Chemistry: molecular biology and microbiology – Virus or bacteriophage – except for viral vector or...
Reexamination Certificate
1995-06-07
2001-09-18
Bui, Phuong T. (Department: 1638)
Chemistry: molecular biology and microbiology
Virus or bacteriophage, except for viral vector or...
C435S005000, C435S236000, C435S239000, C435S325000, C424S093100, C424S207100, C424S204100, C424S229100, C536S023720
Reexamination Certificate
active
06291225
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to a medium for in vitro culture of cells infected by a virus present in individuals suffering from multiple sclerosis, a process for the production of infected cells using said medium and the infected cell lines thus obtained.
DESCRIPTION OF THE PRIOR ART
Multiple sclerosis (MS) is a demyelinizing disease of the central nervous system (CNS) which has been suspected f or several years of being associated with a virus, although the causal agent still has not been determined with certainty.
Several works have supported this hypothesis of a viral etiology of the disease, but none of the known viruses tested has proved to be the causal agent sought.
Consequently, the observation in patients suffering from multiple sclerosis of phenomena comparable to an autoimmunity reaction has led to an “essential” auto-immune etiological hypothesis (Lisak R. P., Zweiman B. New Engl J. Med. 1977; 297, 850-853, and Lassmann H. and Wisniewski H. M. Arch. Neurol. 1979; 36 490-497). However, this auto-immunity directed against certain components of the central nervous system has proven to be not very specific to MS and frequent in inflammations of the CNS, which may or may not be associated with an infection, as was demonstrated by Hirayama M. et al. (Neurology 1986; 36, 276-8) Kenneth G. Warren et al. (Annals of Neurology 1986; 20, 20-25), Suzumura A. et al. (Journal of Neuroimmunology 1986; 11 137-47) and Tourtelotte W. et al. (Journal of Neurochemistry 1986; 46, 1086-93). Furthermore, as noted by E. J. Field (The Lancet 1989; 1, 1272), none of the immunosuppressive therapeutic agents has allowed decisive results against MS to be obtained.
One hypothesis has been put forward, according to which a retrovirus is said to be the cause of the disease. The discovery by A. Gessain et al. (J. Infect. Disease 1988; 1226-1234) of neurological syndromes associated with the HTLV-1 virus, known originally as an agent of T-cell leukemias in adults, has led several authors, such as H. Koprowski et al. (Nature 1985; 318, 154), M. Ohta et al. (J. Immunol. 1986; 137, 3440), E. P. Reddy et al. (Science 1989; 243, 529), S. J. Greenberg et al. (Proc. Natl. Acad. Sci. USA 1989; 86, 2878), J. H. Richardson et al. (Science 1989; 246, 821), S. L. Bauser et al. (Nature 1986; 322, 176) and A. Rarpas et al. (Nature 1986; 322, 177), to investigate an involvement of this human retrovirus in MS, but without success or with results which suggest cross-reactions.
There is furthermore an animal model which is very close to MS and is induced by a retrovirus: the MAEDI-VISNA virus of sheep. It is known that natural infection by this virus causes an ovine disease similar to MS, as reported by Johnson R. T. (Rev. Infect. Dis. 1985; 7, 66-67), Narayan O. and Cork L. C. (Rev. Infect. Dis. 1985; 7, 89-98) and Nathanson N. et al. (Rev. Infect. Dis. 1985; 7, 75-82). Experimental infection of sheep by intraventricular inoculation of neurovirulent strains of the Visna virus has allowed the responsibility of this virus in the origin of this demyelinizing infection of sheep to be established. As explained by Nathanson N. et al. (Rev. Infect. Dis. 1985; 7, 75-82), Roffman P. M. and Panitch H. S. (“Handbook of Clinical Neurology, 12; Viral Diseases” R. R. McKendall, ed., Elsevier Science Publishing, Amsterdam, 1989, 453-466) and A. Haase (Nature 1986; 322, 130-136), it differs slightly from natural infection, but nevertheless remains close to MS. It is moreover interesting to note that in all the works carried out on this subject by the above-mentioned authors, the Visna virus is found in the cells of the plexus choroideus of the brain of infected sheep, which constitutes a site of dormancy and occasional replication of the Visna provirus; the location of these cells at the cephalorrachidian blood/fluid boundary certainly explains this phenomenon.
All these results argue in favor of the role of an unknown retrovirus in MS.
Works by H. Perron et al. (Res. Virol. 1989; 140, 551-561, and “Current concepts in multiple sclerosis” Wiethölter et al., editors Amsterdam, Elsevier, 1991, pages 111-116 and The Lancet 1991; 337, 862-863) have recently allowed isolation of a line of non-lymphoid cells from a lumbar puncture of the cephalorrachidian fluid of a patient suffering from MS and demonstration of the presence of a virus, having the characteristics of a retrovirus and showing in particular an reverse transcriptase activity, in the supernatant of the culture of the cells of this line. Examination of cells of this line by electron microscopy has allowed the demonstration of viral particles having a diameter of between about 110 and 140 nm, the size of the particles varying according to whether they are mature or immature particles. Furthermore, a serological study by the ELISA technique using a cell extract of infected cells of this line has shown, with 40 sera of patients among whom 20 are suffering from MS (certain MS) and 20 are presumed patients (probable MS), 60% of positive results. A comparative study with 40 sera of patients suffering from neurological diseases other than MS gave only 5% of positive results. This line, which the authors have called LM7, is clonal and non-immortal and immunocyto-chemical and ultrastructural study of the line has allowed characterization of its leptomeningeal origin.
However, this virus has proved to be very difficult to study because on the one hand it expresses itself very weakly in vitro in the primary cell line of leptomeningeal origin, and on the other hand this cell line degenerates quite rapidly after about 30 passes due to extinction of its mitotic potency such that it no longer allows viral expression.
In addition, the authors have envisaged a new approach (H. Perron et al., The Lancet, volume 337, 862-863, (1991)), which comprises taking a blood sample from a patient suffering from MS, culturing monocytes and collecting the supernatant to verify expression of a reverse transcriptase activity, either directly in the ultracentrifugation pellet or after equilibrium sedimentation over a sucrose gradient. It has thus been found that there is a reverse transcriptase activity peak in the supernatant of the patients suffering from MS, and that this activity is found in the fraction having a density of about 1.17 g/ml. Examination of the infected cells by electron microscopy has revealed particles similar to retroviruses of 100 to 120 nm which are found in the ultracentrifugation pellets of supernatants of cultures which express an increased reverse transcriptase activity. The centrifugation pellets containing cell debris and, potentially, viral particles were then cultivated on funicular blood cells to demonstrate a viral expression. However, as explained by the authors, a cytopathic effect was observed in the infected funicular blood cells, but is no longer detectable six weeks after inoculation, such that this culture method is not satisfactory for an in-depth study of the characteristics of this virus.
It was thus essential to have available a process for in vitro culture of cells infected by this virus.
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 infec
Perron Herve
Seigneurin Jean-Marie
Bio Merieux
Bui Phuong T.
Oliff & Berridg,e PLC
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