RECOMBINANT PROTECTIVE PROTEIN FROM $I(STREPTOCOCCUS PNEUMONIAE)

Description

FIELD OF THE INVENTION

[0001] The present invention provides amino acid sequences and nucleic acid sequences relating to a protein of Streptococcus pneumonia having a molecular weight of 20 kilo Daltons (kDa). The present invention also pertains to compositions for the treatment and prophylaxis of infection or inflammation associated with bacterial infection.

BACKGROUND OF THE INVENTION

[0002] The middle ear is a sterile, air-filled cavity separated from the outer ear by the eardrum. Attached to the eardrum are three ear bones that vibrate when sound waves strike the eardrum. Vibrations are transmitted to the inner ear, which generates nerve impulses that are sent to the brain. Air may enter the middle ear through the Eustachian tube, which opens in the walls of the nasopharynx.

[0003] The nasopharynx is located posterior to the nasal cavities. The nasopharynx is lined by the respiratory epithelium and stratified squamous epithelium. Beneath the respiratory epithelium, the abundant mucosa-associated lymphoid tissue (MALT) forms the nasopharyngeal tonsil (adenoids).

[0004] Bacterial infection or inflammation of the middle ear is mainly observed in children. Due to the isolation of the middle ear, it is suggested that development of middle ear infections requires the involvement of the nasopharynx and Eustachian tube. Infections with Streptococcus pneumoniae (S. pneumoniae) are one of the major causes of middle ear infections, as well as bacteremia, meningitis, and fatal pneumonia worldwide (Butler, J.C., et al., American Journal of Medicine, 1999, 107:69S-76S). The rapid emergence of multi-drug resistant pneumococcal strains throughout the world has led to increased emphasis on prevention of pneumococcal infections by vaccination (Goldstein and Garau, Lancet, 1997, 350:233-4).

[0005] Protein antigens of S. pneumoniae have been evaluated for protective efficacy in animal models of pneumococcal infection. Some of the most commonly studied vaccine candidates include the the PspA proteins, PsaA lipoprotein, and the CbpA protein. Numerous studies have shown that PspA protein is a virulence factor (Crain, M.J., et al., Infect Immun, 1990, 58:3293-9; McDaniel, L.S., et al., J Exp Med,1984, 160:386-97), but is antigenically variable among pneumococcal strains. Additionally, a recent study has indicated that some antigenically conserved regions of a recombinant PspA variant may elicit cross-reactive antibodies in human adults (Nabors, G.S., et al., Vaccine, 2000, 18:1743-1754). PsaA, a 37 kDa lipoprotein with similarity to other Gram-positive adhesins, is involved in manganese transport in pneumococci (Dintilhac, A., et al., Molecular Microbiology, 1997, 25(4):727-739; Sampson, J.S., et al., Infect Immun, 1994, 62:319-24.) and has been shown to be protective in mouse models of systemic disease (Talkington, D.F., et al., Microb Pathog, 1996. 21:17-22). The surface exposed choline binding protein, CbpA, is antigenically conserved and also is protective in mouse models of pneumococcal disease (Rosenow, C., et al. Molecular Microbiology, 1997, 25:819-29). Since nasopharyngeal colonization is a prerequisite for otic disease, intranasal immunization of mice with pneumococcal proteins and appropriate mucosal adjuvants has been used to enhance the mucosal antibody response and thus, the effectiveness of protein vaccine candidates (Briles, D.E., et al., Infect Immun, 2000, 68:796-800; Yamamoto, M., et al., A. J Immunol, 1998, 161:4115-21).

[0006] The currently available 23-valent pneumococcal capsular polysaccharide vaccine is not effective in children of less than 2 years of age or in immunocompromised patients, two of the major populations at risk from pneumococcal infection (Douglas, R.M., et al., Journal of Infectious Diseases, 1983, 148:131-137). A 7-valent pneumococcal polysaccharide-protein conjugate vaccine, was shown to be highly effective in infants and children against systemic pneumococcal disease caused by the vaccine serotypes and against cross-reactive capsular serotypes (Shinefield and Black, Pediatr Infect Dis J, 2000, 19:394-7). The seven capsular types cover greater than 80% of the disease isolates in the United States, but only 57-60% of disease isolates in other areas of the world (Hausdorff, W.P., et al., Clinical Infectious Diseases, 2000, 30:100-21). Therefore, there is an immediate need for a vaccine to cover most or all of the disease causing serotypes of pneumococci.

[0007] Iron is an essential element for colonization and infection by many pathogenic bacteria. Prevention of the acquisition process should result in a reduction of colonization and a lower disease potential. Iron acquisition complexes in successful pathogens such as, but not limited to, N. gonorrheae, N. meningitidis, M. catarrhalis, and H. influenzae have been evaluated for their vaccine potential by other laboratories (Conte, M.P, et al., Infection and Immunity, 1999, 64:3925; Gray-Owens, S.D., et al. Infection and Immunity, 1995, 64:1201; Luke N.R. et al., Infection and Immunity, 1999, 67:681; Pettersson, A, et al., Infection and Immunity, 1993, 61: 4724). Thus, isolation of the structures responsible for iron acquisition could lead to vaccine candidates.

[0008] The prevention of colonisation of S. pneumoniae as a strategy in the prevention of infections has been previously described. Different surface associated proteins have been described which are involved in the attachment of the bacterium of the mucosal surface in the upper respiratory tract. In particular, Briles et al., (Vaccine, Butterworth Scientific. Guildford, GB, volume 19, 8 December 2000, pages S87-S95) describes that protein PspA has efficacy against otitis media in animals Briles et al., (Vaccine, Butterworth Scientific. Guildford, GB, volume 18, 16 February 2000, pages 1707-1711) shows several pneumococcal proteins that elicit protection in mice including PspA, pneumolysin, PsaA and PspC. Hammerschmidt Sven et al., (Infection and Immunity, volume 67, no 4 April 1999, pages 1683-1687) describes that PspA acts as a human Lactoferrin-binding protein and suggests that S. pneumoniae may use the human Lactoferrin-PspA interaction to overcome the iron limitation at mucosal surfaces due to the presence of Lactoferrin perhaps representing a potential virulence mechanism. Finally, Gosink Khoosheh K et al., (Infection and Immunity, volume 68, no 10, October 2000, pages 590-5695) describes a number of choline binding proteins some of which were found to be involved in colonization of S. pneumoniae.

SUMMARY OF THE INVENTION

[0009] The present invention contemplates an isolated S. pneumoniae surface associated Pneumo Protective Protein (PPP) having an amino acid sequence selected from SEQ ID Nos : 5 and 10-19 or a fragment thereof; the PPP or fragment thereof having the ability to reduce colonization of pneumococcal bacteria, and retain the biological function of interacting with iron.

[0010] The present invention contemplates a recombinant S. pneumoniae surface associated PPP having an amino acid sequence selected from SEQ ID Nos : 5 and 10-19, or a fragment thereof; the PPP or fragment thereof having the ability to reduce colonization of pneumococcal bacteria, and retain the biological function of interacting with iron.

[0011] The present invention contemplates a recombinant S. pneumoniae surface associated PPP having an amino acid sequence selected from SEQ ID Nos : 5 and 10-19, or a fragment thereof; the PPP or fragment thereof having the ability to reduce colonization of pneumococcal bacteria and retain the biological function of interacting with iron; where the PPP has an isoelectric point of about 4.587.

[0012] The present invention contemplates a recombinant S. pneumoniae surface associated PPP having an amino acid sequence selected from SEQ ID Nos : 5 and 10-19, or a fragment thereof; the PPP or fragment thereof having the ability to reduce colonization of pneumococcal bacteria and retain the biological function of interacting with iron, where the PPP has an isoelectric point of about 4.587 and a charge of about -14.214 at pH7.

[0013] The present invention also contemplates an isolated S. pneumoniae surface associated PPP, or a fragment thereof; where the PPP or fragment thereof has an amino acid sequence as depicted in SEQ ID NO: 5, or a fragment thereof; the PPP or fragment thereof having the ability to reduce colonization of pneumococcal bacteria and retain the biological function of interacting with iron.

[0014] The present invention also contemplates a nucleic acid sequence encoding an isolated S. pneumoniae surface associated PPP having an amino acid sequence selected from SEQ ID Nos : 5 and 10-19, or a fragment thereof; where the nucleic acid sequence has a sequence as depicted in SEQ ID NO: 4, or a fragment thereof; the PPP or fragment thereof having the ability to reduce colonization of pneumococcal bacteria and retain the biological function of interacting with iron.

[0015] The present invention also contemplates a cDNA encoding an isolated S. pneumoniae surface associated PPP having an amino acid sequence selected from SEQ ID Nos : 5 and 10-19, or a fragment thereof; where the nucleic acid sequence has a sequence as depicted in SEQ ID NO: 4, or a fragment thereof; the PPP or fragment thereof having the ability to reduce colonization of pneumococcal bacteria and retain the biological function of interacting with iron.

[0016] The present invention contemplates an expression vector comprising a nucleic acid sequence encoding an isolated S. pneumoniae surface associated PPP having an amino acid sequence selected from SEQ ID Nos : 5 and 10-19, or a fragment thereof; the PPP or fragment thereof having the ability to reduce colonization of pneumococcal bacteria and retain the biological function of interacting with iron, where the sequence is operatively associated with an expression control sequence.

[0017] The present invention also contemplates a vector comprising a nucleic acid sequence encoding an isolated S. pneumoniae surface associated PPP having an amino acid sequence selected from SEQ ID Nos : 5 and 10-19, or a fragment thereof; the PPP or fragment thereof having the ability to reduce colonization of pneumococcal bacteria and retain the biological function of interacting with iron, where the sequence is operatively associated with an expression control sequence, and where the PPP has an isoelectric point of about 4.587.

[0018] The present invention further contemplates a vector comprising a nucleic acid sequence encoding an isolated S. pneumoniae surface associated PPP having an amino acid sequence selected from SEQ ID Nos : 5 and 10-19, or a fragment thereof; the PPP or fragment thereof having the ability to reduce colonization of pneumococcal bacteria and retain the biological function of interacting with iron, where the sequence is operatively associated with an expression control sequence, and where the PPP has an isoelectric point of about 4.587 and a charge of about -14.214 at pH 7.

[0019] The present invention also contemplates an expression vector comprising a nucleic acid sequence encoding an isolated S. pneumoniae surface associated PPP , or a fragment thereof; where the PPP or fragment thereof has an amino acid sequence as depicted in SEQ ID NO: 5, or a fragment thereof; the PPP or fragment thereof having the ability to reduce colonization of pneumococcal bacteria and retain the biological function of interacting with iron; and where the nucleic acid sequence is operatively associated with an expression control sequence.

[0020] The present invention also contemplates an expression vector comprising a nucleic acid sequence encoding an isolated S. pneumoniae surface associated PPP having , or a fragment thereof; where the PPP or fragment thereof has an amino acid sequence as depicted in SEQ ID NO: 5, or a fragment thereof; the PPP or fragment thereof having the ability to reduce colonization of pneumococcal bacteria and retain the biological function of interacting with iron; where the amino acid sequence is encoded by the nucleic acid sequence as depicted in SEQ ID NO: 4, or a fragment thereof; and where the nucleic acid sequence is operatively associated with an expression control sequence.

[0021] The present invention contemplates a host cell transfected with an expression vector comprising a nucleic acid sequence encoding an isolated S. pneumoniae surface associated PPP having an amino acid sequence selected from SEQ ID Nos : 5 and 10-19, or a fragment thereof; the PPP or fragment thereof having the ability to reduce colonization of pneumococcal bacteria and retain the biological function of interacting with iron; where the sequence is operatively associated with an expression control sequence.

[0022] The present invention further contemplates a host cell transfected with a vector comprising a nucleic acid sequence encoding an isolated S. pneumoniae surface associated PPP , or a fragment thereof; where the PPP or fragment thereof has an amino acid sequence as depicted in SEQ ID NO: 5, or a fragment thereof; the PPP or fragment thereof having the ability to reduce colonization of pneumococcal bacteria and retain the biological function of interacting with iron, where the sequence is operatively associated with an expression control sequence.

[0023] The present invention also contemplates a method for producing recombinant PPP or fragment thereof, which method comprises isolating the PPP or fragment thereof produced by a host cell transfected with an expression vector and cultured under conditions that provide for expression of the PPP or fragment thereof by the vector, where the vector comprises a nucleic acid sequence encoding an isolated S. pneumoniae surface associated PPP having an amino acid sequence selected from SEQ ID Nos : 5 and 10-19, or a fragment thereof; the PPP or fragment thereof having the ability to reduce colonization of pneumococcal bacteria and retain the biological function of interacting with iron; where the sequence is operatively associated with an expression control sequence.

[0024] The present invention also contemplates a method for producing recombinant PPP or fragment thereof, which method comprises isolating the PPP or fragment thereof produced by host cell transfected with a vector and cultured under conditions that provide for expression of the PPP or fragment thereof by the vector, where the vector comprises a nucleic acid sequence encoding an isolated S. pneumoniae surface associated PPP , or a fragment thereof where the PPP or fragment thereof has an amino acid sequence as depicted in SEQ ID NO: 5, or a fragment thereof; the PPP or fragment thereof having the ability to reduce colonization of pneumococcal bacteria and retain the biological function of interacting with iron, where the sequence is operatively associated with an expression control sequence.

[0025] The present invention also contemplates a composition comprising (1) an isolated S. pneumoniae surface associated PPP having an amino acid sequence selected from SEQ ID Nos : 5 and 10-19, or a fragment thereof; the PPP or fragment thereof having the ability to reduce colonization of pneumococcal bacteria and retain the biological function of interacting with iron; and (2) a pharmaceutically acceptable carrier.

[0026] The present invention also contemplates a composition comprising (1) an isolated S. pneumoniae surface associated PPP , or a fragment thereof; the PPP or fragment thereof having the ability to reduce colonization of pneumococcal bacteria and retain the biological function of interacting with iron, and which PPP has an amino acid sequence as depicted in SEQ ID NO: 5, or a fragment thereof; and (2) a pharmaceutically acceptable carrier.

[0027] The present invention contemplates a composition comprising (1) a nucleic acid sequence encoding an isolated S. pneumoniae surface associated PPP having an amino acid sequence selected from SEQ ID Nos : 5 and 10-19, or a fragment thereof; the PPP or fragment thereof having the ability to reduce colonization of pneumococcal bacteria and retain the biological function of interacting with iron, where the nucleic acid sequence has a sequence as depicted in SEQ ID NO: 4, or a fragment thereof; and (2) a pharmaceutically acceptable carrier.

[0028] The present invention contemplates a composition comprising (1) an expression vector comprising a nucleic acid sequence encoding an isolated S. pneumoniae surface associated PPP having an amino acid sequence selected from SEQ ID Nos : 5 and 10-19, or a fragment thereof; the PPP or fragment thereof having the ability to reduce colonization of pneumococcal bacteria and retain the biological function of interacting with iron, where the sequence is operatively associated with an expression control sequence; and (2) a pharmaceutically acceptable carrier.

[0029] The present invention also contemplates a composition comprising (1) an expression vector comprising a nucleic acid sequence encoding an isolated S. pneumoniae surface associated PPP , or a fragment thereof; where the PPP or fragment thereof has an amino acid sequence as depicted in SEQ ID NO: 5, or a fragment thereof; and retain the biological function of interacting with iron and where the nucleic acid sequence is operatively associated with an expression control sequence; and (2) a pharmaceutically acceptable carrier.

[0030] The present invention also contemplates a composition comprising (1) a host cell transfected with an expression vector comprising a nucleic acid sequence encoding an isolated S. pneumoniae surface associated PPP having an amino acid sequence selected from SEQ ID Nos : 5 and 10-19, or a fragment thereof; the PPP or fragment thereof having the ability to reduce colonization of pneumococcal bacteria and retain the biological function of interacting with iron, where the sequence is operatively associated with an expression control sequence; and (2) a pharmaceutically acceptable carrier.

[0031] The present invention contemplates a composition comprising (1) a host cell transfected with a vector comprising a nucleic acid sequence encoding an isolated S. pneumoniae surface associated PPP , or a fragment thereof; the PPP or fragment thereof having the ability to reduce colonization of pneumococcal bacteria and retain the biological function of interacting with iron, where the sequence is operatively associated with an expression control sequence; where the PPP or fragment thereof has an amino acid sequence as depicted in SEQ ID NO: 5, or a fragment thereof; and a (2) pharmaceutically acceptable carrier.

[0032] The present invention also contemplates an immunogenic composition comprising (i) a S. pneumoniae surface associated PPP having an amino acid sequence selected from SEQ ID Nos : 5 and 10-19, or a fragment thereof; (ii) a pharmaceutically acceptable carrier; and (iii) optionally at least one adjuvant.

[0033] The present invention also contemplates an immunogenic composition comprising (i) a S. pneumoniae surface associated PPP having an amino acid sequence selected from SEQ ID Nos : 5 and 10-19, or a fragment thereof, the PPP having an isoelectric point of about 4.587; (ii) a pharmaceutically acceptable carrier; and (iii) optionally at least one adjuvant.

[0034] The present invention also contemplates an immunogenic composition comprising (i) a S. pneumoniae surface associated PPP having an amino acid sequence selected from SEQ ID Nos : 5 and 10-19, or a fragment thereof, PPP having having an isoelectric point of about 4.587 and a charge of about -14.214 at pH 7; (ii) a pharmaceutically acceptable carrier, and (iii) optionally at least one adjuvant.

[0035] The present invention also contemplates an immunogenic composition comprising (i) a S. pneumoniae surface associated PPP , or a fragment thereof, which PPP or fragment thereof has an amino acid sequence as depicted in SEQ ID NO: 5, or an immunogenic fragment thereof; (ii) a pharmaceutically acceptable carrier; and (iii) optionally at least one adjuvant.

[0036] The present invention also contemplates an immunogenic composition comprising (i) a S. pneumoniae surface associated PPP having an amino acid sequence selected from SEQ ID Nos : 5 and 10-19, or a fragment thereof, the PPP or fragment thereof encoded by a nucleic acid sequence having a sequence as depicted in SEQ ID NO: 4, or an immunogenic fragment thereof; (ii) a pharmaceutically acceptable carrier; and (iii) optionally at least one adjuvant.

[0037] The present invention also contemplates an immunogenic composition comprising (i) a S. pneumoniae surface associated PPP having an amino acid sequence selected from SEQ ID Nos : 5 and 10-19, or a fragment thereof; (ii) a pharmaceutically acceptable carrier; and (iii) optionally at least one adjuvant; where the composition elicits protective immunity from a disease caused by Streptococcus pneumoniae.

[0038] The present invention also contemplates an immunogenic composition comprising (i) a S. pneumoniae surface associated PPP having an amino acid sequence selected from SEQ ID Nos : 5 and 10-19, or a fragment thereof; (ii) a pharmaceutically acceptable carrier; and (iii) optionally at least one adjuvant; where the composition elicits protective immunity from a disease caused by Streptococcus pneumoniae; where the disease is selected from the group consisting of otitis media, rhinosinusitis, bacteremia, meningitis, pneumonia, and lower respiratory tract infection.

[0039] The present invention also contemplates an immunogenic composition comprising (i) a S. pneumoniae surface associated PPP having an amino acid sequence selected from SEQ ID Nos : 5 and 10-19, or a fragment thereof; (ii) a pharmaceutically acceptable carrier; and (iii) optionally at least one adjuvant; where the composition elicits protective immunity from a disease caused by Streptococcus pneumoniae; where the PPP or fragment thereof comprises an amino acid sequence as depicted in SEQ ID NO: 5, or an immunogenic fragment thereof.

[0040] The present invention also contemplates an immunogenic composition comprising (i) a S. pneumoniae surface associated PPP having an amino acid sequence selected from SEQ ID Nos : 5 and 10-19, or a fragment thereof where the PPP or fragment thereof is encoded by a nucleic acid sequence as depicted in SEQ ID NO: 4, or an immunogenic fragment thereof; (ii) a pharmaceutically acceptable carrier; and (iii) optionally at least one adjuvant; where the composition elicits protective immunity from a disease caused by Streptococcus pneumoniae.

[0041] The present invention contemplates an immunogenic composition comprising (i) at least one expression vector encoding a PPP having an amino acid sequence selected from SEQ ID Nos : 5 and 10-19; (ii) a pharmaceutically acceptable carrier; and (iii) optionally at least one adjuvant.

[0042] The present invention contemplates an immunogenic composition comprising (i) at least one expression vector encoding a PPP having an amino acid sequence selected from SEQ ID Nos : 5 and 10-19; (ii) a pharmaceutically acceptable carrier; and (iii) optionally at least one adjuvant; where the pneumococcal bacteria is Streptococcus pneumoniae.

[0043] The present invention contemplates an immunogenic composition comprising (i) at least one expression vector encoding a PPP having an amino acid sequence selected from SEQ ID Nos : 5 and 10-19; (ii) a pharmaceutically acceptable carrier; and (iii) optionally at least one adjuvant; where the composition elicits protective immunity from a disease caused by Streptococcus pneumoniae.

[0044] The present invention contemplates an immunogenic composition comprising (i) at least one expression vector encoding a PPP having an amino acid sequence selected from SEQ ID Nos : 5 and 10-19; (ii) a pharmaceutically acceptable carrier; and (iii) optionally at least one adjuvant; where the composition elicits protective immunity from a disease caused by Streptococcus pneumoniae; where the disease is selected from the group consisting of otitis media, rhinosinusitis, bacterenia, meningitis, pneumonia, and lower respiratory tract infection.

[0045] The present invention contemplates an immunogenic composition comprising (i) at least one expression vector encoding a PPP having an amino acid sequence selected from SEQ ID Nos : 5 and 10-19, where the PPP has an isoelectric point of about 4.587; (ii) a pharmaceutically acceptable carrier; and (iii) optionally at least one adjuvant.

[0046] The present invention contemplates an immunogenic composition comprising (i) at least one expression vector encoding a PPP having an amino acid sequence selected from SEQ ID Nos : 5 and 10-19, where the PPP has an isoelectric point of about 4.587 and has a charge of about 14.214 at pH7; (ii) a pharmaceutically acceptable carrier; and (iii) optionally at least one adjuvant.

[0047] The present invention contemplates an immunogenic composition comprising (i) at least one expression vector encoding a PPP or fragment thereof where expression vector comprises a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 5, or an immunogenic fragment thereof; (ii) a pharmaceutically acceptable carrier; and (iii) optionally at least one adjuvant.

[0048] The present invention contemplates an immunogenic composition comprising (i) at least one expression vector encoding a PPP or fragment thereof where the expression vector comprises a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 5; or an immunogenic fragment thereof; (ii) a pharmaceutically acceptable carrier; and (iii) optionally at least one adjuvant.

[0049] The present invention contemplates an immunogenic composition comprising (i) at least one expression vector encoding a PPP having an amino acid sequence selected from SEQ ID Nos : 5 and 10-19, or fragment thereof, where the expression vector comprises a nucleic acid sequence depicted in SEQ ID NO:4, or an immunogenic fragment thereof; (ii) a pharmaceutically acceptable carrier; and (iii) optionally at least one adjuvant.

[0050] The present invention contemplates a composition for use in inducing an immune response in a mammal; where the composition comprises (i) a S. pneumoniae surface associated PPP having an amino acid sequence selected from SEQ ID Nos : 5 and 10-19, or a fragment thereof; (ii) a pharmaceutically acceptable carrier; and (iii) optionally at least one adjuvant.

[0051] The present invention contemplates a composition for use in inducing an immune response in a mammal; where the composition comprises (i) a S. pneumoniae surface associated PPP or a fragment thereof, which PPP has an amino acid sequence as depicted in SEQ ID NO: 5, or an immunogenic fragment thereof; (ii) a pharmaceutically acceptable carrier; and (iii) optionally at least one adjuvant.

[0052] The present invention contemplates a composition for use in inducing an immune response in a mammal; where the composition comprises (i) at least one expression vector encoding a PPP having an amino acid sequence selected from SEQ ID Nos : 5 and 10-19, where the PPP having an isoelectric point of about 4.582; (ii) a pharmaceutically acceptable carrier; and (iii) optionally at least one adjuvant.

[0053] The present invention contemplates a composition for use in inducing an immune response in a mammal; where the composition comprises (i) at least one expression vector encoding a PPP (ii) a pharmaceutically acceptable carrier; and (iii) optionally at least one adjuvant; wherein said expression vector comprises a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 5, or an immunogenic fragment thereof.

[0054] The present invention contemplates a compound for use in inducing an immune response in a mammal which is infected with pneumococcal bacteria, the effective to inhibit binding of an amino acid sequence as depicted in SEQ ID NO: 5 to induce the immune response in the mammal.

[0055] The present invention also contemplates a method for screening for a compound which induces an immune response in a mammal infected with pneumococcal bacteria, the method comprising comparing a first amount of binding of an amino acid sequence as depicted in SEQ ID NO: 5 in the presence of the compound to a second amount of binding of an amino acid sequence as depicted in SEQ ID NO: 5 not in the presence of the compound; whereby a lower first amount of binding than the second amount binding indicates that the compound may induce the immune response in the mammal.

[0056] The present invention also contemplates a method for diagnosing pneumococcal bacterial infection, the method comprising comparing the level of PPP as depicted in SEQ ID NO: 5, or fragments thereof, in suspect sample to the level of PPP as depicted in SEQ ID NO: 5, or fragments thereof, in a control sample, whereby a higher level of the Pneumo Protective Protein the suspect sample than the level of the Pneumo Protective Protein in the control sample indicates that the suspect sample comprises pneumococcal bacterial infection.

[0057] The present invention contemplates an antibody which binds to Streptococcus pneumoniae PPP, which selectively recognizes an amino acid sequence as depicted in SEQ ID NO: 5, or fragments thereof.

[0058] The present invention also contemplates a chimeric antibody which binds to Streptococcus pneumoniae PPP which selectively recognizes an amino acid sequence as depicted in SEQ ID NO: 5, or fragments thereof.

[0059] The present invention also contemplates a humanized antibody which binds to Streptococcus pneumoniae PPP which selectively recognizes an amino acid sequence as depicted in SEQ ID NO: 5, or fragments thereof.

[0060] The present invention also contemplates an anti-idiotypic antibody which binds to Streptococcus pneumoniae PPP which selectively recognizes an amino acid sequence as depicted in SEQ ID NO: 5, or fragments thereof.

[0061] The present invention also contemplates an antibody which binds to Streptococcus pneumoniae PPP which selectively recognizes an amino acid sequence as depicted in SEQ ID NO: 5, or fragments thereof, where the antibody is conjugated to a pharmaceutically active compound.

[0062] The present invention also contemplates a monoclonal antibody which binds to Streptococcus pneumoniae PPP which selectively recognizes an amino acid sequence as depicted in SEQ ID NO: 5, or fragments thereof.

[0063] The present invention also contemplates a monoclonal antibody which binds to Streptococcus pneumoniae PPP which selectively recognizes an amino acid sequence as depicted in SEQ ID NO: 5, or fragments thereof, where the antibody is humanized.

[0064] The present invention also contemplates a monoclonal antibody which binds to Streptococcus pneumoniae PPP which selectively recognizes an amino acid sequence as depicted in SEQ ID NO: 5, or fragments thereof, where the antibody is anti-idiotypic.

[0065] The present invention also contemplates a monoclonal antibody which binds to Streptococcus pneumoniae PPP which selectively recognizes an amino acid sequence as depicted in SEQ ID NO: 5, or fragments thereof, where the antibody is conjugated to a pharmaceutically active compound.

[0066] The present invention contemplates a use in the manufacture of a medicament for inducing an immune response in a mammal, anti-idiotypic antibody which binds to Streptococcus pneumoniae PPP which selectively recognizes an amino acid sequence as depicted in SEQ ID NO: 5, or fragments thereof which is effective to induce an immune response in the mammal.

[0067] The present invention contemplates a use in the manufacture of a medicament for inducing an immune response in a mammal, of a monoclonal antibody which binds to Streptococcus pneumoniae PPP which selectively recognizes an amino acid sequence as depicted in SEQ ID NO: 5, or fragments thereof, where the antibody is anti-idiotypic;
effective to induce an immune response in the mammal.

[0068] The present invention contemplates a use in the manufacture of a medicament for inducing an immune response in a mammal infected with pneumococcal bacteria, of an antibody which binds to Streptococcus pneumoniae PPP which selectively recognizes an amino acid sequence as depicted in SEQ ID NO: 5, or fragments thereof, where the antibody is conjugated to a pharmaceutically active compound; effective to induce an immune response in the mammal.

[0069] The present invention also contemplates a use in the manufacture of a medicament for inducing an immune response in a mammal infected with pneumococcal bacteria, of a monoclonal antibody which binds to Streptococcus pneumoniae PPP which selectively recognizes an amino acid sequence as depicted in SEQ ID NO: 5, or fragments thereof, where the antibody is conjugated to a pharmaceutically active compound; effective to induce an immune response in the mammal.

BRIEF DESCRIPTION OF THE DRAWINGS

[0070] 

Figure 1. SDS -PAGE gel of DEAE fractions from PBS washes of S. pneumoniae strain 49136. Lane 1 is unstained standards; lane 2 is fraction #8; lane 3 is fraction #9; lane 4 is fraction #10; lane 5 is fraction #11; lane 6 is fraction #12; lane 7 is fraction #13; lane 8 is fraction #19; lane 9 is fraction #15; and lane 10 is fraction #16. The gel in figure1 shows the distinct small molecular weight band in fractions #14 and #15 (lanes 8 and 9) resolved by the gel.

Figure 2. Gel of whole cell lysate of recombinant expression of pLP533 showing expression of the desired product. Lane 1, Biorad prestained markers; Lane 2, uninduced cells; Lane 3, induced cells.

Figure 3. Western blot of whole cell lysates of several serotypes showing cross reactivity and oligomer formation. Lane 1, Biorad Precision prestained markers; lane 2, type 3; lane 3, type 4; lane 4, type 9; lane 5, type 14; lane 6, type 19F; lane 7, type 18C; lane 8, type 5; and lane 9, type 23F.

Figure 4. Reduction of colonization by rPPP1. Bacteria recovered shown as Log10 CFU/gram of tissue. One standard error of the mean is shown. *values are significantly different compared to the control by Tukey-Kramer statistical test.

Figure 5. SDS-PAGE gel shows purification of rPPP1. Lane 1, Bio Rad Precision standards; lane 2, diafiltrate; lane 3, purified rPPP1.

Figure 6. Comparison of sequences of PPP1 from serotypes of S. pneumoniae.

Figure 7. Gel shows amplified PPP1 from in vitro and in vivo cultures.

DETAILED DESCRIPTION

[0071] The proteins and nucleic acids of this invention possess diagnostic, prophylactic and therapeutic utility for diseases caused by Streptococcus pneumoniae infection. They can be used to design screening systems for compounds that interfere or disrupt interaction of proteins associated with S. pneumoniae with iron. The nucleic acids and proteins also can be used in the preparation of compositions against S. pneumoniae infection and/or other pathogens when used to express foreign genes.

[0072] In the present invention, a recombinant 20kDa protein from whole S. pneumoniae that reduces colonization of S. pneumoniae, in an intranasal challenge model, has been identified. The protein described herein has been named Pneumo Protective Protein 1 (PPP1). This protein shows significant homology to a non heme containing ferretin protein from L. innocua, which interestingly, is a member of the Dps family of DNA binding proteins (Pikis, A., et al., J. Infect. Diseases, 1998, 178:700). The ability of this protein to reduce colonization was thus unexpected, due to its predicted location in the cytoplasm.

[0073] Chemical studies indicate that the isolated S. pneumoniae surface associated PPP has a molecular weight of about 20 kDa, where the molecular weight is determined using a 10-20% SDS-PAGE gel. The recombinant PPP is determined to have an isoelectric point of about 4.587. Additionally, the protein has a charge of about -14.214 at pH of about 7.

Streptococcus Pneumoniae

[0074] S. pneumoniae is a species of bacteria which is highly infectious in the human body. There have been more than 80 serotypes identified, to date. Several of these serotypes are etiological agents in a variety of disease states including, but not limited to, pneumonia, meningitis, endocarditis, arthritis, sinusitis, otitis, bronchitis, and laryngitis. Pneumococcal infections have been identified as a leading cause of death in persons with immunocompromised systems, such as those infected with HIV.

[0075] S. pneumoniae is a species of the Streptococcus genus of the Streptococcaceae family. This family comprises Gram-positive, non-motile, spherical or oval cells that do not form endospores. S. pneumoniae have an inorganic terminal electron acceptor for oxidative-metabolism; however, they will grow in the presence of oxygen. This allows S. pneumoniae to grow in a variety of environments and thus it is well adapted to grow in various human tissues. The bacteria is difficult to target with penicillin, since many strains produce a polysaccharide capsule.

[0076] The first step towards pneumococcal infection is colonization of the nasopharynx. Disruption of binding of the pneumococci to human nasopharyngeal/otic cells should result in reduction of colonization and a lower disease potential. Thus, isolation of the structures responsible for pneumococcal binding to human cells could lead to vaccine candidates. Pneumococci have evolved numerous mechanisms for binding to human nasopharyngeal cells, including the PspA, PsaA, and CbpA proteins. Additionally, pneumococci may specifically bind to human nasopharyngeal mucin as a first step in colonization. Thus, identification of the pneumococcal structure(s) responsible for this interaction may identify potential vaccine targets.

Molecular Biology

[0077] Embodiments of this invention relate to isolated polynucleotide sequences encoding the polypeptides or proteins, as well as variants of such sequences. Preferably, under high stringency conditions, these variant sequences hybridize to polynucleotides encoding one or more pneumo protective proteins. More preferably, under high stringency conditions, these variant sequences hybridize to polynucleotides encoding one or more pneumo protective protein sequences, such as the polynucleotide sequence of SEQ ID NO: 4. For the purposes of defining high stringency southern hybridization conditions, reference can conveniently be made to Sambrook et al. (1989) at pp. 387-389, where the washing step is considered high stringency.

[0078] This invention also relates to conservative variants wherein the polynucleotide sequence differs from a reference sequence through a change to the third nucleotide of a nucleotide triplet. Preferably these conservative variants function as biological equivalents to the PPP1 reference polynucleotide sequence. Variants that function as biological equivalents are those that bind to iron.

[0079] The present invention further comprises DNA sequences which, by virtue of the redundancy of the genetic code, are biologically equivalent to the sequences which encode for the PPP1, that is, these other DNA sequences are characterized by nucleotide sequences which differ from those set forth herein, but which encode a protein having the same amino acid sequence as that encoded by the DNA sequence in SEQ ID NO: 4.

[0080] This invention also comprises DNA sequences which encode amino acid sequences which differ from those of the S. pneumonia PPP1, but which are biologically equivalent to those described for this protein (SEQ ID NO: 5). Such amino acid sequences may be said to be biologically equivalent to such PPP1 if their sequences differ only by minor deletions from, insertions into or substitutions to the PPP1 sequence, such that the tertiary configurations of the sequences are essentially unchanged from those of the wild-type protein.

[0081] For example, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, changes which result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, as well as changes based on similarities of residues in their hydropathic index, can also be expected to produce a biologically equivalent product. Nucleotide changes which result in alteration of the N-terminal or C-terminal portions of the protein molecule would also not be expected to alter the activity of the protein.

[0082] One can use the hydropathic index of amino acids in conferring interactive biological function on a polypeptide, as discussed by Kyte and Doolittle (1982), wherein it was determined that certain amino acids may be substituted for other amino acids having similar hydropathic indices and still retain a similar biological activity. Alternatively, substitution of like amino acids may be made on the basis of hydrophilicity, particularly where the biological function desired in the polypeptide to be generated is intended for use in immunological embodiments. See, for example, U.S. Patent 4,554,101, which states that the greatest local average hydrophilicity of a "protein," as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity. Accordingly, it is noted that substitutions can be made based on the hydrophilicity assigned to each amino acid. In using either the hydrophilicity index or hydropathic index, which assigns values to each amino acid, it is preferred to introduce substitutions of amino acids where these values are ± 2, with ± 1 being particularly preferred, and those within ± 0.5 being the most preferred substitutions.

[0083] Furthermore, changes in known variable regions are biologically equivalent where the tertiary configurations of the conserved regions are essentially unchanged from those of PPP1. An alternative definition of a biologically equivalent sequence is one that is still capable of generating a cross-reactive immune response. In particular, the proteins may be modified by lengthening or shortening the corresponding insertion from the gonococcal pilin, as long as the modified protein is still capable of generating a desired immune response.

[0084] Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of structural and biological activity of the encoded products. Therefore, where the terms "pneumo protective protein", or "PPP1", or "PPP" are used in either the specification or the claims, it will be understood to encompass all such modifications and variations which result in the production of a biologically equivalent protein.

[0085] Preferable characteristics of PPP1 described herein, encoded by the nucleotide sequences of this invention, include one or more of the following: (a) being a membrane protein or being a protein directly associated with a membrane; (b) capable of being separated as a protein using an SDS acrylamide gel; and (c) retaining its biological function of interacting with iron.

[0086] The fragments and variant amino acid sequences and variant nucleotide sequences expressing PPP1 are biological equivalents, i.e. they retain the same function of the wild-type PPP1. Such variant amino acid sequences are encoded by polynucleotides sequences of this invention. Such variant amino acid sequences may have about 70% to about 80%, and preferably about 90%, overall similarity to the amino acid sequence of PPP1. In a preferred embodiment, these sequences are shown in Figure 6 and SEQ ID NOs10-19. The variant nucleotide sequences may have either about 70% to about 80%, and preferably about 90%, overall similarity to the nucleotide sequences which, when transcribed, encode the amino acid sequence of PPP1 or a variant amino acid sequence of PPP1. In alternative embodiments, the epitopic region of the protein comprises at least 20 contiguous nucleotides or 8 contiguous amino acids.

[0087] The invention further relates to the overall consensus sequence of PPP1. Deduced amino acid sequences of PPP1 from different serotypes of S. pneumoniae may be compared to determine the conserved sequences. In a one embodiment, 10 different serotypes are compared. The conserved sequence may have many uses such as, but not limited to, determining the minimal requirements needed for protein binding, activity, and/or function. In a preferred embodiment, the consensus sequence of PPP1 is depicted in Figure 6 and SEQ ID NO:20.

[0088] The "isolated" sequences of the present invention are non-naturally occurring sequences. For example, these sequences can be isolated from their normal state within the genome of the bacteria; or the sequences may be synthetic, i.e. generated via recombinant techniques, such as well-known recombinant expression systems, or generated by a machine.

[0089] The invention also provides a recombinant DNA cloning vehicle capable of expressing a PPP1 comprising an expression control sequence having promoter and initiator sequences and a nucleic acid sequence of the present invention located 3' to the promoter and initiator sequences. Cloning vehicles can be any plasmid or expression vector known in the art, including viral vectors (see below). In a further aspect, there is provided a host cell containing a recombinant DNA cloning vehicle and/or a recombinant PPP1 of the present invention. Suitable expression control sequences, host cells and expression vectors are well known in the art, and are described by way of example, in Sambrook et al. (1989).

[0090] Suitable host cells may be selected based on factors which can influence the yield of recombinantly expressed proteins. These factors include, but are not limited to, growth and induction conditions, mRNA stability, codon usage, translational efficiency and the presence of transcriptional terminators to minimize promoter read through. Upon selection of suitable host cells, the cell may be transfected with expression vectors comprising nucleic acid sequences of the present invention. The cells may be transfected using any methods known in the art (see below).

[0091] Once host cells have been transfected with expression vectors of the present invention, cells are cultured under conditions such that polypeptides are expressed. The polypeptide is then isolated substantially free of contaminating host cell components by techniques that are well known to those skilled in the art.

[0092] Depending on the application of the desired recombinant proteins, a heterologous nucleotide sequence may encode a co-factor, cytokine (such as an interleukin), a T-helper epitope, a restriction marker, adjuvant, or a protein of a different microbial pathogen (e.g. virus, bacterium, fungus or parasite), especially proteins capable of eliciting a protective immune response. It may be desirable to select a heterologous sequence that encodes an immunogenic portion of a co-factor, cytokine (such as an interleukin), a T-helper,epitope, a restriction marker, adjuvant, or a protein of a different microbial pathogen (e.g. virus, bacterium or fungus). Other types of non-PPP1 moieties include, but are not limited to, those from cancer cells or tumor cells, allergens, amyloid peptide, protein or other macromolecular components.

[0093] For example, in certain embodiments, the heterologous genes encode cytokines, such as interleukin-12, which are selected to improve the prophylatic or therapeutic characteristics of the recombinant proteins.

[0094] Examples of such cancer cells or tumor cells include, but are not limited to, prostate specific antigen, carcino-embryonic antigen, MUC-1, Her2, CA-125 and MAGE-3.

[0095] Examples of such allergens include, but are not limited to, those described in U. S. Patent Number 5,830,877 and published International Patent Application Number WO 99/51259 and include pollen, insect venoms, animal dander, fungal spores and drugs (such as penicillin). Such components interfere with the production of IgE antibodies, a known cause of allergic reactions.

[0096] Amyloid peptide protein (APP) has been implicated in diseases referred to variously as Alzheimer's disease, amyloidosis or amyloidogenic disease. The β-amyloid peptide (also referred to as Aβ peptide) is a 42 amino acid fragment of APP, which is generated by processing of APP by the β and y secretase enzymes, and has the following sequence:

[0097] Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val Val Ile Ala (SEQ ID NO: 6).

[0098] In some patients, the amyloid deposit takes the form of an aggregated Aβ peptide. Surprisingly, it has now been found that administration of isolated Aβ peptide induces an immune response against the Aβ peptide component of an amyloid deposit in a vertebrate host (See Published International Patent Application WO 99/27944). Such Aβ peptides have also been linked to unrelated moieties. Thus, the heterologous nucleotides sequences of this invention include the expression of this Aβ peptide, as well as fragments of Aβ peptide and antibodies to Aβ peptide or fragments thereof. One such fragment of Aβ peptide is the 28 amino acid peptide having the following sequence (as disclosed in U.S. Patent 4,666,829):

[0099] Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys (SEQ ID NO: 7).

[0100] The heterologous nucleotide sequence can be selected to make use of the normal route of infection of pneumococcal bacteria, which enters the body through the respiratory tract and can infect a variety of tissues and cells, for example, the meninges, blood, and lung. The heterologous gene may also be used to provide agents which are used for gene therapy or for the targeting of specific cells. As an alternative to merely taking advantage of the normal cells exposed during the normal route of pneumococcal infection, the heterologous gene, or fragment, may encode another protein or amino acid sequence from a different pathogen which, when employed as part of the recombinant protein, directs the recombinant protein to cells or tissue which are not in the normal route of infection. In this manner, the protein becomes a targeting tool for the delivery of a wider variety of foreign proteins.

[0101] Molecular weight of proteins may be determined by using any method known in the art. A non-limiting list of methods includes, denaturing SDS-PAGE gel, size exclusion chromatography, and iso-electric focusing. Conditions appropriate for each method (e.g. time of separation, voltage, current, and buffers) can be determined as needed using defined methods in the art. In a preferred embodiment, denaturing SDS-PAGE is used to determine the molecular weight of the proteins. Additionally, the conditions used to determine the molecular weight are preferably, 1 hour separation time at 20 milli Amps and constant current.

[0102] Detection of the proteins can be determined using various methods in the art. These methods include, but are not limited to, Western blotting, coomassie blue staining, silver staining, autoradiography, fluorescent and phosphorescent probing. In a preferred embodiment of this invention, the proteins were detected by Western blotting.

[0103] The terms "pneumo protective protein", "PPP1", and "PPP" in describing embodiments of the invention, infra, includes embodiments that employ fragments, variants and attenuated forms thereof as a replacement for wild-type PPP 1 or as addition thereto, unless specified otherwise.

Viral and Non-Viral Vectors

[0104] Preferred vectors, particularly for cellular assays in vitro and in vivo, are viral vectors, such as lentiviruses, retroviruses, herpes viruses, adenoviruses, adeno-associated viruses, vaccinia virus, baculovirus, alphaviruses and other recombinant viruses with desirable cellular tropism. Thus, a gene encoding a functional or mutant protein or polypeptide domain fragment thereof can be introduced in vivo, ex vivo, or in vitro using a viral vector or through direct introduction of DNA. Expression in targeted tissues can be effected by targeting the transgenic vector to specific cells, such as with a viral vector or a receptor ligand, or by using a tissue-specific promoter, or both. Targeted gene delivery is described in PCT Publication No. WO 95/28494.

[0105] Viral vectors commonly used for in vivo or ex vivo targeting and therapy procedures are DNA-based vectors and retroviral vectors. Methods for constructing and using viral vectors are known in the art (e.g., Miller and Rosman, BioTechniques, 1992, 7:980-990). Preferably, the viral vectors are replication-defective, that is, they are unable to replicate autonomously in the target cell. Preferably, the replication defective virus is a minimal virus, i.e., it retains only the sequences of its genome which are necessary for encapsulating the genome to produce viral particles.

[0106] Examples of alphaviruses include, but are not limited to, Eastern Equine Encephalitis virus (EEE), Venezuelan Equine Encephalitis virus (VEE), Everglades virus, Mucambo virus, Pixuna virus, Western Equine Encephalitis virus (WEE), Sindbis virus, Semliki Forest virus, Middelburg virus, Chikungunya virus, O'nyong-nyong virus, Ross River virus, Barmah Forest virus, Getah virus, Sagiyama virus, Bebaru virus, Mayaro virus, Una virus, Aura virus, Whataroa virus, Babanki virus, Kyzylagach virus, Highlands J virus, Fort Morgan virus, Ndumu virus, and Buggy Creek virus (U.S. Patent No. 6,156,558).

[0107] DNA viral vectors include an attenuated or defective DNA virus, such as but not limited to herpes simplex virus (HSV), papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV), and the like. Defective viruses, which entirely or almost entirely lack viral genes, are preferred. Defective virus is not infective after introduction into a cell. Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells. Thus, a specific tissue can be specifically targeted. Examples of particular vectors include, but are not limited to, a defective herpes virus 1 (HSV 1) vector (Kaplitt et al., Molec. Cell. Neurosci., 1991, 2:320-330), defective herpes virus vector lacking a glyco-protein L gene, or other defective herpes virus vectors (PCT Publication Nos. WO 94/21807 and WO 92/05263); an attenuated adenovirus vector, such as the vector described by Stratford-Perricaudet et al. (J. Clin. Invest., 1992, 90:626-630; see also La Salle et al., Science, 1993, 259:988-990); and a defective adeno-associated virus vector (Samulski et al., J. Virol., 1987, 61:3096-3101; Samulski et al., J. Virol., 1989, 63:3822-3828; Lebkowski et al., Mol. Cell. Biol., 1988, 8:3988-3996).

[0108] Various companies produce viral vectors commercially, including, but not limited to, Avigen, Inc. (Alameda, CA; AAV vectors), Cell Genesys (Foster City, CA; retroviral, adenoviral, AAV vectors, and lentiviral vectors), Clontech (retroviral and baculoviral vectors), Genovo, Inc. (Sharon Hill, PA; adenoviral and AAV vectors), Genvec (adenoviral vectors), IntroGene (Leiden, Netherlands; adenoviral vectors), Molecular Medicine (retroviral, adenoviral, AAV, and herpes viral vectors), Norgen (adenoviral vectors), Oxford BioMedica (Oxford, United Kingdom; lentiviral vectors), and Transgene (Strasbourg, France; adenoviral, vaccinia, retroviral, and lentiviral vectors).

[0109] Adenovirus vectors. Adenoviruses are eukaryotic DNA viruses that can be modified to efficiently deliver a nucleic acid of the invention to a variety of cell types. Various serotypes of adenovirus exist. Of these serotypes, preference is given, within the scope of the present invention, to using type 2 or type 5 human adenoviruses (Ad 2 or Ad 5) or adenoviruses of animal origin (see PCT Publication No. WO 94/26914). Those adenoviruses of animal origin which can be used within the scope of the present invention include adenoviruses of canine, bovine, murine (example: Mav1, Beard et al., Virology, 1990, 75-81), ovine, porcine, avian, and simian (example: SAV) origin. Preferably, the adenovirus of animal origin is a canine adenovirus, more preferably a CAV2 adenovirus (e.g., Manhattan or A26/61 strain, ATCC VR-800, for example). Various replication defective adenovirus and minimum adenovirus vectors have been described (PCT Publication Nos. WO 94/26914, WO 95/02697, WO 94/28938, WO 94/28152, WO 94/12649, WO 95/02697, WO 96/22378). The replication defective recombinant adenoviruses according to the invention can be prepared by any technique known to the person skilled in the art (Levrero et al., Gene, 1991, 101:195; European Publication No. EP 185 573; Graham, EMBO J., 1984, 3:2917; Graham et al., J. Gen. Virol., 1977, 36:59). Recombinant adenoviruses are recovered and purified using standard molecular biological techniques, which are well known to one of ordinary skill in the art.

[0110] Adeno-associated viruses. The adeno-associated viruses (AAV) are DNA viruses of relatively small size that can integrate, in a stable and site-specific manner, into the genome of the cells which they infect. They are able to infect a wide spectrum of cells without inducing any effects on cellular growth, morphology or differentiation, and they do not appear to be involved in human pathologies. The AAV genome has been cloned, sequenced and characterized. The use of vectors derived from the AAVs for transferring genes in vitro and in vivo has been described (see, PCT Publication Nos. WO 91/18088 and WO 93/09239; U.S. Patent Nos. 4,797,368 and 5,139,941; European Publication No. EP 488 528). The replication defective recombinant AAVs according to the invention can be prepared by cotransfecting a plasmid containing the nucleic acid sequence of interest flanked by two AAV inverted terminal repeat (ITR) regions, and a plasmid carrying the AAV encapsidation genes (rep and cap genes), into a cell line which is infected with a human helper virus (for example an adenovirus). The AAV recombinants which are produced are then purified by standard techniques.

[0111] Retrovirus vectors. In another embodiment the gene can be introduced in a retroviral vector, e.g., as described in U.S. Patent No. 5,399,346; Mann et al., Cell, 1983, 33:153; U.S. Patent Nos. 4,650,764 and 4,980,289; Markowitz et al., J. Virol., 1988, 62:1120; U.S. Patent No. 5,124,263; European Publication Nos. EP 453 242 and EP 178 220; Bernstein et al., Genet. Eng.,1985, 7:235; McCormick, BioTechnology,1985, 3:689; PCT Publication No. WO 95/07358; and Kuo et al., Blood, 1993, 82:845. The retroviruses are integrating viruses that infect dividing cells. The retrovirus genome includes two LTRs, an encapsidation sequence and three coding regions (gag, pol and env). In recombinant retroviral vectors, the gag, pol and env genes are generally deleted, in whole or in part, and replaced with a heterologous nucleic acid sequence of interest. These vectors can be constructed from different types of retrovirus, such as, HIV, MoMuLV ("murine Moloney leukaemia virus" MSV ("murine Moloney sarcoma virus"), HaSV ("Harvey sarcoma virus"); SNV ("spleen necrosis virus"); RSV ("Rous sarcoma virus") and Friend virus. Suitable packaging cell lines have been described in the prior art, in particular the cell line PA317 (U.S. Patent No. 4,861,719); the PsiCRIP cell line (PCT Publication No. WO 90/02806) and the GP+envAm-12 cell line (PCT Publication No. WO 89/07150). In addition, the recombinant retroviral vectors can contain modifications within the LTRs for suppressing transcriptional activity as well as extensive encapsidation sequences which may include a part of the gag gene (Bender et al., J. Virol., 1987, 61:1639). Recombinant retroviral vectors are purified by standard techniques known to those having ordinary skill in the art.

[0112] Retroviral vectors can be constructed to function as infectious particles or to undergo a single round of transfection. In the former case, the virus is modified to retain all of its genes except for those responsible for oncogenic transformation properties, and to express the heterologous gene. Non-infectious viral vectors are manipulated to destroy the viral packaging signal, but retain the structural genes required to package the co-introduced virus engineered to contain the heterologous gene and the packaging signals. Thus, the viral particles that are produced are not capable of producing additional virus.

[0113] Retrovirus vectors can also be introduced by DNA viruses, which permits one cycle of retroviral replication and amplifies tranfection efficiency (see PCT Publication Nos. WO 95/22617, WO 95/26411, WO 96/39036 and WO 97/19182).

[0114] Lentivirus vectors. In another embodiment, lentiviral vectors can be used as agents for the direct delivery and sustained expression of a transgene in several tissue types, including brain, retina, muscle, liver and blood. The vectors can efficiently transduce dividing and nondividing cells in these tissues, and maintain long-term expression of the gene of interest. For a review, see, Naldini, Curr. Opin. Biotechnol., 1998, 9:457-63; see also Zufferey, et al., J. Virol., 1998, 72:9873-80). Lentiviral packaging cell lines are available and known generally in the art. They facilitate the production of high-titer lentivirus vectors for gene therapy. An example is a tetracycline-inducible VSV-G pseudotyped lentivirus packaging cell line that can generate virusparticles at titers greater than 106 IU/ml for at least 3 to 4 days (Kafri, et al., J. Virol., 1999, 73: 576-584). The vector produced by the inducible cell line can be concentrated as needed for efficiently transducing non-dividing cells in vitro and in vivo.

[0115] Non-viral vectors. In another embodiment, the vector can be introduced in vivo by lipofection, as naked DNA, or with other transfection facilitating agents (peptides, polymers, etc.). Synthetic cationic lipids can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Felgner, et. al., Proc. Natl. Acad. Sci. U.S.A., 1987, 84:7413-7417; Felgner and Ringold, Science, 1989, 337:387-388; see Mackey, et al., Proc. Natl. Acad. Sci. U.S.A., 1988, 85:8027-8031; Ulmer et al., Science,1993, 259:1745-1748). Useful lipid compounds and compositions for transfer of nucleic acids are described in PCT Patent Publication Nos. WO 95/18863 and WO 96/17823, and in U.S. Patent No. 5,459,127. Lipids may be chemically coupled to other molecules for the purpose of targeting (see Mackey, et. al., supra). Targeted peptides, e.g., hormones or neurotransmitters, and proteins such as antibodies, or non-peptide molecules could be coupled to liposomes chemically.

[0116] Other molecules are also useful for facilitating transfection of a nucleic acid in vivo, such as a cationic oligopeptide (e.g., PCT Patent Publication No. WO 95/21931), peptides derived from DNA binding proteins (e.g., PCT Patent Publication No. WO 96/25508), or a cationic polymer (e.g., PCT Patent Publication No. WO 95/21931).

[0117] It is also possible to introduce the vector in vivo as a naked DNA plasmid. Naked DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, e.g., electroporation, microinjection, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun, or use of a DNA vector transporter (e.g., Wu et al., J. Biol. Chem., 1992, 267:963-967; Wu and Wu, J. Biol. Chem., 1988, 263:14621-14624; Canadian Patent Application No. 2,012,311; Williams el al., Proc. Natl. Acad. Sci. USA, 1991, 88:2726-2730). Receptor-mediated DNA delivery approaches can also be used (Curiel et al., Hum. Gene Ther., 1992, 3:147-154; Wu and Wu, J. Biol. Chem., 1987, 262:4429-4432). U.S. Patent Nos. 5,580,859 and 5,589,466 disclose delivery of exogenous DNA sequences, free of transfection facilitating agents, in a mammal. Recently, a relatively low voltage, high efficiency in vivo DNA transfer technique, termed electrotransfer, has been described (Mir et al., C.P. Acad. Sci., 1988, 321:893; PCT Publication Nos. WO 99/01157; WO 99/01158; WO 99/01175).

Assay System

[0118] Any cell assay system that allows for assessing functional activities of immunogenic compositions and compounds that modulate binding of PPP1 to iron is contemplated by the present invention. In a specific embodiment, the assay can be used to identify compounds that interact with PPP1 to decrease binding of PPP1, described herein, to iron. This can be evaluated by assessing the effects of a test compound on the interaction the protein described herein. A cell assay system that assesses the ability of the compound to elicit opsonophagocytic antibodies against S. pneumoniae may also be utilized (Gray, B.M. 1990. Conjugate Vaccines Supplement p694-697).

[0119] Any convenient method that permits detection of the binding of iron with PPP are contemplated by the present invention. In a preferred embodiment of the invention, protein components of S. pneumoniae can be separated on a polyacrylamide gel and transferred to a solid support. The support then may be probed with a labeled interacting component (e.g. iron). The component may be labeled with any label known in the art including, but not limited to, radioactivity, enzyme-based, dye molecules, or a flourescent or phosphorescent tag. In a preferred embodiment, the label is radioactive. The label may be detected by any means known in the art. For example, autoradiography, scintillation counter, or ultra-violet light. In a preferred embodiment, the radiolabel is detected by autoradiography. Assays that amplify the signals from the probe are also known, such as, for example, those that utilize biotin and avidin, and enzyme-labeled immunoassays, such as ELISA assays.

In Vitro Screening Methods

[0120] Candidate agents are added to assay systems, prepared by known methods in the art, and the level of binding betwen iron and PPP1 is measured. Various in vitro systems can be used to analyze the effects of a compound on iron binding. Preferably, each experiment is performed more than once, such as, for example, in triplicate at multiple different dilutions of compound.

[0121] The screening system of the invention permits detection of binding inhibitors. An inhibitor screen involves detecting interaction of iron and PPP 1 when contacted with a compound that regulates interaction of these proteins. If a decrease in the binding of iron to PPP1 is detected, then the compound is a candidate inhibitor. If no decrease is observed, the compound does not alter the binding of iron to the protein of the present invention.

Immunogenic Compositions

[0122] In further embodiments of this invention PPP1 are employed in immunogenic compositions comprising (i) at least one PPP1; (ii) at least one pharmaceutically acceptable buffer, diluent, or carrier; and (iii) optionally at least one adjuvant. In a preferred embodiment, the immunogenic composition is used as a vaccine. The PPP1 may be recombinantly produced or isolated from a bacterial preparation, according to methods known in the art. Preferably, these compositions have therapeutic and prophylactic applications as immunogenic compositions in preventing, protecting and/or ameliorating pneumococcal infection. In such applications, an immunologically effective amount of at least one PPP1 is employed in such amount to cause a reduction, preferably a substantial reduction, in the course a normal pneumoccocal infection. The proteins may be attenuated. The term "attenuated" refers to a protein that maintains its immunogenic activity, while one or more other functional characteristics are decreased or deleted. For example, the attenuated form of this protein may exhibit diminished binding properties, such as its ability to bind iron. Alternatively, the attenuated form may decrease the ability of S. pneumoniae to bind iron.

[0123] As used herein, the term "effective amount" refers to amount of the immunogen component (i.e. PPP1) described herein to stimulate an immune response, i.e., to cause the production of antibodies and/or a cell-mediated response when introduced into a subject. In a preferred embodiment, the effective amount will decrease the colonization of S. pneumoniae. The term "immunogen component" refers to the ability of this component to stimulate secretory antibody and/or cell-mediated response production in local regions, e.g. nasopharynx, when administered systemically as an immunogenic composition according to the present invention.

[0124] As used herein the term "adjuvant" refers to an agent, compound or the like, which potentiates or stimulates the immune response in a subject when administered in combination with the immunogenic composition. Thus, the immune response, elicited by the immunogenic composition combination, as measured by any convention method known in the art, will generally be greater than that provoked by the immunogenic composition alone.

[0125] The compositions of the invention can include an adjuvant, including, but not limited to aluminum hydroxide; aluminum phosphate; Stimulon™ QS-21 (Aquila Biopharmaceuticals, Inc., Framingham, MA); MPL™ (3-O-deacylated monophosphoryl lipid A; Corixa, Seattle, Washington); RC529 (Corixa) and aminoalkyl glucosamine phosphate compounds as described in PCT Published Application WO 98/50399 (RIBI Immunochem Research); IL-12 (Genetics Institute, Cambridge, MA); N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP); N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor-MDP); N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphos-phoryloxy)-ethylamine (CGP 19835A, referred to a MTP-PE); granulocyte-macrophage colony stimulating factor (GM-CSF) and cholera toxin. Others which may be used are non-toxic derivatives of cholera toxin, including its B subunit (for example, wherein glutamic acid at amino acid position 29 is replaced by another amino acid, preferably, a histidine in accordance with Published International Patent Application WO 00/18434), and/or conjugates or genetically engineered fusions of non-PPP polypeptides with cholera toxin or its B subunit, procholeragenoid, fungal polysaccharides. The adjuvant may be used in its natural form or one can use a synthetic or semi-synthetic version of an adjuvant. Any formulation of the adjuvant may be used depending on the desired response and admininstration method. Various forms of the adjuvant may be used, e.g., a liquid, powder or emulsion.

[0126] The immunogenic composition may be administered as a single bolus dose or as a "series" of administrations over a defined period of time (e.g., one year). When given in later year, such series of administrations is referred to as "booster shots". These administrations increase the antibody levels produced by the previous administration. The immunogenic compound may be administered until sufficient antibody levels have been identified in the subject, so as to induce an immune response upon challenge from the immunogen.

[0127] The formulation of such immunogenic compositions is well known to persons skilled in this field. Immunogenic compositions of the invention may comprise additional antigenic components (e.g., polypeptide or fragment thereof or nucleic acid encoding an antigen or fragment thereof) and, preferably, include a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers and/or diluents include any and all conventional solvents, dispersion media, fillers, solid carriers, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The term "pharmaceutically acceptable carrier" refers to a carrier that does not cause an allergic reaction or other untoward effect in patients to whom it is administered. Suitable pharmaceutically acceptable carriers include, for example, one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antigen. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in immunogenic compositions of the present invention is contemplated.

Compositions

[0128] In further embodiments of this invention, PPP I nucleic acid sequences, amino acid sequences, expression vectors or host cells are employed in compositions comprising (i) at least one PPP1 protein, or nucleic acid encoding an amino acid sequence of a PPP1, or an expression vector or host cell that expresses such nucleic acid arid (ii) at least one of a pharmaceutically acceptable buffer, diluent, or carrier. The PPP1 may be recombinantly produced or isolated from a bacterial preparation, according to methods known in the art. Preferably, these compositions have therapeutic and prophylactic applications. In such applications, a pharmaceutically effective amount of at least one PPP1 is employed in such amount to produce a defined functional activity. As used herein, the term "effective amount" refers to amount of the PPP 1 protein described herein, to produce a functional effect.

[0129] Administration of such compositons or immunogenic compositions may be by any conventional effective form, such as intranasally, parenterally, orally, or topically applied to mucosal surface such as intranasal, oral, eye, lung, vaginal, or rectal surface, such as by aerosol spray. The preferred means of administration is parenteral or intranasal.

[0130] Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like.

[0131] The polynucleotides and polypeptides of the present invention may be administered as the sole active immunogen in an immunogenic composition. Alternatively, however, the immunogenic composition may include other active immunogens, including other immunologically active antigens from other pathogenic species. Preferably, the pathogenic species that provide other immunologically active antigens are bacterial pathogens, e.g., involved in bacterial infections. Indeed, preferably therapeutic use of the PPP antigen of the invention will be as a component of a multivalent vaccine that includes other bacterial antigens from S. pneumonia or other pathogenic bacteria. The other immunologically active antigens may be replicating agents or non-replicating agents. Replicating agents include, for example, attenuated forms of measles virus, rubella virus, variscella zoster virus (VZV), Parainfluenza virus (PIV), and Respiratory Syncytial virus (RSV).

[0132] One of the important aspects of this invention relates to a method of inducing immune responses in a mammal comprising the step of providing to said mammal an immunogenic composition of this invention. The immunogenic composition is a composition which is immunogenic in the treated animal or human such that the immunologically effective amount of the polypeptide(s) contained in such composition brings about the desired response against pneumococcal infection. Preferred embodiments relate to use in the manufacture of a medicament for the treatment, including amelioration, or prevention of pneumococcal infection in a human of an immunologically effective amount of the immunogenic composition. The dosage amount can vary depending upon specific conditions of the individual. This amount can be determined in routine trials by means known to those skilled in the art.

[0133] Certainly, the isolated amino acid sequences for the proteins of the present invention may be used in forming subunit immunogenic compositions. They also may be used as antigens for raising polyclonal or monoclonal antibodies and in immunoassays for the detection of anti-PPP1 protein-reactive antibodies. Immunoassays encompassed by the present invention include, but are not limited to, those described in U.S. Patent No. 4,367,110 (double monoclonal antibody sandwich assay) and U.S. Patent No. 4,452,901 (western blot). Other assays include immunoprecipitation of labeled ligands and immunocytochemistry, both in vitro and in vivo.

Methods of Inducing an Immune Response

[0134] According to the present invention, colonization of S. pneumonia involves PPP1 proteins. The present invention provides use in the manufacture of a medicament to prevent pneumococal infections of a therapeutically effective amount of an immunogenic composition that induces an immune response in the subject. These uses include, but are not limited to, administration of an immunogenic composition comprised of at least one PPP1 protein, variant, fragment or attenuated version thereof, or at least one expression vector encoding the protein variant, fragment or attenuated version thereof..

Methods of Inhibiting Pneumococcal Infection

[0135] The present invention further provides the use in the manufacture of a medicament to induce an immune response in a subject which is infected with pneumococal bacteria of a therapeutically effective amount of a composition or compound that blocks functional effects associated with the PPP1 proteins. These methods include, but are not limited to, administration of a composition comprised of at least one PPP1 protein or fragments thereof or at least one expression vector encoding a PPP1 protein or administration of a compound that blocks, substantially all or at least in part, a function of the PPP1 proteins.

Methods of Diagnosis

[0136] This invention also provides for a method of diagnosing a pneumococcal infection, or identifying a pneumococcal immunogenic compositon strain that has been administered, comprising the step of determining the presence, in a sample, of an amino acid sequence of SEQ ID NO: 5 or any of 10-19. Any conventional diagnostic method may be used. These diagnostic methods can easily be based on the presence of an amino acid sequence or polypeptide. Preferably, such a diagnostic method matches for a polypeptide having at least 10, and preferably at least 20, amino acids which are common to the amino acid sequences of this invention.

[0137] The nucleic acid sequences disclosed herein also can be used for a variety of diagnostic applications. These nucleic acids sequences can be used to prepare relatively short DNA and RNA sequences that have the ability to specifically hybridize to the nucleic acid sequences encoding the PPP 1 protein. Nucleic acid probes are selected for the desired length in view of the selected parameters of specificity of the diagnostic assay. The probes can be used in diagnostic assays for detecting the presence of pathogenic organisms, or in identifying a pneumococcal immunogenic composition that has been administered, in a given sample. With current advanced technologies for recombinant expression, nucleic acid sequences can be inserted into an expression construct for the purpose of screening the corresponding oligopeptides and polypeptides for reactivity with existing antibodies or for the ability to generate diagnostic or therapeutic reagents. Suitable expression control sequences and host cell/cloning vehicle combinations are well known in the art, and are described by way of example, in Sambrook et al. (1989).

[0138] In preferred embodiments, the nucleic acid sequences employed for hybridization studies or assays include sequences that are complementary to a nucleotide stretch of at least about 10, preferably about 15, and more preferably about 20 nucleotides. A variety of known hybridization techniques and systems can be employed for practice of the hybridization aspects of this invention, including diagnostic assays such as those described in Falkow et al., US Patent 4,358,535. Preferably, the sequences recognize or bind a nucleic acid sequence on the PPP 1 protein are consecutive.

[0139] In general, it is envisioned that the hybridization probes described herein will be useful both as reagents in solution hybridizations as well as in embodiments employing a solid phase. In embodiments involving a solid phase, the test DNA (or RNA) from suspected clinical samples, such as exudates, body fluids (e.g., middle ear effusion, bronchoalveolar lavage fluid) or even tissues, is absorbed or otherwise affixed to a selected matrix or surface. This fixed, single-stranded nucleic acid is then subjected to specific hybridization with selected probes under desired conditions. The selected conditions will depend on the particular circumstances based on the particular criteria required (depending, for example, on the G+C contents, type of target nucleic acid, source of nucleic acid, size of hybridization probe). Following washing of the hybridized surface so as to remove nonspecifically bound probe molecules, specific hybridization is detected, or even quantified, by means of the label.

[0140] The nucleic acid sequences which encode the PPP1 protein of the invention, or their variants, may be useful in conjunction with PCR* technology, as set out, e.g., in U.S. Patent 4,603,102. One may utilize various portions of any of the PPP1 protein sequences of this invention as oligonucleotide probes for the PCR* amplification of a defined portion of a PPP I gene, or nucleotide, which sequence may then be detected by hybridization with a hybridization probe containing a complementary sequence. In this manner, extremely small concentrations of the PPP1 nucleic acid sequence may be detected in a sample utilizing the nucleotide sequences of this invention.

[0141] The following examples are included to illustrate certain embodiments of the invention.

Antibodies

[0142] The present invention describes antibodies that may be used to detect the presence of PPP1 proteins present in samples. Additionally, the antibodies (e.g., anti-idiotypic antibodies) may be used to inhibit immune responses to pneumococcal infections.

[0143] According to the invention, PPP 1 protein polypeptides produced recombinantly or by chemical synthesis, and fragments or other derivatives, may be used as an immunogen to generate antibodies that recognize the polypeptide or portions thereof. The portion of the polypeptide used as an immunogen may be specifically selected to modulate immunogenicity of the developed antibody. Such antibodies include, but are not limited to, polyclonal, monoclonal, humanized, chimeric, single chain, Fab fragments, and an Fab expression library. An antibody that is specific for human PPP1 protein may recognize a wild-type or mutant form of the PPP1 proteins. In a specific embodiment, the antibody is comprised of at least 8 amino acids, preferably from 8-10 amino acids, and more preferably from 15-30 amino acids. Preferably, the antibody recognizes or binds amino acids on PPP1 are consecutive.

[0144] Various procedures known in the art may be used for the production of polyclonal antibodies to polypeptides, derivatives, or analogs. For the production of antibody, various host animals, including but not limited to rabbits, mice, rats, sheep, goats, etc, can be immunized by injection with the polypeptide or a derivative (e.g., fragment or fusion protein). The polypeptide or fragment thereof can be conjugated to an immunogenic carrier, e.g., bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH). Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum.

[0145] Monoclonal antibodies directed toward a PPP1 protein, fragment, analog, or derivative thereof, may be prepared by any technique that provides for the production of antibody molecules by continuous cell lines in culture may be used. These include but are not limited to the hybridoma technique originally developed by Kohler and Milstein (Nature 256:495-497, 1975), as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al., Immunology Today 4:72, 1983; Cote et al., Proc. Natl. Acad. Sci. U.S.A. 80:2026-2030, 1983), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96,1985). "Chimeric antibodies" may be produced (Morrison et al., J. Bacteriol. 159:870, 1984; Neuberger et al., Nature 312:604-608, 1984; Takeda et al., Nature 314:452-454, 1985) by splicing the genes from a non-human antibody molecule specific for a polypeptide together with genes from a human antibody molecule of appropriate biological activity.

[0146] In the production and use of antibodies, screening for or testing with the desired antibody can be accomplished by techniques known in the art, e.g., radioimmunoassay, ELISA (enzyme-linked immunosorbant assay), "sandwich" immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc.

[0147] The foregoing antibodies can be used in methods known in the art relating to the localization and activity of the polypeptide, e.g., for Western blotting, imaging the polypeptide in situ, measuring levels thereof in appropriate physiological samples, etc. using any of the detection techniques mentioned above or known in the art. Such antibodies can also be used in assays for ligand binding, e.g., as described in U.S. Patent No. 5,679,582. Antibody binding generally occurs most readily under physiological conditions, e.g., pH of between about 7 and 8, and physiological ionic strength. The presence of a carrier protein in the buffer solutions stabilizes the assays. While there is some tolerance of perturbation of optimal conditions, e.g., increasing or decreasing ionic strength, temperature, or pH, or adding detergents or chaotropic salts, such perturbations will decrease binding stability.

[0148] In a specific embodiment, antibodies that agonize the activity of the PPP 1 protein can be generated. In particular, intracellular single chain Fv antibodies can be used to regulate the PPP1 protein. Such antibodies can be tested using the assays described below for identifying ligands.

[0149] In another specific embodiment, the antibodies of the present invention are anti-idiotypic antibodies. These antibodies recognize and or bind to other antibodies present in the system. The anti-idiotypic antibodies may be monoclonal, polyclonal, chimeric, humanized.

[0150] In another specific embodiment, antibodies of the present invention are conjugated to a secondary component, such as, for example, a small molecule, polypeptide, or polynucleotide. The conjugation may be produced through a chemical modification of the antibody, which conjugates the antibody to the secondary component. The conjugated antibody will allow for targeting of the secondary component, such as, for example, an antibiotic to the site of interest. The secondary component may be of any size or length. In a specific embodiment, the secondary component is a pharmaceutically active compound.

[0151] A further aspect of this invention relates to the use of antibodies, as discussed supra, for targeting a pharmaceutical compound. In this embodiment, antibodies against the PPP1 protein are used to present specific compounds to infected sites. The compounds, preferably an antibiotic agent, when conjugated to the antibodies are referred to as targeted compounds or targeted agents. Methods for generating such target compounds and agents are known in the art. Exemplary publications on target compounds and their preparation are set forth in U.S. Patent Nos. 5,053,934; 5,773,001; and 6,015,562.

EXAMPLES

Materials and Methods

Bacterial Strains and Plasmids

[0152] S. pneumoniae strains utilized in this work were S. pneumoniae CP1200, a nonencapsulated, highly transformable derivative of R36A, a rough variant of D39, a virulent type 2 strain, (Morrison, D.A. et al., J. Bacteriology, 1983, 156:281) was obtained from Margaret Hostetter at Yale University, CT., and S. pneumoniae strain 49136 obtained from the ATCC. S. pneumoniae were grown to log phase (approx O.D. of 0.6-0.8 at 600nm) in Todd Hewitt media (Difco Lab., Detroit, MI) with 0.5% yeast extract (Difco) at 37°C with aeration or on Tryptic Soy (Difco) blood agar plates. Escherichia coli strains used in this study were BL21(DE3), BLR(DE3) (Novagen, Madison, WI), Top10F'(Invitrogen, San Diego, CA), and were grown in SOB media (15) at 37 °C with aeration containing appropriate antibiotics. Plasmids used in this work were PCR2.1 TOPO (Invitrogen) and pET28a (Novagen). Where specified, chloramphenicol was used at 20 µg/ml, ampicillin at 100 µg/ml, streptomycin at 100 µg/ml, and kanamycin at 25 µg/ml. Restriction enzymes were purchased from New England Biolabs (Beverly, MA) and used according to manufactures directions.

Identification of a Surface Associated Protein in Outer Membrane Fractions of S. pneumoniae

[0153] Extraction of surface associated components - Bacteria were grown in 4 liters of Todd Hewitt broth, and harvested by centrifugation at 8000 x g for 30 minutes. The pellet was suspended in ~175 ml of PBS with the aid of a pipette and immediately centrifuged at 20000 x g for 30 min. The wash was filtered through a 0.45m filter (Nalgene, Rochester, NY), dialyzed and lyophilized. Ion-exchange chromatography of surface associated protein components -The PBS extract of S. pneumoniae was dissolved in Tris-HCl, pH 7.6 (10 mM, 100 ml) and subjected to ion exchange chromatography in a column of DEAE-Sepharose CL-6B. After washing the column with the sample buffer, it was eluted first with 200 mM Tris-HCl, pH 7.6 followed by a linear NaCl gradient to a final NaCl concentration of 0.75 M (in 200 mM Tris-HCl, pH 7.6) over 300 ml. Column fractions were analyzed by SDS-PAGE gel. Fractions containing a substantial amount of a surface associated protein of approximately 18- 20kDa were pooled, desalted by Centricon SR3 concentrator and lyophilized.

N-terminal Amino Acid Sequence Analysis by PVDF Blot Excision.

[0154] The sample was diluted to 1 mg/mL total protein and combined 1:1 with 2X Tris-SDS-β-ME sample loading buffer (0.25M Tris-HCl pH6.8, 2% SDS, 10% β-mercaptoethanol, 30% glycerol, 0.01% Bromophenol Blue) (Owl Separation, Portsmouth, NH) and heated at 100°C for 5 minutes. Approximately 10µg of total protein (20uL of heated solution) of sample was loaded in each of ten lanes on a 12 lane, 10cm x 10cm x 1mm, 10-20% gradient acrylamide/bis-acrylamide gel (Zaxis, Hudson, OH). Molecular weight markers (Novex, San Diego, CA) were loaded in the two outermost lanes of each side of the gel. Electrophoresis was carried out on an Owl Separations Mini-Gel rig at a constant amperage of 50 mA for 1 hour in Bio-Rad Tris-Glycine-SDS running buffer. The gel was then rinsed with deionized water and transferred to Millipore Immobilon-P PVDF (polyvinylidene fluoride) using a semi-dry blotting system supplied by Owl Separations at constant amperage of 150 mA for 1 hour. The resulting blot was stained with Amido Black (10% acetic acid, 0.1 % amido black in deionized water) and destained in 10% acetic acid. The protein band was then excised from all ten lanes using a methanol cleaned scalpel or mini-Exacto knife and placed in the reaction cartridge of the Applied Biosystems 477A Protein Sequencer (Foster City, CA). The N-terminal Sequencer was then run under optimal blot conditions for 12 or more cycles (1 cycle Blank, 1 cycle Standard, and 10 or more cycles for desired residue identification). PTH-amino acid detection was done on the Applied Biosystems 120A PTH Analyzer. The cycles were collected both on an analog chart recorder and digitally via the instrument software. N- terminal Amino acid assignment was perfomed by comparison of the analog and digital data to a standard set of PTH-amino acids and their respective retention times on the analyzer (cysteine residues are destroyed during conversion and are not detected).

Subcloning and Expression of the Recombinant 20kDa Surface Associated Proteins

[0155] N-terminal sequence was compared against the NCBI non redundant database located at www.ncbi.nlm.org using the BLAST algorithim developed by Altschul (Altschul, SF, et al., J. Mol-Biol., 1990, 215:403). This showed that the N-terminal sequence had identity to a open reading frame (ORF) in NCBI database. This ORF had been previously sequenced and was listed as an unidentified ORF (Pikis, A. et al., J. Infect. Dis., 1998,178:700). Subsequent BLAST analysis of the unknown ORF against the public release of the S. pneumoniae genome (serotype 4), made available by The Institute for Genomic Research (TIGR, www.tigr.org), showed the ORF to be present in the genome, but unidentified as well. DNA analyses of the unknown ORF in the S. pneumoniae genomic sequence and primer designs were performed using the DNASTAR (Madison, WI) Lasergene DNA and protein analysis software.

[0156] Primers flanking the ORF were designed (SEQ ID NOs: 1 and 2) and subsequently synthesized using the AB1380A DNA synthesizer. To facilitate subcloning the PCR product into the pET28a expression vector, restriction sites were designed into the PCR primers. An Nco 1 site was included in the 5' primer, which allowed both for the ligation into the Nco 1 site of the expression vector and also included an ATG start codon. To maintain the correct reading frame, two extra bases were included in the 5' primer, resulting in the addition of a codon for Leucine. A Sal1 site was included in the 3' primer.

[0157] A PCR fragment of the expected size was generated from CP1200, ligated into the pCR2.1 vector, and used to transform OneShot Top 10F' cells (Invitrogen). Ampicillin resistant transformants were screened screened by restriction digestion of plasmid DNA prepared by alkaline lysis (Birnboim, H.C. and Duly, J., Nuc. Acid Res., 1978 7:1513). A recombinant plasmid, containing the 20kDa gene, was identified. DNA sequence was obtained from the clones using the Applied Biosystems Prism Dye Terminator cycle-sequencing core kit based on the Prism protocol supplied by the vendor. Approximately 1 ug of template DNA and 100 ng of primer were used for each cycling reaction. The reactions were cycled on the GeneAmp PCR Systems 2400 unit, purified using the Prism method, and analyzed on an ABI 373A DNA sequencer (Applied Biosystems).

[0158] The insert containing the r20kDa gene was excised by restriction digestion with Nco1 and Sal1, and separated on a 1.5% Agarose gel. The DNA fragment was cut from the gel and purified away from the agarose by a Bio 101 Spin kit (Vista, CA). The insert was ligated with plasmid vector DNA(pET28a) also digested with Nco1 and Sal1, and was subsequently transformed into Top10F' cells (Invitrogen). The kanamycin resistant transformants were screened by restriction digestion of plasmid DNA prepared by alkaline lysis (Birnboim, H.C. and Duly J., Nuc. Acid Res., 1978 7:1513). A recombinant plasmid was subsequently transformed into BL21 cells (Novagen) to create pLP533 and grown in SOB media supplemented with 30ug/ml kanamycin. Cells were grown to an O.D.₆₀₀ of 0.6, and were subsequently induced with 0.4mM IPTG (Boehringer Mannheim, Indianapolis, IN) for 2-4 hours. Whole cell lysates were prepared and electrophoresed on a 15% SDS-PAGE gel (Laemmli, U.K., Nature,1970, 227:680) to confirm expression of the desired recombinant product.

Purification of the recombinant 20kDa surface associated protein.

[0159] A 250 mL flask containing 50 mL of SOB medium, supplemented with 30 µg/mL kanamycin (Sigma, St. Louis, MO), was inoculated with a scraping from a frozen culture of E. coli pLP533. The culture was incubated at 37° C with shaking at 200 rpm for approximately 16 hours. Subsequently, two 1 liter flasks containing SOB plus 30 ug/ml kanamycin were inoculated with 20 mL of the overnight culture and incubated at 37° C with shaking at 200 rpm. When the culture reached an optical density of OD₆₀₀ 0.7 - 0.8, IPTG (Gold Biotechnology, St. Louis, MO) was added to 0.8 mM. The culture was incubated at the same temperature with shaking for an additional three hours. The cells were then harvested by centrifugation for 15 min. at 7300 xg. The cell pellets were frozen at -20° C and were then thawed and resuspended in 300 mL of 10 mM sodium phosphate pH 6.0 (J.T. Baker, Phillipsburg, PA ). The cell suspension was then passed through a microfluidizer (Microfluidics Corporation, Newton, MA) to lyse the cells. The lysate was centrifuged for 15 min. at 16,000 x g and the resulting supernatant was then centrifuged for 45 min. at 200,000 x g. Supernatants and pellets at each step were assayed by SDS-PAGE. The supernatant was diluted to 500 mL in 10 mM sodium phosphate pH 6.0. The solution was then diafiltered with a 100,000 MW cutoff membrane (Millipore, Bedford, MA) against 1 L of the same buffer and concentrated 2.5 fold. The protein, in the retentate, was loaded onto a 70 mL ceramic hydroxyapatite column (Bio-Rad Laboratories Hercules, CA) in 10 mM sodium phosphate pH 6.0. The column was then washed with 10 column volumes (CV) of the loading buffer. Contaminating proteins were removed by washing the column with 10 CV of 108 mM sodium phosphate pH 6.0. The protein was eluted from the column with a linear gradient over 10 CV from 108 mM to 500 mM sodium phosphate pH 6.0. The peak fractions were run on a 10% - 20% SDS-PAGE gel (Zaxis, Hudson, OH). The fractions containing the protein were pooled and stored at -20° C. The protein was analyzed for homogeneity by SDS-PAGE, and the concentration of protein during purification was determined by the method of Lowry (Lowry, O.H., et al, S. Biol. Chem., 1951, 198:265). Protein concentration prior to immunization was determined using a BCA kit obtained from Pierce Chemicals (Northbrook, IL) and was used according to the manufacturers directions. BSA was used as protein standard.

Polyclonal Antisera for western blot analysis.

[0160] Recombinant protein was used to generate polyclonal antisera in mice. Briefly, 10 µg of r20kDa protein was adjuvanted for each dose as an emulsion with Incomplete Freund's Adjuvant (IFA) (1:1 v/v) and injected subcutaneously into 6-8 week old Swiss Webster mice. The mice were bled and vaccinated at wk 0, boosted at wk4, then exsanguinated at wk 6. Ten mice were vaccinated with the r20kDa protein adjuvanted with IFA. The sera where pooled and used for further analysis.

SDS-PAGE and Western blotting.

[0161] Whole cell lysates were prepared by centrifuging equivalent numbers of pneumococcal cells, based on the OD₆₀₀, in a microcentrifuge for 30 sec. Pneumococcal cell pellets were resuspended in an appropriate volume of loading buffer. Where indicated, samples were boiled for 5 min and separated on a 10% SDS-PAGE gel using the method of Laemmli (Laemmli, Nature, 1970; 227:680). The samples were transferred to nitrocellulose (BioRad, Hercules, CA) using a Biorad Mini Transblot cell (Biorad) and the blots were blocked at room temp for 30 minutes in 5% nonfat milk-PBS (BLOTTO). Pooled mouse antisera were used at a 1:1000 dilution in BLOTTO for 60 minutes, followed by 25 minute washes in PBS- 0.2% Tween80. Goat anti-mouse IgG+M conjugated to alkaline phosphatase (Biosource International, Camarillo, CA) was used to detect bound antibodies at a 1:1000 dilution in BLOTTO. The blots were washed as previously described and detected with NBT and BCIP from BioRad according to the manufacturer's directions.

Intranasal immunization of mice prior to challenge.

[0162] Six-week old, pathogen-free, Balb/c mice were purchased from Jackson Laboratories (Bar Harbor, Maine) and housed in cages under standard temperature, humidity, and lighting conditions. BALB/C mice, at 10 animals per group, were immunized with 5 µg of r20kDa protein. On weeks 0, 2, and 4. On each occasion, 5 µg r20kDa formulated with 0.1 µg of CT-E29H, a genetically modified cholera toxin that is reduced in enzymatic activity and toxicity (Tebbey, P:W., et al., Vaccine, 2000, 18:2723), was.slowly instilled into the nostril of each mouse in a 10 µl volume. Mice immunized with Keyhole Limpet Hemocyanin (KLH)-CT-E29H were used as controls. Serum samples were collected 4 days after the last immunization.

Mouse intranasal challenge model.

[0163] Balb/c mice were challenged on week 4 day 6 with 1X10⁵ CFU's of serotype 3 streptomycin resistant S. pneumoniae. Pneumococci were inoculated into 3 ml of Todd-Hewitt broth containing 100 µg/ml of streptomycin. The culture was grown at 37°C until mid-log phase, then diluted to the desired concentration with Todd-Hewitt broth and stored on ice until use. Each mouse was anesthetized with 1.2 mg of ketamine HCl (Fort Dodge Laboratory, Ft. Dodge, Iowa) by i.p. injection. The bacterial suspension was inoculated to the nostril of anesthetized mice (10 µl per mouse). The actual dose of bacteria administrated was confirmed by plate count. Four days after challenge, mice were sacrificed, the noses were removed, and homogenized in 3-ml sterile saline with a tissue homogenizer (Ultra-Turax T25, Janke & Kunkel Ika-Labortechnik, Staufen, Germany). The homogenate was 10-fold serially diluted in saline and plated on streptomycin containing TSA plates. Fifty µl of blood collected 2 days post-challenge from each mouse was also plated on the same kind of plates. Plates were incubated overnight at 37°C and then colonies were counted.

ELISA Assay for r20kDa protein.

[0164] Antibody titers against r20kDa protein were determined by enzyme-linked immunosorbent assay (ELISA). ELISAs were performed using r20kDa (100 µl per well of a 5 µg/ml stock in PBS, pH7.1) to coat Nunc-Immuno^™ PolySorp Plates. Plates were coated overnight at 4°C. After blocking with 200 µl of PBS containing 5% nonfat dry milk (blocking buffer) for 1 hour at room temperature, the plates were incubated with serial dilutions of test sera diluted in blocking buffer for 1.5 hours at room temperature. The plates were then washed five times with PBS containing 0.1% Tween (PBS-T) and incubated with biotinylated goat anti-mouse IgG or IgA (1:8000 or 1:4000 in PBS; Brookwood Biomedical, Birmingham, AL) for 1 hour at room temperature. After five additional washes with PBS-T, the plates were incubated with streptavidin conjugated horseradish peroxidase (1:10,000 in PBS; Zymed Laboratory Inc., San Francisco, CA) for 1 hour at room temperature. The plates were then washed five times with PBS-T, incubated 20 minutes with 100 µl of ABTS substrate (KPL, Gaithersburg, MD), followed by addition of 100 µl stopping solution (1 % SDS). Absorbance values were read at 405 nm using a VERSAmax microplate reader (Molecular Devices Corp., Sunnyvale, CA). The end point titers of test sera were the reciprocal of the highest mean dilution that resulted in an OD₄₀₅ reading of 0.1. The mean background titers of test sera were quantified by absorbance values read at 405 nm on the wells that had all reagents except sera.

[0165] Statistical Methods. Comparison of nasal colonization among groups was performed using the Tukey-Kramer test (Ludbrook, J., Clin Exp Pharmacol Physiol., 1998, 25:1032). Results were considered significant at p < 0.05.

Sequence heterogeneity of PPP1.

[0166] To examine sequence heterogeneity for the PPP 1 protein, the nucleotide sequence for the gene was compared among 10 different serotypes. Genomic DNA was prepared from overnight cultures of each serotype of S. pneumoniae. Cells were harvested by centrifugation at 1000 x g for 15 minutes at 4 °C and resuspended in 2 ml TE buffer. Cells were lysed by the addition of SDS to 0.3% and Proteinase K (Sigma) to 10 µg/ml. The cells were incubated overnight at 55 °C. Proteins were extracted from the cleared lysate by the addition of an equal volume of phenol/chloroform/ isoamyl alcohol (made by combining a 24:1 mixture of chloroform/isoamyl alcohol with an equal volume of water saturated phenol). The phases were separated by centrifugation at 7500 x g for 10 minutes at room temperature, then the aqueous phase was removed to a new tube. The process was repeated, then the DNA was precipitated from the aqueous phase by the addition of 10.4M NH₄Ac to 20%, and 2.5 volume of ethanol. The genomic DNA was spooled out using a glass rod and resuspended in 200 µl TE buffer. The gene for PPP1 was sequenced from the genomic DNA of serotypes 1,3,4,5,6,7,9,14,18,23F, and CP1200, using the Applied Biosystems Prism Dye Terminator cycle-sequencing core kit based on the Prism protocol supplied by the vendor. Approximately 1 µg template DNA and 100ng of primers were used for each cycling reaction. The reactions were cycled on the Gene Amp PCR Systems 2400 unit, purified using the Prism method, and analyzed on an ABI 373A DNA sequencer (Applied Biosystems). The nucleotide sequences and their predicted amino acid sequences were aligned in the Megalign application of the DNA Lasergene package from DNAstar, using the Clustal W algorithm.

Evaluation of PPP1 message expressed in vivo.

Preparation of RNA from cells grown in vitro

[0167] Various S. pneumoniae serotypes were grown to log phase (O.D.₅₅₀ approx 0.3) in 60 ml THB -0.5%YE at 37 °C with 5% CO₂. The cells were harvested by centrifugation at 1000 x g for 15 minutes at 4 °C. The supernatant was aspirated and the cells were resuspended in 1 ml RNAse later (Ambion, CA) and stored for >1 hr at 4 °C. The cells were then centrifuged in a microfuge for 5 minutes at 8000 x g. The supernatant was aspirated and the cells were resuspended in 100 µl 10%Deoxycholate (DOC). 1100 µl of RNAZOL B (Tel-Test, Inc) was then added and the suspension mixed briefly by inversion. 120 µl of CHCl₃ were then added, the sample mixed by inversion and then centrifuged in a microfuge at full speed for 10 minutes at 4 °C . The aqueous layer was removed and the RNA was precipitated by addition of an equal volume of 2-propanol. The RNA was incubated at 4 °C for > 1 hr and then centrifuged in a microfuge at full speed for 10 minutes at room temperature. The supernatant was aspirated and the RNA was washed with 75% ethanol and recentrifuged for 5 minutes. The supernatant was aspirated and the RNA was resuspended in 50-100 µl nuclease free water. DNA was removed from the RNA by treating the sample with RNAse free DNAase (DNA FREE, Ambion) for 20 minutes at 37 degrees, followed by inactivation of the enzyme by addition of the DNA FREE chleator. The purity and yield of the RNA was assessed by measuring the absorbance at 260 and 280 nm.

Preparation of RNA from cells grown in vivo

[0168] Log phase S. pneumoniae cells were prepared as described above and resuspended to 106 cfu/ml in RPMI media (Celltech) supplemented with 0.4% glucose. 1 ml of the cell suspension was sealed in a PVDF dialysis membrane with a 80,000 MW cutoff (SprectraPor). Two such bags were implanted intraperitoneally in 400g Sprague Dawley rats. The bags remained in the rats for 22 hours, after which the rats were terminated and the bags were harvested. RNA was prepared from the intraperitoneally grown cells as described above.

RT PCR to examine the message for PPP1 in vivo

[0169] Message for the PPP1 gene was amplified out from both RNA prepared from in vitro and in vivo grown cells using RT-PCR. A reverse PCR primer corresponding to the 3' end of the gene was used to generate ds cDNA in the following reaction. 1 µg RNA was incubated with 0.25 µM of the reverse primer: GGG GTC GAC TAA ACC AGG TGC TTG TCC AAG TTC (SEQ ID NO:8) for 3 minutes at 75 °C, then cooled to 44 °C. The message was reverse transcribed using the RETROscript (Ambion) kit according to the manufacturer's directions. ReddyMix (ABgene) was used according to the manufacturer's directions to amplify the PPP1 message from 2-5 µl of the sample, using 0.25 µM of the above reverse primer and the forward primer: GGG GCC ATG GCT GTA GAA TTG AAA AAA GAA (SEQ ID NO:9). 10 µl of the amplified product was electrophoresed on a 2% Agarose gel.

Results

[0170] Identification of the 20kDa surface associated protein- A PBS wash and ion exchange chromatography was used to identify an 20kDa surface associated component of S. pneumoniae (Figure 1). Lane 2-9 in figure 1 represents fraction #8-16 from a DEAE column. There is clearly a major protein band between 15 and 20kDa. The low molecular weight band was resolved on a preparative SDS-PAGE gel and transferred to a PVDF membrane. The PVDF membrane has a high binding capacity, which increases sample recovery and sequencing performance, allowing efficient determination of the amino terminal residues. The amino terminal sequence (SEQ ID NO: 3) of this protein allowed the identification of a corresponding open reading frame in the S. pneumoniae genome (SEQ ID NOs:4 and 5). This ORF showed similarity to similar to non-heme iron-containing ferritin proteins in other organisms, which may indicate similar function in S. pneumoniae (Pikis, A., et al., J. Infectious Diseases,1998,178:700). However, the exact function and cellular location of the proteins in S. pneumoniae is unknown. Subcloning and expression of this ORF provided recombinant material of the expected size (Figure 2).

[0171] Purification of the recombinant 20kDa surface associated protein. Purification was aided by the solubility of the recombinant protein. Significant purification away from cellular membranes was achieved by sequential centrifugations. In addition, the characteristic of oligomer formation was successfully utilized to remove the remaining low molecular weight contaminating proteins by diafiltration. The predicted charge of the protein at neutral pH allowed the protein to be purified to greater than 90% homogeneity on a Hydroxyapatite column, as seen in Figure 5.

[0172] Reactivity of anti-r20kDa surface associated protein sera. Polyclonal antisera to recombinant 20kDa surface associated protein were generated in Swiss Webster mice to evaluate antigenic conservation of the protein among strains. Antisera to the r20kDa protein reacted with proteins of approximately 20, 40, and 80kDa in unheated whole cell lysates of native species (Figure 3), while the major reactive species seen in heated samples is at approximately 20kDa (not shown). These results suggest that this protein is part of a complex of 4 subunits or more.

[0173] Intranasal Challenge. To determine whether i.n. immunization with r20kDa surface associated protein can induce serum immune responses, Balb/c mice were administered 5 µg r20kDa 3 times at biweekly intervals using CT-E29H (0.1 µg/dose) as a mucosal adjuvant. Immune sera collected 4 days after the last booster immunization were tested in the antigen-specific ELISA assays. At 4 days after the last booster immunization, strong, antigen-specific IgG and IgA antibody responses were generated in mice immunized with r20kDa- E29H (Table 6). When compared to the unrelated protein KLH, immunization with r20kDa surface associated protein was able to significantly reduce colonization of type 3 S. pneumoniae the nasopharynx of BALB/C mice. (Figure 4) The results are comparable to the ability of the type 3 conjugate to reduce colonization of the homologous serotype (Henrikson, J, et al. Alcohol Clin Exp Res, 1997, 21:1630).

Antigen specific ELISA titers for r20kDa surface associated protein from S. pneumoniae.

[0174] 

Group	Sera wk4d5 IgG	Sera wk4d5 IgA
5µg r20kDa +0.1µg CT-E29H	79,726	1563
5µg Type-3-Conjugate +0.1µg CT-E29H	<50	<50
5µg KLH+0.1 µg CT-E29H	<50	<50

Note: Endpoint titers determined from pools of 5 BALB/c mice

[0175] Sequence Alignment of the PPP1 protein from 10 serotypes. As shown in Figure 6, the sequence of PPP1 is largely conserved among serotypes. As can be seen, serotype 9 is the most divergent serotype. The PP 1 isolated from this serotype showed 15 amino acid differences from the majority. The remaining serotypes showed less than 5 amino acid differences. An overall consensus sequence of PPP1 is shown in Figure 6 (SEQ ID NO:20).

[0176] RNA Amplification. A discrete band of the expected size is seen in both the in vitro and in vivo samples (Figure 7). The size of the product was estimated to be full length by comparison to Hae III restriction fragments of Lambda DNA.

SEQUENCE LISTING

[0177] 

<110> American Home Products Corporation
Green, Bruce
Masi, Amy

<120> Recombinant Protective Protein from Streptococcus pneumoniae

<130> 0630/2H814

<140> TBA
<141> Concurrently herewith

<150> US 60/258,841
<151> 2000-12-28

<160> 20

<170> PatentIn version 3.1

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ggggtcgact aaaccaggtg cttgtccaag ttc

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Claims

1. An isolated S. pneumoniae surface associated Pneumo Protective Protein (PPP) having an amino acid sequence selected from SEQ ID NOs: 5 and 10-19, or a fragment thereof, wherein said PPP or fragment thereof has the ability to reduce colonization of pneumococcal bacteria and retains the biological function of interacting with iron.

2. A PPP or fragment thereof as defined in claim 1, wherein said PPP or fragment thereof is a recombinant protein.

3. A PPP or fragment thereof as defined in claim 2, said PPP having an isoelectric point of about 4.587.

4. A PPP or fragment thereof as defined in claim 3, said PPP having a charge of about 14.214 at pH 7.

5. A PPP or fragment thereof as defined in claim 1, said PPP having an amino acid sequence as depicted in SEQ ID NO: 5, or a fragment thereof.

6. A nucleic acid sequence encoding a PPP or a fragment thereof as defined in claim 1, wherein said nucleic acid sequence has a sequence as depicted in SEQ ID NO: 4, or a fragment thereof.

7. A nucleic acid as defined in claim 6 which is a cDNA.

8. An expression vector comprising a nucleic acid sequence encoding a PPP or fragment thereof as defined in any of claims 1 to 5 or a nucleic acid sequence of claim 6, wherein said sequence is operatively associated with an expression control sequence.

9. A host cell transfected with the vector as defined in claim 8.

10. A method for producing recombinant PPP or fragment thereof which method comprises isolating said PPP or fragment thereof produced by the host cells as defined in claim 9, wherein the host cells have been cultured under conditions that provide for expression of said PPP or fragment thereof by said vector.

11. A composition comprising a PPP or fragment thereof as defined in any one of claims 1 to 5 and a pharmaceutically acceptable carrier.

12. A composition comprising a nucleic acid sequence encoding a PPP or fragment thereof as defined in claim 6 and a pharmaceutically acceptable carrier.

13. A composition comprising the expression vector as defined in claim 8 and a pharmaceutically acceptable carrier.

14. A composition comprising the host cell as defined in claim 9 and a pharmaceutically acceptable carrier.

15. An immunogenic composition comprising (i) a S. pneumoniae surface associated PPP or fragment thereof according to any one of claims 1 to 5; (ii) a pharmaceutically acceptable carrier, and (iii) optionally at least one adjuvant.

16. A composition as defined in claim 15, said PPP or fragment thereof encoded by a nucleic acid sequence having a sequence as depicted in SEQ ID NO: 4, or an immunogenic fragment thereof.

17. An immunogenic composition comprising (i) at least one expression vector according to claim 8; (ii) a pharmaceutically acceptable carrier; and (iii) optionally at least one adjuvant.

18. A composition as defined in claim 17, wherein said pneumococcal bacteria is Streptococcus pneumoniae.

19. A composition as defined in claim 15, 16 or 18, wherein said composition elicits protective immunity from a disease caused by Streptococcus pneumoniae.

20. A composition as defined in claim 19, wherein said disease is selected from the group consisting of otitis media, rhinosinusitis, bacterenia, meningitis, pneumonia, and lower respiratory tract infection.

21. Use, in the manufacture of a medicament for inducing an immune response in a mammal, of a composition as defined in claim 15 or 17.

22. Use, in the manufacture of a medicament for inducing an immune response in a mammal which is infected with pneumococcal bacteria, of a compound effective to inhibit binding of an amino acid sequence as depicted in SEQ ID NO: 5 to induce said immune response in said mammal.

23. A method for screening for a compound which induces an immune response in a mammal infected with pneumococcal bacteria, said method comprising comparing a first amount of binding of an amino acid sequence as depicted in SEQ ID NO: 5 in the presence of said compound to a second amount of binding of an amino acid sequence as depicted in SEQ ID NO:5 not in the presence of said compound; whereby a lower first amount of binding than said second amount binding indicates that said compound may induce said immune response in said mammal.

24. An in vitro method for diagnosing pneumococcal bacterial infection, said method comprising comparing the level of PPP as depicted in SEQ ID NO: 5, or fragments thereof, in suspect sample to the level of PPP as depicted in SEQ ID NO: 5, or fragments thereof, in a control sample, whereby a higher level of said Pneumo Protective Protein said suspect sample than the level of said Pneumo Protective Protein in said control sample indicates that said suspect sample comprises pneumococcal bacterial infection; wherein said PPP or fragment thereof has the ability to reduce colonization of pneumococcal bacteria and retains the biological function of interacting with iron.

25. An antibody which selectively recognizes an amino acid sequence as depicted in SEQ ID NO: 5, or a fragment thereof.

26. An antibody as defined in claim 25, which is chimeric.

27. An antibody as defined in claim 25, which is a monoclonal antibody.

28. An antibody as defined in claim 25 or 27, which is humanized.

29. An antibody as defined in claim 25 or 27, which is anti-idiotypic.

30. An antibody as defined in claim 25 or 27, which is conjugated to a pharmaceutically active compound.

31. Use, in the manufacture of a medicament for inducing an immune response against pneumococcal infection in a mammal, of an antibody as defined in claim 29.

32. Use, in the manufacture of a medicament for inducing an immune response in a mammal infected with pneumococcal bacteria, of an antibody as defined in claim 30.

Ansprüche

1. Isoliertes, Oberflächen-assoziiertes pneumoprotektives Protein (PPP) aus S. pneumoniae, das eine Aminosäuresequenz, ausgewählt aus SEQ ID NOs.: 5 und 10 bis 19, oder einem Fragment davon, aufweist, wobei das PPP oder Fragment davon die Fähigkeit aufweist, die Kolonisierung von Bakterien vom Pneumokokkentyp zu reduzieren und die biologische Funktion, mit Eisen zu interagieren, behält.

2. PPP oder Fragment davon wie in Anspruch 1 definiert, wobei das PPP oder Fragment davon ein rekombinantes Protein ist.

3. PPP oder Fragment davon wie in Anspruch 7 definiert, wobei das PPP einen isoelektrischen Punkt von etwa 4,587 aufweist.

4. PPP oder Fragment davon wie in Anspruch 3 definiert, wobei das PPP eine Ladung von etwa 14,214 bei einem pH-Wert von 7 aufweist.

5. PPP oder Fragment davon wie in Anspruch 1 definiert, wobei das PPP eine Aminosäuresequenz wie in SEQ ID NO.: 5 dargestellt oder ein Fragment davon aufweist.

6. Nukleinsäuresequenz, die ein PPP oder ein Fragment davon wie in Anspruch 1 definiert kodiert, wobei die Nukleinsäuresequenz eine Sequenz wie in SEQ ID NO.: 4 dargestellt oder ein Fragment davon aufweist.

7. Nukleinsäure wie in Anspruch 6 definiert, die eine cDNA ist.

8. Expressionsvektor, umfassend eine Nukleinsäuresequenz, die ein PPP oder Fragment davon, wie in einem der Ansprüche 1 bis 5 definiert, oder eine Nukleinsäuresequenz nach Anspruch 6, wobei die Sequenz funktionell mit einer Expressionskontrollsequenz assoziiert ist, kodiert.

9. Wirtszelle, die mit dem Vektor wie in Anspruch 8 definiert transfiziert ist.

10. Verfahren zur Herstellung von rekombinantem PPP oder einem Fragment davon, wobei das Verfahren das Isolieren des PPP oder des Fragments davon, das von der Wirtszelle wie in Anspruch 9 definiert hergestellt wird, umfaßt, wobei die Wirtszellen unter Bedingungen kultiviert wurden, welche die Expression des PPP oder des Fragments davon durch den Vektor bereitstellt.

11. Zusammensetzung, umfassend ein PPP oder Fragment davon wie in einem der Ansprüche 1 bis 5 definiert und einen pharmazeutisch verträglichen Träger.

12. Zusammensetzung, umfassend eine Nukleinsäuresequenz, die ein PPP oder ein Fragment davon wie in Anspruch 6 definiert kodiert, und einen pharmazeutisch verträglichen Träger.

13. Zusammensetzung, umfassend den Expressionsvektor wie in Anspruch 8 definiert und einen pharmazeutisch verträglichen Träger.

14. Zusammensetzung, umfassend die Wirtszelle wie in Anspruch 9 definiert und einen pharmazeutisch verträglichen Träger.

15. Immunogene Zusammensetzung, umfassend (i) ein Oberflächen-assoziiertes PPP oder Fragment davon aus S. pneumoniae nach einem der Ansprüche 1 bis 5, (ii) einen pharmazeutisch verträglichen Träger und (iii) gegebenenfalls mindestens ein Adjuvans.

16. Zusammensetzung wie in Anspruch 15 definiert, wobei das PPP oder das Fragment davon von einer Nukleinsäuresequenz, die eine Sequenz wie in SEQ ID NO.: 4 dargestellt oder ein immunogenes Fragment davon aufweist, kodiert wird.

17. Immunogene Zusammensetzung, umfassend (i) mindestens einen Expressionsvektor nach Anspruch 8, (ii) einen pharmazeutisch verträglichen Träger und (iii) gegebenenfalls mindestens ein Adjuvans.

18. Zusammensetzung wie in Anspruch 17 definiert, wobei das Bakterium vom Pneumokokkentyp Streptococcus pneumoniae ist.

19. Zusammensetzung wie in Anspruch 15, 16 oder 18 definiert, wobei die Zusammensetzung protektive Immunität gegen eine Krankheit, die von Streptococcus pneumoniae verursacht wird, hervorruft.

20. Zusammensetzung wie in Anspruch 19 definiert, wobei die Krankheit aus der Gruppe, bestehend aus Otitis media, Rhinosinusitis, Bakterenie, Meningitis, Pneumonie und Infektionen der unteren Atemwege, ausgewählt ist.

21. Verwendung einer Zusammensetzung wie in Anspruch 15 oder 17 definiert in der Herstellung eines Medikaments zur Induktion einer Immunantwort in einem Säugetier.

22. Verwendung einer Verbindung, die wirksam ist, die Bindung einer Aminosäuresequenz wie in SEQ ID NO.: 5 dargestellt zu inhibieren, um die Immunantwort in dem Säugetier hervorzurufen, in der Herstellung eines Medikaments zur Induktion einer Immunantwort in einem Säugetier.

23. Verfahren zum Screenen einer Verbindung, die eine Immunantwort in einem Säugetier hervorruft, das mit Bakterien vom Pneumokokkentyp infiziert ist, wobei das Verfahren das Vergleichen einer ersten Menge an Bindung einer Aminosäuresequenz wie in SEQ ID NO.: 5 dargestellt in der Gegenwart der Verbindung mit einer zweiten Menge an Bindung einer Aminosäuresequenz wie in SEQ ID NO.: 5 dargestellt in Abwesenheit der Verbindung umfaßt, wobei eine niedrigere erste Menge an Bindung als die zweite Menge an Bindung anzeigt, daß die Verbindung die Immunantwort in dem Säugetier induzieren könnte.

24. In vitro Verfahren zur Diagnose einer bakteriellen Infektion vom Pneumokokkentyp, wobei das Verfahren das Vergleichen des Spiegels an PPP wie in SEQ ID NO.: 5 dargestellt oder eines Fragments davon in einer verdächtigen Probe mit dem Spiegel von PPP wie in SEQ ID NO.: 5 dargestellt oder eines Fragments davon in einer Kontrollprobe umfaßt, wobei ein höherer Spiegel des pneumoprotektiven Proteins in der verdächtigen Probe als der Spiegel des pneumoprotektiven Proteins in der Kontrollprobe anzeigt, daß die verdächtige Probe eine bakterielle Infektion vom Pneumokokkentyp umfaßt, wobei das PPP oder das Fragment davon die Fähigkeit aufweist, die Kolonisierung von Bakterien vom Pneumokokkentyp zu reduzieren und die biologische Funktion, mit Eisen zu interagieren, behält.

25. Antikörper, der selektiv eine Aminosäuresequenz wie in SEQ ID NO.: 5 dargestellt oder ein Fragment davon erkennt.

26. Antikörper wie in Anspruch 25 definiert, der chimär ist.

27. Antikörper wie in Anspruch 25 definiert, der ein monoklonaler Antikörper ist.

28. Antikörper wie in Anspruch 25 oder 27 definiert, der humanisiert ist.

29. Antikörper wie in Anspruch 25 oder 27 definiert, der anti-ideotypisch ist.

30. Antikörper wie in Anspruch 25 oder 27 definiert, der an eine pharmazeutisch aktive Verbindung konjugiert ist.

31. Verwendung eines Antikörpers wie in Anspruch 29 definiert in der Herstellung eines Medikaments zur Induktion einer Immunantwort gegen eine Infektion vom Pneumokokkentyp in einem Säugetier.

32. Verwendung eines Antikörpers wie in Anspruch 30 definiert in der Herstellung eines Medikaments zur Induktion einer Immunantwort in einem Säugetier, das mit Bakterien vom Pneumokokkentyp infiziert ist.

Revendications

1. Protéine pneumo-protectrice (PPP, Pneumo Protective Protein) isolée associée à la surface de S. pneumoniae, présentant une séquence d'acides aminés sélectionnée parmi les SEQ. ID n° 5 et 10-19, ou fragment de celle-ci, dans laquelle ladite PPP ou ledit fragment de celle-ci est capable de réduire la colonisation de bactéries pneumococciques et conserve la fonction biologique d'interaction avec le fer.

2. PPP, ou fragment de celle-ci, selon la revendication 1, dans laquelle ladite PPP ou ledit fragment de celle-ci est une protéine recombinante.

3. PPP, ou fragment de celle-ci, selon la revendication 2, ladite PPP présentant un point isoélectrique d'environ 4,587.

4. PPP, ou fragment de celle-ci, selon la revendication 3, ladite PPP présentant une charge d'environ 14,214 à un pH 7.

5. PPP, ou fragment de celle-ci, selon la revendication 1, ladite PPP présentant une séquence d'acides aminés telle que dépeinte dans le SEQ. ID N° 5, ou un fragment de celle-ci.

6. Séquence d'acide nucléique codant pour une PPP, ou un fragment de celle-ci, selon la revendication 1, dans laquelle ladite séquence d'acide nucléique présente une séquence dépeinte dans le SEQ. ID N° 4, ou un fragment de celle-ci.

7. Acide nucléique selon la revendication 6, qui est un ADNc.

8. Vecteur d'expression comprenant une séquence d'acide nucléique codant pour une PPP, ou un fragment de celle-ci, selon l'une quelconque des revendications 1 à 5 ou une séquence d'acide nucléique selon la revendication 6, dans lequel ladite séquence est associée de manière fonctionnelle à une séquence de contrôle de l'expression.

9. Cellule hôte transfectée par le vecteur selon la revendication 8.

10. Procédé de production de PPP recombinante, ou d'un fragment de celle-ci, ledit procédé comprenant l'isolation de ladite PPP ou dudit fragment de celle-ci, produit(e) par les cellules hôtes selon la revendication 9, dans lequel les cellules hôtes sont cultivées dans des conditions qui permettent l'expression de ladite PPP ou dudit fragment de celle-ci par ledit vecteur.

11. Composition comprenant une PPP, ou un fragment de celle-ci, selon l'une quelconque des revendications 1 à 5, et un excipient acceptable sur le plan pharmaceutique.

12. Composition comprenant une séquence d'acide nucléique codant pour une PPP, ou un fragment de celle-ci, selon la revendication 6, et un excipient acceptable sur le plan pharmaceutique.

13. Composition comprenant le vecteur d'expression selon la revendication 8 et un excipient acceptable sur le plan pharmaceutique.

14. Composition comprenant la cellule hôte selon la revendication 9 et un excipient acceptable sur le plan pharmaceutique.

15. Composition immunogène comprenant (i) une PPP associée à la surface de S. pneumoniae, ou un fragment de celle-ci, selon l'une quelconque des revendications 1 à 5 ; (ii) un excipient acceptable sur le plan pharmaceutique, et (iii) en option au moins un adjuvant.

16. Composition selon la revendication 15, ladite PPP, ou ledit fragment de celle-ci, codé(e) par une séquence d'acide nucléique ayant une séquence telle que dépeinte dans le SEQ. ID N° 4, ou un fragment immunogène de celle-ci.

17. Composition immunogène comprenant (i) au moins un vecteur d'expression selon la revendication 8 ; (ii) un excipient acceptable sur le plan pharmaceutique ; et (iii) en option au moins un adjuvant.

18. Composition selon la revendication 17, dans laquelle ladite bactérie pneumococcique est Streptococcus pneumoniae.

19. Composition selon la revendication 15, 16 ou 18, dans laquelle ladite composition provoque l'immunité protectrice d'une maladie provoquée par Streptococcus pneumoniae.

20. Composition selon la revendication 19, dans laquelle ladite maladie est sélectionnée dans le groupe constitué de l'otite moyenne, de la rhinosinusite, de la bactériémie, de la méningite, de la pneumonie et d'une infection des voies respiratoires inférieures.

21. Utilisation, dans la fabrication d'un médicament destiné à induire une réponse immunitaire chez un mammifère, d'une composition selon la revendication 15 ou 17.

22. Utilisation, dans la fabrication d'un médicament destiné à induire une réponse immunitaire chez un mammifère infecté par une bactérie ménococcique, d'un composé efficace pour inhiber la liaison d'une séquence d'acides aminés telle que dépeinte dans le SEQ. ID N° 5 pour induire ladite réponse immunitaire chez ledit animal.

23. Procédé de criblage d'un composé qui induit une réponse immunitaire chez un mammifère infecté par des bactéries pneumococciques, ledit procédé comprenant la comparaison d'une première quantité de liaison d'une séquence d'acides aminés telle que dépeinte dans le SEQ. ID N° 5 en présence dudit composé avec une seconde quantité de liaison d'une séquence d'acides aminés telle que dépeinte dans le SEQ. ID N° 5 en l'absence dudit composé ; où une première quantité de liaison inférieure à ladite seconde quantité de liaison indique que ledit composé peut induire ladite réponse immunitaire chez ledit mammifère.

24. Procédé in vitro de diagnostic d'une infection à bactérie pneumococcique, ledit procédé comprenant la comparaison du niveau de PPP telle que dépeinte dans le SEQ. ID N° 5, ou de fragments de celle-ci, dans un échantillon suspect avec le niveau de PPP telle que dépeinte dans le SEQ. ID N° 5, ou de fragments de celle-ci, dans un échantillon de contrôle, où un niveau de ladite protéine pneumo-protectrice dudit échantillon suspect supérieur au niveau de ladite protéine pneumo-protectrice dans ledit échantillon de contrôle indique que ledit échantillon suspect comprend une infection par une bactérie pneumococcique ; ladite protéine PPP, ou ledit fragment de celle-ci, étant capable de réduire la colonisation des bactéries pneumococciques et de conserver la fonction biologique d'interaction avec le fer.

25. Anticorps qui reconnaît de façon sélective une séquence d'acides aminés telle que dépeinte dans le SEQ. ID N° 5, ou un fragment de celle-ci.

26. Anticorps selon la revendication 25, qui est chimère.

27. Anticorps selon la revendication 25, qui est un anticorps monoclonal.

28. Anticorps selon la revendication 25 ou 27, qui est humanisé.

29. Anticorps selon la revendication 25 ou 27, qui est anti-idiotypique.

30. Anticorps selon la revendication 25 ou 27, qui est conjugué à un composé actif sur le plan pharmaceutique.

31. Utilisation, dans la fabrication d'un médicament destiné à induire une réponse immunitaire contre une infection pneumococcique chez un mammifère, d'un anticorps selon la revendication 29.

32. Utilisation, dans la fabrication d'un médicament destiné à induire une réponse immunitaire chez un animal infecté par une bactérie pneumococcique, d'un anticorps selon la revendication 30.

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