The present invention relates to hyaluronidase enzyme methods, vaccines and compositions for treating or preventing pathogenic infections, such as HIV, in a patient. The invention also relates to methods, vaccines and compositions for providing immunity against HIV infection in a patient comprising treating the patient with HIV virus or HIV infected cells that have been treated with hyaluronidase.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to hyaluronidase compositions and methods for the effective treatment and prevention of pathogen infection, including retroviral infections such as HIV. The invention also relates to novel methods for developing and eliciting immune responses to pathogen infections, including retroviral infections such as HIV.

2. Description of the Related Art

Hyaluronidase enzymes are found throughout the animal kingdom and exist as hydrolytic, transglycosidic and mucolytic proteins. There are essentially two types of hyaluronidase: the non-specific type which breaks down hyaluronic acid, chondroitin and related polysaccharides; and the type which specifically cleaves hyaluronic acid. Hyaluronidases of varying molecular weights, composition, and activity are available from numerous sources including bacteria (via fermentation, etc.) various cell lines (including human) and animal testes.

Hyaluronidase has been used for the treatment of tumors (U.S. Pat. No. 6,193,963); for accelerating the clearance of hemorrhagic blood from the vitreous humor of the eye (U.S. Pat. No. 6,039,943); to reduce intraocular pressure in the eyes of glaucoma patients through degradation of hyaluronan within the vitreous humor (U.S. Pat. No. 4,820,516); as a "spreading agent" to enhance the activity of chemotherapeutics and/or the accessibility of tumors to chemotherapeutics (Czejka, et al., Pharmazie 1990, 45(9), 693 4); in the prevention and treatment of helmith infections (U.S. Pat. No. 5,811,100); and for treating benign prostatic hypertrophy (U.S. Pat. No. 5,116,615).

The primary substrate for the hyaluronidase enzyme is hyaluronic acid. Hyaluronic acid is a well known substance that is found in joint tissue and in the vitreous humor of the eye of mammals. Hyaluronic acid is also an integral part of many cell wall structures and has been extracted from rooster combs, human umbilical cords and bacterial cultures. It is a naturally occurring mucopolysaccharide with a molecular weight generally ranging between 50,000 and 8,000,000 (or possibly higher or lower), depending on the source of the material, the analytical method used in its determination and isolation technique.

The terms "haluronate" or "hyaluronan" and the abbreviation "HA" are often used to mean "hyaluronic acid equivalent" indicating hyaluronic acids of varying molecular weights, any of its salt forms, derivatives, isoforms, and/or ligand complexes.

The HIV virus utilizes hyaluronic acid in the adhesion process enabling the virus to bond to different cell lineages. For example, it has been reported that HIV can bind to hyaluronic acid through acquired CD44 adhesion molecules and its isoforms and retain its biological activity when expressed on the virus (J. Immunol. 1996, 156(4), 1557 1565; AIDS Res. Hum. Retroviruses 1995, 11(9), 1007 1113).

Described herein are hyaluronidase compositions and methods of using said compositions for the prevention and/or treatment of retroviral infections, such as HIV infection, in mammals.

BRIEF DESCRIPTION OF THE INVENTION

The present invention relates to hyaluronidase compositions for the treatment and prevention of retroviral infections. The present invention also relates to methods of using hyaluronidase compositions for treatment and/or prevention of retroviral infections.

Accordingly, in one aspect, this invention relates to compositions and prodrug compositions for providing immunity against HIV infection, for preventing HIV infection and/or for treating HIV infection in a mammal, said compositions comprising hyaluronidase.

In another aspect, this invention relates to methods for providing immunity against HIV infection, for preventing HIV infection and/or for treating HIV infection in a mammal in need of HIV immunity, prevention, or treatment, comprising administering an effective amount of a composition comprising hyaluronidase.

In yet another aspect, this invention relates to compositions for providing immunity against HIV infection, for preventing HIV infection and/or for treating HIV infection in a mammal, said composition comprising hyaluronidase treated virus. The virus can be further attenuated or inactivated, for example, by heat treatment.

In still another aspect, this invention relates to methods for providing immunity against HIV infection, for preventing HIV infection and/or for treating HIV infection in a mammal, comprising administering treatments that consist of hyaluronidase treated virus. The virus can be further attenuated or inactivated, for example, by heat treatment.

The invention described herein demonstrates that antigen presenting cells, such as macrophages and monocytes, will internalize hyaluronidase treated virus and initiate an appropriate and competent immune response to HIV and other retroviruses.

The invention described herein also demonstrates that antigen presenting cells can initiate the same degree of immune response when co-cultured with hyaluronidase treated dead virus or with hyaluronidase treated live virus.

The invention described herein also provides a method for the development of therapeutic and prophylactic vaccines as well as therapeutic parenteral formulations.

The invention described herein further provides for the use of hyaluronidase as a means and method to augment immune response to virus, antigen and/or immunogen.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

As used in this specification, the term "inactivated virus" refers to viral pathogens that have been killed, for example, by heat treatment or chemical treatment.

As used in this specification, the term "attenuated virus" refers to live viral pathogens capable of replication but which are not pathogenic.

This invention relates to hyaluronidase compositions and methods for treating retroviral infections, such as HIV infections, using hyaluronidase, hyaluronidase treated virus, or hyaluronidase treated virally infected cells. The invention also provides compositions and methods for providing immunity against HIV in mammals. The compositions of the present invention are comprised of hyaluronidase enzyme.

One of the problems associated with treating HIV infection is that the antigen presenting cell `presentation` is either missing or infectiously impaired in HIV infection. It is believed that the reason for the poor efficacy of either chemically treated HIV or heat inactivated HIV vaccines, therefore, is the inability of Antigen Presenting Cells to recognize, internalize and phagocytize the killed or denatured virus. Thus, the initial Antigen Presenting Cell step is severely limited or absent altogether. In addition to not recognizing dead or living HIV as an immunogen or antigen, the putative Th1 on/off switch necessary for a cellular response, the CD44 receptor, is not innervated. Also, in order to elicit cellular immunity the APC must internalize, analyze and recognize a "self-identifying marker" such as the non-antigenic or anti-antigenic viral envelope.

This invention provides compositions, vaccine compositions and methods of treatment which overcome the above problems associated with the treatment of HIV. Disclosed herein is the discovery that the hyaluronidase enzyme exhibits adverse and toxic effects against HIV infected cells. This adverse activity results in significantly reduced viral replication within the infected cell. Thus the hyaluronidase enzyme is capable of treating HIV infected cells in an HIV infected patient.

Further, the treatment of the HIV virus itself with the hyaluronidase enzyme creates an effective antigen or immunogen capable of eliciting an immune response against the virus. Thus hyaluronidase is capable of directly reducing or eliminating the HIV virus from an infected patient.

Antigen Presenting Cells (APCs), such as monocytes and macrophages, process hyaluronidase treated HIV infected cells or hyaluronidase treated virus in a different manner than non-hyaluronidase treated infected cells. Hyaluronidase treated cells or virus, unlike non-hyaluronidase treated cells or virus, are more readily recognized and internalized by APCs thus permitting the APCs to stimulate an immune response to the virus. Therefore, the treatment of HIV virus or infected cells with hyaluronidase allows naive immunocytes to recognize the HIV virus and be resistant to HIV challenge. These results permit the development of therapeutic and prophylactic vaccines and therapeutic parenteral formulations.

Without wishing to be bound by any particular theory, it is believed that hyaluronidase is the binding molecule between cellular hyaluronic acid and the APC and signals the APC to become a "host" or "autotropic" APC. Because of this, hyaluronidase can also bridge antigen to APC. As a result, when inactivated virus or virally infected cells are treated with hyaluronidase a proper and competent cell-mediated immune response to HIV infection is elicited.

The compositions of the invention may be formulated as a solution of lyophilized powders for parenteral administration. Powders may be reconstituted by addition of a suitable diluent or other pharmaceutically acceptable carrier prior to use. The liquid formulation is generally a buffered, isotonic, aqueous solution. Examples of suitable diluents are normal isotonic saline solution, standard 5% dextrose in water or in buffered sodium or ammonium acetate solution. Such formulation is especially suitable for parenteral administration, but may also be used for oral administration. It may be desirable to add excipients such as polyvinylpyrrolidone, gelatin, hydroxy cellulose, acacia, polyethylene glycol, mannitol, sodium chloride or sodium citrate.

The compositions of the invention can also be used as a vaccine, by compounding the enzyme with a suitable adjuvant or excipient of the kind conventionally employed in vaccine formulations. Vaccine formulations of the present invention comprise an immunogenic amount of a live or inactivated or attenuated virus treated with hyaluronidase as disclosed herein in combination with a pharmaceutically acceptable carrier. An "immunogenic amount" is an amount of the hyaluronidase treated virus sufficient to evoke an immune response. Exemplary pharmaceutically acceptable carriers include, but are not limited to, sterile pyrogen-free water and sterile pyrogen-free physiological saline solution.

Administration of the vaccines disclosed herein may be carried out by any suitable means, including both parenteral injection (such as intraperitoneal, subcutaneous, or intramuscular injection), and by topical application of the vaccine (typically carried in the pharmaceutical formulation) to an airway surface. Topical application of the vaccine to an airway surface can be carried out by intranasal administration (e.g. by use of dropper, swab, or inhaler which deposits a pharmaceutical formulation intranasally). Topical application of the vaccine to an airway surface can also be carried out by inhalation administration, such as by creating respirable particles of a pharmaceutical formulation (including both solid particles and liquid particles) containing the hyaluronidase treated virus as an aerosol suspension, and then causing the subject to inhale the respirable particles. Methods and apparatus for administering respirable particles of pharmaceutical formulations are well known, and any conventional technique can be employed. The vaccines of the present invention can also be administered intravenously, such as by IV drip.

The dosage ranges for administration of the compounds of the invention are those necessary to produce the desired affect whereby symptoms of infection are ameliorated. For example, as used herein, an anti-HIV effective amount for treating or preventing an HIV infection refers to the amount administered so as to maintain an amount which suppresses or inhibits HIV infection as evidenced by standard assay. The dosage will also be determined by the existence of any adverse side effects that may accompany the compounds. It is always desirable, whenever possible, to keep adverse side effects to a minimum.

One skilled in the art can easily determine the appropriate dosage, schedule, and method of administration for the exact formulation of the composition being used in order to achieve the desired effective concentration in the individual patient. However, the dosage can vary from between about 0.001 mg/kg/day to about 1000 mg/kg/day, but preferably between about 0.001 to about 100 mg/kg/day.

In the compositions, vaccines and methods of the invention, viral immunity which is induced, altered or enhanced by hyaluronidase may be increased or modified by the use of mitogens, phytogens, colony stimulating factors, growth factors, immunomodulating or immunoenhancing cytokines, chemokines and/or soluble substances, interferons, proteglycans, perforins, granzymes, and the like, such as, but not limited to, IL-2 and PHA.
 

Claim 1 of 9 Claims

1. A composition comprising: a) HIV infected cells that have been treated with a bovine hyaluronidase or a hyaluronidase enzyme with comparable activity at a concentration of at least 0.01611 units/mL, and b) optionally a pharmaceutically acceptable carrier or diluent.

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