Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
1998-05-11
2001-09-18
Graser, Jennifer E. (Department: 1645)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C424S130100, C424S134100, C424S135100, C424S136100, C424S137100, C424S139100, C424S141100, C424S150100, C424S164100, C424S169100, C530S388100, C435S069100, C435S070210, C435S069300, C435S243000
Reexamination Certificate
active
06291649
ABSTRACT:
The present invention relates, inter alia, to an antigen useful, e.g., for the immunization of a mammal, an antibody raised against it, and a vaccine produced from it.
Antigens composed of several proteins which together form a distinct phenotype in a pathogenic bacterial strain or species, and which must therefore be assumed to contain a large number of immunogenic determinants, are well known. However, such antigens—and vaccines produced from them—have a number of disadvantages: in particular, they have a tendency to be too selective in that, on immunization, antibodies are formed against each of these immunogenic determinants which together identify the one particular bacterial strain from which the antigen has been derived, but not other bacterial strains of the same species so that immunization is only effected against this particular strain, but not other, closely related strains of the same species.
The present invention is an attempt to overcome these disadvantages by providing an antigen which substantially only comprises the immunogenic determinant(s) which lead to the desired immunity and which is furthermore not limited to one particular strain of the pathogenic bacteria in question.
It has become increasingly clear that the capacity of many pathogenic bacteria to adhere to the surface of cells is of primary importance for the initiation of many infectious diseases (Beachey,
J. Infect. Dis.
143, 1981, pp. 325-345). This adhesion capacity is caused by the presence of receptors on mammalian tissue cells, such as epithelial cells, or on mammalian erythrocytes, which receptors, due to their configuration, form bonds with adhesin polypeptides. (In the present context, the term “adhesin polypeptide” is intended to indicate both a polypeptide specifically required for the adhesion phenotype and, more generally, a polypeptide in whose absence adhesion does not take place (for whatever reason).) Each receptor is assumed to bond with a different adhesin structure. The receptor may be a peptide receptor, such as an amino acid present on a sugar, or—more usually—a carbohydrate such as neuramic acid-(2→3)-galactose, mannose-&agr;-(1→2)-mannose or digalactoside (the &agr;-D-Gal&rgr;-(1→4)-&bgr;-D-Gal&rgr; moiety present in the globoseries of glycolipids which in the present context is occasionally termed the globoside).
In many pathogenic bacteria, the adhesin polypeptide proper is believed to form only part of a larger sequence of polypeptides which are all, in one way or another, related to the adhesion function (e.g. polypeptides which mediate the transport of the adhesin through the cell wall or anchor it to the outer surface of the cell wall and so on), and in accordance with the aim of the present invention, a specific adhesin polypeptide is identified among the other polypeptides of the sequence and used as an antigen. This is thought to constitute a less selective identification marker so that antibodies will not only be raised against the strain from which the antigen is derived but also against the other pathogenic strains of the same bacterial species.
Accordingly, the present invention relates to an antigen which, as its major immunizing component, comprises a determinant of an adhesin polypeptide or an immunogenically active subsequence thereof or a precursor therefor which is convertible to an immunologically active form, antibodies against which determinant react with the adhesin polypeptide produced by pathogenic adhesin-forming bacteria which adhere to mammalian tissue. This antigen may comprise an amino acid sequence of at least 5 amino acids and up to the entire amino acid sequence of the adhesin polypeptide.
The adhesin polypeptide may conveniently be derived from adhesinforming bacteria. This group of bacteria comprises both grampositive and gramnegative bacteria, and the bacterial species of the greatest interest in the present context from which it would be advantageous to derive one or more specific adhesin polypeptides are uropathogenic or enteropathogenic strains of
Escherichia coli
or other enteric bacteria or oral bacteria,
Neisseria gonorrhoeoe, Neisseria meningiditis, Neisseria catarrhalis,
Yersinia spp.,
Pseudomonas aeruginosa
or other Pseudomonas spp.,
Moraxella bovis
or other Moraxella spp.,
Bacteroides nodosus,
Staphylococcus spp., Streptococcus spp. or Bordetella spp. such as
Bordetella pertussis.
Alternatively, the adhesin polypeptide may be prepared synthetically, as described below.
For some pathogenic bacteria in this group, there is evidence that filamentous structures termed pili (fimbriae) projecting from the cell wall are in some way connected with adhesion, and therefore—and because the pili are easily purified—whole pili preparations have been used as antigens in vaccines, e.g. gonococcus pili antigen (tested in US Army field tests).
Previous investigators working with pill preparations sent to great lengths in attempts to prepare “pure” pili protein for protein characterization and immunization, and their efforts were apparently successful in that their preparations only showed one band in SDS gels, (U.S. Pat. No. 4,443,431 (Buchanan T. M. et al); Saliet I. E. & E. C., Gottlisch,
J. Exp. Med.
146, 1977, p. 1169; Klemm P., J. Orskov & F. Orskov,
Infect. and Immunity
36, 1982, p. 462, Schoolnik G. K. et al,
J. Exp. Med.
159, 1984, p. 1351; Svanborg E. C.,
Prog. Allergy
33, 1983 p. 189). The obtained pili protein preparations exhibited at least three functions. The first function was the ability to form polymers, presumably through hydrophobic binding processes; a properly essential for the formation of a pilus filament from monomeric subunits. The second property was the ability to engender antibodies; a property which would be essential for any attempts to use the protein as a vaccine. The third property was the ability to adhere to cell surface receptors. Since the investigators were not able to identify more than one protein in their pili protein preparations as well as in the pili themselves, it was concluded that the pili were polymeric aggregates of identical monomeric protein subunits, each subunit having all three of the above-described functions, (U.S. Pat. No. 4,443.431 (Buchanan T. M. et al); Rothbard J. B.,
PNAS
82, 1985, p. 915). However, as mentioned previously, the intact whole pili from a single species has great antigenic diversity. In addition, it has been demonstrated that when used as a vaccine, intact whole pili of a single antigenic type produce antibody primarily to that single antigenic type rather than shared pili antigens. Previous investigators have chemically cleaved the purified pilus subunit into fragments with the proposed functions: Polymerization function, common antigen function, and binding function. Each individual function was identified with a separate fragment of the purified pilus subunit. Thus, it has been assumed that purified pili protein preparations contain a single protein—the pilin monomer. This pilin monomer has been chemically cleaved and assumed to contain the binding function and the principal antigenisity—the same as the polymerized pure pilus protein.
Extensive studies carried out by the applicants demonstrate, however, that the supposed pure pili protein in fact consists of several protein fractions with separate functions. In fact, the pilus filament is not responsible for the cell surface binding but a minor component considered to be a contaminant which is most likely associated with the filament is what is responsible for the cell surface binding. This unique observation could only be ascribed to the fact that the structural formation of pili and the property of adhesion to digalactoside receptors can be genetically dissociated. Other mutated organisms retaining recognizable pili structures but being unable to adhere further confirmed the observation. The implication of the observation was further that the pilus protein, previously supposed to be pure, must contain at least two fractions, one of which is a structural element involved in th
B{dot over (a)}ga Britt Monika
Goransson Mikael
Lark David Lee
Lindberg Frederik Carl
Lund Bjorn Olof
Cooper Iver P.
Graser Jennifer E.
Symbicom Aktiebolag
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