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
2002-12-17
Smith, Lynette R. F. (Department: 1615)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Amino acid sequence disclosed in whole or in part; or...
C424S184100, C424S185100, C424S244100, C530S300000, C530S324000, C530S350000
Reexamination Certificate
active
06495139
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to choline binding polypeptides and to nucleic acids encoding such polypeptides. The invention also relates to vaccines which provide protection or elicit protective antibodies to bacterial infection, and to antibodies and antagonists against or inhibitors of such polypeptides for use in diagnosis, therapy and passive immune therapy. In particular, the choline binding polypeptides of the invention are useful as vaccines against Streptococcus, particularly pneumococcus. A choline binding polypeptide of the present invention is also useful as a competitive inhibitor of bacterial adhesion, or to discover small molecule antagonists of adhesion.
BACKGROUND OF THE INVENTION
Streptococcus pneumoniae
is a gram positive bacteria which is a major cause of invasive infections such as sepsis, meningitis, otitis media and lobar pneumonia (Tuomanen et al
NEJM
322:1280-1284, 1995). Vaccination has long been an important armament in the arsenal against infectious microorganisms. Prior to the introduction of antibiotics, vaccination was the major hope for protecting populations against viral or bacterial infection. With the advent of antibiotics, vaccination against bacterial infections became less important. However, the emerging problems withantibiotic resistance among such infectious bacteria, including
S. pneumoniae
strains, have raised an urgent need for a better understanding of the pathogenesis of these pathogens and has reestablished the importance of anti-bacterial vaccines (Appelbaum, P.C. (1992)
Clin Infect Dis
15:77-83).
There are over 90 different types of the pneumococcal organism, each with a different chemical structure of the capsular polysaccharide (i.e., each antigenically distinct). The capsular polysaccharide is a principal known virulence factor of the pneumococcus and induces an antibody response in adults. Current vaccines against
S. pneumoniae
employ mixtures of the capsules of the 23 most common serotypes of this bacterium. After several comprehensive studies there is now overwhelming evidence that this vaccine is approximately 60% efficacious for the general population (Shapiro et al.(1991)
NEJM
325:1453-60). Importantly, these vaccines are ineffective in individuals most susceptible to pathological infection—the young, the old, and the immune compromised—because of their inability to elicit a T cell immune response. Conjugation of the capsule to a protein permits protection in the younger age group but is inherently limited in the number of capsules that can be conjugated at one time (approximately 5-8 capsules only).
Exported proteins in bacteria participate in many diverse and essential cell functions such as motility, signal transduction, macromolecular transport and assembly, and the acquisition of essential nutrients. For pathogenic bacteria such as
S. pneumoniae
, many exported proteins are virulence determinants that function as adhesins to colonize and thus infect the host, or as toxins to protect the bacteria against the host's immune system (for a review, see Hoepelman and Tuomanen (1992)
Infect Immun
60:1729-33). One alternative to current vaccines are subunit vaccines in which the antigen, or antigens, include a bacterial surface protein or proteins. These vaccines could overcome the deficiencies of whole bacterial or capsule-based vaccines. Moreover, given the importance of exported or surface proteins to bacterial virulence, these proteins are an important target for therapeutic intervention.
Pneumococci bind avidly to cells of the upper and lower respiratory tract. Like most bacteria, adherence of pneumococci to human cells is achieved by presentation of bacterial surface proteins that bind to eukaryotic cell surface carbohydrates in a lectin-like fashion (Cundell, D. & Tuomanen, E. (1994)
Microb Pathog
17:361-374). Pneumococci bind to non-inflamed epithelium, a process that can be viewed as asymptomatic carriage. It has been proposed that the conversion to invasive disease involves the local generation of inflammatory factors which, activating the human epithelial cell, change the number and type of receptors available on the human cells (Cundell, D. et al. (1995)
Nature,
377:435-438). Presented with an opportunity in this new setting, pneumococci appear to take advantage and engage one of these unregulated receptors, the platelet activating factor (PAF) receptor (Cundell et al. (1995)
Nature,
377:435-438). Within minutes of the appearance of the PAF receptor, pneumococci undergo waves of enhanced adherence and invasion. Inhibition of bacterial binding to activated cells, for instance by soluble receptor analogs, blocks the progression to disease in animal models (Idanpaan-Heikkila, I. et al. (1997)
J. Infect. Dis.,
176:704-712). Particularly effective in this regard are soluble carbohydrates containing lacto-N-neotetraose with or without an additional sialic acid, which prevent pneumococcal attachment to human cells in vitro and prevent colonization in the lung in vivo.
Pneumococci display a family of surface proteins which bind to the bacterial surface by non-covalent association to the cell wall teichoic acid or lipoteichoic acid, specifically through its terminal component phosphorylcholine. The surface of
Streptococcus pneumoniae
is decorated with twelve types of these choline binding proteins (Cbps). The Cbps are represented by a family of molecules which decorate the surface of pneumococcus, each serving a unique function but bound to the surface by a common element. These proteins consist of an N-terminal activity domain and a repeated C-terminal signature choline binding domain that contains two to greater than ten repeats of a 20 amino acid choline binding sequence that binds to phosphoryl choline and that anchors these molecules to the surface of the bacteria. This motif has been identified in the C-terminal regions of a secreted glycoprotein from
Clostridium acetobutylicum
NCIB 88052 [Sanchez-Beato, et al.
, J. Bacteriol.
177:1098-1103 (1995)], toxins A and B from
Clostridium difficile
[Von Eichel-Streiber and Sauerbom
, Gene
96:107-13 (1990); Von Eichel-Streiber et al.,
J. Bacteriol.
174:6707-6710 (1992)], a glucan-binding protein from
Streptococcus mutans
, several glycosyltransferases from
Streptococcus downei
and
S. mutans
, the murein hydrolase (LytA) from pneumococcus and pneumococcal lytic phage [Ronda et al.
, Eur. J. Biochem.
164:621-4 (1987); Diaz et al.
, J. Bacteriol.
174:5516-25 (1992); Romero et al.
, Microb. Lett.
108:87-92 (1993); Yother and White
, J. Bacteriol.
176:2976-85 (1994)], and a surface antigen (PspA) also from pneumococcus. The known pneumococcal Cbp family members are CbpA, LytA and PspA.
The choline binding domain was recognized and fully characterized by Lopez et al. in his studies of the autolytic enzyme, LytA (Ronda et al. (1987)
Eur. J. Biochem,
164:621-624). From studies of the sequences of the recognized choline binding proteins, consensus sequences have been reported, most particularly that of Garcia et al: GWLKDNGSWYYLNANGAMAT (SEQ ID NO:26) (Garcia, P. et al (1990)
Gene
86:81-88; Wren B. et al (1991)
Mol Microbiol
5:797-803); Sanchez-Beato, A. R. et al (1995) J Bacteriol 177:1098-1103).
Teichoic acid (TA), an integral part of the cell wall of
Streptococcus pneumoniae
, contains many terminal phosphorylcholine moieties. Choline affinity chromatography or Mono-Q Sepharose, a close relative of choline, were used to purify the CBPs. Previous studies have shown that PspA, as well as one other surface exposed protein, LytA, the autolytic amidase, bind in a choline-dependent manner. PspA, a protein having a molecular weight of 84 kDa, and which is highly variable, is released from the cell surface with high choline concentration (at least about 2% to about 10%). The function of PspA is unknown. LytA, or autolysin, is a 36 kDa protein, which lyses the pneumococcal cell wall (self lysis), but is not released from the cell by growth in high concentrations of choline,
Gosink Khoosheh
Masure Robert
Tuomanen Elaine I.
Alston & Bird LLP
Baskar Padma
Smith Lynette R. F.
St. Jude Children's research Hospital
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