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Title:  Process and culture medium for the production of cells infected by a multiple sclerosis-associated virus

United States Patent:  6,291,225

Inventors:  Perron; Herve (Grenoble, FR); Seigneurin; Jean-Marie (Bernin, FR)

Assignee:  Bio Merieux (Marcy l'Etoile, FR)

Appl. No.:  485145

Filed:  June 7, 1995

Foreign Application Priority Data:  Apr 03, 1992[FR] (92 04322); Nov 03, 1992[FR] (92 13443)

Abstract

Process for in vitro production of a culture or cell line infected by a viral strain associated with multiple sclerosis (MS), according to which a body sample is taken from an individual suffering from MS, the sample is cultivated in a culture medium which promotes the growth of infected cells to obtain a culture of primary infected cells, and a sample of the culture of primary cells or of a subculture of the latter is cultivated in series, that is to say by successive passages, in the culture medium to obtain the culture or cell line infected by a virus associated with MS. The process includes a procedure in which the culture medium also contains a beta anti-interferon antibody or an antibody which is directed against an antigenically close molecule, the antibody playing an inhibiting role in viral expression and allowing long-lasting expression and propagation of the viral strain in the culture or cell line.

SUMMARY OF THE INVENTION

A hypothesis has been put forward and verified, according to which permissive human plexus choroideus cells could be cells permissive to the virus found in patients suffering from MS. On the basis of this discovery, a process for in vitro culture of cells infected by a virus associated with MS has been developed.

The process comprises culture of plexus choroideus cells obtained after post mortem explantation of human plexus choroideus in a suitable medium comprising amino acids, vitamin factors, inorganic salts and glucose, in total weight concentrations respectively, of between 400 and 2250 mg/l, 3.5 and 130 mg/l, 9100 and 13,000 mg/l and 1000 and 6000 mg/l, to which a growth factor, such as ECGF ("Endothelial Cell Growth Factor"), to promote growth of the cells, and at least one antibiotic are advantageously added, then in bringing the plexus choroideus cells thus cultivated, in their culture medium, into contact with infected primary or derived cells or a supernatant of the culture comprising the virus under the given conditions allowing propagation of the virus from infected cells to cultivated cells, its replication and its expression.

Cells derived from primary cells are understood as meaning any culture obtained directly or indirectly from said primary cells, for example by storage at low temperature or maintenance of the viability of said cells. They may be, for example, reference cells deposited in a collection.

However, the propagation of the virus from a few infected cells, although this exists, remains relatively restricted in the course of successive passages and requires several passages to obtain a sufficient level of expression. Since the life of these cells in a culture Is limited, it is often only at the last passages that expression becomes quantifiable, which considerably limits the usefulness of said procedure.

The works of the present inventors have led them to demonstrate, quite surprisingly, the production of beta-interferon by plexus choroideus cells in response to a viral attack. In fact, only fibroblasts, epithelial cells and macrophages are known to date for the production of interferon (Interferon: Principles and Medical Applications, the University of Texas Medical Branch at GALVESTON, Department of Microbiology, GALVESTON, Tex. 1992). The effects of interferons are well known, and in particular they induce a refractory state of cells to the synthesis and replication of viral material, thus inhibiting propagation and production of the viruses in the culture. It therefore became highly probable that the production of beta-interferon by plexus choroideus cells is a determining limiting factor in the process for in vitro culture of cells infected by a virus present in individuals suffering from multiple sclerosis.

On the basis of this unexpected discovery, the present inventors have developed a new culture medium which can be used in a process for in vitro culture of cells infected by a virus found in patients suffering from MS.

The present invention thus relates to a medium which is suitable for in vitro culture of cells infected by a virus present in individuals suffering from multiple sclerosis, which comprises, in addition to amino acids, vitamin factors, inorganic salts and glucose in total weight concentrations of, respectively, between 400 and 2250 mg/l, 3.5 and 130 mg/l, 9100 and 13,000 mg/l and 1000 and 6000 mg/l, and at least one anti-beta-interferon antibody.

More particularly, the medium comprises at least the following constituents:

one or more amino acids chosen from the following compounds:

arginine: 100 to 500 mg/l, preferably 100 to 300 mg/l

cysteine and/or cystine: 25 to 300 mg/l, preferably

cystine: 25 to 100 mg/l

glutarine: 200 to 1000 mg/l, preferably 200 to 500 mg/l

histidine: 5 to 50 mg/l, preferably 5 to 20 mg/l

isoleucine: 20 to 100 mg/l, preferably 20 to 60 mg/l

leucine: 20 to 100 mg/l, preferably 20 to 60 mg/l

lysine: 20 to 100 mg/l, preferably 20 to 80 mg/l

methionine: 5 to 50 mg/l, preferably 5 to 30 mg/l

phenylalanine: 10 to 70 mg/l, preferably 10 to 50 mg/l

threonine: 15 to 100 mg/l, preferably 15 to 60 mg/l

tryptophan: 2 to 30 mg/l, preferably 2 to 25 mg/l

tyrosine: 10 to 70 mg/l, preferably 10 to 50 mg/l

valine: 10 to 80 mg/l, preferably 10 to 60 mg/l

one or more vitamin factors chosen from the following compounds:

pantothenate; 0.15 to 5 mg/l, preferably the calcium salt: 0.15 to 2 mg/l

choline: 0.5 to 10 mg/l, preferably the chloride

salt: 0.5 to 5 mg/l

folic acid: 0.5 to 10 mg/l, preferably 0.5 to 5 mg/l

inositol: 1 to 70 mg/l, preferably 1 to 50 mg/l nicotinamide and/or niacinamide: 0.5 to 10 mg/l,

preferably nicotinamide: 0.5 to 5 mg/l

pyridoxine and/or pyridoxal: 0.5 to 10 mg/l, preferably

ably pyridoxine/HCl: 0.5 to 5 mg/l riboflavin: 0.05 to 1 mg/l preferably 0.05 to 0.5 mg/l

thiamine: 0.5 to 10 mg/l, preferably 0.5 to 5 mg/l

one or more inorganic salts chosen from the following compounds:

calcium salts: 100 to 200 mg/l, preferably anhydrous CaCl2

potassium chloride: 350 to 450 mg/l

magnesium salts: 40 to 60 mg/l, preferably anhydrous MgSO4

sodium chloride: 6000 to 8000 mg/l

HCO3 salts: 2000 to 3000 mg/l, preferably NaHCO3

HPO4 salts: 600 to 1000 mg/l, preferably anhydrous Na2 HPO4

glucose: 1000 to 6000 mg/l, preferably D-glucose

anti-beta-interferon antibodies: about 10 U/ml

The amino acids are advantageously chosen from among those of the natural L series.

The medium can also comprise at least one antibiotic, preferably a mixture of penicillin and streptomycin, and, if desired, clindamycin to prevent mycoplasmic contaminations.

According to one embodiment of the invention, the medium furthermore comprises at least one growth factor chosen from ECGF ("Endothelial Cell Growth Factor", also called acid FGF) and basic FGF ("Fibroblast Growth Factor") in varying proportions as determined by the expert from his general knowledge of cell cultures and products available to him. By way of example, the concentration of growth factor is between 1 and 50 pg/l of culture medium, in the presence of heparin at a concentration of between 50 and 50 .mu.g/l. The growth factor chosen is advantageously ECGF (10 .mu.g/l, in the presence of heparin as above).

The invention also relates to a process for in vitro production of a culture of infected human plexus choroideus cells, sampled post mortem from the body of an individual or patient suffering from MS, according to which said cells sampled are cultivated in the above-mentioned culture medium and under the given conditions to obtain a first culture of primary plexus choroideus cells, and a sample of said culture of primary cells or of a subculture of the latter is then cultivated in series, that is to say by successive passages, in said culture medium to obtain a culture of infected plexus choroideus cells.

The invention also relates to an infected cell line of plexus choroideus cells obtained in accordance with the process described above, called PLI-2, in accordance with the provisions of the Treaty of Budapest, and deposited at the ECACC on Jul. 22, 1992 under number 92072201, and to the viral strain which it harbors, called POL-2 and deposited at the ECACC the same day under number V92072202. These two depositions were made under the authority of and in accordance with the provisions of the Treaty of Budapest.

The cells of the line PLI-2 are advantageously transfected by an "immediately precocious" gene of a virus of the genus Herpesviridae to increase the viral expression in these cells.

The invention also relates to a process for the production of a viable or continuous infected cell culture or line comprising cells infected by at least one human viral strain associated with MS, which comprises

(a) cultivation of human cells infected by the viral strain to obtain at least one primary or derived culture infected by said strain,

(b) cultivation of permissive human cells, preferably of non-infected human plexus choroideus cells obtained in accordance with the process described above, said permissive cells being capable of becoming infected with and of replicating said viral strain, to obtain at least one permissive culture,

(c) cocultivation of at least one sample of a primary infected culture and one sample of a permissive culture, to obtain a primary derived culture infected by a said viral strain,

(d) cultivation in series, that is to say by successive subcultures, of the first derived infected culture; for this purpose, the stage comprising cocultivation, for example over 5 to 8 days, of a new sample of a non-infected permissive culture and a sample of the first derived infected culture, or of a subculture of the latter, is repeated in the course of time, to obtain a new subculture of the same first derived infected culture constituting a continuous viral culture in non-immortal cells.

Culture derived from the primary culture is understood as meaning any culture or subculture obtained directly or indirectly from said primary culture by storage at low temperature or, for example, by maintaining the viability of said culture. It may be, for example, a reference culture deposited in a collection.

At least any one of stages (a) to (d) is carried out with a culture medium comprising an anti-beta-inter-feron antibody.

The primary infected culture is advantageously obtained from human cells infected by said viral strain resulting from the in vitro production process described above, for example the cell line 92072201 of the ECACC, and/or from human cells infected by said viral strain chosen from the group comprising leptomeningeal cells, plexus choroideus cells, myeloid blood cells, in particular macrophages and monocytes, and lymphocytes.

The permissive culture is preferably obtained from human plexus choroideus cells.

Finally, the invention relates to a viable or continuous viral culture obtained according to the above process.

The cells harboring said continuous viral strain are advantageously transfected by at least one "immediately precocious" gene of a virus of the genus Herpesviridae to increase viral expression in these cells.

The use of human anti-beta-interferon sera or antibodies has allowed increased propagation of viral strains present in cells explanted from anatomical specimens or introduced into non-infected plexus choroideus cells by coculture. It has thus been possible to increase global expression of the virus in plexus choroideus cell cultures and to shorten the time before a detectable signal is obtained after culture of pathological isolates. These effects can be attributed to the neutralization by the beta-interferon antibodies or an antigenically close molecule which plays an inhibiting role in viral expression and is produced by these cells in the presence of the virus.

"Anti-beta-interferon antibody" is understood as meaning any preparation comprising antibodies of mono- or polyclonal origin which may or may not be purified (for example by affinity chromatography) and which recognize epitopes belonging to human beta-interferon or to any analogous antigenic molecule which plays an inhibiting role in viral expression.

According to a particular embodiment of the invention, the primary infected culture is first treated by irradiation, for example by irradiation with X-rays, before being brought into contact with the permissive cultivated cells.

According to a particular embodiment of the invention, several primary cultures infected by viral strains or MS isolates which differ respectively are obtained, and during stage (b), samples of said primary cultures or subcultures of the latter, which differ respectively, are cocultivated. The cell culture of a mixture of viral strains are thus obtained, allowing inter-strain recombination, possible complementation of defective genomes and the emergence of recombinant strains, the suitability of which for certain criteria can be greatly increased. This may allow, in particular, production of a strain highly suitable for in vitro culture or production of replicative strains from defective strains.

The invention also relates to a process for the detection of antibodies directed against a virus associated with MS in a biological fluid.

The invention also relates to a process for the production of a vaccine preparation from infected cells obtained according to the process of the invention or the virus produced by said cells.

The term "infected cells" as used in the present invention refers:

i) to primary infected cells obtained from a culture of cells resulting directly from in vivo or post mortem sampling of biological tissues or fluids from an individual, and to derived cells obtained by passages of these primary cells, and

ii) to secondary infected cells obtained by coculture of primary infected cells and of permissive cells, and to derived cells obtained by passages of these secondary cells.

"Primary cells" is understood as meaning culture cells originating directly from sampling biological tissues or fluids and passed in culture without any coculture or inoculation by viral strains resulting from other cells.

The cells removed in vivo or post mortem can be any cells infected by the virus, for example leptomeningeal cells isolated from the cephalorrachidian fluid of a patient (E. Perron et al., Rea. Virol., 140, 551-561 (1989)), myeloid cells found in the blood, in the cephalorrachidian fluid, in tissues or in bone marrow, in particular macrophages or monocytes (B. Perron et al., The Lancet, volume 337, 862-863, Apr. 10, 1991) and also lymphocytes (S. Haahr et al., The Lancet, volume 337, 863-864, Apr. 6 1991) or analogous cells. Another candidate for preparation of a culture of primary cells is represented by human plexus choroideus cells, which are presumed to be a dormancy site for a virus associated with multiple sclerosis.

The term "macrophage(s)" refers to cells derived directly from blood monocytes, to cells residing in tissues (for example microgliocytes or Kupfer cells) and cells of the reticulo-endothelial system, in particular Langerhans cells.

Permissive cells are cells which can become infected with and allow replication of a given virus with production of viral particles, in particular extracellular particles, which can be studied, for example in respect of their reverse transcriptase activity in supernatants.

The term "passage" refers to a cell culture and corresponds to dissociation of cells from a culture flask for transfer into one or more new flasks.

It is well-known to experts that spontaneous or induced modifications can survive in the karyotype during storage or passages. Cells derived from a reference cell line thus cannot be exactly identical to cultures or starting cells. Furthermore, the genetic variability of retroviruses is well known, and a given retroviral strain may modify its characteristics by spontaneous or induced mutations in the course of cultures.

The invention generally relates to any biological cell material which can be used directly or indirectly for various purposes, for example therapeutic, clinical, diagnostic or analytical purposes, comprising:

either cells sampled from or belonging to a cell culture or line infected by a human viral strain associated with MS and obtained by any of the processes described above, for example the cell line called PLI-2, deposited at the ECACC on Jul. 22, 1992 under number 92072201 in accordance with the provisions of the Treaty of Budapest

or derived cells obtained by modifying the genome of said cells spontaneously or artificially, but without alteration of their phenotype of cells infected by a virus associated with MS.

The virus present in the biological cell material can preferably be transactivated according to the process for transfection described above such that the virus associated with MS is expressed more intensively, more quickly and/or more completely.

The invention also relates to any viral biological material which can be used directly or indirectly for various purposes, in particular for clinical, therapeutic, diagnostic or analytical purposes, this material comprising

either a viral fraction obtained from the biological cell material defined above, if appropriate after transactivation of the viral element, for example obtained by separation of viral particles from the supernatant of infected cells or by antigen fractionation; this viral fraction results, for example, from the viral strain called POL-2, deposited at the ECACC on Jul. 22, 1992 under number V 92072202 in accordance with the provisions of the Treaty of Budapest

or a viral fraction derived from said viral particles, obtained by modifying the genome and/or the envelope and/or the nucleocapsid of viral particles of said fraction spontaneously or artificially.

The invention also relates to a process for detection of the presence of antibodies directed against an MS virus in any biological fluid samples from the human body. For this purpose, it is sufficient to bring a sample of this biological fluid into contact with either an antigenic extract of the biological cell material defined above or an antigenic extract of a viral biological material as defined above, or all or some of an antigenic extract immunologically analogous to said virus which is obtained by chemical synthesis or genetic recombination and comprising at least one epitope of a viral strain associated with MS; the presence of an antibody/epitope complex is then investigated by any suitable means, for example by a chromogenic, chromophoric or radioactive reaction.

The invention also relates to any immunological reagent, in particular monoclonal or polyclonal antibodies, which have an immunological reaction with a naturally occurring or synthetic antigenic extract as defined above.

Finally, the invention also relates to any vaccine preparation comprising:

either an antigenic extract from a biological call material or from a viral biological material as defined above which is dead or inactive or attenuated,

or an immunoreactive compound which induces an immunological reaction analogous to that caused against said antigenic extract.

Claim 1 of 8 Claims

What is claimed is:

1. A viral biological material selected from the group consisting of:

(a) a viral strain POL-2 deposited at the ECACC on Jul. 22, 1992 under number V92072202;

(b) a viral strain obtained by culture of said viral strain POL-2;

(c) a viral strain obtainable from a cell line PLI-2, deposited at the ECACC on Jul. 22, 1992 under number 92072201; and

(d) a viral strain from cells obtained by culture of said cell fine PLI-2.

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