WO2011067758A2 - Immunogenic fragments and multimers from streptococcus pneumoniae proteins - Google Patents

Abstract

The present invention relates to immunogenic fragments, including variants and analogs derived from Streptococcus pneumoniae (S. pneumoniae) proteins, to polypeptide-multimers and fusion proteins comprising such polypeptides, and to vaccines comprising such immunogenic entities. In particular, the present invention relates to the use of such vaccines for eliciting protective immunity to S. pneumoniae.

Description

IMMUNOGENIC FRAGMENTS AND MULTIMERS FROM STREPTOCOCCUS

PNEUMONIAE PROTEINS

FIELD OF THE INVENTION

The present invention relates to the immunogenic polypeptide fragments derived from Streptococcus pneumoniae (S. pneumoniae) cell wall or cell membrane proteins and to their use in protection against infection with the bacteria. In particular, the present invention relates to immunogenic polypeptide fragments and multimers derived from cell wall or cell membrane proteins of S. pneumoniae which exhibit age-dependent antigenicity.

BACKGROUND OF THE INVENTION

Streptococcus pneumoniae belongs to the commensal flora of the human respiratory tract, but can also cause invasive infections such as meningitis and sepsis. Mortality due to pneumococcal infection remains high all over the world, augmented by a wide-spread antibiotic resistance in many pneumococcal strains (Dagan et al., Pneumococcal Infections, in: Feigin R, et al, eds. Textbook of Pediatric Infectious Diseases. 5 ed. Philadelphia: Saunders Co, 2004:1204-58). The current polysaccharide- based vaccines (including polysaccharide conjugates) elicit a strain-specific protection in children and the elderly, who are the main targets for pneumococcal infections. However the available vaccines either do not elicit long lasting protection or are limited in strain coverage. Development of new preventive interventions is hampered due to the incomplete understanding of pneumococcal pathogenesis.

Most children in the developing world become nasopharyngeal carriers of Streptococcus pneumoniae. Many develop pneumococcal disease that can be invasive (such as bacteremia, sepsis or meningitis), or mucosal infections (such as pneumonia and otitis media). S. pneumoniae is the leading cause of non-epidemic childhood meningitis in Africa and other regions of the developing world. Approximately, one million children die from pneumococcal inflicted diseases each year. Specifically, when considering deaths of children under five years of age worldwide, about 20% are from pneumococcal pneumonia. These high morbidity and mortality rates and the persistent emergence of antibiotic-resistant strains of S. pneumoniae heighten the need to develop an effective means of prevention, such as vaccination. The optimal anti-pneumococcal vaccine should be safe, efficacious, wide-spectrum (covering most or all pneumococcal strains), affordable, and available in large quantities.

The search for a wide-range anti-pneumococcal vaccine is ongoing. Indeed, introduction of pneumococcal 7-valent polysaccharide conjugate vaccine reduced significantly the rates of invasive diseases in infants and restricted significantly the rates of invasive diseases in the non-vaccinated members of the community in developed countries (Kyaw et al., N. Engl. J. Med. 2006, 354, 1455-63). However, carriage and diseases resulting from strains not included in the vaccine are on the rise (Musher DM., N. Engl. J. Med. 2006, 354, 1522-4, Huang et al., Pediatrics 2005, 1 16, e408-13). Vaccination with multivalent polysaccharide conjugate vaccines has been shown to be associated with serotype replacement, whereby non-vaccine serotype strains have elevated levels of carriage in populations with reduced incidence of vaccine serotype strains, which means that the effectiveness of conjugate vaccines is expected to diminish over time.

The mucosal epithelial surfaces with their tight junctions constitute the first line of defense that prevents the entry of pathogens and their products. S. pneumoniae adhere to the nasopharyngeal mucosal cells (Tuomanen E. 1999, Curr. Opin. Microbiol., 2:35-9), causing carriage without an overt inflammatory response. For clinical disease to occur, S. pneumoniae have to spread from the nasopharynx into the middle ear or the lungs or cross the mucosal epithelial cell layer and be deposited basally within the submucosa (Ring et al., J. Clin. Invest. 1998, 102:347-60). Molecules involved in adhesion, spread and invasion of S. pneumoniae, include capsular polysaccharides, cell-wall peptidoglycan and surface proteins (Jedrzejas MJ. Microbiol. Mol. Biol. Rev. 2001, 65, 187-207).

It has been observed that the antibody response in infants to S. pneumoniae cell wall or cell membrane proteins increases with age and correlates negatively with morbidity (Lifshitz et al. Clin. Exp. Immunol. 2002, 127, 344-53). To identify these proteins, a longitudinal series of children's sera was utilized to survey which S. pneumoniae cell-wall-associated proteins exhibit age-dependent antigenicity. The identity of these proteins was determined by MALDI-TOF analyses (Ling et al., Clin Exp Immunol 2004, 138, 290-8). WO 2003/082183 to one of the inventors of the present application discloses a defined group of cell wall and cell membrane S. pneumoniae proteins for use as vaccines against said bacteria. The thirty eight identified S. pneumoniae proteins demonstrate age- dependent antigenicity. All proteins tested to date elicit a protective immune response against the bacteria. These proteins are identified for use in vaccines especially in age groups (infants) which do not produce anti-S. pneumoniae antibodies following inoculation with polysaccharide-based vaccines or who do not mount significant antibody responses to these vaccines (elderly).

International Patent Application Publication No. WO 02/077021, assigned to Chiron S.P.A., discloses the sequence of about 2,500 S. pneumoniae genes, and their corresponding amino acid sequences from S. pneumoniae type 4 strain that were identified in silico. The use of a subset of 432 of those sequences as antigens for immunization is also suggested although no guidance in selecting useful proteins as antigens in the production of vaccines are provided.

Multi-epitope vaccines against influenza virus are disclosed in WO 2009/016639.

Multi-epitope DNA vaccines are discussed in Subbramanian et al. (J. Virol. 2003, 77, 10113-10118). Multivalent minigene vaccines containing B-cell, CTL and Th epitopes from several pathogens are described in Ling-Ling and Whitton (J. Virol 1997, 71 2292- 2302).

Thus there is an unmet need to provide immunogenic polypeptide fragments, having broad specificity against a wide range of different S. pneumoniae serotypes and minimal homology with human proteins, for use in improved S. pneumoniae peptide- based vaccines which can induce long-lasting immunological responses and in all age groups, including young children, immunocompromised subjects and elderly people.

SUMMARY OF THE INVENTION

The present invention provides immunogenic polypeptides and vaccines against S. pneumoniae. The polypeptides of the present invention are specific fragments of S. pneumoniae antigens referred to herein as age-dependent proteins.

The antibody response to S. pneumoniae proteins increases with age in infants, and this increase correlates with decreased morbidity. It was previously shown, using sera longitudinally collected from healthy children exposed to bacterial colonization, that there is an age-dependent enhancement of the antibody response to certain S. pneumoniae surface protein antigens. This enhancement, with age, of antibody responses against a set of specific pneumococcal surface proteins is implicated in the development of natural immunity and was used to identify candidate protein antigens (herein "age dependent proteins") for use in vaccine compositions against the bacteria.

The polypeptides of the present invention possess reduced homology to human sequences compared to the intact protein, minimizing the risk of developing autoimmunity against the patient's own proteins. Furthermore, the polypeptides of the present invention have increased sequence identity to many different S. pneumoniae strains making them ideal for wide-spectrum vaccines against the bacteria.

According to the present invention immunogenic protein fragments can be produced recombinantly, as isolated polypeptides or polypeptide-multimers, or as part of a fusion protein, or synthetically by peptide synthesis, or by linking several identical and/or different synthetic polypeptide fragments. Recombinant or synthetic production can be used, according to the present invention, to introduce specific mutations and/or variations in the peptide sequence for improving specific properties such as solubility and stability.

The production of specific fragments from different proteins is more cost- effective than the production of the respective intact proteins. It reduces the protein load and more immunogenic epitopes will be present per microgram of product. Advantageously, it will also be easier to purify in a consistent manner and to characterize analytically, thereby better addressing regulatory requirements. A fragment of an immunogenic protein may comprise several immunogenic epitopes but lack portions of the proteins which are not immunogenic or which confer undesired properties to the protein (e.g. toxicity, binding, cross reactivity to human sequences etc.)

The polypeptides of the present invention can be used in vaccine compositions against S. pneumoniae alone, in mixture with other immunogenic peptides, protein fragments or proteins, as part of a chimeric protein which may be used as an adjuvant, or mixed or formulated with an external adjuvant.

In a first aspect the present invention provides a synthetic or recombinant polypeptide of 51-250 amino acids derived from the sequence of a S. pneumoniae protein selected from the group consisting of: phosphoglucomutase/phosphomannomutase family protein (Accession No. NP_346006, SEQ ID NO:l); elongation factor G/tetracycline resistance protein (tetO), (Accession No. NP_34481 1, SEQ ID NO:2); Aspartyl/glutamyl- tRNA amidotransferase subunit C (Accession No. NP_344960, SEQ ID NO:3); L-lactate dehydrogenase (Accession No. NP 345686, SEQ ID NO:4); glyceraldehyde 3-phosphate dehydrogenase (GAPDH), (Accession No. NP_346439, SEQ ID NO:5); UDP-glucose 4- epimerase (Accession No. NP_346261, SEQ ID NO:6); elongation factor Tu family protein (Accession No. NP_358192, SEQ ID NO:7); Bifunctional GMP synthase/glutamine amidotransferase protein (Accession No. NP_345899, SEQ ID NO:8); glutamate dehydrogenase (Accession No. NP_345769, SEQ ID NO:9); Elongation factor TS (Accession No. NP_346622, SEQ ID NO: 10); phosphoglycerate kinase (TIGR4) (Accession No. AAK74657, SEQ ID NO: 11); 30S ribosomal protein SI (Accession No. NP_345350, SEQ ID NO: 12); 6-phosphogluconate dehydrogenase (Accession No. NP_357929, SEQ ID NO: 13); aminopeptidase C (Accession No. NP_344819, SEQ ID NO: 14); carbamoyl-phosphate synthase (large subunit) (Accession No. NP_345739, SEQ ID NO: 15); PTS system, mannose-specific IIAB components (Accession No. NP_344822, SEQ ID NO: 16); 30S ribosomal protein S2 (Accession No. NP 346623, SEQ ID NO: 17); dihydroorotate dehydrogenase IB (Accession No. NP_358460, SEQ ID NO: 18); aspartate carbamoyltransferase catalytic subunit (Accession No. NP_345741, SEQ ID NO: 19); elongation factor Tu (Accession No. NP_345941, SEQ ID NO:20); Pneumococcal surface immunogenic protein A (PsipA) (Accession No. NP_344634, SEQ ID NO:21); phosphoglycerate kinase (R6) (Accession No. NP_358035, SEQ ID NO:22); ABC transporter substrate-binding protein (Accession No. NP_344690, SEQ ID NO:23); endopeptidase O (Accession No. NP_346087, SEQ ID NO:24); Pneumococcal surface immunogenic protein C (PsipC) (Accession No. NP_345081, SEQ ID NO:25), and variants and analogs thereof.

According to some embodiments, the synthetic or recombinant polypeptide of 51 - 250 amino acids is selected from the group consisting of SEQ ID NOS: 26-75.

According to some embodiments of the present invention the polypeptide consists of 101-250 amino acids. According to other embodiments the polypeptide consists of 51- 100 amino acids.

According to yet some embodiments, a polypeptide according to the present invention consists of a sequence selected from SEQ ID NO:26-75. According to some embodiments, the polypeptides of the present invention share less than 30% sequence identity with the sequence of the homologous human proteins. According to other embodiments, the polypeptides according to the invention share less than 10%) sequence identity with such human proteins. According to yet another embodiment, when aligning the sequence of a polypeptide according to the invention with the corresponding sequence of a human protein, no more than nine contiguous amino acid residues are identical between the two sequences.

Variants of the peptides of the present invention include substitution of one amino acid residue per maximum of each contiguous sequence of nine amino acid residues in a peptide sequence, namely, peptides having about 90% or more identity are included within the scope of the present invention. According to other embodiments, sequences having at least 97% identity to the peptides of the present invention are provided.

According to some embodiments the present invention provides a synthetic or recombinant polypeptide comprising at least one polypeptide fragment of 51-250 amino acids, derived from the sequence of an S. pneumoniae protein associated with an age- dependent immune response, wherein the peptide sequence of 51-250 amino acids is selected from the group consisting of:

DVLVDRMRREFKVEANVGAPQVSYRETFRASTQ 33

ARGFFKRQSGGKGQFGDVWIEFTPNEEGKGFEFE NAIVGGWPREFIPAVEKGLVESMANGVLAGYP MVDV

QNTIVTTVMSNLGFHKALNREGINKAVTAVGDR 34

YWEEMRKSGYNLGGEQSGHVILMDYNTTGDG QLSAVQLTKIMK

QALAEKLDVDARSVHAYIMGEHGDSEFAVWSH 35 NP 345686

ANIAGVNLEEFLKDTQNVQEAELIELFEGVRDAA L-lactate

YTIINKKGATYYGIAVALARITKAILDDENAVLPL dehydrogenase

SVFQEGQYGVENVFIGQPAVVGAHGIVRPVNIPL

NDAETQKMQASAKELQAIIDEAWKNPEFQEASK

N

AFRRIQNVEGVEVTRINDLTDPVMLAHLLKYDTT 36 NP 346439 QGRFDGTVEVKEGGFEVNGKF glyceraldehyde-3 - phosphate dehydrogenase

KVSAERDPEQIDWATDGVEIVLEATGFFAKKEAA 37

EKHLKGGAKKWITAPGGNDVKTWFNTNH

DGSAQRVPTPTGSVTELVAVLEKNVTVDEVNAA 38

MKA A SNES YG YTEDPI VS SDI VGMS YG SLFD ATQ TKVLDVDGKQLVKVVSWYDNEMSYTAQLVRTL EY

VDLAIGHIKALEKVSEKTDVYIYNLGSGEGTSVL 39 NP 346261 QLVNTFESVNKIPIPYKIVPRRSGDVATCYANAD UDP-glucose 4- KAYKELNWRTTKSIEDMCRDTWNWQSK epimerase

K TAVAYNGTRI IMDTPGHADFGGEVERIMKM 40 NP 358192 VDGVVLWDAYEGTMPQTRFVLKKALEQDLVPI elongation factor Tu VWNKIDKPSARP family protein

MAPIFDTIIDHIPAPVDNSDEPLQFQVSLLDYNDF 41

VGRIGIGRVFRGTVKVGDQVTLSKLDGTTKNFRV

TKLFGFFGLERREIQEAKAGDLIAVSGMEDIFVGE

TITPTDAVEALPILfflDEPTLQMTFLVN SPFAGK

EGKWYTSRKVEERLQAELQTDVSLRVDPTDSPD

KWTVSGRGELHLSILIETMRREGYELQVSRPEVIV

KEIDG

KCEPFERVQIDTPEEYQGSVIQSLSERKGEMLDMI 42

STGNGQTRLVFLVPARGLIGYSTEFLSMTRGYGI

MNHTFDQYLPLIPGEIGGRHRGALVSIDAGKATT

YSIMSIEERGTIFVNPGTEVYEGMIIGENSRENDLT

VNITKAKQMTNVRSATKDQTAVIKTPRILTLEES

LEFLNDDEYMEVTPESIRLRKQILNKAEREKANK

KKKSAE

MSNISTDLQDVEKIIVLDYGSQYNQLISRRIREIGV 43 NP 345899 F SELKSHKI S A AE VREVNP VG Bifunctional GMP synthase/ glutamine amidotransferase protein

LLTHKLGGKVVPAGDAGNREYGQSTLTHTPSAL 44

FESTPDEQTVLMSHGDAVTEIPADFVRTGTSADC

YAAIENPDKHIYGIQFHPEVRHSVYGNDILRNFAL 45

NICKAKGDWSMDNFIDMQIKKI

TVGDKRVLLGLSGGVDSSWGVLLQKAIGDQLIC 46 IFVDHGLLRKGEADQVMDMLGGKFGLNIVKAD

AAKPvFLDKLAGVSDPEQKRKIIGNEFVYVFDDEA

SKLKDVKFLAQGTLYTDVIESGTDTAQTIKSHHN VGGLPE

MGEITEEKLETVRESDAILREEIAKAGLDRDIWQ 47

YFTVNTGVRSVGVMGDGRTYDYTIAIRAITSIDG MTADFAKIPWEVLQKISVRIVNEVDHV RIVYDI TSKPPATVEWE

QVNRGYRVQFNSAVGPYKGGLRFHPTVNQGILK 48 NP 345769

FLGFEQIFKNVLTGLPIGGGKGGSDF glutamate

dehydrogenase

LALIMPSGETLEAAYVSATATIGEKISFRRFALIEK 49 NP 346622

TDAQHFGAYQHNGGRIGVISVVE Elongation factor Ts

MAEITAKLVKELREKSGAGVMDAKKALVETDG 50

DIEKAIELLREKGMAKAAKKADRVAAEGLTGVY VNGNVAAVIEVNAETDFVAKNAQFVELV TTAK VIAEGKPAN EE

DEALAKQLSMHIAAMKPTVLSYKELDEQFVKDE 51

LAQLNHVIDQDNESRAMVNKPALPHL YGSKAQ

LTDDVIAQAEADIKAELAAEGKPEKIWD IIPGK

MDRFMLDNTKVDQAYTLLAQVYIMDDSKTVEA

YLESVNASVVEFARFEVGEGIEKA

KLTVKDVDLKGKKVLVRVDFNVPLKDGVITNDN 52 NP 358035

RITAALPTIKYIIEQGGRAILFSHLGRVKEE Phosphoglycerate kinase

ASNVGISANVEKAVAGFLLENEIAYIQEAVETPER 53

PFVAILGGSKVSDKIGVIENLLEKAD

VLIGGGMTYTFYKAQGIEIGNSLVEEDKLDVAKA 54

LLEKANGKLILPVDSKEANAFAGYTEVRDTEGEA

VSEGFLGLDIGPKSIAKFDEALTGAKTVVWNGPM

GVFENPDFQAGTIGVMDAIVKQPGVKSIIGGGDS

AAAAINLGRADKFSWI

MAVISMKQLLEAGVHFGHQTRRWNPKMAKYIF 55 NP 346623

TERNGIHVIDLQQTVKYADQAYDFMRDAAAND 30S ribosomal protein AVVLFVGTKKQAADAVAEEAVRSGQYFINHRW S2

LGGTLTNWGTIQKR1ARLKEIKRMEEDGTFEVLP

KKEVALLNKQR

VDNTPVFSLDLTKDKVTNQKASGRCWMFAALN 56 NP 344819

TFRHKLISQYKLENFELSQAHTFFWDKYEKSNWF Aminopeptidase C

LEQVI

TSDQELTSRKVSFLLQTPQQDGGQWD VVSLFE 57

KYG VVPKS VYPES VS S S S SRELN AILNKLLRQDA QILRDLLVSGADQ

MTVQAKKEDLLQEIFNFLAMSLGLPPRKFDFAYR 58

DKDNNYKSEKGITPQEFYKKYVNLPLEDYVSVIN

APTADKPYGKSYTVEMLGNVVGSRAVRYI VPM

ERLKELAIAQMQAGETVWFGSDVGQLSNRKAGI

LATDVYDFESSMDIKLTQDKAGRLDYSESLMTH

AMVLTGVDLDENGKS

AFAATIGYPVIVRPAFTLGGTGGGMCANEKELRE 59 NP 345739

ITENGLKLSPVTQCLIERSIAGFKEIEYEVMRDSA Carbamoyl phosphate

DNALVVCNMENFDPVGIHTGDSIVFAP synthase large subunit

KLAAKIAVGLTLDEVINPVTGSTYAMFEPALDYV 60 VAKIPRFPFDKFEKGERRLGTQMKATGEVMAIGR

NIEESLLKACRSLE

LIEKWKAQDDRLFYVSEAIRRGYTPEEIAELTKI 61

DIFYLDKLLHIFEIEQELGAHPQDLEVL

MAQVATKLILGQSLSELGYQNGLYPESTRVHIKA 62

PVFSFTKLAKVDSLLGPEMKSTGEVMGSDATLE

KALYKAFEASYLHLPTFGNVVFTIADDAKEEALN

LARRFQNIGYGILATEGTAAFFASHGLQAQPVGK

IGDDDKDIPSFVRKGRIQAIINTVGTKRTADEDGE

QIRRSAIEHGVPLFTALDTANAMLKVLESRSFVTE

AI

SVSDKLYFEPLTFEDVMNVIDLEQPKGVIVQFGG 63

QTAINLAEPLAKAGVTILGTQVADLDRAEDRDLF

EQALKELDIPQPPGQTATNEEEAALAARKIGFPVL

VRPSYVLGGRAMEIVENEEDLRSYMRTAVKASP

DHPVLVDSYIVGQECEVDAISDGK

LLGSIMIKATTLEPRFGNPTPRVAETPAGMLNAIG 64 NP 358460

LQNPGLEVVLAEKLPWLEREYPNLPIIANVAGFS dihydroorotate

KQEYAAVSHGISK dehydrogenase I B

KAIELNISCPNVDHCNHGLLIGQDPDLAYDVVKA 65

AVEASEVPVYVKLTPSVTDIVTVAKAAE

AGASGLTMINTLVGMRFDLKTRKPILANGTGGM 66

SGPAVFPVALKLIRQVAQT TDLPIIGMGGVDS

TVLARRLPSSVNQPKDYASIDAAPEERERGITINT 67 NP 345941

AHVEYETEKRHYAHIDA Elongation factor Tu

MILLSRQVGVKHLIVFMNKVDLVDDEELLELVE 68

MEIRDLLSEYDFPGDDLPVIQGSALKALEGDSKY

EDIVMELMNTVDEYIPEPERDTDKPLLLPVEDVF

SITGRGTVASGRIDRGIVKVNDEIEIVGIKEETQKA

VVTGVEMFRKQLDEGLAGDNVGVLLRGVQRDEI

ERGQVIAKPGSINPHTKFKGEVYILTKEEGGRHTP

FFN YRPQFYFRTTDVTGSIELPAGTEMVMPGDN

VTIDVELIHPIAVEQGTTFSIREGGRTVGSGMVTEI

EA

FNLIAGILEVQSGRIVLDGEENPKGRVSYMLQKD 69 NP-346607

LLLEHKTVLGNIILPLLIQKVDKAEAISRADKILAT ABC transporter, ATP-

FQLTAVRDKYPHELSGGMRQRVALLRTYL binding protein

LLDE AF S ALDEMTKMELHA WYLEIHKQLQLTTLI 70

ITHSIEEALNLSDRIYILKNRPG

MTRYQDDFYDAINGEWQQTAEIPADKSQTGGFV 71 NP 346087

DLDQEIEDLMLATTDKWLAGEEVPEDAILENFVK endopeptidase 0 YHRLVRDFDKREADGITP

SEYAKLYHPYSYEDFKKFAPALPLDDFFKAVIGQ 72

LPDKVIVDEERFWQAAEQFYSEE

KAAYHLAQEPFKQALGLWYAREKFSPEAKADVE 73

KKVATMIDVYKERLL NDWLTPETCKQAIV LN VIKPYIGYPEELPARYKDKVVNETASLFENALAF ARVEIKHSWSKWNQPV

DLHQS S S ANYGGIGA VI AHEI SFIAFDTNG ASFDE 74

NGSLKDWWTESDYAAFKEKTQKVIDQFDGQDS YGATINGKLTVSENVADLGGIAAALEAAKREAD FSAEEFFYNFGRIWRMKGRPEFMKLLASVDVHA PAKLRVNVQVPNFDDFFTTYDVKEGDGMWRSPE ERVIIW

MIGWARENAAEQIKQYQKFTVNISDETSMLAM 75 NP 345081

EQAGFISHQEKLERLGVHYEISERTQ Hypothetical protein

SP 0565

and variants and analogs thereof as defined herein. Each polypeptide is a separate embodiment of the invention.

According to yet other embodiments the present invention provides a synthetic or recombinant peptide of 51-100 amino acids selected from the group of SEQ ID NOS: 26, 27, 28, 29, 30, 31, 36, 37, 39, 40, 43, 44, 45, 48, 49, 52, 53, 56, 57, 59, 60, 61, 64, 65, 66, 67, 69, 70, 71, 72, and 75.

According to yet other embodiments the present invention provides a synthetic or recombinant peptide of 101-250 amino acids selected from the group of SEQ ID NOS: 32, 33, 34, 35, 38, 41, 42, 46, 47, 50, 51, 54, 58, 62, 63, 68, 73, and 74.

The present invention provides, according to some specific embodiments, a synthetic or recombinant polypeptide of 51-250 amino acids derived from the sequence of a Streptococcus pneumoniae (S. pneumoniae) cell wall or cell membrane protein associated with an age-dependent immune response, wherein the cell wall or cell membrane protein associated with an age-dependent immune response is selected from the group consisting of SEQ ID NO:l to SEQ ID NO:25 and the synthetic or recombinant polypeptide is selected from SEQ ID NO: 26 to SEQ ID NO:75, and variants and analogs having at least about 90% sequence identity, or at least about 97% identity to said synthetic or recombinant polypeptide. In another aspect the present invention provides a synthetic or recombinant polypeptide (herein denoted "multimer") comprising a plurality of S. pneumoniae derived polypeptide fragments. The multimer may contain a plurality of repeats not necessarily adjacent, of a specific fragment, a plurality of different fragments, from same or different protein, a plurality of repeats of a plurality of fragments, or a combination of any of these options.

A plurality according to the present invention means that at least two copies of & pneumoniae derived polypeptide or polypeptides fragment or fragments are present in a single polypeptide-based multimer construct.

According to certain embodiments, the synthetic or recombinant multimer comprises a sequence selected from SEQ ID NOs: 76, 78, 80, 82, 84, 86, 88, 90, 92 and 94. According to yet other embodiments, a synthetic or recombinant polypeptide multimer consisting of a sequence selected from SEQ ID NOs: 76, 78, 80, 82, 84, 86, 88, 90, 92 and 94 is provided.

A multimer according to some embodiments is produced as part of a fusion protein comprising a carrier sequence which may, according to some embodiments, serve as an adjuvant. According to other embodiments, the adjuvant provides a scaffold for better expression of the polypeptides. According to yet other embodiments, the adjuvant provides T-helper epitopes to the expressed polypeptides. According to yet other embodiments, the adjuvant or formulation has properties of a delivery system.

According to one embodiment, the fusion protein comprises detoxified pneumolysin or a fragment thereof. According to another embodiment, the fusion protein comprises heat shock protein 60 (hsp60) or a fragment thereof

According to a preferred embodiment, the present invention comprises a multimer comprising multiple copies of a plurality of different S. pneumoniae derived fragments, providing a high -density vaccine. According to the present invention, the multimer can be produced recombinantly, as an isolated polypeptide or as a fusion protein, or synthetically by linking a plurality of synthetic polypeptide fragments, or can be mixed or formulated with an external adjuvant and/or delivery system.

According to some embodiments, the present invention provides a synthetic or recombinant multimer comprising multiple copies of a plurality of S. pneumoniae derived fragments arranged in an alternating sequential polymeric structure (ΧιΧ2Χ3· ..)_η or in a block copolymer structure (Xi)_n(X2)n(¾)n—(Xm)n-

A synthetic or recombinant multimer according to the present invention is selected according to a specific embodiment from the group consisting of: i. B(XiZX₂Z ...X_m)_nB; and ii. B(Xi)_nZ(X₂)_nZ...(X_m)_nB;

wherein B is an optional sequence of 1-4 amino acid residues; n is at each occurrence independently an integer of 2-4; m is an integer of 2-4; each of X\, X₂...X_m is an immunogenic S. pneumoniae derived fragment consisting of 51-250 amino acid residues; Z at each occurrence is a bond or a spacer of 1-20 amino acid residues, and wherein the maximal number of amino acid residues in the multimer is about 1000.

According to certain embodiments, the spacer Z is selected from the group consisting of: Ala, Ala-Ala, Ala-Ala-Ala, Gly, Gly-Gly, Gly-Gly-Gly, Pro, Ser and Lys.

According to some embodiments, at least one amino acid of the spacer induces a specific conformation on a segment of the polypeptide (e.g. one or more proline residue).

According to yet other embodiments, the spacer comprises a cleavable sequence.

According to one embodiment, the cleavable spacer is cleaved by intracellular enzymes. According to a more specific embodiment, the cleavable spacer comprises a proteaseOspecific cleavable sequence. According to some embodiments, at least one fragment or multimer of the present invention is produced as part of a fusion protein comprising a carrier sequence, namely the polypeptide sequences are inserted within a sequence of a carrier polypeptide or are fused to a free amino group or a free carboxy group of a carrier protein sequence, which according to certain embodiments is a S. pneumoniae protein or fragment. According to other embodiments, the carrier protein sequence serves as an adjuvant.

According to certain embodiments, the carrier polypeptide is selected from the group consisting of: detoxified pneumolysin, hsp60 or a fragment thereof.

The present invention provides, according to another aspect, isolated polynucleotide sequences encoding a polypeptide according to any one of SEQ ID NOS: 26-75.

According to certain embodiments, the isolated polynucleotide sequence encoding a multimer according to the present invention comprises a sequence selected from SEQ ID NOs: 77, 79, 81, 83, 85, 87, 89, 91, 93 and 95.

According to some embodiments the present invention provides isolated polynucleotide sequences encoding a chimeric or fusion polypeptide comprising at least one peptide of SEQ ID NOS: 26-75.

According to yet another aspect, the present invention provides vaccine compositions for immunization of a subject against S. pneumoniae comprising at least one synthetic or recombinant polypeptide of 51-250 amino acids derived from an age- dependent S. pneumoniae cell wall or cell membrane protein. According to other embodiments, a vaccine composition according to the present invention further comprises at least one additional S. pneumoniae peptide, polypeptide or protein sequence.

According to some embodiments, the vaccine composition further comprises an adjuvant. According to other embodiments, the vaccine does not contain an adjuvant.

According to some embodiments, the vaccine composition further comprises a delivery system. According to other embodiments, the vaccine does not contain a delivery system.

Pharmaceutically acceptable adjuvants include, but are not limited to water-in-oil emulsion, lipid emulsion, and liposomes. According to specific embodiments the adjuvant is selected from the group consisting of: CCS/C^®, Montanide^®, alum, muramyl dipeptide, Gelvac^®, chitin microparticles, chitosan, cholera toxin subunit B, labile toxin, AS21V, AS02V, Intralipid^®, and Lipofundin^®.

According to some specific embodiments of the preset invention, the adjuvant CCS/C^® is included in the vaccine formulation.

In some embodiments, the vaccine is formulated for intramuscular, intranasal, oral, intraperitoneal, subcutaneous, topical, intradermal and transdermal delivery. In some embodiments, the vaccine is formulated for intramuscular administration. In other embodiments, the vaccine is formulated for oral administration. In yet other embodiments, the vaccine is formulated for intranasal administration. According to some embodiments, formulations for various of these routes of delivery contain delivery systems such as liposomes, ISCOMs, or other macromolecular carriers.

The present invention provides according to a further aspect a method for inducing an immune response and conferring protection against S. pneumoniae in a subject, comprising administering a vaccine composition comprising at least one synthetic or recombinant polypeptide of 51-250 amino acids derived from the sequence of a cell-wall or cell-membrane protein of S. pneumoniae associated with age-dependent immune response, and variants and analogs thereof. According to some preferred embodiments, the composition comprises a multimer or a fusion polypeptide comprising at least one synthetic or recombinant polypeptide, variant or analog of 51-250 amino acids derived from the sequence of a cell-wall or cell-membrane protein of S. pneumoniae associated with age-dependent immune response. The route of administration of the vaccine is selected from intramuscular, oral, intranasal, intraperitoneal, subcutaneous, topical, intradermal, and transdermal delivery. According to preferred embodiments the vaccine is administered by intramuscular, intranasal or oral routes.

According to a further aspect of the present invention, use of a composition comprising at least one synthetic or recombinant S. pneumoniae derived polypeptide of 51-250 amino acids, and variants, analogs, multimers and fusion polypeptides thereof in protection against an S. pneumoniae infection in a subject is provided.

Use of a peptide or polypeptide derived from the sequence of an age-dependent cell-wall or cell-membrane protein of S. pneumoniae, and variants and analogs thereof, for preparation of a vaccine composition for immunization against S. pneumoniae is also within the scope of the present invention. Further aspects provide use of an isolated polynucleotide according to the invention for production of a polypeptide of 51-250 amino acids, and variants, analogs, multimers and fusion polypeptides thereof, and for vaccination against an S. pneumoniae infection in a subject.

Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent from this detailed description to those skilled in the art.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Colonization of CBA/N xid mice following immunization with PS20 emulsified with CFA as adjuvant. CBA/N xid mice (n=3 per group) were immunized subcutaneously (SC) with either the indicated amount of rPS20 or \0\ig non-lectin (NL) protein extract of S. pneumoniae serotype 3 strain WU2 cell wall fraction. The antigens were emulsified with CFA as an adjuvant in the first immunization and with IFA in the two following immunizations. Mice were subsequently challenged intranasally (IN) with S. pneumoniae serotype 3 strain WU2 (7.5* 10⁵ CFU per mouse). Forty eight hours later, mice were sacrificed and nasopharynx (A) and right lobe-lung (B) of each mouse were homogenized and plated on blood agar for bacterial enumeration. Asterisks represent significant values (p value<0.05) compared to control mice immunized with adjuvant only, as indicated by student t-test.

Figure 2. Colonization of CBA/N xid mice following immunization with PS20 formulated in CCS/C as adjuvant. CBA/N xid mice (n=3 per group) were immunized intramuscularly (IM) with the indicated amount of rPS20 formulated with CCS/C liposomes. Mice were also subcutaneously immunized with l(^g non-lectin protein extract of S. pneumonia serotype 3 strain WU2 cell wall fraction emulsified with CFA as a positive control. The CFA emulsified group was emulsified with CFA as an adjuvant in the first immunization and with IFA in the two following immunizations. Mice were subsequently challenged IN with 5". pneumoniae serotype 3 strain WTJ2 (7.5* 10⁵ CFU per mouse). Forty eight hours later, mice were sacrificed and nasopharynx (A) and right lobe- lung (B) of each mouse were homogenized and plated on blood agar for bacterial enumeration. Asterisks represent significant values (p value<0.05) compared to control mice immunized with adjuvant only, as indicated by student t-test.

Figure 3: Survival rate of CBA N xid mice following immunization with PS19 with CFA as adjuvant. CBA/N xid mice (n=7 per group) were immunized SC with the indicated amount of rPS19 emulsified, with CFA as an adjuvant in the first immunization and with IFA in the two following immunizations. Mice were subsequently challenged IN with a lethal dose of S. pneumoniae serotype 3 strain WU2 (3* 10⁶ CFU per mouse). Survival rate were monitored daily in the following 7 days. Mice that survived for more than 7 days were marked as alive.

Figure 4: Survival rate of CBA/N xid mice following immunization with PS19 with CCS/C as adjuvant. CBA/N xid mice (n=7 per group) were immunized IM with the indicated amounts of rPS 19 formulated in CCS/C liposomes (at antigen: adjuvant ratio of 1 :200). Mice were subsequently challenged IN with a lethal dose of S. pneumoniae serotype 3 strain WU2 (3 * 10⁶ CFU per mouse). Survival rates were monitored daily in the following 7 days. Mice that survived for more than 7 days were marked as alive.

Figure 5: Colonization of CBA/N xid mice following immunization with PS19 formulated in CCS/C as adjuvant. CBA/N xid mice (n=3 per group) were immunized IM with the indicated amounts of rPS19 formulated in CCS/C liposomes liposomes (at antigen: adjuvant ratio of 1 :200). Mice were subsequently challenged IN with S. pneumoniae serotype 3 strain WU2 (1.9* 10⁶ CFU per mouse). Forty eight hours later, mice were sacrificed and nasopharynx (A) and right lobe-lung (B) of each mouse were homogenized and plated on blood agar for bacterial enumeration. Asterisks represent significant values (p value<0.05) compared to control mice immunized with adjuvant only, as indicated by student t-test.

DETAILED DESCRIPTION OF THE INVENTION

Novel therapeutic strategies are necessary to counter the prevalence of antibiotic- resistant pneumococci and the limitations of currently available vaccines. Future discovery of therapeutic modalities requires a better understanding of the dynamic interplay between pathogen and host, which leads either to S. pneumoniae clearance or to carriage and disease development. It is suspected that inappropriate or altered immune responses underlie the switch from benign carriage to clinical disease. It has been observed in infants that the antibody response and antibody levels to S. pneumoniae increase with age and correlates negatively with morbidity. The development of a universal vaccine against S. pneumoniae will prevent replacement carriage and disease development, caused by serotypes not included in the conjugate vaccine observed following immunization with the polysaccharide conjugate vaccine. Furthermore, such a vaccine may be used in subjects previously immunized with the polysaccharide vaccine.

As previously described (WO 2003/082183), to identify proteins having vaccine potential, a cell wall fraction was extracted from S. pneumoniae. The proteins (around 150) in the cell wall fraction were screened by 2D-immunoblotting using sera obtained longitudinally from children attending day care centers and sera from healthy adult volunteers. Similarly, membrane extracts were resolved by 2D-PAGE and screened with sera obtained longitudinally from children attending day care centers and sera from healthy adult. Thirty eight proteins exhibited age-dependent antigenicity and are therefore denoted "age-dependent". The sequences of the age-dependent proteins were determined and the proteins were identified (for example SEQ ID NOS 1-25). These proteins were observed to elicit cross-strain protection against lethal intranasal pneumococcal challenge in a mouse model (for example, Ling et al., Clin Exp Immunol 2004, 138, 290-8). The proteins and antibodies raised against them were found to inhibit bacterial adhesion to cultured epithelial cells in vitro. In an attempt to identify immunogenic fragments derived from the above age- dependent proteins and thereby to reduce protein load upon vaccination, an array of polypeptides is prepared and screened with sera obtained longitudinally from infants and healthy adults and from mice immunized with the intact age-dependent proteins.

The polypeptides of the present invention have the advantage of reduced homology or no to human sequences. If a microbial antigen has significant sequence homology to a human protein, then use of such an antigen in a vaccine would entail the risk of eliciting autoimmune responses directed against the particular human protein - an unacceptable outcome. Therefore, it is very important to remove any such sequences - homologous between the microbial antigen and the human protein - from the antigen in order that it would have utility as a vaccine.

It is now disclosed, that certain fragments of 51-250 amino acids, derived from the above 25 age-dependent proteins of S. pneumoniae, lack sequence or structural homology to human proteins, and possess high homology among all available sequenced strains of S. pneumoniae, retain the age-dependency characteristic in children, and can be used in improved vaccines against S. pneumoniae.

These protein fragments, alone, as part of multimeric constructs, conjugated to or mixed with a carrier protein and/or with an adjuvant, are effective in protecting subjects against infection with S. pneumoniae.

For convenience, certain terms employed in the specification, examples and claims are described herein.

The term "antigen presentation" means the expression of antigen on the surface of a cell in association with major histocompatibility complex class I or class II molecules (MHC-I or MHC-II) of animals or with the HLA-I and HLA-II of humans.

The term "immunogenicity" or "immunogenic" relates to the ability of a substance to stimulate or elicit an immune response. Immunogenicity is measured by determining the ability to produce antibodies specific for the substance. The presence of antibodies is detected by methods known in the art, for example using ELISA.

"Amino acid sequence" as used herein refers to an oligopeptide, peptide, polypeptide, or protein sequence, and fragment thereof, and to naturally occurring or synthetic molecules. A "chimeric protein/polypeptide" or a "fusion protein/polypeptide" are used interchangeably and refer to an immunogenic peptide or peptides operatively linked to a polypeptide or protein.

A "multimer" refers to a construct comprising at least two covalently linked, immunogenic protein fragments or analogs thereof according to the invention. The at least two protein fragments or analogs thereof may be identical or different, and the multimer may include at least one sequence of a carrier protein or a protein fragment which is optional functionalized as an adjuvant.

Synthetic peptides

Some of the polypeptides of the present invention may be synthesized chemically using methods known in the art for synthesis of peptides and polypeptides. These methods generally rely on the known principles of peptide synthesis; most conveniently, the procedures can be performed according to the known principles of solid phase peptide synthesis.

As used herein "peptide" indicates a sequence of amino acids linked by peptide bonds. A polypeptide is generally a peptide of about 51 or more amino acids.

Polypeptide analogs and peptidomimetics are also included within the scope of the invention as well as salts and esters of the polypeptides of the invention.

A polypeptide analog according to the present invention may optionally comprise at least one non-natural amino acid and/or at least one blocking group at either the C terminus or N terminus. Salts of the polypeptides of the invention are physiologically acceptable organic and inorganic salts. The design of appropriate "analogs" may be computer assisted.

The term "peptidomimetic" means that a polypeptide sequence according to the invention is modified in such a way that it includes at least one non-peptidic bond such as, for example, urea, carbamate, sulfonamide, hydrazine, or any other covalent bond. The design of appropriate "peptidomimetic" may be computer assisted.

Salts and esters of the polypeptides of the invention are encompassed within the scope of the invention. Salts of the polypeptides of the invention are physiologically acceptable organic and inorganic salts. Functional derivatives of the polypeptides of the invention covers derivatives which may be prepared from the functional groups which occur as side chains on the residues or the N- or C-terminal groups, by means known in the art, and are included in the invention as long as they remain pharmaceutically acceptable, i.e., they do not destroy the activity of the polypeptide and do not confer toxic properties on compositions containing it. These derivatives may, for example, include aliphatic esters of the carboxyl groups, amides of the carboxyl groups produced by reaction with ammonia or with primary or secondary amines, N-acyl derivatives of free amino groups of the amino acid residues formed by reaction with acyl moieties (e.g., alkanoyl or carbocyclic aroyl groups), or O-acyl derivatives of free hydroxyl group (for example that of seryl or threonyl residues) formed by reaction with acyl moieties.

The term "amino acid" refers to compounds which have an amino group and a carboxylic acid group, preferably in a 1,2- 1,3-, or 1,4- substitution pattern on a carbon backbone. oc-Amino acids are most preferred, and include the 20 natural amino acids (which are L-amino acids except for glycine) which are found in proteins, the corresponding D-amino acids, the corresponding N-methyl amino acids, side chain modified amino acids, the biosynthetically available amino acids which are not found in proteins (e.g., 4-hydroxy-proline, 5 -hydroxy-lysine, citrulline, ornithine, canavanine, djenkolic acid, β-cyanolanine), and synthetically derived -amino acids, such as amino- isobutyric acid, norleucine, norvaline, homocysteine and homoserine. β-Alanine and γ- amino butyric acid are examples of 1,3 and 1,4-amino acids, respectively, and many others are well known to the art. Statine-like isosteres (a dipeptide comprising two amino acids wherein the CONH linkage is replaced by a CHOH), hydroxyethylene isosteres (a dipeptide comprising two amino acids wherein the CONH linkage is replaced by a CHOHCH₂), reduced amide isosteres (a dipeptide comprising two amino acids wherein the CONH linkage is replaced by a CH₂NH linkage) and thioamide isosteres (a dipeptide comprising two amino acids wherein the CONH linkage is replaced by a CSNH linkage) are also useful residues for this invention.

The amino acids used in this invention are those that are available commercially or are available by routine synthetic methods. Certain residues may require special methods for incorporation into the peptide, and sequential, divergent or convergent synthetic approaches to the peptide sequence are useful in this invention. Natural coded amino acids and their derivatives are represented by three-letter codes according to IUPAC conventions. When there is no indication, the L isomer was used. Conservative substitutions of amino acids as known to those skilled in the art are within the scope of the present invention, as long as antigenicity is preserved in the substituted peptide. Conservative amino acid substitutions include replacement of one amino acid with another having the same type of functional group or side chain, e.g., aliphatic, aromatic, positively charged, negatively charged. These substitutions may enhance oral bioavailability, penetration into the central nervous system, targeting to specific cell populations and the like. One of skill in the art will recognize that individual substitutions, deletions or additions to peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art.

The following six groups each contain amino acids that are conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

Recombinant production of peptides and polypeptides

The polypeptides of the present invention can be prepared by expression from an expression vector per se or as a chimeric protein. The methods to produce a chimeric or recombinant protein comprising one or more peptides derived from age-dependent proteins of S. pneumoniae are known to those with skill in the art. A nucleic acid sequence encoding one or more polypeptide comprising at least one such peptide can be inserted into an expression vector for preparation of a polynucleotide construct for propagation and expression in host cells.

The term "expression vector" and "recombinant expression vector" as used herein refers to a DNA molecule, for example a plasmid or virus, containing a desired and appropriate nucleic acid sequences necessary for the expression of the recombinant polypeptides for expression in a particular host cell. As used herein, "operably linked" refers to a functional linkage of at least two sequences. Operably linked includes linkage between a promoter and a second sequence, for example, a nucleic acid of the present invention, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence.

The regulatory regions necessary for transcription of the polypeptides can be provided by the expression vector. The precise nature of the regulatory regions needed for gene expression may vary among vectors and host cells. Generally, a promoter is required which is capable of binding RNA polymerase and promoting the transcription of an operably-associated nucleic acid sequence. Regulatory regions may include those 5' non-coding sequences involved with initiation of transcription and translation, such as the TATA box, capping sequence, CAAT sequence, and the like. The non-coding region 3' to the coding sequence may contain transcriptional termination regulatory sequences, such as terminators and polyadenylation sites. A translation initiation codon (ATG) may also be provided.

In order to clone the nucleic acid sequences into the cloning site of a vector, linkers or adapters providing the appropriate compatible restriction sites are added during synthesis of the nucleic acids. For example, a desired restriction enzyme site can be introduced into a fragment of DNA by amplification of the DNA by use of PCR with primers containing the desired restriction enzyme site.

An alternative method to PCR is the use of synthetic gene. The method allows production of an artificial gene which comprises an optimized sequence of nucleotides to be expressed in cells of a desired species (for example, E. coli). Redesigning a gene offers a means to improve gene expression in many cases. Rewriting the open reading frame is possible because of the redundancy of the genetic code. Thus it is possible to change up to about one-third of the nucleotides in an open reading frame and still produce the same protein. For a typical protein sequence of 300 amino acids, there are over 10¹⁵⁰ codon combinations that will encode an identical protein. Using optimization methods such as replacing rarely used codons with more common codons can result in dramatic effects. Further optimizations such as removing RNA secondary structures can also be included. Computer programs are available to perform these and other simultaneous optimizations. A well optimized gene can dramatically improve protein expression. Because of the large number of nucleotide changes made to the original DNA sequence, the only practical way to create the newly designed genes is to use gene synthesis.

An alternative approach is cloning the gene into pET30+ vector omitting the His- tag sequence by the use of Ndel restriction enzyme to produce the first methionine.

An expression construct comprising a polypeptide sequence operably associated with regulatory regions can be directly introduced into appropriate host cells for expression and production of polypeptide per se or as a recombinant fusion protein. The expression vectors that may be used include but are not limited to plasmids, cosmids, phage, phagemids or modified viruses. Typically, such expression vectors comprise a functional origin of replication for propagation of the vector in an appropriate host cell, one or more restriction endonuclease sites for insertion of the desired gene sequence, and one or more selection markers.

The recombinant polynucleotide construct comprising the expression vector and a polypeptide according to the invention should then be transferred into a host cell where it can replicate (for example a bacterial cell), and then be transfected and expressed in an appropriate prokaryotic or eukaryotic host cell. This can be accomplished by methods known in the art. The expression vector is used with a compatible prokaryotic or eukaryotic host cell which may be derived from bacteria, yeast, insects, mammals and humans.

Once expressed by the host cell, the polypeptide or multimer can be separated from undesired components by a number of protein purification methods. One such method uses a polyhistidine tag on the recombinant protein. A polyhistidine-tag consists in at least six histidine (His) residues added to a recombinant protein, often at the N- or C-terminus. Polyhistidine-tags are often used for affinity purification of polyhistidine- tagged recombinant proteins that are expressed in E. coli or other prokaryotic expression systems. The bacterial cells are harvested by centrifugation and the resulting cell pellet can be lysed by physical means or with detergents or enzymes such as lysozyme. The crude lysate contains at this stage the recombinant protein among several other proteins derived from the bacteria and are incubated with affinity media such as NTA-agarose, HisPur resin or Talon resin. These affinity media contain bound metal ions, either nickel or cobalt to which the polyhistidine-tag binds with micromolar affinity. The resin is then washed with phosphate buffer to remove proteins that do not specifically interact with the cobalt or nickel ion. The washing efficiency can be improved by the addition of 20 mM imidazole, and proteins are then usually eluted with 150-300 mM imidazole. The polyhistidine tag may be subsequently removed using restriction enzymes, endoproteases or exoproteases. Kits for the purification of histidine-tagged proteins can be purchased for example from Qiagen.

Another method is through the production of inclusion bodies, which are aggregates of protein that may form when a recombinant polypeptide is expressed in a prokaryote. While the cDNA may properly code for a translatable mRNA, the protein that results may not fold correctly or completely, or the hydrophobicity of the sequence may cause the recombinant polypeptide to become relatively insoluble. Inclusion bodies are easily purified by methods well known in the art. Various procedures for the purification of inclusion bodies are known in the art. In some embodiments the inclusion bodies are recovered from bacterial lysates by centrifugation and are washed with detergents and chelating agents to remove as much bacterial protein as possible from the aggregated recombinant protein. To obtain soluble protein, the washed inclusion bodies are dissolved in denaturing agents and the released protein is then refolded by gradual removal of the denaturing reagents by dilution or dialysis (as described for example in Molecular cloning: a laboratory manual, 3rd edition, Sambrook, J. and Russell, D. W., 2001 ; CSHL Press).

Analytical and large scale purifications for preparative purposes generally utilize three properties to separate proteins. First, proteins may be purified according to their isoelectric points by running them through a pH graded gel or an ion exchange column. Second, proteins can be separated according to their size or molecular weight via size exclusion chromatography or by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) analysis. Thirdly, proteins may be separated by polarity/hydrophobicity via high pressure liquid chromatography or reversed-phase chromatography. Alternatively, proteins are often purified on a small scale by using 20- PAGE and are then analyzed by peptide mass fingerprinting to establish the protein identity. The purified protein is tracked by its molecular mass or other methods known in the art.

In order to evaluate a process of multistep purification, the amount of the specific protein has to be compared to the amount of total protein. The latter can be determined by the Bradford or Lowry total protein assay or by absorbance of light at 280 nm; however some reagents used during the purification process may interfere with accurate quantification. For example, imidazole (commonly used for purification of polyhistidine-tagged recombinant proteins) is an amino acid analogue and at low concentrations will interfere with the bicinchoninic acid (BCA) assay for total protein quantification. Impurities in low-grade imidazole will also absorb at 280 nm, resulting in an inaccurate reading of protein concentration from UV absorbance.

Another method to be considered is Surface Plasmon Resonance (SPR). SPR can detect binding of label free molecules on the surface of a chip. If the desired protein is an antibody, binding can be translated directly to the activity of the protein. One can express the active concentration of the protein as the percent of the total protein. SPR can be a powerful method for quickly determining protein activity and overall yield.

Vaccine Formulation

The vaccines of the present invention comprise at least one immunogenic polypeptide derived from S. pneumoniae age-dependent proteins, and optionally, an adjuvant and/or delivery system. Formulation can contain one or more of a variety of additives, such as adjuvant, delivery system excipient, stabilizers, buffers, or preservatives. The vaccine can be formulated for administration in one of many different modes.

In its preferred embodiment, the vaccine is formulated for parenteral administration, for example intramuscular administration. According to yet another embodiment, the vaccine is formulated for oral administration.

According to yet another embodiment, the vaccine is formulated for intradermal administration. Needles specifically designed to deposit the vaccine intradermally are known in the art, as disclosed for example in US 6,843,781 and US 7,250,036 among others. According to other embodiments, administration is performed with a needleless injector.

According to one embodiment of the invention, the vaccine is formulated for intranasal administration. The vaccine formulation may be applied to the mucosal tissue of the nose in any convenient manner. However, it is preferred to apply it as a liquid stream or liquid droplets to the walls of the nasal passage. The intranasal composition can be formulated, for example, in liquid form as nose drops, spray, or suitable for inhalation, as powder, as cream, or as emulsion. In another embodiment of the invention, the vaccine is formulated for oral administration; the vaccine may be presented, for example, in the form of a tablet or encased in a gelatin capsule or a microcapsule.

The formulation of these modalities is general knowledge to those with skill in the art.

Liposomes provide another delivery system for antigen delivery and presentation. Liposomes are bilayered vesicles composed of phospholipids and other sterols surrounding a typically aqueous center where antigens or other products can be encapsulated. The liposome structure is highly versatile with many types ranging from about 25 nm to about 500 μηι in size. Liposomes have been found to be effective in delivering therapeutic agents to dermal and mucosal surfaces. Liposomes can be further modified for targeted delivery by, for example, incorporating specific antibodies into the surface membrane, or altered to encapsulate bacteria, viruses or parasites. The average survival time or half life of the intact liposome structure can be extended with the inclusion of certain polymers, for example polyethylene glycol, allowing for prolonged release in vivo. Liposomes may be unilamellar or multilamellar. Liposomes may have diverse ionic charges.

The vaccine composition may be formulated by: encapsulating an antigen or an antigen/adjuvant complex in liposomes to form liposome-encapsulated antigen and mixing the liposome-encapsulated antigen with a carrier comprising a continuous phase of a hydrophobic substance. If an antigen/adjuvant complex is not used in the first step, a suitable adjuvant may be added to the liposome-encapsulated antigen, to the mixture of liposome-encapsulated antigen and carrier, or to the carrier before the carrier is mixed with the liposome-encapsulated antigen. The order of the process may depend on the type of adjuvant used. Typically, when an adjuvant like alum is used, the adjuvant and the antigen are mixed first to form an antigen/adjuvant complex followed by encapsulation of the antigen/adjuvant complex with liposomes. The resulting liposome- encapsulated antigen is then mixed with the carrier. The term "liposome-encapsulated antigen" may refer to encapsulation of the antigen alone or to the encapsulation of the antigen/adjuvant complex depending on the context. This promotes intimate contact between the adjuvant and the antigen and may, at least in part, account for the immune response when alum is used as the adjuvant. When another adjuvant is used, the antigen may be first encapsulated in liposomes and the resulting liposome-encapsulated antigen is then mixed into the adjuvant in a hydrophobic substance.

In formulating a vaccine composition that is substantially free of water, antigen or antigen/adjuvant complex is encapsulated with liposomes and mixed with a hydrophobic substance. In formulating a vaccine in an emulsion of water-in-a hydrophobic substance, the antigen or antigen/adjuvant complex is encapsulated with liposomes in an aqueous medium followed by the mixing of the aqueous medium with a hydrophobic substance. In the case of the emulsion, to maintain the hydrophobic substance in the continuous phase, the aqueous medium containing the liposomes may be added in aliquots with mixing to the hydrophobic substance.

In all methods of formulation, the liposome-encapsulated antigen may be freeze- dried before being mixed with the hydrophobic substance or with the aqueous medium as the case may be. In some instances, an antigen/adjuvant complex may be encapsulated by liposomes followed by freeze-drying. In other instances, the antigen may be encapsulated by liposomes followed by the addition of adjuvant then freeze-drying to form a freeze-dried liposome-encapsulated antigen with external adjuvant. In yet another instance, the antigen may be encapsulated by liposomes followed by freeze-drying before the addition of adjuvant. Freeze-drying may promote better interaction between the adjuvant and the antigen resulting in a more efficacious vaccine, as well as maintenance of stability.

Formulation of the liposome-encapsulated antigen into a hydrophobic substance may also involve the use of an emulsifier to promote more even distribution of the liposomes in the hydrophobic substance. Typical emulsifiers are well-known in the art and include mannide oleate (Arlacel™ A), lecithin, Tween™ 80, Spans™ 20, 80, 83 and 85. The emulsifier is used in an amount effective to promote even distribution of the liposomes. Typically, the volume ratio (v/v) of hydrophobic substance to emulsifier is in the range of about 5 : 1 to about 15: 1.

Microparticles and nanoparticles employ small biodegradable spheres which act as depots for vaccine delivery. The major advantage that polymer microspheres possess over other depot-effecting adjuvants is that they are extremely safe and have been approved by the Food and Drug Administration in the US for use in human medicine as suitable sutures and for use as a biodegradable drug delivery system (Langer R. Science. 1990; 249(4976): 1527-33). The rates of copolymer hydrolysis are very well characterized, which in turn allows for the manufacture of microparticles with sustained antigen release over prolonged periods of time (O'Hagen, et al., Vaccine. 1993;l l(9):965-9).

CCS/C^® is a synthetic polycationic sphingolipid derived from D-erythro ceramide to which spermine is covalently attached, thereby forming Ceramide Carbamoyl Spermine (CCS). CCS mixed with cholesterol (CCS/C) self-assembles into liposomes known as VaxiSome. Based on its structure and components (ceramide, C0₂ and spermine), CCS is predicted to be biocompatible and biodegradable. In vitro and in vivo studies suggest that the CCS/C formulation up-regulates levels of CD40 and B7 co- stimulatory molecules, which are essential in antigen presentation and T-helper cell activation. As a result, VaxiSome is a potent liposomal adjuvant/delivery system for stimulating enhanced immune responses via the Thl and Th2 pathways.

Parenteral administration of microparticles elicits long-lasting immunity, especially if they incorporate prolonged release characteristics. The rate of release can be modulated by the mixture of polymers and their relative molecular weights, which will hydrolyze over varying periods of time. Without wishing to be bound to theory, the formulation of different sized particles (1 μπι to 500 μπι) may also contribute to long- lasting immunological responses since large particles must be broken down into smaller particles before being available for macrophage uptake. In this manner a single- injection vaccine could be developed by integrating various particle sizes, thereby prolonging antigen presentation.

In some applications an adjuvant or excipient may be included in the vaccine formulation. Alum for example, is a preferred adjuvant for human use. The choice of the adjuvant will be determined in part by the mode of administration of the vaccine. One preferred mode of administration is intramuscular administration. Another preferred mode of administration is intranasal administration. Non-limiting examples of intranasal adjuvants include chitosan powder, PLA and PLG microspheres, QS-21, AS02V, calcium phosphate nanoparticles (CAP); mCTA/LTB (mutant cholera toxin E112K with pentameric B subunit of heat labile enterotoxin), and detoxified E. coli derived heat- labile toxin. The adjuvant used may also be, theoretically, any of the adjuvants known for peptide- or protein-based vaccines. For example: inorganic adjuvants in gel form (aluminium hydroxide/aluminium phosphate, Warren et al., 1986; calcium phosphate, Relyvelt, 1986); bacterial adjuvants such as monophosphoryl lipid A (Ribi, 1984; Baker et al., 1988) and muramyl peptides (Ellouz et al., 1974; Allison and Byars, 1991; Waters et al., 1986); particulate adjuvants such as the so-called ISCOMS ("immunostimulatory complexes", Mowat and Donachie, 1991 ; Takahashi et al., 1990; Thapar et al., 1991), liposomes (Mbawuike et al. 1990; Abraham, 1992; Phillips and Emili, 1992; Gregoriadis, 1990) and biodegradable microspheres (Marx et al., 1993); adjuvants based on oil emulsions and emulsifiers such as Montanide™ (Incomplete Freund's adjuvant, Stuart-Harris, 1969; Warren et al., 1986), SAF (Allison and Byars, 1991), saponins (such as QS-21 ; Newman et al., 1992), squalene/squalane (Allison and Byars, 1991); synthetic adjuvants such as non-ionic block copolymers (Hunter et al., 1991), muramyl peptide analogs (Azuma, 1992), synthetic lipid A (Warren et al., 1986; Azuma, 1992), synthetic polynucleotides (Harrington et al., 1978) and polycationic adjuvants (WO 97/30721).

Adjuvants for use with immunogens of the present invention include aluminum or calcium salts (for example hydroxide or phosphate salts). A particularly preferred adjuvant for use herein is an aluminum hydroxide gel such as Alhydrogel™. Calcium phosphate nanoparticles (CAP) is another potential adjuvant. The immunogen of interest can be either coated to the outside of particles, or encapsulated on the inside (He et al., 2000, Clin. Diagn. Lab. Immunol., 7,899-903).

Another adjuvant for use with an immunogen of the present invention is an emulsion. A contemplated emulsion can be an oil-in-water emulsion or a water-in-oil emulsion. In addition to the immunogenic chimer protein particles, such emulsions comprise an oil phase of squalene, squalane, peanut oil or the like, as are well known, and a dispersing agent. Non-ionic dispersing agents are preferred, and such materials include mono- and di-C₁₂-C₂₄-fatty acid esters of sorbitan and mannide such as sorbitan mono-stearate, sorbitan mono-oleate and mannide mono-oleate.

Such emulsions are for example water-in-oil emulsions that comprise squalene, glycerol and a surfactant such as mannide mono-oleate (Arlacel™ A), emulsified with the chimer protein particles in an aqueous phase. Alternative components of the oil-phase include alpha-tocopherol, mixed-chain di- and tri-glycerides, and sorbitan esters. Well- known examples of such emulsions include Montanide™ ISA-720, and Montanide™ ISA 703 (Seppic, Castres, France. Other oil-in-water emulsion adjuvants include those disclosed in WO 95/17210 and EP 0 399 843.

The use of small molecule adjuvants is also contemplated herein. One type of small molecule adjuvant useful herein is a 7-substituted-8-oxo- or 8-sulfo-guanosine derivative described in U.S. Pat. No. 4,539,205, U.S. Pat. No. 4,643,992, U.S. Pat. No. 5,01 1,828 and U.S. Pat. No. 5,093,318. 7-allyl-8-oxoguanosine(loxoribine) has been shown to be particularly effective in inducing an antigen-(immunogen-) specific response.

A useful adjuvant includes monophosphoryl lipid A (MPL^®), 3-deacyl monophosphoryl lipid A (3D-MPL^®), a well-known adjuvant manufactured by Corixa Corp. of Seattle, formerly Ribi Immunochem, Hamilton, Mont. The adjuvant contains three components extracted from bacteria: monophosphoryl lipid (MPL) A, trehalose dimycolate (TDM), and cell wall skeleton (CWS) (MPL+TDM+CWS) in a 2% squalene/Tween™ 80 emulsion. This adjuvant can be prepared by the methods taught in GB 2122204B.

Other compounds are structurally related to MPL^® adjuvant called aminoalkyl glucosamide phosphates (AGPs) such as those available from Corixa Corp under the designation RC-529™ adjuvant {2-[(R)-3-tetra-decanoyloxytetradecanoylamino]-ethyl- 2-deoxy-4-0-phosphono-3 -O- [(R)-3 -tetradecanoyloxytetra-decanoyl] -2- [(R)-3 -tetra- , decanoyloxytet-radecanoyl-amino]-p-D-glucopyranoside triethylammonium salt}. An RC-529 adjuvant is available in a squalene emulsion sold as RC-529SE and in an aqueous formulation as RC-529AF available from Corixa Corp. (see, U.S. Pat. No. 6,355,257 and U.S. Pat. No. 6,303,347; U.S. Pat. No. 6,113,918; and U.S. Publication No. 03-0092643).

Further contemplated adjuvants include synthetic oligonucleotide adjuvants containing the CpG nucleotide motif one or more times (plus flanking sequences). The adjuvant designated QS21, available from Aquila Biopharmaceuticals Inc., is an immunologically active saponin fractions having adjuvant activity derived from the bark of the South American tree Quillaja Saponaria Molina (e.g., Quil™ A), and the method of its production is disclosed in U.S. Pat. No. 5,057,540. Derivatives of Quil™ A, for example QS21 (an HPLC purified fraction derivative of Quil™) and other fractions such as QA17 are also disclosed. Semi-synthetic and synthetic derivatives of Quillaja Saponaria Molina saponins are also useful, such as those described in U.S. Pat. No. 5,977,081 and U.S. Pat. No. 6,080,725. The adjuvant denominated MF59 is described in U.S. Pat. No. 5,709,879 and U.S. Pat. No. 6,086,901.

Muramyl dipeptide adjuvants are also contemplated and include N-acetyl- muramyl-L-threonyl-D-isoglutamine (thur-MDP), N-acetyl-nor-muramyl-L-alanyl-D- isoglutamine [CGP 11637, referred to as nor-MDP], and N-acetylmuramyl-L-alanyl-D- isoglutaminyl-L-alanine-2-(r-2'-dipalmityol-s-n-glycero-3-hydroxyphosphoryloxy) ethylamine [(CGP) 1983 A, referred to as MTP-PE]. The so-called muramyl dipeptide analogues are described in U.S. Pat. No. 4,767,842.

Other adjuvant mixtures include combinations of 3D-MPL and QS21 (EP 0 671

948 Bl), oil-in-water emulsions comprising 3D-MPL and QS21 (WO 95/17210, PCT/EP98/05714), 3D-MPL formulated with other carriers (EP 0 689 454 Bl), QS21 formulated in cholesterol-containing liposomes (WO 96/33739), or immunostimulatory oligonucleotides (WO 96/02555). Adjuvant SBAS2 (now AS02) from GlaxoSmithKline containing QS21 and MPL in an oil-in-water emulsion is also useful. Alternative adjuvants include those described in WO 99/52549 and non-particulate suspensions of polyoxyethylene ether (UK Patent Application No. 9807805.8).

The use of an adjuvant that contains one or more agonists for toll-like receptor-4 (TLR-4) such as an MPL^® adjuvant or a structurally related compound such as an RC- 529^® adjuvant or a Lipid A mimetic, alone or along with an agonist for TLR-9 such as a non-methylated oligodeoxynucleotide-containing the CpG motif is also optional.

A heat-shock protein, fragment or peptide is also an optional adjuvant, as a carrier protein or peptide, in a mixture, or as part of a fusion polypeptide expressed or synthesized together with at least one polypeptide according to the invention. For example, U.S. Patent Nos. 5,736,146 and 5,869,058 provide peptides derived from humans and E. coli heat-shock protein 60 (hsp60) as carriers for vaccination against viral and bacterial pathogens. Defined peptides present uniquely effective characteristics in conjugate vaccines due to the following reasons: i. HSP60 epitopes provide natural T-cell help; Humans are born with a high frequency of T cells responsive to HSP60, so no induction is needed and youngsters respond. ii. Defined HSP60-peptide conjugates function as built-in adjuvants activating innate TLR-4 receptors on APC; the HSP60-conjugate vaccine administered in aqueous solution serves as its own adjuvant.

iii. Defined HSP60-peptide conjugates do not induce the production of competing antibodies and therefore do not suppress vaccination responses, even with multiple administrations.

iv. Boosting to the HSP60-epitope occurs naturally, since HSP60 is up-regulated at the site of any immune response (infection or tumor); the vaccination effect does not decline for prolonged periods. Immune memory is robust and effective.

Detoxified pneumolysin, known as a carrier protein and as an adjuvant (for example Michon et al., Vaccine, 18, 1732-1741, 1998), or fragment or analog thereof, can be also used in conjunction or conjugation of the polypeptides of the present invention.

Another type of adjuvant mixture comprises a stable water-in-oil emulsion further containing aminoalkyl glucosamine phosphates such as described in U.S. Pat. No. 6,113,918. Of the aminoalkyl glucosamine phosphates the molecule known as RC-529 {(2-[(R)-3-tetradecanoyloxytetradecanoylamino]ethyl-2-deoxy-4-0-phosphono-3-0- [(R)-3-tetradecanoyloxy-tetradecanoyl]-2-[(R)-3— tetradecanoyloxytetra- decanoylamino]-p-D-glucopyranoside triethylammonium salt.)} is most preferred. A preferred water-in-oil emulsion is described in WO 99/56776.

Adjuvants are utilized in an adjuvant amount, which can vary with the adjuvant, host animal and immunogen. Typical amounts can vary from about 1 μg to about 10 mg per immunization. Those skilled in the art know that appropriate concentrations or amounts can be readily determined.

Vaccine compositions comprising an adjuvant based on oil in water emulsion is also included within the scope of the present invention. The water-in-oil emulsion may comprise a metabolisable oil and a saponin, such as for example as described in US 7,323,182.

According to several embodiments, the vaccine compositions of the present invention may contain one or more adjuvants, characterized in that it is present as a solution or emulsion which is substantially free from inorganic salt ions, wherein said solution or emulsion contains one or more water soluble or water-emulsifiable substances which are capable of making the vaccine isotonic or hypotonic. The water soluble or water-emulsifiable substances may be, for example, selected from the group consisting of: maltose; fructose; galactose; saccharose; sugar alcohol; lipid; and combinations thereof.

The polypeptides, multimers, and fusion proteins of the present invention may comprise according to several specific embodiments a proteosome adjuvant. The proteosome adjuvant comprises a purified preparation of outer membrane proteins of meningococci and similar preparations from other bacteria. These proteins are highly hydrophobic, reflecting their role as transmembrane proteins and porins. Due to their hydrophobic protein-protein interactions, when appropriately isolated, the proteins form multi-molecular structures consisting of about 60-100 nm diameter whole or fragmented membrane vesicles. This liposome-like physical state allows the proteosome adjuvant to act as a protein carrier and also to act as an adjuvant.

The use of proteosome adjuvant has been described in the prior art and is reviewed by Lowell GH in "New Generation Vaccines", Second Edition, Marcel Dekker Inc, New York, Basel, Hong Kong (1997) pages 193-206. Proteosome adjuvant vesicles are described as comparable in size to certain viruses which are hydrophobic and safe for human use. The review describes formulation of compositions comprising non-covalent complexes between various antigens and proteosome adjuvant vesicles which are formed when solubilizing detergent is selectably removed using exhaustive dialysis technology.

Vaccine compositions comprising different immunogenic polypeptides can be produced by mixing or linking a number of different polypeptides according to the invention with or without an adjuvant. In addition, an immunogenic polypeptide according to the present invention may be included in a vaccine composition comprising any other S. pneumoniae protein or protein fragment, including mutated proteins such as detoxified pneumolysin, or they can be linked to or produced in conjunction with any such S. pneumoniae protein or protein fragment.

Vaccine compositions according to the present invention may include, for example, influenza polypeptides or peptide epitopes, conjugated with or coupled to at least one immunogenic S. pneumoniae polypeptide according to the invention. The antigen content is best defined by the biological effect it provokes. Naturally, sufficient antigen should be present to elicit the production of measurable amounts of protective antibody. A convenient test for the biological activity of an antigen involves the ability of the antigenic material undergoing testing to deplete a known positive antiserum of its protective antibody. The result is reported in the negative log of the LD₅₀ (lethal dose, 50%) for mice treated with virulent organisms which are pretreated with a known antiserum which itself was pretreated with various dilutions of the antigenic material being evaluated. A high value is therefore reflective of a high content of antigenic material which has blocked the antibodies in the known antiserum, thus reducing or eliminating the neutralizing effect of the antiserum on the virulent organism. It is preferred that the antigenic material present in the final formulation is at a level sufficient to increase the negative log of LD₅₀ by at least 1 preferably 1.4 compared to the result from the virulent organism treated with untreated antiserum. The absolute values obtained for the antiserum control and suitable vaccine material are, of course, dependent on the virulent organism and antiserum standards selected.

The following method may be also used to achieve the ideal vaccine formulation: starting from a defined antigen, which is intended to provoke the desired immune response, in a first step an adjuvant matched to the antigen is found, as described in the specialist literature, particularly in WO 97/30721. In a next step the vaccine is optimized by adding various isotonic-making substances as defined in the present inventions, preferably sugars and/or sugar alcohols, in an isotonic or slightly hypotonic concentration, to the mixture of antigen and adjuvant, with the composition otherwise being identical, and adjusting the solution to a physiological pH in the range from pH 4.0 to 10.0, particularly 7.0-7.5. Then, the substances or the concentration thereof which will improve the solubility of the antigen/adjuvant composition compared with a conventional, saline-buffered solution are determined. The improvement in the solubility characteristics by a candidate substance is a first indication that this substance is capable of bringing about an increase in the immunogenic activity of the vaccine.

Since one of the possible prerequisites for an increase in the cellular immune response is increased binding of the antigen to APCs (antigen presenting cells), in a next step an investigation can be made to see whether the substance leads to an increase of binding to APCs. The procedure used may be analogous to that described in the definition of the adjuvant, e.g., incubating APCs with fluorescence-labeled peptide or protein, adjuvant and isotonic-making substance. An increased uptake or binding of the peptide to APCs brought about by the substance can be determined by comparison with cells which have been mixed with peptide and adjuvant alone or with a peptide/adjuvant composition which is present in conventional saline buffer solution, using flow cytometry.

The immunomodulatory activity of the formulation is measured in animal tests.

The following examples are presented in order to more fully illustrate some embodiments of the invention. They should, in no way be construed, however, as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.

EXAMPLES

Example 1. Identification of fragments derived from age-dependent proteins of S. pneumoniae

Analysis of sequence homology was performed using the BLAST software (http://blast.ncbi.nlm.nih.gov/Blast.cgi^'). Each of the proteins of SEQ ID NOS 1-25 was compared to the human genome using the program: "human build protein (previous build 35.1)" database and "BLASTP: compare protein sequence". A region with homology of less than nine amino acids is defined as non-homology sequences. To ensure that the sequences are Streptococcus pneumoniae origin, each non-homology sequence was compared to the protein in all S. pneumoniae strains tested October 2008 - March 2009 (http://www.ncbi.nlm.nih.gov/sutils/genom table.cgi . Non-identical amino acids among the known sequences of Streptococci were removed from the non-human sequence homology peptides dividing those sequences to more peptides.

The identified peptide sequences, SEQ ID NOS 26-75 of Table 1, have no homology to human sequences and retain 100% homology to all S. pneumoniae strains (NCBI, March 2009). Table 1.

Protein Peptide Sequence SEQ ID

NO.

NP 346006 MGKYFGTDGVRGEANLELTPELAFKLGRFGGYV 26 phosphoglucomutase/ LSQHETEAPKVFVGRDTRISGEMLESALVAGLLS phosphomannomutase VGIHVYKLGVLAT

family protein

PSAEGLGILVDYPEGLRKYEGYLVSTGTPLDGMK 27 VALDTANGAASTSARQIFADLGAQLTVIGETPDG LNINLNVGSTHPEALQE

QNTIVTTVMSNLGFHKALNREGINKAVTAVGDR 28 YVVEEMRKSGYNLGGEQSGHVILMDYNTTGDG QLSAVQLTKIMK

NPJ44811 RPASDEEPFAALAFKIMTDPFVGRLTFFRVYSGVL 29 Elongation factor G QSGSYVLNTSKGKRERIGRILQMHANSRQEIDTV

YSGDIAAAVGLKDTTTGDSLTDEK

KIILESINVPEPVIQLMVEPKSKADQDKMGIALQK 30 LAEEDPTFRVETNVETG

LKEAAKSAQPAILEPMMLVTITVPEENLGDVMG 31 HVTARRGRVDGMEAHGNSQIVRAYVPLAEMFG YATVLRSASQGRGTFMMVFDHYEDVPKSVQEEI

TVLDSQSGVEPQTETVWRQATEYGVPRIVFANK 32 MDKIGADFLYSVSTLHDRLQANAHPIQLPIGSED DFRGIIDLIKMKAEIYTNDLGTDILEEDIPAEYLDQ AQEYREKL

DVLVDRMRREFKVEANVGAPQVSYRETFRASTQ 33 ARGFFKRQSGGKGQFGDVWIEFTPNEEGKGFEFE NAIVGGVVPREFIPAVEKGLVES ANGVLAGYP MVDV

QNTIVTTVMSNLGFHKALNREGI KAVTAVGDR 34 YVVEEMRKSGYNLGGEQSGHVILMDYNTTGDG QLSAVQLTKIMK

NP 345686 QALAEKLDVDARSVHAYIMGEHGDSEFAVWSH 35

L-lactate ANIAGVNLEEFLKDTQNVQEAELIELFEGVRDAA dehydrogenase YTIINKKGATYYGIAVALARITKAILDDENAVLPL

SVFQEGQYGVENVFIGQPAVVGAHGIVRPVNIPL

NDAETQKMQASAKELQAIIDEAWKNPEFQEASK

N

NP 346439 AFRRIQNVEGVEVTRINDLTDPVMLAHLLKYDTT 36 glyceraldehy de-3 - QGRFDGTVEVKEGGFEVNGKF

phosphate

dehydrogenase

KVSAERDPEQIDWATDGVEIVLEATGFFAKKEAA 37 EKHLKGGAKKVVITAPGGNDVKTVVFNTNH

DGSAQRVPTPTGSVTELVAVLEKNVTVDEVNAA 38 MKAASNESYGYTEDPIVSSDIVGMSYGSLFDATQ TKVLDVDGKQLVKWSWYDNEMSYTAQLVRTL EY

NP 346261 VDLAIGHIKALEKVSEKTDVYIYNLGSGEGTSVL 39 UDP-glucose 4- QLVNTFESVNKIPIPYKIVPRRSGDVATCYANAD

epimerase KAYKELNWRTTKSIEDMCRDTWNWQSK

NP 358192 KNTAVAYNGTRINIMDTPGFLADFGGEVERIMKM 40 elongation factor Tu VDGVVLVVDAYEGTMPQTRFVLKKALEQDLVPI family protein VVVNKIDKPSARP

MAPIFDTIIDHIPAPVDNSDEPLQFQVSLLDYNDF 41

VGRIGIGRVFRGTVKVGDQVTLSKLDGTTKNFRV

TKLFGFFGLERREIQEAKAGDLIAVSGMEDIFVGE

TITPTDAVEALPILHIDEPTLQMTFLVNNSPFAGK

EGKWVTSRKVEERLQAELQTDVSLRVDPTDSPD

KWTVSGRGELHLSILIETMRREGYELQVSRPEVIV

KEIDG

KCEPFERVQIDTPEEYQGSVIQSLSERKGEMLDMI 42

STGNGQTRLVFLVPARGLIGYSTEFLSMTRGYGI

MNHTFDQYLPLIPGEIGGRHRGALVSIDAGKATT

YSIMSIEERGTIFVNPGTEVYEGMI1GENSRENDLT

VNITKAKQMTNVRSATKDQTAVIKTPRILTLEESL

EFLNDDEYMEVTPESIRLRKQILNKAEREKANKK

KKSAE

NP 345899 MSNI STDLQD VEKII VLDYG SQ YNQLI SRRIREIG V 43 Bifunctional GMP F SELKSHKI S A AE VREVNP VG

synthase/ glutamine

amidotransferase

protein

LLTHKLGGKWPAGDAGNREYGQSTLTHTPSAL 44 FESTPDEQTVLMSHGDAVTEIPADFVRTGTSADC

YAAIENPDKfflYGIQFHPEVRHSVYGNDILRNFAL 45 NICKAKGDWSMDNFIDMQIKKI

TVGDKRVLLGLSGGVDSSVVGVLLQKAIGDQLIC 46

IFVDHGLLRKGEADQVMDMLGGKFGLNIVKADA

AKRFLDKLAGVSDPEQKRKIIGNEFVYVFDDEAS

KLKDVKFLAQGTLYTDVIESGTDTAQTIKSHHNV

GGLPE

MGEITEEKLETVRESDAILREEIAKAGLDRDIWQY 47 FTVNTGVRSVGVMGDGRTYDYTIAIRAITSIDGM TADFAKIPWEVLQKISVRIVNEVDHVNRIVYDITS KPPATVEWE

NP 345769 QVNRGYRVQFNSAVGPYKGGLRFHPTVNQGILK 48 glutamate FLGFEQIFKNVLTGLPIGGGKGGSDF

dehydrogenase

NP 346622 LALIMPSGETLEAAYVSATATIGEKISFRRFALIEK 49 Elongation factor Ts TDAQHFGAYQHNGGRIGVISVVE

MAEITAKLVKELREKSGAGVMDAKKALVETDG 50 DIEKAIELLREKGMAKAAKKADRVAAEGLTGVY VNGNVAAVIEVNAETDFVAKNAQFVELVNTTAK VIAEGKPANNEE

DEALAKQLSMfflAAMKPTVLSYKELDEQFVKDE 51

LAQLNHVIDQDNESRAMVNKPALPHLKYGSKAQ

LTDDVIAQAEADIKAELAAEGKPEKIWDKIIPGK

MDRFMLDNTKVDQAYTLLAQVYIMDDSKTVEA

YLESVNASVVEFARFEVGEGIEKA

NP 358035 KLTVKDVDLKGKKVLVRVDFNVPLKDGVITNDN 52

Phosphoglycerate RITAALPTIKYIIEQGGRAILF SULGRVKEE

kinase

ASNVGI S ANVEKA VAGFLLENEI A YIQEA VETPER 53 PFVAILGGSKVSDKIGVIENLLEKAD VLIGGGMTYTFYKAQGIEIGNSLVEEDKLDVAKA 54

LLEKANGKLILPVDSKEANAFAGYTEVRDTEGEA

VSEGFLGLDIGPKSIAKFDEALTGAKTVVWNGPM

GVFENPDFQAGTIGVMDAIVKQPGVKSIIGGGDS

AAAAINLGRADKFSWI

NP 346623 MA VI SMKQLLEAGVHFGHQTRRWNPKMAKYIFT 55

30S ribosomal protein ERNGIHVIDLQQTVKYADQAYDFMRDAAANDA

S2 WLFVGTKKQAADAVAEEAVRSGQYFINHRWL

GGTLTNWGTIQKRIARLKEIKRMEEDGTFEVLPK

KEVALLNKQR

NP 344819 VDNTPVFSLDLTKDKVTNQKASGRCWMFAALN 56 Aminopeptidase C TFRHKLISQYKLENFELSQAHTFFWDKYEKSNWF

LEQVI

TSDQELTSRKVSFLLQTPQQDGGQWDMVVSLFE 57 KYGVVPKS VYPES VS S S S SRELNAILNKLLRQDA QILRDLLVSGADQ

MTVQAKKEDLLQEIFNFLAMSLGLPPRKFDFAYR 58

DKDNNYKSEKGITPQEF YKK YVNLPLED YV S VIN

APTADKPYGKSYTVEMLGNVVGSRAVRYINVPM

ERLKELAIAQMQAGETVWFGSDVGQLSNRKAGI

LATDVYDFESSMDIKLTQDKAGRLDYSESLMTH

AMVLTGVDLDENGKS

NP 345739 AFAATIGYPVIVRPAFTLGGTGGGMCANEKELRE 59 Carbamoyl phosphate ITENGLKLSPVTQCLIERSIAGFKEIEYEVMRDSA synthase large subunit DNALWCNMENFDPVGIHTGDSIVFAP

KLAAKIAVGLTLDEVINPVTGSTYAMFEPALDYV 60 VAKIPRFPFDKFEKGERRLGTQMKATGEVMAIGR NIEESLLKACRSLE

LIEKWKAQDDRLFYVSEAIRRGYTPEEIAELTKI 61 DIFYLDKLLHIFEIEQELGAHPQDLEVL

MAQVATKLILGQSLSELGYQNGLYPESTRVHIKA 62

PVFSFTKLAKVDSLLGPEMKSTGEVMGSDATLEK

ALYKAFEASYLHLPTFGNVVFTIADDAKEEALNL

ARRFQNIGYGILATEGTAAFFASHGLQAQPVGKI

GDDDKDIPSFVRKGRIQAIINTVGTKRTADEDGE

QIRRSAIEHGVPLFTALDTANAMLKVLESRSFVTE

AI

SVSDKLYFEPLTFEDVMNVIDLEQPKGVIVQFGG 63

QTAINLAEPLAKAGVTILGTQVADLDRAEDRDLF

EQALKELDIPQPPGQTATNEEEAALAARKIGFPVL

VRPSYVLGGRAMEIVENEEDLRSYMRTAVKASP

DHPVLVDSYIVGQECEVDAISDGK

NP 358460 LLGSIMIKATTLEPRFGNPTPRVAETPAGMLNAIG 64 dihydroorotate LQNPGLEWLAEKLPWLEREYPNLPIIANVAGFS dehydrogenase IB KQEYAAVSHGISK

KAIELNISCPNVDHCNHGLLIGQDPDLAYDVVKA 65 AVEASEVPVYVKLTPSVTDIVTVAKAAE

AGASGLTMINTLVGMRFDLKTRKPILANGTGGM 66 SGPAVFPVALKLIRQVAQT TDLPIIGMGGVDS

NP 345941 TVLARRLPSSVNQPKDYASIDAAPEERERGITINT 67 Elongation factor Tu AHVEYETEKRHYAHIDA

MILLSRQVGVKHLIVFMNKVDLVDDEELLELVE 68 MEIRDLLSEYDFPGDDLPVIQGSALKALEGDSKY EDIVMELMNTVDEYIPEPERDTDKPLLLPVEDVFS

ITGRGTVASGRIDRGIVKVNDEIEIVGIKEETQKA

VVTGVEMFRKQLDEGLAGDNVGVLLRGVQRDEI

ERGQVIAKPGSINPHTKFKGEVYILTKEEGGRHTP

FFNN YRPQF YFRTTDVTG SIELPAGTEM VMPGDN

VTIDVELIHPIAVEQGTTFSIREGGRTVGSGMVTEI

EA

NP-346607 FNLIAGILEVQSGRIVLDGEENPKGRVSYMLQKD 69 ABC transporter, LLLEHKTVLGNIILPLLIQKVDKAEAISRADKILAT

ATP-binding protein FQLTAVRDKYPHELSGGMRQRVALLRTYL

LLDEAFSALDEMTKMELHAWYLEIHKQLQLTTLI 70 ITHSIEEALNLSDRIYILKNRPG

NP 346087 MTRYQDDFYDAINGEWQQTAEIPADKSQTGGFV 71 endopeptidase 0 DLDQEIEDLMLATTDKWLAGEEVPEDAILENFVK

YHRLVRDFDKREADGITP

SEYAKLYHPYSYEDF FAPALPLDDFFKAVIGQ 72 LPDKVIVDEERFWQAAEQFYSEE

KAAYHLAQEPFKQALGLWYAREKFSPEAKADVE 73 KKVATMIDVYKERLLKNDWLTPETCKQAIVKLN VIKPYIGYPEELPARYKDKVVNETASLFENALAF ARVEIKHSWSKWNQPV

DLHQSSSANYGGIGAVIAHEISHAFDTNGASFDE 74

NGSLKDWWTESDYAAFKEKTQKVIDQFDGQDS

YGATINGKLTVSENVADLGGIAAALEAAKREAD

FSAEEFFYNFGRIWRMKGRPEFMKLLASVDVHA

PAKLRVNVQVPNFDDFFTTYDVKEGDGMWRSPE

ERVIIW

NP 345081 MIG VVAREN AAEQIKQ YQKFTVNI SDETSMLAM 75 Hypothetical protein EQAGFI SHQEKLERLGVHYEI SERTQ

SP 0565

Example 2. Screening the polypeptide fragments and multimers

Polypeptide arrays and polypeptide libraries are used to synthesize the peptides of table 1 and derivatives and analogs of these peptides. The peptides are synthesized using different linkers, matrixes and absorption methods, using methods known in the art (for example US 2002/0006672; Gaseitsiwe et al., Plos One 3, e3840, 1- 8, 2008; Biissow et al., Am J Pharmacogenomics 2001 ; 1, 1-7; Andresen et al., Proteomics 6, 1376-1384, 2006, Jan Marik and Kit S. Lam, Methods in Molecular Biology, vol. 310: Chemical Genomics: Reviews and Protocols, Ed. E. D. Zanders, Humana Press Inc., Totowa, NJ). Polypeptides are obtained for screening either in a solution or absorbed or linked to a matrix. The peptide arrays are screened using sera obtained from infants at various ages as described for example in Ling et al., Clin Exp Immunol 2004, 138, 290-8. According to one specific method, sera are collected longitudinally from healthy children attending day-care centers at different ages (for example 18, 30 and 42 months). Starting at 12 months of age, nasopharyngeal swabs are taken from the children on a bimonthly schedule over the 2.5 years of the study. Pneumococcal isolates are characterized by inhibition with optochin and a positive slide agglutination test (Phadebact, Pharmacia Diagnostics). In addition, sera are collected from healthy adults.

An increase in the antibody response to a polypeptide derived from a bacterial protein which coincides with the diminution in morbidity described in children indicates that the peptide retained the "age-dependent" characteristic of the bacterial protein and it is further synthesized and tested for its ability to elicit protection against S. pneumoniae.

Example 3. Construction of fusion polypeptides and multimers

Artificial genes encoding polypeptides comprising sequences selected to be immunogenic and age dependent with or without carrier polypeptides, are constructed to encode chimeric proteins of up to 1000 amino acids. The structure of the chimeric proteins is constructed to minimize homology to human sequences based on potential neoantigens at the fusion junction of peptides in the construct.

One set of constructs comprises 2-5 different polypeptides, each in 1-5 repeats, with a spacer of 0-20 Glycine and/or Alanine residues between each peptide, and an optional a detoxified pneumolysin as a carrier protein.

The following exemplary recombinant multimeric polypeptides were produced by synthesizing and cloning the optimized DNA sequence into pET30-a+ and subcloning to pET 30a+ Vector to Nde I (ATG) without any tags. DNA sequences include (in italics) the restriction sites 5' Nde I (CAT ) and Bpul 102 I 3' ( TAAGC TTGCT GAGC):

PS19 comprising SEQ ID NOs: 41-42 derived from elongation factor Tu family protein, linked by an Ala- Ala-Ala (aaa) spacer:

508 amino acids sequence (SEQ ID NO:76):

MAPIFDTIIDHIPAPVDNSDEPLQFQVSLLDYNDFVGRIGIGRVFRGTVKVGDQVT LSKLDGTTKNFRVTKLFGFFGLERREIQEAKAGDLIAVSGMEDIFVGETITPTDAV EALPILHIDEPTLQMTFLVNNSPFAGKEGKWVTSRKVEERLQAELQTDVSLRVDP TDSPDKWTVSGRGELHLSILIETMRREGYELQVSRPEVIVKEIDGaaaKCEPFERVQ IDTPEEYQGSVIQSLSERKGEMLDMISTGNGQTRLVFLVPARGLIGYSTEFLSMTR GYGIMNHTFDQYLPLIPGEIGGRHRGALVSIDAGKATTYSIMSIEERGTIFVNPGTE VYEGMIIGENSRENDLTVNITKAKQMTNVRSATKDQTAVIKTPRILTLEESLEFLN DDEYMEVTPESIRLRKQILNKAEREKANKKKKSAECKNTAVAYNGTRINIMDTP GHADFGGEVERIMKMVDGVVLVVDAYEGTMPQTRFVLKKALEQDLVPIVVVN KIDKPSARP

Optimized DNA sequence (SEQ ID NO:77):

CAT AT GGCTC CAATT TTTGA TACTA TTATT GATCA TATTC CAGCT CCAGT

TGATA ACTCT GATGA ACCAC TGCAG TTCCA GGTTT CTCTG CTGGA CTACA

ACGAC TTCGT GGGTC GTATT GGTAT CGGTC GTGTA TTCCG TGGTA CTGTG

AAAGT GGGTG ATCAG GTGAC TCTGA GCAAA CTGGA CGGCA CCACG AAAAA

CTTTC GCGTG ACCAA ACTGT TTGGC TTCTT TGGCC TGGAA CGCCG TGAGA

TTCAG GAAGC TAAAG CAGGT GACCT GATCG CTGTT TCTGG CATGG AAGAC

ATCTT CGTTG GTGAA ACCAT CACCC CAACT GACGC TGTTG AAGCG CTGCC

GATTC TGCAC ATCGA TGAAC CGACC CTGCA GATGA CCTTC CTGGT TAACA

ACTCT CCATT CGCAG GCAAA GAAGG CAAAT GGGTT ACGTC TCGCA AAGTA

GAGGA ACGCC TGCAG GCTGA ACTGC AGACT GATGT AAGCC TGCGT GTAGA

TCCGA CTGAC TCCCC AGACA AATGG ACTGT TTCTG GTCGT GGTGA ACTGC

ACCTG TCTAT CCTGA TCGAA ACGAT GCGTC GCGAA GGTTA CGAAC TGCAG

GTATC CCGTC CGGAA GTGAT CGTCA AAGAA ATCGA CGGTG CTGCA GCGAA

ATGTG AACCG TTCGA ACGCG TGCAG ATTGA TACTC CGGAA GAGTA TCAGG

GCAGC GTTAT CCAGT CTCTG AGCGA ACGTA AAGGC GAAAT GCTGG ACATG

ATCTC CACGG GTAAC GGTCA GACTC GTCTG GTCTT CCTGG TACCA GCTCG

TGGTC TGATC GGCTA CAGCA CTGAA TTCCT GTCCA TGACC CGTGG CTATG

GCATC ATGAA CCACA CCTTC GATCA GTACC TGCCA CTGAT TCCAG GTGAG

ATTGG TGGTC GTCAC CGCGG TGCAC TGGTT TCTAT TGACG CAGGC AAAGC

GACGA CCTAC AGCAT CATGT CCATC GAGGA ACGTG GTACC ATCTT TGTTA

ACCCA GGCAC CGAAG TCTAC GAAGG CATGA TCATT GGTGA GAACT CCCGT

GAAAA CGACC TGACC GTGAA CATTA CCAAA GCGAA ACAGA TGACC AACGT

CCGCT CTGCC ACGAA AGATC AGACT GCGGT GATCA AAACC CCACG TATCC

TGACC CTGGA AGAGT CTCTG GAATT CCTGA ACGAC GATGA GTATA TGGAA

GTCAC CCCAG AATCT ATCCG TCTGC GCAAA CAGAT CCTGA ACAAA GCGGA

GCGTG AGAAA GCGAA CAAAA AAAAA AAATC TGCCG AGTGC AAAAA CACTG CAGTG GCCTA CAACG GTACT CGCAT CAACA TCATG GATAC CCCAG GTCAC GCAGA CTTTG GCGGT GAAGT AGAGC GTATC ATGAA AATGG TTGAT GGCGT TGTAC TGGTA GTTGA CGCGT ATGAA GGCAC CATGC CACAG ACCCG TTTCG TTCTG AAAAA AGCGC TGGAA CAGGA CCTGG TTCCG ATCGT TGTGG TCAAC AAAAT CGACA AACCG TCCGC ACGTC CGTAA TAAGC TTGCT GAGC

PS20 comprising SEQ ID Nos. 58 and 56 derived from the protein Aminopeptidase C, linked by an Ala- Ala-Ala (aaa) spacer:

256 amino acids sequence (SEQ ID NO: 78)

MTVQAKKEDLLQEIFNFLAMSLGLPPRKFDFAYRDKDNNYKSEKGITPQEF YKK YVNLPLEDYVSVINAPTADKPYGKSYTVEMLGNVVGSRAVRYINVPMERLKEL AIAQMQAGETVWFGSDVGQLSNRKAGILATDVYDFESSMDIKLTQDKAGRLDY SESLMTHAMVLTGVDLDENGKSaaaVDNTPVFSLDLTKDKVTNQKASGRCWMF AALNTFRHKLISQYKLENFELSQAHTFFWDKYEKSNWFLEQVI.

Optimized DNA sequence (SEQ ID NO:79)

CATAT GACTG TTCAA GCTAA AAAAG AAGAT CTGCT GCAAG AAATT TTTAA

TTTTC TGGCT ATGTC TCTGG GTCTG CCGCC ACGTA AATTC GACTT CGCAT

ATCGC GATAA AGATA ACAAC TATAA ATCCG AGAAA GGCAT CACGC CGCAG

GAATT CTACA AAAAA TACGT CAACC TGCCA CTGGA GGACT ACGTA TCCGT

GATTA ACGCC CCGAC CGCTG ATAAA CCGTA TGGCA AAAGC TACAC TGTTG

AAATG CTGGG TAACG TCGTT GGTTC TCGTG CTGTG CGCTA CATC A ACGTG

CCGAT GGAAC GTCTG AAAGA ACTGG CTATC GCGCA GATGC AGGCC GGCGA

GACTG TTTGG TTTGG TTCTG ATGTC GGTCA GCTGT CCAAC CGCAA AGCGG

GCATC CTGGC GACCG ATGTC TATGA CTTCG AGTCT AGCAT GGATA TCAAA

CTGAC GCAGG ACAAA GCGGG TCGTC TGGAT TACAG CGAAA GCCTG ATGAC

TCATG CAATG GTCCT GACGG GCGTG GATCT GGACG AAAAC GGCAA ATCTG

CGGCG GCAGT TGACA ACACC CCGGT ATTCT CCCTG GACCT GACCA AAGAT

AAAGT CACGA ACCAG AAAGC CTCTG GCCGT TGCTG GATGT TCGCC GCTCT

GAACA CCTTC CGTCA CAAAC TGATT TCCCA GTATA AACTG GAAAA CTTCG

AGCTG TCTCA GGCCC ACACC TTCTT TTGGG ACAAA TACGA AAAAT CCAAC

TGGTT CCTGG AACAG GTGAT CTAAT AAGCT TGCTG AGC.

PS25 comprising SEQ ID NOs. 32, 33, 34, 29, 30 and 31 derived from Elongation factor G, linked by an Ala- Ala- Ala (aaa) spacer: 551 amino acids sequence (SEQ ID NO:80):

MTVLDSQSGVEPQTETVWRQATEYGVPRIVFANKMDKIGADFLYSVSTLHDRLQ ANAHPIQLPIGSEDDFRGIIDLIKMKAEIYTNDLGTDILEEDIPAEYLDQAQEYREK

LaaaDVLVDRMRREFKVEANVGAPQVSYRETFRASTQARGFFKRQSGGKGQFGD VWIEFTPNEEGKGFEFENAIVGGVVPREFIPAVEKGLVESMANGVLAGYPMVDV

aaaQNTIVTTVMSNLGFHKALNREGINKAVTAVGDRYVVEEMRKSGYNLGGEQS GHVILMDYNTTGDGQLSAVQLTKIMKaaaRPASDEEPFAALAFKIMTDPFVGRLTF FRVYSGVLQSGSYVLNTSKGKRERIGRILQMHANSRQEIDTVYSGDIAAAVGLK DTTTGDSLTDEKaaaKIILESINVPEPVIQLMVEPKSKADQDKMGIALQKLAEEDPT FRVETNVETGaaaLKE AAKS AQP AILEPMMLVTITVPEENLGD VMGHVTARRGRV DGMEAHGNSQIVRAYVPLAEMFGYATVLRSASQGRGTFMMVFDHYEDVPKSV QEEI

Optimized DNA sequence (SEQ ID NO:81):

CAT AT GACTG TTCTG GATTC TCAAT CTGGT GTTGA ACCAC AAACT GAAAC

CGTAT GGCGT CAAGC AACCG AATAC GGTGT TCCGC GTATC GTCTT CGCGA

ACAAA ATGGA CAAAA TCGGT GCGGA TTTCC TGTAC AGCGT TTCTA CCCTG

CATGA TCGCC TGCAG GCAAA CGCAC ACCCG ATTCA GCTGC CAATC GGTAG

CGAGG ATGAC TTCCG TGGCA TCATC GATCT GATCA AAATG AAAGC GGAGA

TCTAC ACCAA CGACC TGGGT ACGGA TATCC TGGAG GAAGA TATCC CGGCT

GAGTA CCTGG ACCAG GCACA GGAAT ATCGC GAAAA ACTGG CTGCA GCCGA

CGTTC TGGTT GACCG TATGC GTCGT GAATT CAAAG TCGAA GCGAA CGTGG

GTGCT CCACA GGTCT CTTAT CGCGA AACTT TTCGC GCAAG CACCC AGGCC

CGTGG CTTCT TTAAA CGCCA GTCTG GCGGT AAAGG CCAGT TTGGC GACGT

TTGGA TCGAA TTCAC CCCAA ACGAG GAAGG CAAAG GCTTC GAGTT CGAAA

ACGCG ATTGT TGGTG GCGTT GTGCC GCGTG AGTTT ATTCC GGCAG TTGAG

AAAGG CCTGG TCGAA TCTAT GGCCA ACGGT GTACT GGCTG GTTAC CCGAT

GGTCG ATGTA GCTGC AGCGC AGAAC ACGAT CGTTA CCACT GTGAT GTCCA

ACCTG GGCTT CCACA AAGCG CTGAA CCGCG AAGGT ATTAA CAAAG CGGTG

ACCGC TGTTG GTGAT CGCTA CGTAG TGGAA GAGAT GCGCA AATCT GGCTA

TAACC TGGGT GGCGA ACAGT CTGGT CACGT GATCC TGATG GACTA TAACA

CTACC GGCGA CGGTC AGCTG TCTGC TGTGC AGCTG ACCAA AATTA TGAAA

GCCGC AGCGC GTCCA GCGTC TGACG AAGAA CCGTT TGCAG CCCTG GCCTT

CAAAA TTATG ACCGA TCCGT TCGTA GGTCG TCTGA CCTTC TTTCG CGTGT ATAGC GGTGT TCTGC AGTCC GGCTC TTACG TGCTG AACAC TTCCA AAGGC

AAACG TGAAC GTATC GGCCG TATTC TGCAG ATGCA CGCGA ACTCT CGCCA

GGAAA TCGAC ACTGT TTACA GCGGT GATAT TGCTG CGGCA GTAGG CCTGA

AAGAC ACGAC CACTG GTGAC TCTCT GACGG ATGAG AAAGC GGCTG CGAAA

ATTAT CCTGG AATCC ATCAA CGTTC CGGAA CCAGT GATCC AGCTG ATGGT

GGAAC CGAAA TCCAA AGCCG ATCAG GACAA AATGG GTATC GCTCT GCAGA

AACTG GCTGA AGAGG ACCCG ACCTT CCGTG TCGAA ACTAA CGTTG AAACC

GGTGC TGCAG CCCTG AAAGA AGCTG CAAAA TCTGC GCAGC CGGCC ATTCT

GGAAC CGATG ATGCT GGTGA CGATT ACCGT TCCGG AAGAG AACCT GGGTG

ACGTT ATGGG TCATG TAACC GCGCG TCGTG GTCGT GTAGA CGGCA TGGAA

GCACA CGGTA ACAGC CAGAT CGTTC GTGCG TATGT TCCGC TGGCT GAAAT

GTTCG GTTAC GCGAC CGTAC TGCGT TCCGC TTCTC AGGGT CGTGG CACCT

TCATG ATGGT GTTCG ATCAC TACGA GGACG TCCCG AAATC CGTAC AGGAA

GAGAT CTAAT AAAAG CTTGC TGAGC

PS26 comprising SEQ ID NOs. 62, 63 and 61 derived from Carbamoyl phosphate synthase large subunit, linked by an Ala-Ala-Ala (aaa) spacer:

495 amino acids sequence (SEQ ID NO:82):

MAQVATKLILGQSLSELGYQNGLYPESTRVHIKAPVFSFTKLAKVDSLLGPEMKS TGEVMGSDATLEKALYKAFEASYLHLPTFGNVVFTIADDAKEEALNLARRFQNI GYGILATEGTAAFFASHGLQAQPVGKIGDDDKDIPSFVRKGRIQAIINTVGTKRTA DEDGEQIRRSAIEHGVPLFTALDTANAMLKVLESRSFVTEAIaaaSVSDKLYFEPLT FEDVMNVIDLEQPKGVIVQFGGQTAINLAEPLAKAGVTILGTQVADLDRAEDRD LFEQALKELDIPQPPGQTATNEEEAALAARKIGFPVLVRPSYVLGGRAMEIVENE EDLRSYMRTAVKASPDHPVLVDSYIVGQECEVDAISDGKAAAFAATIGYPVIVRP AFTLGGTGGGMCANEKELREITENGLKLSPVTQCLIERSIAGFaaaLIEKVVKAQD DRLFYVSEAIRRGYTPEEIAELTKIDIFYLDKLLHIFEIEQELGAHPQDLEVL

Optimized DNA sequence (SEQ ID NO:83):

CATAT GGCTC AAGTT GCTAC TAAAC TGATT CTGGG TCAAT CTCTG TCTGA ACTGG GTTAT CAAAA CGGTC TGTAT CCGGA ATCTA CTCGC GTGCA CATCA AAGCT CCGGT CTTCA GCTTC ACCAA ACTGG CTAAA GTAGA CTCCC TGCTG GGTCC GGAAA TGAAA TCTAC CGGTG AGGTG ATGGG TTCTG ACGCA ACTCT GGAGA AAGCG CTGTA CAAAG CGTTC GAAGC ATCTT ACCTG CACCT GCCGA

CCTTC GGCAA CGTAG TTTTC ACCAT CGCCG ACGAT GCGAA AGAAG AAGCG

CTGAA CCTGG CTCGT CGTTT CCAGA ACATC GGCTA CGGTA TCCTG GCTAC

TGAAG GCACC GCTGC ATTCT TCGCA TCTCA CGGTC TGCAG GCACA GCCGG

TAGGT AAAAT CGGCG ATGAC GATAA AGACA TCCCG AGCTT CGTTC GCAAA

GGTCG TATCC AGGCC AT CAT CAACA CCGTT GGCAC CAAAC GTACC GCCGA

TGAAG ATGGT GAGCA GATTC GTCGT TCCGC TATCG AACAT GGCGT TCCGC

TGTTT ACTGC GCTGG ATACC GCGAA CGCGA TGCTG AAAGT GCTGG AATCC

CGTTC CTTCG TCACC GAAGC TATTG CGGCT GCGTC TGTTA GCGAC AAACT

GTACT TTGAG CCGCT GACGT TTGAG GACGT GATGA ACGTT ATCGA TCTGG

AACAG CCGAA AGGCG TGATC GTACA GTTTG GCGGC CAGAC TGCAA TTAAC

CTGGC TGAAC CACTG GCAAA AGCCG GTGTG ACGAT TCTGG GTACT CAGGT

TGCAG ACCTG GACCG TGCTG AAGAC CGCGA TCTGT TCGAA CAGGC GCTGA

AAGAA CTGGA CATTC CACAG CCGCC AGGTC AGACC GCAAC TAACG AAGAA

GAGGC AGCGC TGGCT GCACG TAAAA TCGGT TTCCC AGTGC TGGTT CGTCC

GAGCT ATGTA CTGGG CGGTC GTGCG ATGGA AATCG TTGAG AACGA AGAAG

ATCTG CGTAG CTATA TGCGC ACGGC AGTTA AAGCG TCCCC AGACC ACCCA

GTCCT GGTAG ATTCC TACAT TGTGG GTCAG GAATG TGAGG TGGAT GCGAT

TTCTG ACGGT AAAGC TGCCG CGTTC GCAGC TACCA TCGGT TATCC AGTGA

TTGTA CGCCC GGCCT TTACG CTGGG CGGTA CCGGC GGTGG CATGT GCGCG

AACGA AAAAG AACTG CGCGA AATCA CCGAG AACGG TCTGA AACTG TCTCC

GGTCA CTCAG TGCCT GATCG AACGT TCTAT TGCGG GTTTC GCGGC TGCCC

TGATT GAAAA AGTCG TGAAA GCTCA GGATG ACCGC CTGTT TTACG TTAGC

GAAGC CATCC GTCGT GGCTA TACCC CAGAG GAAAT CGCCG AGCTG ACCAA

AATCG ACATC TTCTA CCTGG ACAAA CTGCT GCACA TCTTC GAGAT CGAAC

AGGAA CTGGG CGCAC ATCCG CAGGA TCTGG AAGTG CTGTA ATAAA AGCTT

GCTGA GC

PS30 comprising SEQ ID NOs. 50, 51, 49 derived from Elongation factor Ts, and SEQ ID NO. 54 derived from Phosphoglycerate kinase and 57 derived from Aminopeptidase C, linked by an Ala-Ala-Ala (aaa) spacer:

568 amino acids sequence (SEQ ID NO:84):

MAEITAKLVKELREKSGAGVMDAKKALVETDGDIEKAIELLREKGMAKAAKKA DRVAAEGLTGVYVNGNVAAVIEVNAETDFVAKNAQFVELVNTTAKVIAEGKPA NNEEaaaDEALAKQLSMHIAAMKPTVLSYKELDEQFVKDELAQLNHVIDQDNESR AMV KPALPHLKYGSKAQLTDDVIAQAEADIKAELAAEGKPEKIWDKIIPGKMD RFMLDNTKVDQAYTLLAQVYIMDDSKTVEAYLESVNASVVEFARFEVGEGIEKa aaLALIMPSGETLEAAYVSATATIGEKISFRRFALIEKTDAQHFGAYQHNGGRIGVI SVVEaaaVLIGGGMTYTFYKAQGIEIGNSLVEEDKLDVAKALLEKANGKLILPVDS KEANAFAGYTEVRDTEGEAVSEGFLGLDIGPKSIAKFDEALTGAKTVVWNGPMG VFENPDFQAGTIGVMDAIVKQPGVKSIIGGGDSAAAAINLGRADKFSWIaaaTSDQ ELTSRKVSFLLQTPQQDGGQWDMVVSLFEKYGVVPKSVYPESVSSSSSRELNAIL NKLLRQDAQILRDLLVSGADQ

Optimized DNA sequence (SEQ ID NO:85):

CATKT GGCTG AAATT ACTGC TAAAC TGGTT AAAGA ACTGC GTGAA AAAAG

CGGTG CAGGT GTTAT GGACG CGAAA AAAGC TCTGG TTGAG ACCGA TGGTG

ACATC GAGAA AGCGA TTGAG CTGCT GCGTG AGAAA GGCAT GGCGA AAGCG

GCTAA AAAAG CCGAT CGCGT AGCAG CCGAA GGTCT GACTG GTGTA TACGT

CAACG GCAAC GTTGC TGCAG TTATC GAGGT AAACG CTGAG ACTGA CTTCG

TGGCT AAAAA CGCTC AGTTT GTCGA GCTGG TGAAC ACTAC CGCAA AAGTA

ATCGC AGAGG GTAAA CCAGC CAACA ACGAG GAAGC AGCTG CAGAT GAAGC

ACTGG CGAAA CAGCT GTCTA TGCAC ATCGC AGCGA TGAAA CCAAC CGTCC

TGTCC TACAA AGAGC TGGAT GAGCA GTTCG TCAAA GACGA GCTGG CACAG

CTGAA CCACG TAATC GACCA GGACA ACGAA TCTCG TGCGA TGGTC AACAA

ACCAG CTCTG CCACA CCTGA AATAC GGTAG CAAAG CGCAG CTGAC TGATG

ACGTG ATTGC TCAGG CTGAA GCTGA TATCA AAGCC GAGCT GGCGG CAGAA

GGTAA ACCGG AGAAA ATCTG GGACA AAATT ATCCC AGGCA AAATG GATCG

CTTCA TGCTG GACAA CACGA AAGTT GACCA GGCTT ACACG CTGCT CGCAC

AGGTC TACAT CATGG ACGAT AGCAA AACCG TCGAG GCCTA TCTGG AATCC

GTGAA CGCTA GCGTC GTTGA ATTCG CTCGC TTTGA GGTAG GTGAG GGTAT

CGAGA AAGCT GCAGC TCTGG CACTG ATCAT GCCGT CTGGT GAAAC CCTGG

AGGCT GCATA CGTAT CTGCG ACTGC TACCA TCGGT GAGAA AATCA GCTTC

CGTCG CTTCG CACTG ATTGA GAAAA CTGAC GCACA GCACT TCGGT GCATA

CCAGC ACAAC GGCGG CCGTA TTGGT GTTAT CAGCG TTGTC GAAGC TGCAG

CCGTA CTGAT TGGTG GTGGT ATGAC GTACA CCTTC TACAA AGCAC AGGGC

ATCGA GATCG GTAAC TCCCT GGTTG AGGAA GACAA ACTGG ACGTG GCGAA

AGCTC TGCTG GAGAA AGCGA ACGGC AAACT GATTC TGCCA GTCGA TAGCA

AAGAG GCAAA CGCGT TTGCA GGCTA CACTG AAGTA CGTGA TACCG AAGGT GAAGC GGTTT CTGAA GGCTT TCTGG GTCTG GACAT TGGCC CAAAA AGCAT

CGCCA AATTC GACGA AGCAC TGACT GGTGC CAAAA CCGTA GTTTG GAACG

GCCCG ATGGG TGTGT TCGAA AACCC AGATT TCCAG GCAGG TACGA TTGGT

GTAAT GGACG CAATT GTGAA ACAGC CAGGC GTGAA ATCTA TTATC GGCGG

TGGCG ACTCT GCTGC AGCCG CTATC AACCT GGGTC GTGCG GACAA ATTCT

CTTGG ATCGC GGCTG CGACT AGCGA CCAGG AACTG ACCAG CCGTA AAGTT

AGCTT TCTGC TGCAG ACTCC ACAGC AGGAT GGTGG CCAGT GGGAT ATGGT

TGTCA GCCTG TTTGA GAAAT ACGGT GTCGT GCCGA AAAGC GTCTA TCCGG

AATCT GTCTC CTCTA GCTCC TCTCG TGAAC TGAAC GCCAT TCTGA ACAAA

CTGCT GCGTC AGGAT GCACA GATCC TGCGT GACCT GCTGG TATCT GGTGC

GGATC AGTAA ΓΑΑΑΑ GCTTG CTGAG C .

PS31 comprising SEQ ID Nos. 55 derived from 30S ribosomal protein S2,SEQ ID Nos. 38, 36 and 37 derived from glyceraldehyde-3 -phosphate dehydrogenase, and SEQ ID NO. 35 derived from L-lactate dehydrogenase, linked by an Ala- Ala- Ala (aaa) spacer:

542 amino acids sequence (SEQ ID NO:86):

MAVISMKQLLEAGVHFGHQTRRWNPKMAKYIFTERNGIHVIDLQQTVKYADQA YDFMRDAAANDAVVLFVGTKKQAADAVAEEAVRSGQYFINHRWLGGTLTNW GTIQKRIARLKEIKRMEEDGTFEVLPKKEVALLNKQRaaaDGSAQRVPTPTGSVTE LVAVLEKNVTVDEVNAAMKAASNESYGYTEDPIVSSDIVGMSYGSLFDATQTKV LDVDGKQLVKVVSWYDNEMSYTAQLVRTLEYaaaAFRRIQNVEGVEVTRINDLT DPVMLAHLLKYDTTQGRFDGTVEVKEGGFEVNGKFaaaKVSAERDPEQIDWATD GVEIVLEATGFFAKKEAAEKHLKGGAKKVVITAPGGNDVKTVVFNTNHaaaQAL AEKLDVDARSVHAYIMGEHGDSEFAVWSHANIAGVNLEEFLKDTQNVQEAELIE LFEGVRDAAYTIINKKGATYYGIAVALARITKAILDDENAVLPLSVFQEGQYGVE NVFIGQPAVVGAHGIVRPVNIPLNDAETQKMQASAKELQAIIDEAWKNPEFQEAS KN

Optimized DNA sequence (SEQ ID NO:87):

CATAT GGCTG TTATT TCTAT GAAAC AGCTG CTGGA AGCTG GTGTT CATTT TGGCC ATCAG ACTCG CCGTT GGAAC CCAAA AATGG CTAAA TACAT CTTCA CGGAA CGCAA CGGCA TCCAC GTTAT CGATC TGCAG CAGAC GGTGA AATAC GCTGA CCAGG CTTAC GACTT CATGC GTGAC GCTGC AGCCA ACGAC GCAGT TGTGC TGTTT GTTGG CACCA AAAAA CAGGC AGCGG ACGCT GTAGC AGAAG AGGCA GTTCG CTCCG GTCAG TACTT CATC A ACCAC CGTTG GCTGG GCGGT

ACCCT GACTA ACTGG GGCAC GATCC AGAAA CGTAT TGCAC GTCTG AAAGA

GATCA AACGT ATGGA GGAAG ATGGC ACCTT CGAAG TTCTG CCGAA AAAAG

AGGTA GCTCT GCTGA ACAAA CAGCG TGCGG CTGCA GATGG TTCTG CCCAG

CGTGT TCCAA CTCCA ACGGG TTCTG TAACC GAGCT GGTGG CTGTA CTGGA

GAAAA ACGTT ACCGT GGATG AAGTC AACGC TGCAA TGAAA GCTGC CAGCA

ACGAG TCCTA TGGTT ACACT GAAGA TCCGA TTGTT TCCAG CGACA TTGTG

GGTAT GAGCT ACGGT TCCCT GTTCG ACGCC ACTCA GACTA AAGTA CTGGA

CGTGG ACGGT AAACA GCTGG TCAAA GTAGT TAGCT GGTAC GATAA CGAAA

TGAGC TATAC TGCCC AGCTG GTACG CACGC TGGAA TACGC TGCAG CTGCG

TTTCG CCGTA TCCAG AACGT TGAAG GCGTA GAGGT GACTC GCATC AACGA

TCTGA CCGAC CCAGT AATGC TGGCT CACCT GCTGA AATAC GATAC CACTC

AGGGT CGTTT CGATG GCACG GTAGA AGTGA AAGAA GGTGG CTTCG AAGTC

AACGG CAAAT TCGCT GCAGC TAAAG TGTCC GCTGA ACGTG ACCCA GAGCA

GATCG ATTGG GCTAC TGACG GTGTA GAGAT CGTAC TGGAG GCGAC TGGCT

TCTTT GCGAA AAAAG AGGCA GCGGA GAAAC ACCTG AAAGG TGGCG CGAAA

AAAGT CGTAA TCACG GCTCC AGGTG GCAAC GATGT GAAAA CCGTT GTGTT

CAACA CCAAC CACGC TGCTG CCCAG GCTCT GGCTG AGAAA CTGGA CGTCG

ACGCA CGCAG CGTTC ACGCC TACAT CATGG GTGAG CACGG TGACT CTGAA

TTCGC AGTCT GGTCC CACGC AAACA TCGCA GGCGT TAACC TGGAA GAGTT

TCTGA AAGAT ACTCA GAACG TGCAG GAGGC AGAAC TGATC GAACT GTTTG

AAGGC GTTCG TGACG CTGCG TACAC GATTA TCAAC AAAAA AGGCG CGACG

TACTA TGGCA TCGCT GTCGC ACTGG CACGT ATCAC TAAAG CGATC CTGGA

TGACG AAAAC GCTGT TCTGC CACTG TCCGT CTTTC AGGAA GGCCA GTATG

GTGTA GAGAA CGTCT TCATC GGTCA GCCAG CTGTG GTTGG TGCTC ACGGT

ATCGT TCGTC CAGTC AACAT CCCAC TGAAC GATGC CGAAA CCCAG AAAAT

GCAGG CTTCT GCGAA AGAGC TGCAG GCAAT TATCG ATGAG GCGTG GAAAA

ACCCA GAATT CCAGG AAGCC TCCAA AAACT ΑΑΓΑΑ AAGCT TGCTG AGC

PS32 comprising SEQ ID Nos. 26, 27 and 28 derived from phosphoglucomutase/phosphomannomutase family protein, SEQ ID No. 39 derived from UDP-glucose 4-epimerase, SEQ ID Nos. 69 and 70 derived from ABC transporter, ATP- binding protein, and SEQ ID No. 75 derived from Hypothetical protein SP_0565, linked by an Ala-Ala-Ala (aaa) spacer:

570 amino acids sequence (SEQ ID NO:88):

MGKYFGTDGVRGEANLELTPELAFKLGRFGGYVLSQHETEAPKVFVGRDTRISG EMLESALVAGLLSVGIHVYKLGVLATaaaPSAEGLGILVDYPEGLRKYEGYLVST GTPLDGMKVALDTANGAASTSARQIFADLGAQLTVIGETPDGLNINLNVGSTHPE ALQEaaaQNTI VTTVM SNLGFHKALNREGINKA VT A VGDR YV VEEMRKS G YNLG GEQSGHVILMDYNTTGDGQLSAVQLTKIMKaaaVDLAIGHIKALEKVSEKTDVYI YNLGSGEGTSVLQLVNTFESVNKIPIPYKIVPRRSGDVATCYANADKAYKELNW RTTKSIEDMCRDTWNWQSKaaaFNLIAGILEVQSGRIVLDGEENPKGRVS YMLQK DLLLEHKTVLGNIILPLLIQKVDKAEAISRADKILATFQLTAVRDKYPHELSGGMR QRVALLRTYLaaaLLDEAFSALDEMTKMELHAWYLEIHKQLQLTTLIITHSIEEAL NLSDRIYILKNRPGaaaMIGVVARENAAEQIKQYQKFTVNISDETSMLAMEQAGFI SHQEKLERLGVHYEISERTQ

Optimized DNA sequence (SEQ ID NO:89):

CATAT GGGTA AATAT TTCGG TACTG ATGGT GTTCG TGGTG AAGCT AACCT

GGAAC TGACT CCAGA GCTGG CTTTC AAACT GGGTC GTTTT GGTGG CTACG

TCCTG TCTCA GCACG AGACT GAAGC TCCGA AAGTT TTCGT CGGTC GTGAT

ACTCG CATTT CCGGT GAAAT GCTGG AGTCT GCTCT GGTAG CTGGT CTGCT

GTCCG TTGGT ATCCA CGTCT ACAAA CTGGG TGTTC TGGCA ACGGC AGCTG

CACCA AGCGC TGAAG GTCTG GGTAT CCTGG TGGAC TATCC GGAGG GTCTG

CGCAA ATACG AGGGT TACCT GGTAT CTACT GGCAC TCCGC TGGAT GGCAT

GAAAG TGGCT CTGGA TACCG CTAAC GGTGC AGCTT CTACG TCTGC ACGTC

AGATC TTCGC AGATC TGGGC GCACA GCTGA CTGTG ATTGG TGAAA CGCCA

GACGG TCTGA ACATC AACCT GAACG TAGGT TCTAC TCACC CAGAA GCACT

GCAGG AAGCA GCTGC ACAGA ACACG ATCGT GACCA CTGTT ATGAG CAACC

TGGGC TTCCA CAAAG CGCTG AACCG CGAAG GCATC AACAA AGCGG TTACT

GCTGT CGGTG ACCGC TATGT GGTCG AGGAA ATGCG CAAAT CCGGT TACAA

CCTGG GTGGC GAACA GAGCG GTCAC GTCAT CCTGA TGGAT TACAA CACTA

CGGGC GACGG TCAGC TGTCT GCTGT CCAGC TGACC AAAAT CATGA AAGCA

GCTGC GGTCG ATCTG GCTAT TGGTC ACATC AAAGC CCTGG AGAAA GTATC

CGAGA AAACG GACGT GTACA TCTAC AACCT GGGTT CTGGC GAAGG CACGT

CTGTA CTGCA GCTGG TCAAC ACTTT CGAGA GCGTC AACAA AATCC CGATC CCGTA CAAAA TTGTT CCACG TCGCT CTGGC GATGT TGCTA CGTGT TACGC

TAACG CAGAC AAAGC CTACA AAGAG CTGAA CTGGC GTACT ACCAA ATCCA

TTGAG GACAT GTGTC GCGAT ACGTG GAACT GGCAG TCCAA AGCAG CTGCT

TTCAA CCTGA TTGCA GGTAT CCTGG AGGTC CAGTC TGGTC GTATT GTTCT

GGACG GTGAG GAAAA CCCGA AAGGT CGTGT CTCCT ACATG CTGCA GAAAG

ATCTG CTGCT GGAGC ACAAA ACGGT CCTGG GCAAC ATCAT TCTGC CACTG

CTGAT CCAGA AAGTC GACAA AGCAG AGGCT ATCAG CCGTG CGGAC AAAAT

CCTGG CAACC TTTCA GCTGA CTGCA GTACG CGACA AATAC CCACA CGAAC

TGTCC GGTGG CATGC GTCAG CGCGT TGCTC TGCTG CGTAC GTATC TGGCA

GCTGC ACTGC TGGAC GAAGC ATTCT CCGCT CTGGA CGAGA TGACC AAAAT

GGAAC TGCAC GCTTG GTATC TGGAG ATTCA CAAAC AGCTG CAGCT GACGA

CCCTG ATCAT TACTC ACTCC ATCGA AGAGG CTCTG AACCT GTCCG ACCGC

ATCTA CATTC TGAAA AACCG TCCAG GTGCG GCAGC TATGA TTGGT GTCGT

AGCAC GTGAA AACGC AGCCG AGCAG ATCAA ACAGT ACCAG AAATT CACGG

TCAAC ATCAG CGACG AAACG TCTAT GCTGG CAATG GAACA GGCAG GCTTT

ATTAG CCACC AGGAG AAACT GGAGC GTCTG GGTGT ACACT ACGAG ATTTC

TGAGC GCACC CAGTA A T AAA AGCTT GCTGA GC

PS33 comprising SEQ ID NOs. 43, 44, 46 and 47 derived from Bifiinctional GMP synthase/glutamine amidotransferase protein, and SEQ ID NO: 48 derived from glutamate dehydrogenase, linked by an Ala-Ala-Ala (aaa) spacer:

508 amino acids sequence (SEQ ID NO:90):

MSNISTDLQDVEKIIVLDYGSQYNQLISRRIREIGVFSELKSHKISAAEVREVNPVG

aaaLLTHKLGGKVVPAGDAGNREYGQSTLTHTPSALFESTPDEQTVLMSHGDAVT EIPADFVRTGTSADCaaaYAAIENPDKHIYGIQFHPEVRHSVYGNDILRNFALNICK AKGDWSMDNFIDMQIKKIaaaTVGDKRVLLGLSGGVDSSVVGVLLQKAIGDQLIC IFVDHGLLRKGEADQVMDMLGGKFGLNIVKADAAKRFLDKLAGVSDPEQKRKII GNEFVYVFDDEASKLKDVKFLAQGTLYTDVIESGTDTAQTIKSHHNVGGLPEaaa MGEITEEKLETVRESDAILREEIAKAGLDRDIWQYFTVNTGVRSVGVMGDGRTY D YTIAIRAITSIDGMTADF AKIP WEVLQKIS VRIVNEVDH VNRIV YDITSKPP ATVE WEaaaQVNRGYRVQFNSAVGPYKGGLRFHPTVNQGILKFLGFEQIFKNVLTGLPIG GGKGGSDF Optimized DNA sequence (SEQ ID NO:91):

CAT AT GTCTA ACATT TCTAC TGATC TGCAG GATGT TGAAA AAATT ATCGT

CCTGG ACTAC GGTTC CCAGT ACAAC CAGCT GATTT CTCGC CGTAT TCGCG

AGATT GGTGT CTTCT CTGAG CTGAA AAGCC ACAAA ATCTC CGCTG CAGAA

GTTCG CGAAG TTAAC CCAGT TGGTG CAGCC GCTCT GCTGA CGCAC AAACT

GGGTG GCAAA GTTGT GCCAG CTGGC GATGC TGGCA ACCGC GAATA CGGTC

AGAGC ACCCT GACTC ACACC CCATC CGCAC TGTTT GAAAG CACCC CAGAT

GAGCA GACGG TACTG ATGTC TCATG GCGAC GCTGT AACGG AAATC CCAGC

TGACT TCGTC CGTAC TGGCA CTTCT GCTGA CTGCG CAGCT GCGTA CGCTG

CAATC GAAAA CCCGG ACAAA CACAT CTACG GCATC CAGTT TCACC CAGAA

GTACG CCACT CTGTT TACGG TAACG ACATT CTGCG CAACT TCGCT CTGAA

CATCT GCAAA GCCAA AGGTG ACTGG TCCAT GGACA ACTTC ATCGA CATGC

AGATC AAAAA AATCG CAGCG GCTAC TGTGG GTGAC AAACG CGTAC TGCTG

GGTCT GAGCG GCGGT GTAGA TTCTA GCGTC GTGGG TGTTC TGCTG CAGAA

AGCGA TTGGC GATCA GCTGA TTTGT ATCTT CGTAG ACCAT GGCCT GCTGC

GTAAA GGCGA AGCTG ACCAG GTAAT GGATA TGCTG GGCGG TAAAT TCGGT

CTGAA CATCG TCAAA GCCGA CGCAG CCAAA CGTTT CCTGG ACAAA CTGGC

TGGCG TCAGC GACCC AGAAC AGAAA CGTAA AATTA TCGGC AACGA GTTTG

TCTAC GTCTT CGATG ACGAG GCCTC TAAAC TGAAA GACGT GAAAT TTCTG

GCTCA GGGTA CCCTG TATAC CGACG TGATC GAATC CGGTA CGGAT ACTGC

GCAGA CCATC AAATC CCATC ACAAC GTAGG CGGCC TGCCA GAAGC AGCTG

CCATG GGTGA AATCA CCGAA GAGAA ACTGG AAACG GTACG TGAAT CCGAT

GCTAT CCTGC GCGAA GAGAT CGCTA AAGCA GGCCT GGATC GCGAT ATCTG

GCAGT ACTTC ACTGT TAACA CTGGC GTCCG CTCTG TAGGC GTGAT GGGTG

ACGGT CGTAC CTACG ACTAC ACGAT TGCTA TTCGC GCAAT CACTT CCATC

GACGG CATGA CCGCT GACTT CGCCA AAATC CCATG GGAAG TACTG CAGAA

AATCT CCGTG CGCAT CGTGA ACGAG GTCGA TCACG TGAAC CGCAT TGTCT

ATGAC ATCAC CAGCA AACCA CCGGC AACTG TTGAG TGGGA AGCTG CAGCG

CAGGT AAACC GTGGT TATCG TGTCC AGTTC AACTC TGCTG TAGGC CCATA

CAAAG GTGGC CTGCG TTTTC ACCCA ACCGT TAACC AGGGC ATCCT GAAAT

TCCTG GGTTT CGAGC AGATC TTCAA AAACG TTCTG ACGGG TCTGC CGATC

GGCGG TGGCA AAGGT GGCAG CGATT TCTAA TAAAA GCTTG CTGAG C PS34 comprising SEQ ID No. 68 and 67 derived from Elongation factor Tu, SEQ ID NOs. 52 and 53 derived from Phosphoglycerate kinase, and SEQ ID NO: 65 derived from dihydroorotate dehydrogenase IB, linked by an Ala- Ala- Ala (aaa) spacer:

592 AA amino acids sequence (SEQ ID NO:92): MILLSRQVGVKHLIVFMNKVDLVDDEELLELVEMEIRDLLSEYDFPGDDLPVIQG SALKALEGDSKYEDIVMELMNTVDEYIPEPERDTDKPLLLPVEDVFSITGRGTVA SGRIDRGIVKVNDEIEIVGIKEETQKAVVTGVEMFRKQLDEGLAGDNVGVLLRG VQRDEIERGQVIAKPGSINPHTKFKGEVYILTKEEGGRHTPFFNNYRPQFYFRTTD VTGSIELPAGTEMVMPGDNVTIDVELIHPIAVEQGTTFSIREGGRTVGSGMVTEIEa aaTVLARRLPSSVNQPKDYASIDAAPEERERGITINTAHVEYETEKRHYAHIDaaaK LTVKDVDLKGKKVLVRVDFNVPLKDGVITNDNRITAALPTIKYIIEQGGRAILFSH LGRVKEEaaaSNVGISANVEKAVAGFLLENEIAYIQEAVETPERPFVAILGGSKVSD KIGVIENLLEKADaaaKAIELNISCPNVDHCNHGLLIGQDPDLAYDVVKAAVEASE VPVYVKLTPSVTDIVTVAKAAEaaAGASGLTMINTLVGMRFDLKTRKPILANGTG GMSGPAVFPVALKLIRQVAQTTDLPIIGMGGVDS

Optimized DNA sequence (SEQ ID NO:93):

CAT AT GTCTA ACATT TCTAC TGATC TGCAG GATGT TGAAA AAATT ATCGT

CCTGG ACTAC GGTTC CCAGT ACAAC CAGCT GATTT CTCGC CGTAT TCGCG

AGATT GGTGT CTTCT CTGAG CTGAA AAGCC ACAAA ATCTC CGCTG CAGAA

GTTCG CGAAG TTAAC CCAGT TGGTG CAGCC GCTCT GCTGA CGCAC AAACT

GGGTG GCAAA GTTGT GCCAG CTGGC GATGC TGGCA ACCGC GAATA CGGTC

AGAGC ACCCT GACTC ACACC CCATC CGCAC TGTTT GAAAG CACCC CAGAT

GAGCA GACGG TACTG ATGTC TCATG GCGAC GCTGT AACGG AAATC CCAGC

TGACT TCGTC CGTAC TGGCA CTTCT GCTGA CTGCG CAGCT GCGTA CGCTG

CAATC GAAAA CCCGG ACAAA CACAT CTACG GCATC CAGTT TCACC CAGAA

GTACG CCACT CTGTT TACGG TAACG ACATT CTGCG CAACT TCGCT CTGAA

CATCT GCAAA GCCAA AGGTG ACTGG TCCAT GGACA ACTTC ATCGA CATGC

AGATC AAAAA AATCG CAGCG GCTAC TGTGG GTGAC AAACG CGTAC TGCTG

GGTCT GAGCG GCGGT GTAGA TTCTA GCGTC GTGGG TGTTC TGCTG CAGAA

AGCGA TTGGC GATCA GCTGA TTTGT ATCTT CGTAG ACCAT GGCCT GCTGC

GTAAA GGCGA AGCTG ACCAG GTAAT GGATA TGCTG GGCGG TAAAT TCGGT

CTGAA CATCG TCAAA GCCGA CGCAG CCAAA CGTTT CCTGG ACAAA CTGGC

TGGCG TCAGC GACCC AGAAC AGAAA CGTAA AATTA TCGGC AACGA GTTTG TCTAC GTCTT CGATG ACGAG GCCTC TAAAC TGAAA GACGT GAAAT TTCTG

GCTCA GGGTA CCCTG TATAC CGACG TGATC GAATC CGGTA CGGAT ACTGC

GCAGA CCATC AAATC CCATC ACAAC GTAGG CGGCC TGCCA GAAGC AGCTG

CCATG GGTGA AATCA CCGAA GAGAA ACTGG AAACG GTACG TGAAT CCGAT

GCTAT CCTGC GCGAA GAGAT CGCTA AAGCA GGCCT GGATC GCGAT ATCTG

GCAGT ACTTC ACTGT TAACA CTGGC GTCCG CTCTG TAGGC GTGAT GGGTG

ACGGT CGTAC CTACG ACTAC ACGAT TGCTA TTCGC GCAAT CACTT CCATC

GACGG CATGA CCGCT GACTT CGCCA AAATC CCATG GGAAG TACTG CAGAA

AATCT CCGTG CGCAT CGTGA ACGAG GTCGA TCACG TGAAC CGCAT TGTCT

ATGAC ATCAC CAGCA AACCA CCGGC AACTG TTGAG TGGGA AGCTG CAGCG

CAGGT AAACC GTGGT TATCG TGTCC AGTTC AACTC TGCTG TAGGC CCATA

CAAAG GTGGC CTGCG TTTTC ACCCA ACCGT TAACC AGGGC ATCCT GAAAT

TCCTG GGTTT CGAGC AGATC TTCAA AAACG TTCTG ACGGG TCTGC CGATC

GGCGG TGGCA AAGGT GGCAG CGATT TCTAA TAAAA GCTTG CTGAG C

PS35 comprising SEQ ID NOs. 71, 72 and 74 derived from endopeptidase O, and SEQ ID NO: 64 derived from dihydroorotate dehydrogenase IB, linked by an Ala- Ala- Ala (aaa) spacer:

523 AA amino acids sequence (SEQ ID NO:94): MTRYQDDFYDAINGEWQQTAEIPADKSQTGGFVDLDQEIEDLMLATTDKWLAG EEVPEDAILENFVKYHRLVRDFDKREADGITPAAASEYAKLYHPYSYEDFKKFAP ALPLDDFFKAVIGQLPDKVIVDEERFWQAAEQFYSEEAAAKAAYHLAQEPFKQA LGLWYAREKFSPEAKADVEKKVATMIDVYKERLLKNDWLTPETCKQAIVKLNV IKPYIGYPEELPARYKDKVVNETASLFENALAFARVEIKHSWSKWNQPVAAADL HQSSSANYGGIGAVIAHEISHAFDTNGASFDENGSLKDWWTESDYAAFKEKTQK VIDQFDGQDSYGATINGKLTVSENVADLGGIAAALEAAKREADFSAEEFFYNFGR IWRMKGRPEFMKLLASVDVHAPAKLRVNVQVPNFDDFFTTYDVKEGDGMWRS PEERVIIWAAALLGSIMIKATTLEPRFGNPTPRVAETPAGMLNAIGLQNPGLEVVL AEKLPWLEREYPNLPIIANVAGFSKQEYAAVSHGISK

Optimized DNA sequence (SEQ ID NO:95):

CA T AT GATTC TGCTG TCTCG TCAGG TTGGT GTTAA ACATC TGATT GTTTT CATGA ACAAA GTTGA CCTGG TTGAC GATGA AGAAC TGCTG GAGCT GGTGG AAATG GAAAT CCGTG ACCTG CTGTC TGAAT ATGAT TTCCC GGGCG ACGAT CTGCC AGTTA TTCAG GGCTC TGCGC TGAAA GCTCT GGAAG GCGAC TCTAA ATACG AGGAT ATCGT CATGG AACTG ATGAA CACCG TGGAC GAGTA CATTC CGGAA CCGGA ACGTG ATACC GACAA ACCAC TGCTG CTGCC GGTGG AAGAT GTATT TAGCA TCACC GGTCG TGGTA CCGTG GCTAG CGGTC GTATT GACCG TGGTA TCGTA AAAGT AAACG ACGAA ATCGA GATCG TCGGT ATTAA AGAGG AGACC CAGAA AGCCG TTGTG ACCGG TGTGG AAATG TTCCG CAAAC AGCTG GACGA AGGTC TGGCG GGTGA TAACG TAGGT GTACT GCTGC GTGGC GTTCA GCGTG ATGAA ATTGA ACGTG GCCAG GTCAT CGCAA AACCA GGCTC TATCA ACCCG CACAC GAAAT TCAAA GGCGA AGTGT ACATC CTGAC GAAAG AGGAA GGTGG TCGCC ATACC CCATT CTTCA ACAAC TATCG CCCAC AGTTC TATTT CCGTA CCACG GATGT GACGG GTTCC ATTGA ACTGC CAGCT GGTAC GGAAA TGGTT ATGCC GGGCG ACAAC GTTAC CATTG ACGTA GAGCT GATCC ACCCG ATTGC GGTTG AACAG GGCAC TACCT TTTCC ATCCG CGAGG GTGGC CGTAC TGTAG GTTCT GGTAT GGTTA CCGAG ATTGA AGCTG CGGCG ACTGT ACTGG CCCGT CGCCT GCCGT CTTCC GTTAA CCAGC CGAAA GATTA CGCAT CTATC GACGC TGCGC CGGAA GAGCG TGAAC GTGGC ATCAC TATCA ACACC GCACA CGTTG AATAT GAAAC CGAAA AACGC CACTA CGCAC ACATC GATGC AGCTG CTAAA CTGAC TGTGA AAGAC GTCGA CCTGA AAGGC AAAAA AGTAC TGGTG CGTGT GGACT TCAAC GTGCC GCTGA AAGAT GGCGT CATC A CTAAC GACAA CCGCA TTACT GCTGC GCTGC CGACC ATCAA ATATA TCATC GAACA GGGCG GTCGT GCGAT TCTGT TCTCC CACCT GGGTC GCGTA AAAGA AGAAG CGGCA GCGTC TAACG TTGGT ATCTC TGCCA ACGTG GAAAA AGCGG TTGCA GGCTT CCTGC TGGAG AACGA AATCG CCTAC ATCCA GGAAG CCGTC GAAAC TCCGG AACGT CCGTT TGTGG CGATC CTGGG TGGCT CTAAA GTTTC CGACA AAATC GGTGT GATTG AGAAC CTGCT GGAGA AAGCA GACGC TGCCG CGAAA GCGAT CGAAC TGAAC ATCAG CTGCC CAAAC GTCGA CCATT GTAAC CACGG TCTGC TGATC GGTCA GGATC CGGAC CTGGC TTACG ATGTG GTCAA AGCAG CTGTT GAAGC TAGCG AGGTC CCAGT TTACG TCAAA CTGAC CCCGT CTGTT ACCGA TATTG TTACC GTAGC CAAAG CAGCC GAAGC GGCAG CTGGT GCTTC TGGTC TGACG ATGAT CAACA CCCTG GTGGG CATGC GCTTT GATCT GAAAA CCCGT AAACC GATCC TGGCG AACGG TACTG GCGGC ATGTC CGGTC CAGCA GTTTT TCCGG TAGCG CTGAA ACTGA TTCGC CAGGT TGCAC AGACG ACTGA TCTGC CGATC ATCGG TATGG GCGGC GTTGA TAGCT ΑΑΓΑΑ AAGCT TGCTG AGC Following expression in, for example, E. coli and purification, the resulting polypeptides are tested for their vaccine potential in the intranasal and intraperitoneal mouse challenge models to test their vaccine efficacy.

Example 4. Testing the efficacy of the polypeptides and multimers

Immunogenic polypeptides are produced and used individually, as multimers, or in different combinations as parts of fusion polypeptides with or without a carrier or adjuvant sequence, and are tested with or without an external adjuvant for their vaccine potential in several in-vitro, ex-vivo and in-vivo models. Cross protection against capsularly and genetically unrelated bacterial strains is also tested. In certain cases, antibodies produced against selected peptides and polypeptides are used. The following models are used to test the efficacy:

i. In vitro model in which interference of bacterial adhesion to cultured upper respiratory tract epithelial cells and to endothelial cells is tested by the addition of the polypeptides, chimeric polypeptides and antisera against them to the test system;

ii. To evaluate the stage at which the immune system prevents disease development over time, an in vivo test is used: in vaccinated mice, the extent of nasopharyngeal, lung, blood and spleen colonization of S. pneumoniae tagged with luciferase is monitored using the bioluminescence live-imaging system(IVIS live-imaging system).

iii. Ex-vivo immunization with antiserum against the polypeptides - Several hundreds of CFU of S. pneumoniae serotype 3 strain WU2 are neutralized ex-vivo with rabbit antiserum against the polypeptides for 1 hr and used to challenge 7 week old BALB/c female mice. Negative control mice are challenged with S. pneumoniae strain WU2 after neutralization with pre-immune diluted serum. Positive control mice are challenged with S. pneumoniae serotype 3 strain WU2 after neutralization with rabbit anti-Non-lectin protein serum. Survival is monitored for seven days. iv. Mouse model for systemic infection -For systemic S. pneumoniae lethal challenge, mice immunized with a polypeptide formulated with adjuvant and with adjuvant alone as control are inoculated intraperitoneally (i.p.) or intravenous (i.v.) with a lethal dose of S. pneumoniae serotype 3 strain WU2. The inoculum size is determined to be the lowest that cause 100% mortality in the control mice within 96-120 hours. Survival is monitored daily.

v. Mouse models for upper respiratory infections - For respiratory S. pneumoniae lethal challenge, mice immunized with polypeptide in adjuvant, and with adjuvant alone as control, are anaesthetized with isoflurane and inoculated intranasally (IN) with a lethal dose of S. pneumoniae serotype 3 strain WU2 (in 25 μΐ PBS). The inoculum's size is determined to be the lowest that causes 100% mortality in the control mice within 96-120 hours. Survival is monitored daily.

vi. Mouse models for upper respiratory S. pneumoniae colonization - Mice immunized with polypeptide in adjuvant, and with adjuvant alone as control, are anaesthetized with isoflurane, and inoculated IN with a sublethal dose of S. pneumoniae serotype 3 strain WU2 (in 25 μΐ PBS). The nasopharynx and lungs are excised homogenized and plated onto blood agar plates for bacteria enumeration;

vii. Ex-vivo inhibition of nasopharyngeal and lung colonization - To find whether peptides and polypeptides derived from age-dependent S. pneumoniae proteins are capable of inhibiting colonization, mice are inoculated intranasally with S. pneumoniae serotype 3 prior and after treatment ex vivo with antibodies to the polypeptide. Alternatively, the polypeptide is mixed with S. pneumoniae strain WU2 bacteria, and the mixture is inoculated IN with 5xl0⁵ to 5x10⁷ CFU S. pneumoniae. At 3, 6 24 and 48 hours following inoculation, mice are sacrificed, and the nasopharynx and lungs excised homogenized and plated onto blood agar plates for colony number enumeration.

viii. Otitis media models - Otitis media models in chinchilla and the rat (developed according to Chiavolini et al., 2008, Clinical Microbiology Reviews, 21 :666-685; Giebink, G. S. 1999, Microb. Drug Resist., 5:57-72; Hermansson et al., 1988, Am. J. Otolaryngol. 9:97-101 ; and Ryan et al., 2006, Brain Res. 1091 :3-8) are utilized to test the effectiveness of multimers according to the invention. The ability of multimers to protect these animals from developing otitis media following IN challenge is studied.

Example 5. Immunogenicity of PS20 protein formulated with CCS/C or CFA in CBA/N xid mice In order to determine the immunogenicity of the recombinant multimer denoted PS20, CBA/N xid mice (n=3 per group) were immunized with rPS20 emulsified either with CFA or formulated in CCS/C adjuvant. Control mice were immunized with l(^g non-lectin proteins (NL) of S. pneumonia serotype 3 strain WU2 cell wall fraction as a positive control or adjuvant alone as a negative control, or with rPS20 alone to control the adjuvant effect. The CFA groups were injected with CFA as an adjuvant in the first immunization and IFA in the two following immunizations. Mice were subsequently challenged intranasally (IN) with a sub-lethal dose of S. pneumoniae strain WU2 (7.5* 10⁵ CFU per mouse). Forty eight hours later, mice were sacrificed and nasopharynx and right lobe-lung of each mouse were homogenized and plated onto blood agar for CFU enumeration.

Colonization studies with CFA as adjuvant demonstrate that mice immunized with 2C^g of PS20 showed significant reduced colonization in the nasopharynx (Figure 1A, P value<0.01) and moderate, but not statistically significant, reduction in colonies in the lungs (Figure IB). The results demonstrated protection in the 2(^g antigen group in comparison to negative control mice immunized with the adjuvant CFA/IFA alone.

Colonization studies using CCS/C as adjuvant (at antigen : adjuvant ratio of 1 : 100) demonstrate that immunized mice showed reduced colonization in the nasopharynx using 3μg PS20 (P value <0.05, Figure 2A). In the lungs, reduced colonization was observed using 3μg protein and to a lesser extent using 10μg protein (P value<0.05, Figure 2B). No significant protection was observed when the multimers were administered alone without an adjuvant.

Example 6. Immunogenicity of PS19 protein formulated with CCS/C or CFA in CBA/N xid mice

In order to determine the immunogenicity of PS 19, CBA/N xid mice (n=7) were immunized with rPS19 either emulsified with CFA or formulated in CCS/C. Control mice were immunized with adjuvant alone as a negative control. The CFA groups were injected with CFA as an adjuvant in the first immunization and IFA in the two following immunizations. Fourteen days after the last immunization, mice were inoculated IN (3* 10⁶ CFU, of S. pneumoniae serotype 3 strain WU2) and survival was monitored daily for 7 days. For colonization studies, mice (n=3) were subsequently challenged IN with a sub-lethal dose of S. pneumoniae serotype 3 strain WU2 (2* 10⁶ CFU per mouse). Forty eight hours later, mice were sacrificed and nasopharynx and right lobe-lung of each mouse were homogenized and plated onto blood agar for CFU enumeration.

The result demonstrated protection against lethal IN challenge in both CFA and CCS/C groups (Figure 3 and 4, respectively). Reduced colonization was prominent in the nasopharynx (Figure 5A) and lung (Figure 5B) using CCS/C as an adjuvant (at antigen: adjuvant ratio of 1 : 100) compared to adjuvant-alone negative control groups.

While the present invention has been particularly described, persons skilled in the art will appreciate that many variations and modifications can be made. Therefore, the invention is not to be construed as restricted to the particularly described embodiments, and the scope and concept of the invention will be more readily understood by reference to the claims, which follow.

Claims

A synthetic or recombinant polypeptide of 51-250 amino acids derived from the sequence of a Streptococcus pneumoniae (S. pneumoniae) cell wall or cell membrane protein associated with an age-dependent immune response, wherein the cell wall or cell membrane protein associated with an age-dependent immune response is selected from the group consisting of SEQ ID NO:l to SEQ ID NO:25 and the synthetic or recombinant polypeptide of 51-250 amino acids is selected from SEQ ID NO: 26 to SEQ ID NO:75, and variants and analogs of said polypeptides having at least about 90% sequence identity to said synthetic or recombinant polypeptide.

The polypeptide variant or analog according to claim 1 having at least 97% sequence identity to the synthetic or recombinant polypeptide derived from the sequence of a S. pneumoniae cell wall or cell membrane protein associated with an age-dependent immune response.

The polypeptide according to claim 1 consisting of 51-100 amino acids and selected from SEQ ID NOS: 26, 27, 28, 29, 30, 31, 36, 37, 39, 40, 43, 44, 45, 48, 49, 52, 53, 56, 57, 59, 60, 61, 64, 65, 66, 67, 69, 70, 71, 72, and 75, and variants and analogs thereof.

The polypeptide according to claim 1 consisting of 101-250 amino acids and selected from SEQ ID NOS: 2, 33, 34, 35, 38, 41, 42, 46, 47, 50, 51, 54, 58, 62, 63, 68, 73, and 74, and variants and analogs thereof.

The polypeptide according to claim 1 sharing less than 30% sequence identity with human protein sequences.

The polypeptide according to claim 1 sharing less than 10%» sequence identity with human protein sequences.

The polypeptide according to claim 1 conjugated or recombinantly fused to a carrier protein.

The polypeptide according to claim 7 wherein the carrier protein is selected from detoxified pneumolysin or a fragment thereof, and heat shock protein 60 (hsp60) or a fragment thereof.

An isolated polynucleotide sequence encoding a polypeptide according to claim 1 or encoding a fusion protein according to claim 7.

10. A polypeptide multimer comprising a plurality of S. pneumoniae-de^ed polypeptides, analogs or variants according to claim 1.

11. The multimer according to claim 10 comprising a plurality of repeats of a specific peptide.

12. The multimer according to claim 10 comprising a plurality of repeats of at least two different peptides.

13. The multimer according to claim 10 consisting of a maximum of 1000 amino acid residues.

14. The multimer according to claim 10 comprising a sequence selected from SEQ ID NOs:76, 78, 80, 82, 84, 86, 88, 90, 92, 94

15. The multimer according to claim 10 conjugated or fused to or expressed as part of a carrier protein.

16. An isolated polynucleotide sequence encoding a polypeptide according to any one of claims 10-15.

17. The isolated polynucleotide sequence according to claim 16 comprising a sequence selected from SEQ ID NOs: 77, 79, 81, 83, 85, 87, 89, 91, 93 and 95.

18. A vaccine composition for protecting a subject against S. pneumoniae comprising at least one synthetic or recombinant polypeptide according to claim 1 , or at least one polypeptide multimer according to claim 10.

19. The vaccine composition according to claim 18 further comprising an adjuvant and/or delivery system.

20. A method for inducing an immune response and conferring protection against S. pneumoniae in a subject, wherein the method comprises administering to the subject a vaccine composition according to claim 18.

21. The method according to claim 20 wherein the route of administration of the vaccine is selected from intramuscular, intranasal, oral, intraperitoneal, subcutaneous, topical, intradermal, and transdermal delivery.

22. A polypeptide according to any one of claims 1-8 or a multimer according to any one of claims 10-15 for use in immunizing against S. pneumoniae.

23. Use of a polypeptide according to any one of claims 1-8 or a multimer according to any one of claims 10-15 for preparation of a vaccine composition for immunizing against S. pneumoniae.

24. Use of an isolated polynucleotide according to claim 9 or claim 16 for production of a polypeptide.