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• • C—CHEMISTRY; METALLURGY
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  • C12N2740/00—Reverse transcribing RNA viruses
  • C12N2740/00011—Details
  • C12N2740/10011—Retroviridae
  • C12N2740/16011—Human Immunodeficiency Virus, HIV
  • C12N2740/16051—Methods of production or purification of viral material

Definitions

• Monocytoid cellline its production and method for infection with virus, especially HIV.
• the invention is concerned with novel cell lines adapted especially for culturing and studying retroviruses capable of infecting the ceils of these cell lines.
• retroviruses contemplated may be mentioned HIV (Human Immuno Deficiency Virus).
• HIV Human Immuno Deficiency Virus
• Various isolates of HIV have previously been designated LAV (Lymphoadenopathy Associated Virus), HTLV-III (Human T Lymphotropic Virus III) and ARV (AIDS- associated Retrovirus).
• T4 cell tropism of various HIV isolates has been demonstrated in vitro. Virus replication and high reverse transcriptase activity have been observed in populations of T4 cells butnot in the corresponding T8 cells (11). Blocking experiments with monoclonal antibodies to the T4 and T4A antigens (12, 15) and inhibition of syncytia formation of VSV (HTLV-III) pseudotype virus (3) have indicated that the T4 antigen or a part thereof constitutes an important component of the HIV receptor.
• a neoplastic aneuploid T cell line (33) and (ii) a lymphoblastoid B cell line (32) should be employed.
• the subclone H9 has been mentioned as a preferred cell line.
• references 3, 13 and 34 as being of particular relevance.
• References 9, 33, 35 and 36 have been cited as defining the general state of the art.
• the object of this invention is to provide means for promoting the understanding of diseases caused by viruses which are capable of infecting monocytoid cells.
• viruses contemplated in this context may be noted in particular certain retroviruses such as HIV.
• the invention thus provides cell lines (cell populations) the cells of which can be infected in vitro by the virus in question and consequently are potentially utilizable also for virus production in vitro, and furthermore for studying i.a. the AIDS pathogenetic mechanism on a cellular level and for studying the manner in which drugs will affect the course of the disease.
• a first aspect of the invention comprises the cell lines as such, a second aspect comprises the development of said cell lines, and a third aspect comprises the use of said cell lines for in vitro infection and optionally large-scale production of the virus.
• the virus-infected cell line in itself which may be an excellent diagnostic tool (e.g. in radioimmunoprecipitation).
• the cell line of the invention is a stable subclone of a monocytoid wild type and is capable of unlimited growth in vitro.
• the cell lines are characterized in that T4 antigens are expressed on the surface of at least about 5 %, e.g. more than 10 %, of the cells of the population.
• the preferred forms thereof can be infected productively.
• Other characteristic features reside in that class II antigen and Fc receptors may be found present as cell surface antigens on at least some of the cells of the population. These characteristics can be demonstrated if the cell lines of the invention are studied under the conditions set forth in the experimental portion of this application.
• the cell lines of the invention are potentially obtainable by cloning monocytoid cell lines which are derived from cancer tissue and are capable of unlimited growth.
• U-937 which is of histiocytic origin. It was described for the first time by Sundström and Nilsson (28) and has been deposited at the ATCC as No. ATCC CRL 1593.
• Examples of the cell lines of the invention (clone 1, 2 and 16) have been deposited at the European Collection for Animal Cell Cultures, Salisbury, England; they have been given the preliminary designations of clone 1 86121901, clone 2 86121902 and clone 16 86121903.
• Redeposits have been made and given the designations 87100805, 87100806 and 87100807, respectively. They have been accepted to be in accordance with the Budapest Treaty of 1977. U-937 is cytogenetically abnormal. Studies of its karyotype have shown that its chromosomal alterations are progressive. Detailed studies of the chromosome complements in the cell lines of the invention are currently under way and are expected to show that every clone has a distinct karyotype.
• the cloning technique employed is known per se. We have conducted cloning successfully in agar gel. Potentially other techniques too may be employed, such as the so-called limiting dilution technique where the clone cells have been allowed to grow independently of one another. After cloning the proliferating clones fulfilling the characteristics of the present invention are selected.
• the scope of the invention of course comprises also such cell lines as are obtained upon recloning of subclones that have been obtained in a first cloning of the wild type.
• the clones obtainable from the preferred wild type ⁇ -937 will form stably virus-producing lines after viral infection - thus differing from H9 and other T cell lines which show much variation in respect of their virus production.
• the percent amount of cells possessing T4 antigen may vary from as low as 5 % up to 100 % or just below 100 %, e.g. 95 %.
• the cell lines preferred for virus production i.e. those giving a productive infection, will preferably have a T4 content exceeding 50 %, as e.g. exceeding 60 or 70 % although cell lines expressing lower T4-content may also be used.
• the cell lines of the invention are cultured in vitro in a medium such as is commonly employed for U-937 or in some other medium resulting in satisfactory growth. Infection of the lines is performed in a manner such as in common practice in the case of each particular virus employed, the cell line being exposed to a suitable virus inoculum, for instance HIV. In some cases a practical expedient may reside in that, some days after the addition of the first virus inoculum to the cell line, fresh cells are added which are uninfected and have properties as according to the present invention. Cell lines having a T4 content of less than about 50-60 % are suitable in the first place for studies of the virus- cell interaction.
• virus products obtained may be used for developing drugs for the treatment and prevention of the corresponding virus infection, e.g. vaccines, and/or for developing reagents for immunochemically assaying the virus and/or corresponding homologous antibodies.
• the U-937 cell line derived from a histiocytic lymphoma (28) was grown in RPMI 1640 or F-10 supplemented with 10 % fetal on new born calf serum and antibiotics (penicillin 100 IU/ml, streptomycin 50/ug/ml).
• the cell line has been kept in continuous passage since its establishment in 1976 and has retained the basic phenotype corresponding to that of an immature monocyte.
• the cell line is inducible by various agents to phenotypic alterations similar to those of normal monoblasts undergoing differentiation (5, 19, 20).
• ⁇ -937 thus expresses mono ⁇ ytic cell markers with a high degree of accuracy also when induced to further differentiation, and therefore appears to be a very useful model for human monocytic cells (19, 20).
• the dishes contained a bottom layer of Ham's F-10 together with 10 % FCS (GIBCO EUROPE, United Kingdom), glutamine (2 mM), penicillin (100 IU/ml), streptomycin (150 ⁇ g/ml) and a final concentration of 0,5 % Agarose A (Pharmacia AB, Uppsala, Sweden).
• the top layer (1 ml) in which the cells were spread out contained the same medium but with a final concentration of agarose of 0.33 %.
• the petri dishes were incubated for 2-4 weeks in a humified atmosphere (5 % CO 2 in air) at 37oC. During this period the petri dishes were observed for clonal growth of U-937 cells. Suitable clones were picked and the cells were transferred into 96-well plates and later into 24-well plates (Falcon) for further expansion. Two experiments yielded a total of 16 clones.
• T4 and HLA-DR antigens were studied by flow cytometry using monoclonal antibodies directed against the antigens in question (Becton-Dickinson, Monoclonal Center, Mountain View, Ca. (10)). Fc receptor expression was determined using IgG coated erythrocytes. Selected cytoplasmic enzyme markers were studied by cytochemical techniques (29).
• Cells (2 x 10 5 ) were incubated with 2.5 ⁇ g or 12.5 ⁇ g anti-T4 antibody in a total volume of 75 ⁇ g RPMI medium for 20 minutes at 37°C and washed once with said medium. The cells were then resuspended in 125 ⁇ l of virus dilutions and incubated at 37°C for 60 minutes. Following incubation the cells were washed once more with the medium and resuspended in 10 % RPMI medium containing 2 ⁇ g/ml PB at a concentration of 10 5 cells/ml, and cultured in 24-well plates. Anti-T4 antibody at a concentration of 1.25 ⁇ g/ml and 6.25 ⁇ g/ml, respectively, was present for the first 4 days but removed later. The medium was harvested twice a week for reverse transcriptase (RT) determination.
• RT reverse transcriptase
• RT reverse transcriptase
• Southern blotting 10 mg of chromosomal DNA were isolated, cleaved with Sac I, ele ⁇ trophoresed on 0.8 % agarose gel, blotted onto nylon membranes and hybridized with a 32P labeled probe. Cytoplasmic DNA was prepared using the Hirt procedure (8), normalized to mitochondrial DNA and run on 0.8 % agarose gel, then transferred to nylon membranes, and hybridized with a 32P labeled probe.
• HLA-DR and Fc receptors showed a tendency for co-expression.
• the frequency of cells positive for both HLA-DR and Fc receptors was highest in clones 1 and 2, whereas both markers were less frequent in the parental cell line U-937 and clone 16 populations.
• growth properties, morphology, phagocytic activity, capacity of functioning as killer cells in antibody-dependent cellular cytotoxicity assays and expression of the monocyte-associated surface antigens defined by the M3 and OKMl monoclonal antibodies no differences could be demonstrated between the cell lines studied under the culture conditions employed.
• the parental U-937 and clone 4 showed a similar pattern. No RT activity was detected in culture fluids of U-937 during the first six weeks following virus infection. In fact the cultures became RT-positive only two months after infection and then remained positive during the entire period of continued culturing (more than one year). Infection of clone 4 cells was more difficult than infection of the parental line cells since the dose of virus that resulted in a virus-producing infection of all other cell lines (150 x 10 3 cpm in RT activity) failed to productively infect clone 4. During the entire observation period (three months) clone 4 remained RT-negative.
• RT activity 1800 x 10 3 cpm yielded a virus producer culture within one week.
• Clone 4 cells were thus more difficult to infect than the parental line but this difficulty could be overcome by a higher dose of virus for infection.
• the parental line and clone 4 were similar in that no or only a marginal cytopathic effect with occasional syncytia formation could be observed in infected cultures.
• clone 16 was the most sensitive to infection with HTLV-IIIB. RT activity and cytopathic effect were demonstrable already six days after infection. Virus replication and cell lysis were however so pronounced that the cultures died out entirely. It was not possible to obtain virus producing cell lines even when fresh uninfected cells were repeatedly added to the infected culture. With respect to sensitivity to HTLV-IIIB infection, clones 1, 2 and 3 represented intermediates between the two extremes i.e. the parental line and clone 16. In cultures of clone 2 cells the cytopathic effect and RT activity appeared within 10 days after infection.
• Clone 2a Unlike clone 16, addition of uninfected clone 2 cells led to the establishment of a stable virus producer cell line one month later. Clone 2 has during the priority year been recloned and one of the cell lines then obtained (clone 2a) is an even better producer of HIV, showing a minimum of cytopathic effect. Clones 1 and 3 were less sensitive than clone 2 to the cytopathic effect of the virus. A transient cytopathic effect was observed three to four weeks after infection when the cultures were already producing virus (RT-positive culture broths.
• the cell lines differed in the number of free genome copies present in the cytoplasm of infected cells. An estimation of the number of copies could be made in that serial dilutions of the 9 kb fragment inserted by means of SacI into pBHI0-R3 were compared with the high molecular SacI fragments of the infected cells or with Hirt lysate DNA. Clone 16 cells contained approximately 100 copies per cell whereas the parental line contained about one copy per cell, as did the T cell positive control (H9). The other cell lines were intermediate with respect to their contents of copies. The highest molecular weight band (> 12 kb) probably represented HTLV-IIIB DNA that had penetrated into the mitochondria. Proviral DNA could not be demonstrated to be present in uninfected clone 16 cells.
• High molecular DNA that had been digested with SacI showed mainly two bands representing DNA integrated from HTLV-IIIB. Each cell line that had been infected contained approximately one to five integrated copies of proviral DNA from HTLV-IIIB. Two fragments of 5.5 and 3.5 kb resp. corresponded to proviral DNA containing an internal SacI site as well as SacI sites from proviral LTRs. All these bands were to be found also in the infected T cell line employed as control. (Molt-3). The uninfected clone 16 cells were negative with respect to the integrated form of proviral DNA.
• Radioimmunoprecipitation assay (RIPA): Antibodies to virusrelated proteins were identified by RIPA (the antigen was
• HTLV-IIIB which had been obtained from R C Gallo (reverse transcriptase activity was 300 x 10 3 cpm).
• the infected cultures were monitored for cytopathic changes, reverse transcriptase activity (1) and viral antigen expression by means of immunofluorescence with monoclonal antibodies directed against p24 and p19 which in turn were detected by rabbit anti-mouse IgG labeled with fluorescein isothiocyanate (Dakopatts, Glostrup, Denmark).
• the cultures were isotope labeled 7 days after infection when a pronounced cytopathic effect was evident and about 90 % of the cells expressed viral antigen.
• Tris-H 3 PO 4 pH 6.8) Tris-H 3 PO 4 pH 6.8
• the samples were analyzed by PAGE on 9 to 16 % polyacrylamide gradient gels with a 2.25 % stacking gel.
• the gels were fixed for 1 hour in a solution containing 30 % methanol and
• a 14C labeled protein mixture (phosphorylase b 97,000, BSA 69,000, ovalbumin 46,000, carbonic anhydrase 30,000, lactoglobulin A 18,500) was run parallelly on each gel as the molecular weight reference.
• virus-specific proteins could be distinctly identified with the aid of HTLV-IIIB-positive sera. These were gp60, gp120, pr55, gp41, p24, p19. As regards pr55, this protein occurred in variable amounts in different batches so it was not subjected to further study. None of the proteins could be detected, by immunoprecipitation of uninfected cell lysates or by sera not containing HIV-specific antibodies. Antibody-positive control sera with reactivity to several HTLV-IIIB encoded products precipitated the corresponding proteins from virus-infected cells of both clone 16 and HUT-78. The number of bands was the same for each of the two cell types, but the best quality of the precipitates was obtained with clone 16 because cellular proteins from this clone were labeled to a very minor extent.
• An exxlanation of this phenomenon may be that infection of
• HUT-78 gives a continuously virus producing cell line, while the virus is lethal for clone 16 cells.
• two proteins having molecular weights of 160 Kd and 120 Kd could be precipitated.
• the presence of gp41 could not be demonstrated by means of positive sera; but on the other hand such demonstration was readily feasible with the aid of monoclonal antibody directed against gp41 (gift from R C Gallo).
• the identities of p19, p24 and their precursors (pr55 and gp41 for p24) were also checked by using monoclonal antibodies.
• some of the patient sera precipitated proteins of 23 Kd and 27 Kd, these being the products of the sor and orf genes.
• Example 1 Our results show that cells of human monocytoid origin can be infected with HTLV-IIIB and long term producer cell lines can be established.
• the T4 antigen seems to be crucial for enabling HTLV-IIIB to bind to the surface of monocytoid cells, since infection of one of the subclones (clone 16) can be blocked by preincubating the cells with anti-T4 antibodies.
• the T cell leukemia cell line H9 requires a higher concentration of anti-T4 antibodies for blocking the infection than does the monocytoid cell line.
• the results indicate a close similarity between the HTLV-IIIB receptors on lymphoid and monocytoid cells eventhough receptor density may be lower in the monocytoid cell line than in the T cell line.
• clones 1, 2 and 3 are similar in T4 expression (50-60 % in the cases of 1 and 3; 60-70 % in the case of 2), yet the extent of lytic effect and virus replication is most pronounced in clone 2. Since the subclones of U-937 appear to represent cells frozen at different levels of monocyte differentiation they may also differ in their ability to modulate T4 expression after viral infection.
• Example 2 It is inferrable from the results obtained that the cell lines of the present invention may offer major advantages for the production of viral antigens which are useful for detecting HIV-positive sera.
• T-lymphocyte T4 molecule behaves as the receptor for human retrovirus LAV. Nature 312, 767-768. 13. LEVY, J.A. et al, (1985) Virology 147, 441-449.
• lymphadenopathy associated virus LAV

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