United States Patent: 4647773

http://patft.uspto.gov/netacgi/nph-Parser?Sect2=PTO1&Sect2=HITOFF&p=1&u=%2Fnetahtml%2FPTO%2Fsearch-bool.html&r=1&f=G&l=50&d=PALL&RefSrch=yes&Query=PN%2F4647773 Method of continuous production of retroviruses (HTLV-III) from patients with AIDS and pre-AIDS Abstract A cell system is disclosed for the reproducible detection and isolation of human T-lymphotropic retroviruses (HTLV-family) with cytopathic effects (HTLV-III) from patients with the acquired immune deficiency syndrome (AIDS), pre-AIDS and in healthy carriers. One neoplastic aneuploid T-cell line derived from an adult with lymphoid leukemia, and termed HT, was susceptible to infection with the new variants of HTLV, which are transformed and providing T-cell populations which are highly susceptible and permissive from HTLV-III, and convenience for large scale production, isolation and biological detection of the virus. Inventors: Gallo; Robert C. (Bethesda, MD), Popovic; Mikulas (Bethesda, MD) Assignee: The United States of America as represented by the Department of Health (Washington, DC) [*] Notice: The portion of the term of this patent subsequent to May 28, 2002 has been disclaimed. Appl. No.: 06/602,946 Filed: April 23, 1984 Current U.S. Class: 435/239 ; 424/208.1; 435/235.1; 435/372.3; 435/948 Current International Class: C12N 5/06 (20060101); C12N 7/00 (20060101); C12N 007/02 (); C12N 007/00 (); C12N 005/00 (); C12R 001/91 () Field of Search: 435/235,239,240,948,5,29 424/89 128/1T References Cited [Referenced By] U.S. Patent Documents 4464465 August 1984 Lostrom et al. 4520113 May 1985 Gallo et al. Other References Popovic et al, Science, 224(4648):497-500, May 4, 1984. . Gallo et al, “Isolation of Human T-Cell Leukemia Virus in Acquired Immune Deficiency Syndrome (AIDS)”, Science, 220, pp. 865-867 (5-1983). . Barre-Sinoussi et al, “Isolation of a T-Lymphotropic Retrovirus from a Patient at Risk for AIDS”, Science, 220, pp. 868-871 (5-1983). . Marx, “Strong New Candidate for AIDS Agent”, Science, 224, pp. 475-477 (5-1984).. Primary Examiner: Warren; Charles F. Assistant Examiner: Tarcza; John Edward Attorney, Agent or Firm: Roberts, Jr.; John S. Claims We claim: 1. A method for continuous production of HTLV-III virus which comprises infecting in cell culture highly susceptible, permissive cells consisting of a neoplastic aneuploid HT-cell line with said virus, said cells preserve the capacity for permanent growth after the infection with said virus, growing said cells under conditions suitable for cell growth and recovering said virus produced by said cells. 2. The method of claim 1, wherein said virus consists of cytopathic variants of HTLV. 3. The method of claim 1 wherein said infecting comprises cocultivating said virus with said cells to produce a cell line and recovering said cell line. 4. The method of claim 1 wherein said infecting comprises cell-free infection of said cells with said virus. 5. The method of claim 1 wherein said cells are neoplastic aneuploid T-cells derived from an adult with lymphoid leukemia. 6. A process for the continuous production of HTLV-III virus which comprises cocultivating said virus with a target HT-cell to produce an infected cell line, growing said cell line and recovering said virus from supernatants produced by said cell line. 7. The process of claim 6 wherein said target T-cell is a neoplastic aneuploid T-cell susceptible to infection with HTLV-III. 8. A process for the continual production of HTLV-III by infecting T-cells in cultures which comprises cocultivating HTLV-III virus with an HT-clone to produce an infected cell line, said clone being an aneuploid T-cell line derived from lymphoid leukemia, growing said cell line and recovering said virus from supernatants produced by said cell line. 9. The process in claim 8 wherein said clone is a mature T-cell phenotype of OKT3.sup.+ (62%), OKT4.sup.+ (39%) and OKT8.sup.-. 10. A method of producing a cell line containing an antigen of HTLV-III which comprises infecting an HT-cell line derived from lymphoid leukemia and susceptible to infection with HTLV-III, said cell line is capable of continuous large-scale production of HTLV-III, and growing the infected cell line under conditions suitable for growth. 11. The method of claim 10 wherein said cell line is a neoplastic aneuploid T-cell line. 12. The method of claim 10 wherein said HTLV-III are variants of human T-lymphotropic retrovirus, exhibit cytopathic effects and are non-transforming. 13. A cell line containing HTLV-III designated H9/HTLV-III.sub.B, ATCC Accession CRL 8543. 14. A process for producing a cell line H9/HTLV-III.sub.B which comprises infecting a target T-cell with HTLV-III virus to produce a cell line and recovering same, said infecting process overcomes the normal cytopathic effect of HTLV-III and preserves the immortal growth capacity of the target cell. 15. An HT cell line permanently infected with HTLV-III virus, wherein said cell line continually produces said virus. Description The present invention describes a cell system for the reproducible detection and isolation of human T-lymphotropic retroviruses (HTLV-family) with cytopathic or cell killing effects (HTLV-III) from patients with the acquired immune deficiency syndrome (AIDS), pre-AIDS and in healthy carriers. One neoplastic aneuploid T-cell line derived from an adult with lymphoid leukemia, and termed HT, was susceptible to infection with the new variants of HTLV, providing T-cell populations which are highly susceptible and permissive for HTLV-III, and convenience for large scale production, isolation, and biological detection of the virus. BACKGROUND OF THE INVENTION The disclosure of this invention is contained in the following journal articles: Gallo et al., “Detection, Isolation, and Continuous Production of Cytopathic Human T-Lymphotropic Retroviruses (HTLV-III) from Patients with AIDS and pre-AIDS,” Science, in press; and Gallo et al., “Human T-Lymphotropic Retrovirus, HTLV-III, Isolated from AIDS Patients and Donors at Risk for AIDS,” in press. Epidemiologic data strongly suggest that acquired immune deficiency syndrome (AIDS) is caused by an infectious agent which is apparently horizontally transmitted by intimate contact or blood products. Though the disease is manifested by opportunistic infections, predominantly Pneumocystis carcinii pneumonia and Kaposi’s sarcoma, the underlying disorder affects the patient’s cell-mediated immunity with absolute lymphopenia and reduced helper T-lymphocyte (OKT4.sup.+) subpopulation(s). Moreover, before a complete clinical manifestation of the disease occurs, its prodrome, pre-AIDS, is frequently characterized by unexplained chronical lymphadenopathy and/or leukopenia involving a helper T cell subset. This leads to the severe immune deficiency of the patient, suggesting that a specific subset of T-cells is the primary target for an infectious agent. Although patients with AIDS or pre-AIDS are often chronically infected with cytomegalovirus or hepatitis B virus, for various reasons these appear to be opportunistic or coincidental infections apparently not linked to the immunological response deficiency. It is believed that the cause of AIDS may be a virus from the family of human T-cell lymphotropic retroviruses (HTLV) which, prior to the present invention, comprised two major well characterized subgroups of human retroviruses, called human T-cell leukemia/lymphoma viruses, HTLV-I and HTLV-II. The most common isolate, HTLV-I, is mainly obtained from patients with mature T-cell malignancies. Seroepidemiological studies, in vitro biological effects, and nucleic acid hybridization data indicate that HTLV-I is etiologically associated with these malignancies, affecting adults primarily in the south of Japan, the Caribbean and Africa. HTLV of subgroup II (HTLV-II) was first isolated from a patient with a T-cell variant of hairy cell leukemia. To date, this is the only reported isolate of HTLV-II from a patient with a neoplastic disease. Virus isolation and seroepidemiological data show that HTLV of both subgroups can sometimes be found in patients with AIDS. Evidence suggests that the retrovirus(es) of the HTLV family is an etiological agent of AIDS based on the following: (1) there is precedence for an animal retrovirus cause of immune deficiency (feline leukemia virus in cats); (2) retroviruses of the HTLV family are T-cell tropic; (3) they preferentially infect “helper” T-cells (OKT4.sup.+); (4) they have cytopathic effects on various human and mammalian cells as demonstrated by their induction of cell syncytia formation; (5) they can alter some T-cell functions; (6) in some cases infection may result in selective T-cell killing; and (7) they are transmitted by intimate contact or through blood products. The presence of antibodies directed to cell membrane antigens of HTLV infected cells has been shown in sera of more than 40% of patients with AIDS [Essex et al., Science, 220:859 (1983)]. This antigen has since been defined as part of the envelope of HTLV [Schupbach, et al., Science, in press; and Lee, et al., Proc. Nat. Acad. Sci. U.S.A., in press]. The original detection and isolation of the various HTLV isolates were made possible by two earlier developments: the discovery of T-cell growth factor (TCGF), also called Interleukin 2 (Il-2), which enabled the routine selective growth of different subsets of normal and neoplastic mature T-cells [Ruscetti, et al., J. Immunol., 119:131 (1977); and Poiesz, et al., Proc. Nat. Acad. Sci. U.S.A, 77:6134 (1980)] and the development of sensitive assays for detection of retroviruses based on reverse transcriptase assays. The methods of HTLV isolation and transmission involved a cocultivation procedure using permissive T-cells for the virus. The use of normal human T-cells in cocultivation experiments preferentially yielded HTLV of both subgroups with immortalizing (transforming) capability for some of the target T-cells. However, HTLV variants (now termed HTLV-III), lack immortalizing properties for normal T-cells and mainly exhibit cytopathic effects on the T-cells and is now believed to be the cause of AIDS. In fact, such variants were frequently but only transiently detected using these normal T-cells as targets in cocultivation or cell-free transmission experiments. The cytopathic effect was overcome by finding a highly susceptible, permissive cell for cytopathic variants of HTLV, thus preserving the capacity for permanent growth after infection with the virus. The present invention discloses the identification and characterization of this new immortalized T-cell population and its use in the isolation and continuous high- level production of such viruses from patients with AIDS and pre-AIDS. Early experiments identified one neoplastic aneuploid T-cell line, termed HT, derived from an adult with lymphoid leukemia, that was susceptible to infection with the new cytopathic virus isolates. This cell line is a sensitive target for transmission of these virus isolates (HTLV-III) and it allows continuous large-scale virus production and development of specific immunologic reagents and nucleic acid probes useful for comparison of these new isolates among themselves and with HTLV-I and HTLV-II. In addition to their differences in biological effects that distinguish them from HTLV-I and HTLV-II, HTLV-III also differs from these known HTLV subgroups in several immunological assays and in morphology. However, these new retroviruses are T4 lymphotropic and exhibit many properties similar to HTLV-I and II, including similar properties of the reverse transcriptase, cross reactivity of structural proteins as determined by heterologous competition radioimmune assays with patients’ sera and with animal hyperimmune sera, and induction of syncytia. These new retrovirus isolates are collectively designated HTLV-III, together with detectable differences in some of their proteins and genetic information, HTLV-III’s ability to kill T-cells clearly separates these variants from other members of the HTLV family. Read the full patent via the US patent website