Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Patent
1997-07-01
2000-10-10
Salimi, Ali
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
435476, 4352523, 435882, 4242001, 4242111, 530350, 530300, C12N 700, C12N 100, C12N 1500, A61K 39155
Patent
active
061300918
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to a method of recombinant DNA technology enabling point modifications of nucleotides to be introduced by directed mutagenesis into a gene coding for a polypeptide sequence, which is useful, in particular, for obtaining oral vaccines against the respiratory syncytial virus (RSV); it also relates to peptides and recombinant bacteria capable of being obtained by this method, to compositions containing them and also to the corresponding nucleotide sequences.
BACKGROUND OF THE INVENTION
Respiratory syncytial virus (RSV) is the commonest cause of respiratory ailments in newborn infants: bronchopneumopathies (bronchiolitis). The WHO estimates that 50 million individuals are affected by RSV every year, of whom 160,000 die worldwide. There are two subgroups of the virus (subgroups A and B).
RSV is classified in the family Paramyxoviridae, genus Pneumovirus, containing an unsegmented RNA genome of negative polarity coding for 10 specific proteins.
No vaccine against RSV is currently available. Vaccines containing inactivated virus have been shown to be ineffective and have even sometimes aggravated the infections of newborn infants. In the 1960s, the attempts at vaccination with formalin-inactivated RSV led to failure: instead of conferring protection on reinfection due to RSV, the vaccine had the effect of aggravating the ailment in the child.
Application WO 87/04185 proposed the use of RSV structural proteins for the purpose of a vaccine, such as the envelope proteins known as F protein (fusion protein) or G protein, a 22-kD glycoprotein, a 9.5-kD protein or the major capsid protein (N protein).
Application WO 89/02935 describes the protective properties of the whole F protein of RSV, where appropriate modified in monomeric or deacetylated form.
A series of fragments of the F protein has been cloned for the purpose of investigating their neutralizing properties.
However, the immune vaccines tested hitherto have been shown to be ineffective or have induced a pulmonary pathology (bronchiolitis or peribronchitis).
At the present time, no treatment of primary choice exists for infections due to RSV.
RSV infections of the upper airways: treatment is based essentially on symptomatic medication identical to that used for other viral infections.
RSV infections of the lower airways: treatment in newborn infants is based on the maintenance of correct hydration, the suctioning of secretions and the administration of oxygen if necessary. A positive effect has been observed with ribavirin, a nucleotide which is active in vitro against RSV.
In order to facilitate the administration of the vaccine, it would be desirable to have at one's disposal a product which is active via the oral route, generating a good immunity with reduced side effects.
BRIEF SUMMARY OF THE INVENTION
For this reason, the subject of the present invention is a method for obtaining a peptide or a protein, characterized in that: peptide sequence flanked by amino acid residues 130 and 230 of the G protein of the respiratory syncytial virus, or a peptide sequence displaying at least 80% homology with the said peptide sequence, membrane of the bacterium, are introduced into a bacterium which is non-pathogenic for mammals.
It will thus be possible to observe an immune response against the said heterologous polypeptide during its administration, without using an adjuvant.
Advantageously, the heterologous peptide may be expressed fused with a fragment enabling its anchorage in the membrane of the bacterium.
The G protein is a methionine-poor envelope glycoprotein of RSV, of molecular weight between 84 and 90 kD. The sequence of the G protein differs for the subgroups A and B of RSV; the term "sequence of the G protein", when employed in the present application, should be understood to refer to the sequence of either subgroup A or subgroup B where not otherwise specified.
The Applicant has demonstrated that the sequence flanked by amino acids 130 and 230 of the natural G protein is especially suitable for
REFERENCES:
European Journal of Biochemistry, vol. 230, No. 1, May 1995, pp. 38-44; Murby, M. et al.; `Hydrophobicity engineering to increase solubility and stability of a recombinant protein from respiratory synctial virus`.
Journal of Biotechnology, vol. 42, No. 3, Oct. 16, 1995, pp. 207-219; Nguyen Ngoc, T. et al.; `Hydrophobicity engineering to facilitate display of heterologous gene products on Staphylococcus xylosus`.
Journal of Cellular Biochemistry, vol. Supplement O, No. 19A, Jan. 5, 1995, p. 264; Stahl, S. et al.; `Staphylococcus carnosus with surface-displayed recombinant immunogens, a novel live vector for subunit vaccine delivery`.
Journal of Bacteriology, vol. 177, No. 6, Mar. 1995, pp. 1470-1476; Samuelson, P. et al.; `Cell surface display of recombinant proteins on Staphylococcus carnosus`.
Journal of Bacteriology, vol. 174, No. 13, Jul. 1992, pp. 4239-4245; Hansson, M. et al.; `Expression of recombinant proteins on the surface of the coagulase-negative bacterium Staphylococcus xylosus`.
Virology, vo. 185, No. 2, Dec. 1991, pp. 749-757; Trudel, M. et al.; `Protection of Balb-C mice from respiratory syncytial virus derived from the G glycoprotein`.
Gene., vol. 128, No. 1, 1993 Amsterdam NL, pp. 89-94; Nguyen, T. N. et al.; `Cell-surface display of heterologous epitopes on Staphylococcus xylosus as a potential delivery system for oral vaccination`.
Journal of General Virology, vol. 74, No. 3, Mar. 1993 Reading GB, pp. 453-458; Martin-Gallardo, A. et al.; `Expression of the G glycoprotein gene of human respiratory syncytial virus in Salmonella typhimurium`.
Binz Hans
Ngoc Thien Nguyen
Nygren Per Ake
Stahl Stefan
Uhlen Mathias
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