Title:  Immune enhancing agent for treating HIV infected humans

United States Patent:  6,713,064

Issued:  March 30, 2004

Inventors:  Apte; Sateesh N. (2817 Crow Canyon Rd., San Ramon, CA 94583-1639)

Appl. No.:  879099

Filed:  June 19, 1997

Abstract

Human immunodeficiency virus (HIV) is the primary etiologic agent for the acquired immune deficiency syndrome (AIDS). HIV exhibits considerable genotypic and phenotypic variability as manifested by different replicative kinetics, susceptibility to serum neutralization, antiviral drug resistance, cytopathic effects, and host-cell range specificity. Infection by HIV leads to progressive deterioration of cell-mediated immune responses making infected patients susceptible to a variety of opportunistic infections, such as pneumocystis carinii pneumonia (PCP), as well as, to the development of tumors such as Kaposi's sarcoma (KS). While the use of live-attenuated viruses to treat HIV infection has been proposed, to date researchers have been unable to demonstrate their efficacy in a clinical setting. The claimed invention addresses this defect by providing therapeutic compositions and methods of treatment employing viral suspensions prepared from cells transfected with a recombinant HIV-1 nef-deficient molecular clone. The HIV-1 nef gene product is required for efficient viral replication and pathogenicity in vivo. Nef may exert its effects through the downregulation of CD4 by endocytosis and lysosomal degradation, although other activities have also been ascribed to this gene product. Accordingly, this gene product was targeted for inactivation and a recombinant HIV-1_ELI molecular clone was generated containing an NcoI/XhoI nef deletion. Suspensions comprising viral particles generated from cells transfected with the recombinant clone were prepared and administered to HIV-infected patients. Patients receiving these suspensions displayed a statistically significant reduction in viral burden and increase in CD4⁺ lymphocyte counts during the study. The claimed compositions should facilitate the reduction of viral burden and the restoration of CD4⁺ lymphocyte levels in HIV-1-infected subjects.

SUMMARY OF THE INVENTION

A recombinant clone of HIV-1_ELI isolate with its nef open reading frame deleted was constructed from a plasmid vector by endonuclease cleaving at Nco I and Xho I sites and filling in the open ends with an oligonucleotide. The resultant plasmid DNA was screened and transfected by using DEAE dextran into HuT 78 cell line. HIV virus propagation was confirmed by monitoring proteins gp41, p24, p17 and p15, by monitoring reverse transcriptase activity and by electron micoscopic identification of virions. Virus particles were separated from supernatant medium and frozen in liquid nitrogen until use. For treatment of HIV infection, after baseline diagnostic procedures including confirmation of HIV infection and CD4-CD8 cell counts, a skin test for allegic reaction and an informed consent, approximately 200,000,000 virus particles will be injected intravenously. This will be followed by semimonthly monitoring of CD4 counts and a booster dose of another 200,000,000 virus particles intravenously. This will be followed by monthly monitoring of CD4 counts for one year. According to the invention, patients are expected to have a normal CD4 count in 6-9 months and will have restored immune systems in 1 year. For prevention of wild-type HIV infection in high risk populations, approximately 1,000,000 virus particles will be injected subcutaneously, the subjects observed for sufficient time to ensure absence of untoward effects such as an anaphylactic reaction. Immunity in this population will be ascertained by seroconversion and wild-type HIV infection can be diagnosed by utilizing enzyme linked immunosorbent assays for detection of antibodies to the nef gene product.

DESCRIPTION OF THE PREFERRED EMBODIMENT

HuT 78 Cells, a human lymphoid cell line was obtained from the American Type Culture Collection (Rockville, Md.) and propagated in Dulbecco's modified Eagle's medium (Gibco, Grand Island, N.Y.) containing 10% heated (56^oF., 30 minutes) calf serum (Sigma Chemical Company, St. Louis, Mo.) and 10% interleukin 2--a T cell growth factor (Meloy laboratories, Springfield, Va.). Cells were grown on plastic tissue clulture dishes (Falcon) and transferred using trypsin with EDTA (Gibco, Grand Island, N.Y.). This cell line was inoculated with peripheral blood mononuclear cells (PBMCs) from an AIDS patient infected with the HIV-1_ELI strain. The PBMCs were first prepared by banding over Ficoll-diatrizoate (density, 1.077 to 1.080 g/ml at 20^oC.)(Pharmacia LKB Biotechnology, Uppsala, Sweden). The PBMCs were washed with RPMI 1640 medium, stimulated for 5 days with 1 .mu.g/ml of phytohemagglutinin (Sigma Chemical Co., St. Louis, Mo.) and washed free of phytohemagglutinin prior to inoculation. The molecular cloning techniques were used as described by Maniatis T, Fritsch, EF et al (Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). By using a non-cutter restriction endonuclease of HIV-1_ELI (New England Biolabs, Beverly, Mass.) from total cell DNA of the infected cell line, integrated proviral DNA with flanking cellular sequences were cloned into the Xba I site of bacteriophage J1 (Promega Biotec, Madison, Wis.) giving rise to a recombinant phage clone .lambda.HXELI. A vector SP65gpt was constructed by ligating Bam HI-Pvu II fragment of plasmid pSV2gpt into the Bam HI-Pvu II sites of SP65 (Promega Biotec, Madison, Wis.). A 12.5 Kilobase (kb) Hpa I-Xba I fragment of the clone .lambda.HXELI was blunt-ended with Klenow fragment of DNA polymerase I and cloned into similarly blunt-ended Bam HI to Eco RI sites of vector SP65gpt. The resultant clone HXELIgpt had the HIV-1_ELI and xanthine guanine phosphoribosyl transferase (gpt) sequences in identical transcriptional orientation. The provirus containing plasmid vector was digested with Nco I (Boehringer Mannheim Biochemicals, Mannheim, Germany) and Xho I (New England Biolabs, Beverly, Mass.) restriction endonucleases followed by a filling in the ends with an oligonucleotide constructed on a Biosearch Cyclone synthesizer, reverese transcriptase and dNTPs, followed by ligation of the blunt ends. Plasmids were screened by electrophoresis on 0.8% agarose gels (Sigma Chemicals, St. Louis, Mo.) for derivatives of HXELIgpt containing nef deletion. The exact coordinates of the deletion were confirmed by DNA sequencing with chain terminating inhibitors of DNA polymerase -2',3'-dideoxy and arabinonucleoside analogues of the normal deoxynucleoside triphosphate (ddCTP was obtained from Collaborative Research, Inc., Waltham, Mass., araATP and araCTP were obtained from P-L Biochemicals, Inc., Milwaukee, Wis.) as described by Sanger, F, Nicklsen, S et al (Proc Natl Acad of Sci 74:5463-5467, 1977). Heteroduplex DNA was subjected to ethanol precipitation and and resuspended in sterile water. Serial dilutions of DNA were prepared to a final volume of 80 micL. To each sample of DNA was added 20 .mu.L DEAE dextran (molecular weight 5x10⁵) obtained from Pharmacia in a concentration of 2 mg/ml after sterilizing by autoclaving and 100 .mu.L of two-fold concentrated serum-free Dulbecco's modified Eagle's medium (Gibco, Grand Island, N.Y.). The HuT 78 cells described above were transferred to fresh plates 24 hours prior to transfection to ascertain an exponential growth. These growing cells were removed from plates with 0.1% trypsin with EDTA (Gibco, Grand Island, N.Y.) in Tris-buffered isotonic saline at pH 7.2 (Sigma Chemicals, St. Louis, Mo.), mixed with fresh Dulbecco's modified Eagle's medium containing heated calf serum as described above to inactivate the trypsin and counted with a Coulter counter. 6x10⁵ cells were added to 2 ml Dulbecco's modified Eagle's medium containing serum in 12 mmx75 mm clear plastic tubes (Falcon #2058). The tubes were centrifuged at 5000 rpm for 1 minute. The medium was withdrawn carefully using a pipette with an aspirator. A 100 .mu.L sample of the DNA dilution was added to each tube. The tubes were gently shaken and transferred to a 37^oC. CO₂ incubator for 1 hr. The rack was gently shaken every 15 minutes. At the end of the incubation, 2 ml of fresh Dulbecco's modified Eagle's medium containing heated calf serum as described above was added to each tube, the tubes were shaken, centrifuged and the medium aspirated as described above. The cells were then resuspended in 2 ml of fresh Dulbecco's modified Eagle's medium containing heated calf serum as described above and incubated at 37^oC. in a 5% CO₂ incubator. The cultures were monitored for appearance of HIV-1 gag and env products p17, p24 and gp41, reverse transcriptase activity and virions as seen by electron microscopy as is readily known to those knowledgeable in the art. Virus containing supernatant of the cultures was filtered through a millipore filter (filter size 0.45 .mu.m, Millipore Corp., Bedford, Mass.) and placed in sterile vials so as to contain about 200,000,000 virion particles per ml. The sterile vials were stored in liquid nitrogen.

EXAMPLE 1

Two volunteers (S1 and S2), both commercial sex workers in India became HIV positive in 1989. Since then, both have had a downhill course with diarrhoea, weight loss, candida and CMV infections. Their CD4 counts were 327 and 258 respectively. Families and friends of both had deserted them due to their HIV infection and they had almost no support structure left. After a detailed informed consent and a thorough discussion of all the risks involved with the use of the present invention, these individuals were given a physical, confirmatory Western Blot tests to ensure HIV status, baseline CD4-CD8 cell counts and a skin test for sensitivity to the viral suspension, both were given 1 ml of recombinant viral suspension containing approximately 200,000,000 virus particles intravenously. The patients were quarantined in an isolated facility and all personnel coming in contact with them used communicable disease precautions. The patients' CD4 counts were recorded one month after the first injection and they were given a second injection of equal dose intravenously. Their CD4 counts were recorded once again, 4-6 weeks after the booster. The patients started gaining weight in approximately 4-6 weeks after the first injection and their CD4 counts increased as shown in the accompanying table. They became asymptommatic in 3 and 4.5 months respectively.