Soluble recombinant plasmodium falciparum circumsporozoite protein, use in vaccines, methods of making and uses thereof
Inventors
Assignees
United States Department of the Army
Publication Number
US-9919040-B2
Publication Date
2018-03-20
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 present invention provides novel nucleotide sequences and constructs for expressing recombinant Plasmodium falciparum circumsporozoite proteins (rCSP) in bacterial cells, notably E. coli, where the recombinant protein is soluble. The technology includes processes to produce and purify soluble recombinant P. falciparum CSP from E. coli without denaturing or refolding, yielding a protein composition that is highly pure (greater than 95% by weight), with low endotoxin and host cell protein levels, suitable for human vaccine use.
This technology addresses the difficulty in expressing native P. falciparum CSP in E. coli due to factors such as high AT content of the gene, rare codons incompatible with E. coli, complex disulphide bonds, and repetitive sequences that cause genetic rearrangements. Previous efforts resulted only in insoluble protein requiring refolding. The invention overcomes these issues by providing a codon-optimized gene lacking the GPI anchor sequence, constructs with specific N-terminal and C-terminal truncations, and expression in strains such as SHUFFLE™ E. coli that facilitate disulphide bond formation, thereby producing a stable, soluble, near full-length recombinant CSP.
The recombinant proteins have defined sequence characteristics, including lacking approximately the first 20–25 N-terminal amino acids, and reduced numbers of NANP repeats (preferably 18 or 19), and optionally reduced C-terminal residues. The invention also includes vaccines comprising this soluble rCSP combined with one or more adjuvants, processes for large-scale production in animal component-free media under current good manufacturing practices, and demonstrated immunogenicity and protective efficacy in animal models. The vaccines elicit high titer antibodies including those that recognize native CSP on sporozoites and can confer partial to sterile protection against malaria challenge.
Claims Coverage
The patent includes one independent claim focusing on an anti-malaria vaccine and associated methods of immunization. The main inventive features relate to the recombinant circumsporozoite protein composition, its purities, and vaccine formulations with adjuvants.
Recombinant circumsporozoite protein composition
An anti-malaria vaccine comprising a recombinant Plasmodium falciparum circumsporozoite protein (rCSP) comprising SEQ ID NO: 2 or having 95% sequence identity to SEQ ID NO: 2, with specific characteristics such as 278 amino acids and defined numbers of NANP and NVDP repeats.
Inclusion of one or more adjuvants in the vaccine
The vaccine comprises one or more adjuvants, including water-in-oil emulsions containing metabolizable oils and optionally provided in particulate forms such as microparticles or liposomes with adjuvants either inside or on the surface, which may be covalently linked to the rCSP.
Purity and composition parameters of the vaccine
The vaccine has an endotoxin level less than about 5 endotoxin units per microgram of protein, less than about 1 ng/ml of bacterial host proteins, and a protein content greater than 95% recombinant P. falciparum CSP as measured by gel densitometry (optionally greater than 99%).
Structural and sequence modifications to rCSP
The rCSP includes 18 or 19 NANP SEQ ID NO: 13 repeats (preferably 19), 0 to 3 (preferably 3) NVDP SEQ ID NO: 14 repeats, lacks Met1 to Cys25 of the N-terminal region of native protein, and may lack 10–14 C-terminal amino acids of the native protein; specific inclusion of SEQ ID NO: 8 is also claimed.
Method of eliciting an immune response
A method of eliciting an immune response against malaria in an animal or human by administering the vaccine comprising the rCSP with one or more adjuvants, optionally intramuscularly, to induce a broad antibody response.
The claims cover the composition of the recombinant soluble P. falciparum circumsporozoite protein with defined sequence and repeat characteristics, vaccine formulations with specified adjuvants and purity levels, and methods of use for immunization against malaria, highlighting the novel protein expression and vaccine advantages.
Stated Advantages
The recombinant protein is produced as a soluble, near full-length protein in E. coli without requiring denaturing and refolding, overcoming prior expression challenges.
The vaccine comprises highly pure rCSP (>95% purity), low endotoxin levels, and low or undetectable host cell proteins, suitable for human injection and meeting cGMP standards.
The recombinant protein elicits a strong immune response, inducing high titer antibodies that recognize native sporozoite CSP and can confer partial or sterile protection against malaria challenge in animal models.
The production process uses animal component-free media and includes a simplified two-step purification method, facilitating scalable and economical vaccine manufacture.
Documented Applications
Use as a human-grade vaccine against malaria, comprising soluble recombinant P. falciparum circumsporozoite protein combined with adjuvants to elicit protective immunity.
Immunization of animals or humans to induce antibody and cellular immune responses preventing malaria infection or reducing disease severity.
Production of recombinant CSP protein in bacterial hosts for vaccine formulation under current good manufacturing practice (cGMP) compliance.
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