EP0991762A2

EP0991762A2 - SURFACE PROTEIN (SPsA PROTEIN) OF STREPTOCOCCUS PNEUMONIAE, DELETED DERIVATIVES, EXPRESSION SYSTEM FOR SAID PROTEINS AND VACCINE SYSTEM WITH SAID PROTEINS

Info

Publication number: EP0991762A2
Application number: EP98916880A
Authority: EP
Inventors: Gursharan Singh Chhatwal; Sven Hammerschmidt
Original assignee: Helmholtz Zentrum fuer Infektionsforschung HZI GmbH
Current assignee: Helmholtz Zentrum fuer Infektionsforschung HZI GmbH
Priority date: 1997-03-03
Filing date: 1998-03-02
Publication date: 2000-04-12
Also published as: WO1998039450A3; WO1998039450A2; JP2001524073A; DE19708537A1

Abstract

The invention relates to a novel surface protein (SPsA protein) of streptococcus pneumoniae, deleted derivatives of the surface protein, an expression system for the surface protein and its derivatives as well as a vaccine that uses them.

Description

SURFACE PROTEIN (SPSA-PROTEIN) FROM STREPTOCOCCUS PNEUMONIAE DELETED COUPLINGS, EXPRESSION SYSTEM FOR THESE PROTEINS AND VACCINE WITH THE PROTEINS

Introduction :

Streptococcus pneumoniae (pneumococci) are gram-positive bacteria that are surrounded by a capsule polysaccharide and were first isolated from a healthy carrier in 1881 (1). Pneumococci are part of the natural flora of the upper human respiratory tract and colonize the nasopharynx in up to 40% of healthy adults, whereby up to four different serotypes have been demonstrated over several months (2). Streptococcus pneumoniae is a common source of infection in the United States and many other parts of the world. The infections, which are mostly endogenous, and deaths occur especially in very young children under the age of 2, in people over 60 and in immunocompromised people

(3).

Pneumococci are also the second most common cause of bacterial meningitis (after Haemophilus influenzae type b)

(4) and otitis media (otitis media) in children (5). In the United States alone, pneumococci caused 500,000 pneumonia, 7 million cases of otitis media and 40,000 deaths in one year (6.7).

The mortality rate for diseases caused by Streptococcus pneumoniae is, despite the antibiotics available, at a consistently high level. For example, mortality from pneumococcal bacteremia in the past four decades has been between 25 and 29% (8).

Due to the composition of the capsule polysaccharide structures, pneumococci are divided into 90 serotypes (9). The capsular polysaccharide protects the pneumococci from phagocytosis by polymorphonuclear leukocytes (PMN's), prevents activation of the alternative pathway of the complement (10,11,12) and is therefore an important virulence factor of Streptococcus pneumoniae (13,14,15).

The virulence of the strain with respect to the capsule depends on the chemical composition of the polysaccharide and not on the size of the capsule polysaccharide (16).

The vaccine currently used (pneumococcal vaccine) for active immunization contains the unconjugated form of 23 capsule polysaccharides from Streptococcus pneumoniae. This vaccine contains the serotype polysacccharides, which cause 85-90 percent of bacteremic infections. Since there is no stimulation of the T helper cells by a polysaccharide antigen in children and the elderly (17), the use of this vaccine is restricted to adults only.

Another problem with treatment for pneumococcal infections is the increased incidence of antibiotic-resistant strains (penicillin resistance) worldwide (18). In addition, the use of ß-lactam antibiotics in sensitive strains of strepto- coccus pneumoniae in 30% of the treated children to death or permanent, irreversible brain damage remains. The bacteria are lysed by ß-lactam antibiotics and the released cell wall components cause swelling of the brain. The swelling of the brain then leads to dangerously high intercranial pressure, which causes irreversible damage (19).

Due to the increasing antibiotic resistance and the limited effect of the polysaccharide vaccine, there is great interest in finding a pneumococcal protein that can act as a carrier for the capsule polysaccharides or that is a preventive vaccine due to its own strong immunogenicity and ubiquitous occurrence in Streptococcus pneumoniae isolates Can be used.

The proteins currently considered as vaccine candidates are autolysin (20), neuraminidase (21), pneumococcal surface adhesin A (PsaA) (22), pneumococcal surface protein A (PspA) (23) and the pneumolysin (24).

Pneumolysin is an intracellular protein and belongs to the family of thiol-activated toxins (25). This 53 kDa cytoplasmic protein is released when the pneumococci spontaneously lyse under the influence of autolysin. In high concentrations, oligomers of pneumolysin form on mammalian cells and cause cell lysis by increasing the transmembrane pores. In lower concentrations, pneumolysin stimulates the production of inflammatory cytokines (26), destroys monolayer of the epithelial cells of the upper respiratory tract (27) in vitro and reduces the bactericidal activity and migration of the neutrophils (28). Pneumolysin also activates the classic complement pathway in the absence of anti-toxin antibodies (29). The pneumoccocal surface protein A (PspA) is a surface protein with structural and antigenetic variability between the different pneumococcal strains, the function of which has not yet been elucidated. PspA occurs in most clinical isolates (30) and is also important for the development of full virulence in pneumococci (31,32).

Other known virulence factors are an IgAl protease (33) whose gene has been cloned and characterized (34, 35), an inhibitor of elastase (36) and peptide permeases that are homologous to permeases from other streptococci that also colonize the nasopharynx. Of particular interest are the permeases, in which the loss of function results in reduced adherence of the pneumococci to eukaryotic cells

(37). Evidence of direct involvement of these permeases, of which the AmiA permease and the PlpA (permease-like protein A) belong to the family of protein-dependent permeases that are responsible for the transport of small peptides

(38, 39) adherence to eukaryotic cells has so far not been demonstrated. Another potential regulator of pneumococcal adherence is pyruvate oxidase, SpxB, which affects the concentration of acetyl phosphate

(40).

One embodiment of the invention relates to a surface protein of Streptococcus pneunomiae (SpsA protein), which is characterized in that it binds to secretory IgA (slgA).

Another embodiment of the invention relates to a secretory protein from Streptococcus pneumoniae, which is characterized in that it binds to secretory IgA (slgA).

The surface protein or secretory protein of Streptococcus pneumonia e according to the invention can be partially digested and characterized in that it is associated with secretory IgA (slgA) binds.

The invention further relates to a C-terminally deleted derivative of a surface protein or secretory protein according to the invention, which is characterized in that it binds to secretory IgA (slgA).

The invention further relates to a deleted derivative of a surface protein or secretory protein according to the invention, which is characterized in that

- at least the signal sequence and / or

- At least optional repeats of the surface protein or secretory protein are deleted, however

- The secretory IgA (slgA) binding domain is present, so that the descendant binds to secretory IgA (slgA).

The derivative according to the invention can be characterized in that the surface protein or the secretory protein are deleted except for the secretory IgA (slgA) binding domain.

The surface protein according to the invention can be characterized by 523 amino acids according to FIG. 2 (positions 1 to 523).

The C-terminal deleted derivative of the invention

Surface protein can be identified by 324

2 (positions 1 to 324) and

Repeats 1 to 6 (positions 325 to 444) or

Repeats 1 to 7 (positions 325 to 464) or

Repeats 1 to 8 (positions 325 to 484 or to 485).

The invention further relates to an N-terminally deleted derivative of the surface protein, which is characterized in that it (i) in the region of positions 1 to 159 according to FIG. 2 has been deleted by 1 to a maximum of 159 amino acids,

(ii) is not, however, deleted in the range of positions 160 to 523.

The invention further relates to an N-terminal and C-terminally deleted derivative of the surface protein according to the invention, which is characterized in that it

(i) in the region of positions 1 to 159 according to FIG. 2 has been deleted by 1 to a maximum of 159 amino acids,

(ii) is not, however, deleted in the range of items 160 to 324 and, if appropriate

(iii) repeats 1 to 8 (positions 325 to 484 or to 485) or repeats 1 to 7 (positions 325 to 464) or repeats 1 to 6 (positions 325 to 444).

The offspring according to the invention can be characterized in that it is not deleted in the region of positions 174 to 285.

Another embodiment of the invention relates to a <? secretory IgA (slgA) binding protein, which is characterized in that its amino acid sequence is at least 80% identical to that of the surface protein according to the invention or one of its derivatives.

A further embodiment of the invention relates to an expression system, in particular for Escherichia coli, for expressing a surface protein, a secretory protein, a derivative or a protein according to the Invention comprising a DNA sequence encoding the surface protein or the progeny.

Finally, the invention relates to a vaccine for protection against diseases caused by Streptococcus pneunomiae, which can be produced with the aid of a surface protein according to the invention, a secretory protein or a derivative.

The invention is explained in more detail below with reference to figures, experimental results and examples. Show it:

Figure 1: Western blot analysis of Streptococcus pneumoniae ATCC 33400 [serotype 1] (lane 1), NCTC 10319 [serotype 47, R36A] (lane 2), ATCC 11733 [serotype 2, R36A] (lane 3) and ATCC 12213 [serotype 1 , I-192R] (lane 4) with secretory immunoglobulin A. A peroxidase-labeled anti-human IgA antibody was used to detect the binding.

Figure 2: Nucleic acid sequence of the gene spsA and the amino acid sequence of the protein SpsA (Streptococcus pneumoniae secretory IgA binding protein) from Streptococcus pneumoniae ATCC 33400 serotype 1. RBS: Ribosomal binding site; Leader: signal sequence of SpsA (amino acid 1-37); mature protein: SpsA after processing; Repeats: 9 repeating sequences of 20 amino acids each.

Figure 3: Western blot analysis with slgA after cloning of spsA and spsΛ fragments in the expression vector pQE (Pharmacia) and overexpression of the proteins in Escherichia coli M15 [pREP4]. Lane 1: SpsA (AS1-523; pQSHA12); Lane 2: N-terminus of SpsA (AS1-324; pQSHA14); Lane 3: truncated N-terminus of SpsA (AS1-159; pSHA3); Lane 4: Repeats from SpsA (AS325-523; pQSHA30). Figure 4: Southern blot analysis of Streptococcus pneumoniae ATCC 33400 [serotype 1] (lane 1) and Streptococcus pneumoniae ATCC 11733 [serotype 2, R36A] (lane 2) with a 32 _phosphorus- labeled DNA probe from spsA (A), from a 5 '-spsA fragment [ntl-nt476] (B) and pspA (C). nt: nucleotide.

Results:

In binding studies with radioactively labeled human secretory immunoglobulin A (slgA) and in Western blots with human slgA, it was demonstrated that Streptococcus pneumoniae binds secretory IgA. Both clinical isolates and strains from the Type Culture Collections (ATCC or NCTC) of the most common serotypes of clinical isolates were tested for their ability to bind slgA. Of the pneumococcal strains examined, 73% slgA could bind in a Western blot (FIG. 1). Furthermore, after digestion of the pneumococcal proteins with protease, it could be shown in binding studies that the binding of secretory IgA takes place by a protein from Streptococcus pneumoniae.

The gene coding for the slgA binding protein was detected by screening a LambdaZAP expression bank of Streptococcus pneumoniae ATCC 33400 (serotype 1) with secretory IgA. The selected positive clone, pSHAl, has a 5085 bp insert of Streptococcus pneumoniae ATCC 33400 in the phase id pBK-CMV. By constructing deletion clones of pSHAl, the gene coding for the slgA binding protein could be detected in the subclone pSHA2. In contrast to the two other subclones pSHA3 and pSHA4, the subclone pSHA2 showed a binding of the secretory IgA in the Western blot. The sequencing and subsequent analysis of the sequence of the 2204 large insert showed an open reading frame from nucleotide 282 to 1853 in pSHA2 (FIG. 2). This 1572 bp reading frame codes for a 523 amino acid slgA binding protein from pneumococci of serotype 1. The molecular weight of the protein called SpsA (Streptococcus pneumoniae secretory IgA binding protein) is 59151 Da (FIG. 2).

The comparison of the nucleic acid sequence of spsA with the sequences stored in the EMBL database showed a 78.8% identity to the pspA of Streptococcus pneumoniae. A 64.1% identity to PspA was demonstrated at the protein level. The identity is primarily limited to the C-terminus of the two proteins. The C-terminus of the PspA consists of ten repeats, each 20 amino acids long (41). The identity to the C-terminus of SpsA, which consists of nine repeats, is 92.5%.

The repeats of PspA are involved in attaching the protein to the pneumococcal cell wall. This mechanism requires a choline-mediated interaction between the membrane-associated lipoteichoic acid and the repeat region of PspsA (42). To attach to the cell wall, at least six of the 20 amino acid repeats must be expressed, otherwise the PspA is secreted (42, 43).

Since the C-terminus of SpsA, i.e. the region of the repeats of SpsA, 92.5% identical to the repeats of PspA, an involvement of the SpsA repeats in the attachment to the cell wall of the pneumococci can be assumed. This mechanism of SpsA attachment is supported by the fact that computer analysis of the secondary structure of SpsA did not detect a helical transmembrane structure that would be necessary for the protein to be anchored in the gram-positive cell wall.

Furthermore, previous results indicated that the 3 'end of pspA is a conserved sequence that is homologous to other sequences in Streptococcus pneumoniae. These homologous sequences could code for proteins that are similar Attachment mechanisms to the cell wall have, like pneumococcal surface protein A (PspA) (44).

The identity in the N-terminus of the two proteins SpsA and PspA is only 34.5%. The signal sequence (leader) of SpsA is 37 amino acids long and shows a 75.7% identity to the signal sequence of the IgA receptor from Streptococcus agala ctiae (45, 46).

To clarify the binding domain, i) spsA (pQSH12), ii) the N-terminus (pQSH14) and iii) the nucleic acid sequence which codes for the nine repeats of SpsA (pQSH30) were cloned into the expression vector pQE30 and the slgA binding in Western blot examined. Western blot analysis showed binding of the secretory IgA in the N-terminus of SpsA. In addition, the binding domain of SpsA could be restricted to amino acids 160 to 324 by a further subclone of pSHAl, which only expresses amino acids 1 to 159 of the N-terminus of SpsA (pSHA3) and does not bind slgA (FIG. 3).

By Southern blot analysis of Streptococcus pneumoniae ATCC 33400 (Serotypl) and S. pneumoniae ATCC 11733 (serotype 2, R36A) could be demonstrated with a pspA, spsA and a b '-spsA specific DNA probe that spsA is not identical to pspA (FIG. 4).

With these investigations it was possible to prove that the C-terminal sequence of spsA is one of the conserved sequences described which are homologous to pspA and is involved in the attachment of the proteins to the cell wall in Streptococcus pneumoniae.

SpsA therefore codes for a new surface protein of Streptococcus pneumoniae, whose biological function is the binding of secretory immunoglobulin A in the N-terminus of SpsA. The secretory IgA, which consists of an IgA-J-IgA-SC (SC: secretory component) complex, is the most important immunoglobulin in the human respiratory and gastrointestinal tract. The precursor of the secretory IgA, an IgA dimer linked by the 15.6 kDa J chain, is synthesized in B lymphocytes and binds to a poly-immunoglobulin receptor located on the basolateral surface of the epithelial cells. This poly-Ig receptor mediates transcytosis through the cells (47). The C-terminal part of the receptor is separated and becomes the secretory component of the (IgA) 2 ~ J chain complex. After transport to the apical membrane of the cells, the complex fuses with the membrane and is released as secretory IgA (48). The secretory component, which is covalently bound to the complex by a disulfide bridge, protects the synthesized slgA in external liquids from denaturation and proteolysis.

Studies on the adherence of pneumococci to human epithelial cells showed an adherence of more than 100 pneumococci per epithelial cell for strains that had bound slgA in the binding studies, but only an adherence of μ2 pneumococci for strains tested negative in binding studies.

These results indicate that SpsA is involved in pneumococcal adherence to epithelial cells and invasion into epithelial cells.

The secretory IgA binding protein, SpsA, from Streptococcus pneumoniae is therefore a promising new candidate for vaccine development.

Example 1 : This example describes the secretory IgA binding of Streptococcus pneumoniae strains in a Western blot. The proteins of the Streptococcus pneumoniae lysate (ODgQO adjusted ^by l'O and after absorption of the bacteria in 100 μl digestion solution (20% glycerol, 3% SDS, 3% ß-mercaptoethanol, 0.05% bromophenol blue) boiled at 94 ° C. for 10 minutes ) were separated in SDS-PAGE and then transferred to a nitrocellulose membrane. After saturation with 10% skim milk in 0.1 M PBS, the filters were incubated with secretive IgA [1 μg / ml] (Sigma, Munich, Germany) in 0.1 M PBS for 1 hour at room temperature with shaking. After washing three times with 0.1 M PBS, the filters were incubated for 1 hour with a Goat-Anti Human IgA-HRP conjugate antibody (ICN, Eschwege, Germany). The color development was carried out after three washes with 1 mg of 4-chloro-1-naphthol and 0.1% H ₂ 0 ₂ per 1 ml of PBS.

Example 2:

This example describes the binding of 125j _oc j-labeled secretory IgA from Streptococcus pneumoniae strains. 100 ng slgA in 1 ml 0.05 M phosphate buffer pH 7.5 were added after the addition of 20 μg chloramine T with 350 μCi ¹² 5 iodine and the reaction was stopped after 5 minutes by adding 20 μg Na-Metabisul- fit. The labeled proteins were separated from the unlabeled proteins with a PDIO column (Pharmacia, Freiburg, Germany) and frozen at -20 ° C. 250 μl of a pneumococcal suspension (transmission 10% at 600 nm) in PBST (PBS with 0.05% Tween 20) were incubated with 0.023 μCi ¹²⁵ iodine-labeled slgA for 45 minutes and the reaction was stopped with 1 ml PBST. The slgA binding to the Streptococcus pneumoniae strains was measured by measuring the activity of the bacteria in the gamma counter (Packard, Dreieich, Germany).

Example 3: This example describes the cloning of the chromosomal DNA from Streptococcus pneumoniae ATCC 33400 into the vector Lambda ZAP Express ™ and the screening of the gene bank for a Streptococcus pneumoniae secretory IgA binding protein (SpsA). The chromosomal DNA from Streptococcus pneumoniae ATCC 33400 was isolated, partially digested with Sau3A and fractionated according to the size of the DNA fragments in a sodium chloride gradient which was formed by freezing and thawing a 20% strength sodium chloride solution. The ligation of the 2.0 kb to 6.0 kb DNA fragments of the chromosomal DNA in the BamHI-cut Lambda ZAP Express ™ and in vi tro packaging was carried out using a commercial kit according to the manufacturer (Stratagene, Heidelberg, Germany). The phage gene library was plated out without further amplification and the recombinant plaques were examined for the expression of a secretory IgA binding protein. The proteins were transferred to nitrocellulose filters, and after saturation with 10% skim milk in 0.1 M PBS, secretarial IgA [1 μg / ml] (Sigma, Munich, Germany) in 0.1 M PBS for 1 hour Incubated room temperature with shaking. After washing three times with 0.1 M PBS, the filters were incubated for 1 hour with a Goat-Anti Human IgA-HRP conjugate antibody. The color developed after washing three times with 1 mg of 4-chloro-1-naphthol and 0.1% H2O2 in 1 ml of PBS. Positive plaques were isolated and, after amplification, the in vivo excision of the pBK-CMV phagmid was carried out using the Exassist helper phage and XLOLR system according to the manufacturer's instructions (Stratagene, Heidelberg, Germany).

Example 4:

This example describes DNA sequencing and the derivation of the amino acid sequence. The 5.085 kb insert of Streptococcus pneumoniae ATCC 33400 in the phagemid pBK-CMV, called pSHAl, was sequenced by the ABI PRISM ™ Dye Terminator Cycle Sequencing according to the manufacturer (Perkin-Elmer, Germany) with the vector primers T3X (5 ' -AATTAACCCTCACTAAAGGG-3 ') and T7X (5'-TAATACGACTCACTATCGGG-3 ') and the primers derived from the sequence obtained (see table). The translation of the nucleic acid sequence into the amino acid sequence was carried out with the aid of the GeneWorks program, version 2.45 (Intelligence, Montain View, CA).

Table 1:

SH1 5'-ATATACAGTTCATATTGAAGTG-3 '(220-241)

SH2 5'-CAATATGAACTGTATATAAATCG-3 (236-214)

SH5 5'-GAGAGATAGACATAAAGATAC-3 '(553-571)

SH6 5'-GAACTATTTTATTCAAATACTCG-3 '(630-608)

SH8 5'-CAATATAGAACGAGATAAGGC-3 '(470-490)

SH9 5'-GCTGGAAACAAGAAAACGG-3 '(1259-1276)

SH10 5'-AATCTAAACTTCCTACAAGGG-3 '(1962-1942)

SH11 5'-GGTGCTATGAAAGCAAGCC-3 '(1722-1740)

SH17 5'-GAATTCACGAACTGCGACG-3 '(2203-2221)

Example 5:

This example describes the construction of the subclones of pSHAl and their characterization in the Western blot. Subclone pSHA2 (ntl-nt2203 from pSHAl) was obtained by deleting a 2882 bp EcoRI fragment and subclone pSHA3 (ntl-nt757) by deleting a 4328 bp HindiII fragment from pSHAl. Another subclone called pSHA4 (nt2952-nt5085 from pSHAl) was obtained by deleting a 2133 bp SacI fragment.

The characterization of the clones was carried out after separation of the proteins of the cell lysate [ODgQO ^{of λ} r ^ set and according to the bacteria ^¬ acceptance in 100 ul lysis solution (20% glycerol, 3% SDS, 3% ß-mercaptoethanol, 0.05% bromophenol blue) Boiled for 10 minutes at 94 ° C.] of the recombinant E. coli cells in SDS-PAGE and transfer of the proteins to a nitrocellulose membrane in the Western blot with secretory IgA. After saturation with 10% skim milk in 0.1 M PBS, the filters were incubated with secretory IgA [1 μg / ml] (Sigma, Munich, Germany) in 0.1 M PBS for 1 hour at room temperature with shaking. After washing three times with 0.1 M PBS, the filters were incubated for 1 hour with a Goat-Anti Human IgA-HRP conjugate antibody. The color development was carried out after three washes with 1 mg of 4-chloro-1-naphthol and 0.1% H ₂ 0 ₂ per 1 ml of PBS.

Example 6:

This example describes the PCR amplification and cloning of spsA, the 5 'region of spsA (ntl-nt972) and the 3' region (nt973-ntl572) of spsA into the expression vector pQE30 (Pharmacia).

The PCR primers for spsA and the spsΛ fragments were derived from the spsA sequence of Streptococcus pneumoniae ATCC 33400 serotype 1 obtained in pSHAl. The 5 'primer SH22 (5'-GCGCGCG CGCGGATCCTTGTTTGCATCAAAAAGCGAAAG-3') is 39 bp long and begins with a modified start codon of the spsÄ gene (TTG instead of ATG). The 5 'primer for the repeats, SH24 (5' -GCGCGCGCGCGGATCCACAGGCT GGAAACAAGAAAAC-3 '), begins with the initial sequence of the first repeat at nucleotide 973 of the spsÄ gene. The 3 'primer of spsASH23 (CTCAGCTAATTAAGCTTGTTTAGTTTACCCATTCACCATTGGC-3') begins with the stop codon and the 3 'primer of the N-terminus, SH25 (5'-CTCAGCTAATTAAGCTTTTTTTGGAGTAGATG2 nucleotide-3), starts at The primers SH22-SH23 were used to construct pQSH12, the primers SH22-SH25 to construct pQSH14 and the primers SH24-SH23 to construct pQSH30. The 5 'primers contained a B-amtil restriction site for cloning, the 3' primers a HindiII restriction site.

The amplification of the genomic pneumococcal DNA with the 5 'and 3' primers (20 pmol each) was carried out in a thermal cycler (MWG-Biotech, Ebersberg, Germany) in a 100 μl volume with 2.5 units of the Goldstar Taq polymerase according to the manufacturer (Eurogentec, Seraing, Belgium) and 50 ng chromosomal DNA. The samples were denatured at 94 ° C for two minutes and the amplification was carried out in 35 cycles consisting of 1 minute denaturation of the DNA at 94 ° C, 1 minute annealing of the primer at 55 ° C and 2 minutes extension at 72 ° C.

The primers SH22 to SH23 were also used for the amplification and cloning of the spsA genes from Streptococcus pneumoniae serotype 2

(R36A smooth, ATCC 11733 and D39, NCTC 7466) and serotype 47

(R36A rough, NCTC 10319) can be used.

The primers SH22 to SH25 could also be used for the amplification and cloning of the 5 'region of Streptococcus pneumoniae serotype 47 (R36A rough, NCTC 10319).

Example 7:

This example describes the study of the adherence of Streptococcus pneumoniae strains to human epithelial cells.

Confluent HEp-2 laryngeal carcinoma cells or A549 alveolar lung cells (2xl0 ⁵ ), which grew in DMEM / 5% fetal calf serum (FCS), were washed with DMEM / lmM HEPES with 10 ⁷ pneumococci in DMEM / 1 mM HEPES for 1 after washing the cells Infected at 37 ° C for one hour. The cells were then washed three times with PBS and fixed with methanol (-20 ° C., 30 minutes). The extracellular pneumococci were stained with Giemsa for microscopic enumeration according to the manufacturer's instructions (Sigma Diagnostic, Munich, Germany). The number of adherent pneumococci was determined by counting at least 100 epithelial cells.

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Claims

claims

1. Surface protein of Streptococcus pneunomiae (SpsA protein), characterized in that it binds to secretory IgA (slgA).

2. Secretory protein of Streptococcus pneumoniae, characterized in that it binds to secretory IgA (slgA).

3. Partially digested surface protein or secretory protein of Streptococcus pneumoniae according to claim 1 or 2, characterized in that it binds to secretory IgA (slgA).

4. C-terminally deleted derivative of a surface protein or secretory protein according to claim 1 or 2, characterized in that it binds to secretory IgA (slgA).

5. Deleted descendant of a surface protein or sekre ^¬ toric protein according to claim 1 or 2, characterized in that

- at least the signal sequence and / or

- At least optional repeats of the surface protein or secretory protein are deleted, however - The secretory IgA (slgA) binding domain is present, so that the descendant binds to secretory IgA (slgA).

6. derivative according to claim 5, characterized in that the surface protein or the secretory protein are deleted except for the secretory IgA (slgA) binding domain.

7. Surface protein according to claim 1, characterized by 523 amino acids according to FIG. 2 (positions 1 to 523).

8. C-terminally deleted derivative of the surface protein according to claim 1 or 7, characterized by 324 amino acids according to FIG. 2 (positions 1 to 324) and

Repeats 1 to 6 (positions 325 to 444) or repeats 1 to 7 (positions 325 to 464) or repeats 1 to 8 (positions 325 to 484 or to 485).

9. N-terminally deleted derivative of the surface protein according to claim 1 or 7, characterized in that it

(ii) is not, however, deleted in the range of positions 160 to 523.

10. N-terminal and C-terminal deleted derivative of the surface protein according to claim 1 or 7, characterized in that it

(ii) is not, however, deleted in the range of items 160 to 324 and, if appropriate (iii) repeats 1 to 8 (positions 325 to 484 or to 485) or repeats 1 to 7 (positions 325 to 464) or repeats 1 to 6 (positions 325 to 444).

11. derivative according to claim 10, characterized in that it is not deleted in the range of positions 174 to 285.

12. A secretory IgA (slgA) binding protein, characterized in that its amino acid sequence is at least 80% identical to that of the surface protein according to claim 7 or one of its derivatives according to claim 8, 9, 10 or 11.

13. Expression system in particular for Escherichia coli for the expression of a surface protein, a secretory protein, a derivative or a protein according to one of the preceding claims, comprising a DNA sequence encoding the surface protein or the derivative.

14. Vaccine for protection against diseases caused by Streptococcus pneunomiae, which can be produced with the aid of a secretory protein or a surface protein or a derivative according to one of claims 1 to 12.