Expression of pneumococcal surface protein A (PspA)

Abstract

To provide an expression construct capable of stably and constitutively or inducibly expressing a high level of truncated pneumococcal surface protein A1 (PspA1) in bacteria.SOLUTION: The present invention provides an expression construct allowing high-level expression of truncated pneumococcal surface protein A1 (PspA1), as set forth in specific sequences. The present invention provides an expression construct comprising a gene, which encodes truncated PspA1, as set forth in second specific sequences.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to high-level expression of a truncated pneumococcal surface protein A1 (PspA1) in bacteria. [Background technology]

[0002] Streptococcus pneumoniae is an important cause of otitis media, meningitis, bacteremia, and pneumonia, and is a major cause of fatal infections in the elderly and those with underlying medical conditions. An attractive goal of streptococcal vaccination is to reduce carriage in vaccinated populations and subsequently decrease the incidence of pneumococcal disease.

[0003] The pneumococcal polysaccharide vaccine, marketed under the trade name Pneumovax23, is ineffective in children under the age of 2 years. The ineffectiveness of the polysaccharide vaccine in this population is attributed to the immaturity of the pediatric immune system in the expression of B cell receptors. Conjugation of polysaccharides (PS) to carrier proteins changes them from T cell-independent antigens to T cell-dependent antigens. As T cell-dependent antigens, polysaccharides can initiate responses with isotype switching, generation of memory cells and boostable immune responses.

[0004] Membrane proteins are found in and across membranes and play a role in transporting molecules or promoting cell adhesion. Proteins can assist in the movement of substances by facilitating diffusion (i.e., passive transport) or by active transport. Pneumococcal surface protein A (PspA) is a membrane protein and another important virulence factor found associated with the cell wall of all Streptococcus pneumoniae strains.

[0005] Replacement of generic carrier proteins such as tetanus toxoid (TT) or CRM197 by pneumococcal proteins, especially PspA1 or fragments thereof, would also prevent impaired immune responses caused by overusing the same carrier proteins in conjugate vaccines, in addition to expanding the scope of vaccine coverage. Expression plasmids contain regulatory sequences that act as enhancer and promoter regions and are engineered to ensure that genes carried in the expression vector are efficiently transcribed. The goal of a well-designed expression vector is efficient production of proteins, which can be achieved by synthesizing significant amounts of stable messenger RNA. It is possible to design expression vectors that tightly control expression, and proteins can be produced in large quantities only when needed with appropriate expression conditions. If gene expression is not tightly controlled, proteins can also be expressed constitutively.

[0006] Corynebacterium glutamicum is a Gram-positive fermentative bacterium that is widely used in the large-scale production of monosodium glutamate. Corynebacterium glutamicum is classified as a GRAS organism (generally regarded as safe) due to its stable genetic properties and lack of endotoxins. This bacterium is known not to secrete extracellular proteases, making it an attractive platform for the production of heterologous proteins in culture media. This can be achieved using expression plasmid constructs that allow the synthesis of large amounts of recombinant proteins.

[0007] European Patent No. 2310502 describes the use of P in a structure tac The use of a promoter has been disclosed, and an Escherichia Coli strain containing an IPTG-inducible ftsZ and minCDE deletion mutation was grown in LB medium.

[0008] Nokano et al., J Bacteriol. 1984 January;157(1):79-83, disclose the use of a kanamycin resistance gene in a plasmid construct to generate transformed strains that have acquired the trait of resistance to kanamycin.

[0009] Masai et al., Proc Natl Acad Sci USA. 1987 Jul;84(14):4781-5, disclose the RepA protein for initiation of R1 plasmid replication and interaction with the oriR sequence.

[0010] Nayak et al., Infect. Immun. 1998, Nayak, 3744-51, disclose a live oral recombinant Salmonella vaccine strain expressing pneumococcal surface protein A (PspA).

[0011] Nabors et al., Vaccine 18 (2000) 1743-54, disclose the expression of recombinant truncated PspA as a cytoplasmic protein in E. coli. Figueredo et al., Appl Microbiol Biotechnol (2017) 101:2305-2317, disclose the production and purification of untagged recombinant pneumococcal surface protein A (PspA4Pro).

[0012] The documents disclose either genetic elements in expression vectors or expression of pneumococcal surface protein A in E. coli or Salmonella. Summary of the Invention [Problem to be solved by the invention]

[0013] Having identified an immunogenic fragment of the pneumococcal surface protein, the inventors have subsequently made extensive efforts to develop expression constructs that allow stable and constitutive or inducible expression of truncated pneumococcal surface protein A1 (PspA1) at high levels in bacteria.

[0014] Therefore, the present invention aims to overcome the problems of the prior art by preparing expression vectors and recombinant host cells for the expression of truncated pneumococcal surface proteins. Furthermore, the inventors have prepared vaccine compositions comprising the truncated proteins as carrier proteins.

[0015] The primary object of the present invention is to provide expression constructs for high-level expression of truncated pneumococcal surface protein A1 (PspA1) in bacteria. Another object of the present invention is to provide expression constructs that allow stable, constitutive or inducible expression of high levels of truncated pneumococcal surface protein A1 (PspA1) in bacteria. [Means for solving the problem]

[0016] The present invention provides expression constructs that allow for high-level expression of the truncated pneumococcal surface protein A1 (PspA1) shown in SEQ ID NOs:3 and 4. The present invention provides an expression construct comprising a gene encoding the truncated pspA1 shown in SEQ ID NOs:5 and 6.

[0017] The present invention relates to a) a gene encoding a truncated pspA1 as shown in SEQ ID NO: 5 or 6; b) replication origin; c) antibiotic resistance genes, d) a promoter, and e) ribosome binding site The present invention provides an expression construct for high-level expression of a truncated PspA1 (pneumococcal surface protein A1) as set forth in SEQ ID NO: 3 or 4 in bacteria, comprising:

[0018] The present invention is also directed to a method for high-level expression of truncated PspA1 (pneumococcal surface protein A1), which comprises culturing bacteria transformed with an expression construct and purifying the protein expressed thereby. [Brief description of the drawings]

[0019] [Figure 1-1] Panel A is a schematic of pBE31C. Panel B is a domain layout of pspA delineated from the deduced amino acid sequence of Rx1 pspA1. Panel C is a schematic of pBE117. Panel D shows a PCR amplicon containing the RBS, native signal peptide, truncated pspA1, native terminator and trrnB. [Figure 1-2] Same as above. [Diagram 2] FIG. 1 shows protein sequence coverage of truncated PspA1 in peptide fingerprint analysis. [Diagram 3] FIG. 1 shows intact mass analysis of truncated PspA1 expressed in Corynebacterium glutamicum. [Figure 4] FIG. 1 shows truncated PspA1 eluted from a ceramic hydroxyapatite (CHT-II) column. [Diagram 5] FIG. 1 shows truncated PspA1 eluted from an anion exchange column. [Figure 6] FIG. 1 shows truncated PspA1 after diafiltration. [Figure 7] FIG. 2 shows SEC-HPLC chromatograms for the conjugation kinetics of pneumococcal polysaccharide serotypes 3 (A), 6A (B) and 6B (C). [Figure 8] FIG. 1 shows serum antibody titers from rabbits immunized against various conjugates of truncated pspA1 carrier protein and Streptococcus pneumoniae polysaccharides of serotypes 3, 6A and 6B (2.2 mcg). [Figure 9] FIG. 1 shows serum antibody titers from rabbits immunized against various conjugates of truncated pspA1 carrier protein and Streptococcus pneumoniae polysaccharides of serotypes 3, 6A and 6B (4.4 mcg). [Figure 10]Panel A is a schematic of pBE114k, and Panel B shows pBE114k confirmed by restriction digestion. [Figure 11] FIG. 1 shows truncated PspA1 eluted from CHT I chromatography. [Figure 12] FIG. 1 shows truncated PspA1 eluted from Capto Q Impress chromatography. [Figure 13] FIG. 1 shows protein coverage of PspA1 by MALDI. [Figure 14] Intact mass analysis of truncated PspA1 expressed in E. coli. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] The term PspA1 refers to pneumococcal surface protein A1 from Streptococcus pneumoniae. The present invention relates to high-level expression of truncated PspA1 (Pneumococcal surface protein A1) in bacteria. Bacteria suitable for high-level expression of pspA1 are Corynebacterium glutamicum and Escherichia coli.

[0021] In one embodiment, the present invention relates to high-level expression of a truncated PspA1 (pneumococcal surface protein A1) as set forth in SEQ ID NO:3 in Corynebacterium glutamicum.

[0022] In another embodiment, the invention relates to high-level expression of a truncated PspA1 (pneumococcal surface protein A1) as set forth in SEQ ID NO:4 in E. coli. The present invention provides expression constructs that allow for high level expression of the surface protein shown in SEQ ID NO:3.

[0023] The present invention also provides an expression construct that allows for high level expression of the surface protein shown in SEQ ID NO:4. In another embodiment, the present invention relates to an expression construct for high-level expression of a truncated PspA1 (pneumococcal surface protein A1) as set forth in SEQ ID NO:3 in Corynebacterium glutamicum.

[0024] In yet another embodiment, the present invention relates to an expression construct for high-level expression of a truncated PspA1 (pneumococcal surface protein A1) as set forth in SEQ ID NO:4 in E. coli. The present invention relates to i.oriR replication origin, ii. kanamycin resistance gene, iii.P tac promoter, iv. A gene of interest encoding a truncated pspA1 (SEQ ID NO:5) The present invention relates to an expression construct used for high-level expression of a truncated PspA1 in Corynebacterium glutamicum, comprising:

[0025] The present invention also provides i.pUC replication origin, ii. kanamycin resistance gene, iii.P T7 promoter, iv. A gene of interest encoding a truncated pspA1 (SEQ ID NO:6) The present invention relates to an expression construct used for high-level expression of truncated PspA1 in E. coli, comprising:

[0026] In one embodiment, the expression construct for high level expression of truncated pspA1 further comprises a ribosome binding site (RBS). The RBS is included in the forward primer and a short stretch of DNA containing the natural terminator is included in the reverse primer used for further amplification of the truncated pspA1. The RBS (ribosome binding site) is triose phosphate isomerase in the expression construct for expression of truncated PspA1 in Corynebacterium glutamicum.

[0027] In one embodiment of the invention, the expression construct contains a region encoding the PspA1 signal peptide (native), a truncated pspA1, tac It contains the promoter and the ribosome binding site (RBS) of the triosephosphate isomerase gene.

[0028] In a preferred embodiment, the present invention comprises: a. A gene encoding a truncated pspA1 (SEQ ID NO:5); b. oriR origin of replication (SEQ ID NO: 12), c. kanamycin resistance gene (SEQ ID NO:1), dP tac A promoter (SEQ ID NO:2), and e. triosephosphate isomerase ribosome binding site The present invention provides an expression construct for high-level expression of a truncated PspA1 (SEQ ID NO:3) in Corynebacterium glutamicum, comprising:

[0029] In another preferred embodiment, the present invention comprises: a) a gene encoding a truncated pspA1 (SEQ ID NO:6); b) pUC origin of replication, c) kanamycin resistance gene (SEQ ID NO: 1); d) P T7 A promoter (SEQ ID NO: 11), and e) ribosome binding site The present invention provides an expression construct for high-level expression of truncated PspA1 (SEQ ID NO:4) in E. coli, comprising:

[0030] Pneumococcal surface protein A (PspA) is a membrane protein and an important virulence factor found associated with the cell wall of all Streptococcus pneumoniae strains, and is a promising component since it has been shown to be highly immunogenic.

[0031] In a particular embodiment of the present invention, truncated PspA1 is used as a carrier protein. The carrier protein employed in the conjugate vaccine is preferably a protein that is non-toxic, non-reactogenic, and can be obtained in large quantities and with high purity. The carrier protein may also be conjugated to capsular polysaccharides isolated from pathogenic bacteria to enhance the immunogenicity of the polysaccharide. The carrier protein must be amenable to standard chemical conjugation procedures.

[0032] The truncated PspA1 in Corynebacterium glutamicum is secreted into the extracellular medium, which lends itself to efficient purification. The present invention relates to high-level expression of a truncated PspA1 in Corynebacterium glutamicum, where the N-terminal region together with the proline-rich region of the truncated pspA1 gene, together with the upstream region, is amplified from the 23F capsular serotype of Streptococcus pneumoniae.

[0033] In one embodiment of the invention, the expression construct comprises a truncated pspA1, P tac It contains the promoter and the ribosome binding site (RBS) of the triosephosphate isomerase gene. In one embodiment of the invention, the expression construct comprises a truncated pspA1, P T7 It contains a promoter and a ribosome binding site (RBS).

[0034] The expression construct is electroporated into Corynebacterium glutamicum and selected on LB plates containing kanamycin as the selectable marker.

[0035] P tac is a strong hybrid promoter consisting of the −35 region of the trp promoter and the −10 region of the lacUV5 promoter / operator. The pspA sequence was obtained from GenBank and aligned to the protein database. Primers were designed to specifically amplify the coding region of the native signal peptide, the N-terminal region along with the proline-rich region of the pspA genes belonging to families 1 and 2. The required regions of pspA1 and pspA2 were amplified from available clinical isolates of Streptococcus pneumoniae. MHC peptide analysis showed that pspA1 was relatively more immunogenic than pspA2.

[0036] The amino acid sequence encoding the truncated PspA1 expressed and produced in Corynebacterium glutamicum is set forth in SEQ ID NO:3, with an observed intact mass of 35452 daltons.

[0037] The amino acid sequence encoding the truncated PspA1 expressed and produced in E. coli is SEQ ID NO: No. 4 and the intact mass has been observed to be 41,966 daltons. The DNA sequences encoding the truncated pspA1 expressed in Corynebacterium glutamicum and E. coli are set forth in SEQ ID NOs: 5 and 6, respectively.

[0038] Corynebacterium glutamicum (formerly known as Micrococcus glutamicus), used to express truncated pspA1, is a Gram-positive rod-shaped bacterium that is a GRAS organism. Corynebacterium glutamicum is a GRAS organism. The complete genome sequence of Corynebacterium glutamicum ATCC 13032 is available, it can be grown to high cell densities, and it is genetically stable due to the lack of a recombinational repair system. The bacterium has limited restriction-modification systems. It does not exhibit autolysis and can maintain metabolic activity under growth-restrictive conditions. It has low protease activity, which is favorable for recombinant protein production. Its metabolic plasticity and strong secondary metabolic properties, ability to utilize a wide range of carbon sources (pentose, hexose, and other carbon sources), and stress tolerance to carbon sources make it a promising host for heterologous protein production. These physiological properties make Corynebacterium glutamicum easy to manipulate and cultivate in stable industrial conditions, making it a force for successful industrial production. Heterologous expression of proteins such as α-amylase, endoglucanase, endoxylanase, GFP, and xylanase has been reported in Corynebacterium glutamicum.

[0039] In one embodiment of the invention, the yield of truncated PspA1 is about 500 mg / L, about 400 mg / L, about 300 mg / L, about 250 mg / L, about 220 mg / L, about 200 mg / L, about 180 mg / L, about 160 mg / L, about 150 mg / L, about 120 mg / L, about 100 mg / L.

[0040] In yet another embodiment, the present invention provides a pneumococcal conjugate vaccine comprising at least one polysaccharide of a Streptococcus pneumoniae serotype selected from 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19F, 19A, 20A, 20B, 22F, 23A, 23B, 23F, 24B, 24F, 31, 33F, 34, 35B, 35F, 38, 39, and 45 conjugated to a truncated pspA1 of the present invention or a combination of a truncated PspA1 and other carrier proteins such as CRM197, tetanus toxoid, pertussis toxoid, PsaA, etc.

[0041] In yet another embodiment, the present invention provides a Streptococcus bacterium selected from 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19F, 19A, 20A, 20B, 22F, 23A, 23B, 23F, 24B, 24F, 31, 33F, 34, 35B, 35F, 38, 39, and 45 conjugated to a truncated pspA1 of the present invention or a combination of a truncated PspA1 with another carrier protein, such as CRM197, tetanus toxoid, pertussis toxoid, PsaA, etc. The present invention provides a multivalent pneumococcal vaccine composition selected from a 10-valent, 14-valent, 15-valent, 17-valent, 18-valent, 19-valent, 20-valent, 22-valent, 23-valent, 24-valent or 25-valent composition, which comprises a polysaccharide of a S. pneumoniae serotype.

[0042] In a preferred embodiment, the present invention provides a multivalent conjugate vaccine comprising at least three polysaccharides of Streptococcus pneumoniae serotypes 3, 6A and 6B conjugated to a truncated PspA1 of the present invention.

[0043] In one embodiment, the present invention provides Streptococcus pneumoniae serotypes 3, 6A and 6B and C conjugated to a truncated PspA1 of the present invention. RM 197 The present invention provides a multivalent conjugate vaccine comprising polysaccharides of Streptococcus pneumoniae serotypes 1, 4, 5, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F conjugated to

[0044] The present invention provides 2.2 μg or 4.4 μg of each of the pneumococcal polysaccharides of serotypes 3, 6A and 6B, respectively, conjugated to the truncated PspA1 of the invention, and CRM 197 The present invention provides a formulation containing about 2.2 μg of each of pneumococcal polysaccharides of serotypes 1, 4, 5, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F conjugated to, respectively.

[0045] In another embodiment, the invention provides a pneumococcal vaccine composition as a single 0.5 mL dose providing about 2.2 to 4.4 μg of one or more pneumococcal polysaccharides, about 1 μg to about 50 μg of a truncated PspA1 of the invention conjugated to each of the one or more pneumococcal polysaccharides, about 0.2 mg to about 1 mg of an aluminium phosphate adjuvant and an excipient.

[0046] In another embodiment, the invention provides a pneumococcal vaccine composition as a single 0.5 mL dose, the single dose comprising about 2.2 to 4.4 μg of one or more pneumococcal polysaccharides, about 1 μg to about 30 μg of a truncated PspA1 of the invention conjugated to each of the one or more pneumococcal polysaccharides, about 1 μg to about 30 μg of a CRM conjugated to each of the one or more pneumococcal polysaccharides, 197 about 1 μg to about 30 μg, about 0.2 mg to about 1 mg of aluminum phosphate adjuvant, and excipients.

[0047] In another embodiment, the present invention also provides a vaccine for the prevention of invasive disease caused by Streptococcus pneumoniae by administering a conjugate vaccine prepared by conjugating a truncated PspA1 of the present invention with a pneumococcal polysaccharide. EXAMPLES

[0048] The following examples are provided to illustrate the present invention and are for illustrative purposes only and should not be construed as limiting the scope of the present invention. Example 1 Recombinant expression of truncated PspA1 in Corynebacterium glutamicum.

[0049] Construction of pBE31C A synthetic plasmid carrying the 2.692 Kb pNG2 oriR sequence was used to generate a small plasmid, pBE30, which is stably replicating and has a broad host range. Using this plasmid DNA as a template, the 1.8 Kb oriR region was amplified with primer pEP-F1 (5'-GCG CGG ACT AGT AGA TCT ATG GTA AAT CTG CGC AGA CAG-3') and pEP-R1 (5'-GCG CGG ACT AGT GAA TTC GGT GAG GTT ATG GCG-3') was used for amplification.

[0050] At the same time, 1.033 Kb of kanR sequence was amplified using pUC4-KIXX template DNA and Kan-F2 (5'-AAG GTC CCG GGA TGG CGA TAG CTA GAC TGG GCG GT-3') and Kan-R2 (5'-AAG GTC CCG GGG GTT GGG CGT CGC TTG GTC GG-3') primers. Both the kanR gene amplicon and the oriR amplicon were blunt-end ligated to generate the pBE30 vector. In addition, tandem tac A 0.851 Kb expression cassette containing the lac UV5 promoter, multiple cloning sites, the lacZα component and the TrrnB terminator sequence was cloned into P tac -F1(5'-GG AGC ACT AGT CTG AAA TGA GCT GTT GAC AAT TAA TC-3') and P tac -R1(5'-GG AGC ACT AGT TTT AAA CAT GAG CGG ATA CAT ATT TGA A-3') primer was used for amplification. The template DNA used for amplification of the expression cassette was pMMB206 (ATCC37808). The expression cassette was then cloned into the unique SpeI site engineered into the pBE30 plasmid. The resulting plasmid is called pBE31C (Figure 1A; SEQ ID NO: 14).

[0051] Construction of pBE117 The truncated pspA1 gene (N-terminal region together with the proline-rich region Figure 1B) together with the upstream region was amplified from 23F capsular serotype of Streptococcus pneumoniae. It was cloned in a TA vector (pTZ57R / T obtained from Fermentas) using primers PSPAF1_FP (5'ATG AAT AAG AAA AAA ATG ATT TTA ACA AGT CTA GCC 3') and PSPAF1_RP (5'CGA GAG AGA TCT AAA TTA AAA TGT CAA ATG TTC TTA ACA TGC TTT AAT TTT TAT TTT GGT GC 3') and the verified sequence. This was called pTZ-pspA1. The native terminator sequence was included in the reverse primer PSPAF1_RP. The clade confirmation region was mapped to the resulting truncated pspA1 sequence to confirm that it belongs to family 1 of PspA proteins.

[0052] To express truncated pspA1 in Corynebacterium glutamicum, tac The promoter (SEQ ID NO: 2) and the ribosome binding site (RBS) of the triosephosphate isomerase gene belonging to Corynebacterium (SEQ ID NO: 7) were selected. The pspA1 gene together with the whole cassette including the RBS, the native signal peptide (SEQ ID NO: 8), the truncated pspA1 gene, and the native terminator (SEQ ID NO: 10) were amplified using primers SDTICGR0949_FP5 (5'GAG CGA TGG ATC CTA GAA AGG TGT GTT TCA CCC ATG AAT AAG AAA AA 3') and PSPA2_2RP (5'TCA AAT GTT CTT AAC ATG CTT TAA TTT TTA TGG TGC The pspA1 gene was amplified using TER FP2 (5'ATG TTA AGA ACA TTT GAC ATT TTA ATT TCG GCA CTG GCC GTC GTT 3') and pTZ-pspA1 as template. The RBS was included in the forward primer SDTICGR0949_FP5. The rrnB terminator (SEQ ID NO: 13) region was amplified from pBE31C available in-house and linked to the gene encoding truncated pspA1 using splicing by overlap extension (SOE) PCR. The rrnB gene was amplified using TER FP2 (5'ATG TTA AGA ACA TTT GAC ATT TTA ATT TCG GCA CTG GCC GTC GTT 3') and TER_RP3 (5'GCG ATA TGG ATC CCA TGA GCG GAT ACA 3'). PSPA2_2RP and TER FP2 were designed to have a 17-base overlap. Amplicons pspA1 and rrnB were both added at a 1:1 molar ratio and used as templates for SOE PCR with primers SDTICGR0949_FP5 (5'GAG CGA TGG ATC CTA GAA AGG TGT GTT TCA CCC ATG AAT AAG AAA AA 3') and TER_RP3 (5'GCG ATA TGG ATC CCA TGA GCG GAT ACA 3'). The resulting construct, which contains the RBS, the native signal peptide (SEQ ID NO: 8), the truncated pspA1 gene, the native terminator (SEQ ID NO: 10), the rrnB terminator, The entire amplicon was digested with the restriction enzymes added to the forward (SDTICGR0949_FP5) and reverse primers (TER_RP3) and cloned into the expression vector pBE31C designed for the genus Corynebacterium. The resulting clone was designated expression construct pBE117 (Figure 1C, SEQ ID NO: 15). Hereafter, the expression vector together with the truncated pspA1 will refer to the expression construct. The orientation of the insert (Figure 1D) was confirmed by PCR analysis. The sequence of the truncated pspA1 gene together with its expression cassette was confirmed by DNA sequencing.

[0053] Expression of truncated PspA1 The expression construct was electroporated into Corynebacterium glutamicum ATCC13032 and selected on LB plates containing kanamycin as a selectable marker. Twenty recombinant Corynebacterium glutamicum colonies were picked and analyzed by PCR. Five colonies were selected for constitutive expression of truncated pspA1. The recombinant colonies together with Corynebacterium glutamicum ATCC13032 (as a negative control) were inoculated into 10 ml of Terrific broth containing a final kanamycin concentration of 25 μg / ml and incubated at 35 °C with shaking at 200 rpm. After 16 h, a secondary inoculation was performed with 10 ml of the same medium as above to a final OD of 0.1. The cultures were incubated for 18–20 h at 35 °C with shaking at 200 rpm. After 18 h, the culture supernatants were examined for expression of truncated pspA1. 30 μl of the supernatant was subjected to 12% SDS-PAGE and analyzed for truncated pspA1 expression. A prominent band was observed at approximately 45 kDa.

[0054] Western analysis using an N-terminal epitope-specific pspA polyclonal antibody (SantaCruz) confirmed the expression of truncated pspA1. Expression analysis of recombinant clone 5 was scaled up to 500 ml, and expression of truncated pspA1 was confirmed at least three times. Truncated pspA1 was first purified to nearly 99% purity from shake flask experiments using CHT1 and Capto Q impress. Expression was then scaled up to 1.5 L after confirming consistent expression of truncated PspA1 in C. glutamicum.

[0055] Purification and validation of truncated PspA1 800 ml of culture supernatant containing truncated PspA1 (0.66 mg / ml) from 1.6 L obtained from the upstream was dialyzed through 10 kDa and concentrated to 260 ml (1.92 mg / ml). 70 ml of this dialyzed concentrate was purified using CHT1 type and Capto Q impress. The final yield of truncated pspA1 was 162 mg / L.

[0056] MALDI of gel plugs containing purified truncated PspA1 from SDS-PAGE MS / MS analysis yielded 233 distinct hit scores for pneumococcal surface protein A. Sequence coverage of PspA protein was shown to be 31% (Figure 2) with NCBI protein id ABY67187.1. Intact molecular weight analysis showed that the molecular weight of the expressed truncated pspA1 was 35.4 kDa. This mass is consistent with the theoretical molecular weight of truncated pspA1. The peak at 17725.4 is that of a molecule with two charges, so in m / z, the peak appears at half the intact mass of truncated pspA1 (Figure 3).

[0057] Example 2 Production of truncated PspA1 Corynebacterium glutamicum ATCC13032 carrying an expression construct containing a truncated pspA1 (hereafter referred to as pspA1) gene was isolated from a stock. The culture was incubated in LB medium from the fermentation vessel (5 L; CSTR). This was used to inoculate the fermentation vessel (5 L; CSTR). Parameters monitored during the production process were pH, DO, temperature (ΔT), carbon source, and metabolites. A process automation technology (PAT) based feeding strategy was employed to perform short fed-batch truncated PspA1 fermentation. No induction-based control of the product (truncated PspA1) was performed since this is a growth-associated product. The harvest OD during harvest in semi-synthetic medium was approximately 90 (OD 600nm The cells were harvested, washed with PBS, and then disrupted using a French press.

[0058] Purification of truncated PspA1 16.2 L of spent medium with a total protein concentration of 0.8 mg / mL was harvested from a 20 L fermentation batch. The 16.2 L of spent medium was purified using a 10 kDa 0.5 ml 2The mixture was concentrated using a cassette to 2.6 L (3.6 mg / mL) and then diafiltered against 20 mM potassium phosphate pH 6.8, conductivity 3.2 ms / cm. The 2.6 L was split into two lots, Lot 1 of 1.4 L and Lot 2 of 1.2 L, for further purification. 500 ml of CHT I resin was loaded onto a HiScale50 / 40 column. The resin was washed with sterile distilled water and then equilibrated with 8 column volumes (CV) of 20 mM potassium phosphate pH 6.8 (Buffer A). 1400 mL (3.6 mg / mL) of spent media concentrate (Lot 1) was applied to the column and the flow-through was collected. The column was washed with 5 column volumes of Buffer A. PspA1 was eluted with a step gradient using 250 mM potassium phosphate pH 6.8 (Buffer B). The step gradient included 5 CV of step-40% B, 5 CV of step-80% B, and 3 CV of 0.5 M potassium phosphate buffer. The flow rate was maintained at 80 mL / min throughout the run. PspA1 was collected in fraction 1 of step-80% B, with a fraction volume of 1250 mL (Figure 4).

[0059] Capto Q Impress was used as the second chromatographic step for PspA1 purification. 250 ml of Capto Q Impress resin was packed into an XK50 / 20 column. The resin was washed with sterile distilled water and then equilibrated with 5 column volumes (CV) of 20 mM potassium phosphate and 100 mM NaCl, pH 6.8 (buffer A). 1250 mL of the CHT I fraction was diluted to 2300 mL with 20 mM potassium phosphate, pH 6.8, applied to the column and the flow-through fraction was collected. The column was washed with 5 column volumes of buffer A. PspA1 was eluted using 12 CV of buffer B (20 mM potassium phosphate, pH 6.2 with 1 M NaCl) with a linear gradient from 0 to 40% B and a final step of 3 CV of 100% B. Fractions of 250 mL were collected. The flow rate was maintained at 40 mL / min. PspA1 was collected in a linear gradient of 40% B, and the pooled fraction volume was 1250 mL (FIG. 5).

[0060] Capto Q fractions 4, 5, 6, 7 and 8 were pooled and concentrated / diafiltered with 20 mM potassium phosphate pH 6.8. The final yield from lot 1 was 100 mL of pspA1 at a protein concentration of 15.5 mg / mL (1550 mg total PspA1). A similar process was performed for lot 2, and the final yield from lot 2 was 70 mL of pspA1 at a concentration of 16 mg / mL (1120 mg total PspA1). The purified PspA1 from both lot 1 and lot 2 is 2670 mg for a 16.2 L batch (yield of purified PspA1 is 164 mg / L) (Figure 6).

[0061] Example 3 Construction of pBE114K The expression vector pRSET A (a commercially available vector from Invitrogen) was modified by removing the ampicillin resistance gene using DraI restriction digestion and ligating to a SmaI digested kanamycin coding gene (obtained from pUC4 KIXX). This modified vector was designated pRSET-km. The truncated PspA1 was expressed as pRSET -km's P T7 The pspA1 gene was expressed in E. coli under the promoter (SEQ ID NO: 11). A single-stranded DNA containing the natural terminator was included in the reverse primer used for further amplification of pspA1. The entire truncated pspA1 gene was then amplified with its natural terminator, digested with the restriction enzymes added to the forward and reverse primers, and cloned into the pRSET-km vector. The resulting clone was designated pBE114k (Figure 10A). Hereafter, together with truncated PspA1, expression vector refers to the expression construct. The expression construct pBE114k was confirmed by restriction digestion (Figure 10B). The sequence of the truncated PspA1 gene, together with its expression cassette, was confirmed by DNA sequencing.

[0062] Expression of truncated PspA1 pBE114k was transformed into E. coli DH5α-T1R chemically competent cells (obtained from Invitrogen) and selected on LB plates containing kanamycin as a selectable marker. 40 recombinant E. coli colonies were picked and analyzed by PCR. All 40 colonies were selected for inducible expression of truncated PspA1. The recombinant colonies along with E. coli (as a negative control) were inoculated into 10 ml of Terrific Broth containing a final kanamycin concentration of 25 μg / ml and incubated at 37°C with shaking at 200 rpm. At mid-logarithmic growth phase, IPTG 1 mM was added to induce expression of PspA1 in E. coli (pBE114k). After induction, the culture was incubated at 30°C for 16 h with shaking at 200 rpm. After 16 h, the culture supernatant was examined for expression of truncated PspA1. Cells were harvested by centrifugation, lysed, subjected to 12% SDS-PAGE, and analyzed for truncated PspA1 expression. A prominent band was observed at approximately 65 kDa. Expression of truncated PspA1 was confirmed by Western analysis using an N-terminal epitope-specific PspA polyclonal antibody (SantaCruz). Expression analysis of 29 recombinant clones confirmed expression of truncated PspA1 at least three times. Truncated PspA1 was initially purified from shake flask experiments using CHT1 and Capto Q impress.

[0063] Purification and validation of truncated PspA1 40 grams (wet weight) of cell pellet of E. coli (pBE114k) was harvested and resuspended in 400 ml of 20 mM potassium phosphate buffer pH 6.8 containing 1 mg / ml lysozyme and 1 mM PMSF. The cell suspension was lysed three times at 1000 psi using a high pressure homogenizer. 400 ml of cell lysate with a total protein concentration of 4 mg / ml was diluted to 750 ml with 20 mM potassium phosphate buffer pH 6.8 and purified using CHT I resin followed by Capto Q Impress as a second chromatography run.

[0064] 250 ml of CHT I resin was loaded into a HiScale50 / 20 column. The resin was washed with sterile distilled water and then equilibrated with 5 column volumes (CV) of buffer A. 750 mL of diluted cell lysate was applied to the column and the flow-through was collected. The column was washed with 5 column volumes of buffer A. PspA1 was eluted with a step gradient using 250 mM potassium phosphate pH 6.8, conductivity 29.5 ms / cm (buffer B). The step gradient included 5 CV of step-50% buffer B, 5 CV of step-80% buffer B, and the column was stripped with 3 CV of 0.5 M potassium phosphate buffer pH 6.8, conductivity 48 ms / cm (buffer C). The flow rate was maintained at 40 mL / min throughout the run. PspA1 protein peak fractions were collected manually and the final volume of the elution fraction pool of step-80%B 5, 6 and 7 was 350 mL (Figure 11).

[0065] Capto Q Impress was used as the second chromatographic step for PspA1 purification. 60 ml of Capto Q Impress resin was packed into an XK26 / 20 column. The resin was washed with sterile distilled water and then loaded with 5 column volumes (CV) of 20 mM phosphorus. The column was equilibrated with potassium phosphate and 100 mM NaCl, pH 6.8, conductivity 13.6 ms / cm (buffer A). 350 mL of the CHT I fraction was diluted to 700 mL with 1 mM potassium phosphate, pH 6.8, applied to the column and the flow-through was collected. The column was washed with 5 column volumes of buffer A. PspA1 was eluted using a linear gradient of 10 CV of buffer B (20 mM potassium phosphate, pH 6.2, conductivity 89 ms / cm, containing 1 M NaCl), and fractions in the linear gradient from 0 to 40% B were collected manually, with 4 to 8, 10, 11 being 60 mL fractions and 9, 12 being 100 mL fractions (Figure 12). A final step of 3 CV of 100% B, 180 mL, was collected as fraction 13. The flow rate was maintained at 10 mL / min. The PspA1 protein was collected in fraction 9 of a 40% B linear gradient, 100 mL.

[0066] Nine Capto Q fractions were collected and concentrated / diafiltered with 20 mM potassium phosphate pH 6.8. The final recovery yield was 100 mL of PspA1 at 5 mg / mL protein concentration (500 mg total PspA1 protein) from 400 mL of cell lysate at 4 mg / mL concentration. The % recovery is 31.25.

[0067] MALDI of gel plugs containing purified truncated PspA1 from SDS-PAGE MS / MS analysis yielded 223 distinct hit scores for pneumococcal surface protein A. Sequence coverage of the PspA protein was shown to be 39% (Figure 13) for NCBI protein id WP_050210652.1.

[0068] Intact molecular weight analysis showed that the molecular weight of the expressed truncated PspA1 was 41.96 kDa. Intact mass analysis of truncated PspA1 expressed in E. coli showed 41966 Da (41.9 kDa), which was consistent with the theoretical molecular weight of truncated PspA1. The peak at 20982.7 Da is that of a molecule with two charges, so in m / z, the peak appears at half the intact mass of truncated pspA1 (Figure 14).

[0069] Example 4 Conjugation of individual pneumococcal polysaccharides to carrier proteins to form polysaccharide-truncated PspA1 conjugates Activation of serotype 3 and conjugation with truncated pspA1 Approximately 1:1 ratio of sized serotype 3 (PS 6.0 mL, concentration 10 mg / mL) and CDAP (100 mg / mL (w / v) solution in acetonitrile) were mixed in a glass vial and stirred for 1 min. The pH of the pneumococcal polysaccharide serotype 3 was adjusted to 9.25 with 0.2 M triethylamine and stirred for 3 min at room temperature (RT). Truncated PspA1 (4.0 mL at a concentration of 15.0 mg / mL) was added to the activated serotype 3 in a 1:1 ratio (truncated PspA1:serotype 3).

[0070] The pH of the reaction was adjusted to about 9.05 with 0.2 M triethylamine, and the reaction was continued for 5 hours at room temperature with stirring, and finally the reaction was stopped by adding an excess concentration of glycine. The reaction mixture was diafiltered using a 100 kDa MWCO membrane and purified by size exclusion chromatography; in chromatogram A, the solid line is polysaccharide, the dashed line is truncated pspA1, and the 5 hour reaction is represented by the dotted line (Figure 7). Fractions were analyzed by the SEC-MALLS anthrone method, and fractions containing the conjugate were pooled and filter-sterilized through a 0.2μ filter. Hereafter, this material is referred to as monovalent conjugate bulk (serotype 3-truncated PspA1 conjugate).

[0071] Activation of serotype 6A and conjugation with truncated PspA1 Approximately 1:1 ratio of sized serotype 6A (PS 6.0 mL, concentration 10 mg / mL) and CDAP (100 mg / mL (w / v) solution in acetonitrile) were mixed in a glass vial and stirred for 1 min. The pH of pneumococcal polysaccharide serotype 6A was adjusted to 0.2 M triethylamine. The pH was adjusted to 9.25 with 5% CO and stirred at room temperature (RT) for 3 min. Truncated PspA1 (4.0 mL at a concentration of 15.0 mg / mL) was added to the activated serotype 6A in a 1:1 ratio (truncated PspA1:serotype 6A).

[0072] The pH of the reaction was adjusted to about 9.05 with 0.2 M triethylamine, and the reaction was continued for 5 hours at room temperature with stirring, and finally the reaction was stopped by adding an excess concentration of glycine. The reaction mixture was diafiltered using a 100 kDa MWCO membrane and purified by size exclusion chromatography; in chromatogram B, the solid line is polysaccharide, the dashed line is truncated PspA1, and the 5 hour reaction is represented by a dotted line (Figure 7). Fractions were analyzed by SEC-MALLS, anthrone method, and fractions containing the conjugate were pooled and filter-sterilized through a 0.2μ filter. Hereafter, this material is referred to as monovalent conjugate bulk (serotype 6A-truncated PspA1 conjugate).

[0073] Activation of serotype 6B and conjugation with truncated PspA1 Approximately 1:1 ratio of sized serotype 6B (PS 6.0 mL, concentration 10 mg / mL) and CDAP (100 mg / mL (w / v) solution in acetonitrile) were mixed in a glass vial and stirred for 1 min. The pH of pneumococcal polysaccharide serotype 6B was adjusted to 9.25 with 0.2 M triethylamine and stirred for 3 min at room temperature (RT). Truncated PspA1 (4.0 mL at a concentration of 15.0 mg / mL) was added to the activated serotype 6B in a 1:1 ratio (truncated PspA1:serotype 6B).

[0074] The pH of the reaction was adjusted to about 9.05 with 0.2 M triethylamine, and the reaction was continued for 5 hours at room temperature with stirring, and finally the reaction was stopped by adding an excess concentration of glycine. The reaction mixture was diafiltered using a 100 kDa MWCO membrane and purified by size exclusion chromatography; in chromatogram C, the solid line is polysaccharide, the dashed line is truncated PspA1, and the 5 hour reaction is represented by a dotted line (Figure 7). Fractions were analyzed by SEC-MALLS, anthrone method, and fractions containing the conjugate were pooled and filter-sterilized through a 0.2μ filter. Hereafter, this material is referred to as monovalent conjugate bulk (serotype 6B-truncated PspA1 conjugate).

[0075] Example 5 Immunogenicity testing of conjugate vaccines Two formulations containing 2.2 μg or 4.4 μg of serotypes 3, 6A, and 6B conjugated to truncated PspA1, respectively, were 197 The conjugates were absorbed onto Al-hydrogels.

[0076] Groups of seven rabbits weighing 1.5 to 2 kg were immunized with the above conjugate preparations. Serum samples were analyzed before and after immunization. Serum obtained from immunized rabbits was analyzed for the presence of polysaccharide-specific antibodies by indirect ELISA.

[0077] The serum antibody titers of immunized rabbits were evaluated by indirect ELISA. Specific polysaccharide-coated microtiter plates were reacted with serum antibodies. Rabbit sera from pre-immunization, first and second doses were used for analysis, and the Y-axis indicates the antibody titer, which is calculated by the ELISA OD. 450 is obtained using the reciprocal of the highest dilution where exceeds the cutoff. Serum antibody titers of rabbits before immunization were below the detection limit. Open bars indicate titers after the first vaccination and closed bars indicate antibody titers after the second vaccination (Figures 8 and 9). There was a dose-dependent increase in both truncated pspA1-conjugated and CRM197-conjugated serotype titers. The titers of CRM197-conjugated polysaccharide were not inhibited by the presence of truncated PspA1 conjugates and vice versa. This indicates that truncated PspA1 could be used as an alternative carrier protein for polysaccharide-protein conjugate vaccines. In one aspect, the present invention may be as follows. [Embodiment 1] A nucleic acid encoding a truncated pneumococcal surface protein A1 (PspA1), comprising the nucleotide sequence of SEQ ID NO:5 or SEQ ID NO:6. [Aspect 2] An expression construct comprising the nucleic acid described in aspect 1. [Embodiment 3] An expression construct that enables high-level expression of the truncated pneumococcal surface protein A1 (PspA1) shown in SEQ ID NO:3 or SEQ ID NO:4. [Embodiment 4] a. A gene encoding a truncated pspA1 as shown in SEQ ID NO: 5 or SEQ ID NO: 6; b. Replication starting point, C antibiotic resistance genes, d. a promoter, and e ribosome binding site 3. An expression construct for high-level expression of a truncated PspA1 (pneumococcal surface protein A1) as shown in SEQ ID NO:3 or SEQ ID NO:4, comprising: [Aspect 5] The expression construct according to aspect 4, wherein the antibiotic resistance gene is a kanamycin resistance gene. [Aspect 6] a.oriR origin of replication, B. kanamycin resistance gene, cP tac Promoters, and d. A gene of interest encoding a truncated pspA1 as set forth in SEQ ID NO:5. 5. The expression construct of embodiment 4, comprising: [Aspect 7] a. pUC origin of replication, B. kanamycin resistance gene, cP T7 Promoters, and d. A gene of interest encoding a truncated pspA1 as set forth in SEQ ID NO:6 5. The expression construct of embodiment 4, comprising: [Aspect 8] The expression construct according to aspect 7, further comprising a triosephosphate isomerase ribosome binding site as shown in SEQ ID NO:7. [Aspect 9] a. A gene encoding a truncated pspA1 (SEQ ID NO: 5), b. oriR origin of replication (SEQ ID NO: 12), c. kanamycin resistance gene (SEQ ID NO:1), dP tac A promoter (SEQ ID NO:2), and e. triosephosphate isomerase ribosome binding site An expression construct for high-level expression of a truncated PspA1 (SEQ ID NO:3) in Corynebacterium glutamicum, comprising: [Aspect 10] a. A gene encoding a truncated pspA1 (SEQ ID NO: 6), b.pUC replication origin, c. kanamycin resistance gene (SEQ ID NO:1), dP T7 A promoter (SEQ ID NO: 11), and e ribosome binding site An expression construct for high-level expression of truncated PspA1 (SEQ ID NO: 4) in E. coli, comprising: [Aspect 11] A recombinant host cell comprising the expression vector according to any one of aspects 2 to 10. [Aspect 12] A recombinant host cell according to aspect 11, wherein the host is Corynebacterium glutamicum or Escherichia coli. [Aspect 13] A method for high-level expression of Streptococcus pneumoniae truncated PspA1 (pneumococcal surface protein A1), comprising the steps of culturing a bacterium transformed with an expression construct according to any one of aspects 2 to 10, and purifying the protein expressed thereby. [Aspect 14] A method for high-level expression and purification of truncated PspA1 described in aspect 13, wherein the yield of truncated pspA1 is about 500 mg / L, about 400 mg / L, about 300 mg / L, about 250 mg / L, about 220 mg / L, about 200 mg / L, about 180 mg / L, about 160 mg / L, about 150 mg / L, about 120 mg / L, or about 100 mg / L. [Embodiment 15] A pneumococcal conjugate vaccine comprising at least one polysaccharide of a Streptococcus pneumoniae serotype selected from 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19F, 19A, 20A, 20B, 22F, 23A, 23B, 23F, 24B, 24F, 31, 33F, 34, 35B, 35F, 38, 39, and 45, conjugated to a truncated PspA1 as set forth in SEQ ID NO:3 or SEQ ID NO:4, or a combination of a truncated PspA1 and another carrier protein selected from the group including CRM197, tetanus toxoid, pertussis toxoid, and PsaA. [Aspect 16] The vaccine composition according to Aspect 15, wherein the vaccine is a multivalent vaccine selected from a 10-valent, 14-valent, 15-valent, 17-valent, 18-valent, 19-valent, 20-valent, 22-valent, 23-valent, 24-valent, and 25-valent vaccine. [Embodiment 17] Streptococcus pneumoniae serotypes 3, 6A and 6B conjugated to the truncated PspA1 shown in SEQ ID NO: 3 or SEQ ID NO: 4, and CRM 197 16. The multivalent conjugate vaccine according to embodiment 15, comprising at least three polysaccharides from Streptococcus pneumoniae serotypes 1, 4, 5, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F conjugated to [Aspect 18] Use of a truncated pneumococcal surface protein A1 (PspA1) comprising the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4 as a carrier protein in a conjugate vaccine.

Claims

1. 1. A multivalent pneumococcal conjugate vaccine composition comprising capsular polysaccharides derived from two or more Streptococcus pneumoniae serotypes selected from the group consisting of: 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19F, 19A, 20A, 20B, 22F, 23A, 23B, 23F, 24B, 24F, 31, 33F, 34, 35B, 35F, 38, 39 and 45, a capsular polysaccharide from at least one pneumococcal serotype conjugated to a truncated pspA1 having the sequence shown in SEQ ID NO:3 or SEQ ID NO:4; a capsular polysaccharide from at least one pneumococcal serotype is conjugated to CRM197; the pneumococcal capsular polysaccharide serotype conjugated to the truncated pspA1 having the sequence shown in SEQ ID NO:3 or SEQ ID NO:4 is different from the pneumococcal capsular polysaccharide serotype conjugated to CRM197; The composition above.

2. 2. The multivalent pneumococcal conjugate vaccine composition of claim 1, wherein the at least one capsular polysaccharide from a Streptococcus pneumoniae serotype conjugated to a truncated pspA1 having the sequence of SEQ ID NO:3 or SEQ ID NO:4 is selected from the group consisting of 3, 6A and 6B.

3. 2. The multivalent pneumococcal conjugate vaccine composition of claim 1, wherein the vaccine is a multivalent vaccine selected from 10-valent, 14-valent, 15-valent, 17-valent, 18-valent, 19-valent, 20-valent, 22-valent, 23-valent, 24-valent and 25-valent.

4. 2. The multivalent pneumococcal conjugate vaccine composition of claim 1, wherein the multivalent vaccine is a 15-valent composition comprising pneumococcal capsular polysaccharides selected from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F.

5. Capsular polysaccharides from at least three Streptococcus pneumoniae serotypes 3, 6A and 6B conjugated to a truncated PspA1 as shown in SEQ ID NO:3 or SEQ ID NO:4, and CRM １９７ 2. The multivalent pneumococcal conjugate vaccine composition of claim 1, comprising capsular polysaccharides from Streptococcus pneumoniae serotypes 1, 4, 5, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F conjugated to

6. The composition comprises: 2.2 to 4.4 μg each of capsular polysaccharides from Streptococcus pneumoniae serotypes 3, 6A, and 6B conjugated to truncated PspA1; and CRM １９７ 2.2 μg each of capsular polysaccharides from Streptococcus pneumoniae serotypes 1, 4, 5, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F conjugated to; The multivalent pneumococcal conjugate vaccine composition of claim 5, comprising

7. The pneumococcal vaccine composition comprises: about 2.2 to 4.4 μg of one or more pneumococcal capsular polysaccharides; from about 1 μg to about 30 μg of a truncated PspA1 having the sequence of SEQ ID NO:3 or SEQ ID NO:4 conjugated to each of one or more pneumococcal capsular polysaccharides; about 0.2 mg to about 1 mg of aluminum phosphate adjuvant; and Excipients; 2. The multivalent pneumococcal conjugate vaccine composition of claim 1, which is a 0.5 mL single dose comprising:

8. The composition comprises a CRM conjugated to each of one or more pneumococcal capsular polysaccharides. １９７ 8. The multivalent pneumococcal conjugate vaccine composition of claim 7, further comprising about 1 μg to about 30 μg of

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