Retrovirus associated with lymphadenopathies and adapted to continuous lines of lymphoblastoid B cells, capable of producing retrovirus continuously and process for their production

Claim 2

A continuous cell line from a culture of human B lymphoblastoid cells, wherein the cells have the following characteristics: (a) Lymphadenopathy Associated Virus (LAV) is not cytotoxic to the cells; (b) When the cells are cultured, supernatant from the cells exhibits reverse transcriptase activity; (c) The cells do not immunologically react with monoclonal antibodies to Leu-3 cells; (d) When the cells are cultured in the absence of T cell growth factor (TCGF), infected cells continuously produce B-LAV retrovirus, which is a retrovirus having the essential antigenic characteristics of LAV.

Claim 3

Cell line as claimed in claim 1, wherein the cells do not contain the genome of Epstein-Barr virus (EBV).

Claim 4

Cell line as claimed in claim 1, wherein the cells contain at least part of the genome of Epstein-Barr virus (EBV).

Claim 5

Cell line as claimed in claim 1, wherein the cells are derived from a Burkitt lymphoma.

Claim 6

Cell line as claimed in claim 1, wherein the cells contain Epstein-Barr nuclear antigen (EBNA).

Claim 7

A process for producing a continuous cell line of B lymphoblastoid cells, wherein the process comprises: (a) providing a cell culture medium comprising human B lymphocytes; (b) contacting the B lymphocytes in the culture medium with an oncogenic transforming agent under conditions to form a continuous cell line of transformed B lymphoblastoid cells; and (c) contacting the transformed B lymphoblastoid cells with Lymphadenopathy Associated Virus (LAV) under conditions in which the transformed B lymphoblastoid cells are infected with LAV; wherein the B lymphoblastoid cells after infection with LAV produce B-LAV, which is a retrovirus having the essential antigenic characteristics of LAV.

Claim 8

A process for producing a continuous cell line of B lymphoblastoid cells, wherein the process comprises: (a) providing a cell culture medium comprising human B lymphocytes; (b) contacting the B lymphocytes in the culture medium with an oncogenic transforming agent under conditions to form a continuous cell line of transformed B lymphoblastoid cells; and (c) contacting the transformed B lymphoblastoid cells with Lymphadenopathy Associated Virus (LAV) under conditions in which the transformed B lymphoblastoid cells are infected with LAV; wherein the B lymphoblastoid cells after infection with LAV produce B-LAV, which is a retrovirus having the essential antigenic characteristics of LAV.

Claim 9

A process as claimed in claim 6, wherein the transformed B lymphoblastoid cells are subject to multiple infections with LAV.

Claim 10

A process for producing a continuous cell line of B lymphoblastoid cells, wherein the process comprises: (a) providing a cell culture medium comprising a mixture of human T lymphocytes and human B lymphocytes; (b) contacting the T lymphocytes and B lymphocytes in the culture medium with Epstein-Barr virus as a transforming agent under conditions to form a continuous cell line of transformed B lymphoblastoid cells; and (c) contacting the transformed B lymphoblastoid cells with Lymphadenopathy Associated Virus (LAV) under conditions in which the transformed B lymphoblastoid cells are infected with LAV; wherein the B lymphoblastoid cells after infection with LAV produce B-LAV, which is a retrovirus having the essential antigenic characteristics of LAV.

Claim 11

A process for producing a continuous cell line of B lymphoblastoid cells, wherein the process comprises: (a) providing a cell culture medium comprising a mixture of human T lymphocytes and human B lymphocytes; (b) contacting the T lymphocytes and B lymphocytes in the culture medium with Epstein-Barr virus as a transforming agent under conditions to form a continuous cell line of transformed B lymphoblastoid cells; and (c) contacting the transformed B lymphoblastoid cells with Lymphadenopathy Associated Virus (LAV) under conditions in which the transformed B lymphoblastoid cells are infected with LAV; wherein the B lymphoblastoid cells after infection with LAV produce B-LAV, which is a retrovirus having the essential antigenic characteristics of LAV.

Claim 12

A process as claimed in claim 8, wherein the transformed B lymphoblastoid cells are subject to multiple infections with LAV.

Claim 13

A process for producing a continuous cell line of B lymphoblastoid cells, wherein the process comprises: (a) providing a cell culture medium comprising a mixture of human T lymphocytes and human B lymphocytes; (b) contacting the T lymphocytes and B lymphocytes with a mitogen under conditions to facilitate blastic transformation of the B lymphocytes; (c) contacting the lymphocytes from step (b) with Epstein-Barr virus (EBV) as a transforming agent under conditions to form a continuous cell line of transformed B lymphoblastoid cells; (d) contacting the transformed B-lymphoblastoid cells with Lymphadenopathy Associated Virus (LAV) under conditions in which the transformed cells are infected with LAV; and (e) cultivating the resulting LAV-infected cells to obtain a sustained level of reverse transcriptase activity in the culture; wherein the B lymphoblastoid cells after infection with LAV produce B-LAV, which is a retrovirus having the essential antigenic characteristics of LAV.

Claim 14

A process for producing a continuous cell line of B lymphoblastoid cells, wherein the process comprises: (a) providing a cell culture medium comprising a mixture of human T lymphocytes and human B lymphocytes; (b) contacting the T lymphocytes and B lymphocytes with a mitogen under conditions to facilitate blastic transformation of the B lymphocytes; (c) contacting the lymphocytes from step (b) with Epstein-Barr virus (EBV) as a transforming agent under conditions to form a continuous cell line of transformed B lymphoblastoid cells; (d) contacting the transformed B-lymphoblastoid cells with Lymphadenopathy Associated Virus (LAV) under conditions in which the transformed cells are infected with LAV; and (e) cultivating the resulting LAV-infected cells to obtain a sustained level of reverse transcriptase activity in the culture; wherein the B lymphoblastoid cells after infection with LAV produce B-LAV, which is a retrovirus having the essential antigenic characteristics of LAV.

Claim 15

A process as claimed in claim 10, wherein the transformed B lymphoblastoid cells are subject to multiple infections with LAV.

Claim 16

Process as claimed in claim 11, wherein the T lymphocytes and the B lymphocytes are from the same human donor.

Claim 17

Process as claimed in claim 11, wherein the nitrogen is Protein A of Staphylococcus Aureus.

Claim 18

Process as claimed in claim 11, which comprises the additional steps of: cultivating the LAV-infected B lymphoblastoid cells with T cell growth factor (TCGF) to obtain a culture medium that exhibits reverse transcriptase activity; after reverse transcriptase activity declines, contacting the culture medium with fresh T lymphocytes to proliferate viral infection; and cultivating the resulting cells under conditions to obtain a sustained level of reverse transcriptase activity in the culture medium.

Claim 19

Process as claimed in claim 14, wherein all of the lymphocytes are from the same human donor.

Claim 20

Process as claimed in claim 15, wherein the fresh lymphocytes are activated with phytohemaglutinin prior to contact with the culture medium.

Claim 21

Process as claimed in claim 15, wherein the B lymphocytes are derived from umbilical cord lymphocytes.

Claim 22

Process as claimed in claim 11, wherein the B lymphocytes are derived from a Burkitt lymphoma.

Claim 23

A retrovirus termed B-LAV, which is comprised of antigen and the antigen is immunologically recognized by sera of a patient afflicted with Lymphadenopathy Syndrome (LAS) or Acquired Immune Deficiency Syndrome (AIDS), and wherein the retrovirus: (a) exhibits reverse transcriptase activity with a strong affinity for poly(adenylate-oligodeoxy-thymidylate)[poly(A)-oligo(dT) 12-18 ] with Mg 2+ ; (b) exhibits tropism for T-lymphocytes; (c) exhibits preferential tropism for Leu-3 cells; (d) is cytopathic to Leu-3 cells infected with the retrovirus; (e) induces the production of p25 viral protein of Lymphadenopathy Associated Virus (LAV) in T-lymphocytes infected with LAV; (f) exhibits slight antigenic homology with the virus of infectious anemia of the horse (VAIC), in that antibody against VAIC immunoprecipitates p25 viral protein of LAV; and (g) is capable of infecting continuous B lymphoblastoid cell lines having C.N.C.M. Deposit Accession Nos. I-300, I-301, I-302, and I-303, and of being replicated by said cell lines when cultured; wherein the retrovirus is in biologically pure form.

Claim 24

A retrovirus termed B-LAV, which is comprised of antigen and the antigen is immunologically recognized by sera of a patient afflicted with Lymphadenopathy Syndrome (LAS) or Acquired Immune Deficiency Syndrome (AIDS), and wherein the retrovirus: (a) exhibits reverse transcriptase activity with a strong affinity for poly(adenylate-oligodeoxy-thymidylate)[poly(A)-oligo(dT) 12-18 ] with Mg 2+ ; (b) exhibits tropism for T-lymphocytes; (c) exhibits preferential tropism for Leu-3 cells; (d) is cytopathic to Leu-3 cells infected with the retrovirus; (e) induces the production of p25 viral protein of Lymphadenopathy Associated Virus (LAV) in T-lymphocytes infected with LAV; (f) exhibits slight antigenic homology with the virus of infectious anemia of the horse (VAIC), in that antibody against VAIC immunoprecipitates p25 viral protein of LAV; and (g) is capable of infecting continuous B lymphoblastoid cell lines having C.N.C.M. Deposit Accession Nos. I-300, I-301, I-302, and I-303, and of being replicated by said cell lines when cultured; wherein the retrovirus is in biologically pure form.

Claim 25

Retrovirus as claimed in claim 19, wherein the retrovirus has an average diameter of 139 nanometers and an eccentric nucleus having an average diameter of 41 nanometers.

Claim 26

A method of amplifying a retrovirus, wherein the retrovirus is B-LAV, said method comprising: (a) contacting a continuous cell line of human B lymphoblastoid cells as claimed in claim 19 with B-LAV; and (b) culturing the cells in suspension in the absence of T cell growth factor (TCGF) and under conditions in which the retrovirus proliferates.

Claim 27

Method as claimed in claim 21, wherein the method includes the further step of separating B-LAV from the cultured cells in step (b).

Claim 28

Method as claimed in claim 21, wherein the cell line has the identifying characteristics of C.N.C.M. Deposit Accession No. I-300.

Claim 29

Method as claimed in claim 21, wherein the cell line has the identifying characteristics of C.N.C.M. Deposit Accession No. I-301.

Claim 30

Method as claimed in claim 21, wherein the cell line has the identifying characteristics of C.N.C.M. Deposit Accession No. I-302.

Claim 31

Method as claimed in claim 21, wherein the cell line has the identifying characteristics of C.N.C.M. Deposit Accession No. I-303.

Claim 32

A retrovirus having the identifying characteristics of C.N.C.M. Deposit Accession No. I-299.

Claim 33

A retrovirus having the identifying characteristics of C.N.C.M. Deposit Accession No. I-299.

Claim 34

A continuous cell line of human B lymphoblastoid cells having the identifying characteristics of C.N.C.M. Deposit Accession No. I-300.

Claim 35

A continuous cell line of human B lymphoblastoid cells having the identifying characteristics of C.N.C.M. Deposit Accession No. I-300.

Claim 36

A continuous cell line of human B lymphoblastoid cells having the identifying characteristics of C.N.C.M. Deposit Accession No. I-301.

Claim 37

A continuous cell line of human B lymphoblastoid cells having the identifying characteristics of C.N.C.M. Deposit Accession No. I-301.

Claim 38

A continuous cell line of human B lymphoblastoid cells having the identifying characteristics of C.N.C.M. Deposit Accession No. I-302.

Claim 39

A continuous cell line of human B lymphoblastoid cells having the identifying characteristics of C.N.C.M. Deposit Accession No. I-302.

Claim 40

A continuous cell line of human B lymphoblastoid cells having the identifying characteristics of C.N.C.M. Deposit Accession No. I-303.

Claim 41

A continuous cell line of human B lymphoblastoid cells having the identifying characteristics of C.N.C.M. Deposit Accession No. I-303.