Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
1998-09-02
2003-10-21
Falk, Anne-Marie (Department: 1632)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C435S320100
Reexamination Certificate
active
06635624
ABSTRACT:
BACKGROUND OF THE INVENTION
The present application relates to a vector for immunization against hepatitis.
It is also related to a composition containing this vector.
Immunization by injection of bare DNA into muscle tissues has been the object of several studies since the beginning of the 1990s.
Thus, ULMER et al. (Science, 259, 1745-1749, 1993) obtained protection against the Influenza virus by induction of the cytotoxic T lymphocytes through injection of a plasmid coding for the Influenza A nucleoprotein into the quadriceps of mice. The plasmid used carries either the Rous sarcoma virus promoter or the cytomegalo virus promoter.
RAZ et al. (Proc. Natl. Acad. Sci. USA, 90, 4523-4527, 1993) injected vectors comprising the Rous sarcoma virus promoter and a gene coding for interleukin-2, interleukin-4 or the &bgr;1-type transforming growth factor (TGF-&bgr;1). The humoral and cell immune responses of the mice to which these plasmids have been intramuscularly administered are improved.
WANG et al. (Proc. Natl. Acad. Sci. USA, 90, 4156-4160, 1993) injected a plasmid carrying a gene coding for the envelope protein of the HIV-1 virus into mice muscles. The plasmid injection was preceded by treatment with bupivacaine in the same area of the muscle. The authors demonstrate the presence of antibodies capable of neutralizing the HIV-1 virus infection. However, it will be noted that DNA was injected twice a week for a total of four injections.
DAVIS et al. (Compte-Rendu du 28ème Congrès Europèen sur le muscle, Bielefeld, Germany, 21-25 September 1992) injected plasmids carrying a luciferase or &bgr;-galactosidase gene coding by pretreating the muscles with sucrose or a cardiotoxin. The authors observed the expression of luciferase or &bgr;-galactosidase.
More recently, an article published in Science et Avenir (September 1993, pages 22-25) indicates that WHALEN et DAVIS succeeded in immunizing mice against the hepatitis B virus by injecting pure DNA from the virus into their muscles. An initial injection of snake venom toxin, followed 5 to 10 days later by a DNA injection, is generally cited. It is specified that this is not a practical method.
These studies were preceded by other experiments in which various DNAs were injected, in particular into muscle tissues. Thus, the PCT/US application No. 90/01 515 (published under No. WO-90/11 092) discloses various plasmid constructions which can be injected in particular into muscle tissues for the treatment of muscular dystrophy. However, this document specifies that DNA is preferentially injected in liposomes.
This also applies to Canadian patent CA-362.966 (published under No. 1.169.793) which discloses the intramuscular injection of liposomes containing DNA coding in particular for HBs and HBc antigens. The results described in this patent mention the HBs antigen expression. The presence of anti-HBs antibodies was not investigated.
International application PCT/FR 92/00 898 (published under No. WO-93/06 223) discloses viral vectors which can be conveyed to target cells by blood. These vectors are thus recognized by the cell receptors, such as the muscle cells, and can be used in the treatment of muscular dystrophy or of thrombosis.
This application does not relate to immunization against viruses such as, for example, that of hepatitis B.
Thus, it arises from the state of the art cited that although immunization techniques against hepatitis by injection of bare DNA are already known, these techniques had many disadvantages which made their implementation impractical.
Furthermore, the bare DNA used to vaccinate the mice was pure DNA from the virus. This type of treatment can not be considered for human vaccination due to the risks involved for the patients.
Finally, the earliest experiments in which the injected DNA is contained in liposomes did not demonstrate any immune response.
The applicant has therefore aimed at discovering new vector constructions allowing immunization against hepatitis without having a detrimental effect on human health.
He has further aimed at finding an additive for compositions containing the constructions which would allow an effective degeneration of muscle tissue before the DNA injection, and compatible with the requirements of human health.
The applicant has surprisingly shown that it is possible to achieve an effective and durable level of antibodies much greater than the level permitting to obtain in man an efficient and durable immune protection against infection by the hepatitis virus, by administering by intramuscular injection a vector with defined construction, and a substance capable of inducing a coagulating necrosis of the muscle fibres.
The present application thus relates to a nucleotide vector comprising at least:
a gene or a complementary DNA coding for at least a part of the virus protein, and
a promoter allowing the expression of this gene in muscle cells.
Said vector may not replicate in these cells.
It may also be replicative, allowing to obtain a high number of copies per cell and to enhance the immune response.
The vector is also chosen in order to avoid its integration into the cell's DNA, such integrations being known to activate the oncogens and induce cell canceration.
The vector according to the present invention is advantageously a plasmid of partly bacterial origin and notably carrying a bacterial replication origin and a gene allowing its selection, such as a gene for resistance to an antibiotic.
This vector may also be provided with a replication origin allowing it to replicate in the muscle cells of its host, such as the replication origin of the bovine papilloma virus.
The gene or the complementary DNA included in this vector advantageously codes for a structure protein of a virus but it can also code for a regulatory protein.
The gene or complementary DNA carried by this vector can code for a least a portion of a hepatitis virus protein, in particular hepatitis B, and preferentially the protein HBs, in one of its forms S, S-preS2 or S-preS2-preS1, in which case the gene is gene S.
The virus may also be responsible for another hepatitis such as a hepatitis A or of a non-A, non-B hepatitis, such as a hepatitis C, E or delta.
The gene or protein sequences for these hepatitis viruses are described or may be deduced from the following documents:
patent FR-79 21 811, patent FR 80.09.039,
patent EP-81.400.634, patent FR 84.03.564,
patent EP 91.830.479 and the article by Najarian et al. (Proc. Natl. Acad. Sci. USA, 1985, 82, 2627-2631).
The vector may also include genes coding for at least a portion of the gp160 protein of HIV-1 virus associated with the p25 protein, and/or the p55 protein, and/or the p18 protein or at least a gene coding for the Rev protein of HIV-1 virus.
The vector may also include instead of a virus protein, a protein from a pathogenic micro-organism such as a protein from the bacterium causing diphtheria, whooping cough, listeriosis, the tetanus toxin etc.
The promoter carried by this vector is advantageously the promoter for the cytomegalovirus (CMV). It may however be any other promoter which allows the efficient expression of the gene in the muscle cells.
It may thus be:
an internal or endogenic promoter, that is a promoter of the virus from which the gene is taken; such a promoter may be completed by a regulatory element of the muscle or another tissue, in particular an activating element,
a promoter from a gene of a cytoskeleton protein, in particular desmine as described by BOLMONT et al. (Journal of submicroscopic cytology and pathology, 1990, 22, 117-122) et ZHENLIN et al. (Gene, 1989, 78, 243-254).
the promoter from the virus HBV surface genes.
Generally, the promoter may be heterologous to the host, that is not naturally found in the host, but it is advantageously homologous, while being originally active in a tissue other than the muscle tissue.
In addition to the promoter, the vector may include a terminal transcription sequence, situated downstream of the gene.
Such vector may be the pCMV/HBS or pRCCMV-HBS plasmid, having the SEQ ID No. 1 sequen
Davis Heather Lynn
Michel Marie-Louise
Whalen Robert Gerald
Falk Anne-Marie
Institut Pasteur
Wolf Greenfield & Sacks P.C.
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