Expression of pneumococcal surface protein a (PSPA)
Inventors
Sriraman, Rajan • Matur, Ramesh Venkat • Mantena, Narender Dev • Datla, Mahima • Kamireddy, Swetha
Assignees
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Publication Number
US-11725029-B2
Publication Date
2023-08-15
Expiration Date
Abstract
The present invention relates to expression of Pneumococcal Surface Protein A (PspA). The invention represents an advancement in the field of genetic engineering and vaccine technology. The invention discloses expression vectors and recombinant host cells for expression of truncated PspA peptide. The invention also discloses vaccine compositions comprising the truncated peptides as carrier protein.
Core Innovation
The invention relates to nucleic acids encoding a truncated pneumococcal surface protein A1 (PspA1) and to expression constructs for high level expression of the truncated PspA1. In particular, the truncated PspA1 is defined by the nucleotide sequence of SEQ ID NO:5 and the amino acid sequence as set forth in SEQ ID NO:3. The disclosed constructs include a gene encoding the truncated PspA1, an origin of replication, an antibiotic resistance gene, a promoter, and a ribosomal binding site.
Expression of the truncated PspA1 is disclosed in Corynebacterium glutamicum and Escherichia coli using expression constructs that comprise an oriR/oriR-like replication origin, kanamycin resistance, promoters including P_tac or P_T7, together with ribosomal binding site elements including a triose phosphate isomerase RBS. The truncated PspA1 is produced and then analyzed by SDS-PAGE, Western blot, and intact mass and MALDI MS/MS sequence coverage, and the results include intact mass values reported for each host.
The disclosed truncated PspA1 is also used as a carrier protein for conjugate pneumococcal vaccines, including monovalent and multivalent conjugates. The document discloses vaccine conjugate formulations and includes immunogenicity data using rabbit immunogenicity results that show dose-dependent antibody titers and reports no mutual inhibition between truncated PspA1 conjugates and CRM197-conjugated components.
Claims Coverage
The partial claim set includes two independent claims covering a nucleic acid encoding a truncated PspA1 defined by SEQ ID NO:5 and an expression construct for high level expression of a truncated PspA1 defined by SEQ ID NO:3/SEQ ID NO:5, with additional dependent claims specifying major construct elements, host/production constraints, and optional quantitative yield levels.
Nucleic acid encoding a truncated PspA1 defined by SEQ ID NO:5
A nucleic acid encoding a truncated pneumococcal surface protein A1 (PspA1) consisting of the nucleotide sequence of SEQ ID NO:5.
Expression construct for high level expression of truncated PspA1 with defined SEQ ID elements
An expression construct for high level expression of a truncated pneumococcal surface protein A1 (PspA1) having an amino acid sequence as set forth in SEQ ID NO:3, comprising a gene encoding the truncated PspA1 having a nucleotide sequence as set forth in SEQ ID NO:5, an origin of replication, an antibiotic resistance gene, a promoter, and a ribosomal binding site.
Construct elements including oriR origin, kanamycin resistance, P_tac promoter and triose phosphate isomerase RBS
An expression construct including an oriR origin of replication with SEQ ID NO:12, a kanamycin resistance gene with SEQ ID NO:1, and a P_tac promoter with SEQ ID NO:2, further comprising a triose phosphate isomerase ribosomal binding site.
Recombinant hosts Corynebacterium glutamicum or Escherichia coli carrying the construct
A recombinant host cell where the host organism is Corynebacterium glutamicum or Escherichia coli.
High level expression yielding specified truncated PspA1 production levels
A method in which 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.
Overall, the claim coverage centers on nucleic acids and expression constructs defining a specific truncated PspA1 sequence, including core expression elements and recombinant hosts, with optional quantitative yield levels; the partial content also ties the truncated PspA1 to carrier use in pneumococcal conjugate vaccine contexts.
Stated Advantages
Dose-dependent rabbit antibody titers are reported for truncated PspA1 conjugates.
No mutual inhibition between truncated PspA1 conjugates and CRM197-conjugated components is reported.
Documented Applications
Use of truncated PspA1 as a carrier protein for conjugate pneumococcal vaccines, including monovalent and multivalent conjugates.
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