# Patent

## Title
Antigenic protein fragments of Streptococcus pneumoniae

## Abstract

Antigenic protein fragments of Streptococcus pneumoniae to be used for the preparation of a medicament for the prevention and the treatment of bacterial infections and a method for the detection thereof, and related compositions using said epitopes, are disclosed.

## Background
This application is the U.S. national phase of International Application No. PCT/EP2009/053121 filed 17 Mar. 2009, which designated the U.S. and claims priority to EP Application No. 08425177.6 filed 19 Mar. 2008, the entire contents of each of which are hereby incorporated by reference.
The present invention refers to the field of infectious diseases and more in particular it refers to antigenic protein fragments of Streptococcus pneumoniae to be used for the preparation of a medicament for the prevention and the treatment of bacterial infections and to a method for the detection thereof, and related compositions using said epitopes.
BACKGROUND OF THE INVENTION
Streptococcus pneumoniae is a major cause of invasive diseases such as meningitis, septicaemia and pneumonia. Approximately, one million children under 5 years of age die of pneumococcal disease annually (Jaffar S., et al., Vaccine, 1999; 18(7-8):633-40).
In countries where the incidence of Neisseria meningitidis and Haemophilus influenzae infections has drastically decreased through the introduction of vaccines against meningococci group C and H. influenzae type B, S. pneumoniae has become the major cause of meningitis and septicemia in children. In addition, the morbidity by S. pneumoniae through respiratory tract infections such as otitis media and sinusitis is enormous. Thirty to 50% of all patients with otitis media and a substantial percentage of cases of sinusitis and pneumonia are caused by pneumococci. Risk groups for serious pneumococcal disease include children under the age of 2 years, elderly and patients with immunodeficiencies (Pichichero M. E., et al., Pediatr. Infect. Dis. J., 1997; 16(1):72-4).
Nasopharyngeal colonization by S. pneumoniae is common: probably all humans are colonized with this organism at least once early in life. Especially in circumstances of crowding, as in day-care centers, nursing homes, hospitals and jails, the risk of colonization with pneumococci is high (Kellner J. D., et al., Arch. Pediatr. Adolesc. Med., 1999; 153(5):495-502; Nuorti J. P., et al., N. Engl. J. Med., 1998; 338(26):1861-8; Principi N., et al., Pediatr. Infect. Dis. J., 1999; 18(6):517-23).
Colonization is not usually followed by disease, since this is prevented by the innate and adaptive immune system. However, disturbance of homeostasis between host and pathogen, for example through viral infections, malnutrition or local damage of the mucosa, is associated with the development of invasive diseases (Hament J. M., et al., FEMS Immunol, Med, Microbiol. 1999; 26(3/4):189-95; Mulholland K., Vaccine, 1999; 17(Suppl 1):S79-84; Plotkowski M. C., et al., Am. Rev. Respir. Dis., 1986; 134(5):1040-4).
Despite the availability of various potential control measures, bacterial infections persist as major causes of morbidity and mortality. For example, despite the long-standing use of the 23-valent pneumococcal vaccine in specific at-risk group over the age of 2 years, the pneumococcus (Streptococcus pneumoniae) remains the leading cause of community-acquired pneumonia, otitis media and meningitis (Fedson D. S., Vaccine, 1999; 30; 17 Suppl. 1:S85-90. Review; Tuomanen E. I., Vaccine, 2000; 8; 19 Suppl. 1:S38-40. Review).
The recent introduction of the 7-valent conjugate pneumococcal vaccine into the childhood immunization schedules of some countries will likely reduce pneumococcal disease. However, this vaccine covers infections caused by only some pneumococcal serotypes and the replacement over time of these serotypes by resistant ones is a likely possibility. Problems exist also with therapeutic interventions, since many invasive S. pneumoniae strains are resistant to beta-lactam and other antibiotics (Neuman M. I., et al., J. Emerg. Med., 2007; 32(4):349-57).
In the last years, many pneumococcal proteins, including pneumolysin, surface protein A (PspA), surface adhesin A (PsaA), surface protein C (PspC), neuraminidase and autolysin, have been proposed as potential vaccine candidates (Briles D. E., et al., Vaccine, 2000; 8; 19 Suppl. 1:S87-95. Review).
In addition, intensive research is aimed towards discovery of novel targets for antibiotic treatment to overcome drug resistance (Bogaert D., et al., Vaccine, 2004; Sep. 28; 22(29-30):4014-20).
In the past years, one of the inventors and colleagues applied the technology of phage display to the identification of antigens of Toxoplasma gondii (Beghetto E., et al., Int. J. Parasitol., 2001; 31(14):1659-68; Beghetto E., et al., Int. J. Parasitol., 2003; 33(2):163-73; WO03/080839) and tumors (WO03/010199; WO03/011903).
In 2006 the present inventors focused their attention on the identification of pneumococcal proteins by using the technology of phage display.
The library screening allowed the isolation of phage clones carrying three distinct antigenic regions of a hypothetical pneumococcal protein, encoded by the open reading frame (ORF) spr0075 in the S. pneumoniae R6 strain genome sequence. This was the first identified S. pneumoniae gene product, having an antigenic function during infection (Beghetto et al., FEMS Microbial Lett., 2006; 262:14-21).
The spr0075 ORF in the R6 genome of S. pneumoniae encodes a putative protein of 1161 aa (GenBank accession no. NP357669), having an expected molecular mass of 123 kDa. Analysis of the Spr0075 protein sequence reveals the presence of: (a) a putative signal peptide, located between amino acids 1 and 40 (putative cleavage site aa 41), (b) six adjacent repeated regions (152 aa long) and (c) an LPxTG anchoring motif (Schneewind O Mihaylova-Petkov D & Model P 1993), in the C-terminal region (residues 1148-1152 aa). The spr0075 gene from the R6 strains is well preserved among several strains (type 19F, 6B, 2, 4, 23F), although the number of repeated regions may vary.
The protein Spr0075 is encoded by an spr0075 ORF in the R6 genome sequence (Hoskins J., et al., J. Bacteriol., 2001; 183:5709-5717) located between nucleotides 80186 and 83671.
Antigenic regions of Spr0075 protein reacted with more than 60% of sera, indicating a broad recognition of this protein antigen.
The analysis of virulence was conducted comparing FP242, the isogenic encapsulated mutant strain wherein spr0075 was deleted, with the wild type D 39.
Female CD mice, 5-8 week old were intravenously injected with 100 μl of PBS containing 70,000 CFU of D39 and the percentage of survival was followed up to 10 days. The results in FIG. 1 show that the virulence of the spr0075 deletion mutant was comparable to the wild type.
The efficacy of antipneumococcus capsular polysaccharide-based vaccines has been extensively debated, as the protection elicited by capsule polysaccharides is stringently serotype-specific (Hausdorff et al., 2005) and often unable to induce long-term memory response.
In the last generation of vaccines (Prevnar/Prevenar, 7-valent pneumococcal conjugate vaccine), purified capsular polysaccharides of seven S. pneumoniae strains were coupled with a protein carrier, in order to exceed the above limitations. The vaccine is effective in 97% of invasive diseases caused by vaccine serotypes and offers some protection against otitis media and pneumococcal carriage (Bogaert et al., 2004b).
Owing to the limited serotype coverage, risks of serotype replacement and the high cost of pneumococcal glycoconjugated vaccines, great interest in the development of formulations based on pneumococcal protein antigens has emerged in the last decade (Bogaert et al., 2004b).
It is strongly felt the need of vaccine compositions based on new antigen fragments, capable to recognize several serotypes and induce immune response to species with high variability or to different bacterial types.
In view of the prior art and starting from the results obtained on spr0075, the present inventors deeply investigated the genoma of pneumococcus by using the phage display technique, in order to find new antigens with the desired properties.
Surprisingly the inventors identified the sequences defined as spr1370, spr1875 and spr1120.
Said sequences are virulence factors isolated from Streptococcus pneumoniae and conserved in other bacteria. A virulence factor is a protein indispensable for bacteria propagation in the host.
Object of the present invention are antigen fragments and/or fragments containing an epitope with the following amino acid sequence:







SEQ SPM4


(SEQ ID NO: 1)


FISQAVAKYPTLLESLPVKDSGARYRLEGYLFPATYSIKESTTIESLI


DEMLAAMDKNLSLYYSTIKSKNLTVNELLTIASLVEKEGAKTEDRKLI


AGVFYNRLNRDMPLQSNIAILYAQGKLGQNISLAEDVAIDTNIDSPYN


VYKNVGLMPGPVDSPSLDAIESSINQTKSDNLYFVADVTEGKVYYANN


QEDHDRN


 


SEQ Spr1370


(SEQ ID NO: 2)


MSEKSREEEKLSFKEQILRDLEKVKGYDEVLKEDEAVVRTPANEPSAE


ELMADSLSTVEEIMRKAPTVPTHPSQGVPASPADEIQRETPGVPSHPS


QDVPSSPAEESGSRPGPGPVRPKKLEREYNETPTRVAVSYTTAEKKAE


QAGPETPTPATETVDIIRDTSRRSRREGAKPAKPKKEKKSHVKAFVIS


FLVFLALLSAGGYFGYQYVLDSLLPIDANSKKYVTVGIPEGSNVQEIG


TTLEKAGLVKHGLIFSFYAKYKNYTDLKAGYYNLQKSMSTEDLLKELQ


KGGTDEPQEPVLATLTIPEGYTLDQIAQTVGQLQGDFKESLTAEAFLA


KVQDETFISQAVAKYPTLLESLPVKDSGARYRLEGYLFPATYSIKEST


TIESLIDEMLAAMDKNLSLYYSTIKSKNLTVNELLTIASLVEKEGAKT


EDRKLIAGVFYNRLNRDMPLQSNIAILYAQGKLGQNISLAEDVAIDTN


IDSPYNVYKNVGLMPGPVDSPSLDAIESSINQTKSDNLYFVADVTEGK


VYYANNQEDHDRNVAEHVNSKLN


 


SEQ SPM8


(SEQ ID NO: 3)


GVKESSNIASYEDLKGKTVGVKNGTASQTFLTENQSKYGYKIKTFADG


SSMDDSLNTGAIDAVMDDEPVLKYSISQGQKLKTPISGTPIGETAFAV


KKGANPELIEMF


 


SEQ Spr1120


(SEQ ID NO: 4)


MKKKFLAFLLILFPIFSLGIAKAETIKIVSDTAYAPFEFKDSDQTYKG


IDVDIINKVAEIKGWNIQMSYPGFDAAVNAVQAGQADAIMAGMTKTKE


REKVFTMSDTYYDTKVVIATTKSHKISKYDQLTGKTVGVKNGTAAQRF


LETIKDKYGFTIKTFDTGDLMNNSLSAGAIDAMMDDKPVIEYAINQGQ


DLHIEMDGEAVGSFAFGVKKGSKYEHLVTEFNQALSEMKKDGSLDKII


KKWTASSSSAVPTTTTLAGLKAIPVKAKYIIASDSSFAPFVFQNSSNQ


YTGIDMELIKAIAKDQGFEIEITNPGFDAAISAVQAGQADGIIAGMSV


TDARKATFDFSESYYTANTILGVKESSNIASYEDLKGKTVGVKNGTAS


QTFLTENQSKYGYKIKTFADGSSMDDSLNTGAIDAVMDDEPVLKYSIS


QGQKLKTPISGTPIGETAFAVKKGANPELIEMFNNGLANLKANGEFQK


ILDKYLASESSTASTSTVDETTLWGLLQNNYKQLLSGLGITLALALIS


FAIAIVIGIIFGMFSVSPYKSLRVISEIFVDVIRGIPLMILAAFIFWG


IPNFIESITGQQSPINDFVAGTIALSLNAAAYIAEIVRGGIQAVPVGQ


MEASRSLGISYGKTMRKIILPQVTKLMLPNFVNQFVIALKDTTIVSAI


GLVELFQTGKIIIARNYQSFKMYAILAIFYLVIITLLTRLAKRLEKR


IR


 


SEQ R4


(SEQ ID NO: 5)


EQIQNDLTKTDNKTSYTVQYGDTLSTIAEALGVDVTVLANLNKITNMD


LIFPETVLTTTVNEAEEVTEVEIQTPQADSSEEVTTATADLTTNQVTV


DDQTVQVADLSQPIAEAPKEVASSSEVTKTVIASEEVAPSTGTSVPEE


QTAETSSAVAEEAPQET


 


SEQ Spr1875


(SEQ ID NO: 6)


MKKRMLLASTVALSFAPVLATQAEEVLWTARSVEQIQNDLTKTDNKTS


YTVQYGDTLSTIAEALGVDVTVLANLNKITNMDLIFPETVLTTTVNEA


EEVTEVEIQTPQADSSEEVTTATADLTTNQVTVDDQTVQVADLSQPIA


EAPKEVASSSEVTKTVIASEEVAPSTGTSVPEEQTAETSSAVAEEAPQ


ETTPAEKQETQTSPQAASAVEATTTSSEAKEVASSNGATAAVSTYQPE


ETKIISTTYEAPAAPDYAGLAVAKSENAGLQPQTAAFKEEIANLFGIT


SFSGYRPGDSGDHGKGLAIDFMVPERSELGDKIAEYAIQNMASRGISY


IIWKQRFYAPFDSKYGPANTWNPMPDRGSVTENHYDHVHVSMNG






(wherein SEQ SPM4, SEQ SPM8 and SEQ R4 are the amino acid sequences of the fragments identified by using the technology of phage display while SEQ Spr1370, SEQ Spr1120 and SEQ Spr1875 are the amino acid sequences of the corresponding Open Reading Frame)

and the corresponding coding nucleotide sequence:








SEQ SPM4


(SEQ ID NO: 7)


TTTATCAGTCAAGCAGTAGCGAAATATCCTACTTTACTGGAAAGTTTG


CCTGTAAAAGACAGCGGTGCGCGTTATCGTTTGGAAGGATACCTTTTC


CCAGCTACATACTCTATCAAGGAAAGCACAACTATTGAGAGCTTGATT


GATGAGATGTTAGCTGCTATGGATAAGAACCTATCTCTTTACTATAGT


ACTATCAAATCTAAAAACTTGACTGTCAATGAGTTGTTGACCATTGCT


TCCTTGGTCGAAAAAGAAGGTGCCAAGACAGAAGATCGTAAGCTCATT


GCAGGTGTATTCTACAATCGTTTGAATCGTGATATGCCACTTCAAAGT


AATATTGCAATCTTGTATGCCCAAGGAAAACTGGGGCAAAATATCAGT


CTAGCTGAGGATGTTGCGATTGATACCAACATTGATTCACCTTATAAT


GTTTATAAAAATGTAGGTCTCATGCCTGGTCCAGTCGATAGTCCAAGT


CTGGATGCGATTGAGTCAAGCATCAATCAAACTAAGAGCGATAACCTC


TACTTTGTAGCAGATGTCACAGAAGGCAAGGTCTACTATGCTAACAAT


CAAGAAGACCACGACCGCA


 


SEQ SPM8


(SEQ ID NO: 8)


GGTGTCAAAGAATCAAGTAATATTGCTTCTTATGAAGATCTAAAAGGA


AAGACAGTCGGTGTTAAAAACGGAACTGCTTCTCAAACCTTCCTAACA


GAAAATCAAAGCAAATACGGCTACAAAATCAAAACCTTTGCTGATGGT


TCTTCAATGGATGACAGTTTAAACACTGGTGCCATTGATGCCGTTATG


GATGATGAACCTGTTCTCAAATATTCTATCAGCCAAGGTCAAAAATTG


AAAACTCCAATCTCTGGAACTCCAATCGGTGAAACAGCCTTTGCCGTT


AAAAAAGGAGCAAATCCAGAACTGATTGAAATGTTC


 


SEQ R4


(SEQ ID NO: 9)


GAGCAAATCCAAAACGATTTGACTAAAACGGACAACAAAACAAGTTAT


ACCGTACAGTATGGTGATACTTTGAGCACCATTGCAGAAGCCTTGGGT


GTAGATGTCACAGTGCTTGCGAATCTGAACAAAATCACTAATATGGAC


TTGATTTTCCCAGAAACTGTTTTGACAACGACTGTCAATGAAGCAGAA


GAAGTAACAGAAGTTGAAATCCAAACACCTCAAGCAGACTCTAGTGAA


GAAGTGACAACTGCGACAGCAGATTTGACCACTAATCAAGTGACCGTT


GATGATCAAACTGTTCAGGTTGCAGACCTTTCTCAACCAATTGCAGAA


GCTCCAAAAGAAGTAGCATCAAGTTCAGAAGTTACAAAGACAGTGATT


GCTTCTGAAGAAGTGGCACCATCTACGGGCACTTCTGTCCCAGAGGAG


CAAACGGCCGAAACAAGCAGTGCAGTTGCAGAAGAAGCTCCTCAGGAA


ACG






and the hybridizing nucleotide sequences, also under stringent hybridization, thereof. In this contest the terms “hybridization” and “stringent” refer to the conventional hybridization techniques well known to the person skilled in this field (Buzdin A and Lukyanov S (eds) Nucleic Acids Hybridization Kluwer Academic Publishers Netherlands 2007).

Another object of the present invention is a method for the identification of the amino acid sequences above disclosed comprising the following steps:

    
    
        a) obtaining a serum pool from subjects immunized with a killed bacterial strain;
        b) administering to subjects the serum pool obtained in step a) to give immunized subjects;
        c) collecting the sera from said immunized subjects obtained in step b), and
        d) undergoing the sera of step c to phage display technique.)
    
    


In the context of the above method according to the present invention, a “subject” is for example a laboratory animal, such as a mouse.
Another object of the present invention is a method for the identification of the above antigen fragments and/or fragments containing epitopes by means of selection of libraries of cDNA or DNA fragments of Streptococcus pneumoniae with sera of subjects immunized with the killed Streptococcus pneumoniae. 
A further object of the present invention is the use of said antigen fragments as active agents for the diagnosis of pneumococcal infections, in particular Streptococcus pneumoniae infections, Streptococcus gordonii infections, Streptococcus sanguinis infections, Streptococcus thermophilus infections, Streptococcus suis infections, Streptococcus agalactiae infections, Streptococcus pyogenes infections, Streptococcus mutans infections, Enterococcus faecalis infections, Enterococcus faecium infections, Rhodococcus sp. infections.
It is also object of the present invention, the use of said antigen fragments for the preparation of a medicament, preferably for the prevention or the treatment of pneumococcal infections, such as the ones listed above.
Are object of the present invention also the specific ligands such as natural host ligands (eg complement and other opsonins) or artificial ligands such as peptides selected with the above antigen using random peptide libraries and any molecules that bind to the above epitopes and the anti-epitope antibodies raised against said epitopes, and the use of at least one of said ligands and/or at least one of said antibodies for the preparation of means for the diagnosis of pneumococcal infections, such as the ones listed above.
Another object of the present invention is a method for the diagnosis of pneumococcal infections comprising the selection of sera of subjects affected by or suspected of being affected by said infection with the above antigen fragments and/or with at least one of the above ligands and/or at least one of the above antibodies and a diagnostic kit for pneumococcal infections.
A further object of the present invention is a pharmaceutical composition, particularly in the form of a vaccine, containing at least one of the above antigen fragments or one of the above sequences. Said composition is suitable for human and/or veterinary use.



These and other objects will be illustrated here below in detail, also by means of examples and figures, wherein:
FIG. 1 shows comparative data on the virulence of wild type D39 and FP242.
FIG. 2 shows the lethality induced by spr1370, spr1875 and spr1120 mutants.
FIG. 3 shows gene bank database sequence comparison of spr1370 (SEQ ID NOS 16-25 are disclosed respectively in order of appearance).
FIG. 4 shows gene bank database sequence comparison of spr1875 (SEQ ID NOS 26-36 are disclosed respectively in order of appearance).
FIG. 5 shows gene bank database sequence comparison of spr1120 (SEQ ID NOS 37-44 are disclosed respectively in order of appearance).
FIG. 6 shows the strong immunoprotective activities of R4, a polypeptide encoded by spr1875.



DETAILED DESCRIPTION OF THE INVENTION
All the definitions used herein are part of the common knowledge of a person skilled in this art and reference is made to the general scientific literature. Specific reference can be made to WO02/37115, WO03/080839, WO03/010199, WO03/011903 and WO2004/056851, which disclose and refer to the phage display technique
The present inventors identified three new pneumococcal gene products by using the phage display technology that can be efficiently used as targets for drug treatment and immune-based measures to control bacterial infections, as well as means to diagnose pneumococcal disease. To reach this objective, they used bacteriophage lambda display library of pneumococcal whole genome for the screening with immune sera. They identified three previously unknown pneumococcal protein fragments encoded by open the reading frames (ORF), hereinafter defined as spr1370, spr1875 and spr1120.
Said protein fragments contained B-cell epitopes, and thus can be used for diagnostic purposes and immuno-based strategies for the prevention and treatment of bacterial infections.
Moreover, they showed that the entire products of the corresponding genes may likely represent important targets for drug therapy and prophylaxis.
Since these gene products are highly conserved among bacterial pathogens, they can be effectively used for controlling a number of different infectious diseases caused by microbes with similar sequences, as identified by homology searches in nucleic acid data bases using servers such as clustalW (www.ebi.ac.uk/clustalw/).
The selection from the desired phage display library is performed as known in the art from the above cited references (WO02/37115, WO03/080839, WO03/010199; WO03/011903 and WO2004/056851).
Briefly, to select the bacterial gene product from a display library, a serum pool is obtained from animals, preferably mice, immunized with the killed strain, resuspended and administered to animals, preferably via subcutaneous injection. Then, the sera from immunized animals are collected and used for the library screening.
The display library is affinity-selected using the immune serum pool and the resulting phage population is analyzed by phage ELISA. At the end of the selection procedures, the phage clones bearing protein sequences that matched with the genome sequence of the bacterial strain are identified.
The genome sequences above identified are then molecularly characterized by comparative analysis with other strains in gene bank databases.
The genome sequences identified, and the sequences that hybridize under stringent conditions, encode for amino acid sequences containing epitopes, generating an antibody response. Such amino acid sequences and fragments can be used for the preparation of pharmaceutical compositions, preferably vaccines.
The preparation of pharmaceutical compositions and vaccines is within the framework of general knowledge; for further reference purposes, the reader is referred to the patent literature cited and incorporated by reference in the present description. See for example WO2007/081583 and WO2007/071786 and the references cited therein.
The diagnostic method for detecting bacterial infections comprises the following steps:

    
    
        a) contacting a biological sample of a subject with at least one peptide of the present invention;
        b) detecting antigen-antibody complex formation.
    
    


Preferably, the biological sample is collected from the subject before executing step a).
The diagnostic kits which are object of the present invention are known to the expert in the field but, by the way of an example, the reader is referred to U.S. Pat. No. 6,265,176 and WO01/63283.
The following examples further illustrate the present invention.
EXAMPLES
Example 1
Selection from S. pneumoniae Lambda Display Library
To select pneumococcal gene products from a display library, a serum pool obtained from five mice (6-week-old CBA/Jico mice) immunized with the killed S. pneumoniae D39 strain was used. Briefly, 107 CFU were re-suspended in Freund's adjuvant and administered to animals via subcutaneous injection at days 0 and 21. At day 35, sera from immunized mice were collected and used for library screening. Construction of the pneumococcal library has been previously described (Beghetto E., et al., Int. J. Parasitol., 2003; 33(2):163-73; Minenkova O., et al., Int. J. Cancer, 2003; 10; 106(4):534-44; Beghetto E., et al., FEMS Microbiol. Lett., 2006; 262(1):14-21).
In order to identify encoded protein fragments, the display library was affinity-selected using the immune serum pool. Three rounds of affinity-selection were performed and the resulting phage population was analyzed, after every round of selection, for its immunoreactivity by phage ELISA (Beghetto E., et al., FEMS Microbiol. Lett., 2006; 262(1):14-21). At the end of the selection procedures, phage clones bearing distinct protein regions that matched the genome sequence of S. pneumoniae R6 (GenBank accession no. AE007317) were identified.
Example 2
Molecular Characterization of the Gene Products
Three protein fragments named SPM4, R4, SPM8, encoded by regions of ORF spr1370, spr1875, and spr1120 respectively, were identified.

    
    
        ORF spr1370 consists of 1653 nucleotides and encodes for a hypothetical protein of 551 aa. The protein has a calculated molecular mass of 60.8 k Da and does not have a secretory signal peptide. Comparative analysis of the R6 strain-spr1370 gene with sequences from different pneumococci reveals that the protein is present in all the investigated strains belonging to different serotypes (19F, 6B, 2, 4, 23F).
        ORF spr1120 matches with the sequence of an ABC transporter membrane-spanning permease-glutamine transport gene. It encodes for a 731 aa-protein with a signal peptide of 67 aa length. The protein has a calculated molecular mass of 78.3 kDa.
        The 1140 nucleotide-long ORF spr1875 encodes for a 380 aa protein with a 25 aa-secretory signal peptide.
    
    


Example 3
Construction of the spr1370, spr1875 and spr1120 Gene Knockout Mutants
S. pneumoniae mutants were constructed by gene SOEing (Horton R. M., et al., Biotechniques, 1990; 8(5):528-35), as previously described (Iannelli F., et al., J. Bacteriol., 1999 April; 181(8):2652-4). In the mutants, the sprx gene was replaced by an antibiotic-resistance cassette (Pearce B. J., et al., Res Microbiol., 2002; 153(4):243-7), for this purpose, six oligonucleotide primers for each mutant were used. A first pair of primers was used to amplify the 5′ flanking region, a second pair was used to amplify the 3′ flanking region, and the last pair was used to generate an erythromycin resistance cassette.
The primers used are here listed:









1370-1:



(SEQ ID NO: 10)



AAGTCAAGAGAAGAAGAGAA;



 



1370-2:



(SEQ ID NO: 11)



ATCATCAACAATCACAAATCACTTTAGGCTTAGCGGGTTTTGCT;



 



1370-3:



(SEQ ID NO: 12)



AGCTTCCAAGGAGCTAAAGAGGTTCTATCAAGGAAAGCACAACT;



 



1370-4:



(SEQ ID NO: 13)



TTGATTGTTAGCATAGTAGACC;



 



IF188:



(SEQ ID NO: 14)



AAGTGATTTGTGATTGTTGATG;



 



IF189:



(SEQ ID NO: 15)



ACCTCTTTAGCTCCTTGGAAG.






The whole fragment, assembled by PCR reaction (Horton R., et al., Gene., 1989; 77:61-68), was used to transform S. pneumoniae D39 strain cells. The mutant construction was verified by PCR and sequencing. The spr1370, spr1875, and spr1120-deficient strains were named TF137, TF187 and TF112, respectively. Pneumococcal strains were grown in Todd-Hewitt (TH) broth in a 7% CO2-enriched atmosphere at 37° C. Where necessary, streptomycin (500 μgml−1) and erythromycin (1 μgml−1) were used to select mutants.
Example 4
In Vivo and In Vitro Studies
Streptococcus pneumoniae D39 (wild type) and TF137, TF187 and TF112 mutant strains, were grown to mid log phase (OD600nm=0.4) in 20 ml of TH broth. Importantly, no differences were found between any of the mutant strains and the parental one in their ability to grow in vitro. Cells were collected and washed twice, and then resuspended in PBS in a final volume of 2 ml. Six to eight-week old female CD1 mice were inoculated with diluted samples containing the indicated CFU by intravenous injection. Serial dilutions of the inoculums were plated on TH+1.5% agar, and incubated at 37° C. in 7% CO2 to verify bacterial colony forming units (CFU/ml).
FIG. 2 shows the virulence of TF137, TF187 and TF112 mutants compared to that of the D39 wild-type strain. Inoculation of 3×104 CFU of the wild-type strain resulted in rapid death of 50% of animals (panel A), while 7×104 CFU were sufficient to produce 100% lethality (panel B). In striking contrast the TF137 mutant, where the spr1370 gene was deleted, was totally impaired in causing lethality, even at doses of 2×107CFU. Only at doses of 2×108 half of the infected animals died (panel D). These data indicated that the virulence of the spr1370 mutant was approximately 4 orders of magnitude lower than that of the wild-type strain.
The figure also shows lethality of mice that were inoculated the TF187 and the TF112 strains, which were also considerably less virulent than the D39 wild-type strain. For example with the TF112 strain, in which the spr1120 gene was deleted, lethality was observed only at doses of 2×106 CFU or higher (panels C and D). The TF187 strain was also less virulent than the D39 mutant, although it did cause lethality at doses of 7×104 CFU or higher.
In further experiments (not shown) groups of mice were inoculated with 1×107 CFU of D39 or the TF137 mutant and sacrificed after 24 and 48 hours to examine the presence of bacteria in the blood and kidneys. While high bacterial counts were observed in mice inoculated with the D39 strain, no bacteria were observed in TF137-inoculated animals, confirming the inability of pneumococci to survive in vivo in the absence of the spr1370 gene.
Example 5
Gene Bank Sequence Comparison
The comparison with the sequences of other strains in the gene bank databases showed that spr1370 is conserved in Streptococcus gordonii, Streptococcus sanguinis, Streptococcus mutans, Streptococcus thermophilus, Streptococcus suis, and Streptococcus agalactiae (FIG. 3).
The comparison with the sequences of other strains in the gene bank databases showed that spr 1875 is conserved in Streptococcus sanguinis, Streptococcus thermophilus, Streptococcus pyogenes, Streptococcus gordonii, Streptococcus agalactiae, Streptococcus suis (FIG. 4).
The comparison with the sequences of other strains in the gene bank databases showed that spr 1120 is conserved in Streptococcus sanguinis, Streptococcus mutans, Streptococcus suis, Streptococcus agalactiae, Streptococcus pyogenes, Streptococcus thermophilus, Lactococcus lactis, Streptococcus gordonii, Enterococcus faecalis, Enterococcus faecium, Rhodococcus sp (FIG. 5).
Example 6
Immunoprotective Activities of the R4 Antigenic Polypeptide Encoded by spr1875
FIG. 6 shows the ability of a polypeptide (designated as R4 and encoded by spr1875) to protect, after immunization, mice against lethal pneumococcal infection. The R4 sequence was cloned into an expression vector and R4 was produced recombinantly fused to glutathione S transferase (GST), as previously described (Beghetto et al, FEMS Microbiol Lett, 2006; 262:1421). Mice were immunized with R4-GST for 3 times at days intervals in Freund's adjuvant and challenged intravenously with a lethal dose of the D39 S. pneumoniae strain. Control animals received GST only. While 13 out of 18 (72%) of the latter mice died, only 5 of the 18 mice immunized with R4-GST succumbed to infection (p<0.02 by Fisher exact test; FIG. 6A). Moreover, it has been shown that the R4 polypeptide is expressed on the surface of S. pneumoniae, i.e. it is in a position to be targeted by protective antibodies. This is evidenced in FIG. 6B by the ability of sera from R4-GST immunized animals to stain the D39 surface by indirect immunofluorescence, according to a previously described flow cytometry protocol (Grifantini R., et al., Nat. Biotechnol., 2002; 20(9):914-21). This example demonstrates that spr1875 and its products, with special reference to R4, are suitable candidates for vaccines against S. pneumoniae and other gram positive bacteria. It is of particular interest, in this context, that spr1875 is present in all of the pneumococcal strains whose genome has been sequenced. Moreover, as mentioned above, spr1875 is conserved in Streptococcus sanguinis, Streptococcus thermophiles, Streptococcus pyogenes, Streptococcus gordonii, Streptococcus agalactiae and Streptococcus suis. Therefore spr1875 (and homologous genes and gene products present in streptococci different from S. pneumoniae) could be used in the formulation of vaccines directed against the said and, possibly, additional bacterial pathogens.
Results
The data clearly established that the spr1370, spr1120 and the spr1875, encode for products that are required for in vivo growth of Streptococcus pneumoniae and for its ability to cause disease. Therefore these genes and their products are novel and important targets for the prevention or the therapy of pneumococcal diseases. Since at least portions of spr1120 and spr1875 are also present in other bacteria, these antigens may, in addition, be useful in the control of infections caused by bacteria different from pneumococci.
A pneumococcal strain devoid of the 1370 gene was almost completely unable to replicate in vivo. This mutant did not cause any lethality and no bacteria were detected at any time point in the blood or the organs of infected mice when using inocula lower than 2×108, i.e. at an extremely high dose. These striking results indicate that most likely the 1370 gene encodes of an important virulence factor, enabling S. pneumoniae to resist to antibacterial host defenses. Alternatively the 1370 gene product may be required for the synthesis of an essential nutritional factor, which is available in vitro cultures but not in vivo.
Similar considerations also apply to the products of the other discovered genes (spr1875, spr1120) that were shown here to play essential or important roles in S. pneumoniae virulence.
For example, the spr1875-encoded polypeptide R4 was capable of markedly protecting, after immunization, experimental animals against infection by S. pneumoniae. This underscores the utility of the genes and gene products described here, e.g. in the form of vaccines, for the control of infections by pathogenic bacteria expressing said genes, including S. pneumoniae.

## Claims

1. An isolated polypeptide consisting of amino acids 34-194 of SEQ ID NO: 6.

2. A vaccine composition comprising an isolated polypeptide consisting of amino acids 34-194 of SEQ ID NO: 6 and pharmaceutically acceptable carriers, or excipients and optionally adjuvants.

3. A kit comprising an isolated polypeptide consisting of amino acids 34-194 of SEQ ID NO: 6.