Saturday, May 28, 2011

United States Patent Trademark Office: Method of CREATING THE AIDS VIRUS.

Please keep in mind that this information is for the public to see. You can go to any Patented website and put in the United States Patent number 4,647,773. Millions of people around the world has died because of AIDS but yet it has been man made and it has been suppressed by our Government. I am not just saying this, the proof is here from what I see in the United States Patented website(s). The Patented information is proving this for us.
Please also read the United States Patented information on the CURE of AIDS. YES, this is Patented information.

http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO1&Sect2=HITOFF&d=PALL&p=1&u=%2Fnetahtml%2FPTO%2Fsrchnum.htm&r=1&f=G&l=50&s1=4,647,773.PN.&OS=PN/4,647,773&RS=PN/4,647,773

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United States Patent 4,647,773
Gallo ,   et al. March 3, 1987

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




4464465August 1984Lostrom et al.
4520113May 1985Gallo 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.

STATEMENT OF DEPOSIT

A cell line corresponding to the present invention, and denoted H9/HTLV-III.sub.B, has been deposited in the ATCC (under ATCC No. CRL 8543) on April 19, 1984, prior to the filing of this patent application. This deposit assures permanence of the deposit and ready accessibility thereto by the public. H9 is a representative and preferred cell line in accordance with the invention.

UTILITY STATEMENT

The cell line which is a product of the present invention (H9/HTLV-III.sub.B) is presently useful for the production of vaccines for the relief of AIDS and for the detection of antibodies to the virus in blood samples.

GENERAL DESCRIPTION

A susceptible cell line HT was tested for HTLV before in vitro infection and it was negative by all criteria, including lack of proviral sequences. Continuous production of HTLV-III is obtained after repeated exposure of parental HT cells (3.times.10.sup.6 cells pretreated with polybrene) to concentrated culture fluids containing HTLV-III harvested from short term cultured T-cells (grown with TCGF) which originated from patients with pre-AIDS or AIDS. The concentrated fluids were first shown to contain particle associated reverse transcriptase (RT). When cell proliferation declined, usually 10 to 20 days after exposure to the culture fluids, the fresh (uninfected) HT parental cells are added to cultures. Culture fluids from the infected parental cell line was positive for particulate RT activity and about 20% of the infected cell population was positive in an indirect immune fluorescent assay (IFA) using serum from a hemophilia patient with pre-AIDS (patient E.T.). Serum from E.T. also contained antibodies to proteins of disrupted HTLV-III but did not react with proteins of HTLV-I or HTLV-II infected cells.

SPECIFIC DISCLOSURE

As has been mentioned above, an aneuploid HT-cell line exhibited the desired prerequisites for the continuous propagation of HTLV-III. This cell line is a neoplastic aneuploid T-cell line derived from an adult patient with lymphoid leukemia, selected for its mature T-cell phenotype [OKT3.sup.+ (62%), OKT.sup.4 + (39%) and OKT8.sup.- ], as determined by cytofluorometry using a fluorescence-activated cell sorter. Cultures of these cells are routinely maintained in RPMI/1640 with 20% fetal calf serum and antibiotics. These cultures are shown in Example 1, Table 1. Clone H9 is preferred, with Clone H4 being secondarily preferred.

HTLV-III culture fluids are isolated from cultured cells of patients with acquired immune deficiency syndrome (AIDS). Peripheral blood leukocytes from these patients are banded in Ficoll-Hypaque, incubated in growth media (RPMI 1640, 20% fetal bovine serum 0.29 mg/ml glutamine) containing 5 .mu.g/ml phytohemagglutinin (PHA-P) for 48 hours, at 37.degree. C. in a 5% CO.sub.2 atmosphere. The leukocytes are then refed with growth medium containing 10% purified T cell growth factor (TCGF); optionally, some of the cells also received rabbit antibody to alpha interferon. Cells and growth media from these lymphocytes are then assayed for the presence of HTLV subgroups I-III. Samples exhibiting more than one of the following were considered positive: repeated detection of a Mg.sup.++ dependent reverse transcriptase activity in supernatant fluids; virus observed by electron microscopy; intracellular expression of virus-related antigens detected with antibodies from sero-positive donors or with hyperimmune serum; or transmission of particles, detected by reverse transcriptase assays or by electron microscopic observation, to fresh human cord blood, bone marrow, or peripheral blood T-lymphocytes. All isolates not classified as either HTLV-I or HTLV-II by immunological or nucleic acid analysis were classified as HTLV-III. The cells in the HTLV-III producing cell cultures, characterized using established immunological procedures, are predominantly T-lymphocytes (E rosette receptor, OKT/3 and Leu/1 positive, with a T4 phenotype (OKT4, leu 3a positive). This process is also described by Gallo, et al., in Science, 220:865-867 (1983).

The infection of parental HT cells as well as other cloned cell populations occurs by exposure of these cells to concentrated or nonconcentrated culture fluids (cell-free infection) from T-cell cultures from AIDS or pre-AIDS patients, or by cocultivation; that is, HT cells are infected by exposure to HTLV-III containing cultures. The usual cell-free infection procedure is as follows: 2 to 5.times.10.sup.6 cells are treated with polybrene (2 .mu.g/ml) or DEAE dextran for 30 minutes in CO.sub.2 incubator at 37.degree. C., and then exposed to the virus inoculum (0.1 to 1 ml) for one hour in the incubator (CO.sub.2 /37.degree. C.). The cells are kept in suspension by shaking at regular intervals. After one hour of incubation a regular growth medium is added. The positivity of infected cultures for HTLV-III is assessed after one, two, and three weeks of cultivation.

The infection of HT cells (clones) is also obtained by cocultivation procedure--HT cells are mixed in various proportions (usually 1:5) with short- term cultured T-cells (about 5 to 20 days) from AIDS or pre-AIDS patients. The positivity for HTLV-III was scored by the detection of viral antigens or viral nucleic acid sequences in the infected recipient cells at various intervals (7, 14, 21 days, etc.) after cocultivation. The mixed cultures are maintained in growth medium for several months.

EXAMPLE 1

As shown in Table 1 below, single cell HT clones were isolated as described by Popovic, et al., in Neoplasma, 18:257 (1971), and Bach, et al., Immunol. Rev., 54:5 (1981) from a long-term cultured aneuploid HT-cell line exhibiting mature T-cell phenotype (OKT3.sup.+ [62%], OKT4.sup.+ [39%] and OKT8.sup.-) as determined by cytofluorometry using a fluorescence-activated cell sorter. The cultures were routinely maintained in RPMI/1640 with 20% fetal calf serum and antibiotics. The terminal cell density of the parental cell culture, seeded at a concentration of 2.times.10.sup.5 cells/milliliter of culture media, was in the range 10.sup.6 -1.5.times.10.sup.6 cells/ml after 5 days of culture.

For detection of multinucleated cells, cell smears were prepared from cultures 6 and 14 days after infection and stained with Wright-Giemsa. Cells having more than 5 nuclei were considered as multi-nucleated cells. Cloned cells from uninfected cultures also contained some multi-nucleated giant cells as well; however, the arrangement of multiple nuclei in a characteristic ring formation was lacking and the number of these cells was much less (0.7% to 10%).

Immunofluorescence positive cells were washed with phosphate-buffered saline (PBS) and resuspended in the same buffer at concentration 10.sup.6 cells per milliliter. Approximately 50 .lambda. of cell suspension were spotted on slides, air dried, and fixed in acetone for 10 min. at room temperature. Slides were stored at -20.degree. C. until use. Twenty microliters of either hyperimmune rabbit antiserum to HTLV-III (diluted 1/2000 in PBS) or serum from the patient (E.T.) diluted 1/8 in PBS was applied to cells and incubated for 50 min. at 37.degree. C. The fluorescein-conjugated antiserum to rabbit or human immunogloblin G was diluted and applied to the fixed cells for 30 min. at room temperature. Slides then were washed extensively before microscopic examinations. The uninfected parental cell line as well as the clones were consistently negative in these assays.

To determine reverse transcriptase activity, virus particles were precipitated from cell-free supernatant as follows: 0.4 ml of 4M NaCl and 3.6 ml of 30% (wt/vol.) polyethylene glycol (Carbowax 6000) were added to 8 ml of harvested culture fluids and the suspension was placed on ice overnight. The suspension was centrifuged in a Sorvall RC-3 centrifuge at 2000 rpm at 4.degree. C. for 30 min. The precipitate was resuspended in 300 .mu.l at 50% (vol/vol) glycerol (25 mM Tris-HCl, pH 7.5/5 mM dithiothreitol/150 mM KCl/0.025% Triton X-100. Particles were disrupted by addition of 100 .mu.l of 0.9% Triton X-100/1.5M KCl. Reverse transcriptase (RT) assays were performed as described by Poiesz, et al., Proc Nat. Acad. Sci. U.S.A., 77:7415 (1980) and expressed in cpm per milliliter culture medium.

TABLE 1 __________________________________________________________________________ Response of Cloned T-Cell Populations to HTLV-III Infection Clones Characteristics H3 H4 H6 H9 H17 H31 H35 H38 __________________________________________________________________________ Total cell number (.times. 10.sup.6) 6 days after infection 1 1.5 1.5 0.3 0.4 0.3 0.5 1.8 14 days after infection 2.2 7.3 7.5 10.0 4.7 5.0 4.5 3.2 Multinucleated cells (%) 6 days after infection 24 42 32 7 13 14 30 45 14 days after infection 45 48 45 30 22 45 60 60 Immunofluorescence positive cells (%) 6 days after infection Rabbit antiserum to HTLV-III 55 56 32 32 39 21 10 87 Patient serum (E.T.) 56 29 21 ND ND ND ND 73 14 days after infection Rabbit antiserum to HTLV-III 50 74 60 97 71 40 20 80 Patient serum 45 47 56 78 61 43 22 89 Reverse transcriptase activity (.times. 10.sup.4 cpm/ml) 6 days after infection 2.4 1.8 2.1 4.1 2.6 1.4 1.7 2.5 14 days after infection 16.2 18.1 16.1 20.2 17.1 13.4 15.1 18.2 __________________________________________________________________________ ND = not done

EXAMPLE 2

As shown in Table 2 below, cocultivation with H4 recipient T-cell clone was performed with fresh mononuclear cells from peripheral blood of patients RF and SN, respectively. In the case of patients BK and LS cocultivation was performed with T-cells grown in the presence of exogenous TCGF (10% V/V) for 10 days. The ratio recipient/donor (patients') cells was 1:5. The mixed cultures were maintained in RPMI/1640 (20% FCS and antibiotics) in the absence of exogenous TCGF. Cell-free infection of H9 T-cell clone was performed using concentrated culture fluids harvested from T-cell cultures of the patient WT. The T-cell cultures were grown in the presence of exogenous TCGF for two weeks before the culture fluids were harvested and concentrated. Cells of H9 clones were pretreated with polybrene (2 .mu.g/ml) for 20 min. and 2.times.10.sup.6 cells were exposed for one hr. to 0.5 ml of 100-fold concentrated culture fluids positive for particulate RT activity.

HTLV-III virus expression in both cocultured and cell-free infected cell cultures were assayed approximately one month after in vitro cultivation. There was considerable fluctuation in HTLV-III expression (see Table 2). For details of both reverse transcriptase (RT) assays and indirect immunofluorescence assays (IFA) see Example 1.

TABLE 2 __________________________________________________________________________ Isolation of HTLV-III from Patients with Pre-AIDS and AIDS Virus Expression IFA with RT Activity Rabbit Serum Human Serum (ET) Patient Diagnosis Origin (.times. 10 cpm) (% Positive) (% Positive) EM __________________________________________________________________________ RF AIDS Haiti 0.25 80 33 ND (heterosexual) SN Hemophiliac U.S. 6.3 10 ND + (lymphadenopathy) BK AIDS U.S. 0.24 44 5 + (homosexual) LS AIDS U.S. 0.13 64 19 + (homosexual) WT Hemophiliac U.S. 3.2 69 ND ND (lymphadenopathy) __________________________________________________________________________ RT = reverse transcriptase IFA = immunofluorescence assays EM = electron microscopy ND = not done

EXAMPLE 3

To select for high permissiveness for HTLV-III and to preserve permanent growth and continuous production of virus, extensive cloning of the HT parental T-cell population was performed. A total of 51 single-cell clones was obtained by both capillary and limited dilution techniques using irradiated mononuclear cells from peripheral blood of a healthy donor as a feeder. The growth of these cell clones was compared after HTLV-III infection. A representative example of response to virus infection of 8 T-cell clones which are susceptible to and permissive for HTLV-III is shown in Table 1. In parallel experiments, 2.times.10.sup.6 cells of each T-cell clone were exposed to 0.1 ml of concentrated virus. Then cell growth and morphology, expression of cellular viral antigen(s), and RT activity in culture fluids were assessed 6 and 14 days after infection. Although all 8 clones were susceptible to and permissive for the virus, there were considerable differences in their ability to proliferate after infection. The cell number decreased by 10% to 90% from the initial cell count within 6 days after infection, and a high proportion of multinucleated (giant) cells were consistently found in all 8 infected clones. The percentage of T-cells positive for viral antigen(s) determined by immunofluorescent assays with serum from AIDS patient (E.T.) and with hyperimmune rabbit serum raised against the whole disrupted HTLV-III ranged from 10% to over 80%. Fourteen days after infection, the total cell number and the proportion of HTLV-III positive cells increased in all 8 clones. The virus positive cultures consistently showed round giant cells which contained numerous nuclei. These multinucleataed giant cells are similar to those induced by HTLV-I and HTLV-II except that the nuclei exhibit a characteristic ring formation. Electron microscopic examinations showed that the cells released considerable amounts of virus.

EXAMPLE 4

To determine whether HTLV-III is continuously produced by the infected T-cells in long-term cultures, both virus production and cell viability of the infected clone, H4, were followed for several months. Although the virus production fluctuated, culture fluids harvested from the H4/HTLV-III cell cultures at approximately 14-day intervals consistently exhibited particulate RT activity which has been followed for over 5 months. The viability of the cells ranged from 65% to 85% and doubling time of the cell population, which is called H4/HTLV-III, was approximately 30-40 hours. Thus, this permanently growing T-cell population can continuously produce HTLV-III.

The yield of virus produced by H4/HTLV-III cells was assessed by purification of concentrated culture fluids through a sucrose density gradient and assays of particulate RT activity in each fraction collected from the gradient. The highest RT activity was found at density 1.16 g/ml, similar to other retroviruses.


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United States Patent Trademark Office: Method of CURING AIDS with Tetrasilver Tetroxide Molecular Crystal Devices

Please keep in mind that this information is for the public to see. You can go to any Patented website and put in the United States Patent number 5,676,977. Millions of people around the world has died because of AIDS but yet there is a cure that has been suppressed by our Government. I am not just saying this, the proof is here from what I see in the United States Patented website(s). The Patented information is proving this for us.
Please also read the United States Patented information on the creation of AIDS. YES, this is Patented information.

http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO1&Sect2=HITOFF&d=PALL&p=1&u=%2Fnetahtml%2FPTO%2Fsrchnum.htm&r=1&f=G&l=50&s1=5676977.PN.&OS=PN/5676977&RS=PN/5676977

Please click on the link so you can go to the United States Patented website and see for yourself that this info is true. 




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United States Patent 5,676,977
Antelman October 14, 1997

Method of curing AIDS with tetrasilver tetroxide molecular crystal devices



Abstract
The diamagnetic semiconducting molecular crystal tetrasilver tetroxide (Ag.sub.4 O.sub.4) is utilized for destroying the AIDS virus, destroying AIDS synergistic pathogens and immunity suppressing moieties (ISM) in humans. A single intravenous injection of the devices is all that is required for efficacy at levels of about 40 PPM of human blood. The device molecular crystal contains two mono and two trivalent silver ions capable of "firing" electrons capable of electrocuting the AIDS virus, pathogens and ISM. When administered into the bloodstream, the device electrons will be triggered by pathogens, a proliferating virus and ISM, and when fired will simultaneously trigger a redox chelation mechanism resulting in divalent silver moieties which chelate and bind active sites of the entities destroying them. The devices are completely non-toxic. However, they put stress on the liver causing hepatomegaly, but there is no loss of liver function.

Inventors: Antelman; Marvin S. (Rehovot, IL)
Assignee: Antelman Technologies Ltd. (Providence, RI)
Appl. No.: 08/658,955
Filed: May 31, 1996



Related U.S. Patent Documents








Application NumberFiling DatePatent NumberIssue Date
310859Sep., 1994




Current U.S. Class: 424/618 ; 514/495
Current International Class: A61K 33/38 (20060101); A61K 033/38 ()
Field of Search: 424/618 514/495



References Cited [Referenced By]




U.S. Patent Documents




4415565November 1983Wysor
4915955April 1990Gomori
4952411August 1990Fox, Jr. et al.
5073382December 1991Antelman
5078902January 1992Antelman
5089275February 1992Antelman
5211855May 1993Antelman
5223149June 1993Antelman
5336499August 1994Antelman
5571520November 1996Antelman



Other References


"Is The AIDS Virus A Science Fiction?" by Peter H. Duesberg and Bryan J. Ellison, Policy Review, Summer 1990, pp. 40-51..

Primary Examiner: Hulina; Amy
Attorney, Agent or Firm: Salter & Michaelson



Parent Case Text




This application is a continuation-in-part of patent application Ser. No. 08/310,859 filed Sep. 22, 1994, now abandoned.



Claims




What is claimed is:

1. A method of treating AIDS-afflicted humans comprising injecting a multitude of tetrasilver tetroxide molecular crystals into the bloodstream of the human subject.

2. A method for increasing white blood cell counts in AIDS-afflicted humans comprising injecting a multitude of tetrasilver tetroxide molecular crystals into the bloodstream of the human subject.

3. Methods of treating AIDS-affilicted humans according to claims 1-2 where the concentration of said molecular crystals is approximately 40 PPM of the total blood weight of the human subject.



Description




BACKGROUND OF THE INVENTION

The present invention relates to the employment of molecular crystals as anti-AIDS devices, but more particularly to the molecular crystal semiconductor tetrasilver tetroxide Ag.sub.4 O.sub.4 which has two monovalent and two trivalent silver ions per molecule, and which through this structural configuration enables intermolecular electron transfer capable of killing viruses and binding them to the resulting silver entity so that a single intravenous injection will completely obliterate acquired immune deficiency syndrome (AIDS) in humans. Furthermore, said devices are capable of killing pathogens and purging the bloodstream of immune suppressing moieties (ISM) whether or not created by the AIDS virus (HIV); so as to restore the immune system.

The present invention is based on concepts previously elucidated in applicant's U.S. Pat. No. 5,336,499 which discloses the destruction and inhibition of bacteria, algae and the AIDS virus in nutrient life supporting systems by using said silver oxide devices. Example 3 of said patent discloses that 18 PPM of said crystal devices could totally suppress the AIDS virus (page 6, line 5). Subsequent to the filing of the aforementioned patent, further testing revealed complete 100% destruction of the AIDS virus in vitro at 20 PPM, and the fact that said devices were harmless when ingested and inhaled, being non-toxic.

Encouraged by these evaluations and successes, applicant obtained permission to evaluate the crystals in vitro against murine acquired immune deficiency syndrome (MAIDS). Only one facility in the State of Israel is licensed for these evaluations, namely, the Kaplan Hospital in Rehovot, Israel, which is affiliated with the Hebrew University-Hadassah Medical School where said evaluations were done.

The initial evaluations entailed experimenting with various silver moieties cited in applicant's aforementioned patent, concentrations, non-reactive buffers and modes of administration. After about 18 months of judicious efforts and initial failures, success was finally achieved in destroying the MAIDS virus in C57BL mice with a single intravenous injection. The results of this test program comprise Example 5 of U.S. Pat. No. 5,336,499. After success with mice, the inventor was able to test the efficacy of said devices on two select etiological groups of terminal AIDS patients in a clinic in Tegucigalpa, Honduras, Central America.

The AIDS patients comprised the etiological subgroups, Candidiasis and Wasting Syndrome. Current indicator diseases for diagnosing AIDS which have been expanded by the CDC, fall into the following five major categories with the approximate percent distribution among AIDS patients:

______________________________________ 1. P. carinii pneumonia 51% 2. Wasting syndrome 19% 3. Candidiasis 13% 4. Kaposi's sarcoma 11% 5. Dementia 6% ______________________________________

This invention concerns itself with the treatment and cure of candidiasis and wasting syndrome AIDS patients with Tetrasil*. These two groups account for approximately one third of AIDS cases.

Stedman's Medical Dictionary (Williams & Wilken's 26th Ed., 1995) defines wasting syndrome "as a condition of 10% weight loss in conjunction with diarrhea or fever . . . Associated with AIDS (p. 1744)."

OBJECTS OF THE INVENTION

The main object of the invention is to provide for a molecular scale device of a single tetrasilver tetroxide crystalline molecule capable of restoring the immunity of AIDS afflicted humans of the two AIDS etiological subgroups, candidiasis and wasting syndrome.

Another object of the invention is to provide for immunity restoration in said AIDS afflicted humans through a single injection.

Another object of this invention is to destroy ISM in humans manifesting AIDS diseases of said AIDS etiological subgroups irrespective as to whether said ISM was HIV induced, since it is known that humans may manifest AIDS and still be HIV negative, and thus restore the immune system in said humans.

Another object of this invention is to destroy the AIDS virus when present in the systems of said AIDS afflicted humans.

SUMMARY OF THE INVENTION

This invention relates to a molecular scale device not only capable of destroying the AIDS virus, but of purging the human bloodstream of pathogens and restoring immunity to AIDS patients of the candidiasis and wasting syndrome categories. Said molecular device consists of a single crystal of tetrasilver tetroxide (Ag.sub.4 O.sub.4). The crystal lattice of this molecule has a unique structure since it is a diamagnetic semiconducting crystal containing two mono and two trivalent silver ions, which in effect are capable of "firing" electrons under certain conditions which will destroy AIDS viruses, other pathogens and immune suppressing moieties (ISM), not only through the electrocution mode, but also by a binding process which occurs simultaneously with electron firing, namely, binding and chelation of divalent silver, i.e., the resulting product of the electron transfer redox that occur when the monovalent silver ions are oxidized and the trivalent ions are reduced in the crystal. The binding/chelation effect occurs at active sites of the AIDS virus, pathogens and ISM. Because of the extremely minute size of a single molecule of this crystal, several million of these devices may be employed in concert to destroy a virus colony to purge a life support system of ISM and pathogens with the consumption of only parts per trillion of the crystal devices. Thus an optimum of 40 PPM of the devices by weight of human blood was found to be sufficient to completely obliterate AIDS. This concentration is slightly over double of the optimum concentration recommended in applicant's aforementioned U.S. patent for the destruction of the human AIDS virus in vitro. Other details concerning the structure of the crystal and its mechanism against pathogens, the AIDS virus and ISM would analogously hold here, and have already been further elucidated in said patent.

The actual destruction of pathogens, ISM and the AIDS virus is effectuated by injection of a suspension of these devices in distilled or deionized water with a non-reacting electrolyte directly, i.e. intravenously, into the bloodstream. A single injection is all that is required under these conditions. Accordingly, humans injected in this manner, upon being inspected after three weeks or more had elapsed and compared with similar humans that had been given placebos, were completely cured of AIDS. The control group still manifested AIDS. Accordingly, the tetrasilver tetroxide device performed in concert with and in full conformity with the ultimate objects of this invention. Furthermore, three out of four wasting syndrome terminal patients and four out of the five candidiasis terminal patients were still alive in 1995 after a year and a half had elapsed from their initial injection. By that time all the AIDS patients had been released from the clinic and allowed to return home.

Other objects and features of the present invention shall become apparent to those skilled in the art when the present invention is considered in view of the accompanying examples. It should, of course, be recognized that the accompanying examples illustrate preferred embodiments of the present invention and are not intended as a means of defining the limits and scope of the present invention.

EXAMPLE 1

Five patients afflicted with AIDS of the candidiasis etiological category were segregated for Tetrasil treatment. The rationale for selecting them was based on facts presented in an article by Peter H. Duesberg and Brian J. Ellison entitled "Is The AIDS Virus A Science Fiction?" (Policy Review, Summer 1990 pp. 40-51). Only the factual presentations of the article were utilized and the hypothesis of the authors was ignored. The facts presented in the article related to the method of selecting AIDS patients based on the five aforementioned etiological subgroups targeted by the CDC, and the evidence presented, that there is AIDS without HIV as well as with it so that an anti-viral agent in most instances will not necessarily restore the immunity system.

Evaluations with Tetrasil were conducted on AIDS patients at Lucha Contra el Sida, Comayaguela, Honduras. The patients two weeks prior to inoculation were removed from their AZT, AIDS therapy. Tetrasil was administered at approximately 40 PPM of blood volume per patient as a suspension in a proprietary buffer solution (pH=6.5), supplied by Holipharm Corporation.

The results of evaluations with candidiasis are tabulated in Table I under its disease category. All patients evaluated were terminal. Some, however, were in moderate (m) condition and others in poor (p) as designated in the Table. The I and F designations refer to initial and final values as shown. WBC indicates white cell blood count. The H column, following CD 8, indicates whether hepatomegaly occurred. This was an unfortunate consequence of the treatment which resulted in enlarged livers in all patients except the second one. Despite hepatomegaly, there was no interference with liver function.

The onset of hepatomegaly was not spontaneous and varied from patient to patient, being in the range of 4-16 days.

It should also be noted that shortly after injection of Tetrasil there were indications of fever (symbolized by T in the Ag.sub.4 O.sub.4 column), sometimes accompanied by fatigue (F). The body temperature was invariably 38.5.degree. C. (101.3.degree. F.). This was indicative of restoration of the immune response of the body, since normally the body will destroy pathogens when the immune system is functional by raising the temperature. The patient who died; first responded favorably to Diflucan, which previously gave no response. He was cured of his candidiasis, but unfortunately succumbed to his previous body damage. All the other candidiasis syndrome people who previously did not respond to the indicated medications subsequently responded after the Tetrasil treatment. Further evidence of the recovery of the AIDS patients manifested itself 30 days after the initial injection when white blood cell counts were taken. They are shown in Table I under the WBC column, which gives the initial and final WBC. All candidiasis patients showed a dramatic increase in their white blood cell counts, indicative of the restoration of their immunity systems.

EXAMPLE 2

The above protocol of Example 1 was repeated with AIDS patients exhibiting wasting syndrome. The results of their treatment are tabulated in Table I under the disease category of said syndrome. It should be noted that two of the four wasting syndrome patients showed improved white blood counts. The female patient, whose condition improved from poor and terminal to be among the living, showed a decrease in the WBC. However, she showed an increase in body temperature which was indicative of immune response. The test results indicate that one cannot rely on a single factor to indicate the demise of AIDS. The usual HIV marker CD 4 initial and final are irrelevant. ISM suppression appears to be more critical than the destruction of HIV. AIDS was suppressed, any permanent damage that had been done to the patients in the course of their succumbing to AIDS was not obviously cured or corrected by said crystal device treatment, rather said injury persisted and the patient was improved with respect to AIDS but still suffered from said permanent injury or impairment previously inflicted.

TABLE I __________________________________________________________________________ Response of AIDS Patients to Single 40 PPM Ag.sub.4 O.sub.4 Inoculation Date Weight DISEASE PATIENT Inoc. WBC CD 4 DEATH Lbs. Group Sex Age Medictn 1994 I F I F CD 8 H 1944 I F Ag.sub.4 O.sub.4 __________________________________________________________________________ Candidiasis M p 28 Diflucan 5/5 1,200 4,200 41 -- 221 + 6/11 82 76 T F m 33 " 5/5 6,000 6,700 554 872 394 - 98 98 T F m 33 Ketaconzl 5/27 2,600 3,850 248 181 951 + 123 123 T M p 62 " 6/2 3,300 3,700 89 237 59 + 105 92 F F m 31 Pentamidn 6/2 2,400 3,050 9 181 65 + 121 118 Pain Wasting M m 27 5/27 3,600 4,600 39 14 709 + 119 120 T Syndrome M m 28 5/27 2,750 -- 10 -- 60 + 7/19 121 119 T, F F p 43 5/27 3,600 2,700 68 246 248 + 101 98 T, F M m 19 5/10 3,850 5,400 137 36 48 + 103 106 T, F __________________________________________________________________________

As this invention may be embodied in several forms without departing from the spirit or essential characteristics thereof, the present embodiments are therefore illustrative and not restrictive, since the scope of the invention is defined by the appended claims rather than by the description preceding them, and all changes that fall within the metes and bounds of the claims or that form their functional as well as conjointly cooperative equivalents, are therefore intended to be embraced by these claims.



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Definition of United States Patent:

Patent

From Wikipedia, the free encyclopedia
 
A patent (play /ˈpætənt/ or /ˈpeɪtənt/) is a set of exclusive rights granted by a state (national government) to an inventor or their assignee for a limited period of time in exchange for a public disclosure of an invention.
The procedure for granting patents, the requirements placed on the patentee, and the extent of the exclusive rights vary widely between countries according to national laws and international agreements. Typically, however, a patent application must include one or more claims defining the invention which must be new, non-obvious, and useful or industrially applicable. In many countries, certain subject areas are excluded from patents, such as business methods, treatment of the human body[citation needed], and mental acts. The exclusive right granted to a patentee in most countries is the right to prevent others from making, using, selling, or distributing the patented invention without permission.[1] It is just a right to prevent others' use. A patent does not give the proprietor of the patent the right to use the patented invention, should it fall within the scope of an earlier patent.
Under the World Trade Organization's (WTO) Agreement on Trade-Related Aspects of Intellectual Property Rights, patents should be available in WTO member states for any inventions, in all fields of technology,[2] and the term of protection available should be the minimum twenty years.[3] Different types of patents may have varying patent terms (i.e., durations).

Tuesday, May 24, 2011

FLASHBACK TO 12/26/01: Report – Bin Laden Already Dead


(FOXNEWS)  http://www.foxnews.com/story/0,2933,41576,00.html 
Originally reported on Wednesday, December 26, 2001. Usama bin Laden has died a peaceful death due to an untreated lung complication, thePakistan Observer reported, citing a Taliban leader who allegedly attended the funeral of the Al Qaeda leader.
“The Coalition troops are engaged in a mad search operation but they would never be able to fulfill their cherished goal of getting Usama alive or dead,” the source said.
Screen capture of the article just in case in goes into the memory hole. FJ 2011
Bin Laden, according to the source, was suffering from a serious lung complication and succumbed to the disease in mid-December, in the vicinity of the Tora Bora mountains. The source claimed that bin Laden was laid to rest honorably in his last abode and his grave was made as per his Wahabi belief.
About 30 close associates of bin Laden in Al Qaeda, including his most trusted and personal bodyguards, his family members and some “Taliban friends,” attended the funeral rites. A volley of bullets was also fired to pay final tribute to the “great leader.”
The Taliban source who claims to have seen bin Laden’s face before burial said “he looked pale … but calm, relaxed and confident.”
Asked whether bin Laden had any feelings of remorse before death, the source vehemently said “no.” Instead, he said, bin Laden was proud that he succeeded in his mission of igniting awareness amongst Muslims about hegemonistic designs and conspiracies of “pagans” against Islam. Bin Laden, he said, held the view that the sacrifice of a few hundred people in Afghanistan was nothing, as those who laid their lives in creating an atmosphere of resistance will be adequately rewarded by Almighty Allah.
When asked where bin Laden was buried, the source said, “I am sure that like other places in Tora Bora, that particular place too must have vanished.”
EDITORS NOTE:  Let us not forget what Benazir Bhutto said not long before she was killed…

Iran can prove Bin Laden was dead long before US raid – Iranian minister


Osama Bin Laden (AFP Photo / HO / DoD)
Osama Bin Laden (AFP Photo / HO / DoD)

Iranian Intelligence Minister Heydar Moslehi claims that Osama Bin Laden died from an illness before the US raid on his compound in Abbottabad. Iran has documents to prove it, he said.
­"We have credible information that Bin Laden died some time ago of a disease," Moslehi said on the sidelines of a cabinet meeting on Sunday, as quoted by ISNA news agency.

"If the US military and intelligence apparatus have really arrested or killed Bin Laden, why don't they show him [his body]? Why have they thrown his corpse into the sea?" Moslehi asked rhetorically, FARS news agency reports.

Moslehi labeled the US raid in Abbottabad as a “PR campaign”, created to divert the attention of its citizens from domestic problems, such as the “fragile” state of the US economy.

"We believe that what the US is seeking, by dictated media programs, to overshadow the recent awakening in the region through the release of such reports," ISNA quoted Moslehi as saying.

Bin Laden was killed on May 2 in the Pakistani town of Abbottabad, north of the capital Islamabad, during a special operation carried out by US Navy Seals. President Barack Obama announced the news in a special address to the nation.

The news of “justice having been done” sparked mass celebrations in the United States and then ignited a new wave of conspiracy theories after Washington refused to release Bin Laden’s postmortem photographs, claiming that they might instigate a new wave of radicalism.

Sunday, May 22, 2011

The Royal Cover Up?

PRINCESS DIANA'S JEWISH FATHER..........

PRINCESS DIANA'S JEWISH MOTHER..........

PRINCESS DIANA'S JEWISH HALF-BROTHERS...........

PRINCESS DIANA'S JEWISH HALF SISTER...........

PRINCESS DIANA'S JEWISH SON.............

PRINCE WILLIAM'S JEWISH BRIDE..........

Click on the link below and you won't believe the "Royal Family Tree".

http://helpfreetheearth.com/news305_wedding.html

wildi