Plasmodium falciparum circumsporozoite vaccine gene optimization for soluble protein expression
Inventors
Assignees
United States Department of the Army
Publication Number
US-9115205-B2
Publication Date
2015-08-25
Expiration Date
2031-10-18
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Abstract
The present invention provides novel nucleotide sequence and other constructs used for expression of novel recombinant P. falciparum circumsporozoite proteins in bacterial cells such as E. coli. Processes are provided for producing a soluble recombinant P. falciparum CSP from E. coli. Methods to produce a human-grade, highly immunogenic anti-malaria vaccine based on CSP are shown. The novel recombinant P. falciparum circumsporozoite protein by itself or in combination with other malaria antigens or adjuvants can form the basis of an effective malaria vaccine.
Core Innovation
The invention provides novel recombinant Plasmodium falciparum circumsporozoite proteins (rCSP) along with nucleotide sequences enabling expression of these proteins in bacterial cells such as E. coli as soluble proteins. A process is included for producing soluble recombinant CSP without requiring denaturation or refolding, resulting in purified protein greater than 95% pure and with low endotoxin and host cell protein levels. This advance permits production of a human-grade, highly immunogenic anti-malaria vaccine based on soluble CSP expressed in E. coli.
The problem solved by the invention stems from prior difficulties in expressing soluble full-length or near full-length P. falciparum CSP in E. coli due to issues such as high AT content, rare codons, complex tertiary structure including disulfide bonds, and extensive repetitive sequences causing genetic instability and insolubility. Previously, only insoluble CSP was produced in E. coli requiring extensive refolding. The invention overcomes these challenges by designing novel synthetic genes, optimizing the N- and C-termini, adjusting the number of repetitive NANP motifs, and using specialized E. coli strains (e.g., SHUFFLE™) that enable correct disulfide bond formation and soluble expression.
The technology includes recombinant CSP proteins characterized by a truncated N-terminus (lacking Met1 to Cys25), a reduced number of NANP repeats (preferably 18 or 19), possible inclusion of 0 to 3 NVDP repeats, and a C-terminus truncated to omit the GPI anchor region. Expression vectors and bacterial host cells transformed therewith are provided, along with methods for inducing expression and purifying soluble CSP from cell lysate supernatant without denaturation, yielding highly pure protein suitable for vaccine use. Vaccines comprising these recombinant proteins formulated with adjuvants exhibit strong immunogenicity and confer partial or full protection against malaria challenge.
Claims Coverage
The patent contains multiple independent claims describing a soluble recombinant P. falciparum circumsporozoite protein, processes for producing this protein in E. coli, and features of the purified protein and purification methods.
Soluble recombinant Plasmodium falciparum circumsporozoite protein
A soluble recombinant P. falciparum circumsporozoite protein comprising SEQ ID NO:2 or a 278 amino acid protein with at least 95% sequence identity thereto, characterized by lacking Met1 to Cys25 of the native protein's N-terminus, containing 18 or 19 NANP repeats (preferably 19), 0 to 3 NVDP repeats (preferably 3), and a C-terminus lacking 10 to 14 native residues ending at serine; optionally comprising the sequence of SEQ ID NO:8.
Process for producing soluble recombinant P. falciparum CSP in E. coli
Providing E. coli cells containing nucleotide sequences expressing the recombinant CSP; inducing expression; lysing cells; purifying the CSP from the lysate supernatant without denaturing and refolding.
Culturing conditions with animal-free media
Culturing the E. coli cells in media substantially free of animal-derived components, comprising ingredients including Phytone, yeast extract, ammonium sulfate, potassium phosphate monobasic, sodium phosphate dibasic, MgSO4, glycerol, dextrose, and kanamycin.
Purification method with two chromatographic steps
Purification consisting essentially of nickel affinity column purification followed by a Q-sepharose anion exchange column, without any other chromatographic separations, optionally including filtering the purified protein.
High purity protein with low endotoxin and host proteins
Purified recombinant CSP protein comprises at least about 90% purity by gel densitometry (preferably 95% or higher), less than 1 ng/ml E. coli host proteins, and less than about 5 endotoxin units per microgram protein.
Enhanced solubility compared to wildtype CSP
The recombinant protein exhibits greater solubility when expressed in E. coli relative to wildtype CSP.
The claims cover the novel soluble recombinant CSP protein with defined sequence modifications, an E. coli based production process using animal-free media and specific purification methods yielding highly pure, low endotoxin vaccine-grade protein with enhanced solubility and immunogenicity.
Stated Advantages
Expression of a near full-length soluble recombinant P. falciparum CSP in E. coli without the need for denaturation and refolding.
Production of highly pure, structurally homogeneous protein suitable for human vaccine use with low endotoxin and host-cell protein contamination.
The vaccine induces high-titer, repeat-specific antibodies and confers partial or full sterile protection against malaria challenge.
Use of animal-free media and cGMP-compliant fermentation and purification enabling scalable, economical manufacture of human-grade vaccine antigen.
Improved immunogenicity over yeast-derived CSP vaccines by avoiding post-translational modification, truncation or glycosylation.
Documented Applications
Use as an anti-malaria vaccine component to elicit immune responses in humans or animals.
Vaccination to provide partial or full protective immunity against malaria infection.
Production of recombinant soluble P. falciparum circumsporozoite protein in E. coli for vaccine manufacture.
Combination vaccine formulations incorporating CSP with other malaria antigens or adjuvants.
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