Soluble recombinant plasmodium falciparum circumsporozoite protein

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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.

This application claims the benefit of priority from U.S. Provisional Patent Application No. 61/394,048 entitled "Plasmodium Falciparum Circumsporozoite Vaccine Gene Optimization for Soluble Protein Expression" filed on October 18, 2010, which is incorporated by reference in its entirety.

The present invention was made with support from the United States Government and, specifically, the Walter Reed Army Institute of Research, and, accordingly, the United States government has certain rights in this invention.

The present technology is directed generally to recombinant Plasmodium falciparum circumsporozoite proteins, to methods for production of those proteins, and to vaccines including those proteins, among other aspects.

The present technology provides novel recombinant Plasmodium falciparum circumsporozoite proteins (rCSP), along with nucleotide sequences that express the recombinant P. falciparum CSP in bacterial cells, such as E. coti, as a soluble protein. The present technology also provides processes of expressing and purifying a soluble recombinant CSP protein without denaturing or refolding the protein. The purified protein produced by the present technology can be greater than 95% pure (that is, the soluble protein present in the composition is greater than 95% rCSP by weight) and contain low levels of endotoxin and low or undetectable levels of host cell proteins when analyzed by current techniques.

As one aspect of the present technology, novel recombinant Plasmodium falciparum circumsporozoite proteins are provided. The recombinant P. falciparum circumsporozoite proteins are characterized by an N-terminal region that lacks twenty to twenty-five N-terminus amino acid residues of native P. falciparum circumsporozoite protein; a reduced number of NANP repeats compared to native P. falciparum circumsporozoite protein; and at least 85% homology to SEQ ID NO:2, alternatively at least 90% homology to SEQ ID NO:2, alternatively at least 95% homology to SEQ ID NO:2. Preferably the recombinant P. falciparum circumsporozoite proteins comprise the peptide sequence of SEQ ID NO:2 or SEQ ID NO: 8. In some embodiments, the protein lacks Meti to Cys25 of the N-terminal region of native P. falciparum circumsporozoite protein. In some embodiments, the protein has 18 or 19 NANP repeats, preferably 19 NANP repeats, and/or has 0 to 3 NVDP repeats, preferably 3 NVDP repeats. In some embodiments, the recombinant P. falciparum CSP has a C-terminal region, preferably one that lacks ten to fourteen C-terminus amino acid residues of native P. falciparum circumsporozoite protein, more preferably, the protein ends at Ser383.

As another aspect of the present technology, nucleotide sequences are provided which encode a recombinant P. falciparum CSP as described in the preceding paragraph or elsewhere in this specification. Suitable nucleotide sequences include nucleotide sequences comprising SEQ ID NO: l or sequences that are at least 85% homologous to SEQ ID NO: l, alternatively at least 90% homologous to SEQ ID NO: l ; alternatively at least 95% homologous to SEQ ID NO: l . The nucleotide sequences can include at least one expression tag, such as the sequence of SEQ ID NO:5.

As another aspect of the present technology, novel expression vectors are provided for E. coli comprising a nucleotide sequence which encodes a recombinant P. falciparum CSP as described herein. The expression vectors can be stably cloned into a bacterial cell. A suitable bacterial cell can be transformed with such an expression vector. Preferably the bacterial cell is an E. coli cell, more preferably the SHUFFLE™ strain of E. coli. Surprisingly, the transfected E. coli cell expresses a recombinant P. falciparum CSP as a soluble protein.

As yet another aspect of the present technology, anti-malaria vaccines suitable for human administration are provided. The vaccines comprise a recombinant Plasmodium falciparum CSP as described herein, and one or more adjuvants. Preferred embodiments of the vaccines have an endotoxin level less than about 5 endotoxin units per microgram of protein, and/or less than about 1 ng/ml of bacterial host proteins. In some embodiments, the vaccines have a soluble protein content, and the soluble protein content is greater than 95%, alternatively greater than 99%, pure recombinant P. falciparum CSP as measured by gel densitometry.

As another aspect of the present technology, methods of eliciting an immune response against malaria in an animal or human comprise administering a vaccine or rCSP as described herein. Methods of immunizing an animal or human against malaria or a pathogen that causes malaria are also provided. The methods comprise administering to the animal or human a vaccine or rCSP as described herein. In these methods, the vaccine can be administered intramuscularly or by another route.

As still another aspect of the present technology, processes of producing recombinant P. falciparum CSP are provided, including processes of producing rCSP in soluble form from E. coli. The processes comprise the steps of providing cells, preferably bacterial cells such as E. coli, containing a nucleotide sequence that expresses one of the recombinant P. falciparum CSPs described herein (such as a transfected E. coli that expresses the peptide sequence of SEQ ID NO:2). The cells may be provided in a cell culture. The processes also comprise inducing expression of the recombinant CSP in the cells, and collecting the cells after a period of protein expression, such as by centrifuging to obtain a pellet containing the cells. The processes also comprise lysing the cells to obtain a cell lysate, collecting supernatant from the cell lysate, and purifying the recombinant P. falciparum CSP from the supernatant of the cell lysate without denaturing and refolding the recombinant P. falciparum CSP. Preferably, the bacterial cell is cultured in that is media free or substantially free of animal-derived components, such as media containing one or more or all of Phytone, yeast extract, ammonium sulfate, potassium phosphate monobasic, sodium phosphate dibasic, MgSC^, glycerol, dextrose or kanamycin. In some embodiments, the recombinant P. falciparum CSP includes one or more expression tags at one or both ends to facilitate the recovery or purification of the protein. The processes can include one or more purification steps, such as purifying the soluble protein over an affinity column and purifying the soluble protein over an anion exchange column, for example, a nickel affinity column and a Q- sepharose anion exchange column. The present technology also includes purified protein made by the processes described herein.