WO1987005911A1 - Immunosuppressive factor from human immunodeficiency virus (hiv) or hiv infected cells or cell lines, its use, corresponding antibodies and their use - Google Patents
Immunosuppressive factor from human immunodeficiency virus (hiv) or hiv infected cells or cell lines, its use, corresponding antibodies and their use Download PDFInfo
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- WO1987005911A1 WO1987005911A1 PCT/DK1987/000035 DK8700035W WO8705911A1 WO 1987005911 A1 WO1987005911 A1 WO 1987005911A1 DK 8700035 W DK8700035 W DK 8700035W WO 8705911 A1 WO8705911 A1 WO 8705911A1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/16011—Human Immunodeficiency Virus, HIV
- C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
- C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
Definitions
- the invention concerns a immunosuppressive factor purified from human immunodeficiency virus (HIV) or from cells or cell lines infected with HIV, the use of this factor, at least one part thereof or analogs thereof, i. a. for prophylaxis or treatment of healthy or HIV infected persons and for the pro ⁇ duction of antibodies, and the use of such anti ⁇ bodies, i. a. for detecting this factor or a part thereof in serum, body fluids or tissues of HIV in ⁇ fected persons.
- LAV/HTLV-III is used alternatively with the disigna- tion HIV; its definition will be given below.
- Retroviral infections are accompanied by immuno- suppression in a variety of species, e. g. cats (Na- ture, 274, (1978), L. E. Mathes, R. G. Olsen, L. Y. Hedebrand, 0. S. Hoover and J. P. Schaller, 687-689) and mice (Cancer Res., 37, (1977), A. K. Fowler, D. R. Twardi , C. D. Reed, 0. S. Weislow and A. Hellman, 4529-4531).
- the im unosuppression has been attributed to a hydrophobic 15,000 dalton transmembrane envelope protein, pl5E (Cancer. Res., 39, (1979), L.
- LAV/HTLV-III lymphadenopathy-associated virus
- III type III
- CD4-positive cluster differentiation antigen
- the receptor is a marker for a definite type of cells; 4 indicates that the marker is found on a helper cell.
- AIDS immnunodeficiency syndrome
- helper lymphocytes cannot be the sole explanation for the immunodeficiency of AIDS because the deficency may be severe even when there are still significant mumbers of helper cells left (N. Engl. J. Med., 313, (1985), H. C. Lane, J. M. Depper and W. C. Geene; Scand. J. Immunol., 21, (1985), B. Hofmann, N. ⁇ dum, P. Platz, L. P. Ryder, A. Svejgaard and J. 0.
- T-suppressor lymphocytes are also abnormal (Scand. J. Immunol. (in press), B. Hofmann, N. ⁇ dum, B. Jacobsen, P. Platz, L. P. Ryder, J. 0. Nielsen, J, Gerstoft and A. Svejgaard) although LAV/HTLV-III is not tropic for these cells.
- H9 is the designation of a cell line of T-helper cells.
- a LAV/HTLV-III extract (sucrose gradient purified virions from a culture medium of LAV/HTLV-III infect ⁇ ed H9 cells) and that from non-infected H9 cells were prepared by means of Triton-X 100 (the designation of a detergent from Sigma Chemical Co. , St. Louis, MO, US), heat treated, and dialyzed against isotonic NaCl solution.
- Figure 1 shows that the LAV/HTLV-III extract suppressed the mitogen (antigen, data not shown) responses of normal peripheral mononuclear cells (PBMC) by 63-90% when added in a dilution 1:500 at the beginning of the culture period.
- PBMC peripheral mononuclear cells
- the LAV/HTLV-III extract also suppressed a range of different cell lines from man and mouse including B-cell (bursa Fabricii cell) lines.
- a human IL-2 (interleukin-2) cell line was especially susceptible.
- the suppressive action on PBMC was shown not to be due to direct cytotoxicity.
- the effect of the HTLV-III extract could not not be neutralized by the addition of large ammounts of IL-2, but as shown in table 2 below the addition of a serum from a patient with Kaposi's sarcoma and normal immunoresponses could neutralize the inhibition while that form another patient with opportunistic infections could not.
- Serum from a goat immunized with the LAV/HTLV-III extract could also neutralize the inhibition, while serum taken before immunization could not.
- HTLV-III extract might be cy- totoxic was investigated by incubating PBMC with and without HTLV-III extract for one hour at 24°C and subsequently counting of eosin-stained cells. There were similar low numbers of dead cells in the two samples. Moreover, there were 62% +/-7 and 66% +/- 9 viable cells in six PBMC cultures stimulated with PHA for three days with and without HTLV-III extract, respectively. Accordingly, the suppressive action on PBMC is probably not due to direct cytotoxicity. In contrast when HTLV-III extract was added to a human IL-2-dependent T-cell line (Int. Archs. Allergy Appl. Immun., (in press), E.
- the effect of the LAV/HTLV-III extract on the IL-2 receptor expression was investigated by double immu- nofluorescens studies on a FACS (flourescens-acti- vated cell sorter) Analyzer using anti-Tac (Tac: T-cell activation, now designated as IL-2 receptor) (anti-IL-2 receptor), anti-CD4, and anti-CD8 antibodies and PBMC cultured with a suboptimal concentration of PHA for 72 hours with and without LAV/HTLV-III extract. About 6% of the CD4-positive and 4% of the CD8-positive lymphocytes became Tac positive in cultures without LAV/HTLV-III extract, whereas no cells of either subset became positive in the presence of this extract.
- the pi5 retroviral protein has been shown to interfere with IL-2, but there is controversy as to whether IL-2 can reverse the impaired immune response in animals (J. Immunol., 134, (5), (1985), C. G. Orosz, N. E. Zinn, R. G. Olsen and L. E. Mathes, 3396-3403; J. Immunol., 130 (5), (1983), M. A. Wainberg, S. Vydelignu and R. G. Margolese, 2372-2378).
- pl5E also alters the function og monocytes and macrophage (J. Clin. Invest., 68, (1981), G. Ciancolo, J. Hunter, J. Silva, J. S. Haskill and R.
- pl5E and HTLV-III extract act by the same mechanism and a homology and a homology beween HTLV-III and the immunosuppressive, synthetic peptide of 17 a ino acids which is homologous to pl5E og gp21 of HTLV-I and II (Nature, 311,, (1984), G. J. Ciancolo, R. J. Kipnis and R. Snyderman, 515; Science, 230, (1985), G. J. Ciancolo, T. D. Copeland, S. Oroszlan and R. Snyderman, 453-455; J. Immunol., 134, (5), (1985), C. G. Orosz, N. E. Zinn, R. G. Olsen,L. E. Mathes, 3396-3403).
- HTLV-III extract In order to investigate whether the suppressive effect of HTLV-III extract was confined to T-lympho- cutes, other cell populations were cultivated with HTLV-III extract and extract from non-infected cells (table 1 below). Three Tac-negative EBV-transformed lymphoblastoid cell lines (EBV: Epstein-Barr virus) were all clearly suppressed by the HTLV-III extract, but not with extract from the non-infected H9 cell. Thus, the suppressive action of the LAV/HTLV-III extract is not confined to T-lymphocytes but also involves the proliferation of B-lymphocytes (Science, 220, (1983), Z. Trainin, D. Wernicke. H. Unger-Waron and M. Essex, 858-859).
- phadex G-100 is a definite derivative of starch from Pharmacia, Uppsala, SE). The suppressive activity was recovered in the fractions with molecular weight of about 70,000.
- the suppressive factor in the extract from LAV/HTLV-III-infected cells constitute parts of the virus, but our results cannot rule out that the factor is a host factor produced by the H9 cells in response to the LAV/HTLV-III infection. In either case, if this factor is released by infected cells also in vivo, it may explain the severely suppresssed mitogen response for PMBC from AIDS pati ⁇ ents. In particular, the irreversible nature of the suppression may ensure that a few LAV/HTLV-III infected cells may suffice to suppress many T-lympho- cytes.
- the purified factor may possibly be used in transplantations and in the treatment of cancer (e. g . cases of leukemia).
- Suppressive factor recovered form raw LAV/HTLV-III extract.
- the molecular weight was found to be 65,000-70,000 by separtion on a Se- phadex column. It is possible that there exists a subunit of 25,000-30,000.
- the factor as such is a glycoprotein determined by lentile column fractionation. The factor will lose the activity on boiling.
- Serum constituted 50 ⁇ l og the 170 pi per well. Serum A was obtained from an Aids patient with Kaposi's sarcoma and serum B from an AIDS patient with P. carinii infection. Legend to figure 1
- the HTLV-III extract was prepared from sucrose gradient purified virions from the culture medium of HTLV-III infected H9 cells which were disrupted by 0.6 M KC1 and 0.5% Triton-X 100 and clarified by centrifugation at 40,000 rpm. Further, the extract was heat inactivated for 30 min. at 56°C and dialyzed against PBS (PBS: phosphate-buffered saline) at pH 7.2 for 120 hours.
- PBS phosphate-buffered saline
- the protein contents were 0.6 mg/ml for the HTLV-III extract and 80 mg/ml for the H9 extract.
- PBMC from six healthy blood donors were isolated by Lymphoprep (Nyegaard and Co., Oslo, NO) density gradient centrifugation. Cell cultures were prepared in triplicate in 350 ul round-bottom " micf ⁇ titre plates containing 50,000 PBMC per well.
- the amounts of mitogen and antigen added per well were 2 ug of PHA ( ⁇ PHA + HTLV-III extract, Difco, Detroit, MUCH., US), 20 ul og 1:500, 20 ul of a 1:50 suspen ⁇ sion of PWM (PWN: pokeweed mitogen) ( % PWM + HTLV-III extract, Gibco, Grand Island, N.Y., US) and 20 ul of a 1:100 dilution of HSV antigen (HSV: herpes simplex virus) ( T HSV + HTLV-III extract, kindly supplied by Bodil Norrild, Inst. of Microbiology, Copenhagen, Denmark. The total volume per well was 170 ⁇ l.
- the growth medium used was RPMI 1640 (RMPI 1640 is the designation for a medium from Gibco Laboratories, Grand Island, NY, US containing L-menthionin and dialyzed "fetal bovie serum"). The total volume per well was 170 ul.
- the growth medium used was RPMI 1640 (Gibco) supplemented with the antibiotics penicillin, streptomycin, heparin, L-glu- tamin, and 15% heat inactived pooled human male serum. 20 ul of HTLV-III extract was added in the dilutions indicated at the abscissa. The final dilution of extract from non-infected cells was 1:500 ( PHA + extract from non-infected cells).
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Abstract
Immunosuppressive factor purified from human immunodeficiency virus (HIV) or from cells and cell lines, such as lymphocytes, macrophages, lymphoblastic cell lines of T-lymphocytic origin, macrophage cell lines or hybridoma cell lines infected with HIV suppresssing the mitogen and antigen response of normal lymphocytes stimulated with mitogens or antigens as well as the growth of IL-2 dependent T-cell lines, being a glycoprotein of molecular weight of 65,000 to 70,000 daltons, losing its activity on boiling, and possibly containing a subunit having a molecular weight of 25,000 to 30,000 daltons. A process for the production of the mentioned factor is given. The mentioned factor, at least one part thereof or directly indicated synthetic analogs of this factor may be used for detecting antibodies in HIV infected persons - i.e. persons that may come to suffer from the acquired immunodeficiency syndrome, also known as AIDS - by neutralizing the effect of these antibodies, in immunological methods, for producing poly- or monoclonal antibodies in animals, for purifying antibodies originating from HIV infected persons - and the antibodies resulting from this production and/or purification, for bringing about immunity for the purpose of prophylaxis or treatment (vaccination) in healthy or HIV infected persons. The antibodies so provided may be used for detecting this factor or its indicated part in serum, body fluids and tissues of HIV infected persons or for neutralizing the whole or at least one part of this factor in HIV infected persons for the purpose of treatment. A filter containing AIDS antibodies for use in plasmaphoretic treatment is mentioned.
Description
i.
IMMUNOSUPPRESSIVE FACTOR FROM HUMAN IMMUNODEFICIENCY VIRUS (HIV) OR HIV INFECTED CELLS OR CELL LINES, ITS USE, CORRESPONDING ANTIBODIES AND THEIR USE.
The invention concerns a immunosuppressive factor purified from human immunodeficiency virus (HIV) or from cells or cell lines infected with HIV, the use of this factor, at least one part thereof or analogs thereof, i. a. for prophylaxis or treatment of healthy or HIV infected persons and for the pro¬ duction of antibodies, and the use of such anti¬ bodies, i. a. for detecting this factor or a part thereof in serum, body fluids or tissues of HIV in¬ fected persons. (In what follows the designation LAV/HTLV-III is used alternatively with the disigna- tion HIV; its definition will be given below. )
Retroviral infections are accompanied by immuno- suppression in a variety of species, e. g. cats (Na- ture, 274, (1978), L. E. Mathes, R. G. Olsen, L. Y. Hedebrand, 0. S. Hoover and J. P. Schaller, 687-689) and mice (Cancer Res., 37, (1977), A. K. Fowler, D. R. Twardi , C. D. Reed, 0. S. Weislow and A. Hellman, 4529-4531). For feline and mur-ine leukemia virus, the im unosuppression has been attributed to a hydrophobic 15,000 dalton transmembrane envelope protein, pl5E (Cancer. Res., 39, (1979), L. E. Mathes, R. G. Olsen, L. C. Hebrand, E. A. Hoover, J. P. Schaller, P. W. Adams and W. S. Nichols, 950-955; J. Exp. Med., 158, (1983), G. J. Ciancolo, M. E. Lostrom, M. Tam and R. Snyderman, 885-890; J. Immu¬ nol., 134, (5), (1985), C. G. Orosz, N. E. Zinn, R. G. Olsen and L. E. Mathes, 3396-3403), which suppresses the blastogenic response in cat, mouse,
and human lymphocytes (Cancer Res., 39, (1979), L. C. Hedebrand, R. C. Olsen, L. E. Mathes and W. S. Nichols, 443-447; J. Immunol., 131, (4), (1983), E. A. Copeland, J. J. Rinehart, M. Lewis, L. Mathes, R. Olsen, and A. Sagone, 2017-2020). Recently, similar immunosuppressive properties have been demonstrated for a synthetic peptide of 17 aminoacids which is homologous to parts of pl5E and to gp21 (gp21: glycoprotein of 21,000 daltons) of human T-cell (thymus cell) leukemia virus HTLV-I and II (HTLV: Human T-cell leukemia virus or human T-cell lymphotropic virus; I and II: type I and II) (Nature, 311, (1984), G. J. Ciancolo, R. J. Kipnis and R. Snyderman, 515; Science, 230, (1985), G. J. Ciancolo, T. D. Copeland, S. Oroszlan and R. Snyderman, 453-455). Another human retrovirus, LAV/HTLV-III (LAV: lymphadenopathy-associated virus; III: type III) infects'and destroys CD4-positive (cluster differentiation antigen; it is a receptor on the membrane of the cell. The receptor is a marker for a definite type of cells; 4 indicates that the marker is found on a helper cell.) T-helper lymophcytes and causes acquired immnunodeficiency syndrome (AIDS) (J. Immunol., 135, (5), (1985), J. S. McDougal, A. Mawle, S. P. Cort, J.. K. A. Nicolson, G. D. Cross, J. A. Scheppler-Campbell , D. Hicks and J. Sligh, 3151-3162; Nature, 319, D. Klatzmann and L. Montagnier, 10-11). However, the destruction of helper lymphocytes cannot be the sole explanation for the immunodeficiency of AIDS because the deficency may be severe even when there are still significant mumbers of helper cells left (N. Engl. J. Med., 313, (1985), H. C. Lane, J. M. Depper and W. C. Geene; Scand. J. Immunol., 21, (1985), B.
Hofmann, N. ødum, P. Platz, L. P. Ryder, A. Svejgaard and J. 0. Nielsen, 235-243), and because CD8-positive (8 indicates that the marker is found on a suppressor cell.) T-suppressor lymphocytes are also abnormal (Scand. J. Immunol. (in press), B. Hofmann, N. ødum, B. Jacobsen, P. Platz, L. P. Ryder, J. 0. Nielsen, J, Gerstoft and A. Svejgaard) although LAV/HTLV-III is not tropic for these cells. Here we report that a purified preparation of LAV/HTLV-III virus suppresses the in vitro mitogen and antigen responses of normal lymphocytes while an extract of non-infected H9 (H9 is the designation of a cell line of T-helper cells. The meaning of H and 9 are not known) cells does not. A similar observation has independently been done b others (Proc. Natl. Acad. Sci., 82, (1985), S. Pahwa, R. Pahwa, C. Saxinger, R. Gallo and R. Good, 8198-8202). This observation may in part explain the severe immunodeficiency of AIDS. Furthermore, sera with neutralizing effect on the suppressive factor have been found among anti-LAV/HTLV-III antibody positive individuals without immunodeficiency. The perspectives of this finding are that antibodies against this LAV/HTLV-III associated factor might be used in the treatment of AIDS patients. Moreover, further charactriztion of the factor might be important in the development of a vaccine.
A LAV/HTLV-III extract (sucrose gradient purified virions from a culture medium of LAV/HTLV-III infect¬ ed H9 cells) and that from non-infected H9 cells were prepared by means of Triton-X 100 (the designation of a detergent from Sigma Chemical Co. , St. Louis, MO, US), heat treated, and dialyzed
against isotonic NaCl solution. Figure 1 shows that the LAV/HTLV-III extract suppressed the mitogen (antigen, data not shown) responses of normal peripheral mononuclear cells (PBMC) by 63-90% when added in a dilution 1:500 at the beginning of the culture period. An extract from the non-infected cells was not suppressive in a dilution of 1:500. The LAV/HTLV-III extract did not suppress the response when added at the third day of culture, i. e. 24 hours before harvesting (relative PHA (phyto- hemmaglutinin) response: 108%). PBMC preincubated for one hour at 37°C with a 1:500 dilution of the extract form infected cells and washed twice showed 64% +/-15 of the response of cells not preincubated with the extract indicating that the suppression was at least in part irreversible or otherwise stated not e'asily reversible. The LAV/HTLV-III extract also suppressed a range of different cell lines from man and mouse including B-cell (bursa Fabricii cell) lines. A human IL-2 (interleukin-2) cell line was especially susceptible. The suppressive action on PBMC was shown not to be due to direct cytotoxicity. The effect of the HTLV-III extract could not not be neutralized by the addition of large ammounts of IL-2, but as shown in table 2 below the addition of a serum from a patient with Kaposi's sarcoma and normal immunoresponses could neutralize the inhibition while that form another patient with opportunistic infections could not. Serum from a goat immunized with the LAV/HTLV-III extract could also neutralize the inhibition, while serum taken before immunization could not.
The possibilty that the HTLV-III extract might be cy-
totoxic was investigated by incubating PBMC with and without HTLV-III extract for one hour at 24°C and subsequently counting of eosin-stained cells. There were similar low numbers of dead cells in the two samples. Moreover, there were 62% +/-7 and 66% +/- 9 viable cells in six PBMC cultures stimulated with PHA for three days with and without HTLV-III extract, respectively. Accordingly, the suppressive action on PBMC is probably not due to direct cytotoxicity. In contrast when HTLV-III extract was added to a human IL-2-dependent T-cell line (Int. Archs. Allergy Appl. Immun., (in press), E. Langhoff, J. Ladefoged and N. ødum; Clinical Immunology and Immunopathology, (sub¬ mitted), E. Langhoff, B. Hofmann, N. ødum, J. Ladefoged, P. Platz, L. P. Ryder and A. Svejgaard), a 96% inhibition was observed (table 1 below) and a count of dead cells showed that there was no viable cells after 24 hours. This effect of the HTLV-III extract could not be neutralized by the addition of large amounts of IL-2, but as shown in table 2 below, the addition of serum from a patient with Kaposi's sarcoma could neutralize the inhibition while that from another patient with opportunistic infections could not. The mere observation of neutralization by serum is a strong argument against the assumption that the suppression might be due to cytotoxicity.
The effect of the LAV/HTLV-III extract on the IL-2 receptor expression was investigated by double immu- nofluorescens studies on a FACS (flourescens-acti- vated cell sorter) Analyzer using anti-Tac (Tac: T-cell activation, now designated as IL-2 receptor) (anti-IL-2 receptor), anti-CD4, and anti-CD8
antibodies and PBMC cultured with a suboptimal concentration of PHA for 72 hours with and without LAV/HTLV-III extract. About 6% of the CD4-positive and 4% of the CD8-positive lymphocytes became Tac positive in cultures without LAV/HTLV-III extract, whereas no cells of either subset became positive in the presence of this extract. A curious and unexplained observation was the presence of 64% CD4-positive and 61% CD8-positive cells in the cultures with LAV/HTLV-III extract, indicating that some cells must carry both markers. When IL-2 recpetors had developed in response to PHA, subsequent additon of HTLV-III extract had no effect on this expression.
Based on these experiments, we conclude that the LAV/HTLV-III extract exerts its effect at an early stage of T-cell activation before IL-2 receptors become expressed, perhaps already at the membrane level, but further experiments are required to determine the precise nature of the suppressive mechanisme.
The pi5 retroviral protein has been shown to interfere with IL-2, but there is controversy as to whether IL-2 can reverse the impaired immune response in animals (J. Immunol., 134, (5), (1985), C. G. Orosz, N. E. Zinn, R. G. Olsen and L. E. Mathes, 3396-3403; J. Immunol., 130 (5), (1983), M. A. Wainberg, S. Vydelignu and R. G. Margolese, 2372-2378). However pl5E also alters the function og monocytes and macrophage (J. Clin. Invest., 68, (1981), G. Ciancolo, J. Hunter, J. Silva, J. S. Haskill and R. Snyderman, 831-844; J. Immunol., 124,
(6), (1980), G. J. Ciancolo, T. J. Matthews, D. P. Bolognesi and R. Synder, 2900-2905) and thus its effect cannot be confined to IL-2 alone. It is tempting to speculate that the pl5E as a transme - brane protein may interfere with transmembrane signalling in general. It is unknown whether pl5E and HTLV-III extract act by the same mechanism and a homology and a homology beween HTLV-III and the immunosuppressive, synthetic peptide of 17 a ino acids which is homologous to pl5E og gp21 of HTLV-I and II (Nature, 311,, (1984), G. J. Ciancolo, R. J. Kipnis and R. Snyderman, 515; Science, 230, (1985), G. J. Ciancolo, T. D. Copeland, S. Oroszlan and R. Snyderman, 453-455; J. Immunol., 134, (5), (1985), C. G. Orosz, N. E. Zinn, R. G. Olsen,L. E. Mathes, 3396-3403).
In order to investigate whether the suppressive effect of HTLV-III extract was confined to T-lympho- cutes, other cell populations were cultivated with HTLV-III extract and extract from non-infected cells (table 1 below). Three Tac-negative EBV-transformed lymphoblastoid cell lines (EBV: Epstein-Barr virus) were all clearly suppressed by the HTLV-III extract, but not with extract from the non-infected H9 cell. Thus, the suppressive action of the LAV/HTLV-III extract is not confined to T-lymphocytes but also involves the proliferation of B-lymphocytes (Science, 220, (1983), Z. Trainin, D. Wernicke. H. Unger-Waron and M. Essex, 858-859).
Preliminary attempts were made to partially characterize the suppressive factor(s) by separating the HTLV-III extract of a Sephadex G-100 column (Se-
8
phadex G-100 is a definite derivative of starch from Pharmacia, Uppsala, SE). The suppressive activity was recovered in the fractions with molecular weight of about 70,000.
It seems likely that the suppressive factor in the extract from LAV/HTLV-III-infected cells constitute parts of the virus, but our results cannot rule out that the factor is a host factor produced by the H9 cells in response to the LAV/HTLV-III infection. In either case, if this factor is released by infected cells also in vivo, it may explain the severely suppresssed mitogen response for PMBC from AIDS pati¬ ents. In particular, the irreversible nature of the suppression may ensure that a few LAV/HTLV-III infected cells may suffice to suppress many T-lympho- cytes. The prelimnary observation that a serum from a patient with Kaposi's sarcoma but without oppor¬ tunistic ■'"infections neutralized the suppression of the HTLV-III extract while that from a patient with opportunistic infections did not has several implications. Firstly, it indicates the existance in some patients of antibodies that can neutralize the suppressive factor(s). The presence of such antibodies may prevent or delay immunosuppression in LAV/HTLV Ill-infected individuals and may thus be of pronogstic value. Secondly, it makes the suppresive factor(s) candidate(s) for the development of a vaccine. Finally, it supports the assumption of a viral origin of the suppresive factor.
Perspectives related to the subject matter of the present invention
Treatment of AIDS patients:
I Infusion of serum or purified immunoglobulin originating from LAV/HTLV-III infected healthy individuals where the neutralizing antibody has been shown to be presnt in the serum by means the assay mentioned below as item 4 under the heading "Findings",
II infusion of serum from experimental animals im¬ munized with LAV/HTLV-III extract or the puri- fied suppressive factor,
III infusion of monoclonal antibodies directed against the suppressive factor and
IV plamaphoresis of AIDS patients where the blood passes through a filter in which antibodies against the suppressive factor (monoclonal, from immunized animals, from infected indivi¬ duals) are bound.
Vaccination:
Purification of the suppressive factor and use of the purified suppressive factor or a synthetic protein or synthetic peptide having the same effect for the active immunization of healthy individuals (i. e. vaccinating these individuals) .
Immunosuppressivum:
Purification of the suppressive factor and use of the purified factor or a synthetic peptide having the same sequence of amino acids or parts of the suppressive factor as an immunosup¬ pressivum. The purified factor may possibly be used in transplantations and in the treatment of cancer (e. g . cases of leukemia).
ELISA test:
i Use of the suppressive factor for performing a test for the presence of neutralzing antibodies and
ii use of a monoclonal antibody or antibodies ori¬ ginating from animals after immunization for performing a test for the presence of the fac- tor.
(ELISA is short for enzyme-linked immunosorbent assay. )
Findings
1 Suppressive factor recovered form raw LAV/HTLV-III extract. The molecular weight was found to be 65,000-70,000 by separtion on a Se- phadex column. It is possible that there exists a subunit of 25,000-30,000. The factor as such is a glycoprotein determined by lentile column
fractionation. The factor will lose the activity on boiling.
2 Some individuals that have been infected with LAV/HTLV-III and that are clinically healthy are having antibodies in their serum against the suppressive fact.
3 Neutralizing antibodies against the factor are found in a goat after immunization with
LAV/HTLV-III extract and
4 Assay for the suppressive factor and neutraliz¬ ing antibodies by means of a human IL-2 depen- dent cell line has been found.
Experiments
The results of some experiments related to the subject matter of the invention are shown below in tables 1 and 2, each being accompanied by a legend, as well as in figure 1; the legend to figure 1 is found immediately after the legend to table 2.
It should be noted that the results in these tables and in this figure and accompanying information are given only for the purpose of illustration and are thus not to be construed as limitations in any sense or respect.
Table 1
No. Cell Stimulus Percent of the response of type after addition of exp. day 0 24 timer
ning
HTLV- H9 HTLV- H9
III III
6 PBMC PHA 36 145 108 nd
+/-7 +/-51 +/-39
6 PBMC PHA+IL-2 ' 20~- nd 72 nd +/-15 +/-12
Human IL-2
2 de¬ IL-2 4 93 32 nd pend, cell line
B-
3 cell none 55 89 line +/-2 +/-5
Legend to table 1:
Suppression of the transformation response in dif¬ ferent cell types after addition of HTLV-III extract or H9 extract. The responses are given as % of the responses of cultures without addition of extracts. The results are given as mean relative responses +/- standard errors of the mean. Cultures were prepared as described below in the the legend to figure 1. Interleukin-2 (IL-2) (Lymphocult, Biotest, Frank¬ furt, FRG) was added to a concentration 10 % by volume. A human IL-2 dependant T-cell line (for specifications, see Int. Archs. Allergy Appl. Immun. , (in press), E. Langhoff, J. Ladefoged and N. ødum; Clinical Immunology and Immunopathology, (submitted), E. Langhoff, B. Hofmann, N-. ødum, J. Ladefoged, P. Platz, L. P. Ryder"and A. Svejgaard) was used with 10,000 cells per well. The B-cell lines were Epstein-Barr virus transformed and spontaneously proliferating. There were 50,000 lymphoblastoid cells per well. The HTLV-III and the H9 extract was added in a dilution of 1:500.
Table 2
Addition IL-2 HTLV-III Patient Response, cpm
of extract serum x 103
+ - - 10,9
+ + - 0,5
+ - A 13,0
+ + A 13,4
+ - B 15,4
+ + B 0,6
Legend to table 2:
Neutralization of the effect of LAV/HTLV-III extract on a human IL-2 dependent cell line by addition of serum from AIDS patients. Cultures were prepared as described below in the legend to figure 1. Serum constituted 50 μl og the 170 pi per well. Serum A was obtained from an Aids patient with Kaposi's sarcoma and serum B from an AIDS patient with P. carinii infection.
Legend to figure 1
The HTLV-III extract was prepared from sucrose gradient purified virions from the culture medium of HTLV-III infected H9 cells which were disrupted by 0.6 M KC1 and 0.5% Triton-X 100 and clarified by centrifugation at 40,000 rpm. Further, the extract was heat inactivated for 30 min. at 56°C and dialyzed against PBS (PBS: phosphate-buffered saline) at pH 7.2 for 120 hours. The unifected H9 extract was prepared by disrupting PBS washed H9 cells by freeze-thawing three times in KCl/Triton buffer and heat inactivation and dialysis as with the HTLV-III extract. The protein contents were 0.6 mg/ml for the HTLV-III extract and 80 mg/ml for the H9 extract. PBMC from six healthy blood donors were isolated by Lymphoprep (Nyegaard and Co., Oslo, NO) density gradient centrifugation. Cell cultures were prepared in triplicate in 350 ul round-bottom "micfόtitre plates containing 50,000 PBMC per well. The amounts of mitogen and antigen added per well were 2 ug of PHA ( ■ PHA + HTLV-III extract, Difco, Detroit, MUCH., US), 20 ul og 1:500, 20 ul of a 1:50 suspen¬ sion of PWM (PWN: pokeweed mitogen) ( % PWM + HTLV-III extract, Gibco, Grand Island, N.Y., US) and 20 ul of a 1:100 dilution of HSV antigen (HSV: herpes simplex virus) ( T HSV + HTLV-III extract, kindly supplied by Bodil Norrild, Inst. of Microbiology, Copenhagen, Denmark. The total volume per well was 170 μl. The growth medium used was RPMI 1640 (RMPI 1640 is the designation for a medium from Gibco Laboratories, Grand Island, NY, US containing L-menthionin and dialyzed "fetal bovie serum"). The total volume per well was 170 ul. The growth medium
used was RPMI 1640 (Gibco) supplemented with the antibiotics penicillin, streptomycin, heparin, L-glu- tamin, and 15% heat inactived pooled human male serum. 20 ul of HTLV-III extract was added in the dilutions indicated at the abscissa. The final dilution of extract from non-infected cells was 1:500 ( PHA + extract from non-infected cells). One uCi H -lablled thymidine was added to the cultures stimulated PHA after 72 hours and to cultures stimulated with PWM or HSV for 120 hours. After a further 24 hours, all cultures were harvested on glass fibre filter with an automatic harvesting machine (Skatron, Lierbyen, NO) and the incorporated radioactivity was measured in a liquid scincillation counter (Beckmann LS 1800) after addition of 1,5 ml scincillation fluid. The results of six experiments are given as mean responses relative to those in cultures without the addition of extract and with standard errors of the mean. Values for cultures without HTLV-III added: PHA 47.0 x 103 +/- 16 cpm; PWM 63 x 103 +/-14 cpm and HSV 20 x 103 +/-8 cpm.
Claims
1. I munosuppresive factor purified from human immu¬ nodeficiency virus (HIV) or from cells and cell lines, such as lymphocytes, macrophages, lymphobla¬ stic cell lines of T-lymphocytic origin, macrophage cell lines or hybridoma cell lines infected with HIV, characterized in that it suppresses the mitogen and antigen response of normal lymphocytes stimulat¬ ed with mitogens or antigens, that it suppresses the growth of IL-2 dependent T-cell lines, that it is a glycoprotein, that it has a molecular weight of ap- proximatively 65,000 to approximatively 70,000, espe¬ cially of approximatively 68,000, daltons, that it will lose its activity on boiling, and that it pos¬ sibly contains a subunit having a molecular weight of approximatively 25,000 to appromatively 30,000 daltons, and preferably in that the said cells are disrupted by a solution of a salt and a detergent, preferably by 0.6 M KCl and 0.5% Triton-X 100, and next clarified, preferably by centrifugation, more preferred at 40.000 rpm, then the thus set free virions are preferably purified by a sucrose gradient and the resulting extract is heat inacti¬ vated, preferably for 30 min. and/or at 56°C, and finally dialyzed against a salt solution, preferably a phosphate-buffered saline, at a pH of preferably 7.2 for a period of time, preferably for 120 hours.
2. Process for the production of an immunosup- pressive factor from human immunodeficiency virus (HIV) or from cells and cell lines, such as lymphocytes, macrophages, lymphoblastic cell lines of T-lymphocytic origin, macrophage cell lines or hybridoma cell lines infected with HIV, characterized in that the said cells are disrupted by a solution of a salt and a detergent, preferably by 0.6 M KCl and 0.5% Triton-X 100, and next clarified, preferably by centrifugation, more preferred at 40.000 rpm, then the thus set free virions are preferably purified by a sucrose gradient and the resulting extract is heat inacti¬ vated, preferably for 30 min. and/or at 56°C, and finally dialyzed against a salt solution, preferably a phosphate-buffered saline, at a pH of preferably 7.2 for a period of time, preferably for 120 hours.
3. Use of the whole or at least one part of the fac¬ tor according to claim 1 or a synthetic protein or synthetic peptide having the same polypeptide skel¬ eton as the factor or polypeptides - produced while using DNA sequences specific to the factor - for the detection of antibodies in HIV-infected persons directed against antigenic parts of the factor by means of the neutalizing effect of these antibodies on the biological effect according to claim 1 of the factor or in immunological methods comprising ELISA, Western, RIPA and other known immunological methods.
4. Use of the whole or at least one part of the fac¬ tor according to claim 1 and/or the synthetic protein or synthetic peptide having the same polypeptide skeleton as the, factor and indicated in claim 3 or the polypeptides - produced while using DNA sequences specific to the factor - and indicated in claim 3 for the production of polyclonal and monoclonal antibodies directed against antigenic parts of the factor in animals immunized with the whole factor, at least one part of the factor, or a factor produced by synthsis, viz. the synthetic protein, the synthetic peptide and the polypeptides and all being indicated above in this claim and use of this factor for the purification of antibodies directed against this same factor and originating from persons infected with HIV.
5. Use of the whole or at least one part of the fac¬ tor according to claim " 1 and/or the synthetic protein or synthetic peptide having the same polypeptide skeleton as the factor and indicated in claim 3 or the polypeptides - produced while using DNA sequences specific to the factor - and indicated in claim 3 for bringing about immunity for the purpose of prophylaxis or treatment (vaccination) directed against the factor in healthy or in HIV infected persons.
6. Antibodies corresponding to the immunosuppressive factor according to claim 1, characterized in that they result from the use according to claim 4 and/or claim 5.
7. Use of the antibodies resulting from the use ac¬ cording to claims 3 and/or 4 and directed against the whole or at least one part of the factor according to claim 1 and/or the synthetic protein or synthetic peptide having the same polypeptide skel¬ eton as the factor and indicated in claim 3 or the polypeptides - produced while using DNA sequences specific to the factor - and indicated in claim 2 specific to the factor - and indicated in claim 2 for the detection of the whole or at least one part of the factor according to claim 1 in serum, body fluids and tissues originating from HIV infected persons.
8. Use of polyclonal and monoclonal antibodies di¬ rected against the whole or at least one part of the factor according to claim 1 and/or the synthetic protein or synthetic peptide having the same polypeptide skeleton as the factor and indicated in claim 3 or the polypeptides - produced while using DNA sequences specific to the factor - and indicated in claim 3 whether such antibodies as a part of the use according to claim 4 are produced in animals or purified from serum or body fluids orginating from HIV infected persons for the neutralization of the whole or at least one part of the factor in HIV in¬ fected persons for the purpose of treatment.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DK155886A DK155886D0 (en) | 1986-04-04 | 1986-04-04 | VACCINE AND PROCEDURE FOR PREPARING THIS |
| DK1558/86 | 1986-04-04 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO1987005911A1 true WO1987005911A1 (en) | 1987-10-08 |
Family
ID=8106049
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/DK1987/000035 WO1987005911A1 (en) | 1986-04-04 | 1987-04-03 | Immunosuppressive factor from human immunodeficiency virus (hiv) or hiv infected cells or cell lines, its use, corresponding antibodies and their use |
Country Status (3)
| Country | Link |
|---|---|
| AU (1) | AU7289587A (en) |
| DK (1) | DK155886D0 (en) |
| WO (1) | WO1987005911A1 (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0280468A2 (en) * | 1987-02-19 | 1988-08-31 | Nissin Shokuhin Kabushiki Kaisha | Methods and materials for HIV detection and therapy |
| FR2661834A1 (en) * | 1990-05-09 | 1991-11-15 | Merieux Inst | PROCESS FOR PRODUCING RETROVIRUS IMMUNOGENS AND VACCINES AGAINST RETROVIRAL INFECTIONS, IN PARTICULAR HIV, AND IMMUNOGENES AND VACCINES OBTAINED |
| US5140105A (en) * | 1987-02-19 | 1992-08-18 | Nissin Shokuhin Kabushiki Kaisha | Methods and materials for HIV detection |
| US5169752A (en) * | 1987-02-19 | 1992-12-08 | Nissin Shokuhin Kabushiki Kaisha | Methods and materials for HIV detection |
| US5180660A (en) * | 1987-02-19 | 1993-01-19 | Nissin Shokuhin Kabushiki Kaisha | Methods and materials for HIV detection |
| US5217895A (en) * | 1987-02-19 | 1993-06-08 | Nissin Shokuhin Kabushiki Kaisha | Monoclonal anti-idiotypic antibodies specific for anti-T4 antibodies and cross-reactive with HIV |
| US5696081A (en) * | 1989-03-14 | 1997-12-09 | Board Of Regents, The University Of Texas System | UVB-induced factor for immunosupression |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0280468A2 (en) * | 1987-02-19 | 1988-08-31 | Nissin Shokuhin Kabushiki Kaisha | Methods and materials for HIV detection and therapy |
| EP0280468A3 (en) * | 1987-02-19 | 1990-09-05 | Nissin Shokuhin Kabushiki Kaisha | Methods and materials for hiv detection and therapy |
| EP0468601A1 (en) * | 1987-02-19 | 1992-01-29 | Nissin Shokuhin Kabushiki Kaisha | Methods and materials for HIV detection and therapy |
| US5140105A (en) * | 1987-02-19 | 1992-08-18 | Nissin Shokuhin Kabushiki Kaisha | Methods and materials for HIV detection |
| US5169752A (en) * | 1987-02-19 | 1992-12-08 | Nissin Shokuhin Kabushiki Kaisha | Methods and materials for HIV detection |
| US5180660A (en) * | 1987-02-19 | 1993-01-19 | Nissin Shokuhin Kabushiki Kaisha | Methods and materials for HIV detection |
| US5217895A (en) * | 1987-02-19 | 1993-06-08 | Nissin Shokuhin Kabushiki Kaisha | Monoclonal anti-idiotypic antibodies specific for anti-T4 antibodies and cross-reactive with HIV |
| US5696081A (en) * | 1989-03-14 | 1997-12-09 | Board Of Regents, The University Of Texas System | UVB-induced factor for immunosupression |
| FR2661834A1 (en) * | 1990-05-09 | 1991-11-15 | Merieux Inst | PROCESS FOR PRODUCING RETROVIRUS IMMUNOGENS AND VACCINES AGAINST RETROVIRAL INFECTIONS, IN PARTICULAR HIV, AND IMMUNOGENES AND VACCINES OBTAINED |
| EP0459842A1 (en) * | 1990-05-09 | 1991-12-04 | PASTEUR MERIEUX SERUMS ET VACCINS, Société Anonyme : | Process for the production of retroviral immunogenes and vaccines against retroviral infections, especially HIV, and immunogens and vaccines thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| AU7289587A (en) | 1987-10-20 |
| DK155886D0 (en) | 1986-04-04 |
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