Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Virus or component thereof
Reexamination Certificate
1995-06-07
2001-02-06
Woodward, Michael R. (Department: 1631)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Virus or component thereof
C424S204100, C424S231100, C435S005000, C435S007100, C435S007200, C530S352000, C530S360000, C530S388300, C530S388150, C530S403000
Reexamination Certificate
active
06183754
ABSTRACT:
The invention relates to human anticytomegalovirus monoclonal antibodies (HCMV) and to a process for the in vitro diagnosis of infections induced by human cytomegaloviruses. It relates more particularly to antibodies of this type which are capable of simultaneously detecting human cytomegaloviruses (CMV), human cells infected by CMV and a polypeptide, particularly a protein induced by HCMV and having protein-kinase activity.
The invention relates also to the aforesaid polypeptide having the aforesaid protein-kinase activity.
It is known that CMV is the cause of numerous clinical infections, ranging between benign infectious manifestations, and congenital manifestations, for example, particularly severe malformations. CMV has also been recognised as a cause of morbidity and mortality in persons who have undergone organ transplants, for example, of the kidney. The detection of CMV is a problem which poses itself therefore particularly acutely. In addition, the recognition and isolation of vaccinating polypeptides or proteins, or which can be rendered vaccinating with respect to CMV would open up serious possibilities of prevention of said complications against which at the present time, few means of treatment are available.
Various authors have already brought their attention to bear on diagnosis techniques, which could be based on the use of monoclonal antibodies with respect to the human cytomegalovirus. The monoclonal antibodies obtained hitherto, did not yet contribute the expected solution for performing a diagnosis, which is both easy and reliable, of infection with cytomegamoviruses.
In fact, it is known that CMV (a member of the family of viruses of herpes) induces in the infected cells a considerable number of polypeptides, at various stages of the infectious process, and at various levels of the infected cells. L. N. PEREIRA and Coll. (“Infection and Immunity”), June 1982, p. 924-932., report that they have produced a large number of hybridomas secretors of monoclonal antibodies, capable of recognising in previously unfixed cells, polypeptides, or glycoproteins induced by the CMV in cultures of these cells. The differences observed by PEREIRA and Coll. at the level of the relative behaviour of different antibodies with respect to the virus itself, infected cells and proteins, glycoproteins, or polypeptides, isolatable from extracts of these cells, has led them to formulate the hypothesis, that the conformation of the viral glycoproteins recognised by certain of these antibodies at the membranal surface of the cells infected by the CMV's, could be different from the conformation of the envelope of the virion. The utilization of certain of the antibodies isolated to perform diagnosis operations of the above-indicated type, has been evoked by these authors. It follows however from the foregoing and from the experimental results provided, that the monoclonal antibodies judged the most interesting, were also capable of precipitating several polypeptides at the same time. The use of these antibodies to recognise antigenic determinants, or specific epitopes, could not therefore be contemplated by reason of the non-discriminating character of the recognition.
L. C. GOLDSTEIN and Coll. (“Infections and Immunity”), October 1982, p. 273-281) also described a certain number of monoclonal antibodies which can recognise certain polypeptides localised in the nuclei, or in cytoplasmic inclusions of cells infected by CMV. These monoclonal antibodies have been selected by their capacity to react with cells infected by CMV, and previously fixed with absolute methanol and dried in air. It does not however follow from the article that these monoclonal antibodies were adapted to recognise still intact infected cells.
Lastly, C. AMEDEI and Coll. (Ann. Virol. (Institut Pasteur), described a battery of 24 monoclonal antibodies directed against human cytomegalovirus. The larger number of monoclonal antibodies obtained, arises apparently from the techniques which have been used to detect them. In particular, detection has employed immunofluorescent techniques of detection of antibodies retained on previously infected cells, which had been fixed with acetone and dried in air. The same monoclonal antibodies are revealed to be practically devoid of activity with respect to cells obtained from identical cell lines, previously infected by the same virus, when these infected cells had been fixed beforehand with methanol. Perhaps there should be sought in this observation, the cause of the low number of hybridomas secreting monoclonal antibodies selective against cells infected with HCMV, which had been isolated by L. C. GOLDSTEIN and Coll.
Among these monoclonal antibodies, some have been found to show neutralising properties with respect to several strains of virus. These monoclonal antibodies have moreover been the subject of a French Patent Application No. 83 05384, filed Mar. 31, 1983. These particular monoclonal antibodies have been adapted for the carrying out of in vitro diagnosis tests of an infection with HCMV. However, the Patent Application and indeed, moreover, the above-mentioned article, have not recognised among the various hybridomas described, that which is established in the following to secrete monoclonal antibodies, particularly effective for diagnosis operations, and suitable to permit the isolation of a polypeptide inducible by HCMV's and endowed with biological properties permitting a particularly refined diagnosis operation.
It is an object of the invention to provide a method of diagnosis employing monoclonal antibodies resulting from a selection amongst all of those which have been described in the state of the art. The use of these selected antibodies in in vitro diagnosis tests, is both easy and accurate. They recognise a protein induced by HCMV's in still intact infected cells in immunofluorescence tests. These antibodies are capable of providing positive responses during almost the whole of the infectious cycle. The diagnosis can be improved and confirmed in dosage tests of the enzymatic activity. The infected cells produce polypeptides, proteins of glycoproteins, which are induced by the HCMV's. These polypeptides are moreover bearers of an epitope, or continuous sequential antigenic determinant. By the expression “bearer of an epitope or antigenic determinant”, must be understood a determinant whose recognition by the antibodies is not connected with the particular conformation of this site on the natural polypeptides, proteins or glycoproteins of the virus, or induced by the latter in the cells that it infects.
The invention results from the discovery which the inventors have made, that the hybridoma secreting monoclonal antibodies previously described, complies with all of these conditions. More particularly, the invention relates to the use of monoclonal antibodies produced by the secreting hybridomas concerned for the detection in vitro of a sequential antigenic determinant borne by a protein, of which the molecular weight is in the vicinity of 68,000, isolatable without degradation from extracts of cells infected by a CMV strain, this protein possessing in addition, protein-kinase properties.
In other words, the human anticytomegalovirus monoclonal antibody employed in the process according to the invention for the in vitro diagnosis of infections induced by human cytomegaloviruses and for the detection of a protein-kinase inducible by human cytomegaloviruses in human cells, and particularly in human cell lines, can be defined by the combination of its capacities:
to give rise to reactions detectable by immunofluorescence, on culture cells of human origin, previously infected with HCMV and fixed with acetone
to react with essentially a single viral polypeptide induced by HCMV's in cells of human origin previously infected the them, this polypeptide having a molecular weight of the order of 68,000, the bearer of a continuous sequential epitope, this polypeptide appearing in the nuclei and then diffusing, at least in part, into the cytoplasm
Amadei Clairo
Barzu Octavian
Boue Andre
Horaud Florian
Michelson Susan
Burns Doane Swecker & Mathis L.L.P.
Institut Pasteur
Woodward Michael R.
Zeman Mary K
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