Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-11-05
2003-07-01
Smith, Lynette R. F. (Department: 1645)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C514S04400A, C435S006120
Reexamination Certificate
active
06586580
ABSTRACT:
This invention relates to a novel protein designated Rib (and subfragments, variants and multiples thereof) which confers immunity to most invasive strains of the group B Streptococcus, DNA sequences encoding the protein or functional fragments or domains of the protein, DNA sequences which hybridize under stringent conditions to the DNA encoding the protein, a procedure for purification of the protein, antibodies specific to the protein, a reagent kit and a pharmaceutical composition comprising the protein or fragments thereof.
During the last three decades, the group B Streptococcus has emerged as a major cause of neonatal disease in the Western world. In the United States alone, there are about 10,000 cases per year of invasive disease caused by this bacterium. These infections have an overall mortality of about 20%, and many of the infants that survive have permanent neurological sequelae. In view of these findings, a large effort has been made to find methods of prevention and treatment and to analyze the mechanisms by which group B streptococci cause infections.
About 20% of all women are vaginal carriers of the group B Streptococcus, and vertical transmission from the maternal genital tract is probably the most common source of infection in neonatal disease caused by this bacterium. However, only 1 to 2% of the infants that are colonized by the group B Streptococcus at birth are afflicted by serious infection. Other factors than exposure to the bacterium during birth must therefore contribute to the development of neonatal disease. Mothers of infected infants have significantly lower levels of antibodies to the type III capsule, which implies that these antibodies are important for protection against neonatal disease (Baker, C. J. and D. L. Kasper, N. Engl. J. Med. 1976, 294:753).
Group B streptococcal strains are divided into four major serotypes (Ia, Ib, II, and III) based on the structure of the polysaccharide capsule (Baker, J Inf Dis 1990. 161: 917). Serotypes I, II, and III occur in roughly equal proportions among strains in the normal flora, but type III accounts for about two-thirds of all isolates from invasive infections. Since the capsule is a known virulence factor, it has been studied in considerable detail, in particular in type III strains. Efforts have been made to develop a vaccine, in which the type III polysaccharide capsule would be an essential component. However, use of the polysaccharide capsule as a vaccine may give problems due to crossreactions with human tissues (Pritchard et al., Infect Immun 1992. 60: 1598). It would therefore be very valuable if one could develop a vaccine based on proteins rather than on polysaccharides.
The group B Streptococcus can also cause mastitis in cows, a bovine disease that is of considerable economical importance. Development of a vaccine against group B streptococcal infections is therefore of interest also in veterinary medicine.
Two group B streptococcal cell surface proteins have previously been studied in detail: the alpha and beta proteins. These proteins confer protective immunity to strains expressing the proteins, but they are of limited interest for group B streptococcal disease, since they are usually not expressed by type III strains, which cause the majority of serious infections.
The present invention relates to a streptococcal cell surface protein, and variants and subfragments thereof. This protein, which is designated protein Rib, was isolated from a group B streptococcal strain of serotype III as a distinct 95 kD protein. Protein Rib is expressed by almost all group B streptococcal strains of serotype III and by a few strains of other serotypes such as II. A method has been devised to purify protein Rib and it has been demonstrated that antibodies to this protein protect against lethal infection with strains expressing protein Rib.
The invention also relates to naturally occurring and artificially modified variants, subfragments and multiples of the Rib protein which have the ability to protect against infections caused by protein Rib expressing bacteria, i.e., especially group B streptococcal strains of serotype III.
The invention also relates to a vector, such as a plasmid, a cosmid or a phage, containing the genetic code for protein Rib and variants, subfragments and fragments thereof, suitable for insertion in a non-human host organism and expression from said host. The invention particularly relates to three phage clones, lambda Rib1-3, lambda Rib1-5 and lambda Rib1-7, having deposit numbers DSM 9039, DSM 9040 and DSM 9041, respectively.
The invention also relates to a DNA sequence encoding protein Rib and variants, subfragments fragments and multiples thereof, that may be inserted in a suitable vector, such as a plasmid, a cosmid or a phage. The DNA sequence can be obtained from the deposited phages lambda Rib1-3, lambda Rib1-5 och lambda Rib1-7.
The Rib protein is expressed by different type III strains. Extracts prepared from several different strains that were analyzed by Western blotting, using anti-Rib serum for the analysis, showed that almost all extracts contained protein Rib, but the molecular mass of the protein varied between 65 and 125 kD (data not shown). This result was not unexpected, since size variation has previously been described also for other group B streptococcal proteins, the alpha and beta proteins.
The available data suggest that the protein may consist of multiples of units, each unit corresponding to a molecular mass of about 9 kD. The invention therefore includes subfragments and multiples of the 95 kD protein or of a basic unit with the same characteristics. Variants include substitution or insertions of amino acids without changing the ability to protect against infections caused by bacterias expressing the protein.
Group B streptococcal strains are well known and may be isolated from the blood of infected human beings. The BM110 strain used by the inventors was obtained from Dr. S. Mattingly (University of Texas, San Antonio, Tex.). All strains referred to herein are obtainable from the inventors at the University of Lund and the Lund University Hospital (Doctor Gunnar Lindahl, Department of Medical Microbiology, Sölvegatan 23, S 22362 Lund, Sweden).
Protein Rib may be isolated from group B streptococcal strains of serotype III, preferably from strain BS30 or BM110. The invention concerns a process for purification of protein Rib.
The protein may be isolated by the following procedure: A Streptococcus Group B strain expressing the protein is cultivated, the medium and/or the microorganism are isolated, the bacteria are digested with an enzyme, preferably mutanolysin, a protease inhibitor is optionally added, the digested bacteria are separated from the supernatant and protein Rib is extracted from the supernatant. The media can be any media suitable for cultivation of streptococci; such as Todd-Hewitt broth (Oxoid) and the cells are preferably cultivated 1-30, especially 12-20 hours. The digestion with an enzyme, preferably mutanolysin, is performed without shaking for 1-30, especially 10-20, preferably 15-18 hours at 20-40° C., preferably 37° C. The protein may be isolated from the medium, and in such a case there is no need for digestion with the enzyme which is used to break the cell walls. A protease inhibitor such as benzamidine chloride, iodoacetic acid or phenylmethyl sulfonyl fluoride is added to prevent the action from proteases which may contaminate the mutanolysin or may be present in the microorganisms.
The protein can be purified by ion exchange chromatography, preferably anion exchange chromatography and gel filtration, and fractions containing the protein collected according to established practice within the art.
The invention especially concerns a substantially pure protein Rib or subfragments thereof. With the expression “substantially pure” we understand a substance that does not contain pharmaceutically harmful substances.
The invention also concerns antibodies corresponding to protein Rib and subfragments, variants or multiples thereof
Lindahl Gunnar
Stalhammar-Carlemalm Margaretha
Stenberg Lars
Birch & Stewart Kolasch & Birch, LLP
Lindahl Gunnar
Portner Ginny Allen
Smith Lynette R. F.
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