically corrected with background subtraction, as well as normalization for the non-purposeful photobleaching rate calculated from the whole cell membrane.
Electron microscopy: Electron microscopy was carried out essentially as previously described for retroviruses (Raviv et al., Journal of Virology. 79: 12394-12400 (2005)). In short live or INA-inactivated influenza viruses were applied to 300-mesh, nickel electron microscopy grids pre-coated with formvar and carbon, treated with 1% glutaraldehyde in PBS for 10 min, rinsed in distilled water, and negatively stained with 1% uranyl acetate. Stained grids were examined with a JEOL 1200 EX transmission electron microscope at 80 kV.
Protective Immunization Studies: To assess the ability of INA-inactivated influenza virus to induce protective heterosubtypic immunity, mice were vaccinated just once with live A/Aichi/68 X-31 (X31, H3N2), INA-treated X31, or B/Ann Arbor (B/AA) influenza virus. Five week old, female BALB/c mice (Harlan) were acclimated for 3 weeks. Animals were bled by tail vein nick, and 6 days later were anesthetized via intraperitoneal (i.p.) injection of Avertin. The animals were then immunized or infected with 30 μl solution as indicated in Table 3 and described below. As negative controls, mice were given PBS (naïve) or infected with influenza B/AA intranasally. B/AA influenza was used as a non-specific viral infection control. As positive controls, mice were infected intranasally with live X31. The first experimental group consisted of mice immunized with INA-treated X31 delivered subcutaneously, the standard route of immunization for traditional influenza vaccines. The second experimental group was comprised of animals immunized intranasally with INA-treated X31, with the expectation that this route of delivery would induce a potent mucosal immune response that would provide protection against lethal, heterosubtypic challenge.
The immunization protocols utilized are summarized in Table 3.
After immunization/infection, the mice were allowed to rest for 4 weeks and then bled to provide serum for determining antibody responses. One week later, all groups of mice were anesthetized and challenged intranasally with 10 LD50 of influenza A/PR/8/34 (PR8) in 50 μl volume. The mice were then monitored daily for mortality and weighed every 2-3 days until all animals had either succumbed to infection or recovered most of their initial body weight.
Results
INA reduced influenza viral infectivity without affecting neuraminidase activity: Influenza infectivity is facilitated by viral hemagglutinin (HA) which is an integral membrane protein of the viral envelope whose transmembrane segment's integrity is essential for full fusion and infectivity. Neuraminidase is also an integral membrane protein of the viral envelope only that its catalytic site is located on the hydrophilic segment that protrudes outside the membrane.
INA treatment of influenza reduced the infectivity of the influenza virus to zero in a dose-dependent manner while the catalytic activity of the viral envelope enzyme neuraminidase was not affected (
In order to test if the INA effects the ability of the virus to fuse with the target cell membrane, fusion was measured directly by two different methods: Dequenching of R-18 and photosensitized labeling. Dequenching measures the pH dependent mixing of lipids from the viral envelope with lipids of the target cell and this function was not affected by INA relative to non-treated viruses (
These results suggest that INA may have a general effect on the translational mobility of proteins in the membrane. To test this hypothesis the diffusion of proteins and lipids in the HeLa cell membrane was measured after INA treatment by fluorescence recovery after photobleaching (FRAP). For these experiments CD4 conjugated to GFP (CD4-GFP) was used as the fluorescently-labeled transmembrane protein and DiO as the lipid fluorescent probe. The results presented in
It was also established visually that the influenza virus maintains its structural integrity after INA treatment. For that purpose, INA inactivated and control viruses were subjected to negative staining and visualized by an electron microscope at low resolution. After examining ten different EM imaging fields for each type of virus the INA inactivated and control viruses appeared indistinguishable and structurally intact (data not shown). All experiments were repeated three times except for the photosensitized labeling that was repeated twice.
INA treatment protects animals against influenza infection and death: ELISA assays at day 28 post-vaccination showed that serum antibody titers specific for X31 were equivalent in mice receiving either live or INA-treated X31. These results indicate that the INA inactivation procedure did not reduce influenza viral antigenicity.
Moreover, when challenged intranasally with 10 LD50 of heterologous influenza virus (A/PR/8, H1N1), these live or INA-treated X31 nasally immunized animals exhibited minimal weight loss and 100% survived (
These results demonstrate that INA-treatment of influenza virus generates an effective vaccine composition that, when administered to mammals, protects those mammals against influenza virus infection and the symptoms associated with influenza infection.
All patents and publications referenced or mentioned herein are indicative of the levels of skill of those skilled in the art to which the invention pertains, and each such referenced patent or publication is hereby incorporated by reference to the same extent as if it had been incorporated by reference in its entirety individually or set forth herein in its entirety. Applicants reserve the right to physically incorporate into this specification any and all materials and information from any such cited patents or publications.
The specific methods and compositions described herein are representative of preferred embodiments and are exemplary and not intended as limitations on the scope of the invention. Other objects, aspects, and embodiments will occur to those skilled in the art upon consideration of this specification, and are encompassed within the spirit of the invention as defined by the scope of the claims. It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, or limitation or limitations, which is not specifically disclosed herein as essential. The methods and processes illustratively described herein suitably may be practiced in differing orders of steps, and that they are not necessarily restricted to the orders of steps indicated herein or in the claims. As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to “a host cell” includes a plurality (for example, a culture or population) of such host cells, and so forth. Under no circumstances may the patent be interpreted to be limited to the specific examples or embodiments or methods specifically disclosed herein. Under no circumstances may the patent be interpreted to be limited by any statement made by any Examiner or any other official or employee of the Patent and Trademark Office unless such statement is specifically and without qualification or reservation expressly adopted in a responsive writing by Applicants.
The terms and expressions that have been employed are used as terms of description and not of limitation, and there is no intent in the use of such terms and expressions to exclude any equivalent of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention as claimed. Thus, it will be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.
The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.
Other embodiments are within the following claims. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.
This application is a U.S. national stage filing from International Application No. PCT/US2007/007338 filed Mar. 23, 2007 and published as WO 2008/054481 on May 8, 2008, which claims priority to U.S. Provisional Application Ser. No. 60/785,781 filed Mar. 24, 2006, the contents of which are specifically incorporated herein their entirety. This application is also a continuation-in-part of co-pending U.S. patent application Ser. No. 11/525,250 filed Sep. 21, 2006, which is a U.S. national stage filing from International Application No. PCT/US2005/009559 filed Mar. 22, 2005 and published as WO 2005/093049 on Oct. 6, 2005, which claims priority to U.S. Provisional Application Ser. No. 60/555,268, filed Mar. 22, 2004; the contents of which applications and publications are also specifically incorporated herein in their entireties.
The invention described herein was developed with support from the National Cancer Institute. The United States Government may have certain rights in the invention.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US2007/007338 | 3/23/2007 | WO | 00 | 5/18/2009 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2008/054481 | 5/8/2008 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 5663043 | Zepp et al. | Sep 1997 | A |
| Number | Date | Country |
|---|---|---|
| WO-9941360 | Aug 1999 | WO |
| WO 2005093049 | Jun 2005 | WO |
| WO-2005093049 | Oct 2005 | WO |
| WO-2008054481 | May 2008 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 20090297558 A1 | Dec 2009 | US |
| Number | Date | Country | |
|---|---|---|---|
| 60785781 | Mar 2006 | US | |
| 60555268 | Mar 2004 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 11525250 | Sep 2006 | US |
| Child | 12225551 | US |
