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Title:  Synthetic genes for malarial proteins and methods of use
United States Patent: 
7,078,507
Issued: 
July 18, 2006

Inventors:
 Narum; David (Gaithersburg, MD); Liang; Hong (Gaithersburg, MD); Fuhrmann; Steve (Germantown, MD); Sim; B. Kim Lee (Gaithersburg, MD)
Assignee:
  The United States of America as represented by the Secretary, Department of Health and Human Services (Washington, DC)
Appl. No.:
 293913
Filed: 
November 12, 2002


 

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Abstract

Synthetic gene sequences encoding erythrocyte binding protein of a malaria pathogen for the expression of the erythrocyte binding protein. The codon composition of the synthetic gene sequences approximates the mammalian codon composition. The synthetic gene sequences are useful for incorporation into the DNA vaccine vectors, for the incorporation into various expression vectors for production of malaria proteins, or both. The synthetic genes may be modified to avoid post-translational modification of the encoded protein in hosts. Administration of the synthetic gene sequences, or the encoded protein, as an immunization agent is useful for induction of immunity against malaria, treatment of malaria, or both.

DETAILED DESCRIPTION OF THE INVENTION

Synthetic gene sequences encoding malaria pathogen erythrocyte-binding protein (EBP) and methods of producing of such synthetic gene sequences are provided. Compositions and methods of eliciting immune response to the protein encoded by the synthetic gene sequences are also provided. Methods of large-scale production of the malaria pathogen erythrocyte-binding protein encoded by synthetic gene sequences are described. Methods of modification of the EBP synthetic gene sequences to alter the properties of the resulting protein are also provided, along with the synthetic gene sequences and expression vectors for such altered EBP. These compositions and methods can be used for treating and preventing malarial diseases and infection.

As used herein, the term "synthetic" is defined to mean laboratory produced in a manner known to one skilled in the art. The term "gene segment" and "gene sequence" are used interchangeably and are defined to mean a portion of a nucleotide sequence that transcribes RNA, either m, r, or t, used in the production of polypeptides or used directly. The term "isolated" refers to a composition that is substantially or essentially free from at least some of the components that normally accompany it in its native state. Thus, the isolated proteins of this invention do not contain some of the materials normally associated with their in situ environment. Typically, the isolated proteins of the invention are at least about 80% pure, usually at least 90% pure and preferably at least 95% pure as measured by band intensity on a Coomassie-stained gel. The term "host" is defined to mean an organism that can be infected by a pathogen, and the term "pathogen" is defined to mean any organism that causes a deviation in a normal state. The term "ligand" is defined to mean a molecule that will bind to a complementary site on a given structure. The term "codon" is defined to mean the nucleotide triplet that specifies the amino acid to be inserted in a specific position in a forming polypeptide during translation. The term "altered" is used interchangeably with the term "mutated" to refer to the changes in the nucleotide sequence. The term "improved immunogenicity" is defined herein as the following two characteristics possessed, separately or together, by a vaccine: (a) the ability of a vaccine to induce, at any single dose, higher antibody titers than a reference vaccine; (b) the ability of a vaccine to induce antibody titers comparable to that of a reference vaccine, wherein the administered dose of the vaccine of "improved immunogenicity" is reduced 10 or more fold compared to the dose of the reference vaccine.

The term "sequence homology" is also known in the art as "sequence similarity" between two or more sequences. A high degree of "sequence homology" is at least approximately 60% amino acid similarity between the two amino acid sequences. "Sequence similarity" can be determined by a number of commercially available or freeware programs which are widely used in the art. Examples of such programs are available from the World Wide Web (WWW) "Sequence Analysis Tools" server of National Institutes of Health (NIH) in the United States of America (Bethesda, Md.). They are also available from the WWW server of Institut Suisse de Bioinformatique (ISB) "ExPASy Proteomic Tools" in Switzerland.

The term "large-scale protein production" is used to refer to the production of proteins by a method such as liquid fermentation on a scale over one liter.

Additionally, the terms "a", "an" and "the" as used herein are defined to mean "one or more" and include the plural unless the context is inappropriate. The terms "polypeptide", "peptide" and "protein", as used herein, are interchangeable and are defined to mean a biomolecule composed of two or more amino acids linked by a peptide bond.

According to the present invention, a synthetic gene segment of a synthetic nucleotide sequence encodes an amino acid sequence of erythrocyte-binding protein of a malaria pathogen. In the preferred embodiment of the present invention, the erythrocyte-binding protein is of a malarial pathogen Plasmodium falciparum. In particular, the present invention describes the synthetic gene segment encoding the amino acid sequence of the 616 amino acids of the receptor-binding domain (RII) of the P. falciparum EBA-175 as described in Sim, et al. (1994) and incorporated herein by reference. The 5' cysteine-rich region of the protein, designated region II ("RII"), was chosen for synthesis because it binds to its receptor--sialic acids on glycophorin A--for invasion of erythrocytes by the malarial pathogen, as described in Narum, et al (2000) and incorporated herein by reference. The scheme of the gene structure of the EBA-175 is shown in FIG. 1, panel A. The region RII was chosen because antibodies directed against RII block invasion of P. falciparum strains, which have the capability to invade erythrocytes by distinct pathways, as described in Narum, et al (2000). The use of this particular receptor-binding protein from this particular malaria pathogen is for illustration purposes only and it should be understood that any pathogen ligand capable of invading a host's cell by distinct pathways may be substituted. The ligands include, but are not limited to, P. vivax and P. knowlesi Duffy binding proteins, particularly their receptor-binding (RII) regions, and any malaria pathogen protein containing sequences highly similar to RII region.

Methods commonly known or used in the art can be used to identify the sequences of the malaria pathogen ligands for the production of synthetic genes. One exemplary method is to utilize similarity searches of the malaria pathogen sequences. Searchable databases of Plasmodium genomes, such as the Plasmodium falciparum genome database internet server at TIGR Institute, are becoming readily available.

Once the receptor-binding protein is selected from a desired malaria pathogen, a host species is selected from a group that can be infected by the pathogen. In the most preferred embodiment of the present invention, the host species is Homo sapiens (humans).

The present invention includes a method of producing a synthetic gene segment of a malaria pathogen erythrocyte-binding ligand. The codon usage of highly expressed genes in the host is compared to that of the selected pathogen genes. This comparison can be performed, for example, with the help of the CodonW software by John Peden, available as freeware from the internet. The most frequently used codons for encoding a particular amino acid of the host are used to synthesize a gene segment encoding the receptor-binding protein. The method involves identifying nucleotide sequence of a native gene segment from a pathogen that encodes for a desired erythrocyte-binding ligand, comparing the codons most frequently used by the malaria pathogen and the host, designing the synthetic gene sequence by substituting the nucleotides in the native malaria pathogen gene to approximate the codon composition of the mammalian host, and synthesizing a synthetic gene sequence. One skilled in the art could produce a synthetic gene by obtaining the long oligomers through a commercial source, anneal them together to form a double strand and then connect them in the proper sequence, as described generally in the manuals common in the art, such as Sambrook, J., et al., 2000, which is incorporated herein by reference in its entirety. Protein expression elicited from a synthetic sequence can be tested in vitro and in vivo using those methods known and used in the art.

A synthetic gene segment, as described herein, is useful in the production of DNA vaccines, protein vaccines, and therapeutic compositions. As used herein, the term "DNA vaccine" includes compositions comprising a synthetic gene sequence of the present invention and which is used for immunization of individuals prior to or following infection of a malarial pathogen. As used herein "protein vaccines" includes compositions comprising a protein encoded by a synthetic gene sequence of the present invention and is used for immunization of individuals prior to or following infection of a malarial protein. Accordingly, the present invention includes DNA and protein vaccines that inhibit binding of an EBA-175 protein to an erythrocyte. Additionally included in the present invention are DNA and protein vaccines that inhibit malarial invasion into an erythrocyte.

The present invention includes a method for producing a DNA vaccine comprising a synthesized gene sequence as described above. A synthetic gene sequence is cloned into a vector using molecular biology techniques that are known and used in the art, and described generally in the manuals common in the art, such as Sambrook, et al. (2000). In a preferred embodiment, the vector is a DNA vaccine vector. Standard techniques of molecular biology known and used in the art for preparing and purifying DNA constructs enable the preparation of the DNA vaccines of this invention. While standard techniques of molecular biology are therefore sufficient for the production of the products of this invention, the specific constructs disclosed herein provide novel synthetic gene segments.

In other embodiment of the present invention the synthetic gene sequences are used for production of malaria erythrocyte-binding protein. In a preferred embodiment of the present invention, the synthetic gene sequences are introduced into the expression vectors for the yeast Pichia pastoris. Pichia pastoris expression vectors for production of the recombinant erythrocyte-binding proteins encoded by the synthetic gene sequences are described. Using procedures common in the art, the Pichia pastoris expression vectors are introduced into an appropriate strain of Pichia pastoris. The method of expression of erythrocyte-binding protein in Pichia pastoris is optimized according to a procedure provided herein, which is useful for robust large-scale production of the recombinant erythrocyte-binding protein. The present invention encompasses a method for large-scale production, including expression and purification, of the erythrocyte-binding protein. This large-scale production can be used to produce clinical grade material to administer to humans for prevention and treatment of malaria, including, but not limited to as protein vaccines or as a part of the DNA prime/protein boost immunization regimens. Such immunization regimens with the RII protein have been shown to protect Aotus monkeys from malaria in pre-clinical trials.

Other uses of the recombinant malaria-binding proteins encoded by the synthetic gene sequences are contemplated herein. One such use for raising the antibodies or serum for administration to animals or humans with the purpose of passive immunization and treatment of malarial disease.

In another embodiment of the present invention, the synthetic gene sequences encoding EBA-175 RII protein are altered to affect the properties of the encoded erythrocyte-binding protein. In the preferred embodiment of the present invention the amino acid sequences that determine the attachment of polysaccharides ("glycosylation") to the polypeptide chain are replaced with the amino acids with similar properties ("conserved substitution") except for the ability to guide glycosylation. In particular, N-glycosylation sites in the synthetic gene of the EBA-175 RII region are altered. Pichia pastoris and mammalian expression vectors encoding such altered synthetic gene sequences are described. The altered EBA-175 RII protein is expressed in both P. pastoris and mammalian cells and compared to the non-altered protein encoded by the non-mutated synthetic gene sequence. The altered EBA-175 RII proteins are not N-glycosylated either in P. pastoris or mammalian expression system.

In the preferred embodiment of the present invention, the properties of the non-N-glycosylated RII EBA protein are tested. In the most preferred embodiment, the erythrocyte-binding properties, the ability to inhibit the growth of malaria parasite, and, in particular, the immunogenic properties of the non-glycosylated protein were tested. The immune response induced by administration of the non-N-glycosylated protein to the mammals was tested. The immune response induced by the administration of DNA vaccine encoding the non-glycosylated protein to the mammals was also tested. The comparison of the properties of the N-glycosylated and non-N-glycosylated malaria proteins encoded by synthetic genes, especially their immunogenic properties resulted in the understanding that N-glycosylation of the EBP protein does not affect its properties important for its use in the anti-malaria vaccine compositions and vaccination methods. Therefore, a foremost concern in the development of anti-malaria vaccine methods and compositions was eliminated.

The present invention includes a method of inducing an immune response to a pathogen by administering an amount of a synthetic gene segment or a protein encoded by the synthetic gene segment, or a combination of both, to a potential host in a sufficient amount to induce an immune response. In a preferred embodiment the pathogen is a malarial pathogen namely a species of Plasmodium. In a further embodiment, the species of Plasmodium is falciparum. In a still further embodiment of the present invention, the potential host is a mammal, namely a human. Also included in the present invention is a method of treating malaria. This embodiment of the invention requires selecting an infected host and administering a sufficient dosage of a synthetic gene segment or a protein encoded by the synthetic gene, or a combination of both, to inhibit the malarial pathogen.

In a preferred embodiment, a DNA or protein vaccine for passive or active immunization against malaria is combined with a pharmaceutically acceptable carrier to facilitate administration. The preferred dose for human administration of the DNA vaccine is 10.sup.2 to 10.sup.4 TCID.sub.50/person. (TCID is an abbreviation for tissue culture infectious doses). The preferred dose for human administration of the protein vaccine for active or passive immunization is from 0.001 mg/kg to 10 mg/kg. The dose should be adjusted to suit the individual to whom the composition is administered and will vary with certain factors such as age, weight and metabolism of the individual. The vaccines may additionally contain stabilizers or physiologically acceptable preservatives.

The vaccine can be administered by any appropriate route, including but not limited to, orally, parenterally, intravenously, intradermally, intramuscularly, subcutaneously, or topically, in liquid or solid form, in a single dose or a dose repeated after a certain time interval. The vaccine may be provided to the physician in a lyophilized form, reconstituted in an appropriate solvent such as deionized water or saline and administered as a single injection or a series of injection at a time intervals necessary for the achievement of the immune response or treatment of the disease. The different variations of vaccines may be combined in a single dose or a vaccination regimen to achieve the desired anti-malarial effect.

Preferably, the administration of the DNA vaccine will result in in vivo protein expression of the proteins encoded by the open reading frames contained in the expression vector construct. Most preferably, the administration of the DNA vaccine will result in the induction of immunity against the erythrocyte binding proteins, which are encoded by the synthetic genes incorporated into the vaccine. Preferably, the administration of the protein vaccine will result in induction in the host of the immunity of the protein component of the vaccine. Preferably, the administration of the antibody vaccine will result acquisition by the host of immunity against the malarial disease.

 

Claim 1 of 3 Claims

1. An isolated synthetic nucleotide sequence encoding a domain of the erythrocyte-binding protein of a malaria pathogen that infects humans, wherein codon usage of the synthetic nucleotide sequence is altered compared to a naturally occurring sequence of the erythrocyte-binding protein in order to approximate codon usage of a host of the malaria pathogen, wherein the host is a human, and wherein the domain of the erythrocyte-binding protein is the region II of in 3D7 (NIH) Plasmodium falciparum EBA-175 protein.

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