Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Separation or purification
Reexamination Certificate
1999-05-12
2001-09-18
Wortman, Donna C. (Department: 1645)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Separation or purification
C530S412000, C530S422000, C530S424000
Reexamination Certificate
active
06291654
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to
Streptococcus pneumoniae
and in particular this invention relates to the identification of an
S. pneumoniae
protein that is implicated in
S. pneumoniae
virulence and is capable of binding the complement protein, C3.
BACKGROUND OF THE INVENTION
Respiratory infection with the bacterium
Streptococcus pneumoniae
(
S. pneumoniae
) leads to an estimated 500,000 cases of pneumonia idand 47,000 deaths armually. Those persons at highest risk of bacteremic pneumococcal infection are infants under two years of age and therelderly. In these populations,
S. pneumoniae
is the leading cause of bacterial pneumonia and meningitis. Moreover,
S. pneumoniae
is the major bacterial cause of ear infections in children of all ages. Both children and the elderly share defects in the synthesis of protective antibodies to pneumococcal capsular polysaccharide after either bacterial colonization, local or systemic infection, or vaccination with purified polysaccharides.
S. pneumoniae
is the leading cause of invasive bacterial respiratory disease in both adults and children with HIV infection and produces hematogenous infection in these patients (Connor et al.
Current Topics in AIDS
1987;1:185-209 and Janoffet al.
Ann. Intern. Med
. 1992;1 17(4):314-324).
Individuals who demonstrate the greatest risk for severe infection are not able to make antibodies to the current capsular polysaccharide vaccines. As a result, there are now four conjugate vaccines in clinical trial. Conjugate vaccines consist of pneumococcal capsular polysaccharides coupled to protein carriers or adjuvants in an attempt to boost the antibody response. However, there are other potential problems with conjugate vaccines currently in clinical trials. For example, pneumococcal serotypes that are most prevalent in the United States are different from the serotypes that are most common in places such as Israel, Western Europe, or Scandinavia. Therefore, vaccines that may be useful in one geographic locale may not be useful in another. The potential need to modify currently available capsular polysaccharide vaccines or to develop protein conjugates for capsular vaccines to suit geographic serotype variability entails prohibitive financial and technical complications. Thus, the search for immunogenic, surface-exposed proteins that are conserved worldwide among a variety of virulent serotypes is of prime importance to the prevention of pneumococcal infection and to the formulation of broadly protective pneumococcal vaccines. Moreover, the emergence of penicillin and cephalosporin-resistant pneumococci on a worldwide basis makes the need for effective vaccines even more exigent (Baquero et al.
J. Antimicrob. Chemother
. 1991;28S;31-8).
Several pneumococcal proteins have been proposed for conjugation to pneumococcal capsular polysaccharide or as single immunogens to stimulate immunity against
S. pneumoniae
. Surface proteins that are reported to be involved in adhesion of
S. pneumoniae
to epithelial cells of the respiratory tract include PsaA, PspC/CBP112, and IgA1 proteinase (Sampson et al.
Infect. Immun
. 1994;62:319-324, Sheffield et al.
Microb. Pathogen
. 1992; 13: 261-9, and Wani. et al.
Infect. Immun
. 1996; 64:3967-3974). Antibodies to these adhesins could inhibit binding of pneumococci to respiratory epithelial cells and thereby reduce colonization. Other cytosolic pneumococcal proteins such as pneumolysin, autolysin, neuraninidase, or hyaluronidase are proposed as vaccine antigens because antibodies could potentially block the toxic effects of these proteins in patients infected with
S. pneumoniae
. However, these proteins are typically not located on the surface of
S. pneumoniae
, rather they are secreted or released from the bacterium as the cells lyse and die (Lee et al.
Vaccine
1994; 12:875-8 and Berry et al.
Infect Immun
. 1994; 62:1101-1108). While use of these cytosolic proteins as immunogens might ameliorate late consequences of
S. pneumoniae
infection, antibodies to these proteins would neither promote pneumococcal death nor prevent pneumococcal colonization.
A prototypic surface protein that is being tested as a pneumococcal vaccine is the pneumococcal surface protein A (PspA). PspA is a heterogeneous protein of about 70-140 kDa. The PspA structure includes an alpha helix at the amino terminus, a proline-rich sequence in the mid-portion of the protein. and terminates in a series of choline-binding repeats at the carboxy-terminus. Although much information regarding its structure is available PspA is not structurally conserved among a variety of pneumococcal serotypes, and its fanction is entirely unknown (Yother et al.
J. Bacteriol
. 1992;1 74:601-9 and Yother
J. Bacteriol
. 1994; 176:2976-2985). Studies have confirmed the immunogenicity of PspA in animals (McDaniel et al.
Microb. Pathogen
. 1994; 17:323-337). Despite the immunogenicity of PspA, the heterogeneity of PspA, its existence in four structural groups (or clades), and its uncharacterized function complicate its ability to be used as a vaccine antigen.
In patients who cannot make protective antibodies to the type-specific polysaccharide capsule, the third component of complement, C3, and the associated proteins of the alternative complement pathway constitute the first line of host defense against
S. pneumoniae
infection. Because complement proteins cannot penetrate the rigid cell wall of
S. pneumoniae
, deposition of opsonic C3b on the pneumococcal surface is the principal mediator of pneumococcal clearance. Interactions of pneumococci with plasma C3 are known to occur during pneurnococcal bacteremia, when the covalent binding of C3b, the opsonically active fragment of C3, initiates phagocytic recognition and ingestion (Johnston et al.
J. Exp. Med
1969;129:1275-1290, Hasin HE,
J. Immunol
. 1972; 109:26-31 and Hostetter et al.
J. Infect. Dis
. 1984; 150:653-61). C3b deposits on the pneumococcal capsule, as well as on the cell wall. This method for controlling
S. pneumoniae
infection is fairly inefficient and could be beneficially amplified by the presence of antibodies to surface components of
S. pneumoniae
. There currently exists a strong need for methods and therapies to limit
S. pneumoniae
infection.
SUMMARY OF THE INVNETION
The present invention relates to the identification and purification of an about 90 kDa to about 110 kDa (±5 kDa) protein, as determined following electrophoresis on a 15% SDS-PAGE gel. The protein is named PbcA and is sisolatable from
S. pneumoniae
strains that are capable of binding to human complement protein C3. The protein, PbcA, comprises an amino terminus containing region comprising SEQ ID NO:1 and is capable of binding but not cleaving or degrading the human complement protein C3. The protein also comprises a proline rich region and in one embodiment is a surface exposed protein of
S. pneumoniae.
This invention also relates to the production of antibodies specifically recognizing PbcA. In one embodiment the antibodies are polyclonal and in another embodiment the antibodies are monoclonal. The antibodies can be produced by immunizing a mammal with all or a portion of PbcA. In one embodiment, the monoclonal antibodies are rodent derived.
In another aspect of this invention a method is provided for generating an immune response to
S. pneumoniae
in vivo comprising the steps of: administering a protein or an immunogenic fragment of a protein from
S. pneumoniae
to an animal wherein the amino terminus containing region of the protein comprises SEQ ID NO:1. In one embodiment, the protein is capable of binding but not cleaving or degrading the human complement protein C3. Preferably, the method further comprises detecting an immune response to
S. pneumoniae
in the mammal. Preferably, the immune response comprises the production of antibodies to
S. pneumoniae
. The animal can be a mouse, rat, chinchilla, a rabbit or a human and the method can further comprise the steps of isolating antibody producing cells from the mammal and preparin
Cheng Qi
Hostetter Margaret K.
Mueting Raasch & Gebhardt, P.A.
Regents of the University of Minnesota
Wortman Donna C.
Zeman Robert A.
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