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
1999-04-06
2003-01-07
Minnifield, Nita (Department: 1645)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Amino acid sequence disclosed in whole or in part; or...
C424S184100, C424S234100, C424S237100, C424S244100, C530S300000, C530S350000, C514S002600, C514S008100
Reexamination Certificate
active
06503511
ABSTRACT:
This invention relates generally to the field of bacterial antigens and their use, for example, as immunogenic agents in humans and animals to stimulate an immune response. More specifically, it relates to the vaccination of mammalian species with a polypeptide comprising an alpha helix-forming polypeptide obtained from a choline binding polypeptide as a mechanism for stimulating production of antibodies that protect the vaccine recipient against infection by pathogenic bacterial species. Further, the invention relates to antibodies and antagonists against such polypeptides useful in diagnosis and passive immune therapy with respect to diagnosing and treating such pneumococcal infections.
In a particular aspect, the present invention relates to the prevention and treatment of pneumonococcal infections such as infections of the middle ear, nasopharynx, lung and bronchial areas, blood, CSF, and the like, that are caused by pneumonococcal bacteria. In this regard, certain types of
Streptococcus pneumoniae
are of particular interest.
S. pneumoniae
is a gram positive bacteria which is a major causative agent in invasive infections in animals and humans, such as sepsis, meningitis, otitis media and lobar pneumonia (Tuomanen, et al. NEJM 322:1280-1284 (1995)). As part of the infective process, pneumococci readily bind to non-inflamed human epithelial cells of the upper and lower respiratory tract by binding to eukaryotic carbohydrates in a lectin-like manner (Cundell et al., Micro. Path. 17:361-374 (1994)). Conversion to invasive pneumococcal infections for bound bacteria may involve the local generation of inflammatory factors which may activate the epithelial cells to change the number and type of receptors on their surface (Cundell, et al., Nature, 377:435-438 (1995)). Apparently, one such receptor, platelet activating factor (PAF) is engaged by the pneumococcal bacteria and within a very short period of time (minutes) from the appearance of PAF, pneumococci exhibit strongly enhanced adherence and invasion of tissue. Certain soluble receptor analogs have been shown to prevent the progression of pneumococcal infections (Idanpaan-Heikkila et al., J. Inf. Dis., 176:704-712 (1997)).
A family of choline binding proteins (CBPs), which are non-covalently bound to phosphorylcholine, are present on the surface of pneumococci and have a non-covalent association with teichoic acid or lipoteichoic acid. An example of such family is choline binding protein A (CbpA), an approximately 75 kD weight type of CBP which includes a unique N-terminal domain, a proline rich region, and a C-terminal domain comprised of multiple 20 amino acid repeats responsible for binding to choline. A segment of the N-terminal portion of CbpA protein forms an alpha helix as part of its three-dimensional structure.
Accordingly, it is an object of the present invention to provide a polypeptide having broad protection against pneumococcal infections.
DEFINITIONS
In order to facilitate understanding of the description below and the examples which follow certain frequently occurring methods and/or terms will be described.
“Plasmids” are designated by a lower case p preceded and/or followed by capital letters and/or numbers. The starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accord with published procedures. In addition, equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan.
“Digestion” of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA. The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements were used as would be known to the ordinarily skilled artisan. For analytical purposes, typically 1 &mgr;g of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 &mgr;l of buffer solution. For the purpose of isolating DNA fragments for plasmid construction, typically 5 to 50 &mgr;g of DNA are digested with 20 to 250 units of enzyme in a larger volume. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer. Incubation times of about 1 hour at 37° C. are ordinarily used, but may vary in accordance with the supplier's instructions. After digestion the reaction is electrophoresed directly on a polyacrylamide gel to isolate the desired fragment.
Size separation of the cleaved fragments is performed using 8 percent polyacrylamide gel described by Goeddel, D. et al., Nucleic Acids Res., 8:4057 (1980).
“Oligonucleotides” refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthesized. Such synthetic oligonucleotides have no 5′ phosphate and thus will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.
“Ligation” refers to the process of forming phosphodiester bonds between two double stranded nucleic acid fragments (Maniatis, T., et al., Id., p. 146). Unless otherwise provided, ligation may be accomplished using known buffers and conditions with 10 units to T4 DNA ligase (“ligase”) per 0.5 &mgr;g of approximately equimolar amounts of the DNA fragments to be ligated.
“HPS portion” as used herein refers to an amino acid sequence as set forth in SEQ ID NO:2 for a choline binding protein (“CBP”) of a pneumococcal bacteria that may be located amino terminal with respect to the proline rich portion of the overall amino acid sequence for such CBP.
The terms “identity”, “% identity” or “percent identity” as utilized in this application refer to a calculation of differences between two contiguous sequences which have been aligned for “best fit” (to provide the largest number of aligned identical corresponding sequence elements, wherein elements are either nucleotides or amino acids) and all individual differences are considered as individual difference with respect to the identity. In this respect, all individual element gaps (caused by insertions and deletions with respect to an initial sequence (“reference sequence”)) over the length of the reference sequence and individual substitutions of different elements (for individual elements of the reference sequence) are considered as individual differences in calculating the total number of differences between two sequences. Individual differences may be compared between two sequences where an initial sequences (reference sequence) has been varied to obtain a variant sequence (comparative sequence) or where a new sequence (comparative sequence) is simply aligned and compared to such a reference sequence. When two aligned sequences are compared all of the individual gaps in BOTH sequences that are caused by the “best fit” alignment over the length of the reference sequence are considered individual differences for the purposes of identity. If an alignment exists which satisfies the stated minimum identity, then a sequence has the stated minimum identity to the reference sequence. For example, the following is a hypothetical comparison of two sequences having 100 elements each that are aligned for best fit wherein one sequence is regarded as the “reference sequence” and the other as the comparative sequence. All of the individual alignment gaps in both sequences are counted over the length of the reference sequence and added to the number of individual element substitution changes (aligned elements that are different) of the comparative sequence for the total number of element differences. The total number of differences (for example 7 gaps and 3 substitutions) is divided by the total number of elements in the length of the reference sequence (100 elements) for the “percentage difference” (10/100). The resulting percentage difference (10%) is subtracted from 100% identity to provide a “% ident
Johnson Leslie S.
Koenig Scott
Wizemann Theresa M.
Grant Alan J.
MedImmune, Inc.
Minnifield Nita
Olstein Elliot M.
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