Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Virus or component thereof
Reexamination Certificate
2000-09-01
2002-06-25
Salimi, Ali R. (Department: 1648)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Virus or component thereof
C424S211100, C530S350000, C530S300000, C536S023720
Reexamination Certificate
active
06410030
ABSTRACT:
The present invention relates to polypeptides which can be used especially in the preparation of immunogens and the obtainment of vaccine against respiratory syncytial virus (RSV) and to nucleotide sequences enabling them to be obtained. The invention likewise relates to an immune adjuvant protein extracted from
Klebsiella pneumoniae
, to compositions comprising the immunogenic polypeptides, possibly associated with such an adjuvant protein, and to their preparation process.
Respiratory syncytial virus (RSV) is the most frequent cause of respiratory illnesses in the newborn: bronchopneumopathies (bronchiolites). The WHO estimates each year 50 million cases of RSV attacks, from which 160,000 die in the entire world. There are two subgroups of the virus (subgroups A and B).
RSV is classified in the Paramyxoviridae family, a type of pneumovirus comprising a nonsegmented RNA genome, of negative polarity, coding for 10 specific proteins.
There is at present no vaccine available against RSV. Inactivated virus vaccines have been shown to be inefficaceous and have sometimes even aggravated the infections of nursing infants. In the 60's, vaccination attempts with formalin-inactivated RSV resulted in failure: instead of conferring protection at the time of reinfection due to RSV, the vaccine had the effect of aggravating the illness in the child.
The Application WO 87/04185 proposed to use structural proteins of RSV with a view to a vaccine, such as the envelope proteins called protein F (fusion protein) or protein G, a 22 Kd glycoprotein, a 9.5 Kd protein, or the major capsid protein (protein N).
The Application WO 89/02935 describes the protective properties of the entire protein F of RSV, possibly modified in monomeric or deacetylated form.
A series of fragments of protein F have been cloned with a view to investigating their neutralizing properties.
However, the immune vaccines tested to date have been shown to be inefficaceous or have induced a pulmonary pathology (bronchiolitis or peribronchitis).
At the present time, there is no in-depth treatment of infections due to RSV.
Infections due to RSV of the upper airways: treatment relies essentially on symptomatic medications identical to those for other viral infections.
Infections due to RSV of the lower airways: treatment in nursing infants relies on the maintenance of correct hydration, the aspiration of the 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.
It is for these reasons that an object of the present invention is a polypeptide which is useful especially in immunogen production, characterized in that it is carried by the peptide sequence between the amino acid residues 130 and 230 of the sequence of respiratory syncytial virus protein G, or by a sequence having at least 80% homology with said peptide sequence. This sequence differs slightly for the subgroups A and B of human RSV, or for bovine RSV. The invention comprises the sequences originating from human RSV subgroup A and B, or bovine RSV.
Protein G is an RSV envelope glycoprotein, of molecular weight of between 84 and 90 Kd, which is low in methionine.
The Applicant has demonstrated that the sequence between amino acids 130 and 230 of natural protein G is particularly appropriate for inducing an efficaceous protection against infection by RSV. The invention comprises the sequences originating from human RSV subgroup A or B, or bovine RSV.
More particularly, the present invention relates to polypeptides, which are useful especially as immunogenic element included in the above and which comprise the peptide sequence between the amino acid residues numbered 174 and 187 of RSV protein G (human, subgroups A and B, or bovine) or a sequence having at least 80% homology with the corresponding sequence.
Other peptide sequences adapted to the preparation of an immunogen included in said sequence of RSV protein G are formed by the sequence between the amino acid residues numbered 171 and 187 of human or bovine RSV protein G, or a sequence having at least 80% homology with the corresponding sequence. Other peptides of interest according to the present invention are carried by the sequence between the nucleotides numbered 158 and 190 of RSV protein G or a sequence having at least 80% homology with the corresponding sequence.
According to another method of carrying it out, the invention relates to peptides useful for the preparation of an immunogen and which have a sequence corresponding to the sequence between the amino acid residues numbered 140 and 200 of human or bovine RSV protein G, or a sequence having at least 80% homology with the corresponding sequence. Sequences starting with amino acid 140 of said RSV protein G and whose C-terminal end corresponds respectively to the amino acid 198, 196, 194, 192 or 190, as well as sequences having at least 80% homology with the sequence carried by these fragments, are particularly advantageous.
Among the variants of the above sequences, polypeptides may be mentioned which comprise a sequence in which:
a) the Cys amino acid in positions 173 and/or 186 has been replaced by an amino acid not forming a disulfide bridge, in particular serine, and/or
b) the amino acids in positions 176 and 182 are capable of forming a covalent bridge other than a disulfide bridge, especially aspartic acid and ornithine.
Thus the polypeptide sequence 130-230 of RSV subgroup A can be used complete, in its native form. This sequence corresponds to the written sequence Seq id No. 1 (or G2A).
In the same way, it is possible to use the complete polypeptide sequence 130-230 of RSV subgroup B in its native form. This sequence corresponds to the written sequence Seq id No. 2 (G2B).
The sequence id No. 1 will be written G2A in the remainder of the application.
The sequence id No. 2 will be written G2B in the remainder of the application.
Sequences having at least 80% homology with G2A or G2B are also appropriate.
The sequence between the amino acids 130 and 230 can be modified by the replacement of the cysteine residue in positions 173 and 186 by serine residues to obtain a peptide retaining good immunogenic properties, owing to maintenance of the loop formed by the Cys residues in positions 176 and 182. The amino acid and nucleotide sequences of this polypeptide for subgroup A are represented in seq id No. 3 (G2A&dgr;Cys).
For subgroup B, the amino acid and nucleotide sequences are represented in seq id No. 4 (G2B&dgr;Cys).
The peptide sequences will be written G2A&dgr;Cys and G2B&dgr;Cys.
According to another aspect, an object of the invention is a polypeptide which is useful for the preparation of immunogen, characterized in that it consists in the peptide sequence between the amino acid residues numbered 174 and 187 of RSV protein G or a sequence having at least 80% homology with said peptide sequence.
In this last sequence the peptide 174-187 subgroup A can have the sequence:
Seq id No. 5:
Ser Ile Cys Ser Asn Asn Pro Thr Cys Trp Ala Ile Cys Lys.
The peptide 174-187 subgroup B can have the sequence:
Seq id No. 6:
Ser-Ile-Cys-Gly-Asn-Asn-Gln-Leu-Cys-Lys-Ser-Ile-Cys-Lys.
The Cys residue in position 186 can also be replaced by a serine residue, so as to obtain the following sequence:
Seq id No. 7 for subgroup A:
Ser Ile Cys Ser Asn Asn Pro Thr Cys Trp Ala Ile Ser Lys.
Seq id No. 8 for subgroup B:
Ser-Ile-Cys-Gly-Asn-Asn-Gln-Leu-Cys-Lys-Ser-Ile-Ser-Lys.
In the sequence between residues 174 and 187 of the immunogenic peptide, according to one of the variants of the invention, the amino acid residues in positions 176 and 182 are respectively replaced by an aspartic acid and an ornithine, so as to obtain one of the following sequences:
Seq id No. 9 for subgroup A:
Ser Ile Asp Ser Asn Asn Pro Thr Orn Trp Ala Ile Cys Lys
Seq id No. 10 for subgroup B
Ser-Ile-Asp-Gly-Asn-Asn-Gln-Leu-Orn-Lys-Ser-Ile-Cys-Lys.
Seq id No. 11 for subgroup A:
Ser Ile Asp Ser Asn Asn Pro Thr Orn Trp Ala Ile Ser Lys.
Seq id No. 12 for subgroup B:
Ser-Ile-Asp-Gly-Asn-Asn-Gln-Leu-Orn
Baussant Thierry
Binz Hans
N'Guyen Ngoc Thien
Trudel Michel
Pierre Fabre Medicament
Salimi Ali R.
The Firm of Hueschen and Sage
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