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Title:  Recombinant multivalent malarial vaccine against Plasmodium falciparum

United States Patent:  6,828,416

Issued:  December 7, 2004

Inventors:  Lal; Altaf A. (Atlanta, GA); Shi; Ya Ping (Atlanta, GA); Hasnain; Seyed E. (New Delhi, IN)

Assignee:  The United States of America as represented by the Department of Health and (Washington, DC)

Appl. No.:  763397

Filed:  February 16, 2001

PCT Filed:  August 19, 1999

PCT NO:  PCT/US99/18869

371 Date:  February 16, 2001

102(e) Date:  February 16, 2001

PCT PUB.NO.:  WO00/11179

PCT PUB. Date:  March 2, 2000

Abstract

A recombinant protein is provided which comprises peptides derived from different stages in the life cycle of parasite Plasmodium falciparum. The protein is useful as a reagent and, when combined with a pharmaceutically-acceptable vehicle or carrier, is useful as a vaccine against the material parasite Plasmodium falciparum. A genetic construct used to produce this recombinant protein vaccine is also described. In addition, antibodies to this recombinant protein are provided which are useful for the detection and measurement of peptides derived from different stages in the life cycle of the parasite Plasmodium falciparum.

Description of the Invention

FIELD OF THE INVENTION

The present invention relates generally to the development and use of a gene encoding a recombinant protein useful as a multivalent and multistage malaria vaccine and more specifically relates to a recombinant antigenic protein useful for preventing or treating P. falciparum malarial infections.

BACKGROUND OF THE INVENTION

Malaria is a parasitic infection known to be produced by the Plasmodium species P. falciparum, P. vivax, P. ovale, and P. malariae. Humans become infected following the bite of an infected anopheline mosquito, the host of the malarial parasite. Malaria occasionally occurs in humans following a blood transfusion or subsequent to needle-sharing practices as used by drug addicts.

When an infected anopheline mosquito bites an individual, sporozoites present in the mosquito's saliva are injected into the blood. The initial development of parasites occurs in the liver and is referred to as the liver stage, or the hepatic or exoerythrocytic phase. In this phase, the sporozoite grows and divides, producing numerous tissue merozoites. These merozoites rupture the hepatocyte and enter the circulation. Some merozoites attach to receptor sites on red blood cells, penetrate the plasmalemma and begin a development phase known as the asexual, erythrocytic cycle. Within the erythrocyte, the parasite is recognizable as a ring-stage trophozoite. These trophozoites enlarge, divide and attain the schizont stage. After successive nuclear divisions, the erythrocyte ruptures, releasing merozoites which attach to receptors on erythrocytes and thus begin another erythrocytic cycle. In P. vivax and P. ovale, hepatic parasites persist and may lead to a relapse of the disease months or years after the initial infection.

Some merozoites that enter red blood cells develop into male and female gametocytes. When a mosquito bites an individual possessing erythrocytic gametocytes and ingests them, the gametocytes are fertilized in the stomach of the mosquito and mature into sporozoites that migrate to the salivary glands. In this manner, the mosquito is capable of biting and infecting another individual.

Malaria is one of the most common infections of humans. It is estimated that malaria parasites cause about 300-500 million illnesses and 3 million deaths each year. Most of the severe morbidity and mortality occurs in children and pregnant women, and is caused by P. falciparum (World Health Organization (1989) Weekly Epidemiol. Res. 32, 241-247). While sub-Saharan Africa accounts for more that 90% of these cases, malaria is a serious public health problem for nonimmune individuals and servicemen and servicewomen traveling through and/or stationed in malarious regions of the world. Clinical manifestations of malarial infection which may occur include blackwater fever, cerebral malaria, respiratory failure, hepatic necrosis, and occlusion of myocardial capillaries. An effective vaccine that prevents or reduces infection and minimizes morbidity and mortality will be a very useful tool for the control and prevention of this disease.

The development of an effective malaria vaccine represents one of the most promising approaches for providing cost-effective intervention along with other control measures currently available. Over the last decade there has been considerable progress in the understanding of immune mechanisms involved in protection against parasites and clinical illness. Several malarial antigens have been identified for their ability to confer protection against malaria.

Three main types of malarial vaccines are currently under research and development, based on stages of the parasite's life cycle. The three vaccines are generally directed to the following stages in the life cycle: 1) blood stage, including the asexual blood stage; 2) the sexual stages; and 3) preerythrocytic stages, including the liver stage. Antigens from each of these stages have been identified, the most promising being antigens from the following proteins: circumsporozoite protein (CSP) and SSP-2 protein of the sporozoite stage; the antigen (LSA-1) of the liver stage; the merozoite surface protein-1 (MSP-1), merozoite surface protein-2 (MSP-2), the rhoptry associated protein-1 and -2 (RAP-1 and RAP-2), the erythrocyte binding antigen-175 (EBA-175) and apical membrane antigen-1 (AMA-1) of the asexual blood stage; and the ookinete antigen Pfs 25 and the gamete specific antigen Pfg27 of the sexual stage.

Therefore, what is needed is a single vaccine that provides immunogenicity or confers immunity against various stages in the life cycle of the malarial parasite, particularly P. falciparum, to treat, minimize or prevent infection and reduce associated morbidity and mortality.

SUMMARY OF THE INVENTION

An antigenic recombinant protein, method of making the protein, genetic construct encoding the protein, antibodies to the protein, pharmaceutical composition containing the protein, and a method for the treatment, prevention or reduction of malarial infection by administering the protein to a human or animal are provided. The protein and anti-protein antibodies are useful as research or diagnostic reagents for the detection of the Plasmodium species P. falciparum in a biological sample. When administered to human or nonhuman animals, the protein is effective against malaria by conferring immunogenicity or immunity against various stages in the life cycle of the malarial parasite P. falciparum.

The antigenic recombinant protein is prepared by constructing a gene that encodes stage-specific antigenic determinants. The gene is added to a vector and is then expressed in a suitable expression system, such as a baculovirus system, to produce a single protein that confers immunity against different stages in the malarial life cycle of P. falciparum, or provides immunogenicity against epitopes from different stages in the life cycle of the parasite. In the present invention, these stages are the sporozoite stage, the liver stage, the blood stage and the sexual stage (also known as the gametocyte stage). By using a combination of antigens or epitopes derived from different stages in the life cycle of a malarial parasite, the protein constitutes an efficacious, cost-effective, and sustainable multicomponent vaccine for use in malaria control programs. The protein, in a pharmaceutically acceptable carrier, specifically provides a multivalent and multistage vaccine for malaria caused by the parasite P. falciparum.

The immunogenic regions of the various stage-specific antigens of P. falciparum used to construct the gene encoding the antigenic recombinant protein are selected based on immune response studies in clinically immune adults and in vitro immune response studies using peptides and/or antibody reagents. The resulting synthetic gene is sequence-confirmed and expressed in a baculovirus expression system. The preferred antigenic fragments used to make the coding sequences used in construction of the gene are shown in Table 1. The nucleotide sequence of the preferred gene is shown in SEQ ID NO:1. The amino acid sequence of the preferred recombinant protein encoded by the gene, referred to herein as CDC/NIIMALVAC-1, is shown in SEQ ID NO. 2. The recombinant protein in a pharmaceutically acceptable carrier is useful as a multivalent, multistage vaccine for P. falciparum malaria.

The vaccine described herein is a cost-effective, health-promoting intervention for controlling, preventing or treating the incidence of malaria. The vaccine is useful for reducing sickness, morbidity, mortality and the cost of medical care throughout the world. Similarly, the vaccine is useful for preventing or reducing malarial infection in U.S. citizens and military personnel traveling or living in regions of the world where malaria is present. The vaccine is also useful for decreasing the severity of the malarial disease process when administered after initial infection with P. falciparum.

The vaccine is immunogenic as confirmed by its ability to elicit immune responses against both the vaccine protein and the P. falciparum parasite. In vitro tests of protection conferred by the vaccine against blood stage malarial parasites reveal that antibodies against this vaccine inhibit reproductive growth of P. falciparum. The vaccine also induces multiple layers of immunity to different stages in the parasitic life cycle of P. falciparum.

It is therefore an object of the present invention to provide a multivalent, multistage vaccine against malaria.

Another object of the present invention is to provide a multivalent, multistage vaccine against malaria caused by P. falciparum.

Yet another object of the present invention is to provide a vaccine against malaria that is effective in inhibiting reproductive growth of the parasite within a human or animal after initial infection.

Still another object of the present invention is to provide a gene useful as a DNA vaccine, or for production of a recombinant protein in various expression systems, the recombinant protein containing antigenic epitopes to various stages of a malarial Plasmodium species, particularly P. falciparum.

Another object of the present invention is to provide a vector comprising a gene useful for production of a recombinant protein in various expression systems, the protein containing antigenic epitopes to various stages of a malarial Plasmodium species, particularly P. falciparum. This vector may be used for a variety of purposes including but not limited to administration to animals and humans, and for transfection of cells.

Yet another object of the present invention is to provide a recombinant protein containing antigenic epitopes to various stages of P. falciparum that may be used as a reagent or a multivalent, multistage antimalarial vaccine.

It is another object of the present invention to provide a method for conferring immunity against different stages in the life cycle of the malarial parasite, P. falciparum.

Another object of the present invention is to provide a method of vaccination against malaria caused by infection with P. falciparum.

It is another object of the present invention to provide a method to reduce morbidity and mortality associated with malarial infection by preventing malarial infection and also ameliorating the morbidity and mortality associated with malaria after initial infection with the parasite, P. falciparum.

Another object of the present invention is to provide antibodies against a recombinant protein containing antigenic epitopes to various stages of P. falciparum, that are useful as research or diagnostic reagents for the detection and measurement of P. falciparum in a biological sample.

Yet another object of the present invention is to provide a more effective, simpler and economical vaccine for conferring immunogenicity to different stages in the life cycle of P. falciparum than prior art vaccines.

An advantage of the anti-malaria vaccine of the present invention is that it confers immunogenicity against several stages or all stages in the life cycle of P. falciparum with administration of a single vaccine, as opposed to multiple injections for each stage of the life cycle of the parasite.

DETAILED DESCRIPTION OF THE INVENTION

An antigenic recombinant protein containing immunogenic malarial epitopes from different stages of the malarial parasite life cycle; a method of making the protein, including a genetic construct from which the protein is produced; antibodies to the protein; a pharmaceutical composition containing the protein, useful as a malarial vaccine; and a method for treating, preventing or reducing malarial infection by administering the composition to a human or animal are described herein. The genetic construct includes coding sequences for different peptide fragments obtained from different stages in the life cycle of a malarial parasite, preferably P. falciparum. The genetic construct also includes epitopes chosen to enhance recognition by cells of the immune system of the protein expressed from the genetic construct. A preferred genetic construct includes coding sequences for a signal peptide, for a polyhistidine sequence useful for purification of the protein, a universal T-helper epitope, and at least one epitope from each stage in the life cycle of P. falciparum. The preferred genetic construct has the nucleotide sequence of SEQ ID NO:1, or a nucleotide sequence having conservative nucleotide substitutions, as defined in the definitions, that do not significantly alter the function of the expressed recombinant protein in an adverse manner.

The genetic construct is expressed in an expression system, such as a baculovirus expression system, to produce a recombinant protein. The preferred protein is the protein referred to herein as CDC/NIIMALVAC-1, which has the amino acid sequence set forth in SEQ ID NO:2, or an amino acid sequence having amino acid substitutions as defined in the definitions that do not significantly alter the function of the recombinant protein in an adverse manner. The protein is combined with a pharmaceutical carrier and is used as a multivalent vaccine to confer immunity to the different stages in the life cycle of the malarial parasite, P. falciparum, when combined with a pharmaceutically acceptable carrier and administered in an effective amount to a human or animal. The present invention specifically provides a multivalent and multistage vaccine useful for preventing and treating malaria caused by P. falciparum. The present invention also provides polyclonal and monoclonal anti-protein antibodies produced after immunization with the recombinant protein which are useful, as is the protein, as research or diagnostic reagents in an assay for the detection or monitoring of malarial infection, particularly to detect malarial infection caused by P. falciparum. The antibodies are also useful for inducing passive immunization. Some of these results have been published by Shi et al., Proc. Natl. Acad. Sci. USA 96:1615-1620, the entirety of which is herein incorporated by reference.

Antigenic Peptide Production

When the antigenic epitope peptides are relatively short in length (i.e., less than about 50 amino acids), they are often synthesized using standard chemical peptide synthesis techniques. Solid phase synthesis, in which the C-terminal amino acid of the sequence is attached to an insoluble support followed by sequential addition of the remaining amino acids in the sequence, is a preferred method for the chemical synthesis of the antigenic epitopes described herein. Techniques for solid phase synthesis are known to those skilled in the art.

Alternatively, the antigenic epitopes described herein are synthesized using recombinant nucleic acid methodology. Generally, this involves creating a nucleic acid sequence that encodes the peptide or polypeptide, placing the nucleic acid in an expression cassette under the control of a particular promoter, expressing the peptide or polypeptide in a host, isolating the expressed peptide or polypeptide and, if required, renaturing the peptide or polypeptide. Techniques sufficient to guide one of skill through such procedures are found in the literature.

While the antigenic epitopes are often joined directly together, one of skill will appreciate that the antigenic epitopes may be separated by a spacer molecule such as, for example, a peptide, consisting of one or more amino acids. Generally, the spacer will have no specific biological activity other than to join the antigenic epitopes together, or to preserve some minimum distance or other spatial relationship between them. However, the constituent amino acids of the spacer may be selected to influence some property of the molecule such as the folding, net charge, or hydrophobicity.

Once expressed, recombinant peptides, polypeptides and proteins can be purified according to standard procedures known to one of skill in the art, including ammonium sulfate precipitation, affinity purification through columns or other methods commonly known, column chromatography, gel electrophoresis and the like. Substantially pure compositions of about 50 to 95% homogeneity are preferred, and 80 to 95% or greater homogeneity are most preferred for use as therapeutic agents.

One of skill in the art will recognize that after chemical synthesis, biological expression or purification, the antigenic peptide epitopes, polypeptides and proteins may possess a conformation substantially different than the native conformations of the constituent peptides. In this case, it is often necessary to denature and reduce the polypeptide and then to cause the polypeptide to refold into the preferred conformation. Methods of reducing and denaturing proteins and inducing refolding are well known to those of skill in the art.

Recombinant Protein Production

The method of producing the recombinant protein CDC/NIIMALVAC-1 involves the following steps: 1) selecting antigenic components, preferably antigenic peptides, from different stages in the life cycle of P. falciparum, that are involved in conferring immunologic protection; 2) optionally selecting a signal peptide sequence, such as melittin, optionally selecting other protein or peptide epitopes useful as T-cell helpers such as tetanus toxoid, and optionally selecting protein or peptide epitopes from P. falciparum involved in T-cell and B-cell recognition; 3) generating gene fragments comprised of nucleotide sequences that are complementary to the selected protein fragments; 4) assembling the gene fragments to create a gene, preferably a gene having the nucleotide sequence of SEQ ID NO:1, that encodes a novel recombinant protein, preferably the protein referred to herein as CDC/NIIMALVAC-1 having the amino acid sequence of SEQ ID NO:2; 5) cloning the gene into an expression vector so that it may be expressed in an expression system; and 6) expressing the recombinant protein in the expression system. The expressed recombinant protein is then recovered and purified. This protein is combined with a pharmaceutically acceptable vehicle or carrier and is administered as a multivalent, antimalarial vaccine to humans and nonhuman animals. The vaccine is administered in an amount effective to confer immunity against infection caused by P. falciparum, and particularly, to confer immunogenicity or immunity against different stages in the life cycle of P. falciparum.

Compared to vaccines directed to a single stage in the life cycle of malaria, the multivalent and multistage P. falciparum vaccine of the present invention induces multiple "layers" of immunity which significantly increase the chances of neutralizing all stages in the life cycle of the malaria parasite. The method of the present invention permits synthesis of a gene that contains coding sequences for several protective/immunodominant malarial epitopes of the malarial parasite P. falciparum.

A potential concern with the design of a synthetic gene encoding multiple epitopes is that the tandem arrangement of epitopes in the recombinant protein may induce antigenic competition, thus rendering immunizations ineffective in inducing immune responses. As shown in the examples and figures herein, the results from an immunization study involving the administration of CDC/NIIMALVAC-1 to mice and rabbits alleviate this concern. CDC/NIIMALVAC-1 is immunogenic. The recombinant protein antigen is recognized by antibodies directed against the B-cell epitopes of the construct. The vaccine is also antigenic since the immunization of rodents and rabbits induced antibodies that react with the protein vaccine, and also with the sporozoite and infected red blood cells. In addition, results of the experiments performed to evaluate the protective effects of immunization with CDC/NIIMALVAC-1 show that murine and rabbit antibodies against CDC/NIIMALVAC-1 inhibit parasite growth, as determined by the growth inhibition assay (GIA) and the antibody-dependent cellular inhibition (ADCI) assay.

The data set forth in the examples demonstrate that the protein, CDC/NIIMALVAC-1, in the multicomponent P. faliciparum vaccine, induces "multiple layer" of immunity, and that anti-CDC/NIIMALVAC-1 antibodies recognize different stages of the life cycle of the malarial parasite.

Construction of the Recombinant Gene

Immunogenic regions of various stage-specific antigens are identified by immune response studies in clinically immune adults and immune response studies performed in vitro using peptides and antibody reagents. Short, single-stranded DNA fragments complementary to the different epitopes are synthesized by methods known to those skilled in the art. Different DNA fragments are annealed to create a synthetic multicomponent gene by a three step polymerase chain reaction (PCR) amplification process as shown in FIG. 2. The principle behind the use of overlapping long oligonucleotides or gene fragments in the three round PCR procedure is that the sense strand and anti-sense strands of the nucleotide sequences are complementary at overlapping regions and act as primers after annealing.

Table 1 presents amino acid sequences of the twelve B-cell and nine T-cell epitopes derived from nine stage-specific vaccine candidate antigens of P. falciparum used in the development of the protein CDC/NIIMALVAC-1. One universal T-cell epitope, from tetanus toxoid is also incorporated. A sequence for the melittin signal peptide, used for enhancement of protein secretion in the baculovirus expression system, is added to the N terminus. A sequence of six histidines is inserted immediately C-terminal to the melittin signal peptide sequence to facilitate purification of expressed recombinant CDC/NIIMALVAC-1 on a nickel column. Corresponding nucleotide sequences for the melittin signal peptide sequence, the six histidine residues and the epitopes from P. falciparum are constructed. Restriction enzyme sites BamHI and NotI are designed at the flanking end to facilitate cloning in baculovirus transfer vector. Twelve overlapping single stranded oligonucleotides, each 125-145 nucleotides in length and spanning the entire synthetic gene, are synthesized and the vaccine antigen gene assembled.

The order of different epitopes is chosen such that the final protein has: 1) a random balance of B- and T-cell epitopes; and 2) an overall hydrophilic structure and water solubility. The vaccine antigen gene is ligated into a transfer vector, preferably a baculovirus transfer vector, such as pBacPAK8, and the recombinants are used to transform host cells such as Escherichia coli XL-Blue component cells. For example, lipofectin-mediated transfection and in vivo homologous recombination were used to introduce the vaccine antigen gene from pBacPAK8 into Autographa californica nuclear polyhedrosis virus (AcNPV, strain E2 ) at the polyhedrin locus of the genome.

The synthetic gene is cloned, and the recombinant virus containing CDC/NIIMALVAC-1 gene produced and grown in confluent monolayer cultures of an Sf21 insect cell line. The expressed recombinant protein is then purified, preferably using affinity chromatography techniques, and its purity and specificity determined by known methods. Alternatively, the synthetic gene may be employed as a DNA vaccine.

A variety of expression systems may be employed for expression of the recombinant protein. Such expression methods include, but are not limited to the following: bacterial expression systems, including those utilizing E. coli and Bacillus subtilis; vaccinia virus systems; yeast expression systems; cultured insect and mammalian cells; and other expression systems known to one of ordinary skill in the art.

Purification and Characterization of the Expressed Protein

The expressed protein contains epitopes from the sporozoite stage, liver stage, blood stage and sexual stage (also known as the gametocyte stage) of the malarial parasite P. falciparum, as well as a melittin signal peptide, a polyhistidine sequence and an amino acid sequence from tetanus toxoid. Although the antigens (epitopes) listed in Example 1 and Table 1 are the preferred antigens, it is to be understood that other antigens derived from these different stages in the life cycle of P. falciparum may be employed and are within the scope of the present invention. It is also to be understood that amino acid substitutions, as described elsewhere herein, may be made for amino acids in the peptide epitopes listed in Table 1, and are within the scope of the present invention. The order of the arrangement of these epitopes may be important in producing an efficacious recombinant protein for use as an antimalarial vaccine against P. falciparum. Various arrangements of these epitopes are considered within the scope of the present invention, provided that the arrangements generate an immune response in the recipient to epitopes derived from different stages in the life cycle of P. falciparum. A preferred order of these epitopes is presented in FIG. 1. The expressed protein, herein referred to as CDC/NIIMALVAC-1, is immunogenic when administered in combination with a carrier and adjuvants to mice and rabbits. Antibodies produced against the recombinant protein CDC/NIIMALVAC-1 recognize epitopes in the sporozoite stage, liver stage, blood stage and sexual stage of the malarial parasite P. falciparum.

Antibody Production

The protein is combined with a pharmaceutically acceptable carrier or vehicle to produce a pharmaceutical composition, and is administered to animals for the production of polyclonal antibodies. The preferred animals for antibody production are rabbits and mice. Other animals may be employed for immunization with the recombinant protein. Such animals include, but are not limited to the following; sheep, horses, pigs, donkeys, cows, monkeys and rodents, such as guinea pigs, and rats. Monoclonal antibodies can then be produced using hybridoma technology in accordance with methods well known to those skilled in the art, as taught by Mason et al. (Techniques in Immunocytochemistry, Vol. 2, Bullock & Petrusz eds., Academic Press, pp. 175-216, 1983). The antibodies are useful as research or diagnostic reagents or can be used for passive immunization. The pharmaceutical composition used for generation of antibodies may contain an adjuvant.

The antibodies useful as research or diagnostic reagents may be employed for detection of malarial infection in a biological sample, especially infection caused by P. falciparum. Such capability is useful for early detection of disease so that the vaccine may be administered to ameliorate disease progression. This capability is also useful for detecting the malarial parasite in the blood, especially blood collected for blood banks, so that malarial transmission through this mode is reduced or eliminated. Other biological samples which can be examined for infection are samples of human and animal livers, and also mosquitoes. Detection may be achieved through the use of ELISA, radioimmunoassay or other assays or methods as commonly known to one of ordinary skill in the art.

The CDC/NIIMALVAC-1 protein may be labeled through commonly known isotopic and non-isotopic methods, including but not limited to the following: radiolabeling, biotin-avidin, fluorescent molecules, chemiluminescent molecules and systems, ferritin, colloidal gold, and other methods known in the art of labeling proteins. The anti-CDC/NIIMALVAC-1 antibodies may be used in combination with labeled CDC/NIIMALVAC-1 protein to detect epitopes in P. falciparum.

The anti-CDC/NIIMALVAC-1 antibodies may also be administered directly to humans and animals in a passive immunization paradigm to confer immunity in the recipient to malaria.

Method of Administration

The protein is combined with a pharmaceutically acceptable carrier or vehicle for administration as a vaccine to humans or animals. The terms "pharmaceutically acceptable carrier" or "pharmaceutically acceptable vehicle" are used herein to mean any liquid including, but not limited to, water or saline, a gel, salve, solvent, diluent, fluid ointment base, liposome, micelle, giant micelle, and the like, which is suitable for use in contact with living animal or human tissue without causing adverse physiological responses, and which does not interact with the other components of the composition in a deleterious manner.

The vaccine formulations may conveniently be presented in unit dosage form and may be prepared by conventional pharmaceutical techniques. Such techniques include the step of bringing into association the active ingredient and the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers. Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets commonly used by one of ordinary skill in the art.

Preferred unit dosage formulations are those containing a dose or unit, or an appropriate fraction thereof, of the administered ingredient. It should be understood that in addition to the ingredients particularly mentioned above, the formulations of the present invention may include other agents commonly used by one of ordinary skill in the art.

The vaccine may be administered through different routes, such as oral, including buccal and sublingual, rectal, parenteral, aerosol, nasal, intramuscular, subcutaneous, intradermal, and topical. The vaccine may be administered in different forms, including but not limited to solutions, emulsions and suspensions, microspheres, particles, microparticles, nanoparticles, and liposomes. It is expected that from about 1 to 5 dosages may be required per immunization regimen. Initial injections may range from about 1 .mu.g to 1 mg, with a preferred range of about 10 .mu.g to 800 .mu.g, and a more preferred range of from approximately 25 .mu.g to 500 .mu.g. Booster injections may range from 1 .mu.g to 1 mg, with a preferred range of approximately 10 .mu.g to 750 .mu.g, and a more preferred range of about 50 .mu.g to 500 .mu.g.

The volume of administration will vary depending on the route of administration. Intramuscular injections may range from about 0.1 ml to 1.0 ml.

The vaccine may be stored at temperatures of from about 4oC. to -100oC. The vaccine may also be stored in a lyophilized state at different temperatures including room temperature. The vaccine may be sterilized through conventional means known to one of ordinary skill in the art. Such means include, but are not limited to filtration, radiation and heat. The vaccine may also be combined with bacteriostatic agents, such as thimerosal, to inhibit bacterial growth.

Vacciniation Schedule

The vaccine of the present invention may be administered to humans, especially individuals traveling to regions where malaria is present, and also to inhabitants of those regions. The optimal time for administration of the vaccine is about one to three months before the initial infection. However, the vaccine may also be administered after initial infection to ameliorate disease progression, or after initial infection to treat the disease.

Adjuvants

A variety of adjuvants known to one of ordinary skill in the art may be administered in conjunction with the protein in the vaccine composition. Such adjuvants include, but are not limited to the following: polymers, co-polymers such as polyoxyethylene-polyoxypropylene copolymers, including block co-polymers; polymer P1005; Freund's complete adjuvant (for animals); Freund's incomplete adjuvant; sorbitan monooleate; squalene; CRL-8300 adjuvant; alum; QS 21, muramyl dipeptide; CpG oligonucleotide motifs and combinations of CpG oligonucleotide motifs; trehalose; bacterial extracts, including mycobacterial extracts; detoxified endotoxins; membrane lipids; or combinations thereof.

Claim 1 of 7 Claims

What is claimed is:

1. A single recombinant protein comprising peptides from two or more stages in a life cycle of Plasmodium falciparum wherein each peptide comprises an antigenic epitope comprising the amino acid sequence as forth as SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25.


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