Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Amino acid sequence disclosed in whole or in part; or...
Reexamination Certificate
1993-11-24
2001-08-07
Scheiner, Laurie (Department: 1648)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Amino acid sequence disclosed in whole or in part; or...
C424S192100, C424S193100, C424S268100, C424S272100, C530S350000, C530S395000, C530S403000, C435S007100, C435S007900, C435S007920, C435S810000
Reexamination Certificate
active
06270771
ABSTRACT:
The parasites responsible for malaria in man, including in particular
Plasmodium falciparum
and
Plasmodium vivax
to mention only the principal ones among them, exhibit different morphologies in the human host and express different antigens as a function of their localization in the organism of the infected host. The morphological and antigenic differences shown by these parasites during their life cycle in man makes it possible to define at least four distinct stages of development.
The very first stage of development of the parasite in man corresponds to the sporozoite form introduced into the blood of the host by bites of insect carriers of the parasite. The second stage corresponds to the passage of the parasite into the liver and to the infection of the hepatic cells in which the parasites develop to form the hepatic schizonts which burst to release the hepatic merozoites. The third stage is characterized by the infection of the blood erythrocytes by the asexual forms (merozoites) of the parasite; this erythrocytic form of development of the parasite corresponds to the pathogenic phase of the disease. The fourth stage corresponds to the formation of the sexual forms (or gametocytes) which will become the extracellular gametes in the mosquito.
It is known that very many studies have been undertaken in order to isolate from the strains of parasites infective for a human host polypeptide fractions to provide for the needs of the in vitro diagnosis of malaria by detection of the corresponding antibodies, on the one hand, and to attempt to vaccinate against malaria, on the other.
For example, libraries of cloned cDNAs derived from the sporozoites of
Plasmodium falciparum
have been established by ENEA et al. (1984), Science, vol. 225, 628-630. It was recognized that these libraries included clones capable of expressing immunogenic polypeptides containing repetitive units of 4 amino acids specific for the circumsporozoite antigen (of
P. falciparum
).
However, little work has been done on the hepatic forms of the parasites responsible for malaria. The morphology of the hepatic forms was described for the first time in 1948 on biopsies taken from infected human volunteers (Trans. Roy. Soc. Trop. Med. Hyg., 41, 785 (1948). It was possible to describe an antigen specific for the hepatic stage of
P. falciparum
in the liver of South American monkeys insensitive to the blood forms of the parasite, but in which the hepatic forms can develop (Am. J. Trop. Med. Hyg., 33 (3) 336-341 (1984).
The detection of the localization of the liver-specific antigens (designated hereafter by LS antigens for “Liver Stage” antigens) was carried out by immunofluorescence throughout the maturation steps of the schizont. They are localized at the periphery of the parasite 5 to 40 microns in size; subsequently they are distributed between the cytomers or packets of merozoites, when the schizonts attain between 50 and 100 microns. They are different from the surface antigens of the sporozoites and the antigens shared by the schizonts of the blood and the liver which give an internal immunofluorescence image of the parasite.
Although it is now possible to culture hepatic forms of
P. falciparum
in human hepatocytes (Science, 227, 440 (1985)), the low numbers of mature forms of the parasite obtained by the in vitro and in vivo culture methods do not allow the biochemical analysis of the antigen produced at the hepatic stage.
It has also been observed that the individuals suffering from malaria possess a very high level of antibodies directed against the LSA. The LS antigens seem to be very potent immunogens, among the most potent of all of the antigens synthesized at the various stages of development of the parasite.
One of the objectives of the present invention is precisely to provide novel compositions for the vaccination of humans against the malaria caused by
P. falciparum.
The objective of the invention is also the in vitro diagnosis of the infection of an individual by
P. falciparum
under more sensitive conditions than present methods allow.
A molecule expressed specifically during the hepatic phase has been identified by screening a library of genomic DNA cloned in an expression vector with polyclonal sera (GUERIN-MARCHAND, C. et al.; Nature, 329, 164-167 (1987)). This molecule represents a part of an antigen called LSA (Liver Stage Specific antigen), and is constituted of repetitive motifs of 17 amino acids and seems to be very immunogenic under the natural conditions of exposure to the disease.
These repetitive motifs [SEQ ID NO: 1] of 17 amino acids are represented by the formula:
Leu-Ala-Lys-Glu-Lys-Leu-Gln-X-Gln-Gln-Ser-Asp-Leu-Glu-Gln-Glu-Arg
in which X is Glu or Gly.
The subject of the invention is peptide sequences specific for the hepatic stages of
P. falciparum
which bear epitopes capable of stimulating the T lymphocytes (in particular the cytotoxic lymphocytes).
The invention relates more particularly to molecules or to peptide or polypeptide compositions characterized by the presence in their structure of one or more peptide sequences bearing all or part of one or more T epitope(s) (epitopes implicated in the stimulation of the T lymphocytes) and, optionally, other epitopes, in particular B epitopes (epitopes corresponding to the antibodies produced by B lymphocytes), characteristic of the proteins resulting from the infectious activity of
P. falciparum
in the liver cells.
REFERENCES:
patent: 5599542 (1997-02-01), Marchand et al.
patent: 5602031 (1997-02-01), Marchand et al.
patent: 0 407 230 (1991-01-01), None
patent: 88/05785 (1988-08-01), None
patent: 90/06130 (1990-06-01), None
patent: 92/05193 (1992-04-01), None
Nature,“A Liver-Stage-Specific Antigen ofPlasmodium falciparumCharacterized by Gene Cloning”, C. Guerin-Marchand, et al, vol. 329, pp. 164-167, Sep. 10. 1987.
The Journal of Immunology,“Secondary Structure and Immunogenicity of Hybrid Synthetic Peptides Derived from TwoPlasmodium falciparumPre-Erythrocytic Antigens”, J. Londono et al.; vol. 145, No. 5, pp. 1557-1563, Sep. 1, 1990.
Chemical Abstracts,“Non-CS Pre-erythrocytic Protective Antigens”, M. Hollingdalte, et al, No. 113: 189314w, pp. 534, 1990.
Bulletin of the World Health Organization,“How to SelectPlasmodium falciparumPre-erythrocytic Antigens in an Expression Library Without Defined Probe”, C. Marchand et al.; vol. 68, pp. 158-164, 1990.
Druilhe Pierre
Guerin-Marchand Claudine
Burns Doane , Swecker, Mathis LLP
Institut Pasteur
Scheiner Laurie
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