Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1994-11-23
2001-05-01
Scheiner, Laurie (Department: 1648)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S025300, C435S005000, C435S006120, C435S069100, C435S069300, C424S189100
Reexamination Certificate
active
06225458
ABSTRACT:
The invention relates to a process for the production of a DNA (desoxyribonucleic acid) comprising the genome characteristic of that of the B hepatitis virus. It also relates to DNAs -of which a fragment is constituted by a double strand DNA corresponding to that of viral B hepatitis. Lastly it relates to vectors and compositions including such DNAs, for taking advantages of their biological properties.
B hepatitis is a frequent viral disease, more particularly in tropical Africa, in Southeast Asia and in the Far East where about 10% of the people are carriers of the surface viral antigen also designated as HBs antigen.
Though the infection is often manifested by an acute form without sequelae, it can also be at the origin of a chronic hepatitis, of cirrhosis and even of fatal hepatic necrosis. This explains the importance of studies devoted to the biology of the virus, and the recent development of a vaccine whose efficiency has been demonstrated on patients and members of the personnel of hemodialysis centers (Ph. MAUPAS, A. GOUDREAU, P. COURSAGET, J. DRUCKER and Ph. BAGROS, Intervirol., 10, 1978, 196-208). The Dane particle D. S. DANE, C. H. CAMERON and M. BRIGCS, Lancet. 1970, p. 695-698) is at present considered as the etiological viral agent. This particle, which can be detected by observation with the electron microscope, has a diameter of 42 nm. The patient's serum in the preicteric phase contains up to 10
9
or even 10
10
of it per milliliter. It possesses an envelope (Australia antigen or HBs antigen), a capsid (HBc antigen), an endogenic polymerase and a DNA molecule (J. L. Melnick, G. R. DREESMAN and F. B. HOLLINGER, Sc. amer., 237, 1977, p. 44-52). Under observation with the electron microscope, the genome appears as a bicatenary DNA ring possessing a monocatenary region, whose length varies from one molecule to the next (J. SUMMERS, A. O'CONNELL and I. MILLMAN, Proc. Nat. Acad. Sc., 72, 1975, p 4597-4601, hereafter referred to as “SUMMERS” and W. S. ROBINSON, Ann. rev. Microbiol., 1977, 31:357-377, hereafter referred to as “robinson”. This ring is constituted by two intertwined linear molecules of unequal lengths (as shown diagrammatically in FIG.
1
). It is the smallest viral genome known in mammals. The longest strand contains about 3,200 bases. The endogen polymerase DNA can be used to repair in vitro the single strand region (b
1
in
FIG. 1
) of the shortest strand (T. A. LANDERS. H. B. GREENBERG and W. S. ROBINSON, J. Virol., 23, 1977, p. 368-376, hereafter referred to as “LANDERS”).
Yet when the genome is repaired in vitro, as disclosed in the articles of SUMMERS, ROBINSON and LANDERS, small strands of DNA are formed in situ along the monocatenary region of the genome. However, these new strands do not connect to form a single strand along the full length of the monocatenary region, SUMMERS also discloses that the DNA polymerase reaction on the Dane particle resulted in a “fully double-stranded product consist(ing of a series of newly synthesized strands 50-500 nucleotides in length duplexed with the unlabeled strand”. LANDERS discloses that the “DNA polymerase reaction “closes the single stranded region, resulting in molecules with uniform double-stranded length and a remaining S1-susceptible site (a nick or gap)”. ROBINSON noted, regarding the nature of the DNA product of the in vitro endogenous polymerase reaction, that the ends of the repaired portions of the open strand do not appear to be joined to make closed circular DNA, although the single-stranded gaps appear to be closed by the endogenous DNA polymerase reaction. The circular molecules after the DNA polymerase reaction as before have a nuclease S1-sensitive site, and no closed circular DNA is detected by alkaline sucrose gradient sedimentation or equilibrium centrifugation in CsCl density gradient containing ethidium bromide.
Thus it was clearly established in the prior art that in vitro polymerase-repaired DNA derived from the genomes of hepatitis B virus still contained monocatenary regions, i.e. nuclease S1 susceptible sites. As a matter of fact it was well known in the art that the nuclease S1 enzyme can cleave DNA only in areas of monostranded portions and not in the double-stranded portions.
Study of the virus is however at present made particularly difficult by reason of the difficulties of supplies of serum containing Dane particles. Even a rich serum does not permit the preparation of large amounts of DNA (of the order of 1 &ggr; of DNA per volume of 500 ml of serum). It is hence necessary to collect serums of various origin corresponding to several genetic variants (J. P. SOULIER and A. M. COUROUCE-PAUTY, Vox Sang. 25, 1973, p. 212-235), which renders precarious a study of the primary structure of the genome. The presence of the single strand region makes difficult, moreover the establishment of a physical map by restriction enzymes.
The problem of the isolation of relatively large amounts of viral particles attains a still increased importance, when it is desired to have available sufficient amounts of viral particles, more particularly of their HBs antigens, which appear to carry a surface antigen (protein) having vaccinating properties. The present methods of vaccination, if they have demonstrated their efficiency, are not however absolutely devoid of drawbacks. In particular, preparations of HBs, used as a vaccine, may contain antigen components coming from hepatic cells, which can be the origin of an auto-immune response (B. S. BLUMBERG, Science, 197, 1977, p. 17-24).
It is therefore an object of the invention notably to overcome these difficulties more particularly to provide a process enabling the production of DNA of B hepatitis virus (or of the Dane particle), in sufficient amounts for the realization of the above-mentioned studies, and in a state of purity such that its use can be contemplated; even for therapeutic uses.
The invention takes advantage of the fact that the DNA of the Dane particle possesses, after in vitro “repair” in the presence of precursor nucleotides and of a polymerase, a single recognition site with regard to certain endonucleases, notably restriction enzymes, such as the enzyme EcoRI or Xho.
The process of producing a DNA comprising the genome characteristic of that of the DNA of the B hepatitis virus is characterized by the cloning in a bacterium of a double strand DNA, formed from the B hepatitis virus DNA, notably after repair of the latter in vitro as indicated above. This double strand DNA will be denoted below as DNA-HVB. Preferably, the polymerase used is endogenous polymerase of the B hepatitis virus itself.
Preferably, the DNA to be cloned has, previously, been cleaved by an endonuclease such as defined above, notably by the restriction enzyme EcoRI.
To carry out the cloning, recourse is advantageously had to a vector, notably a phage or plasmid, in which the double strand DNA, previously cleaved at its single site, will have first been inserted.
By way of example of a phage enabling the easy cloning of the double strand DNA first opened by EcoRI, may be mentioned &lgr;gtWES. &lgr;B (P. LEDER, D. TIEMEIER and L. ENQUIST, SCIENCE, 196 (1977) pp. 175-177) which only comprises two EcoRI sites (EcoRI &lgr;1 and EcoRI &lgr;2). The latter enable the insertion of the whole of the DNA of the B hepatitis virus in the genome of this phage, instead and in place of the fragment inside this virus and previously situated between these two EcoRI sites.
It is naturally self-evident that any other vector comprising two EcoRI sites, or even a single EcoRI site, in a part unessential for its own replication, may be used for the same purposes.
Thus the cloning process according to the invention can include the following essential steps:
the repair of the DNA of the B viral hepatitis, in the presence of precursor nucleotides and of a polymerase to form DNA-HVB;
the cleavage of the DNA-HVB by the enzyme selected, notably EcoRI;
the cleavage of the DNA of the vector, recovery of the portions of this DNA (two or three according as the vector includes one
Charnay Patrick
Fritsch Alex
Pourcel Christine
Tiollais Pierre
Finnegan Henderson Farabow Garrett & Dunner L.L.P.
Institut Pasteur
Scheiner Laurie
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