Field of the Invention The present invention relates to the treatment and prevention of HIV infection and AIDS by administration of antigens derived from sarcoidosis-infected tissue.

Background of the Invention Sarcoidosis, also called sarcoid of Boeck, is a chronic, progressive, systemic granulomatous reticulosis of unknown etiology which may involve almost any organ or tissue. The disorder is characterized by the presence in all affected organs or tissues by the accumulation of non-necrotizing epithelioid cell tubercles. The disease can be diagnosed by intracutaneous injection of a suspension of human sarcoidal spleen tissue known as the Kveim antigen (Siltzbach, J. Am. Med. ABSOC , 178:476-482, 1961) .

The Kveim antigen has been used as a diagnostic test to determine the presence of sarcoidosis in a patient. The traditional Kveim test for sarcoidosis is based on the skin's reactivity to injected sarcoid tissue. An intradermal injection of the test material is administered, and, if a noncaseating granuloma appears on the skin at the site in 4 to 8 weeks, the so-called Kveim reaction is positive and is evidence that the recipient has sarcoidosis. The amount of Kveim antigen injected in a diagnostic test is small; roughly two spleens supply between 2,000 and 3,000 Kveim test dosages (Siltzbach, J. Am. Med. Assoc , 178:476-482, 1961; de Bois et al., Lancet, 342:173, 1993).

Acquired immune deficiency syndrome (AIDS) is caused by a retrovirus, the human immunodeficiency virus (HIV) . HIV infection is characterized by a dramatic decrease in T lymphocytes (T _h cells) carrying the CD4 antigen, although HIV- infected individuals can generate new T lymphocytes until late in the progress of the disease. The decline in the population of T _h cells renders the HIV-infected individual susceptible to a large number of opportunistic infections, any of which may prove fatal. To date, there are no effective treatments for

AIDS or the HIV infection.

There are few reports of coexistence of sarcoidosis and HIV. As of May 1995, only nine cases had been reported (Granieri et al. , Southern Med. J. , 88:591-595, 1995; Newman et al., Chest, 102:1899-1901, 1992). The difference between the immune responses seen in HIV-infected patients and those with sarcoidosis is striking in that they are almost diametrically opposed. It is believed that sarcoid activates the immune system by markedly increasing the number of CD4* T helper cells.

This activation is often expressed as a comparison of the ratio of lymphocyte or T-cell subsets. T _h cells have been defined above as expressing the CD4 surface antigen. Another large T cell subset are the killer lymphocytes, so called because they attack infected tissue. This cell subset carries a surface antigen called CD8. The CD4 ⁺ /CD8 ⁺ ratio, or count, is the parameter characteristic of AIDS and sarcoidosis.

This distinction is most striking in pulmonary sarcoidosis cases, where researchers find a dramatic increase in this ratio in patients with sarcoidosis, and a profound decrease in the ratio in HIV positive patients. In the bronchoalveolar lavage (BAL) fluid of healthy, nonsmoking individuals, the CD4 ⁺ /CD8 ⁺ ratio is approximately 1.6. In those with pulmonary sarcoidosis, there is a relative increase of CD4* over CD8 ⁺ cells and the CD4* to CD8 ⁺ ratio can rise to 10:1. In contrast, studies of BAL fluid in HIV-infected persons lacking clinical evidence of lung disease, show that 70% have a change in the amount of lymph cells due to a marked decrease in CD4 ⁺ cells and increase in CD8 ⁺ cells leading to a correspondingly lowered CD4 ⁺ /CD8 ⁺ ratio (Chosia et al. , Pne mol . Alergol . Pol . , 59 (9-10) :23-27; Newman et al. , Chest, 102:1899-1901, 1992; Hunninghake, Ann. Intern. Med. , 94:73-94, 1981; Murray et al. , Am. Rev. Respir. Dis . , 141:1356-1372, 1990) . There is an urgent need for a safe, effective prevention and treatment of HIV infection and AIDS which is provided by the present invention.

Summary of the Invention One embodiment of the present invention is a method of treating HIV-1 infection and AIDS in a patient in need thereof, comprising the step of administering to the patient an effective CD4 ⁺ -increasing amount of a sarcoid antigen in a pharmaceutically acceptable carrier. Preferably, the effective CD4 ⁺ -increasing amount results in an absolute CD4 ⁺ count of greater than 500/mm ³ and a CD4 ^* /CD8 ⁺ ratio of about 1.5. Advantageously, the sarcoid antigen is Kveim antigen. In another aspect of this preferred embodiment, the Kveim antigen is administered in an amount ranging from about 0.10 ml to about 1.00 ml; more preferably, the amount is between about 0.20 and about 0.40 ml; most preferably, the amount is about 0.25 ml. Preferably, the administering step is intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous; most preferably, the administering step is intramuscular.

Another embodiment of the invention is a method for preventing HIV-1 infection and AIDS in an individual at risk therefor, comprising the step of administering to the individual an effective CD4 ⁺ -increasing amount of a sarcoid antigen in a pharmaceutically acceptable carrier. Preferably, the sarcoid antigen is Kveim antigen. Advantageously, the Kveim antigen is administered in an amount ranging from about 0.10 ml to about 1.00 ml; more advantageously, the amount is between about 0.20 and about 0.40 ml; most advantageously, the amount is about 0.25 ml. According to another aspect of this embodiment, the administering step is intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous. Most preferably, the administering step is intramuscular.

The present invention also provides a vaccine composition for preventing HIV-1 infection and AIDS, comprising a sarcoid antigen in a pharmaceutically acceptable carrier. Preferably, the sarcoid antigen is Kveim antigen. Another embodiment of the invention is a method of increasing the CD4 ⁺ count in an individual in need thereof, comprising the step of administering to the individual an

effective CD4 ^* -increasing amount of a sarcoid antigen in a pharmaceutically acceptable carrier. Preferably, the effective CD4 ⁺ -increasing amount results in an absolute CD4* count of greater than 500/mm ³ and an effective CD4 ⁺ -increasing amount results in a CD4 ⁺ /CD8 ⁺ ratio of about 1.5. Advantageously, the sarcoid antigen is Kveim antigen, preferably, the Kveim antigen is administered in an amount ranging from about 0.10 ml to about 1.00 ml; more preferably, the amount is between about 0.20 and about 0.40 ml; most preferably, the amount is about 0.25 ml. In another aspect of this embodiment, the administering step is intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous. Preferably, the administering step is intramuscular.

Detailed Description of the Preferred Embodiments The present invention relates to the immunotherapeutic treatment and prevention of HIV infection and AIDS in a human by administration of antigens derived from sarcoidosis- infected tissues. These sarcoid antigens will effectively stimulate the immune system, resulting in an elevation in the absolute CD4 ⁺ count or in the CD4 ⁺ /CD8 ⁺ ratio and, consequently, exert both a protective effect in high risk individuals by acting as a therapeutic vaccine; and will promote improvement in the health of HIV-infected individuals and those with AIDS by preventing many opportunistic infections.

HIV-1 inhibits cytokine (i.e. interleukin) production; conversely, sarcoid antigen stimulates the production of cytokines, particularly interleukin-12 (IL-12) . Thus, sarcoid antigens will stimulate IL-12 production which will be measured and used to assess the status of HIV-1 infection. The HIV-1 viral load/burden will also be determined during the course of treatment with sarcoid antigen by measuring mRNA levels using, for example, an HIV mRNA assay kit available from Hoffmann-LaRoche. The total body viral load/burden will progressively decrease during sarcoid antigen treatment.

The introduction of immunogenic sarcoid antigen into a patient infected with HIV will "trick" the immune system into

responding to a perceived sarcoidosis disease process, thus stimulating the immune response by elevating the T _h cell population to offset or block the progression of HIV infection and AIDS. The sarcoid antigen will also stimulate recruitment of CD4* cells from the circulating blood pool into the unhealthy organs affected by HIV.

The sarcoid antigen may be administered in a number of ways, as long as it can enter the bloodstream by the selected route. Preferred routes of administration include intramuscular, intravenous, intraarterial, intraperitoneal and subcutaneous. Intramuscular administration is particularly preferred. Although any sarcoid antigens capable of eliciting an increase in the absolute CD4 ⁺ count CD4 ⁺ /CD8* ratio or IL-12 levels are contemplated for use in the present invention, the Kveim antigen is preferred.

It will be appreciated that the amount and frequency of administration of sarcoid antigens will vary depending on the particular antigen employed, the progression of the disease and the physical characteristics of the patient. In a preferred embodiment, up to five intramuscular injections of Kveim antigen, described below, are sequentially administered over a six month period in an amount ranging from about 0.10 ml to about 1.00 ml per injection. Booster administrations may be required at 6 month to one year intervals. In another preferred embodiment, up to four injections are administered during a 30 day period. For example, the Kveim antigen may be administered at day 0 followed by booster injections at days 4, 6 and 14. In a particularly preferred embodiment, the amount of Kveim antigen administered is between about 0.20 ml and 0.40 ml. In a most preferred embodiment, the amount of antigen administered is about 0.25 ml. The antigen is typically administered with a conventional amount of an adjuvant such as alum or Freund's complete adjuvant, although any conventional adjuvant known to one skilled in the art may be utilized. The Kveim antigen is used as a standardized suspension which is not usually quantitated. The antigen is used to test for sarcoidosis in a patient. If

a granuloma forms after injection into a patient having sarcoidosis, then the Kveim preparation is active.

The patient's blood is drawn weekly during the treatment and the absolute CD4 ^* count and CD4 ⁺ /CD8 ⁺ ratio are monitored by standard flow cytometry methods such as fluorescence- activated cell sorting (FACS) using monoclonal antibodies directed to the CD4 and CD8 cell surface proteins. The sarcoid antigen administration is continued until a CD4 ⁺ /CD8 ⁺ ratio of approximately 1.5 is obtained and/or the absolute CD4 ⁺ count is greater than 500/mm ³ . Alternatively, the antigen may be administered even after this ratio has been reached in order to keep the T _h cell count sufficiently high.

The particular sarcoid antigen administered can be any antigen capable of elevating the absolute CD4 ⁺ count and/or CD4 ⁺ /CD8 ⁺ ratio when administered to an AIDS or HIV-infected individual. One particular sarcoid antigen, the Kveim antigen, is a suspension of extracts of sarcoid spleen or lymph node known to contain sarcoid granulomas. Once isolated, the suspension is sterilized by phenolization, heated by steam pressure and autoclaved as described in Example 1. The suspension may be stored at -20°C for one week, then irradiated and heat-sealed.

The antigen preparation for parenteral administration may be in the form of a sterile injectable preparation, such as a sterile aqueous or oleaginous suspension. The suspension may be formulated according to methods well known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally- acceptable diluent or solvent, such as a solution in 1,3- butanediol. Suitable diluents include, for example, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils may be employed conventionally as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid may likewise be used in the preparation of injectable

preparations.

Kveim antigen suspension is obtained from sarcoid- infected human spleens as described below.

Example 1 Preparation of Kveim antiσen suspension

Kveim antigen suspension is prepared according to the preferred method of Teirstein (in Sarcoidosis, Lieberman, J. , ed. , pp.104-105, Grune & Stratton, San Diego, 1985). Sarcoidosis-infected human spleen removed for clinical reasons is stored at -20°C or lower and thawed as required. All steps are performed using sterile buffers and instruments in an enclosed cabinet or hood to prevent contamination. All solutions are kept at 4°C, and the KS suspension is kept on ice unless otherwise noted. The spleen is thawed and the cortex removed and discarded. A 100 gram section of spleen is homogenized in 5 volumes of phosphate-buffered saline (PBS) . The homogenate is subjected to two cycles of freeze-thaw and homogenized a second time. The homogenate is then centrifuged at 10,000 rpm for 30 minutes and the supernatant discarded. The pellet is washed by resuspension in 2-3 volumes PBS and centrifugation as above. This washing/centrifugation is repeated 3-4 times until the sample is free of hemoglobin. The pellets are resuspended in 3 volumes PBS and again homogenized. The suspension is forced through a sterile 40 mesh filter using a 50 ml syringe without a needle. The mesh screen is rinsed with a small volume of PBS and any remaining large solids are discarded. The sample is centrifuged at 15,000 rpm for 45 minutes and the supernatant discarded. The pellet is resuspended in 2 volumes sterile water and heated to 65°C for two hours with intermittent agitation. The suspension is centrifuged at 15,000 rpm for 45 minutes to remove soluble lipids, resuspended in PBS to 5-6 ml/ml solids, incubated at 65°C for one hour and sonicated for 1-2 minutes. The suspension is passed through a 100 mesh filter and any large solids are discarded. The suspension is then subjected to phenolization (0.25%) and exposure to gamma irradiation (Munro et al. , Thorax, 42:321-331, 1987).

The Kveim antigen suspension is then used to treat HIV-i infected individuals and individuals with AIDS as described in the following examples.

Example 2 Immunotherapeutic Treatment of HIV infection and AIDS

Blood is drawn from HIV-infected individuals or individuals with AIDS and their pretreatment CD4 ⁺ /CD8* ratio and absolute CD4 ⁺ count determined by FACS. On day zero, the individuals are intramuscularly administered 0.25 ml of the Kveim suspension in saline produced according to Example 1 in Freund's complete adjuvant. An equal number of HIV-infected individuals are administered only saline. Booster injections of Kveim antigen or saline are administered at days 4, 6 and 14. At day 21, blood is drawn from both experimental groups and the absolute CD4 ^* count and CD4 ⁺ /CD8 ⁺ ratio are determined. Individuals who receive the Kveim antigen exhibit significantly increased CD4 ⁺ counts and/or increased CD4*/CD8 ⁺ ratios in comparison to individuals who receive only saline.

Example 3 Prevention of HIV infection and AIDS by

Therapeutic Vaccination

HIV-1 negative individuals, including those at high risk for HIV-1 infection and HIV-1 negative individuals exhibiting illnesses characteristic of HIV infection or AIDS, are administered a vaccine comprising the Kveim antigen as described in Example 2. High-risk individuals include those practicing unprotected sex with individuals whose HIV status is unknown, intravenous drug users and those receiving blood transfusions prior to 1985. The individuals are monitored over time for appearance of the HIV-1 virus or AIDS by conventional blood detection methods by changes in CD4 ⁺ /CD8 ⁺ ratios and in the absolute CD4 ⁺ count, or development of opportunistic infections and AIDS-defining diseases.