WO2001055181A2 - Recombinant multivalent malarial vaccines against plasmodium vivax - Google Patents

Abstract

Recombinant proteins are provided which comprise peptides derived from different stages in the life cycle of the parasite Plasmodium vivax. The proteins are useful as reagents and, when combined with a pharmaceutically acceptable vehicle or carrier, are useful as vaccines against the malarial parasite Plasmodium vivax. Genetic constructs used to produce these recombinant protein vaccines are also described. In addition, antibodies to these recombinant proteins are provided which are useful for the detection and measurement of peptides derived from different stages in the life cycle of the parasite Plasmodium vivax.

Description

RECOMBINANT MULTIVALENT MALARIAL VACCINES AGAINST PLASMODIUM VIVAX

STATEMENT OF GOVERNMENT INTEREST

This invention was made by the Centers for Disease Control and Prevention, an agency of the United States Government Therefore the United States Government may have certain rights in this invention

FIELD The present disclosure relates generally to the development and use of synthetic genes encoding recombinant proteins useful as multivalent and multistage malaria vaccines and more specifically relates to recombinant anttgenic proteins useful for reducing, preventing and/or treating P vivax malarial infections

BACKGROUND

Malaria is a parasitic infection known to be produced by the Plasmodium species P falcψarum 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 occasionall occurs in humans following a blood transfusion or subsequent to needle-sharing practices used b> 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 inside of hepatocytes, 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 parasite nuclear divisions, the erythrocyte ruptures, releasing merozoites that attach to receptors on erythrocytes and thus begin another erythrocytic cycle In P vivax and P ovale, hepatic parasites persist and ma\ 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 three million deaths each year. Much 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). 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.

Many attempts to develop malaria vaccines have focused on P. falciparum, in view of its public health impact, especially in sub-Saharan Africa. However, the extent of morbidity due to P vivax and its effect on public health impact are also very significant. P vivax has a worldwide prevalence rate second only to P falciparum. At least 35-50 million people are infected by P vivax each year, with the majority of infections occurring in Asia, South America, and Oceania. P vivax is also responsible for more malaria cases in the U.S. than any other malarial parasite. Although it rarely causes mortality, P vivax is a significant cause of morbidity and economic loss. Recent data indicates that P vivax infections are increasingly prevalent among Asian and South American populations. The situation is worsening due to the emergence of chloroquine-resistant vivax parasites and the capacity of this pathogen to relapse. These changes in the epidemiology of P vivax malaria have created a need for vaccines that may control and prevent P vivax infections.

The development of an effective malaria vaccine against P vivax represents one of the most promising approaches for providing cost-effective intervention along with other control measures currently available. Over the last decade considerable progress has been made 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. These three vaccine types are directed individually to blood stage (including the asexual blood stage) parasite stages expressed in mosquito vectors, or erythrocytic stages (including the liver stage). Antigens from each of these stages have been identified, including antigens from the following proteins: circumsporozoite protein (CSP) (Yang, et al, 1996 Vaccine, 15:377-386) and thrombospondin related anonymous protein (TRAP) of the sporozoite stage; the merozoite surface protein- 1 (MSP-1 ) (Yang, et al., 1996 Parasite Immunology; 18:547-558; Yang, et al., 1999 Infection and Immunity, 67:342-349, Collins et al , \ 999 Am. J Tropical Medicine and Hygiene, 60:350-356), Duffy antigen binding protein (DBP) and apical membrane antigen- 1 (AMA- 1 ) of the asexual blood stage; and the ookinete antigen Pvs 25 of the mosquito stage. However, individual vaccines based on antigens related to these specific stages are ineffective against other stages in the parasitic life cycle, thereby allowing the parasite to flourish in these other stages. SUMMARY OF THE DISCLOSURE

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 vivax, to treat, minimize or prevent infection and reduce associated morbidity and mortality Antigenic recombinant proteins, methods of making the proteins, genetic constructs encoding the proteins, antibodies to the proteins, pharmaceutical compositions containing the proteins, and a method for the treatment, prevention or reduction of malarial infection by administering either of the proteins to a human or animal are provided herein The proteins and anti- protein antibodies are also useful as research or diagnostic reagents for the detection of the Plasmodium species P vivax in a biological sample When administered to human or nonhuman animals, the proteins are each effective against malaria by conferring immunogenicity or immunity against multiple stages in the life cycle of the malarial parasite P vivax

This disclosure therefore provides novel recombinant proteins that can serve as multivalent, multistage vaccines against malaria, for instance malaria caused by P vivax Such recombinant proteins comprise antigenic epitopes to multiple stages of a parasite, which proteins may be used as reagents or as multivalent, multistage antimalaπal vaccines Representative examples of such proteins include at least one lmmunogenic peptide from each of at least two different life cycles of a parasite, such as P vivax

Also provided herein are vaccines against malaria that are effective in inhibiting reproductive growth of the parasite within a human or animal after initial infection As used herein, the term "inhibit" is not an absolute, but rather is a relative term in comparison to a condition without treatment

Further embodiments are synthetic genes useful as DNA vaccines, or for production of recombinant proteins in various expression systems, the recombinant proteins containing antigenic epitopes to various stages of a malarial Plasmodium species, particularly P vivax

Also provided are vectors comprising such synthetic genes These vectors may be used for a variety of purposes including, but not limited to, administration to animals and humans, and for transfection of cells

Further embodiments disclosed herein are methods for conferring immunity in a subject against different stages in the life cycle of a malarial parasite, as well as methods of vaccination against malaria caused by infection with a malarial parasite, such as P vivax Also provided are methods to reduce morbidity associated with malarial infection by preventing, reducing, or ameliorating malarial infection and/or by ameliorating the morbidity associated with malaria after initial infection with a parasite, such as P vivax Also provided are antibodies against the described recombinant proteins containing antigenic epitopes to various stages of /³ vivax Such antibodies are useful, for instance, as research or diagnostic reagents for the detection and'or measurement of f vivax in a biological sample Yet further embodiments are more effective, simpler and economical vaccines for conferring immunogenicity to different stages in the life cycle of P vivax than previously known vaccines

An advantage of certain disclosed anti-malarial recombinant proteins is that they confer immunogenicity against two or more stages, for instance all stages, in the life cycle of /⁵ vivax with administration of a single recombinant protein

These and other features and advantages will become apparent after a review of the following detailed description of the disclosed embodiments

BRIEF DESCRIPTION OF THE FIGURES FIG 1 's a schematic map of the synthetic gene, ViVacl (SEQ ID NO 1 ), encoding production of the recombinant protein ViVaclp (SEQ ID NO 2) Locations of epitopes in ViVac lp are indicated by the codes that correspond to codes in Table 2

FIG 2 is a schematic map of the synthetic gene, VιVac2 (SEQ ID NO 3), encoding production of the recombinant protein VιVac2p (SEQ ID NO 4) Locations of epitopes in VιVac2p are indicated by the codes that correspond to codes in Table 3

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids as defined in 37 C F R 1 822 Only one strand of each nucleic acid sequence is shown but the complementary strand is understood as included by any reference to the displayed strand In the accompanying sequence listing

SEQ ID NO 1 shows the nucleic acid sequence of the synthetic gene ViVac l and the amino acid sequence of the corresponding protein encoded thereby (ViVaclp) SEQ ID NO 2 shows the amino acid sequence of ViVaclp

SEQ ID NO 3 shows the nucleic acid sequence of the synthetic gene VιVac2 and the amino acid sequence of the corresponding protein encoded thereby (VιVac2p) SEQ ID NO 4 shows the amino acid sequence of ViVaclp SEQ ID NO 5 shows the amino acid sequence of the six-histidme epitope tag

SEQ ID NO 6 shows the amino acid sequence of the CSP-2Ra epitope SEQ ID NO 7 shows the amino acid sequence of the P2 epitope SEQ ID NO 8 shows the amino acid sequence of the P30 epitope SEQ ID NO 9 shows the amino acid sequence of the CSP-2Rb epitope SEQ ID NO 10 shows the nucleic acid sequence of the synthetic gene VιVac2bιas As with

SEQ ID NO 3, this nucleic acid molecule encodes the recombinant protein referred to herein as VιVac2p (SEQ ID NO 4) DETAILED DESCRIPTION

I. Terms Unless otherwise noted, technical terms are used according to conventional usage

Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes \ published by Oxford University Press, 1994 (ISBN 0-19-854287-9), Kendrew et al (eds ), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd , 1994 (ISBN 0-632-02182- 9), and Robert A Meyers (ed ), Molecular Biology and Biotechnology a Comprehensive Desk Reference, published by VCH Publishers, Inc , 1995 (ISBN 1-56081-569-8)

In order to facilitate review of the various embodiments of the invention, the following explanations of specific terms are provided

The terms "a," "an," and "the" as used herein are defined to mean one or more and include the plural unless the context is inappropriate

The phrase "injectable composition" as used herein refers to a pharmaceutically acceptable fluid composition including at least one active ingredient, e g , a therapeutically effective recombinant multivalent protein or nucleic acid encoding such a protein The active ingredient is usually dissolved or suspended in a physiologically acceptable carrier, and the composition can additionally include minor amounts of one or more non-toxic auxiliary substances, such as emulsifying agents, preservatives, and pH buffering agents and the like Such injectable compositions that are useful for use with the nucleotides and proteins provided herein are conventional, appropriate formulations are well known in the art

An "isolated" biological component (such as a nucleic acid molecule, protein or organelle) has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, / e , other chromosomal and extra-chromosomal DNA and RNA, proteins and organelles Nucleic acids and proteins that have been "isolated" include nucleic acids and proteins purified by standard purification methods The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids

The term "multivalent" as used herein is defined to mean more than one epitope The term "multistage" as used herein is defined to mean more than one stage in the life cycle of a parasite such as P vivax In this particular parasite, for instance, these stages include the sporozoite stage, the liver stage, the blood stage and the parasite stages expressed in mosquito vectors

A first nucleic acid sequence is "operably linked" with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame The term "parenteral" as used herein refers to methods of administration of a composition outside of the intestine, e g , not via the alimentary tract Generally, parenteral formulations are those that will be administered through any possible mode except ingestion This term especially refers to injections, whether administered intravenously, intrathecally, intramuscularly, lntrapeπtoneally, or subcutaneously, and various surface applications including intranasal, mtradermal, and topical application, for instance 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, hposome, 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 pharmaceutically acceptable carriers useful with molecules provided herein are conventional Martin, Remingto 's Pharmaceutical Sciences, published by Mack Publishing Co , Easton, PA, 19th Edition, 1995, describes compositions and formulations suitable for pharmaceutical delivery of the nucleotides and proteins herein disclosed

In general, the nature of the carrier will depend on the particular mode of administration being employed For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle For solid compositions (e g , powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate In addition to biologically- neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non- toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate

The term "purified" does not require absolute purity, rather, it is intended as a relative term Thus, for example, a purified protein preparation is one in which the protein referred to is more pure than the protein in its natural environment within a cell or within a production reaction chamber (as appropriate)

"Peptides", "polypeptides" and "oligopepttdes" are chains of ammo acids (typically L-amino acids) whose alpha carbons are linked through peptide bonds formed by a condensation reaction between the carboxyl group of the alpha carbon of one amino acid and the amino group of the alpha carbon of another ammo acid The terminal amino acid at one end of the chain (; e , the amino terminal) has a free amino group, while the terminal amino acid at the other end of the chain (/ e , the carboxy terminal) has a free carboxyl group As such, the term "amino terminus" (abbreviated N- terminus) refers to the free alpha-amino group on the amino acid at the ammo terminal end of the peptide, or to the alpha-amino group (imino group when participating in a peptide bond) of an amino acid at any other location within the peptide The term "carboxy terminus" (abbreviated C-terminus) refers to the free carboxyl group on the am o acid at the carboxy terminal end of a peptide, or to the carboxyl group of an amino acid at any other location within the peptide

Typically, the amino acids making up a peptide are numbered in order, starting at the amino terminus and increasing in the direction toward the carboxy terminus of the peptide Thus, when one amino acid is said to "follow" another, that amino acid is positioned closer to the carboxy terminal end of the peptide than the preceding amino acid A "promoter" is an array of nucleic acid control sequences that direct transcription of a nucleic acid A promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element A promoter also optionally includes distal enhancer or repressor elements that can be located as much as several thousand base pairs from the start site of transcription A "recombinant" nucleic acid is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence This artificial combination can be accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e g , by genetic engineering techniques A "recombinant multivalent protein" as the phrase is used herein refers to antigenic recombinant proteins containing immunogenic malarial epitopes from at least two different stages of the life cycle of the malarial parasite Plasmodium vivax Recombinant multivalent proteins can serve as multivalent, multistage vaccines against malaria, for instance malaria caused by P vivax Two representative examples of such antigenic recombinant proteins are ViVac lp and VtVac2p The term "residue" is used herein to refer to an amino acid (D or L) or an amino acid mimetic that is incorporated into a peptide by an amide bond As such, the amino acid may be a naturally occurring amino acid or, unless otherwise limited, may encompass known analogs of natural amino acids that function in a manner similar to the naturally occurring amino acids (/ e , ammo acid mimetics) Moreover, an amide bond mimetic includes peptide backbone modifications well known to those of ordinary skill in the art

Alternatively, the coding region may be altered by taking advantage of the degeneracy of the genetic code to alter the coding sequence such that, while the nucleotide sequence is substantially altered, it nevertheless encodes a protein having an amino acid sequence substantially similar to or even identical to the disclosed prototypical protein sequences (ViVaclp and VιVac2p) For example, the 18th amino acid residue from the amino-terminus of the ViVaclp protein is alanine Because of the degeneracy of the genetic code, this residue could be encoded by any of four nucleotide codon triplets - GCT, GCG, GCC and GCA Thus, the nucleotide sequence of the provided multivalent synthetic gene ViVacl could be changed at this position to any of the three alternative codons without affecting the amino acid composition or characteristics of the encoded protein

Based upon the degeneracy of the genetic code, variant DNA molecules may be derived from the cDNA and gene sequences disclosed herein using standard DNA mutagenests techniques as described above, or by synthesis of DNA sequences Thus, this disclosure also encompasses nucleic acid sequences which encode an immunogenic, multivalent protein compπsing antigenic epitopes from multiple stages of the life cycle P vivax parasite, but which vary from the native (i e , P vivax) nucleic acid sequence by virtue of the degeneracy of the genetic code This degeneracy can be used, by way of example, to adapt the codon usage in a construct to more closely fit the bias of an organism in which the construct will be expressed SEQ ID NO 10 shows an example of a synthetic gene as provided herein, wherein the codon bias has been altered by using the degeneracy of the genetic code In that particular example, the codon bias has been modified to fit that of/⁵ pastons

Furthermore, one of ordinary skill in the art will recognize that individual substitutions, deletions or additions in the amino acid sequence of the protein, or in the nucleotide sequence encoding for the amino acids in the protein, which alter, add or delete a single amino acid or a small percentage of amino acids (in some instances less than 5%, or even less than 1%) in an encoded sequence are conservatively modified variations, wherein the alterations result in the substitution of an amino acid with a chemically similar amino acid, and so long as the resultant variant still retains a substantial proportion of an immunostimulatory property (e g , a protective immune response in a subject) of the base protein Envisioned are molecules in which there is no more than one substitution, no more than about three substitutions, or about 5, 10, or even 20 substitutions, so long as the resultant variant retains a substantial proportion (e g , at least 20%, at least 30%. at least 50%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or more) of an immunostimulatory property of the base protein Some variant embodiments are expected to have greater immunostimulatory properties than the protein from which they are derived

Conservative amino acid substitution tables providing functionally similar amino acids are well known to one of ordinary skill in the art The following six groups are examples of amino acids that are considered to be conservative substitutions for one another

1) Alanine (A), Seπne (S), Threonine (T),

2) Aspartic acid (D), Glutamic acid (E),

3) Asparagine (N), Glutamine (Q),

4) Argtnine (R), Lysine (K),

5) Isoleuctne (I), Leucine (L), Methionine (M), Vahne (V), and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W) The phrase "sequence identity" refers to the similarity between two nucleic acid sequences, or two amino acid sequences, and is expressed in terms of the similarity between the sequences Sequence identity is frequently measured in terms of percentage identity (or similarity or homology), the higher the percentage, the more similar the two sequences are Methods of alignment of sequences for comparison are well known in the art Various programs and alignment algorithms are described in Smith & Waterman Adv Appl Math 2 482, 1981 , Needleman & Wunsch J Mol Biol 48 443, 1970, Pearson & Lipman Proc Nad Acad Sci USA 85 2444, 1988, Higgins & Sharp Gene, 73 237-244, 1988, Higgins & Sharp CABIOS 5 151-153, 1989, Corpet e/ α/ N c Acids Res 16 10881 -90, 1988, Huang et al Computer Appls in the Biosaences 8, 155-65. 1992, and Pearson et al Meth Mol Bio 24, 307-31. 1994 Altschul e/ α/ (J Mol Biol 215 403-410, 1990), presents a detailed consideration of sequence alignment methods and homology calculations

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al J Mol Biol 215 403- 410, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx

An alternative indication that two nucleic acid molecules are closely related is that the two molecules hybridize to each other under stringent conditions Stringent conditions are sequence- dependent and are different under different environmental parameters Generally, stringent conditions are selected to be about 5° C to 20° C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH The T_m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence remains hybridized to a perfectly matched probe or complementary strand Conditions for nucleic acid hybridization and calculation of stringencies can be found in Sambrook et al ((1989) In Molecular Cloning A Laboratory Manual, CSHL, New York) and Tijssen (( 1993) Laboratory Techniques in Biochemistry and Molecular Biology— Hybridization uith Nucleic Acid Probes Part I, Chapter 2, Elsevier, New York) Nucleic acid molecules that hybridize under stringent conditions to a multivalent multistage lmmunogen-encoding sequence (e g , a VιVac2p encoding sequence, such as shown in SEQ ID NO 3 or 10) will typically hybridize to a probe based on either an entire multivalent multistage lmmunogen-encoding sequence or selected portions of the encoding sequence under wash conditions of 2 x SSC at 50° C

Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences, due to the degeneracy of the genetic code As explained in more detail herein, it is understood that changes in nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid molecules that all encode substantially the same protein The term "specific binding agent" as used herein refers to an agent that binds substantially only to a defined target Thus a protein-specific binding agent binds substantially only the specified protein As used herein, the term "protein specific binding agent' includes anti-protein antibodies (and functional fragments thereof) and other agents (such as soluble receptors) that bind substantially only to the specified protein

Anti-protein antibodies (such as antι-VιVac2p antibodies) may be produced using standard procedures described in a number of texts, including Harlow and Lane (Antibodies, A Laboratory Manual, CSHL, New York, 1988) The determination that a particular agent binds substantially only to the specified protein, or component epitopes thereof, may readily be made by using or adapting routine procedures One suitable in vitro assay makes use of the Western blotting procedure (described in many standard texts, including Harlow and Lane (Antibodies, A Laboratory Manual, CSHL, New York, 1988)) Western blotting may be used to determine that a given protein binding agent, such as an anti- VιVac2p monoclonal antibody binds substantially only to the specified protein

Shorter fragments of antibodies can also serve as specific binding agents For instance, Fabs, Fvs, and single-chain Fvs (SCFvs) that bind to VιVac2p would be VιVac2p-specιfic binding agents These antibody fragments are defined as follows ( 1 ) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain, (2) Fab', the fragment of an antibody molecule obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain, two Fab' fragments are obtained per antibody molecule, (3) (Fab')2, the fragment of the antibody obtained by treating whole antibody with the enzyme pepsin without subsequent reduction, (4) F(ab')2, a di er of two Fab' fragments held together by two disulfide bonds, (5) Fv, a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains, and (6) single chain antibody ("SCA"), a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule Methods of making these fragments are routine

The term "subject" as used herein refers to living multi-cellular vertebrate organisms, a category that includes both human and non-human mammals Similarly, the term "subject" includes both human and veterinary subjects

A "therapeutical ly effective amount of a substance" is a quantity of that substance (for instance, a recombinant multivalent protein) sufficient to achieve a desired effect in a subject being treated For instance, this can be the amount necessary to stimulate an immune response in the subject (an immunologically effective amount), such as a cellular or humoral immune response Immune responses can be measured using known techniques, including using those methods described herein A "transformed" cell is a cell into which has been introduced a nucleic acid molecule by molecular biology techniques As used herein, the term transformation encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporatton, pofection, and particle gun acceleration

The term "vaccine" is used herein to mean a composition useful for stimulating a specific immune response in a vertebrate The term "vector" as used herein refers to a nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell A vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication A vector may also include one or more selectable marker genes and other genetic elements known in the art

Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety In case of conflict, the present specification, including explanations of terms, will control In addition, the materials, methods, and examples are illustrative only and not intended to be limiting

// Recombinant Multivalent Malarial Vaccines against Plasmodium vivax

Provided herein are antigenic recombinant proteins containing lmmunogenic malarial epitopes from different stages of the life cycle of the malarial parasite Plasmodium vivax Two representative examples of such antigenic recombinant proteins are ViVacl p and VιVac2p, which are single proteins containmg epitopes from different stages of the Plasmodium life cycle Also provided herein are methods of making such proteins, including genetic constructs from which the proteins are produced, antibodies to the proteins, pharmaceutical compositions containing the proteins, useful as malarial vaccines, and methods for treating, preventing or reducing malarial infection by administering either composition to an animal (such as a human)

The disclosed antigenic recombinant proteins are prepared by constructing a synthetic "gene" that encodes multiple 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 recombinant protein that confers increased immunity against different stages in the malarial life cycle of /⁵ vivax, or provides immunogenicity against epitopes from different stages in the life cycle of the parasite In specific embodiments, these stages including two or more of the sporozoite stage, the liver stage, the blood stage and the parasite stages expressed in mosquito vectors (also known as the gametocyte stage, sometimes called the ookinete stage) By using a combination of antigens or epitopes derived from different stages in the life cycle of the malarial parasite P vnax. the resultant recombinant protein provides 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 treatment and/or prevention of malaria caused by the parasite P vivax The lmmunogenic regions of the various stage-specific antigens of /⁵ vivax used to construct synthetic genes encoding the antigenic recombinant proteins are selected based on immunization studies in animals, immune response studies in clinically immune adults, in vitro studies using peptides or antibody reagents, or results of studies with their counterparts in P falciparum The resulting synthetic genes are sequence-confirmed and expressed in a baculovirus or other expression system (e g , Pichia pastoris)

Genetic constructs provided herein include coding sequences for one or more different peptide fragments obtained from two or more different stages in the life cycle of a malarial parasite, such as P vivax Certain examples of the genetic constructs also include epitopes chosen to enhance recognition, by cells of the immune system, of the protein expressed from the genetic construct Examples of such genetic constructs include coding sequences for a polyhistidine sequence useful for purification of the recombinant protein, a universal T-helper epitope, and epitopes from at least one stage in the life cycle of /⁵ vivax

Certain example genetic constructs may additionally include a transmission-blocking antigen An example of a transmission-blocking antigen is Pvs25, which is expressed on the parasite ookinetes in mosquitoes Antibodies against Pvs25 are useful to prevent the penetration of the parasite through the gut wall and the resultant formation of oocysts The end result of the transmission-blocking antigen is the lack of development of infective stages in mosquitoes

The antigenic fragments used to make the coding sequences of a specific example synthetic gene, referred to herein as ViVac l , are shown in Table 2 The nucleotide sequence of the ViVacl synthetic gene is shown in SEQ ID NO 1 The deduced amino acid sequence of the recombinant protein encoded by the synthetic gene, referred to herein as ViVacl p, is shown in SEQ ID NO 2 The recombinant protein in a pharmaceutically acceptable carrier is useful as a multivalent vaccine for P vivax malaria

A second set of antigenic fragments has been used to make a second example synthetic gene, referred to herein as VιVac2 The antigenic fragments used to make the coding sequences of this synthetic gene are shown in Table 3 The nucleotide sequence of VιVac2 is shown in SEQ ID NO 3 The deduced amino acid sequence of a prototypical red recombinant protein encoded by the gene, referred to herein as VιVac2p, is shown in SEQ ID NO 4 The recombinant protein in a pharmaceutically acceptable carrier is useful as another multivalent vaccine for P vivax malaria In certain embodiments, the codon bias of nucleic acid construct is altered from the native P vivax codon bias to that of another species For instance, the codon bias is adapted to reflect the bias of an organism in which the construct is expressed for production of an lmmunogenic protein (such as P pastons, E coli, or another organism) One example of such recombinant construct is SEQ ID NO 10, encoding VιVac2p (SEQ ID NO 4), but in which the codon bias of the nucleic acid sequence has been altered to more closely fit that of/⁵ pastons

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 malaiial disease process when administered after initial infection by P vivax

In certain embodiments, the recombinant proteins may be selected based on the know n immunogenicity of individual peptide components of these proteins (see, e g , Yang, et al , 1996 Vaccine, 15 377-386, Yang, et al , 1996 Parasite Immunology, 18 547-558. Yang, et al , 1999 Infection and Immunity 67 342-349, Collins et al , 1999 Am J Tropical Medicine and Hygiene, 60 350-356) Immunogenicity of the individual and/or combined proteins readily can be confirmed by testing the ability of the vaccine to elicit immune responses against both the vaccine protein and the P vivax parasite In vitro tests of protection conferred by the vaccine against blood stage malarial parasites will reveal that antibodies against this vaccine inhibit reproductive growth of /⁵ vivax The vaccine also induces multiple layers of immunity to different stages in the parasitic life cycle of /⁵ vivax

One specific genetic construct has the nucleotide sequence shown in SEQ ID NO 1 Other examples of nucleotide sequences have conservative nucleotide substitutions thereof, and when expressed retain at least a substantial portion of the immunogenicity of the corresponding prototype recombinant protein A specific example of a genetic construct has the nucleotide sequence shown in SEQ ID NO 3 Also disclosed are nucleotide sequences having conservative nucleotide substitutions thereof, which when expressed retain at least a substantial portion of the immunogenicity of the corresponding prototype recombinant protein

Other nucleotide sequences encode one of the provided recombinant proteins, but have a nucleic acid sequence that reflects the codon bias of a specific species These sequences can be derived, for instance, from the ViVac l or VιVac2 sequence by substitutions that do not change any amino acid residues in the encoded protein One representative construct having conservative nucleotide substitutions is SEQ ID NO 10, wherein the codon bias has been changed to a P pastons bias, without changing the amino acid sequence of the encoded protein (which corresponds to VιVac2p, SEQ ID NO 4) The genetic constructs may be expressed in an expression system, such as a baculovirus expression system, to produce recombinant proteins Two examples of such proteins are the proteins referred to herein as ViVac lp and VιVac2p which have the amino acid sequences set forth in SEQ ID NO 2 and SEQ ID NO 4, respectively Additional amino acid sequences include derivatives of these two prototypical sequences that have at least one or more amino acid substitutions, and which retain at least a substantial portion of the immunogenicity of the corresponding prototype recombinant protein The lmmunogenic proteins, such as ViVac lp and/or VιVac2p, may be combined with a pharmaceutical carrier and used as a multivalent vaccine to confer immunity to multiple stages in the life cycle of the malarial parasite, P vivax when administered in an effective amount to a human or animal Specific examples are disclosed of multivalent and multistage vaccines useful for preventing and treating malaria caused by P vivax Also disclosed are examples of polyclonal and monoclonal anti-protein antibodies produced after immunization with the described recombinant proteins, which are useful (as are the proteins) as research or diagnostic reagents in assays for the detection or monitoring of malarial infection, particularly to detect malarial infection caused by P vivax The antibodies are also useful for inducing passive immunization in an animal subject

Though particular examples of recombinant proteins are provided herein, other recombinant proteins are enabled that can also be used to stimulate immune responses that are protective against parasite infection, such as P vivax infection Such proteins may vary from specific provided examples based on the specific epitopes selected for inclusion in the construct, the order in which these epitopes are assembled, and other modifications

A Antigenic Peptide Production

When the antigenic epitope peptides are relatively short in length (/ e , less than about 50 amino acids), they are often synthesized using standard chemical, peptide synthetic 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 representative method for the chemical synthesis of the antigenic epitopes described herein Techniques for solid phase synthesis are known to those ordinarily skilled in the art

Alternatively, the antigenic epitopes described herein are synthesized using recombinant nucleic acid methodology Generally, this methodology 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, renatuπng the peptide or polypeptide Techniques sufficient to guide one of ordinary 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 Such a spacer may have no specific biological activity other than to join two antigenic epitopes together, or to preserve some minimum distance or other spatial relationship between them However, the constituent am o acids of the spacer may be selected to influence some property of the molecule such as the folding, net charge, or hydrophobicity, or to facilitate isolation of the resultant recombinant protein

Once expressed, recombinant peptides, polypeptides and proteins can be purified according to standard procedures known to one of ordinary skill in the art, including ammonium sulfate precipitation, affinity purification through columns or other methods commonly known, column chromatography, gel electrophoresis and the like In certain examples, substantially pure compositions will be of at least about 50% homogeneity, in other examples, great homogeneity is desired, for instance at least about 60%, at least about 70%, at least about 80%. or even 95% or greater homogeneity Generally, higher levels of homogeneity (e g , at least about 80%) are appropriate for use as therapeutic agents

One of ordinary 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 that is substantially different from the native conformation of one or more of the constituent peptides In this case, it is often beneficial to denature and/or reduce the polypeptide and then to cause or allow the polypeptide to refold into a more nearly native conformation Methods of reducing, denaturing, and refolding proteins are well known to those of ordinary skill in the art

B Recombinant Protein Production

A disclosed example of a method of producing the recombinant proteins, including but not limited to ViVaclp and VιVac2p, involves the following steps 1 ) selecting antigenic components, such as antigenic peptides, from different stages in the life cycle of /⁵ v;v<x , and which are capable of conferring lmmunologic protection, 2) (a) optionally selecting a signal peptide sequence, (b) optionally selecting other protein or peptide epitopes useful as T-cell helpers (e g , tetanus toxoid), and (c) optionally selecting protein or peptide epitopes from P vivax involved in T-cell and B-cell recognition, 3) generating genetic fragments comprised of nucleotide sequences that encode the selected protein fragments, 4) assembling the genetic fragments to create a novel synthetic gene that encodes a novel recombinant protein, 5) cloning the synthetic gene into an expression vector so 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 The protein is individually, or together with another recombinant protein or other pharmaceutical agent, combined with a pharmaceutically acceptable vehicle or carrier and administered as a multivalent, antimalaπal vaccine to humans and nonhuman animals The vaccines can be individually administered in an amount effective to confer increased immunity against infection caused by P vivax, and particularl , to confer simultaneous immunogenicity or immunity against different stages in the life cycle of/⁵ vivax

Two different synthetic genes ViVac l and VιVac2. having the nucleotide sequences of SEQ ID NO 1 or SEQ ID NO 3, and that encode the novel recombinant proteins referred to herein as ViVaclp and VιVac2p having the amino acid sequences of SEQ ID NO 2 and SEQ ID NO 4, respectively, serve as prototypical examples of the multivalent immunostimulatory proteins, which can serve as vaccines in animal subjects Likewise, the synthetic gene VιVac2bιas is another prototypical example of the provided synthetic genes, in VιVac2bιas, the codon bias of the synthetic gene has been modified from that of/⁵ vivax to that of /⁵ pastons, an organism in which the synthetic gene can be beneficially expressed to produce the corresponding recombinant protein VιVac2p

An example of a process of making the synthetic gene encoding ViVaclp involves the following steps 1 ) DNA fragments coding for five antigens (CSP-1 , AMA-1, TRAP, DBP and MSPl-19kD) are amplified by using the polymerase chain reaction (PCR) with primers with restriction enzyme site tails, 2) CSP-1 and AMA-1 are digested using the enzymes BamHI, Mlul and Spel, and then the pFastBac vector is digested with BamHI and Spel, these three fragments are then ligated together to form Plasmid 1 (PI), with the insert fragment (CSP-1 and AMA-1 ) called FI , 3) in a similar enzymatic digestion method, DBP(II) and MSPl -19kD are digested and ligated together (to create a fragment now termed F2) into pFastBac vector, called Plasmid 2 (P2), 4) F2, comprising DBP and MSPI -19kD, is cut from Plasmid 2 and ligated with the sequence encoding TRAP into pFastBac vector, forming Plasmid 3, and 5) FI is then removed from Plasmid 1 , Plasmid 3 is opened by digestion with BamHI and Mlul, and finally FI is ligated into Plasmid 3 This resultant new plasmid is Plasmid 4 (P4) The pFastBac- 1 vector with this recombinant target gene can be a donor vector for recombinant protein expression

An example of a process of making the gene encoding for VιVac2p involves the following steps 1 ) A DNA fragment coding for Pvs25 is amplified by PCR using primers with restriction enzyme (Mlul) site tails, 2) Pvs25 and Plasmid 4 (see above) are digested using Mlul, and 3) Pvs25 is ligated into the Plasmid 4 and the target gene is sequenced to verify the correct direction of the recombinant gene This pFastBac-recombinant (called P5), as donor vector, can be transformed into DHlOBac cells for recombmant Bacmid DNA and then into a Baculovirus expression system for protein expression The system is based on the site-specific transposition of an expression cassette from a donor plasmid into a baculovirus shuttle vector (Bacmid) that is propagated in E coli (Polayer et al , Focus 18 10-13, 1996) The construction of genes with different codon biases is known (see, for instance, Withers-

Martinez et al , "PCR-based gene synthesis as an efficient approach for expression of the A+T-πch malaria genome " Protein Engineering, 12(12) 11 13-1 120, 1999, and Pan et al , "Vaccine candidate MSP-1 from Plasmodium a redesigned 4917 bp polynucleottde enables synthesis and isolation of full-length protein from Escheπchia coli and mammalian cells " Nucleic Acids Research 27(4) 1094- 1 103, 1999) By way of example, production of the synthetic gene VιVac2bιas proceeds from the basis of the VιVac2 synthetic gene (described above) Once the sequence of a synthetic gene is known using the native P vivax sequences instances of rare codon in the synthetic gene are identified and correlated with to highly expressed codon usages according to a P pastons codon usage table The full length coding sequences (in the case of VιVac2, 5 6 kb) can then be divided into several shorter fragments (for instance, fragments of about 1 kb, thus 5 fragments for VιVac2) through the insertion of restriction enzymes sites Overlapping oligonucleotides are then synthesized for each fragment, and the fragments amplified using PCR or a similar technique, for instance using Pfu polymerase and Tag polymerase The individual fragments can be sequenced to confirm the fidelity of the process Each fragment is then cloned into a single vector (e g . pPCZ) assembled in the appropriate locations to recreate the synthetic gene, for expression in P pastons

With the provision herein of a P vivax native coding sequence (VιVac2) and the equivalent coding sequence adapted for expression in P pastons (VιVac2bιas), one of ordinary skill can compare these sequences to determine which codons are preferentially used in these tw o species Alternatively, standard prepared codon usage tables are available, see for instance, Table 1 , which shows a representative P pastons codon usage analysis (adapted from material on the Internet at www kazusa or p/codon/cgt-bin/showcodon cgι°specιes=Pιchιa+pastorιs+[bgpln]) This web-site also provides codon usage tables for thousands of other species

Table 1 Pichia pastoris: uuu 23. ■ 5( 735)¹ UCU 23. 7( 741) UAU 15. 4( 483) UGU 8. 4( 264) UUC 17. • 7( 554) UCC 16 .5( 515) UAC 16 ■ 7( 521) UGC 4 6( 143)

UUA 14. ■ 9( 465) UCA 16 ■ 0( 500) UAA 0 ■ 8( 24) UGA 0 4( 11)

UUG 31. • 9( 996) UCG 7 .0( 219) UAG 0 • 5( 15) UGG 9 • 4( 293)

CUU 16. ■ 7 ( 522) ecu 15 • 4( 481) CAU 10 • 6( 333) CGU 6 • 7( 211) CUC 8 , .K 254) CCC 6 9( 215) CAC 8 ■ 4 ( 263) CGC 2 4( 76)

CUA 11. .6( 363) CCA 17 0( 532) CAA 24 ■ 2 ( 756) CGA 4 K 128)

CUG 16. .4( 512) CCG 4 .K 129) CAG 15 .3( 478) CGG 1 • 9( 60)

AUU 29 .5( 924) ACU 22 • 8( 714) AAU 25 • 0( 782) AGU 12 .4( 387) AUC 18. .7( 584) ACC 13 ■ K 409) AAC 24 • 9( 780) AGC 7 5( 234)

AUA 12 ■ 3 ( 384) ACA 14 -7( 459) AAA 31 • K 974) AGA 19 5( 611)

AUG 20 •K 627) ACG 6 ■ 5 ( 204) AAG 34 ■ 2 ( 1070) AGG 7 2( 225)

GUU 26 • 7( 834) GCU 28 • 4( 887) GAU 37 .0( 1157) GGU 26 4( 826) GUC 14 ■ 3( 448) GCC 16 ■ K 502) GAC 26 3( 823) GGC 8 6( 268)

GUA 10 • 5( 328) GCA 17 • 0( 533) GAA 41 .0( 1283) GGA 19 4( 607)

GUG 13 • 0( 406) GCG 3 .5( 110) GAG 28 • 9( 905) GGG 6 ■ 2( 194)

¹ Fields [triplet] [frequency per thousand] ([number])

Compared to vaccines directed to a single stage in the life cycle of a malaria organism, the multivalent and multistage P vivax vaccines described herein induce multiple "layers^' of immunity , which significantly increases its effectiveness for neutralizing all stages in the life cycle of the malaria parasite, P vivax The methods provided herein permit production of synthetic genes that - I f

contain coding sequences for several protective/immunodominant malarial epitopes of the malarial parasite P vivax

C Alteration of Codon Bias in the Synthetic Genes In specific examples of the provided multicomponent synthetic genes, the codon bias of the sequences that encode one or more of the lmmunogenic peptides, or of the entire synthetic gene, are changed from the native bias (for instance, P vivax for those epitopes derived from the parasite) to that of another organism For instance, in some embodiments the codon bias is changed to be more similar to that of an organism m which the synthetic gene is to be expressed In specific examples, thus, the codon bias may be altered to that of P pastons, E coli, or an animal or mammalian bias Such alteration of codon bias can increase the efficiency of production of the recombinant protein in the expression system of choice, by increasing the recognition of the coding sequence See, for instance, the abstract of Zhao and Huo, Sheng Wu Kung Cheng Hsueh Pao, 16 308-31 1. 2000 ("Synonymous Codon Usage in Pichia pastons^'"), de la Cruz et al , J Biol Chem , 262 1 1935-1 1939, 1987 ("Sequence variation in putative functional domain of the circumsporozoite protein of

Plasmodium falciparum"), Kane, Curr Opm Biotech , 6 494-500. 1995 ("Effects of rare codon clusters on high-level expression of heterologous proteins in Eschenchia coli^"7) Codon preferences for may different species are known to those of ordinary skill in the art, as are methods of determining the codon bias of other organisms (Moπyama and Powell, Nucl Acid Res , 26 3188- 3193, 1998, Comeron and Agude, J Mol Evol , 47 268-274. 1998, Wang et al , Mol Biotechnol , 10 103-106, 1998, Kar n et al , Mol Mwrobiol , 29 1341-1355, 1998, Chen and Cheng, Int J Parasitol , 29 445-449, 1999, Mclntosh et al , Mol Biochem Parasitol , 95 69-80, 1998, Zhang et al , Gene, 105 61-72, 1991 , Bennetzen and Hall, J Biol Chem , 257(6) 3026-3031. 1981) It may be particularly advantageous to alter the codon bias of sequences derived from Plasmodium species, as it is known that Plasmodium has a particularly distinct pattern of codon usage that may not be optimally recognized in certain expression systems Other systems are also available to overcome issues related to codon bias, see, for instance, Baca and Hoi, Int J Parasitol , 30 1 13-1 18. 2000 ("Overcoming codon bias a method for high-level overexpression of plasmodium and other AT-πch parasite genes in Eschenchia coli"), in which the authors express in the recombinant expression system tRNAs that are biased towards the codon preferences of parasite genes

D Construction of the Recombinant Synthetic Genes lmmunogenic regions of various stage-specific antigens may be identified using known techniques, such as by immunization studies in animals, immune response studies in clinically immune adults, and immune response studies performed in vitro using peptides or antibody reagents Short, single-stranded DNA fragments encoding for the lmmunogenic epitopes are synthesized by methods known to those skilled in the art The resultant DNA fragments can be annealed by methods known to those skilled in the art to create a multicomponent synthetic gene, for instance using a three-step PCR amplification process The principle behind the use of overlapping long oligonucleotides or gene fragments in the 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

The overall length of a given multivalent protein depends on the number of individual epitopes contained therein At least one epitope from each of at least two life cycle stages is included in the multivalent recombinant proteins Longer recombinant proteins are contemplated, in which more than two epitopes are included, examples of such longer proteins are described In certain embodiments, the recombinant multivalent proteins will include at least one epitope from each of the live cycle stages of the parasite Other examples of recombinant multivalent proteins will include more than one epitope from a single life cycle stage, with at least one additional epitope from at least one further life cycle stage

Table 2 presents amino acid sequences of the B-cell and T-cell epitopes derived from stage- specific vaccine candidate antigens of P vivax that were used in the development of the protein

ViVacl p Two universal T-cell epitopes from tetanus toxoid were also incorporated A sequence of six histidines was also inserted at the C-terminal to facilitate purification of expressed recombinant ViVacl p on a nickel column Corresponding nucleotide sequences for the six histidine residues and the epitopes from P vivax were constructed Restriction enzyme sites BamHI and Not I were designed at the flanking ends to facilitate cloning in the baculovirus transfer vector BamHI and Notl sites were used for cloning to the vector BamHI was located in front of start codon (ATG) and Notl behind the stop codon (TAA) These bases encoding for BamHI. stop codon (TAA) and Not I were not expressed (thus, a total of 17 nucleotides were not expressed)

PCR primers with restriction enzyme tails were synthesized and five antigen-encoding fragments were amplified by PCR Next, by cutting at the designed restriction enzyme sites, vaccine antigen-encoding ("gene") fragments were obtained and assembled in sequence, as shown in Figure 1

Table 3 presents amino acid sequences of the B-cell and T-cell epitopes derived from stage- specific vaccine candidate antigens of/⁵ vivax that were used in the development of the protein VιVac2p Two universal T-cell epitopes from tetanus toxoid were also incorporated A sequence of six histidines was inserted at the N-terminal to facilitate purification of expressed recombinant VιVac2p on a nickel column Corresponding nucleotide sequences for the six histidine residues and the genes from P vivax were constructed Restriction enzyme sites BamHI and Notl were designed at the flanking ends to facilitate cloning in the baculovirus transfer vector BamHI and Notl sites were used for cloning to the vector BamHI was located in front of start codon (ATG) and Notl behind the stop codon (TAA) These bases encoding the BamHI restriction site, stop codon and Not I restriction site were not expressed PCR primers with restriction enzyme tails were synthesized and 5 antigen-encoding fragments were amplified by PCR Next, by cutting at restriction enzyme sites, vaccine antigen antigen-encoding ("gene") fragments were obtained and assembled in sequence, as shown in Figure 2 In certain embodiments (typified by ViVac lp and VιVac2p), the order of different epitopes is chosen such that the final recombinant protein has 1 ) a random balance of B- and T-cell epitopes, and 2) an overall hydrophihc structure and water solubility In this example, the recombinant multivalent synthetic genes can be ligated into a transfer vector, such as a baculovirus transfer vector, for instance pFastBac, and the recombinant vector used to transform host cells, such as Eschenchia coli DH l OBac competent cells or other host cells Merely by way of example, lipofectin-mediated transfection and in vivo homologous recombination have been used to introduce the provided prototypical vaccine antigen genes from PfastBac into Autographa californica nuclear polyhedrosis virus (AcNPV, strain E2) at the polyhedπn locus of the genome

The synthetic genes are cloned, and the recombinant viruses containing at least one multivalent recombinant synthetic gene (such as the ViVacl or VιVac2 synthetic genes) are produced and grown in confluent monolayer cultures of, for instance, an Sf9 insect cell line The expressed recombinant proteins are then purified, for instance using affinity chromatography techniques, and their purity and specificity determined by known methods Alternatively, the synthetic genes may be employed as DNA vaccines A variety of expression systems may be employed for expression of the recombinant proteins 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, including those utilizing S cerevisiae or P pastons, cultured insect and mammalian cells, and other expression systems known to one of ordinary skill in the art

E Purification and Characterization of Expressed Recombinant Proteins

The recombinant protein expressed using art-known techniques, such as the baculovirus expression system, may be purified on a nickel affinity column if there is a six-histidine tag incorporated into the protein (for instance, at or near one end of the protein) In embodiments provided herein, the expressed protein contains epitopes from the sporozoite stage, liver stage blood stage and ookinete stage of the malarial parasite P vivax, a polyhistidine sequence and amino acid sequences from tetanus toxoid

Although the antigens (epitopes) listed in Examples 1 and 2, and Tables 2 and 3 are the prototypical antigens, it will be understood that other antigens derived from the different stages in the life cycle of P vivax may be employed and are within the scope of the provided disclosure 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 Tables 2 and 3, and are within the scope of the present disclosure It is also understood that the nucleic acid sequence encoding the individual peptides can vary without necessarily varying the ammo acid sequence of the peptide itself, either by merely taking advantage of the redundancy of the genetic code or to take advantage of a codon bias of a particular species The order of the arrangement of epitopes within a synthetic multivalent protein in some embodiments will produce particularly efficacious recombinant protein for use as an antimalaπal vaccine against P vivax Various arrangements of these epitopes are considered within the scope of the present disclosure, provided that the arrangements generate an immune response in the recipient to epitopes derived from different stages in the life cycle of /⁵ vivax An example of a prototypical order of these epitopes is presented in Figures 1 (ViVac lp) and 2 (VιVac2p) The expressed proteins, such as the prototypical proteins herein referred to as ViVac l p and VιVac2p, are believed to be lmmunogenic when administered in combination with a carrier and adjuvants to animals, such mice and rabbits, as well as to humans such as human subjects in danger of being infected by P vivax or already so infected

F Antibody Production

The recombinant proteins provided herein (such as ViVac l p and VιVac2p) can be individually combined with a pharmaceutically acceptable carrier or vehicle to produce different pharmaceutical compositions, and then can be administered to animals for the production of polyclonal antibodies The prototypical 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 be produced using hybridoma technology in accordance with methods well known to those ordinarily skilled in the art, for example using methods 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, which are useful as research or diagnostic reagents, may be employed for detection of malarial infection in a biological sample, especially infection caused by P vivax Such capability is useful for early detection of disease so that vaccine may be administered to ameliorate disease progression This capability is also useful for detecting the malarial parasite in the blood, such as blood collected for blood banks, so that malarial transmission through transfusion is reduced or eliminated Other biological samples that can be examined for infection are samples of human and animal livers, and also mosquitoes Detection may be achieved through the use of immunocytochemistry, ELISA, radioimmunoassay, or other assays or methods as commonly known to one of ordinary skill in the art Antibodies specific to a recombinant multivalent protein (such as anti-ViVaclp and anti- VιVac2p antibodies) may be labeled through commonly known isotopic and non-isotopic methods These methods include, but are not limited to the following methods radiolabeling, biotin-avidin, fluorescent molecules, chemiluminescent molecules and systems, ferπtin, colloidal gold, and other methods known to one of ordinary skill in the art of labeling antibodies By way of example, these labeled antibodies also may be used to detect peptides used to make the recombinant protein, such as the ViVacl p and VιVac2p proteins, or for detection of malarial infection in a biological sample, especially infection caused by P vivax The recombinant proteins (such as ViVaclp and VιVac2p) also may be detected and optionally measured using labeled antibodies The recombinant proteins (including the prototypical ViVacl p and VιVac2p proteins) also 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, ferntin, colloidal gold, and other methods known to one of ordinary skill in the art of labeling proteins In certain specific embodiments, the anti-ViVaclp and antι-VιVac2p antibodies may be used in combination with labeled ViVac lp and VιVac2p proteins to detect epitopes in P vivax

The antibodies, such as anti-ViVac l p and antι-VιVac2p antibodies, also may be administered directly to humans and animals in a passive immunization paradigm to confer increased immunity in the recipient to malaria

G Method of Administration

The recombinant multivalent proteins are individually combined with a pharmaceutically acceptable carrier or vehicle for administration as an immunostimulatory composition or a vaccine to humans or animals In some embodiments, more than one protein may be combined to form a single vaccine preparation

The vaccine formulations may be conveniently presented in unit dosage form and prepared using conventional pharmaceutical techniques Such techniques include the step of bringing into association the active ingredient and the pharmaceutical carπer(s) or excιpιent(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 bacteπostats 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-dπed (lyophi zed) condition requiring only the addition of a 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

In certain embodiments, 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, formulations encompassed herein may include other agents commonly used by one of ordinary skill in the art

The compositions provided herein, including those for use as vaccines, may be administered through different routes, such as oral, including buccal and sublingual. rectal, parenteral, aerosol, nasal, intramuscular, subcutaneous, intradermal, and topical They may be administered in different forms, including but not limited to solutions, emulsions and suspensions, microspheres, particles, microparticles. nanoparticles, and ltposomes It is expected that from about 1 to 5 dosages may be required per immunostimulation or immunization regimen Initial injections may range from about 1 μg to 1 mg, with some embodiments having a range of about 10 μg to 800 μg, and still other embodiments a range of from approximately 25 μg to 500 μg Booster injections may range from 1 μg to 1 mg, with other embodiments having a range of approximately 10 μg to 750 μg, and still others a range of about 50 μg to 500 μg

The volume of administration will vary depending on the route of administration By way of example, intramuscular injections may range from about 0 1 ml to 1 0 ml Those of ordinary skill in the art will know appropriate volumes for different routes of administration The compositions provided herein, including those formulated to serve as vaccines, may be stored at temperatures of from about -100° C to 4° C They may also be stored in a lyophi zed state at different temperatures, including higher temperatures such as room temperature The preparation 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 preparations also may be combined with bacteπostatic agents, such as thimerosal, to inhibit bacterial growth

H Vaccination Schedule

Embodiments of the vaccines provided herein may be administered to humans, especially individuals traveling to regions where malaria is present, and also to inhabitants of those regions In specific embodiments, the optimal time for administration of the vaccine is about one to three months before the initial infection or exposure to the infectious agent (/ e , P vivax) However, the vaccine also may be administered after initial infection/exposure 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 proteιn(s) in the provided 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.

The invention is further illustrated by the following non-limiting Examples.

Example 1

Development, Synthesis and Cloning of the ViVacl and ViVacl Synthetic Genes

ViVacl A recombinant multivalent and multistage vaccine against P. vivax was designed to contain six histidine (His) residues for purification of the protein, two tetanus toxoid universal T- helper epitopes (P2 and P30), and neutralizing domains from CSP (CSP- 1 , CSP-2R), MSP 1 - 19kD, AMA-1, TRAP, and DPB(II) vaccine antigens (see Table 2). These malarial functional antigen domains were obtained from different stages of the life cycle of P. vivax including the following stages: the sporozoite stage (CSP-1 , CSP-2R and TRAP); liver stage (CSP-1 , CSP-2R, and TRAP); and blood stage (AMA- 1 , MSP-1, and DPB(II)). The synthetic gene (SEQ ID NO: 1 ) encoding the recombinant protein ViVac lp (SEQ ID NO: 2) was assembled, cloned, and expressed in a baculovirus system.

ViVacl A recombinant multivalent and multistage vaccine against P. vivax was designed to contain six histidine (His) residues for purification of the protein, two tetanus toxoid universal T- helper epitopes (P2 and P30), and neutralizing domains from CSP (CSP-1. CSP-2R), MSP-1 19kD, AMA-1 , TRAP, DPB(II) and Pvs25 vaccine antigens (see Table 3). These malarial peptide epitopes were obtained from different stages of the life cycle of P. vivax including the following stages: the sporozoite stage (CSP-1 , CSP-2R and TRAP); liver stage (CSP-1, CSP-2R, and TRAP); blood stage (AMA- 1 , MSP-1 , and DPB(II)) and ookinete stage (Pvs25). The synthetic gene (SEQ ID NO: 3) encoding the recombinant protein ViVac2p (SEQ ID NO: 4) was assembled, cloned, and expressed in a baculovirus system.

A schematic map shown in Figure 1 provides the locations of epitopes in ViVac l p indicated by the codes corresponding to the codes in Table 2. A schematic map shown in Figure 2 provides the locations of epitopes in ViVac2p indicated by the codes corresponding to the codes in Table 3. The arrangement of the immunogenic epitopes in each recombinant protein reflects a random balance of position of B cell and T cell epitopes.

Corresponding nucleotide sequences for the six histidine residues and the epitopes from P. vivax were constructed. The nucleotides GGATCC at the 5' ends and the nucleotides GCGGCCGC at the 3' ends of both the ViVac l and ViVac2 synthetic genes (SEQ ID NOs' 1 and 3, respectively) are start and stop codons, respectively. Restriction enzyme sites BamH\ and Not I were designed at the flanking end to facilitate cloning in baculovirus transfer vector. Fragments coding for CSP-1, TRAP, MSP-1 , AMA-1, and DBP were amplified by PCR using primers with restriction enzyme site tails. The fragments were assembled together by restriction digestion to produce the synthetic gene encoding the multivalent vaccine antigen.

The resultant multivalent synthetic genes were ligated separately into different baculovirus transfer vectors, pFastBac- 1 , and the recombinant constructs used to transform Eschenchia coli DHlOBac competent cells Lipofectin-mediated transfection and in vivo homologous recombination were used to introduce vaccine antigen gene from pFastBac- 1 into Autographa californica nuclear polyhedrosis virus (AcNPV, strain E2) at the polyhedrin locus of the genome

Table 2 : ViVaclp components, sequences and locations

1 Location of immune epitopes in ViVaclp is presented in the first column and indicated by the codes

histidine residues were incorporated at N-terminus The order of antigens from top to bottom is the same as the order from the N-terminus to the C-terminus in the recombinant protein

2 In the column entitled "Stage". B represents Blood stage. L represents Liver stage S represents Sporozoite stage and TT represents tetanus toxoid

3 Abbreviations CSP. circumsporozoite protein MSP. merozoite surface protein AMA-1 apical membrane antigen- 1 TRAP, thrombospondin related anonymous protein. CTL. cytotoxic T lymphocyte and DBP(Il). Duffy antigen binding protein region II

4 In the column entitled "Ammo acid sequence^" either the single letter ammo acid sequence of an immune epitope is provided or it is described in terms of its location by numbers indicating its position in the ammo acid sequence of the recombinant protein ViVaclp

5 SEQ ID NO 5

6 SEQ ID NO 6

7 SEQ ID NO 7

8 SEQ ID NO 8

9 SEQ ID NO 9 Table 3 : ViVac2p components, sequences and locations

1 Location of immune epitopes in VιVac2p is presented in the first column and indicated by the codes Six histidine residues were incorporated at N-terminus The order of antigens from top to bottom is the same as the order from the N-terminus to the C-terminus in the recombinant protein

2 In the column entitled "Stage^". B represents Blood stage. L represents Liver stage S represents Sporozoite stage. O represents oo inete stage, and TT represents tetanus toxoid

3 Abbreviations CSP. circumsporozoite protein. MSP. merozoite surface protein. AMA-1. apical membrane antigen- 1 TRAP, thrombospondin related anonymous protein. CTL. cytotoxic T lymphocyte, DBP(II). Duffy antigen binding protein, region II. and Pvs25. Plasmodium vivax ookinete surface antigen 2S kDa

4 In the column entitled amino acid sequence, either the single letter amino acid sequence of an immune epitope is provided or it is described in terms of its location by numbers indicating its position in the amino acid sequence of the recombinant protein VιVac2p

5 SEQ ID NO- 5

6 SEQ ID NO 6

7 SEQ ID NO 7

8 SEQ ID NO 8

9 SEQ ID NO 9

Example 2

Expression of the Recombinant Proteins, ViVaclp and ViVac2p, in a Baculovirus System and Purification of the Protein Antigens The synthetic ViVacl or ViVac2 genes described in Example 1 were cloned, and recombinant viruses containing the ViVacl or ViVac2 genes were produced and grown in confluent monolayer cultures of Sf9 insect cell line, as described by Chatterjee, U. et al , (1996) Gene 171 :209- 213. The synthetic genes are approximately 5.4 kb and 6.0 kb in length, as determined on an agarose gel with corresponding DNA standards run in another lane. Each baculovirus-expressed, recombinant protein, ViVac lp and ViVac2p, was purified from

Sf9 cells at 72 hours post-infection using TALON™ metal affinity resin according to the manufacturer's instructions (CLONTECH, Palo Alto, CA). The purity and specificity of the expressed recombinant proteins were determined using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. The results show that expression of the synthetic genes in the baculovirus expression vector system produced 200 kDa and 225 kDa proteins referred to herein as ViVaclp and VιVac2p, respectively A dot blot assay using monoclonal antibodies to CSP- 1 showed binding to ViVaclp, indicated that the recombinant protein ViVaclp was successfully expressed in the baculovirus system

Example 3

Immunization of Mice and Rabbits against the Expressed Protein and Demonstration of Immunoreactivity against Different Stages in the Life Cycle of? vivax

As described in Example 2, the baculovirus-expressed recombinant protein was purified from cell pellets harvested 72 hours after infection The expressed proteins ViVac lp and VιVac2p were purified on nickel affinity columns using TALON™ metal affinity resin according to manufacturer's instructions (as in Example 2) The purity and specificity of the proteins were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis as described in Example 2 The isolated protein was characterized, using chromatographic techniques, as approximately 200 kDa

Six- to eight-week-old mice were immunized intraperitoneally with purified protein, either

ViVacl p or VιVac2p in Freund's complete adjuvant Booster immunizations of mice were performed using purified protein in Freund's incomplete adjuvant A total of four immunizations were given to each mouse at three-week intervals (; e , at weeks 0, 3, 6, and 9) Blood was removed and sera collected 7 to 10 days after each immunization until weeks 33-51 Sera were stored at -20° C The sera from mice were used in antibody and immunofluorescence (IFA) assays

Four-month-old female New Zealand white rabbits (Jackson Laboratory, Bar Harbor, ME) weie immunized intramuscularly with the purified protein, either ViVacl p or VιVac2p, in vehicle One rabbit was immunized with the protein in Freund^'s adjuvant Another rabbit was immunized with the protein in an adjuvant that may be used in humans, specifically the nonionic block copolymer PI 005 in water-in-oil emulsion Another rabbit was immunized with the protein in the human usable adjuvant, aluminum hydroxide (rabbit 1015) A total of four immunizations were given to each rabbit, each immunization occurring at three-week intervals (/ e . at weeks 0, 3, 6, and 9) Blood was removed and sera collected 7 to 10 days after each immunization until weeks 33-51 Sera were stored at -20° C Sera from rabbits were used in the antibody and IFA assays Sera were collected from the blood of each rabbit during weeks 10 to 16, and were pooled

Total IgGs were purified using ammonium sulfate (Sigma Chemical Co , St Louis, MO) precipitation followed by DEAE (Pierce, Rockford, IL) batch purification according to known methods as taught by Holhngdale, M R et al , (J Immunol 132, 909-913, 1984) Following dialysis against PBS, the purified antibodies were used for immunoelectron microscopy, antibody affinity testing, and in an in vitro protection assay Purified antibodies from rabbits and mice may be used for other purposes as described elsewhere in the application, and for purposes known to one of ordinary skill in the art Serum antibody titers against the vaccine antigen and individual peptide epitopes were quantitated using ELISA Microtiter plates were coated with the vaccine antigen or peptides in borate buffer solution (BBS) overnight at 4°C and then blocked with BBS containing 5% nonfat lyophi zed milk The plates were washed four times with sodium phosphate-buffered saline (PBS, pH 7 4) containing 0 5 M NaCl, 0 5% bovine serum albumin, 0 0005% Tween 20, and 0 05% thimerosal (PBS-T) The rabbit sera are diluted serially in PBS-T containing 1 5 % nonfat milk, added into microtiter plates, and incubated at room temperature for one hour The unbound antibodies were removed by four washes with PBS-T Bound antibodies were detected with peroxidase-conjugated goat anti-rabbit antibodies The secondary antibody was allowed to bind for 1 hour, the wells were washed with PBS-T, 100 ml of 3,3',5,5'-tetramethylbenzιdιne were added, and 10 minutes later the reaction was stopped with 50 ml of 1 M phosphoric acid The plates were read at an absorbence of 450 nm Responses agamst sporozoites, asexual blood-stage parasites, and gametocytes were determined by indirect immunofluorescence (IFA)

The immunogenicity of ViVaclp and VιVac2p can be confirmed by their ability to elicit immune responses against a) ViVaclp and VιVac2p, respectively, b) individual epitopes found in ViVac lp and VιVac2p, and c) different stages in the life cycle of the P vivax parasite Antibody is purified from the rabbit immunized with ViVaclp or VιVac2p in copolymer adjuvant The immunoreactivity of this purified rabbit antiserum against antigens present in the different stages of the life cycle of the malarial parasite, P vivax, may be evaluated using ultrastructural immunocytochemistry To study ultrastructural localization of antibody reactivities with various stages of/⁵ vivax, sporozoite, exoerythrocytic (EE)-ιnfected hepatocyte, gametocyte stage III-IV, and asexual blood stage parasites are chosen for immunoelectron microscopy Briefly, sections are incubated for 24 hours at 4°C with antibody diluted 1 800 for determination of reactivity with sporozoites, or with antibody diluted 1 200 for determination of reactivities with other stages of parasites This step is followed by a one-hour incubation at 25°C with gold-labeled, goat anti-rabbit IgG antibody Method specificity is confirmed by incubating control sections with preimmune rabbit serum instead of the primary antibody, with the colloidal gold probe, or with colloidal gold alone Immunoreactivities of antibodies with parasites is examined in a Zeiss CEM902 electron microscope as taught by Aikawa, M , & Atkinsen, C T (1990) Adv Parasitol 29, 151-214

Immunoelectron micrographs of different stages in the life cycle of the P vivax parasite should demonstrate that the rabbit antiserum from rabbits, which received copolymer as adjuvant, contain antibodies which are immunoreactive to the sporozoite stage, blood stage, gametocyte stage III-IV, and asexual blood stage Gold particles should be found on the surface and in the cytoplasm of the sporozoite, in the paras ltophorous vacuole membrane (PVM) and cytoplasm of blood stage, in the cytoplasm in the gametocyte stage III-IV, in rhoptry and surface of merozoite of the blood stage and in the cytoplasm of trophozoite of the blood stage Immunogenicity of ViVaclp and ViVaclp

Antibody titers against the vaccine antigens, either ViVacl p or VιVac2p, and peptides complementary to immune epitopes (see Tables 2 and 3) are measured by ELISA after each immunization of the rabbits described above The vaccine antigens are believed to induce high titers and prolonged antibody responses against the vaccine in rabbits immunized with different adjuvants Overall, the rabbits receiving Freund's adjuvant should have higher antibody levels to the vaccine antigen compared to those receiving copolymer or alum adjuvants

The binding affinities of the vaccine-elicited antibodies to the vaccine antigen itself are investigated in a Biacore assay employing a surface-plasmon resonance detector (Biocore, Inc Piscataway, NJ) Purified vaccine antigen is covalently immobilized in a "Cl" (short-chain carboxymethyl-dextran) sensor cell by standard carbodiimide/N-hydroxysuccinimide methods Mobile-phase analyte consists of purified IgG preparations diluted to 50 μg of protein per ml m 10 mM HEPES buffer, pH 7 4, plus 150 mM NaCl After establishing a stable baseline signal with buffer, the association reaction is initiated by switching to the analyte stream, and the resonance signal is followed in time, as taught by Wohlhueter, R M et al (J Immunol 153, 181-189. 1994) A flow rate of 5 μl/min is used throughout Association rate curves observed with different IgG preparations are adjusted to a common baseline and superimposed Initial velocities of association of the high-affinity components are estimated by measuring the initial linear slopes of the curves Antigen-antibody binding shows multiple kinetic components distinguishable into fast (450-500 seconds) and slow (800-1400 seconds) components Quantitatively, total IgG from the rabbits immunized in the presence of copolymer adjuvant should contain much higher levels of high affinity antibodies This conclusion is also supported by the biological analysis described below

Example 4

Hepatocyte Invasion Inhibition Assay

Inhibition of sporozoite invasion (ISI) assays are conducted using hepatocytes to determine the inhibitory effects of antibodies as previously described by Holhngdale, M R et al , ( 1984) J Immunol 132, 909-913 Briefly, the purified antibodies are added at two different final concentrations (25 and 50 μg/ml) into the HepG2-A16 hepatoma cells, and then about 30.000 P vivax sporozoites are added The cells are incubated at 37°C in 5% C0₂ for 3 hours, rinsed two times with phosphate-buffered saline (PBS), and fixed with methanol Sporozoites that have entered hepatoma cells are visualized by immunocytochemical staining with a monoclonal antibody to P vivax sporozoites (Jones, T R, Yuan, et al , Am J Trop Med Hyg 1991 , 47-837-843), peroxidase- conjugated, goat anti-mouse immunoglobulin and the substrate 3,3-dιamιnobenzιdιne All cultures are done in triplicate and the numbers of sporozoites that have invaded the hepatoma cells are determined by light microscopy Transmission-blocking assays are performed by membrane feeding assays as taught by Wtzel B & Kumar, N (1991) Proc Natl Acad Sci USA, 88, 9533-9536 In this assay, P vivax (Sal) gametocytes are used to infect Anopheline (An) stephensi mosquitoes Various IgG preparations are tested at final concentrations ranging from 125 to 500 μg/ml to determine their ability to prevent transmission of the P vivax gametocytes to mosquitoes, as judged by the reduction of developing oocysts of malarial parasites in the midgut of mosquitoes

Example 5 Test of Immunogenicity and Protective Efficacy of ViVaclp and Vύ aclp Ϋ vivax Vaccines in a Monkey Model System

Monkeys (Saimiri or other types of monkeys) receive about 100 μg of either the ViVaclp and iVaclp vaccines described in Example 3 per animal for initial immunization and approximately 200 μg per animal for booster injection Animals are immunized with vaccine alone, and also in the presence of one of the following different adjuvants alum, copolymer P1005 in saline, copolymer

P1005 in water-in-oil, QS21 (Acquila Biopharmaceuticals, Boston, MA), Freund's complete adjuvant followed by Freund's incomplete adjuvant, and SBAS2 (SmithK ne-Beecham, Belgium) The non- lmmunized group serves as a control After four immunizations, animals are challenged with live parasites and the course of parasitemia is monitored in immunized and non-immunized animals using techniques known to one of ordinary skill in the art Blood samples are collected periodically throughout the trial to evaluate humoral and cellular immune responses to ViVaclp and VιVac2p and to various peptides contained within ViVaclp and VιVac2p

Example 6

Determination of Immunogenicity of Vi aclp and ViVaclp in Inbred Mice

Six- to eight-week-old female, mice from inbred strains, such as C57BL/6 (H-2^b), B 10 BR (H-2^k), and B10 D2 (H-2^d), are immunized subcutaneously with about 10 μg of purified ViVac lp or VιVac2p with alum adjuvant, or with a nonionic copolymer adjuvant PI 005 in a water-in-oil emulsion Control mice receive only either adjuvant A booster dose ( 10 μg/mouse) is given two weeks after the primary immunization Mice are bled at different time intervals, and cellular and humoral immune responses were determined according to published techniques (Lai, A A , et al , (1996) Infect Immun 64, 1054-1059, Coligan, J E , et al , Current protocols in immunology (1996), Vol 1 , pp 2 1 2-2 1 6, pp 3 12 1-3 1 4, pp 6 8 1 -6 8 3, and Vol 2, pp 7 10 1-7 10 6, National Institutes of Health, John Wiley & Sons, Inc ) Example 7

Immunogenicity of ViVaclp and Vι Vac2p in Outbred Mice Using Different Adjuvant-Vaccine Formulations

Outbred ICR mice are immunized subcutaneously with 10 μg of ViVac lp or VιVac2p (two injections total) at two-week intervals in the presence of CpG oligonucleotides, QS21 , copolymer and alum adjuvants Control mice are given adjuvant alone Four days after the second immunization, spleens are collected from two immunized mice and one control mouse The T cell responses to the vaccine and the synthetic peptides corresponding to the epitopes included in the vaccine are evaluated using the in vitro proliferative assay The results are presented as stimulation index (SI) SI values of greater than two are considered positive Total IgG antibody levels against the vaccine antigen in mice at day 45 and day 60 are determined using ELISA

Example 8 Determination of Immune Responses to ViVaclp and ViVaclp in Human Individuals Naturally Exposed to Malaria

Lymphocyte proliferation, cytokine, and antibody responses to ViVac l p and VιVac2p are tested in non-immune children and clinically immune adults from western Kenya, a malaria holoendemic area Finger prick samples of hepannized blood are used in this study The serum samples are used in determining the antibody response against the vaccine antigen and/or peptides in the vaccine antigen using ELISA methodology In the case of T-cell proliferation assays, peripheral blood mononuclear cells (PBMCs) from these individuals are used The PBMCs are cultured in the presence of vaccine antigen. ViVacl p or VιVac2p The T-cell proliferation is measured quantitatively and the cell culture supernatant is used for measuring cytokine levels using published techniques (Lai, A A , et al , (1996) Infect Imm n 64, 1054-1059, Co gan, J E , et al , Current protocols in immunology (1996), Vol 1 , pp 2 1 2-2 1 6, pp 3 12 1 -3 1 4, pp 6 8 1-6 8 3, and Vol 2 pp 7 10 1-7 10 6, National Institutes of Health, John Wiley & Sons, Inc )

All cited patents, publications, and abstracts are incorporated herein by reference in their entirety It will be apparent that the precise details of the compositions and methods described may be varied or modified without departing from the spirit of the described invention We claim all such modifications and variations that fall within the scope and spirit of the claims below

Claims

What is claimed is

1 A recombinant multivalent protein that stimulates an immune response to

Plasmodium vivax, wherein the protein comprises antigenic determinants, fragments thereof, or conservative substitutions thereof, derived from more than one stage in a life cycle of a Plasmodium vivax parasite

2 A pharmaceutical composition comprising an immunogenically effective amount of the recombinant multivalent protein of claim 1

3 A vaccine against malaria comprising the recombinant multivalent protein of claim 1

4 The recombinant multivalent protein of claim 1, wherein the antigenic determinants, fragments thereof, or conservative substitutions thereof, are selected from the group consisting of CSP-1 , Pvs25, CSP-2R, AMA-1 , P2, P30, TRAP, CSP-2R, DBP(II), MSPl -19kD, and combinations thereof

5 The recombinant multivalent protein of claim 1 , wherein the protein comprises a sequence as shown in SEQ ID NO 2 or SEQ ID NO 4

6 The recombinant multivalent protein of claim 1 , wherein the protein comprises the amino acid sequence shown in SEQ ID NO 2

7 The recombinant multivalent protein of claim 1 , wherein the protein comprises the amino acid sequence shown in SEQ ID NO 4

8 An isolated nucleic acid molecule encoding the recombinant multivalent protein of claim 1 9 The nucleic acid molecule of claim 6, comprising a nucleotide sequence as shown in SEQ ID NO 1 , SEQ ID NO 3, SEQ ID NO 10, a fragment thereof, or a conservative substitution thereof

10 A recombinant nucleic acid molecule comprising a promoter sequence operably linked to the nucleic acid molecule of claim 8 1 1 A vector comprising the recombinant nucleic acid molecule of claim 10

12 A nucleic acid molecule comprising nucleotides encoding a protein comprising antigenic determinants, fragments thereof, or conservative substitutions thereof, wherein the antigenic determinants, or fragments thereof, are derived from more than one stage in a life cycle of a parasite, wherein the parasite is Plasmodium vivax 13 A method of eliciting an immune response in an animal, comprising introducing into the animal a composition comprising the recombinant multivalent protein of claim 1 14 The method of claim 13, wherein the protein comprises the amino acid sequence shown in SEQ ID NO 2, SEQ ID NO 4, fragments thereof, or conservative substitutions thereof

15 The method of claim 13, wherein the immune response is a protective immune response that confers increased resistance to infection by Plasmodium parasites 16 The method of claim 15, wherein the Plasmodium parasites are P vivax

17 A method of generating antibodies specific for the recombinant multivalent protein of claim 1 , compπsing introducing into an animal a composition comprising the recombinant multivalent protein

18 The method of claim 17, wherein the recombinant multivalent protein comprises the amino acid sequence shown in SEQ ID NO 2, SEQ ID NO 4, fragments thereof, or conservative substitutions thereof

19 The method of claim 18, further comprising purifying the antibodies from the animal

20 Use of antibodies produced by the method of claim 17 for detection or measurement of antigenic epitopes derived from one or more stages in a life cycle of a parasite wherein the parasite is Plasmodium vivax

21 A method of immunizing an animal comprising administrating to the animal an lmmunogenic effective amount of the recombinant synthetic protein of claim 1 in a pharmaceutically acceptable carrier 22 The method of claim 21 , further comprising administrating an adjuvant

23 The method of claim 21 , wherein the effective amount of the protein is about 1 μg to 1 mg

24 The method of claim 21 , wherein the protein is administered about one to five times to the same animal 25 The method of claim 21 , wherein the animal is a human

26 The method of claim 21 , which provides protection against or amelioration of infection by a Plasmodium vivax parasite

27 A vaccine composition for therapeutic or prophylactic treatment of an animal subject against infection by Plasmodium vivax, comprising the recombinant multivalent protein of claim 1 in an amount sufficient to mediate an immune response when administered to the animal, and a pharmaceutically acceptable carrier or diluent