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  Pharmaceutical Patents  

 

Title:  Vaccines for blocking transmission of plasmodium vivax
United States Patent: 
7,407,658
Issued: 
August 5, 2008

Inventors:
 Kaslow; David C. (Wayne, PA), Tsuboi; Takafumi (Ehime, JP), Torii; Motomi (Ehime, JP)
Assignee:
  The United States of America as represented by the Secretary of the Department of Health and Human Services (Washington, DC)
Appl. No.:
 11/611,779
Filed:
 December 15, 2006


 

Web Seminars -- Pharm/Biotech/etc.


Abstract

The present invention relates novel methods and compositions for blocking transmission of Plasmodium vivax which cause malaria. In particular, Pvs25 and Pvs28 polypeptides, variants, including deglycosylated forms, and fusion proteins thereof, are disclosed which, when administered to a susceptible organism, induce an immune response against a 25 kD and 28 kD protein, respectively, on the surface of Plasmodium vivax zygotes and ookinetes. This immune response in the susceptible organism can block transmission of malaria.

Description of the Invention

BRIEF SUMMARY OF THE INVENTION

The present invention relates to methods for preventing transmission of malaria, particularly Plasmodium vivax. The invention relates to methods for eliciting an immune response against parasites responsible for malaria. These methods comprise administering to a susceptible organism a pharmaceutical composition comprising Pvs28 polypeptides (such as SEQ ID NO:2), including partially or completely deglycosylated Pvs28 polypeptides, Pvs25 polypeptides(such as SEQ ID NO:4), variants thereof, or Pvs25/Pvs28 fusion proteins(such as SEQ ID NO:5), in an amount sufficient induce an immune response against a 25 kD and 28 kD protein, respectively, on the surface of Plasmodium vivax zygotes and ookinetes. The immune response in the susceptible organism can block transmission of malaria.

The invention also relates to methods of preventing transmission of malaria comprising administering to a susceptible organism a pharmaceutical composition comprising a recombinant virus or expression cassette encoding a Plasmodium vivax polypeptide, including Pvs28 polypeptides (including partially or completely deglycosylated Pvs28 polypeptides), Pvs25 polypeptides, or Pvs25/Pvs28 fusion proteins, in an amount sufficient to block transmission of the disease.

The invention further relates to pharmaceutical compositions comprising a pharmaceutically acceptable carrier and a Pvs28 polypeptide(including partially or completely deglycosylated Pvs28 polypeptides), a Pvs25 polypeptide, or a Pvs25/Pvs28 fusion protein, as described herein.

The invention also relates to isolated nucleic acids comprising nucleotide sequences encoding Pvs28 polypeptides (including partially or completely deglycosylated Pvs28 polypeptides), Pvs25 polypeptides, or Pvs25/Pvs28 fusion proteins. These nucleic acids may be isolated from, for instance, P. vivax. The sequences are typically contained in an expression vector for recombinant expression of the proteins. The sequences can also be incorporated into recombinant viruses, vectors or expression cassettes for use as nucleic acid vaccines, including "naked DNA" vaccines, for recombinant expression of the proteins in vivo. In another embodiment, the nucleic acids of the invention comprise a pharmaceutical excipient and are injected into a host, e.g., as "naked" DNA vectors or "expression cassettes" injected into muscle, to express recombinant protein in vivo, to induce transmission blocking antibodies against encoded polypeptides.

The invention also provides a composition comprising an isolated nucleic acid molecule encoding a Plasmodium vivax Pvs28 polypeptide lacking at least one N-linked glycosylation site. In alternative embodiments, the nucleic acid encodes a polypeptide comprising a sequence as set forth in SEQ ID NO:2, excepting that the amino acid residue corresponding to residue 130 of SEQ ID NO:2 is not an asparagine residue; and the amino acid residue corresponding to residue 130 of SEQ ID NO:2 is glutamine.

The invention also provides a composition comprising an isolated Plasmodium vivax Pvs28 polypeptide lacking at least one N-linked glycosylation site. In alternative embodiments, the polypeptide comprises a sequence as set forth in SEQ ID NO:2, excepting that the amino acid residue corresponding to residue 130 of SEQ ID NO:2 is not an asparagine residue; and, the amino acid residue corresponding to residue 130 of SEQ ID NO:2 is glutamine.

The invention further provides a method of inducing a transmission blocking immune response in a mammal, comprising administering a partially or completely deglycosylated Pvs28 polypeptide, or a nucleic acid encoding such a polypeptide, to a mammal.

Pvs28 (including partially or completely deglycosylated Pvs28) as an immunogenic carrier is provided for by the invention. Pvs28, administered with a second composition, provides a superior antigenic response to the second composition. Thus, the invention relates to an immunogenic composition capable of eliciting an immunogenic response directed to an epitope comprising an isolated Pvs28 and an isolated molecule comprising the epitope. The invention is also directed to methods of eliciting an immunogenic response directed to an epitope comprising administering an isolated Pvs28 and an isolated molecule comprising the epitope. The Pvs28 and the second molecule can be chemically linked or joined together as recombinant fusion proteins.

In one embodiment, the Pvs28-containing fusion protein is a Pvs25-Pvs28 fusion protein. The Pvs28 polypeptide can be designed to be partially or completely deglycosylated, as described herein. The sexual stage malarial proteins Pvs25 and Pvs28, in the form of a Pvs25-Pvs28 fusion protein, will generate transmission-blocking antibodies against both Pvs25 and Pvs28. These fusion proteins have enhanced antigenic properties, as compared to use of either alone as an immunogen.

The invention also provides for Pvs25/Pvs28, Pvs25, partially or completely deglycosylated Pvs28, and Pvs28 fusion proteins, and the nucleic acids encoding such polypeptides, further comprising non-malarial sequences. For example, a Pvs25, Pvs28, or Pvs25/Pvs28 polypeptide of the invention can further comprise epitope tags, enzyme cleavage sequences, leader sequences, sequences which cause the polypeptides to be transported to a particular intracellular organelle, and the like. For example, as discussed below, inclusion of yeast alpha mating pheromone signal sequence in a fusion protein of the invention allows for secretion of the expressed Pvs25 or Pvs28. These fusion proteins can provide for simplified manufacturing of Pvs25-Pvs28 antigens.

In one class of embodiments, the Pvs25-Pvs28 fusion protein includes an N terminal Pvs25 domain and a C terminal Pvs28 domain. This arrangement of Pvs25 and Pvs28 in a fusion protein provides superior antigenic and transmission blocking properties for the fusion protein. In one preferred embodiment, the C terminal Pvs28 domain includes the carboxyl terminal region of Pvs28. Exemplar fusion proteins are provided in the examples set forth herein, and conservative modifications thereof.

Typically, the Pvs25-Pvs28 fusion proteins of the invention include a flexible linker separating the Pvs25 and Pvs28 domains. An exemplar flexible linker is the amino acid sequence GGGPGGG (SEQ ID NO:15).

In one embodiment, the fusion proteins (as Pvs25 and Pvs28) are produced recombinantly. The recombinant proteins of the invention can be expressed, e.g., in vitro, in prokaryotic or in eukaryotic systems. In alternative embodiments, bacterial, yeast, insect, plant, mammalian, or other expression systems can be used.

In another embodiment, a nucleic acid encoding a fusion protein of the invention is optimized for expression in a particular expression system, such as preferred codon usage in bacteria or partial or complete deglycosylation by mutation for yeast expression, thereby facilitating recombinant expression and manufacturing of the polypeptide of the invention. For example, Pvs25 and Pvs28 consist of four epidermal growth factor-like (EGF) domains (similar domains are found in the related Pfs25 and Pfs28 Plasmodium polypeptides). These EGF domains comprise structural domains in the molecules. In alternative embodiments, the immunogen includes one or more domains in a variety of permutations and orientations. As domains may require disulfide bonds to create and maintain structural integrity, alternative embodiments encompass various expression systems that faithfully recreate these disulfide linkages.

In another embodiment, the Pvs25, Pvs28 or Pvs25-Pvs28 fusion protein sequences can be mutated or altered, e.g., using site-specific mutational methodologies. For example, in one embodiment, the Pvs25 and Pvs28 sequences are mutated to eliminate one, several or all potential glycosylation sites. Such mutations can facilitate recombinant expression and manufacturing of the polypeptides of the invention, as in yeast expression systems. The partially or completely deglycosylated Pvs polypeptides of the invention are, in some circumstances, better immunogens, i.e., administration of these forms enhance the antigenicity of the polypeptide. For example, in one embodiment, an amino acid residue at position 130 of Pvs28 is altered to remove a potential glycosylation site.

In other embodiments, the different domains of the immunogenic composition are joined, or linked, together by chemical means. In further embodiments, the domains of the immunogenic compositions are derived from natural sources.

The Pvs25-Pvs28 fusion protein, when administered to a mammal, elicits the production of at least two classes of antibodies: antibodies which specifically bind to Pvs25, and antibodies which specifically bind to Pvs28. In preferred embodiments, the administration of the fusion proteins of the invention elicit a transmission blocking immune response. Immunological enhancers and pharmaceutically acceptable carriers are optionally added to the fusion protein to enhance the immunogenicity of the fusion protein and to facilitate delivery of the fusion protein to a mammal. For example, in alternative embodiments, adjuvants such as alum are added.

Immunogenic compositions comprising the fusion proteins of the invention elicit transmission blocking antibodies in a variety of mammals, including humans and other primates, and mice and other rodents.

Cells expressing the nucleic acids and polypeptide of the invention are a feature of the invention. For example, recombinant cells such as yeast cells can be used to express the Pvs25-Pvs28 fusion protein of the invention. Cell lines containing a nucleic acid encoding the immunogenic polypeptides and fusion proteins in an expression vector are also disclosed.

The invention provides methods of inducing a transmission blocking antibody in a mammal. In the methods, the Pvs25-Pvs28 fusion protein, or a nucleic acid encoding the fusion protein is administered to a mammal, which produces transmission blocking antigens. Administration is typically performed intramuscularly, intradermally, or subcutaneously. An adjuvant such as alum is optionally administered with the fusion protein or nucleic acid.

DETAILED DESCRIPTION

The present invention relates to novel compositions and methods for blocking transmission of malaria, particularly Plasmodium vivax. The invention provides agents which are capable eliciting antibodies and antiserum (generated by administration of the compositions of the invention) which, when ingested by the mosquito, are capable of inhibiting the life cycle of the disease-causing parasite in the mosquito midgut. The agents include Plasmodium vivax Pvs 25 and Pvs28 polypeptides (including partially and completely deglycosylated forms), nucleic acids encoding these polypeptides, and fusion proteins thereof, that are useful for inducing antibodies that block transmission of the parasite. The invention also provides the isolated antibodies generated by these polypeptides. These nucleic acid and polypeptide compositions are useful as vaccines against malaria.

This invention further relates to an immunogenic composition capable of eliciting an immunogenic response directed to an epitope comprising an isolated Pvs25 or Pvs28 and an isolated molecule comprising the epitope. The invention is also directed to methods of eliciting an immunogenic response directed to an epitope comprising administering an isolated Pvs28 and an isolated molecule comprising the epitope. In one embodiment, the Pvs28 is acting as an immunogenic carrier to a hapten epitope to elicit, stimulate or augment a humoral immune response to the epitope.

The fusion proteins of the invention (optionally used with an adjuvant such as alum) can be used to block transmission of a number of parasites associated with malaria. Examples of parasites whose transmission is blocked by the materials and compositions of the invention include the causative parasites for malaria. Four species of the genus Plasmodium infect humans, P. vivax, P. ovale, P. malariae, and P. falciparum. P. falciparum is the most prevalent cause of malaria in humans: Other Plasmodium species infect other animals. For instance, P. gallinaceum is responsible for avian malaria.

The present invention relates to recombinant viruses and vaccines comprising nucleic acid sequences which encode malaria parasite (Plasmodium vivax) Pvs25 and Pvs28 polypeptides, including fusion proteins and deglycosylated forms (SEQ ID NOs: 1 to 5). These polypeptides are naturally expressed by Plasmodium during its mosquito-infective, sexual stage. Because naturally expressed Pvs polypeptides are expressed in malaria parasite oocytes and zygotes, recombinant forms can be used to induce an immune response to the sexual stage of the parasite.

These Pvs25- and Pvs28-expressing malaria parasite sexual stages occur only in the mosquito host and not in the human. This invention includes compositions and methods for eliciting human antibodies which, when ingested by the mosquito during its feeding process, block the development of malaria in the mosquito. Blocking the sexual development of the malaria parasite in the mosquito reduces the vector's ability to further transmit the disease to a second human host.

The human antiserum generated by the compositions and methods of the invention, when ingested by the mosquito, significantly reduces the numbers of malaria parasite oocysts within the insect. Significant public health benefits are attained by the vaccines' ability to elicit antibodies which, upon mosquito ingestion, significantly decrease the number of oocysts capable of maturing into infectious sporozoites. A vaccine is still very useful when it generates an antiserum that decreases the numbers of oocysts in the mosquito, thus reducing the numbers of parasites transmitted by the mosquito. To be useful, it is not necessary that the ingested antisera render the mosquito completely incapable of transmitting the malaria parasite to a second person (i.e., completely inhibit sexual development of all oocysts).

The use of sexual stage polypeptides as a transmission blocking antigens are described, e.g., in U.S. Pat. No. 5,217,898 to Kaslow and Barr directed to Pfs25 as a transmission blocking antigen, and U.S. Pat. No. 5,527,700 to Kaslow and Duffy, directed to Pfs28 as a transmission blocking antigen.

The Pvs25-Pvs28 fusion proteins of the invention have several surprising properties. The fusion protein is more efficient in producing transmission blocking antibodies, e.g., in mice, than Pvs25 or Pvs28 alone. This is true despite the fact a mixed dose of Pvs25 and Pvs28 will not induce a higher level of transmission blocking antibody activity than either Pvs25 or Pvs28 alone. Second, less fusion protein is required as an immunogen than either Pvs25 or Pvs28 alone. Third, titers of transmission blocking antibodies will remain high for a longer period of time when the antigen is a Pvs25-Pvs28 fusion protein than either Pvs25 or Pvs28 alone. In a preferred aspect, the invention provides a nucleic acid with yeast preferred codons for encoding and expressing the fusion protein in yeast.

Pvs28 and Pvs25 Polypeptides

The present invention includes immunogenic Pvs 25 and Pvs28 polypeptides and fragments derived from these proteins, and partially or completely deglycosylated forms of these polypeptides, that are useful for inducing an immune response when the proteins are injected into a human or other host animal. An exemplary polynucleotide sequence for a Pvs25 of the invention is shown in SEQ ID NO:3, FIG. 3 (see Original Patent). An exemplary amino acid sequence for a Pvs25 polypeptide of the invention is shown in SEQ ID NO:4, FIG. 4 (see Original Patent). An exemplary polynucleotide sequence for a Pvs28 of the invention is shown in SEQ ID NO:1, FIG. 1 (see Original Patent). An exemplary amino acid sequence for a Pvs28 polypeptide of the invention is shown in SEQ ID NO:2, FIG. 2 (see Original Patent).

In another embodiment, the immunogenic composition, comprising an isolated Pvs28 and an isolated molecule comprising the epitope, is capable of eliciting or augmenting an immunogenic response directed to the epitope. The Pvs28 can act as a immunological "carrier" to boost, augment or increase the cellular or humoral response to the epitope. The antibodies that arise from the immune response block transmission of the parasite by interfering with the portion of the parasite's life cycle that occurs in the mosquito. For example, purified polypeptides having an amino acid sequence substantially identical to a subsequence of Pvs28 may be used; including partially or completely deglycosylated forms of Pvs28.

The antibodies or T cells that arise from administration of Pvs28, Pvs25 or Pvs28-Pvs25 fusion proteins (e.g., as in a polypeptide vaccine, or a vaccine comprising nucleic acid encoding these polypeptides, such as a virus or vector) generate an immune response by blocking transmission of the parasite malaria by interfering with the portion of the parasite's life cycle that occurs in the mosquito. Pvs 25 and Pvs28 are similar in structure to other known ookinete antigens such as Pfs25 and Pfs28, respectively. All four proteins comprise a putative secretory signal sequence, followed by four EGF-like domains and a terminal hydrophobic transmembrane region without a cytoplasmic tail. Although the four proteins share the six-cysteine motif of the EGF-like domains, the functions of these proteins may be very different. EGF-like domains have been recognized in a range of proteins that have diverse functions (Davis (1990) New Biol. 2:410-419).

Included among the polypeptides of the present invention are proteins that are variants of the native proteins constructed by in vitro or in vivo techniques, including recombinant or synthetic techniques. One skilled in the art will appreciate, for instance, that for certain uses it would be advantageous to produce a Pvs25 or a Pvs28 polypeptide that is lacking one of its structural characteristics. For example, one may remove the transmembrane domain to obtain a polypeptide that is more soluble in aqueous solution.

Alternatively, the invention provides partially and completely deglycosylated variants, such as the genetically engineered Pvs28 of the invention in which the amino acid at position 130 does not encode an asparagine, and thus cannot be a putative site for N-linked glycosylation. In an exemplary sequence, the nucleic acid of the invention was modified to encode glutamine, and the Pvs28 variant polypeptide of the invention was modified to be glutamine at residue 130. However, any putative amino acid site of N-- or O-linked glycosylation (and the nucleic acid which encodes such a site, or motif) can be modified to alternatively be (or encode, in the case of the nucleic acid) any amino acid residue incapable of acting as a glycosylation signal.

The Pvs28 and Pvs25 proteins of the invention may be purified from parasites isolated from infected host organisms. For a review of standard techniques see, e.g., Methods in Enzymology, "Guide to Protein Purification", M. Deutscher, ed. Vol. 182 (1990); Scopes, R. K., Protein Purification: Principles and Practice, 2nd ed., Springer Verlag, (1987). For instance, Pvs25 and Pvs28 polypeptides can be purified using affinity chromatography, SDS-PAGE, and the like. Illustrative examples of methods for purifying Pvs25, Pvs28 and fusion proteins thereof of the invention are described below. Methods for purifying desired proteins are well known in the art and are not presented in detail here.

Solubility Fractionation

If the protein mixture is complex, an initial salt fractionation can separate many of the unwanted host cell proteins (or proteins derived from the cell culture media) from the recombinant protein of interest. The preferred salt is ammonium sulfate. Ammonium sulfate precipitates proteins by effectively reducing the amount of water in the protein mixture. Proteins then precipitate on the basis of their solubility. The more hydrophobic a protein is, the more likely it is to precipitate at lower ammonium sulfate concentrations. A typical protocol is to add saturated ammonium sulfate to a protein solution so that the resultant ammonium sulfate concentration is between 20-30%. This will precipitate the most hydrophobic of proteins. The precipitate is discarded (unless the protein of interest is hydrophobic) and ammonium sulfate is added to the supernatant to a concentration known to precipitate the protein of interest. The precipitate is then solubilized in buffer and the excess salt removed if necessary, either through dialysis or diafiltration. Other methods that rely on solubility of proteins, such as cold ethanol precipitation, are well known to those of skill in the art and can be used to fractionate complex protein mixtures.

Size Differential Filtration

If the size of the protein of interest is known or can be estimated from the cDNA sequence, proteins of greater and lesser size can be removed by ultrafiltration through membranes of different pore size (for example, Amicon or Millipore membranes). As a first step, the protein mixture is ultrafiltered through a membrane with a pore size that has a lower molecular weight cut-off than the molecular weight of the protein of interest. The retentate of the ultrafiltration is then ultrafiltered against a membrane with a molecular cut off greater than the molecular weight of the protein of interest. The recombinant protein will pass through the membrane into the filtrate. The filtrate can then be chromatographed.

Column Chromatography

Proteins can be separated on the basis of their size, net surface charge, hydrophobicity and affinity for ligands. In addition, antibodies raised against proteins can be conjugated to column matrices and the proteins immunopurified. All of these general methods are well known in the art. See Scopes (1987) supra. Chromatographic techniques can be performed at any scale and using equipment from many different manufacturers (e.g., Pharmacia Biotech). Protein concentrations can be determined using any technique, e.g., as in Bradford (1976) Anal. Biochem. 72:248-257.

Amino Acid Sequence Determination

Illustrative amino acid sequences of the Pvs28 and Pvs25 and fusion proteins of this invention can be determined by, for example, Edman degradation, a technique which is well known in the art. In addition to the internal sequencing (see also Hwang (1996) J. Chromatogr. B. Biomed. Appl. 686:165-175), N-terminal sequencing can be performed by techniques known in the art. For C-terminal sequence determination, a chemical procedure for the degradation of peptides and analysis by matrix-assisted-laser-desorption ionization mass spectrometry (MALDI-MS) can be used Thiede (1997) Eur. J. Biochem. 244:750-754.

Molecular Weight/Isoelectric Point Determination

The molecular weight of a protein can be determined by many different methods, all known to one of skill in the art. Some methods of determination include: SDS gel electrophoresis, native gel electrophoresis, molecular exclusion chromatography, zonal centrifugation, mass spectroscopy, and calculation from sequencing. Disparity between results of different techniques can be due to factors inherent in the technique. For example, native gel electrophoresis, molecular exclusion chromatography and zonal centrifugation depend on the size of the protein. The proteins that are cysteine rich can form many disulfide bonds, both intra- and intermolecular. SDS gel electrophoresis depends on the binding of SDS to amino acids present in the protein. Some amino acids bind SDS more tightly than others, therefore, proteins will migrate differently depending on their amino acid composition. Mass spectroscopy and calculated molecular weight from the sequence in part depend upon the frequency that particular amino acids are present in the protein and the molecular weight of the particular amino acid. If a protein is glycosylated, mass spectroscopy results will reflect the glycosylation but a calculated molecular weight may not.

The isoelectric point of a protein can be determined by native gel (or disc) electrophoresis, isoelectric focussing or in a preferred method, by calculation given the amino acid content of the protein (see, for example, Wehr (1996) Methods Enzymol. 270:358-374; Moorhouse (1995) J. Chromatogr. A. 717:61-69, describing capillary isoelectric focusing).

Pvs25-Pvs28 Fusion Proteins

The present invention includes immunogenic polypeptides which comprise polypeptide subsequences derived from both Pvs28 and Pvs25, including the exemplary fusion protein of the invention SEQ ID NO:5 (see FIG. 5 (see Original Patent)) and deglycosylated forms. These polypeptides are useful for inducing an immune response when the fusion protein is injected into a human, mouse or other host animal. The antibodies that arise from the immune response block transmission of the malarial parasite by interfering with the portion of the parasite's life cycle that occurs in the mosquito.

The fusion proteins typically include an immunogenic domain, or epitope, from a Pvs25 and an immunogenic domain, or epitope from a Pvs28 (including deglycosylated forms). The immunogenic domains, or epitopes, are peptide and polypeptide subsequences of the corresponding polypeptides which are sufficient to elicit an immunogenic response (antibody or T cell response) against the domain when administered to a mammal (e.g., a mouse or a human). In one embodiment, the immunogenic domain can elicit the production of an antibody which recognizes the corresponding full length protein. For example, if the immunogenic domain is a Pvs25 subsequence, the domain (epitope) elicits the production of an antibody which specifically binds to Pvs25. Similarly, if the immunogenic domain is a Pvs28 subsequence, the domain preferably elicits the production of an antibody which specifically binds to Pvs28.

To elicit the production of an antibody, the immunogenic domain is typically at least about 3-10 amino acids in length, because the protein recognition site on an antibody typically recognizes an amino acid of about 3-10 amino acids in length. More often, the immunogenic domain is longer than 10 amino acids, and the domain optionally includes the full length sequence of the corresponding protein (i.e., in one embodiment, the Pvs25-Pvs28 fusion protein comprises the complete sequence of both Pvs25 and Pvs28). Ordinarily, only a fraction of the full length protein is included. In one embodiment, about 10% of the full length Pvs25 is included in the fusion protein. In another embodiment, about 20% of the full length Pvs25 is included in the fusion protein. In yet another embodiment, about 30% of the full length protein is included. In still another embodiment, about 40% of the full length Pvs25 is included in the fusion protein. Optionally, as much as about 50% of the full length Pvs25 is included in the fusion protein. Occasionally, as much as about 60% of the full length Pvs25 is included in the fusion protein. In some embodiments, as much as about 70% of the full length Pvs25 is included in the fusion protein. In one class of embodiments, as much as about 80% of the full length Pvs25 is included in the fusion protein. As much as about 90% of the full length Pvs25 is optionally included in the fusion protein. As already mentioned, the entire full length Pvs25 protein is optionally incorporated into the fusion protein.

Similarly, in one embodiment, about 10% of the full length Pvs28is included in the fusion protein. In another embodiment, about 20% of the full length Pvs28 is included in the fusion protein. In yet another embodiment, about 30% of the full length protein is included. In still another embodiment, about 40% of the full length Pvs28 is included in the fusion protein. Optionally, as much as about 50% of the full length Pvs28 is included in the fusion protein. Occasionally, as much as about 60% of the full length Pvs28 is included in the fusion protein. In some embodiments, as much as about 70% of the full length Pvs28 is included in the fusion protein. In one class of embodiments, as much as about 80% of the full length Pvs28 is included in the fusion protein. As much as about 90% of the full length Pvs28 is optionally included in the fusion protein. As already mentioned, the entire full length Pvs28 protein is optionally incorporated into the fusion protein.

The portion of the Pvs25 or Pvs28 protein from which the immunogenic domain, or epitope, is selected is optionally optimized for maximum immunogenicity for the induction of transmission blocking vaccines. Any combination of Pvs25 and Pvs28 subsequences (epitopes) can be combined. Any combination of complete or partially deglycosylated subsequences can be combined. In alternative embodiments, the Pvs25 and Pvs28 epitopes can be in alternating or sequential patterns. For example, in one embodiment, the carboxyl terminal portion of Pvs28 is included. Embodiments also include those derived from fusion proteins in which about 10-20 amino acids are deleted or added to the particular Pvs25 or Pvs28 subsequences described. The added or deleted amino acids are added or deleted by reference to the corresponding full length sequence, e.g., where the subsequence is derived from Pvs25, a 10-20 amino acid sequence derived from Pvs25 is optionally added to either end of the subsequence.

The fusion proteins optionally includes additional features such as a flexible linker between Pvs25 and Pvs28 domains. The linkers can facilitate the independent folding of the Pvs25 and Pvs28 proteins. Preferred flexible linkers are amino acid subsequences which are synthesized as part of a recombinant fusion protein. In one embodiment, the flexible linker is an amino acid subsequence comprising a proline such as Gly.sub.3-Pro-Gly.sub.3 (SEQ ID NO:15). In other embodiments, a chemical linker is used to connect synthetically or recombinantly produced Pvs25 and Pvs28 subsequences. Such flexible linkers are known to persons of skill in the art. For example, poly(ethylene glycol) linkers are available from Shearwater Polymers, Inc. Huntsville, Ala. These linkers optionally have amide linkages, sulfhydryl linkages, or heterofunctional linkages.

In addition to flexible linkers, the fusion proteins optionally include polypeptide subsequences from proteins which are unrelated to Pvs25 or Pvs28, e.g., a sequence with affinity to a known antibody to facilitate affinity purification, detection, or the like. Such detection- and purification-facilitating domains include, but are not limited to, metal chelating peptides such as polyhistidine tracts and histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp, Seattle Wash.). The inclusion of a cleavable linker sequences such as Factor Xa or enterokinase (Invitrogen, San Diego Calif.) between the purification domain and Pvs25 or Pvs28 protein(s) may be useful to facilitate purification. One such expression vector provides for expression of a fusion protein comprising the sequence encoding a Pvs25 or Pvs28 of the invention, or a fusion protein thereof, and nucleic acid sequence encoding six histidine residues followed by thioredoxin and an enterokinase cleavage site (for example, see Williams (1995) Biochemistry 34:1787-1797). The histidine residues facilitate detection and purification while the enterokinase cleavage site provides a means for purifying the desired protein(s) from the remainder of the fusion protein. Technology pertaining to vectors encoding fusion proteins and application of fusion proteins are well described in the patent and scientific literature, see e.g., Kroll (1993) DNA Cell. Biol., 12:441-53).

An exemplary fusion of Pvs25 to Pvs28 by a flexible linker is represented by the polypeptide of FIG. 5, SEQ ID NO:5, whose individual domains are:

a Pvs25 sequence (with or without a signal sequence or anchor) -- see Original Patent.

with a flexible linker, e.g.: GGGPGGG (SEQ ID NO:15); and

a Pvs28 sequence (with or without signal sequence or anchor) -- see Original Patent.

The fusion protein (and a Pvs25 or Pvs28 polypeptide) can also include a secretory signal sequence, e.g., in mammalian cell expression: Ig secretion signal or tPA signal sequence; or a pre-pro secretion signal, e.g., in yeast: alpha-factor.

Included among the polypeptides of the present invention are fusion proteins that have subsequences which are homologues or allelic variants of Pvs28 or Pvs25. Such homologues, also referred to as Pvs28 or Pvs25 polypeptides, respectively, include variants of the native proteins constructed by in vitro techniques, and proteins from parasites related to P. vivax and P. falciparum. For example, one skilled in the art will appreciate that for certain uses it is advantageous to produce a Pvs28 or Pvs25 polypeptide subsequence that is lacking a structural characteristic; e.g., one may remove a transmembrane domain (to obtain a polypeptide that is more soluble in aqueous solution) or a glycosylation site (to obtain a polpeptide that is more antigenic under certain conditions).

One of skill will appreciate that many conservative variations of the fusion proteins and nucleic acid which encode the fusion proteins yield essentially identical products. For example, due to the degeneracy of the genetic code, "silent substitutions" (i.e., substitutions of a nucleic acid sequence which do not result in an alteration in an encoded polypeptide) are an implied feature of every nucleic acid sequence which encodes an amino acid. As described herein, sequences are preferably optimized for expression in a particular host cell used to produce the fusion protein (e.g., yeast). Similarly, "conservative amino acid substitutions," in one or a few amino acids in an amino acid sequence are substituted with different amino acids with highly similar properties (see, the definitions section, supra), are also readily identified as being highly similar to a particular amino acid sequence, or to a particular nucleic acid sequence which encodes an amino acid. Such conservatively substituted variations of any particular sequence are a feature of the present invention.

One of skill will recognize many ways of generating alterations in a given nucleic acid sequence, which optionally provides alterations to an encoded protein. Such well-known methods include site-directed mutagenesis, PCR amplification using degenerate oligonucleotides, exposure of cells containing the nucleic acid to mutagenic agents or radiation, chemical synthesis of a desired oligonucleotide (e.g., in conjunction with ligation and/or cloning to generate large nucleic acids) and other well-known techniques. See, Giliman and Smith (1979) Gene 8:81-97; Roberts et al. (1987) Nature 328:731-734 and Sambrook, Innis, Ausbel, Berger, Needham VanDevanter and Mullis (below).

Most commonly, amino acid sequences are altered by altering the corresponding nucleic acid sequence and expressing the polypeptide. However, polypeptide sequences are also optionally generated synthetically on commercially available peptide synthesizers to produce any desired polypeptide (see, Merrifield, and Stewart and Young, supra).

One can select a desired nucleic acid or polypeptide of the invention based upon the sequences and constructs provided and upon knowledge in the art regarding malaria generally. The life-cycle, genomic organization, developmental regulation and associated molecular biology of malaria strains have been the focus of research since the advent of molecular biology.

Moreover, general knowledge regarding the nature of proteins and nucleic acids allows one of skill to select appropriate sequences with activity similar or equivalent to the nucleic acids, vectors and polypeptides disclosed herein. The definitions section herein describes exemplar conservative amino acid substitutions.

Finally, most modifications to nucleic acids and polypeptides are evaluated by routine screening techniques in suitable assays for the desired characteristic. For instance, changes in the immunological character of a polypeptide can be detected by an appropriate immunological assay. Modifications of other properties such as nucleic acid hybridization to a target nucleic acid, redox or thermal stability of a protein, hydrophobicity, susceptibility to proteolysis, or the tendency to aggregate are all assayed according to standard techniques.

Pvs25 Pvs28 and Pvs25-Pvs28 Nucleic Acids

Another aspect of the present invention relates to the cloning and recombinant expression (using expression cassettes, plasmids, vectors, recombinant viruses, and the like) of Pvs 25 and Pv28 proteins, variants (i.e. deglycosylated forms) construction of Pvs25-Pvs28 fusion proteins, as described above. The recombinantly expressed proteins can be used in a number of ways. For instance, they can be used as transmission-blocking vaccines or as immunogens to raise antibodies, as described below. In addition, oligonucleotides from the cloned genes can be used as probes to identify homologous, allelic and variant species of Pvs polypeptides in Plasmodium vivax, Plasmodium sp., and in other species.

Thus, the invention relies on routine techniques in the field of recombinant genetics, well known to those of ordinary skill in the art and well described in the scientific and patent literature, e.g., basic texts disclosing the general methods of use in this invention include Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152 Academic Press, Inc., San Diego, Calif. (Berger); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Publish., Cold Spring Harbor, NY 2nd ed. (1989) (Sambrook); and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (1995 Supplement). Product information from manufacturers of biological reagents and experimental equipment also provide information useful in known biological methods. Such manufacturers include, e.g., the SIGMA chemical company (Saint Louis, MO), R&D systems (Minneapolis, Minn.), Pharmacia LKB Biotechnology (Piscataway, N.J.), CLONTECH Laboratories, Inc. (Palo Alto, Calif.), Chem Genes Corp., Aldrich Chemical Company (Milwaukee, Wis.), Glen Research, Inc., GIBCO BRL Life Technologies, Inc. (Gaithersberg, Md.), Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland), Invitrogen, San Diego, Calif., and Applied Biosystems (Foster City, Calif.), as well as many other commercial sources known to one of skill.

In summary, the manipulations necessary to prepare nucleic acid segments encoding the polypeptides and introduce them into appropriate host cells involve 1) purifying the polypeptide from the appropriate sources, 2) preparing degenerate oligonucleotide probes corresponding to a portion of the amino acid sequence of the purified proteins, 3) screening a cDNA or genomic library for the sequences which hybridize to the probes, 4) constructing vectors comprising the sequences linked to a promoter and other sequences necessary for expression and 5) inserting the vectors into suitable host cells or viruses.

After isolation of the desired protein as described above, the amino acid sequence of the N-terminus is determined and degenerate oligonucleotide probes, designed to hybridize to the desired gene, are synthesized. Amino acid sequencing is performed and oligonucleotide probes are synthesized according to standard techniques as described, e.g., in Sambrook or Ausubel.

Genomic or cDNA libraries are prepared according to standard techniques as described, e.g., in Sambrook or Ausubel. To construct genomic libraries, large segments of genomic DNA are generated by random fragmentation and are ligated with vector DNA to form concatemers that can be packaged into the appropriate vector. Two kinds of vectors are commonly used for this purpose, bacteriophage lambda vectors and plasmids.

To prepare cDNA, mRNA from the parasite of interest is first isolated. Eukaryotic mRNA has at its 3' end a string of adenine nucleotide residues known as the poly-A tail. Short chains of oligo d-T nucleotides are then hybridized with the poly-A tails and serve as a primer for the enzyme, reverse transcriptase. This enzyme uses RNA as a template to synthesize a complementary DNA (cDNA) strand. A second DNA strand is then synthesized using the first cDNA strand as a template. Linkers are added to the double-stranded cDNA for insertion into a plasmid or phage vector for propagation in E. coli.

cDNA can also be prepared using PCR (see below for further discussion PCR). PCR is used to produce high-quality cDNA from nanograms of total or poly A+ RNA. For example, the CapFinder.TM. PCR cDNA Synthesis Kit (Clonetech, Palo Alto, Calif.) was used to identify and isolate cDNA from Plasmodium. This technique utilizes long-distance PCR (Barnes (1994) Proc. Natl. Acad. Sci. USA 91:2216-2220, Cheng (1994) Proc. Natl. Acad. Sci. USA 91:5695-5699) to generate high yields of representative, double-stranded cDNA. See also, e.g., Zhu (July 1996) CLONTECHniques XI(3):12-13; CLONTECHniques (October 1995) X(4):2-5; and CLONTECHniques (January 1996) XI(1):2-4.

Identification of clones in either genomic or cDNA libraries harboring the desired nucleic acid segments is performed by either nucleic acid hybridization or immunological detection of the encoded protein, if an expression vector is used. The bacterial colonies are then replica plated on solid support, such as nitrocellulose filters. The cells are lysed and probed with either oligonucleotide probes described above or with antibodies to the desired protein.

Other methods well known to those skilled in the art are used to identify desired genes, i.e., various species of Pvs25 and Pvs28 of the invention. For example, the presence of restriction fragment length polymorphisms (RFLP) between wild type and mutant strains lacking a Pvs25 or Pvs28 polypeptide can be used.

Oligonucleotides can be used to identify and detect Pvs25 and Pvs28 using a variety of hybridization techniques and conditions. For example, amplification techniques, such as the polymerase chain reaction (PCR) can be used to amplify the desired nucleotide sequence. One of skill in the art will appreciate that, whatever amplification method is used, if a quantitative result is desired, care must be taken to use a method that maintains or controls for the relative frequencies of the amplified nucleic acids. Suitable amplification methods include, but are not limited to: polymerase chain reaction, PCR (PCR PROTOCOLS, A GUIDE TO METHODS AND APPLICATIONS, ed. Innis, Academic Press, N.Y. (1990) and PCR STRATEGIES (1995), ed. Innis, Academic Press, Inc., NY ("Innis "); and, U.S. Pat. Nos. 4,683,195 and 4,683,202 describe this method), ligase chain reaction (LCR) (Wu (1989) Genomics 4:560; Landegren (1988) Science 241:1077; Barringer (1990) Gene 89:117); transcription amplification (Kwoh Proc. Natl. Acad. Sci. USA, 86:1173 (1989)); and, self-sustained sequence replication (Guatelli (1990) Proc. Natl. Acad. Sci. USA, 87:1874); Q Beta replicase amplification (Smith (1997) J. Clin. Microbiol. 35:1477-1491, automated Q-beta replicase amplification assay; Burg (1996) Mol. Cell. Probes 10:257-271) and other RNA polymerase mediated techniques (e.g., NASBA, Cangene, Mississauga, Ontario); see also Berger (1987) Methods Enzymol. 152:307-316, Sambrook, and Ausubel, as well as Mullis (1987) U.S. Pat. Nos. 4,683,195 and 4,683,202; Arnheim (1990) C&EN 36-47; Lomell J. Clin. Chem., 35:1826 (1989); Van Brunt, Biotechnology, 8:291-294 (1990); Wu (1989) Gene 4:560; Sooknanan (1995) Biotechnology 13:563-564. Methods for cloning in vitro amplified nucleic acids are described in Wallace, U.S. Pat. No. 5,426,039.

The invention provides for amplification and manipulation or detection of the products from each of the above methods to prepare DNA encoding a Pvs25 or Pvs28 protein specie. In PCR techniques, oligonucleotide primers complementary to the two borders of the DNA region to be amplified are synthesized an used (see, e.g., Innis). PCR can be used in a variety of protocols to amplify, identify, isolate and manipulate nucleic acids encoding Pvs25 or Pvs 28. In these protocols, appropriate primers and probes for identifying and amplifying DNA encoding Pvs25 or Pvs 28 polypeptides and fragments thereof are generated that comprise all or a portion of any of the DNA sequences listed herein. PCR-amplified sequences can also be labeled and used as detectable oligonucleotide probes, but such nucleic acid probes can be generated using any synthetic or other technique well known in the art, as described above. The labeled amplified DNA or other oligonucleotide or nucleic acid of the invention can be used as probes to further identify and isolate Pvs25 or Pvs 28 protein isoforms or alleles or Pvs25 or Pvs 28 from various cDNA or genomic libraries.

The present invention also provides RACE-based methods for isolating Pvs25 or Pvs 28 nucleic acids from any organism (RACE is another PCR-based approach for DNA amplification). Briefly, this technique involves using PCR to amplify a DNA sequence using a random 5' primer and a defined 3' primer (5' RACE) or a random 3' primer and a defined 5' primer (3' RACE). The amplified sequence is then subcloned into a vector where can be sequenced and manipulated using standard techniques. The RACE method is well known to those of skill in the art and kits to perform RACE are commercially available, e.g. Gibco BRL, Gaithersburg, Md., #18374-058 (5' RACE) or #18373-019 (3' RACE), see also Lankiewicz (1997) Nucleic Acids Res 25:2037-2038; Frohman (1988) Proc. Natl. Acad. Sci. USA 85:8998; Doenecke (1997) Leukemia 11:1787-1792.

For 5' RACE, a primer, the gene-specific primer, is selected near the 5' end of the known sequence oriented to extend towards the 5' end. The primer is used in a primer extension reaction using a reverse transcriptase and mRNA. After the RNA is optionally removed, the specifically-primed cDNA is either: 1) "tailed" with deoxynucleotide triphosphates (dNTP) and dideoxyterminal transferase, then a primer that is complimentary to the tail with a 5' end that provides a unique PCR site and the first gene-specific primer is used to PCR amplify the cDNA. Subsequent amplifications are usually performed with a gene-specific primer nested with respect to the first primer, or 2) an oligonucleotide that provides a unique PCR site is ligated to an end of the cDNA using RNA ligase; then a primer complimentary to the added site and the first gene-specific primer is used to PCR amplify the cDNA, with subsequent amplifications usually performed with a gene-specific primer nested with respect to the first primer. Amplified products are then purified, usually by gel electrophoresis then sequenced and examined to see contain the additional cDNA sequences desired.

For 3' RACE, an oligo dT-primer is annealed to the poly-A tails of an mRNA and then extended by a reverse transcriptase. Usually the oligo dT primer has a 5' end that provides a unique PCR site. The RNA is then removed, optionally, or dissociated, and the cDNA is amplified with a primer to the oligo dT tail and a gene-specific primer near the 3' end of the known sequence (oriented towards the 3' end). Subsequent amplifications are usually performed with a gene-specific primer nested with respect to the first primer. Amplified products are then purified, usually by gel electrophoresis then sequenced and examined to see contain the additional cDNA sequences desired.

Sequences amplified by PCR can be purified from agarose gels and cloned into an appropriate vector according to standard techniques.

Standard transfection methods are used to produce prokaryotic, mammalian, yeast or insect cell lines which express large quantities of the Pvs25 or Pvs 28 polypeptide, which is then purified using standard techniques, as described above. See, e.g., Colley (1989) J. Biol. Chem. 264:17619-17622; and Scopes, supra.

The polypeptides of the present invention can be readily designed and manufactured utilizing various recombinant DNA or synthetic techniques well known to those skilled in the art. For example, the polypeptides can vary from the naturally-occurring sequence at the primary structure level by amino acid, insertions, substitutions, deletions, and the like. These modifications can be used in a number of combinations to produce the final modified protein chain.

The amino acid sequence variants can be prepared with various objectives in mind, including immunogenicity, facilitating purification, and preparation of the recombinant polypeptide. Design of completely or partially deglycosylated polypeptides improve the antigenicity of the immunogenic composition, as discussed above. Modified polypeptides can also be useful for modifying plasma half life, improving therapeutic efficacy, and lessening the severity or occurrence of side effects during therapeutic use. The amino acid sequence variants are usually predetermined variants not found in nature but exhibit the same, or improved (in the case of deglycosylation variants) immunogenic activity as naturally occurring, Pvs25 and Pvs28 polypeptides. For instance, polypeptide fragments comprising only a portion (usually at least about 60-80%, typically 90-95%) of the primary structure may be produced. For use as vaccines, polypeptide fragments are typically preferred so long as at least one epitope capable of eliciting transmission blocking antibodies remains. In the construction of deglycosylation variants, amino acid motifs which act as N-linked or O-linked glycosylation signals (which are well known in the art, see, e.g., Kakinuma (1997) J Biol Chem 272:28296-28300) are modified to forms (motif variants) that are not recognized as glycosylation sites in the expression systems in which the recombinant form is produced.

The nucleotide sequences used to express the polypeptides of the invention and to transfect the host cells can be modified according to standard techniques to yield Pvs25-Pvs28, Pvs25 or Pvs28 polypeptides, fusion proteins, variants or fragments thereof, with a variety of desired properties. For example, the invention also provides for Pvs25 and Pvs28 which have been modified in a site-specific manner to modify or delete any or all functions or epitopes. Site-specific mutations can be introduced into Pvs25 and Pvs28-encoding nucleic acid by a variety of conventional techniques, well described in the scientific and patent literature. For example, one rapid method to perform site-directed mutagenesis efficiently is the overlap extension polymerase chain reaction (OE-PCR) (Urban (1997) Nucleic Acids Res. 25 :2227-2228). Other illustrative examples include: site-directed mutagenesis by overlap extension polymerase chain reaction (OE-PCR), as in Urban (1997) Nucleic Acids Res. 25:2227-2228; Ke (1997) Nucleic Acids Res 25:3371-3372, and Chattopadhyay (1997) Biotechniques 22:1054-1056, describing PCR-based site-directed mutagenesis "megaprimer" method; Bohnsack (1997) Mol. Biotechnol. 7:181-188; Ailenberg (1997) Biotechniques 22:624-626, describing site-directed mutagenesis using a PCR-based staggered re-annealing method without restriction enzymes; Nicolas (1997) Biotechniques 22:430-434, site-directed mutagenesis using long primer-unique site elimination and exonuclease III. See Gillman (1979) Gene 8:81-97; Roberts (1987) Nature 328:731-734.

In general, modifications of the sequences encoding the homologous polypeptides may be readily accomplished by a variety of well-known techniques, such as site-directed mutagenesis, described above. One of ordinary skill will appreciate that the effect of many mutations is difficult to predict. Thus, most modifications are evaluated by routine screening in a suitable assay for the desired characteristic. For instance, the effect of various modifications on the ability of the polypeptide to elicit transmission blocking can be easily determined using the mosquito feeding assays, described below. In addition, changes in the immunological character of the polypeptide can be detected by an appropriate competitive binding assay. Modifications of other properties such as redox or thermal stability, hydrophobicity, susceptibility to proteolysis, or the tendency to aggregate are all assayed according to standard techniques.

The particular procedure used to introduce the genetic material into the host cell for expression of the Pvs 25 and Pvs28 polypeptide is not particularly critical. Any of the well known procedures for introducing foreign nucleotide sequences into host cells may be used. These include the use of calcium phosphate transfection, spheroplasts, electroporation, liposomes, microinjection, plasma vectors, viral vectors and any of the other well known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell (see, e.g., Sambrook, Ausubel, supra). It is only necessary that the particular procedure utilized be capable of successfully introducing at least one gene into the host cell which is capable of expressing the gene.

The particular vector used to transport the genetic information into the cell is also not particularly critical. Any of the conventional vectors used for expression of recombinant proteins in prokaryotic and eukaryotic cells may be used.

Expression vectors for mammalian cells typically contain regulatory elements from eukaryotic viruses. SV40 vectors include pSVT7 and pMT2. Vectors derived from bovine papilloma virus include pBV-1MTHA, and vectors derived from Epstein Bar virus include pHEBO, and p2O5.

Other exemplary vectors include pMSG, pAV009/A.sup.+, pMTO10/A.sup.+, pMAMneo-5, bacculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV-40 early promoter, SV-40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.

The expression vector typically contains a transcription unit or expression cassette that contains all the elements required for the expression of the Pvs28 or Pvs25 polypeptide DNA in the host cells. A typical expression cassette contains a promoter operably linked to the DNA sequence encoding a Pvs28 or Pvs25 polypeptide and signals required for efficient polyadenylation of the transcript. The term "operably linked" as used herein refers to linkage of a promoter upstream from a DNA sequence such that the promoter mediates transcription of the DNA sequence. The promoter is preferably positioned about the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting. As is known in the art, however, some variation in this distance can be accommodated without loss of promoter function.

The DNA sequence encoding the Pvs28 or Pvs25 polypeptide will typically be linked to a cleavable signal peptide sequence to promote secretion of the encoded protein by the transformed cell. Additional elements of the cassette may include selectable markers, enhancers and, if genomic DNA is used as the structural gene, introns with functional splice donor and acceptor sites.

Enhancer elements can stimulate transcription up to 1,000 fold from linked homologous or heterologous promoters. Enhancers are active when placed downstream from the transcription initiation site. Many enhancer elements derived from viruses have a broad host range and are active in a variety of tissues. For example, the SV40 early gene enhancer is suitable for many cell types. Other enhancer/promoter combinations that are suitable for the present invention include those derived from polyoma virus, human or murine cytomegalovirus, the long term repeat from various retroviruses such as murine leukemia virus, murine or Rous sarcoma virus and HIV. See, Enhancers and Eukaryotic Expression, Cold Spring Harbor Pres, Cold Spring Harbor, N.Y. 1983.

In addition to a promoter sequence, the expression cassette should also contain a transcription termination region downstream of the structural gene to provide for efficient termination. The termination region may be obtained from the same gene as the promoter sequence or may be obtained from different genes.

If the mRNA encoded by the structural gene is to be efficiently translated, polyadenylation sequences are also commonly added to the vector construct. Two distinct sequence elements are required for accurate and efficient polyadenylation: GU or U rich sequences located downstream from the polyadenylation site and a highly conserved sequence of six nucleotides, AAUAAA, located 11-30 nucleotides upstream. Termination and polyadenylation signals that are suitable for the present invention include those derived from SV40, or a partial genomic copy of a gene already resident on the expression vector.

Pvs25 or Pvs28 coding sequences can be inserted into a host cell genome becoming an integral part of the host chromosomal DNA, using for example, retroviral vectors such as SIV or HIV, see for example, Naldini (1996) Science 272:263-267; Vanin (1997) J. Virol. 71:7820-7826; Zufferey (1997) Nat. Biotechnol. 15:871-875, describing attenuated lentiviral vector gene delivery in vivo; Feng (1997) Nat. Biotechnol. 15:866-870, describing stable in vivo gene transduction via adenoviral/retroviral chimeric vector.

Nucleic acids of the invention can used in DNA immunization techniques. Coding sequence is operably linked to expression cassettes or vectors and injected directly as "naked" DNA into the host. The DNA can be injected intramuscularly or intradermally. See. e.g., Donnelly (1995) Ann. NY Acad. Sci. 772:40-46; Corr (1997) J. Immunol. 159:4999-5004; Manickan (1997) J. Clin. Invest. 100:2371-2375. Variations of this technique use cationic liposome-entrapped DNA vaccines (see Gregoriadis (1997) FEBS Lett. 402:107-110); immunization with naked plasmid DNA transfected in dendritic cells (Manickan (1997) J. Leukoc. Biol. 61:125-132); and, cutaneous genetic immunization with naked DNA (Condon (1996) Nat. Med. 2:1122-1128).

Yeast expression systems, being eukaryotic, provide an attractive alternative to bacterial systems for some applications; for an overview of yeast expression systems, see. e.g., Protein Engineering Principles and Practice, eds. Cleland et al., Wiley-Liss, Inc. p 129 (1996), Barr (1988) J. Biol. Chem. 263: 16471-16478, or U.S. Pat. No. 4,546,082. A variety of yeast vectors are publicly available. For example, the expression vector pPICZ B (Invitrogen, San Diego, Calif.) has been modified to create expression vectors of the invention to express the Pvs25 or Pvs28 of the invention in yeast, such as S. cerevisiase and Pichia pastoris. Yeast episomal plasmids comprising inducible promoters can be used for the intracellular expression of the Pvs25 or Pvs28 proteins of the invention. Vectors include the pYES2 expression vector (Invitrogen, San Diego, Calif.) and pBS24.1 (Boeke (1984) Mol. Gen. Genet. 197:345); see also Jacobs (1988) Gene 67:259-269.

One embodiment uses the yeast expression vector comprising the Recombinant Protein Expression Unit called YEpRPEU-1, -2 and -3; and pIXY154 (Immunex Corp.). pIXY154 and YEpRPEU-3 have been used to express Pvs25, Pvs28 and Pvs28-Q130, amutagenized form of Pvs28 which eliminates all, several, or, one potential N-linked glycosylation site, as discussed herein.

Yeast promoters for yeast expression vectors suitable for the expression of a Pvs25 or Pvs28 include the inducible promoter from the alcohol dehydrogenase gene, ADH2, also called the yeast alcohol dehydrogenase II gene promoter (ADH2P) (La Grange (1997) Appl. Microbiol. Biotechnol. 47:262-266). In one embodiment, the ADH2 promoter is modified to include a tract of poly A to enhance the ADH2 promoter in the expression of the polypeptides of the invention. Suitable promoters to use also include the ADH2/GAPDH hybrid promoter as described, e.g., in Cousens (1987) Gene 61:265-275.

In another embodiment, the Pvs25 or Pvs28 to be expressed can also be fused at the amino terminal end to the secretion signal sequence of the yeast mating pheromone alpha-factor (MF alpha 1S) and fused at the carboxy terminal end to the alcohol dehydrogenase II gene terminator (ADH2T), see van Rensburg (1997) J. Biotechnol. 55:43-53. The yeast alpha mating pheromone signal sequence allows for secretion of the expressed Pvs25 or Pvs28. In one embodiment, sequences are added after the KEX-2 cleavage site to enhance cleavage of the alpha factor leader; preferred embodiments include addition of the sequence EAEA (SEQ ID NO:22) and EAEAEAEAK (SEQ ID NO:23).

Yeast cell lines suitable for the present invention include e.g., BJ 2168 (Berkeley Yeast Stock Center) as well as other commonly available lines. For example, the yeast can be a Pichia sp., Hansenula sp., Torulopsis sp., Saccharomyces sp., or a Candida sp. The yeast can specifically be a Pichia pastoris, Hansenula polymorpha, Torulopsis holmil, Saccharomyces fragilis, Saccharomyces cerevisiae, Saccharomyces lactis, or a Candida pseudotropicalis. In other embodiments, Saccharomyces cerevisiae cell lines XV2181 from Immunex; and, 2905/6, VQ1 and VK1 which we have developed as our own yeast expression hosts.

Any of a number of other well known cells and cell lines can be used to express the polypeptides of the invention. For instance, prokaryotic cells such as E. coli can be used. Eukaryotic cells include, Chinese hamster ovary (CHO) cells, COS cells, mouse L cells, mouse A9 cells, baby hamster kidney cells, C127 cells, PC8 cells, and insect cells.

Following the growth of the recombinant cells and expression of the Pvs25 or Pvs28 polypeptide, the culture medium is harvested for purification of the secreted protein. The media are typically clarified by centrifugation or filtration to remove cells and cell debris and the proteins are concentrated by adsorption to any suitable resin such as, for example, CDP-Sepharose, Asialoprothrombin-Sepharose 4B, or Q Sepharose, or by use of ammonium sulfate fractionation, polyethylene glycol precipitation, or by ultrafiltration. Other routine means known in the art may be equally suitable. Further purification of the Pvs25 or Pvs28 or fusion polypeptide can be accomplished by standard techniques, for example, affinity chromatography, metal affinity chromatography (IMAC) (see, e.g., Govoroun (1997) J. Chromatogr. B. Biomed. Sci. Appl. 698:35-46; Froelich (1996) Biochem. Biophys. Res. Commun. 229:44-49), ion exchange chromatography, sizing chromatography or other protein purification techniques to obtain homogeneity, as described above. The purified proteins are then used to produce pharmaceutical compositions, as described below.

Transmission-Blocking Antibodies

A further aspect of the invention includes antibodies against Pvs25 or Pvs28 polypeptides. The antibodies are useful for diagnostic purposes or for blocking transmission of parasites. The antibodies of the invention may be polyclonal or monoclonal. Typically, polyclonal sera are preferred.

Antibodies are typically tetramers of immunoglobulin polypeptides. As used herein, the term "antibody" refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes. Immunoglobulin genes include those coding for the light chains, which may be of the kappa or lambda types, and those coding for the heavy chains. Heavy chain types are alpha, gamma, delta, epsilon and mu. The carboxy terminal portions of immunoglobulin heavy and light chains are constant regions, while the amino terminal portions are encoded by the myriad immunoglobulin variable region genes. The variable regions of an immunoglobulin are the portions that provide antigen recognition specificity. The immunoglobulins may exist in a variety of forms including, for example, Fv, Fab, and F(ab).sub.2, as well as in single chains, e.g., Huston et al., Proc. Natl. Acad. Sci. USA, 85:5879-5883 (1988) and Bird et al., Science 242: 423-426, 1988. See, generally, Hood et al., Immunology, Benjamin, N.Y., 2nd ed. (1984), and Hunkapiller (1986) Nature, 323:15-16. Single-chain antibodies, in which genes for a heavy chain and a light chain are combined into a single coding sequence, may also be used.

Use of the Polypeptides or Nucleic Acids of the Invention to Induce Immune Responses.

The immunoglobulins, nucleic acids, and polypeptides of the present invention are also useful as prophylactics, or vaccines, for blocking transmission of malaria or other diseases caused by parasites. Compositions containing the immunoglobulins, polypeptides, nucleic acids or a cocktail thereof are administered to a subject, giving rise to an anti-Pvs25 or anti-Pvs28 polypeptide immune response in the mammal entailing the production of anti-Pvs25 or anti-Pvs28 polypeptide immunoglobulins. The Pvs25 or Pvs28 polypeptide-specific immunoglobulins then block transmission of the parasite from the subject to the arthropod vector, preventing the parasite from completing its life cycle. An amount of prophylactic composition sufficient to result in a titer of antiserum which, upon ingestion by the mosquito, is capable of blocking transmission or is capable of decreasing ability of the oocyte to mature in the mosquito (resulting in fewer infective particles passed to the mosquitoes' next target bloodmeal), is defined to be an "immunologically effective dose."

The isolated nucleic acid sequences coding for Pvs25 or Pvs28 polypeptides can be used in viruses to transfect host cells in the susceptible organism, particularly, a human. Live attenuated viruses, such as vaccinia or adenovirus, are convenient alternatives for vaccines because they are inexpensive to produce and are easily transported and administered. Vaccinia vectors and methods useful in immunization protocols are well known in the art and are described, e.g., in U.S. Pat. No. 4,722,848.

Suitable viruses for use in the present invention include, but are not limited to, pox viruses, such as, canarypox and cowpox viruses, and vaccinia viruses, alpha viruses, adenoviruses, and other animal viruses. The recombinant viruses can be produced by methods well known in the art: for example, using homologous recombination or ligating two plasmids together. A recombinant canarypox or cowpox virus can be made, for example, by inserting the gene encoding the Pvs25 or Pvs28, or other homologous polypeptide into a plasmid so that it is flanked with viral sequences on both sides. The gene is then inserted into the virus genome through homologous recombination.

A recombinant adenovirus virus can be produced, for example, by ligating two plasmids each containing 50% of the viral sequence and the DNA sequence encoding the Pvs25 or Pvs28 polypeptide. Recombinant RNA viruses such as the alpha virus can be made via a cDNA intermediate using methods known in the art.

The recombinant virus of the present invention can be used to induce anti-Pvs25 or anti-Pvs28 polypeptide antibodies in mammals, such as mice or humans. In addition, the recombinant virus can be used to produce the Pvs25 or Pvs28 polypeptides by infecting host cells which in turn express the polypeptide.

The nucleic acids can also be used to produce other recombinant microorganisms such as bacteria, yeast, and the like. For instance, BCG (Bacille Calmette Guerin) vectors are described, e.g., in Stover (1991) Nature 351:456-460. A wide variety of other vectors useful for therapeutic administration or immunization of the peptides of the invention, e.g., Salmonella typhi, Saccharomyces vectors and the like, will be apparent to those skilled in the art from the description herein.

The DNA encoding the polypeptides of the invention can also be administered to the patient. Typically, an expression cassette suitable for driving expression in human cells is prepared. This approach is described, for instance, in Wolff (1990) Science 247:1465-1468; U.S. Pat. Nos. 5,580,859 and 5,589,466.

The present invention also relates to host cells infected with the recombinant virus of the present invention. The host cells of the present invention are preferably eukaryotic, such as yeast cells, or mammalian, such as BSC-1 cells. Host cells infected with the recombinant virus express the Pvs25 or Pvs28 polypeptides on their cell surfaces. In addition, membrane extracts of the infected cells induce transmission blocking antibodies when used to inoculate or boost previously inoculated mammals.

In the case of vaccinia virus (e.g., strain WR), the sequence encoding the Pvs25 or Pvs28 polypeptides can be inserted into the viral genome by a number of methods including homologous recombination using a transfer vector, pTKgpt-OFIS as described in Kaslow et al., Science 252:1310-1313, 1991.

The Pvs25 or Pvs28 polypeptides or nucleic acids of the present invention can be used in pharmaceutical and vaccine compositions that are useful for administration to mammals, particularly humans, to block transmission of a variety of infectious diseases. The compositions are suitable for single administrations or a series of administrations. When given as a series, inoculations subsequent to the initial administration are given to boost the immune response and are typically referred to as booster inoculations.

The pharmaceutical compositions of the invention are intended for parenteral, topical, oral or local administration. Preferably, the pharmaceutical compositions are administered parenterally, e.g., intravenously, subcutaneously, intradermally, or intramuscularly. Thus, the invention provides compositions for parenteral administration that comprise a solution of the agents described above dissolved or suspended in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers may be used, e.g., phosphate buffered saline, water, buffered water, 0.4% saline, 0.3% glycine, hyaluronic acid and the like. These compositions may be sterilized by conventional, well known sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.

For solid compositions, conventional nontoxic solid carriers may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. For oral administration, a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95% of active ingredient and more preferably at a concentration of 25%-75%.

For aerosol administration, the polypeptides or nucleic acids are preferably supplied in finely divided form along with a surfactant and propellant. The surfactant must, of course, be nontoxic, and preferably soluble in the propellant. Representative of such agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixed or natural glycerides may be employed. A carrier can also be included, as desired, as with, e.g., lecithin for intranasal delivery.

In therapeutic applications, Pvs25 or Pvs28 polypeptides or nucleic acids of the invention are administered to a patient in an amount sufficient to prevent parasite development in the arthropod and thus block transmission of the disease. An amount adequate to accomplish this is defined as a "therapeutically effective dose." Amounts effective for this use will depend on, e.g., the particular polypeptide or virus, the manner of administration, the weight and general state of health of the patient, and the judgment of the prescribing physician.

The vaccines of the invention contain as an active ingredient an immunogenically effective amount of the Pvs25 or Pvs28 polypeptides, nucleic acids, or recombinant virus as described herein. Useful carriers are well known in the art, and include, e.g., thyroglobulin, albumins such as human serum albumin, tetanus toxoid, polyamino acids such as poly(D-lysine:D-glutamic acid), influenza, hepatitis B virus core protein, hepatitis B virus recombinant vaccine and the like. The vaccines can also contain a physiologically tolerable (acceptable) diluent such as water, phosphate buffered saline, or saline, and further typically include an adjuvant. Adjuvants such as incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide, or alum are materials well known in the art.

Vaccine compositions containing the polypeptides or nucleic acids of the invention are administered to a patient to elicit a transmission-blocking immune response against the antigen and thus prevent spread of the disease through the arthropod vector. Such an amount is defined as an "immunogenically effective dose." In this use, the precise amounts again depend on the patient's state of health and weight, the mode of administration, and the nature of the formulation.

As noted above, the Pvs25 or Pvs28 polypeptides of this invention may also be used to make monoclonal antibodies. Such antibodies may be useful as potential diagnostic or therapeutic agents. The polypeptides themselves may also find use as diagnostic reagents. For example, a polypeptide of the invention may be used to diagnose the presence of antibodies against P. vivax in a patient. Alternatively, the polypeptides can be used to determine the susceptibility of a particular individual to a particular treatment regimen, and thus may be helpful in modifying an existing treatment protocol or in determining a prognosis for an infected individual.
 

Claim 1 of 17 Claims

1. A composition comprising an isolated Pvs25 polypeptide having at least 95% amino acid sequence identity to SEQ ID NO:4, wherein the Pvs25 polypeptide induces production of antibodies in a susceptible mammal against a 25 kD protein on the surface of Plasmodium vivax zygotes and ookinetes, wherein the antibodies against the 25 kD protein block the transmission of P. vivax from a mosquito.

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