WO2006019427A2 - Compositions and methods for prophylaxis, treatment and detection of malarial infections - Google Patents

Abstract

Chimeric immunogenic polypeptides are disclosed. The chimeric immunogenic polypeptides comprise peptide eptitopes derived from different stages in the life cycle of the malarial parasite, and, in particular, the Plasmodium falciparum parasite. In some embodiments, spacer amino acid sequences are inserted between epitopes. The disclosed chimeric immunogenic polypeptides can be used to stimulate an immune response to the P. falciparum parasite in a subject.

Description

COMPOSITIONS AND METHODS FOR PROPHYLAXIS, TREATMENT AND DETECTION

OF MALARIAL INFECTIONS

FEELD

[0001] The present invention relates to compositions and methods for prophylaxis, therapy and detection of malarial infections. More specifically, the invention relates to compositions and methods for prophylaxis, treatment and detection of Plasmodium falciparum {P. falciparum) infections.

BACKGROUND

[0002] Malaria is a serious, sometimes fatal, disease caused by protozoal Plasmodium (P.) parasites. Symptoms of malaria include fever, chills, headache, sweating, muscle aches, and tiredness. Nausea, vomiting, diarrhea, anemia, and jaundice also may occur. The appearance of symptoms is dependent on the species of Plasmodium, but can vary from 7 days to 10 months after an initial infection.

[0003] Malaria was successfully eliminated from many countries in temperate climates during the mid 20th century, but recently malaria is re-emerging and has once again becoming a serious health threat to humans. The reemergence of malaria is due largely to the spread of drug-resistant parasite strains, decay of healthcare infrastructure and difficulties in implementing and maintaining mosquito vector control programs in many developing countries. Today, approximately 40% of the world's population, mostly in the world's poorest countries, is at risk of malaria. The World Health Organization estimates that malaria infects an estimated 300-500 million people and kills about 1.5- 3 million people worldwide each year. About 1,200 cases of malaria are also diagnosed in the United States each year, mainly in travelers from areas at high risk for malaria.

[0004] Four species of the protozoal Plasmodium (P.) parasites are commonly known to cause malaria: P. falciparum, P. vivax, P. malariae and P. ovale. The most severe form of malaria is caused by P. falciparum, which results in the majority of clinical cases and nearly all of the deaths and serious morbidity. If left untreated, P. falciparum infections result in a mortality rate as high as 20%.

[0005] Human infection occurs following the bite of malaria-infected female mosquitoes, most commonly female Anopheles mosquitoes. Malaria also occasionally occurs in humans following a blood transfusion or subsequent to needle sharing by intravenous drug users.

[0006] The life cycle of malaria is complex. Initially, a malaria-infected female Anopheles mosquito bites an animal or human and injects the infective sporozoite form of the parasite into the blood stream. Within minutes, these sporozites invade hepatocytes in the liver. The initial development of parasites occurs in the liver and is referred to as the liver stage, or the hepatic or exoerythrocytic stage. In this stage, which occurs over approximately one week, malaria parasites undergo asexual multiplication, producing tens of thousands of merozoite forms of the parasite that ultimately cause the hepatocyte to rupture. Merozoites are then released from the ruptured hepatocyte into the blood stream. Circulating merozoites attach to receptor sites on the surface of erythrocytes (red blood cells) and begin a development stage known as the asexual, erythrocytic stage. Within the erythrocyte, the parasite is recognizable as a ring stage trophozoite. These trophozoites enlarge, divide and attain the schizont stage. During the schizont stage, successive nuclear divisions occur, ultimately resulting in the rupture of the erythrocyte, and release of merozoites into the blood stream. The circulating merozoites then attach to receptors on erythrocytes and begin another erythrocytic cycle. Each erythrocytic cycle last approximately 48-72 hours, with production of 16-20 additional merozoites per red blood cell. Some invading merozoites do not divide, but differentiate into male and female sexual forms. These sexual forms are taken from the blood stream by a feeding female Anopheles mosquito and fertilization occurs in the mosquito midgut to form zygotes. These zygotes further differentiate into motile forms, called ookinetes, migrate through the mosquito gut wall and divide within oocysts on the external gut wall to form thousands of sporozoites. The infective sporozoites move to the salivary gland, where they await injection into another human host, thus completing the life cycle.

[0007] In P. vivax and P. ovale infections, hepatic parasites persist, which may lead to a relapse of the disease months or years after the initial infection.

[0008] Traditionally, efforts to control malaria have concentrated on limiting the population of mosquitoes by spraying insecticides, or on chemical prophylactic measures that limit the progress of an infection. However, these methods of controlling malaria are no longer adequate because of the spread of drag-resistant parasites and insecticide-resistant mosquitoes. Recently, much attention has been focused on the development of an effective malaria vaccine, since vaccines have historically been the most promising approach for cost-effective control of infectious diseases. However, the complex life cycle and distinctive life stages of malaria parasites complicate the development of a malaria vaccine.

[0009] Administration of combinations of malarial epitopes is one approach to inducing protective immune responses to the various life stages of the malarial parasite. Combinations of epitopes that have been investigated include simple mixtures of epitopic peptides, mixtures of fusion proteins where each fusion protein includes a different epitope, and single polypeptide constructs that include multiple epitopes contiguously joined in a single amino acid chain. However, these combinations appear to lack stability and/or are inefficiently processed by the immune system, leading to immune responses to only a fraction of the epitopic sequences administered to a subject and insufficient protection from blood stage forms of the malarial parasite. To date, no effective malaria vaccine has been commercially available.

SUMMARY

[0010] Chimeric polypeptides are disclosed that are useful for prophylaxis, treatment and detection of malarial infections. The chimeric polypeptides include a novel combination of epitopes from the blood and sporozoite stages of the malaria parasite. In particular embodiments, the chimeric polypeptides further include one or more additional epitopes, which may be from one or more life stages of the malaria parasite including the blood, liver and sporozoite stages. The chimeric polypeptides may further include spacer amino acids or spacer amino acid sequences between the epitopes that, in some instances, improve stability and immune system processing of the chimeric polypeptide. Fusion proteins including the disclosed chimeric polypeptides also are provided.

[0011] Recombinant polynucleotides (such as recombinant DNA, cDNA and RNA sequences) are disclosed that code for the disclosed chimeric polypeptides and fusion proteins. Also disclosed are binding agents, such as antibodies, antigen presenting cells (APCs) and immune system effector cells (such as T-cells) that are produced using the chimeric polypeptides, fusion proteins and/or the recombinant polynucleotides.

[0012] The polypeptides and polynucleotides can also be incorporated into pharmaceutical compositions. Such pharmaceutical compositions include the disclosed chimeric polypeptides, fusion proteins, recombinant polynucleotides, binding agents, APCs, and/or immune system cells and a pharmaceutically acceptable carrier. In particular embodiments, the pharmaceutical compositions further include an immunostimulant such as an adjuvant.

[0013] In addition, methods are disclosed for inducing an immune response to a malarial parasite in an animal, for detecting the presence or absence of a malarial parasite in an animal, and for following the progression of a malarial infection in an animal. Such methods employ the disclosed chimeric polypeptides, fusion proteins, polynucleotides and binding agents.

[0014] The foregoing and other embodiments, features and advantages will become more apparent from the following detailed description of several embodiments that proceeds with reference to the accompanying figures and the sequence listing.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIG. 1 is a schematic map of the recombinant chimeric polypeptide FALVAC- 1A-His showing locations of various epitopes and spacers.

[0016] FIG. 2 is a digital representation of a SDS polyacrylamide gel which shows the reproducibility of FALVAC- 1A-His production and purification by the methods described in the Examples. Approximately 3 μg/lane of FALVAC-I A-His isolated from six different bacterial cultures was run on precast 4 ~ 15% gradient bis-Tris gels and stained with Gelcode Blue Stain Reagent. M, Molecular weight standards; Lane 1, F071A; Lane 2, F072A; Lane 3, F134A; Lane 4, F135A; Lane 5, F136A; Lane 6, F137A.

[0017] FIG.3 is a MALDI-TOF mass spectrum of FALVAC-1A-His. The sample was mixed with sinapinic acid matrix, spotted onto a stainless steel target and allowed to dry. The spectrum was collected in positive linear mode using delayed extraction and a nitrogen laser.

[0018] FIG. 4 is far ultraviolet circular dichroism spectra of native FALVAC-1A-His (closed diamonds) and denatured FALVAC-I A-His (closed triangles). The buffer background (10 mM sodium phosphate, 500 mM NaCl pH 7.2) was subtracted from FALVAC-I A-His spectra captured in that buffer. Chemical denaturation was accomplished by addition of 4 M guanidinium HCl. The CDSSTR program within the "CDPro" suite was used to estimate secondary structure contributions as discussed in the Examples.

[0019] FIG. 5 shows reverse-phase HPLC tracings of FALVAC- 1 A (Lot F 134A) in native (top) and reduced (bottom) states, respectively. As described in more detail in the Examples, samples were acidified and injected on a C_]8 reverse phase column and eluted between 5%-90% buffer B (0.1%TFA/80% acetonitrile/H₂0) over 35 minutes. The reduced sample was incubated at 37⁰C for 30 minutes with 2M urea plus 10OmM DTT prior to acidification and injection. [0020] FIG. 6 is two graphs ((A) and (B)) showing the time courses of rabbit antibody responses to FALVAC-I A-His. Two rabbits in each group were immunized at weeks 0, 4, 8 and 25 with 50 μg FALVAC-IA-His formulated with W/O copolymer emulsion (♦), QS-21 (α), ISA-720 (O) and aluminum phosphate (A), and bled out after the final boost. Rabbits in the same group are shown by a dashed and a solid line having the same symbol. One rabbit received W/O copolymer emulsion (0) alone, and another aluminum phosphate (■) alone. ELISA titers against FALVAC-IA were determined by titration. Curves for each rabbit are shown.

[0021] FIG. 7 is a graph showing antibody-dependent cellular inhibition (ADCI) activity of final bleed anti-F ALV AC-I A-His rabbit sera. The rabbits were immunized as described in Example 4. The number of separate ADCI determinations (n) for each rabbit serum and the animal number are shown. "Pos" is the human ADCI positive control.

[0022] FIG. 8 is a graph showing the time course of mouse (strain C57BL/6 (H-2b)) antibody responses to FALVAC-I A-His (as measured by ELISA). Mice were immunized as described in Example 10 with FALVAC-IA-His formulated with W/O copolymer emulsion (♦), QS-21 (■), ISA-720 (o), aluminum phosphate (A), or no adjuvant (□).

[0023] FIGs. 9 is a bar graph showing IFN-G ELISPOT results from C57BL/6 mice immunized (s.c.) on days 0 and 14 with 10 μg FALVAC-IA-His formulated with the indicated adjuvant or PBS alone (no antigen). The spleen cells were harvested on day 23 and stimulated in vitro with culture medium (clear bars), Concanavalin A (ConA, stippled bars), bovine serum albumin (BSA; vertically dashed bars), 0.2 μg FALVAC-IA-His (diagonally hatched bars), or 0.1 μg FALVAC-IA-His (wavy line bars).

[0024] FIG. 10 is a bar graph showing IL-4 ELISPOT results from C57BL/6mice immunized (s.c.) on days 0 and 14 with 10 μg FALVAC-IA-His formulated with the indicated adjuvant or PBS alone (no antigen). The spleen cells were harvested on day 23 and stimulated in vitro with culture medium (clear bars), Concanavalin A (Con A; stippled bars), bovine serum albumin (BSA; vertically dashed bars), 0.2 μg FAL VAC-I A-His (diagonally hatched bars), or 0.1 μg FALVAC-IA-His (wavy line bars).

[0025] FIG. 11 is a bar graph showing the serological responses of Aotus monkeys. The animals were immunized intramuscularly with one of the following formulations on days 0, 28, and 84: (i) Montanide ISA-720 alone; (ii)-Montanide ISA 720 plus 50 ug FALVAC-IA; (iii) 50 ug QS-21 alone; (iv) 50 ug QS-21 plus 50 ug FALVAC-IA; (v) 200 ug AlPO₄ alone; or (vi) 200 ug AlPO₄ plus 50 ug FALVAC-IA. The animals were bled on day 98 and antibody responses determined by ELISA against FALVAC-IA (stippled bars) and IFA against P. falciparum sporozoites (horizontal dashed bars) and blood stages (diagonally hatched bars), respectively. The bars represent the geometric mean titer of the 6 animals in each group.

[0026] FIG. 12 is a graph showing the time course of Aotus cellular proliferative responses to FALVAC-IA. The animals were immunized intramuscularly with one of the following formulations on days 0, 28, and 84: (i) Montanide ISA-720 alone (α); (ii) Montanide ISA 720 plus 50 ug FALVAC-IA (■); (iii) 50 ug QS-21 alone (o); (iv) 50 ug QS-21 plus 50 ug FALVAC-IA (•); (v) 200 ug AlPO₄ alone (0); or (vi) 200 ug AlPO₄ plus 50 ug FALVAC-IA (♦). The monkeys were bled on days O, 28, 56, 98 and 168, and isolated peripheral blood mononuclear cells were cultured with FALVAC-IA to determine lymphocyte proliferation by tritiated thymidine incorporation. The curves are the geometric mean Experimental/Control stimulation values for the monkeys in each group.

SUMMARY OF THE SEQUENCE LISTING

[0027] The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter codes 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:

[0028] SEQ ID NO: 1 is a nucleic acid sequence encoding a representative FALVAC-I A-His chimeric polypeptide that includes an optional polyhistidine affinity tag.

[0029] SEQ ID NO: 2 is an amino acid sequence of a representative FALVAC-1A-His chimeric polypeptide having an optional polyhistidine affinity tag to aid in its purification.

[0030] SEQ ID NO: 3 is an amino acid sequence of a representative P. falciparum sporozoite stage p592 epitope.

[0031] SEQ ID NO: 4 is an amino acid sequence of a representative P. falciparum sporozoite stage p593 epitope.

[0032] SEQ ID NO: 5 is an amino acid sequence of a representative P. falciparum sporozoite stage p594 epitope.

[0033] SEQ ID NO: 6 is an amino acid sequence of a representative P. falciparum blood stage p597 epitope.

[0034] SEQ ID NO: 7 is an amino acid sequence of a representative P. falciparum blood stage p598 epitope.

[0035] SEQ ID NO: 8 is an amino acid sequence of a representative P. falciparum liver stage p595 epitope.

[0036] SEQ ID NO: 9 is an amino acid sequence of a representative P. falciparum sporozoite stage p519 epitope.

[0037] SEQ ID NO: 10 is an amino acid sequence of a representative P. falciparum blood stage p600 epitope.

[0038] SEQ ID NO: 11 is an amino acid sequence of a representative P. falciparum blood stage p601 epitope.

[0039] SEQ ID NO: 12 is an amino acid sequence of a representative P. falciparum blood stage p545 epitope.

[0040] SEQ ID NO: 13 is an amino acid sequence of a representative P. falciparum liver stage p596 epitope.

[0041] SEQ ID NO: 14 is an amino acid sequence of a representative P. falciparum blood stage p606 epitope. [0042] SEQ ID NO: 15 is an amino acid sequence of a representative P. falciparum blood stage p543 epitope.

[0043] SEQ ID NO: 16 is an amino acid sequence of a representative P. falciparum blood stage p544 epitope.

[0044] SEQ ID NO: 17 is an amino acid sequence of a representative P. falciparum blood stage p546 epitope.

[0045] SEQ ID NO: 18 is an amino acid sequence of a representative P. falciparum blood stage p603 epitope.

[0046] SEQ ID NO: 19 is an amino acid sequence of a representative P. falciparum blood stage p602 epitope.

[0047] SEQ ID NO: 20 is an amino acid sequence of a representative P. falciparum blood stage p607 epitope.

[0048] SEQ ID NO: 21 is an amino acid sequence of a representative P. falciparum blood stage p604 epitope.

[0049] SEQ ID NO: 22 is an amino acid sequence of a representative P. falciparum blood stage p605 epitope.

[0050] SEQ ID NO: 23 is an amino acid sequence of a representative P. falciparum blood stage p599 epitope.

[0051] SEQ ID NO: 24 is a FALVAC-IA forward primer.

[0052] SEQ ID NO : 25 is a FALVAC- 1 A reverse primer.

[0053] SEQ ID NOs: 26-31 are amino acid sequences of exemplar reverse-turn spacers; GPGPG (SEQ ID NO: 26) (see also, e.g, residues 18-22, 93-97, 140-144, 215-219, or 314-318 of SEQ ID NO: 2); PGPG (SEQ ID NO: 27); GGPGG (SEQ ID NO: 28); APAPA (SEQ ID NO: 29); GGGG (SEQ ID NO: 30); and APGPA (SEQ ED NO: 31). Other representative reverse-turn sequences (which need not be listed in the Sequence Listing) are shown throughout the specification.

DETAILED DESCRIPTION

I. Introduction

[0054] The disclosure generally relates to compositions and methods for the prophylaxis, therapy and diagnosis of malarial infections, for example, P. falciparum infections. The compositions include chimeric polypeptides that include epitopes of P. falciparum proteins linked into a single polypeptide chain. In particular, the disclosed chimeric polypeptides include a novel combination of one sporozoite stage epitope and four blood stage epitopes (p545, p546, p594, p606 and p607) that provides increased protection against blood stage forms of the parasite and invasion of the parasite into blood cells. It is during the blood stage of malaria (when infected red blood cells that are incubating thousands of parasites literally explode and release more parasites into the blood stream) that the symptoms of malaria occur. These symptoms include fever and flu-like symptoms such as chills, headache, muscle aches and fatigue. Immunity is slow to develop without stimulation of a subject's immune system, and left untreated, malaria may be fatal, taking its greatest toll in children. [0055] Also disclosed are polynucleotides encoding the disclosed chimeric polypeptides. Further, binding agents such as antibodies; antigen presenting cells (APCs); and/or immune system cells (such as T cells) are described. Fusion proteins comprising the disclosed chimeric polypeptides are also included in the disclosure, and such fusion proteins may or may not include additional immunogenic peptide sequences.

[0056] In general, polypeptides (including fusion proteins) and polynucleotides as described herein are isolated.

[0057] In one embodiment, a chimeric polypeptide includes at least four blood stage epitopes such as p545, p546, p606 and p607 and at least one sporozoite stage epitope such as p594, in any order, is provided. Chimeric polypeptides containing functional variants of these epitopes and functional variants of the chimeric polypeptide in which the order of the epitopes (or their functional equivalents) along the chimeric polypeptide's sequence is altered, which variants maintain the function (such as antigenicity or immunogenicity) of the epitopes or chimeric polypeptide also are contemplated. For example, such functional variants of the epitopes can include sequences having at least 90%, such as at least 95% or at least 98% sequence identity to each of the p545, p546, p594, p606 and p607 epitopes (SEQ ID NOs: 12, 17, 5, 14, and 20, respectively). However, in particular examples, the epitopes are those shown in SEQ ID NOs: 5, 12, 17, 14 and 20. Functional variants also include chimeric polypeptides in which the position of the p545, p546, p594, p606 and p607 epitopes in polypeptide sequence varies in any order. In more particular embodiments, these epitopes are arranged in the order shown in SEQ ID NO: 2.

[0058] In particular embodiments, the chimeric polypeptide may further include one or more other blood stage sporozoite, and/or liver stage epitopes. For example, the chimeric polypeptide may further include one or more (such as at least 3, at least 7 or at least 11) additional blood stage epitopes, one or more (such as at least 1, at least 2 or at least 3) additional sporozoite stage epitopes, and one or more additional (such as at least 1 or at least 2) epitopes from the liver stage.

[0059] In more particular embodiments, the chimeric polypeptide may include one or more of (such as at least 5, at least 10, at least 15 or all 16 of) the p519, a p543 epitope, a p544 epitope, a p592 epitope, a p593 epitope, a p595 epitope, a p596 epitope, a p597 epitope, a p598 epitope, a p599 epitope, a p600 epitope, a p601 epitope, a p602 epitope, a p603 epitope, a p604 epitope, a p605 epitope, including functional variants thereof. Functional variants of these epitopes can, for example, include sequences having at least 90%, at least 95% or at least 98% sequence identity to SEQ ID NOs: 3, 4, 6, 7, 8, 9, 10, 11, 13, 14, 16, 18, 19, 21, 22 and 23. In particular embodiments, the chimeric polypeptide combines the epitopic peptide sequences set forth as SEQ ID NOs: 3-23, or sequences having at least 90%, at least 95% or at least 98% sequence identity to SEQ ID NOs: 3-23. In other more particular embodiments, the disclosed chimeric polypeptide includes residues 3-374 of SEQ ID NO: 2, residues 1-374 of SEQ ID NO: 2, the entire sequence set forth as SEQ ID NO: 2, or sequences at least 90%, at least 95% or at least 98% identical thereto.

[0060] Disclosed chimeric polypeptides may further include one or more spacers, such as GPGPG (SEQ ID NO: 26), KAA and KG spacers. The chimeric polypeptides may also further include an affinity or targeting sequence, for example, a poly-histidine affinity tag. Spacers may be included to promote folding of the chimeric polypeptide which may improve stability of the chimeric polypeptide in solution, such that pharmaceutical compositions including the chimeric polypeptide are able to be stored for longer periods of time, or, for example, stored without constant refrigeration, without a substantial loss in activity or function. For example, the GPGPG spacer (SEQ ID NO: 26) and other related turn-inducing spacers, or KG and other related flexible spacers, can promote folding of the polypeptide chain. Others spacers, such as KAA and related spacers can aid in stimulating an immune response to the epitopes in the chimeric polypeptide, particularly by the Th-I pathway. Typically, such spacers will be from 1-10 amino acids in length, such as between 2 and 7 amino acids in length, such as between 2 and 5 amino acids in length. In particular embodiments, the chimeric polypeptide includes at least 6 spacers, such as at least 8, at least 10 or at least 12 spacers separating adjacent epitopes. As used herein "separating adjacent epitopes" refers to spacers that are positioned between the epitopic peptide sequences in the chimeric polypeptide along the sequence of the chimeric polypeptide. For example, in a chimeric polypeptide such as NH₂-p545- spacer-p546-spacer-p606-spacer-p607-COOH, each of the spacers is "separating adjacent epitopes," and since there are multiple different epitopes that are adjacent and separated by spacers, the chimeric polypeptide also includes "spacers separating multiple different adjacent epitopes."

[0061] Also disclosed are polynucleotides coding for the disclosed immunogenic chimeric polypeptides (and functional variants thereof), antibodies that bind such chimeric polypeptides, combinations of antibodies that bind more than about 75% of the epitope sequences in a disclosed chimeric polypeptide, and cells that express polynucleotides that code for the disclosed chimeric polypeptides.

[0062] Pharmaceutical compositions are also disclosed. In one embodiment, the pharmaceutical composition includes a disclosed chimeric polypeptide and a pharmaceutically acceptable carrier. Such pharmaceutical compositions may further include an immunostimulant such as an adjuvant. In another embodiment, the pharmaceutical composition includes a binding agent that specifically binds a disclosed chimeric polypeptide and a pharmaceutically acceptable carrier. The binding agent in such pharmaceutical compositions may be, for example, an antibody or an immune system effector cell.

[0063] A method for stimulating an immune response in a subject is disclosed. In one embodiment, the method includes administering to the subject an effective amount of a pharmaceutical composition comprising a disclosed chimeric polypeptide. A method for treating a subject with a malarial infection is also provided. In this method an effective amount of a pharmaceutical composition comprising a binding agent that specifically binds to a disclosed chimeric polypeptide is administered to the subject.

[0064] A method for detecting a malarial infection in a subject is also provided. In one embodiment, a method for detecting a malarial infection includes contacting a biological sample obtained from a subject with a binding agent that specifically binds to a disclosed chimeric polypeptide. The level of a polypeptide that binds to the binding agent is detected, and compared to a predetermined level of the polypeptide to determine if the subject has a malarial infection. //. Abbreviations and Terms

[0065] ADCI Antibody dependent cellular inhibition

[0066] CTL Cytotoxic T lymphocyte

[0067] CSP Circumsporozoite protein

[0068] IFN-G Interferon gamma

[0069] IL-4 Interleukin-4

[0070] ISI Inhibition of sporozoite invasion

[0071] GIA Growth inhibition assay

[0072] SSP Sporozoite surface protein

[0073] MSP-I Merozoite surface protein- 1

[0074] MSP-2 Merozoite surface protein-2

[0075] LSA-I Liver stage antigen- 1

[0076] RAP-I Rhoptry associated protein- 1

[0077] EBA-175 Erythrocyte-binding antigen- 175

[0078] AMA-I Apical membrane antigen- 1

[0079] Th-I Helper T cells- 1

[0080] Th-2 Helper T cells-2

[0081] IPTG Isopropyl thiogalactoside

[0082] APC Antigen presenting cell

[0083] mL milliliter

[0084] mg milligram

[0085] μg or ug microgram

[0086] ELISA Enzyme-linked immunoassay

[0087] IFA Immunofluorescence assay

[0088] MHC Major Histocompatibility Complex

[0089] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skilled in the art to which this invention belongs. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, Oxford University Press, 1994; Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, Blackwell Science Ltd., 1994; and Robert A. Meyers (ed.)_; Molecular Biology and Biotechnology: A Comprehensive Desk Reference, VCH Publishers, Inc., 1995. In order to facilitate review of the various embodiments of the invention, the following explanations of specific terms are provided:

[0090] Active immunity refers to immunity mediated by the immune system of an animal. Active immunity is conferred by exposure to an immunogenic agent, either upon initial exposure, or by previous exposure to the immunogenic agent through, for example, immunization or previous infection.

[0091] Adjuvant: A compound, composition, or substance that when used in combination with an immunogenic agent augments or otherwise alters or modifies a resultant immune response. For example, an adjuvant may increase the titer of antibodies induced in an animal by the immunogenic agent. In another example, if the antigenic agent is a multivalent antigenic agent, an adjuvant may alter the particular epitopic sequences that are specifically bound by antibodies induced in an animal.

[0092] Administer (or Administration): Provided or given to an animal or human by any effective route, for example, but not limited to, oral, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, or intravenous), sublingual, buccal, rectal, transdermal, intranasal, vaginal or inhalation routes.

[0093] Amino acid (or Residue or Amino acid residue): Used interchangeably to refer to an amino acid (D or L) or an amino acid mimetic that is incorporated into a peptide by an amide bond or amide bond mimetic. 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 (that is, amino acid mimetics). Moreover, an amide bond mimetic includes peptide backbone modifications well known to those of oridinary skill in the art. The twenty naturally occurring amino acids and their single-letter and three-letter designations are commonly known in the art.

[0094] Animal: Living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds. The term mammal includes both human and non-human mammals (for example, veterinary mammals such as dogs, cats, pigs, horses and cattle).

[0095] Antibody: An intact immunoglobulin or an antigen-binding portion thereof, which immunoglobulin or antigen-binding portion can specifically bind to an antigen. Antigen-binding portions (also called, antibody fragments) may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins. Antigen-binding portions include, inter alia, Fab, Fab', F(ab')₂, Fv, dAb (Fd), and complementarity determining region (CDR) fragments, single-chain antibodies (scFv), chimeric antibodies, diabodies and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide. A Fab fragment is a monovalent fragment consisting of the VL, VH, CL and CHl domains; an F(ab')₂ fragment is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; an Fd fragment consists of the VH and CHI domains; an Fv fragment consists of the VL and VH domains of a single arm of an antibody; and a dAb fragment consists of a VH domain (see, e.g., Ward et al., Nature, 341:544-546, 1989).

[0096] Antigen: A compound, composition, or substance that can stimulate an immune response, such as antibody production or a T-cell response, in an animal, including compositions that are injected or absorbed into an animal. An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous immunogens. The term "antigen" includes all related antigenic epitopes.

[0097] Antigenicity refers to the ability to specifically bind to the antigen binding site of an antibody or T-cell receptor for the antigen.

[0098] Cellular immunity; or Cell mediated immunity: An immune response mediated by cells or the products they produce, such as cytokines, rather than by an antibody. It includes, but is not limited to, delayed type hypersensitivity and cytotoxic T cells.

[0099] Chimeric polypeptide (or chimera): A chimeric polypeptide is a polypeptide with a sequence of amino acids that includes multiple, different subsequences of two or more different protein sequences. Chimeric polypeptides also can include spacer amino acids or spacer amino acid sequences that separate the subsequences and/or have other structural roles in the chimera (such as, providing secondary and tertiary structure to the chimeric polypeptide and/or aiding in epitope processing of one or more subsequences of the chimera). Exemplar chimeric polypetides including antigenic or immunogenic peptide sequences from proteins expressed during one or more life stages of P. falciparum are described throughout this specification.

[0100] Conservative substitutions: A conservative substitution is an amino acid substitution that does not substantially affect the charge, hydrophobicity, and/or function of a protein, chimeric polypeptide or peptide (as further described below). A "conservative variant" is a peptide, chimeric polypeptide or protein with a sequence that is derived from the sequence of another peptide, chimeric polypeptide or protein by one or more conservative substitutions of amino acids. Conservative variants can substantially retain the charge, hydrophobicity, or function of the peptide, chimeric polypeptide or protein from which they are derived.

[0101] Degenerate variant: A polynucleotide encoding a peptide, chimeric polypeptide or protein that includes a sequence that is degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included in the invention as long as the amino acid sequence of the peptide, chimeric polypeptide or protein encoded by the nucleotide sequence is unchanged.

[0102] Effective amount: An amount or concentration of a disclosed composition that is effective to produce a protective immune or therapeutic response with respect to the disease malaria, as more fully described below.

[0103] Encode (or Encoded protein): A polynucleotide is said to "encode" a polypeptide if, in its native state or when manipulated by methods well known to those of ordinary skill in the art, it can be transcribed and/or translated to produce the mRNA for and/or the polypeptide or a fragment thereof. The anti-sense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom. The polypeptide is said to be the "encoded protein" of the polynucleotide.

[0104] Epitope (or Antigenic epitope): An antigenic determinant. These are particular chemical groups or peptide sequences that are antigenic (specifically recognized by an antibody or a T-cell receptor) and/or are immunogenic (elicit a specific immune response). An antibody specifically binds a particular epitope. Where the epitope is a peptide sequence, the epitope may be referred to as an "epitopic peptide." Such sequences typically have a minimum sequence of about 6-8 amino acids, which sequence is immunogenic when removed from its natural context (for example, in a malarial parasite protein), or when introduced into a heterologous polypeptide (such as a disclosed chimeric polypeptide). Also, such sequences typically have a maximum sequence of about 100 amino acids, for example, about 50, 25 or 18 amino acids in length. Examples of epitopes include P. falciparum epitopes, which are described throughout this specification. Specific exemplar P. falciparum epitope sequences are not intended to be limiting because an epitope can be defined by antigenicity or immunogenicity. In addition, a given epitope may vary among P. falcipai-um strains. Such epitope variants may differ in both the number and composition of the constituent amino acids. Thus, alternative sequences that are functional variants of a specifically disclosed P. falciparum epitope sequence also are contemplated (as described in additional detail below).

[0105] Epitope processing: An in vivo process by which molecules such as proteins and polypeptides are processed so that individual component epitopes are presented to the immune system to generate an immune response. Epitope processing is important, for example, in inducing an immune response by cytotoxic T lymphocytes. In a particular embodiment, a multi-stage, multivalent chimeric polypeptide including P. falciparum epitopes is processed to yield the component epitopes for presentation by antigen-presenting cells.

[0106] Expression cassette (or Recombinant expression cassette): A nucleic acid construct, generated recombinantly or synthetically, with nucleic acid elements that are capable of effecting expression of a structural gene in hosts compatible with such sequences. Expression cassettes include at least promoters and optionally, transcription termination signals. Typically, the recombinant expression cassette includes a nucleic acid to be transcribed (for example, a nucleic acid encoding a desired polypeptide), and a promoter. Additional factors necessary or helpful in effecting expression may also be used as described herein. For example, an expression cassette can also include nucleotide sequences that encode a signal sequence that directs secretion of an expressed protein from the host cell. Transcription termination signals, enhancers, and other nucleic acid sequences that influence gene expression, can also be included in an expression cassette.

[0107] Expression control sequences: Nucleic acid sequences that regulate the expression of a nucleic acid sequence to which it is operably linked. Expression control sequences are operably linked to a nucleic acid sequence when the expression control sequences control and regulate the transcription and, as appropriate, translation of the nucleic acid sequence. Thus expression control sequences can include appropriate promoters, enhancers, transcription terminators, a start codon (that is, ATG) in front of a protein-encoding gene, splicing signal for introns, and maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons. The term control sequences is intended to include, at a minimum, components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences. Expression control sequences can include a promoter.

[0108] Expression system: A combination of a host and a vector which provides a genetic context for making a cloned gene function, that is to produce peptide, in the host cell. A variety of expression systems may be employed for expression of the recombinant protein and are known by persons of ordinary skill in the art. Such expression methods include, but are not limited to the following: Bacterial expression systems, including those utilizing Eschericia coli (E. coli) and Bacillus subtilis (B. subtilis); vaccinia virus systems; yeast expression systems; cultured insect and mammalian cells; and other expression systems known to one of ordinary skill in the art.

[0109] Functional variant: Sequence alterations in an epitope, wherein the epitope with the sequence alterations retains a function or property (such as antigenicity or imrnunogenicity) of the unaltered epitope. For example, a functional variant of an epitope can specifically bind an antibody that binds an unaltered form of the epitope or stimulates T-cell proliferation to an extent that is substantially the same as the unaltered form of the epitope. This and other examples are described in more detail below.

[0110] Humoral immunity refers to immunity that can be transferred with immune serum from one animal to another. Typically, humoral immunity refers to immunity resulting from the introduction of specific antibodies and/or stimulation of the production of specific antibodies.

[0111] Immune (or immunity): The state of being able to mount a protective response upon exposure to an immunogenic agent. The protective response may, for example, be antibody mediated or immune-cell mediated, and may be directed toward a particular pathogen such as a malarial parasite (or a particular life-stage thereof). Immunity may be acquired actively (such as by exposure to an immunogenic agent, either naturally or in a pharmaceutical composition) or passively (such as by administration of antibodies).

[0112] Immune reactive (or immunogenic): Terms used to describe agents that elicit an immune response by an animal's immune system. Immunogenic agents include antigens such as epitopic polypeptide sequences and chimeric polypeptides comprising the same.

[0113] Immune response refers to a response to an immunogenic agent in an animal that provides protection for the animal from the immunogenic agent and/or the source of the immunogenic agent. For example, the response may protect an animal from infection by a pathogen, or interfere with the progression of an infection by a pathogen. An immune response may be active and involve stimulation of the animal's immune system, or be a response that results from passively acquired immunity. Examples of immune responses include the response of a cell of the immune system, such as a B cell, a T cell, or a monocyte, to an immunogenic stimulus. The response may be specific for a particular antigen (an "antigen- specific response"). In a particular embodiment, an immune response is a T cell response, such as a CD4+ response or a CD8+ response. In another particular embodiment, the response is a B cell response, and results in the production of specific antibodies to the immunogenic agent.

[0114] Immunogenicity refers to the ability of an agent to induce a humoral or cellular immune response. Immunogenicity can be measured, for example, by the ability to bind to an appropriate MHC molecule (such as an MHC Class I or II molecule) and to induce a T-cell response or to induce a B-cell or antibody response, for example, a measurable cytotoxic T-cell response or a serum antibody response to a given epitope. Immunogenicity assays are well-known in the art and are described, for example, in Paul, Fundamental Immunology, 3rd ed., 243-247 (Raven Press, 1993) and references cited therein.

[0115] Isolated: An "isolated" biological component (such as, a nucleic acid, peptide or protein) has been substantially separated, produced apart from, or purified away from other biological components present in a mixture of such components formerly including the isolated component (such as, an intact cell, cell lysate, or reaction mixture). Nucleic acids, peptides and proteins which have been "isolated" include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids, peptides and proteins prepared by recombinant expression in a host cell as well as chemically synthesized peptides, proteins and nucleic acids. Samples of isolated biological components include samples of the biological component wherein the biological component represents greater than 90% (for example, greater than 95%, such as greater than 98%) of the sample.

[0116] Isopropyl thiogalactoside (IPTG): An artificial inducer of the Lac operon. It is used to induce the lac operon because, in contrast to allolactose, which is the natural inducer of the operon, IPTG cannot be hydrolyzed and broken down by the cell. Hence, its concentration does not change during induction of expression.

[0117] Junctional epitope: An epitope that comprises some portion of two or more epitopes in close proximity. Functional epitopes may create undesired irnmunodominance effects, redirecting the immune response to the combination of epitopes rather than the desired epitopes. For example, a linear arrangement of two peptide epitopes in a polypeptide chain can result in a junctional epitope between them, and recognition of the two component epitopes by the immune system may be masked. Alternatively, folding of polypeptide chain including a number of epitopes can create a junctional epitope by bring two or more epitopes in close proximity.

[0118] Lymphocytes are a type of white blood cell that is involved in the immune defenses of the body. B-cells and T-cells are two main types of lymphocytes.

[0119] Mimetic refers to a molecule (such as an organic chemical compound) that mimics the activity of another molecule.

[0120] Multistage refers to more than one stage in the life cycle of an organism. For example, multistage may refer to the life stages of P. falciparum, which include the sporozoite stage, the liver stage, the blood (erythrocytic) stage and the sexual stage.

[0121] Multivalent refers to a molecule comprising more than one epitope.

[0122] The terms nucleic acid or nucleic acid molecule are used interchangeably to refer to a polymer of nucleotides (polynucleotides) and includes both sense and antisense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above. A nucleotide refers to a ribonucleotide, deoxynucleotide or a modified form of either type of nucleotide. Polynucleotides may include naturally-occurring, modified or non-naturally-occurring nucleotides linked together by naturally- occurring and/or non-naturally-occurring nucleotide linkages.

[0123] The nucleic acid molecules may be modified chemically or biochemically or may contain non-natural or derivatized nucleotide bases. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with analogs, and internucleotide linkage modifications. The term "nucleic acid molecule" also includes any topological conformation, including single-stranded, double-stranded, partially duplexed, triplexed, hairpinned, circular and padlocked conformations. Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions. Such molecules are known and include, for example, molecules in which peptide linkages are substituted for phosphate linkages in the backbone.

[0124] Nucleotide refers to a subunit of DNA or RNA consisting of a nitrogenous base (adenine, guanine, thymine, or cytosine in DNA; adenine, guanine, uracil, or cytosine in RNA), a phosphate molecule, and a sugar molecule (deoxyribose in DNA and ribose in RNA). [0125] Passive immunity refers to immunity acquired by the introduction by immune system components into an animal rather than by stimulation

[0126] Polypeptide refers to polymers of amino acids (typically L-amino acids) or amino acid mimetics linked through peptide bonds or peptide bond mimetic to form a chain or peptide sequence. The terminal amino acid at one end of the chain typically has a free amino group (the amino-terminus), while the terminal amino acid at the other end of the chain typically has a free carboxyl group (the carboxy terminus). Typically, the amino acids making up a peptide, polypeptide or protein are numbered in order, starting at the amino terminal amino acid and increasing in the direction toward the carboxy terminal amino acid of the peptide. Thus, when one amino acid is said to "follow" another, that amino acid is positioned closer to the carboxy terminal of the peptide than the preceding amino acid. In particular examples, the polypeptides are 2-25 amino acid residues in length, 25-400 amino acid residues in length, or more than 200 amino acid residues in length. A "peptide" typically refers to smaller polypeptides, for example those having between 2 and 25 amino acid residues, for example no more than 4, 8, 10, 12, 14, 16, 18, or 20 amino acid residues. "Polypeptides" may comprise naturally occurring sequences joined in a non-natural polymer. "Protein," as used herein, typically refers to amino acid polymers having 200 or more amino acid residues, especially if the polymer is naturally occurring. Non-natural combinations of naturally- and/or non-naturally occurring sequences of amino acids may also be referred to as "fusion proteins."

[0127] Peptide Modifications refer to synthetic and recombinant embodiments of the peptides described herein, including analogues (non-peptide organic molecules), derivatives (chemically functionalized peptide molecules obtained starting with the disclosed peptide sequences) and conservative variants or optical isomers thereof (homologs).

[0128] Prophylaxis or treating a disease: "Prophylaxis" of a disease refers to inhibiting the partial or full development of a disease, for example, inhibiting the progression of a P. falciparum infection in a subject exposed to the P. falciparum parasite. "Treating" refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition, for example, a sign or symptom associated with a P. falciparum infection or a pathological condition associated with such an infection.

[0129] Protein expression: Production of proteins from encoding genes or nucleic acid molecules using a protein expression system, for example a prokaryotic, an eukaryotic, or a cell-free expression system.

[0130] Purified: The term purified does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified peptide preparation is one in which the peptide or protein is more enriched than the peptide or protein is in its natural environment within a cell. In one embodiment, a preparation is purified such that the purified protein or peptide represents at least 50% of the total peptide or protein content of the preparation.

[0131] Quantitating: Determining a relative or absolute quantity of a particular component in a sample. In the context of quantitating antibodies in a sample of a subject's blood to detect a malarial infection, quantitating refers to determining the quantity of antibodies using an antibody assay, for example, an ELISA-assay or a T-cell proliferation assay. [0132] Recombinant: 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 is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques. Similarly, a recombinant protein is one encoded for by a recombinant nucleic acid molecule.

[0133] Reverse Turn: A region of a biological polymer, such as a polypeptide or a polynucleotide, where the backbone chain is folded back on itself (such as at an angle greater than 90 degrees). Reverse turns may be stabilized, for example, by hydrogen bonding. For example, a reverse turn in a polypeptide may be stabilized by a hydrogen bond from one main chain carbonyl oxygen to the main chain N-H group that is 3 residues along the sequence (for example, O(i) to N(i+3), or O(i) to N(i-3)). Reverse turns may lead to a more compact tertiary structure for a polypeptide chain, thereby increasing stability.

[0134] Spacer: A spacer amino acid or a spacer amino acid sequence. A spacer serves to join peptide sequences, such as epitopic peptide sequences, in a single polypeptide, for example, a chimeric polypeptide. A "spacer amino acid" is a single amino acid that serves to link two peptide subsequences in a single polypeptide sequence. For example, the amino acid glycine (G) can be placed between two epitopes in a disclosed chimeric polypeptide to provide flexibility of the chimeric polypeptide so that it can more readily fold or rotate about the polypeptide chain in solution and assume a stable secondary and/or tertiary structure. A "spacer amino acid sequence" (or spacer peptide sequence) is two or more amino acids that serve to link two peptide subsequences in a single polypeptide sequence. Examples of spacer amino acid sequences include, but are not limited to, GPGPG (an exemplar reverse turn-inducing sequence; SEQ ID NO: 26), KAA, RAA, CAA, NAA, GAA, and/or KG. Such spacer sequences can facilitate folding of a polypeptide into a stable secondary and/or tertiary structure, and/or serve to improve epitope processing of epitopes in the resulting polypeptide. Spacers that facilitate folding of a polypeptide into a stable secondary and/or tertiary structure may be referred to as "structure-stabilizing spacers."

[0135] Specifically bind: Refers to the situation where two components bind to one another to the substantial exclusion of binding with other components that may be present. In general, two compounds are said to "specifically bind" when the binding constant for complex formation between the components exceeds about 10⁴ L/mol, for example, exceeds about 10⁶ L/mol, exceeds about 10⁸ L/mol, or exceeds about 10¹⁰ L/mol. The binding constant for two components may be determined using methods that are well known in the art.

[0136] Stage, Cycle, or Phase: An identifiable (such as by morphological, etiological, histological, symptomatic or pathological means) period in the growth and/or sexual development of an organism, especially the growth and/or sexual development of a parasite. For example, when referring to the life of the malaria parasite, the terms "stage," "cycle" or "phase" are used interchangeably with reference to the progression of forms and the forms themselves that accompany the development and progression of a malaria infection and transmission thereof from one organism to another. Examples of stages of the life cycle of a malaria parasite (for example, P. falciparum) include the sexual stage, the blood stage, the liver stage and the sporozoite stage.

[0137] Subject: A living multi-cellular vertebrate ("animal"), and a category that includes both human and veterinary subjects, including human and non-human mammals.

[0138] Substantially identical: In the context of two nucleic acids or polypeptides, substantially identical refers to two or more sequences or subsequences that have at least 60%, for example, at least 80%, such as at least 90%, 95%, or 98% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using sequence comparison algorithms or by visual inspection. In one embodiment the substantial identity exists over a region of the sequences that is at least about 50 residues in length, in another embodiment over a region of at least about 100 residues, and in yet another embodiment the sequences are substantially identical over at least about 150 residues.

[0139] Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in Smith and Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J. MoI. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sd. U.S.A. 85:2444, 1988; Higgins and Sharp, Gene 73:237-244, 1988; Higgins and Sharp, CABIOS 5:151-153, 1989; Corpet et ah, Nucleic Acids Research 16:10881-10890, 1988; Pearson and Lipman, Proc. Natl. Acad. Sd. U.S.A. 85:2444, 1988; and Altschul et ah, Nature Genet. 6:119-129, 1994. In addition, the NCBI Basic Local Alignment Search Tool (BLAST™) (Altschul et ah, J. MoI. 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.

[0140] Unit dose: A physically discrete unit containing a predetermined quantity of an active material calculated to individually or collectively produce a desired effect such as an immunogenic effect, possibly in association with a diluent; carrier or vehicle. A single unit dose or a plurality of unit doses can be used to provide the desired effect, such as an immunogenic effect.

[0141] Vaccine: An immunogenic composition that may, for example, be administered to an animal or a human to confer immunity, such as active immunity, to a disease or other pathological condition. Vaccines can be used either prophylactically or therapeutically. Thus, vaccines can be used reduce the likelihood of infection or to reduce the severity of symptoms of a disease or condition or limit the progression of the disease or condition.

[0142] Vector: 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 the host cell, such as an origin of replication. A vector may also include one or more therapeutic genes and/or selectable marker genes and other genetic elements known in the art. A vector can transduce, transform or infect a cell, thereby causing the cell to express nucleic acids and/or proteins other than those native to the cell. A vector optionally includes materials to aid in achieving entry of the nucleic acid into the cell, such as a viral particle, liposome, protein coating or the like. In one embodiment, a vector is a viral vector. Viral vectors include, but are not limited to, retroviral and adenoviral vectors. [0143] The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Hence, "comprising A or B" means "including A or B," or "including A and B." It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. 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. 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.

///. Chimeric Polypeptides

[0144] Disclosed herein are chimeric polypeptides, which include antigenic or immunogenic peptide sequences that together can induce an antibody (and/or other immune) response to two or more life stages of P. falciparum. In some embodiments, a disclosed chimeric polypeptide includes epitopes from one or more P. falciparum life stages; such as two or more, or three or more life stages. In particular embodiments, epitopes from all life stages are included in a disclosed chimeric polypeptide.

[0145] In some embodiments, immunogenic peptides of a chimeric polypeptide are subsequences of proteins expressed during the P. falciparum life cycle. Some such proteins are expressed during one, two, three or more P. faciparum life stages. In some examples, antigenic or immunogenic peptides are subsequences of the P. falciparum proteins AMA-I, CSP, EBA-175, LSA-I, LSA-2, MSP-I, MSP-2, or RAP-I. Non-limiting exemplar immunogenic peptides from these proteins (refer also to Table 1) include: p519 NANPNANPNANP (SEQ ID NO: 9) and functional variants thereof. This is a protective epitope as shown by partial protection from human vaccination studies (Herrington, et ah, Nature, 328:257-259, 1987; Ballou et ah, Lancet, 1(8545): 1277-81, 1987; and Hoffman et al, Am J Trop Med Hyg., 51:603-612, 1994). Fab fragments of anti-CSP repeat antibodies neutralize the infectivity of sporozoites in chimpanzees (Nardin et ah, J Exp Med, 156:20-30, 1982). This sequence is also recognized by mice of H2^b background as a T-helper epitope (Good et al, Ann. Rev. Immunol., 6:663-688, 1998). Immunization in the rodent malaria and P. vivax systems with homologous CS repeat sequences has conferred protection against sporozoite infection. Functional variants of this epitope can, for example, bind specifically with antibodies described by Nardin et al. {J Exp Med, 156:20-30, 1982) or confer protection against sporozoite infection in the rodent system described by Good et al. {Ann. Rev. Immunol., 6:663-688, 1998). p543 SNTFINNA (SEQ ID NO: 15) and functional variants thereof. Mice immunized with this B-cell epitope/carrier conjugates were shown to be protected against blood stage challenge with P. berghei challenge (Saul et ah, J. Immunol., 148:208-11, 1992) and P. yoeliiyoelii 265BY strain (Lougovskoi et ah, Vaccine, 18:920-930, 2000). Functional variants of this epitope can, for example, also protect against blood stage challenge in the assays described by Saul et al. (J. Immunol., 148:208-11, 1992) and Lougovskoi et al. (Vaccine, 18:920-930, 2000). p544 GQHGHMGH (SEQ ID NO: 16) and functional variants thereof. Mice immunized with diphtheria conjugates of this B-cell epitope were protected against blood stage P. berghei challenge (Saul et al, J. Immunol., 148:208-11, 1992). Functional variants of this epitope can, for example, also protect against blood stage challenge in the assay described by Saul et al (J. Immunol., 148:208-11, 1992). p545 KNTLTPLEELYPT (SEQ ID NO: 12) and functional variants thereof. Monoclonal antibodies that specifically bind this epitope have been shown to inhibit growth of blood stage parasites (Harnyuttanakorn et al, MBP, 55:177-86, 1992). Functional variants of this epitope can, for example, specifically bind to the antibodies described by Harnyuttanakorn et al (MBP, 55:177-86, 1992). p546 TLTKEYEDIVLKSHMNRESDD (SEQ ID NO: 17) and functional variants thereof. This peptide is a ligand for glycophorin A and sera against it blocks blood stage parasite growth in vitro (Jackobsen et al., Infect. Immun., 66:4203-4207, 1998). Functional variants of this epitope, can, for example, be bound by the sera described by Jackobsen et al. (Infect. Immun., 66:4203-4207, 1998), or are specifically bound by glycophorin A. pS92 KPKHKKLKQPGDGNP (SEQ ID NO: 3) and functional variants thereof. This epitope is a CSP region I conserved epitope. Antibodies to this epitope have been shown to inhibit invasion of sporozoites in vifro (Aley et al, J. Exp. Med., 164:1915, 1986; Ying et al, Exp Parasitol, 85:168- 82, 1997). Antibodies against this epitope have been found in clinically immune individuals under natural conditions of exposure. Functional variants of the epitope will, for example, be specifically bound by the antibodies described in Aley et al. (J. Exp. Med., 164:1915, 1986) and Ying et al (Exp Parasitol, 85:168-82, 1997). p593 KPKDELDYENDIEKKICKMEKCS (SEQ ID NO: 4) and functional variants thereof. Tins sequence includes a Th3R epitope and contains a human CTL epitope restricted by HLA B35. Natural immune response studies in The Gambia and western Kenya have identified this as a CTL determinant (Hill et al, Nature, 360:434-439, 1992; Udhayakumar et al, Eur. J. Immunol, 27:1952-1957, 1997). Human irradiated sporozoite vaccination studies have also identified this CTL epitope (Malik et al, PNAS, 88:3300, 1991). This sequence also contains a CTL epitope recognized by B10.BR (H-2^K) mice (Malik et al, Inf& Immunity, 63:1955-1959, 1995). p594 SVFNWNS (SEQ ID NO: 5) and functional variants thereof. This conserved universal T epitope recognizes 7 different HLA-DR molecules (Sinigaglia et al, Nature, 336:778, 1988). Natural immune response studies have shown this epitope to be highly immunogenic (Sinigaglia et al., Eur. J. Immunol, 18:633, 1988; Flanagan et al, J. Immunol, 167:4729-37, 2001). This sequence is part of the RTS,S vaccine and elicits cytokine and proliferative responses in vaccinated individuals (Lalvani et al, JID, 180:1656-64, 1999; Bojang et al, Lancet, 358:1927-34, 2001). Functional variants of this epitope can, for example, bind the HLA-DR molecules described by Sinigaglia et al (Eur. J. Immunol, 18:633, 1988). p595 KPIVQYDNF (SEQ ID NO: 8) and functional variants thereof. This HLA-B53 restricted epitope has been implicated as a protective epitope based on the Gambian field study by Hill et al, Nature, 360:534-439, 1992, and has been shown to be reactive in naturally exposed individuals by Aidoo et al, Lancet, , 345:1003-1007, 1995. Functional variants of this epitope can, for example, bind HLA-B53 in a soluble DR molecule binding assay. p596 KPNDKSLY (SEQ ID NO: 13) and functional variants thereof. Monoclonal antibodies recognizing this epitope were shown to inhibit growth of Pf blood stage parasites (Harnyuttanakorn et al., MBP, 55:177-86, 1992). Functional variants of this epitope can, for example, bind to the antibodies described in by Harnyuttanakorn et al. (MBP, 55:177-86, 1992). p597 NSGCFRHLDEREECKCLL (SEQ ID NO: 6) and functional variants thereof. In an antibody response study, using overlapping peptides and sera from clinically immune and aparasitemic Kenyan individuals, this epitope was also found to be highly reactive (in frequency and magnitude). Functional variants of this epitope can, for example, react with sera from clinically immune individuals. p598 EDSGSNGKKITCECTKPDS (SEQ ID NO: 7) and functional variants thereof. In an antibody response study, using overlapping peptides and sera from clinically immune and aparasitemic Kenyan individuals, this epitope was also found to be highly reactive (in frequency and magnitude). Functional variants of this epitope can, for example, react with sera from clinically immune individuals. p599 GISYYEKVLAKYKDDLE (SEQ ID NO: 23) and functional variants thereof. This peptide showed a high level of responder frequency among 17 peptides tested from the C-terminal region of MSP-I in naturally exposed Kenyan population (Udhayakumar et al., J. Immunol., 154:6022- 6030, 1995). Functional variants of this epitope can, for example, be identified using the assays described by Udhayakumar et al. (J. Immunol, 154:6022-6030, 1995). p600 DGNCEDIPHVNEFSAIDL (SEQ ID NO: 10) and functional variants thereof. In an antibody response study, using overlapping peptides and sera from clinically immune and aparasitemic Kenyan individuals, this epitope was also found to be highly reactive (in frequency and magnitude). Functional variants of this epitope can, for example, react with sera from clinically immune individuals. p601 GNAEKYDKMDEPQHYGKS (SEQ ID NO: 11) and functional variants thereof. In an antibody response study, using overlapping peptides and sera from clinically immune and aparasitemic Kenyan individuals, this epitope was found to be highly reactive (in frequency and magnitude). Functional variants of this epitope can, for example, react with sera from clinically immune individuals. p602 DQPKQYEQHLTDYEKIKEG (SEQ ID NO: 19) and functional variants thereof. This peptide shows a high level of responder frequency and magnitude of response in a T-cell proliferative assay among 17 peptides selected from the AMA-I protein and tested in Kenyan population. (LaI A et al., Infect. Immun., 64:1054-1059, 1996). Functional variants of this epitope can, for example, elict a T-cell proliferative response in the assay described by LaI et al. (Infect. Immun., 64:1054-1059, 1996). p603 EFTYMINFGRGQNYWEHPYQKS (SEQ ID NO: 18) and functional variants thereof. This peptide shows a high level of responder frequency and magnitude of response in a T-cell proliferative assay among 17 peptides selected from the AMA-I protein and tested in Kenyan, population. (LaI A et al., Infect Immun., 64:1054-1059, 1996). Functional variants of this epitope can, for example, elict a T-cell proliferative response in the assay described by LaI et al. (Infect. Immun., 64:1054-1059, 1996). p604 SSPSSTKSSSPSNVKSAS (SEQ ID NO: 21) and functional variants thereof. This peptide induces a T-cell proliferative response in the Kenyan population (unpublished). A functional variant of this peptide can, for example, induce a T-cell proliferation response in a T-cell proliferation assay. p605 LATRLMKKFKAEIRDFF (SEQ ID NO: 22) and functional variants thereof. This peptide induces a T-cell proliferative response in the Kenyan population (unpublished). A functional variant of this peptide can, for example, induce a T-cell proliferation response in a T-cell proliferation assay. p606 LMIKEHILAIAIYESRILKR (SEQ ID NO: 14) and functional variants thereof. This peptide is recognized by monoclonal antibodies specific for the EBA-RII region of the EBA- 175 protein, and maybe part of the binding domain. Functional variants of this epitope can, for example, be specifically bound by monoclonal antibodies specific for the EBA-RII region of the EBA- 175 protein. p607 RDEWWKVIKKDVWNVISWVF (SEQ ID NO: 20) and functional variants thereof. This peptide is recognized by monoclonal antibodies specific for the EBA-RII region of the EBA- 175 protein, and may be part of the binding domain. Functional variants of this epitope can, for example, be specifically bound by monoclonal antibodies specific for the EBA-RII region of the EBA- 175 protein.

[0146] Some embodiments of disclosed chimeric polypeptides include the p545, p546, p594, p606 and p607 epitopes in any order. In other examples, chimeric polypeptides can include these epitopic peptide sequences or sequences having at least 75%, at least 90%, at least 95% or at least 98% identity to SEQ ID NOs: 5, 12, 14, 17 and 20. Polypeptides as described herein may be of any length (for example, between about 50 and 2000 amino acids in length, for example between about 100 and 1000 amino acids in length such as between about 150 and 500 amino acids in length). Additional sequences derived from the native proteins from which epitopes are derived and/or heterologous sequences may be included in the sequenceofthechimericpolypeptide,andsuchsequencesmay(butneednot)possessfurtherimmunogenic orantigenicproperties.

[0147] Alternativeembodimentsincludefunctionalvariantsthatincludepermutationsofthe positionsorindividualepitopesequenceswithinthechimericpolypeptidesequence. Forexample,chimeric polypeptidescomprisingthep545,p546,p594,p606,andp607epitopescanincludethesesequencesinany orderfromtheN-terminusofthepolypeptidetotheC-terminusofthepolypeptide. Forexample,theorder oftheseepitopicsequencesinthechimericpolypeptidemaybep594,p545,p606,p546,p607;p594,p545, p606,p607,p546;p594,p545,p546,p607,p606;p594,p545,p546,p606,p607;p594,p545,p607,p546, p606;p594,p545,p607,p606,p546;p545,p594,p606,p546,p607;p545,p594,p606,p607,p546;p545, p594,p546,p607,p606;p545,p594,p546,p606,p607;p545,p594,p607,p546,p606;p545,p594,p607, p606,p546;p594,p607,p545,p606,p546;p594,p607,p545,p546,p606,p594,p607,p606,p545,p546; p594,p607,p606,p546,p545;p594,p607,p546,p545,p606;p594,p607,p546,p606,p545,p607,p594, p545,p606,p546,p607,p594,p545,p546,p606,p607,p594,p606,p545,p546;p607,p594,p606,p546, p545;p607,p594,p546,p545,p606;p607,p594,p546,p606,p545;p546,p607,p594,p545,p606;p546, p607,p594,p606,p545;p546,p607,p545,p594,p606;p546,p607,p545,p606,p594;p546,p607,p606, p594,p545;p546,p607,p606,p545,p594;p607,p546,p594,p545,p606;p607,p546,p594,p606,p545; p607,p546,p545,p594,p606;p607,p546,p545,p606,p594;p607,p546,p606,p594,p545;p607,p546, p606,p545,p594;p545,p606,p594,p546,p607;p545,p606,p594,p607,p546;p545,p606,p546,p594, p607;p545,p606,p546,p607,p594;p545,p606,p607,p594,p546,p545,p606,p607,p546,p594;p606, p545,p594,p546,p607;p606,p545,p594,p607,p546;p606,p545,p546,p594,p607;p606,p545,p546, p607,p594;p606,p545,p607,p594,p546;p606,p545,p607,p546,p594;p606,p546,p594,p545,p607; p606,p546,p594,p607,p545;p606,p546,p545,p594,p607;p606,p546,p545,p607,p594;p606,p546, p607,p594,p545;p606,p546,p607,p545,p594;p546,p606,p594,p545,p607;p546,p606,p594,p607, p545;p546,p606,p545,p594,p607;p546,p606,p545,p607,p594;p546,p606,p607,p594,p545;p546, p606,p607,p545,p594;p594,p606,p545,p546,p607;p594,p606,p545,p607,p546;p594,p606,p546, p545,p607;p594,p606,p546,p607,p545;p594,p606,p607,p545,p546;p594,p606,p607;p546,p545; p606,p594,p545,p546,p607;p606,p594,p545,p607,p546;p606,p594,p546,p545,p607;p606,p594, p546,p607,p545;p606,p594,p607,p545,p546;p606,p594,p607;p546,p545;p594,p546,p545,p606, p607;p594,p546,p545,p607,ρ606;p594,p546,p606,p545,p607;p594,p546,p606,p607,p545;p594, p546,p607,p545,p606;p594,p546,p607,p606,p545;p546,p594,p545,p606,p607;p546,p594,p545, p607,p606;p546,p594,p606,p545,p607;p546,p594,p606,p607,p545;p546,p594,p607,p545,p606; p546,p594,p607,p606,p545;p545,p546,p594,p606,p607;p545,p546,p594,p607,p606;p545,p546, p606,p594,p607;p545,p546,p606,p607,p594;p545,p546,p607,p594,p606;p545,p546,p607,p606, p594;p546,p545,p594,p606,p607;p546,p545,p594,p607,p606;p546,p545,p606,p594,p607;p546, p545,p606,p607,p594;p546,p545,p607,p594,p606;p546,p545,p607,p606,p594;p545,p607,p594, p606,p546;p545,p607,p594,p546,p606;p545,p607,p606,p594,p546;p545,p607,p606,p546,p594; p545,p607,p546,p594,p606;p545,p607,p546,p594,p606;p607,p545,p594,p606,p546;p607,p545, p594,p546,p606;p607,p545,p606,p594,p546;p607,p545,p606,p546,p594;p607,p545,p546,p594, p606;p607,p545,p546,p594,p606;p606,p607,p594,p545,p546;p606,p607,p594,p546,p545;p606, p607, p545, p594, p546; p606, p607, p545, p546, p594; p606, p607, p546, p594, p545; p606, p607, p546, p545, p594; p607, p606, p594, p545, p546; p607, p606, p594, p546, p545; p607, p606, p545, p594, p546; p607, p606, p545, p546, p594; p607, p606, p546, p594, p545; or p607, p606, p546, p545, p594. As described below, spacer amino acid sequences and additional immunogenic or non-immunogenic polypeptide sequences may be present in the chimeric polypeptides at any position relative to the epitopic peptide sequences.

[0148] Similarly, chimeric polypeptides that include the p545, p546, p594, p606, and p607 epitopes and one or more of (such as at least 5 of, at least 10 of, at least 15 of, or all 16 of) a ρ519, a p543 epitope, a p544 epitope, a p592 epitope, a p593 epitope, a p595 epitope, a p596 epitope, a p597 epitope, a p598 epitope, a p599 epitope, a p600 epitope, a p601 epitope, a p602 epitope, a p603 epitope, a ρ604 epitope, or a ρ605 epitope are possible in multiple combinations of order of epitopes chosen from the group of a p519 epitope, a p543 epitope, a p544 epitope, a p592 epitope, a p593 epitope, a p595 epitope, a p596 epitope, a p597 epitope, a ρ598 epitope, a p599 epitope, a p600 epitope, a p601 epitope, a p602 epitope, a p603 epitope, a p604 epitope, and a p605 epitope. In particular embodiments, all of these optional epitopes are included in the chimeric polypeptide. As described below, spacer amino acid sequences and additional immunogenic or non-immunogenic polypeptide sequences may be present in the chimeric polypeptides at any position relative to the epitopic peptide sequences.

[0149] Particular examples of chimeric polypeptides with one or more epitopes in addition to the p545, p546, p594, p606, and ρ607 epitopes include chimeric polypeptides in which the order of chimeric polypeptides are: p545, p546, p594, p606, p607, p602; p602, p545, p546, p594, p606, p607, p602; p545, p546, p594, p606, p607, p602; p545, p546, p594, p606, p607, p600, p599, p545, p544, p598; p599, p545, p544, p598, p545, p546, p594, p606, p607, p600, p599, p545, p544, p598; p545, p546, p594, p606, p607, p600, p599, p545, p544, p598, p519, p543, p592, p593, p595; p599, p545, p544, p598, p545, p546, p594, p606, p607, p600, p519, p543, p592, p593, p595; p599, p545, p544, p598, p545, p546, p594, p606, p607, ρ600, p519, p543, p592, p593, p595, p603, p604, p605, p596, p597; p544, p598, p545, p546, p594, p599, p545, p606, p607, p600, p519, p543, p592, p593, p595, p603, p604, p605, p596, p597; p544, p598, p545, p546, p594, p599, p545, p606, p607, p600, p519, p543, p592, p593, p595, p603, p604, p605, p596, p597, p602; and p544, p598, p545, p546, p594, p544, p598, p545p599, p545, p606, p607, p600, p519, p543, p592, p593, p595, p603, p604, p605, p596, p597, p602, p595, p603, p604.

[0150] One or more spacer amino acid sequences and/or additional peptide sequences (both immunogenic and/or non-immunogenic) may be present between epitopes of any disclosed chimeric polypeptide. In some embodiments, the spacer amino acid is from 2 to about 25 amino acids in length; for example, from 2 to about 15, from 2 to about 10, from 2 to about 7, or from 2 to about 5 amino acids in length. Such spacers and additional sequences may be located in any position(s) relative to any included epitope(s) (such as, the ρ594, p545, p606, ρ546, or p607 epitopes). In some embodiments, one or more spacers are between one or more pairs of epitopes in a chimeric polypeptide. In other embodiments, two or more spacers are between two or more pairs of epitopes in a chimeric polypeptide. In still further embodiments, three or more, such as five or more, seven or more or nine or more spacers are between pairs of epitopes in a chimeric polypeptide. [0151] Spacer sequences can, for example, facilitate folding of a disclosed chimeric polypeptide into a stable secondary and/or tertiary structure, and/or serve to improve epitope processing of epitopes in the resulting polypeptide. Spacers can include, without limitation, reverse-turn sequences, which permit a chimeric polypeptide chain to make a fold or bend. For example, the peptide sequence GPGPG (SEQ ID NO: 26) readily folds to provide a reverse turn in a polypeptide chain. The combination of small, flexible glycine residues and the rigid, direction altering proline residues bends a polypeptide back on itself to provide the reverse turn. Other amino acid sequences, naturally occurring or otherwise, that induce or permit such turns are also contemplated; including, for example, GPG, PGP, PGPG (SEQ ID NO: 27), GGPGG (SEQ ID NO: 28), APAPA SEQ ID NO: 29, GGG, GGGG (SEQ ID NO: 30) and APGPA (SEQ ID NO: 31). Thus, for example, when such amino acid sequences are used as spacers in a chimeric polypeptide, the chimeric polypeptide can fold at the position of such spacer(s). Other exemplar spacers (such as, KG or other glycine-containing spacers that do not also include structurally rigid prolines) can introduce flexibility into a peptide chain; so, for example, the peptide chain can rotate about its long axis. Reverse turns and flexibility of the peptide chain can permit the peptide chain to assume lower energy (hence, more stable) secondary and tertiary structures. Still other spacers, such as KAA, CAA, NAA, and/or GAA, can facilitate epitope processing (such as by improving processing efficiency by the Th-I pathway of CTL epitopes immediately preceeding such spacers; Livingston et al., Vaccine, 19:4652-4660, 2001). In some exemplar chimeric polypeptides, one or more spacer amino acid sequences, such as GPGPG (SEQ ID NO: 26), KAA or KG spacers, may be located between some or all of the epitopic peptides. In some such embodiments, the chimeric polypeptides are fusion proteins of the chimeric polypeptide with a fusion partner.

[0152] Particular non-limiting examples of chimeric polypeptides with one or more epitopes in addition to the p545, p546, p594, p606, and p607 epitopes and one or more spacers (such as 3 or more, 6 or more, 9 or more, or 12 or more spacers) include chimeric polypeptides in which the order of chimeric polypeptides are: p545, spacer, p546, spacer, p594, spacer, p606, p607, p602; p602, spacer, p545, p546, spacer, p594, spacer, p606, spacer, p607, p602; p545, spacer, p546, p594, spacer, p606, p607, spacer, p602; p545, spacer, p546, spacer, p594, p606, p607, spacer, p600, p599, p545, p544, p598; p599, spacer, p545, spacer, p544, spacer, p598, p545, spacer, p546, spacer, p594, p606, spacer, p607, spacer, p600, p599, spacer, p545, spacer, p544, p598; p545, spacer, p546, p594, p606, spacer, p607, p600, p599, spacer, p545, p544, ρ598, p519, p543, p592, p593, ρ595; p599, spacer, p545, spacer, p544, spacer, p598, spacer, p545, spacer, p546, spacer, p594, spacer, p606, spacer, p607, spacer, p600, spacer, p519, spacer, p543, spacer, p592, spacer, p593, spacer, p595; ρ599, p545, spacer, p544, p598, p545, p546, ρ594, p606, p607, p600, spacer, p519, ρ543, ρ592, p593, p595, spacer, p603, p604, ρ605, p596, p597; p544, p598, p545, p546, p594, p599, p545, p606, p607, p600, p519, p543, p592, p593, p595, p603, p604, p605, p596, p597; p544, spacer, p598, p545, p546, p594, p599, spacer, p545, p606, p607, spacer, p600, p519, p543, spacer, p592, p593, p595, p603, p604, spacer, p605, p596, p597, p602; and p544, p598, spacer, p545, p546, spacer, p594, p544, p598, spacer, p545p599, p545, spacer, p606, p607, p600, spacer, p519, p543, p592, p593, p595, p603, p604, p605, p596, p597, p602, p595, p603, p604. [0153] Chimeric polypeptides also can include comprise a signal (or leader) sequence at the N- terminus which co-translationally or post-translationally directs transfer of the polypeptide. The chimeric polypeptides may also be conjugated to a linker or other sequence for ease of synthesis, purification or identification of the polypeptide (such as poly-His or poly-Arg), or to enhance binding of the chimeric polypeptide to a solid support. For example, a chimeric polypeptide may be conjugated to an immunoglobulin Fc region (see also the description of fusion proteins below).

[0154] In some embodiments, a chimeric polypeptide does not include one or more of a melittin signal peptide, a p547 epitope, a p589 epitope and a p591 epitope as described in WO 00/11179.

[0155] Immunogenicity of a disclosed chimeric polypeptide or epitope can be confirmed according to the methods described, for example, in the Examples or elsewhere in this specification.

[0156] Also disclosed herein are variants of disclosed chimeric polypeptides, which polypeptide variants may include, for example, variants of one or more described epitopes (such as, p592, p593, p594, p597, p598, p595, p519, p600, p601, p545, p596, p606, p543, p544, p546, p603, p602, p607, p604, p605, or p599). A variant of a chimeric polypeptide (or of an epitope) substantially retains the function, such as antigenicity or immunogenicity (such as determined in one or more ELISA assays and/or a T-cell reactivity assay), of the chimeric polypeptide (or epitope) from which it is derived. As contemplated herein, variants include, without limitation, sequence variants of, peptide derivatives of, or fusion proteins including a disclosed chimeric polypeptide (or, as applicable, an epitope thereof).

[0157] In some examples, a variant of a chimeric polypeptide (or epitope thereof) is characterized by possession of at least 50% sequence identity counted over a full-length alignment with the reference amino acid sequence (e.g., a chimeric polypeptide or epitope). One useful alignment tool for determining sequence identity is the NCBI Blast 2.0 software, gapped blastp, set to default parameters. For comparisons of amino acid sequences of greater than 30 amino acids, the Blast 2 sequences function is employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1). When aligning short peptides (fewer than around 30 amino acids), the alignment is performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gapl penalties). Polypeptides with even greater similarity to a reference sequence will show increasing percentage of identifies when assessed by this method, such as at least 60%, at least 65%, at least 75%, at least 80%, at least 90%, or at least 95%, or at least 98%, or at least 99% sequence identity. When less than the entire sequence is being compared for sequence identity (for example, sequence identity of individual epitopes), homologs and variants will typically possess at least 75% sequence identity over short windows of 10-20 amino acids, and may possess sequence identifies of at least 85% or at least 90%, or at least 95%, or at least 98%, or at least 99% depending on their similarity to the reference sequence. Methods for determining sequence identity over such short windows are described at the website that is maintained by the National Center for Biotechnology Information in Bethesda, Maryland. One of ordinary skill in the art will appreciate that these sequence identity ranges are provided for guidance only; it is entirely possible that strongly significant homologs could be obtained that fall outside of the ranges provided. [0158] In some embodiments, a disclosed chimeric polypeptide includes polypeptide sequences that have at least 90%, at least 95% or at least 98% sequence identity to the chimeric polypeptide set forth as residues 1-374 of SEQ ID NO: 2, or sequences that have at least 90%, at least 95% or at least 98% sequence identity to the chimeric polypeptide set forth as residues 3-374 of SEQ ID NO: 2, or sequences that have at least 90%, at least 95% or at least 98% sequence identity to the chimeric polypeptide set forth as SEQ ID NO: 2. In other embodiments, a disclosed chimeric polypeptide includes one or more variants of any of p592, p593, p594, p597, p598, p595, p519, p600, p601, p545, p596, p606, p543, p544, p546, p603, p602, p607, p604, p605, or p599. Each such epitope variant will independently have at least 80%, at least 90%, or at 95%, or at least 98%, or at least 99% sequence identity to its respective epitope reference sequence. In particular embodiments, at least two, at least four, at least six, at least eight, or all of the epitopes of a disclosed chimeric polypeptide are variants of p592, p593, p594, p597, p598, p595, p519, p600, p601, p545, p596, p606, p543, p544, p546, p603, p602, p607, p604, p605, or p599.

[0159] Those of ordinary skill in the art will also recognize that individual substitutions which alter a single amino acid or a small percentage of amino acids (typically less than 5%, more typically less than 1%) in a polypeptide sequence (such as, an encoded polypeptide sequence) are "conservative substitutions" where the alterations result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following five groups each contain amino acids that are conservative substitutions for one another:

Aliphatic: Glycine (G), Alanine (A), Valine (V), Leucine (L), Isoleucine (I);

Aromatic: Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

Sulfur-containing: Methionine (M), Cysteine (C);

Basic: Arginine (R), Lysine (K), Histidine (H);

Acidic: Aspartic acid (D), Glutamic acid (E), Asparagine (N), Glutamine (Q).

[0160] See also, Creighton, Proteins, W.H. Freeman and Company, 1984, for additional groupings of amino acids as conservative substitutions for one another.

[0161] In one embodiment, a conservative substitution is an amino acid substitution in an antigenic epitope of a P. falciparum peptide that does not substantially affect the ability of an antibody that specifically binds to the unaltered epitope to specifically bind the epitope including the conservative substitution. Thus, in some embodiments, a conservative variant of an epitope is also a functional variant of the epitope.

[0162] In another embodiment, one or more conservative substitutions are made in one or more antigenic P. falciparum sequences (epitopes) that are included in a single chimeric polypeptide, such that the substitution or substitutions do not substantially affect the ability of antibodies to specifically bind to the antigenic sequences. In other words, the conservative substitutions provide functional variants. Such conservative substitutions that provide functional variants can be identified based upon an ELISA assay that compares a level of specific binding of an antibody that specifically binds a particular antigenic peptide to a level of specific binding of the antibody to a corresponding peptide with the substitution(s) to determine if the substitution(s) does not substantially affect specific binding of the substituted peptide to the antibody. [0163] In some embodiments, one conservative substitution is included in one or more epitopic peptide subsequences of a chimeric polypeptide. In other embodiments, two conservative substitutions or less are included in an individual antigenic peptide subsequence or an entire chimeric polypeptide. In further embodiments, three conservative substitutions or less are included in an individual antigenic peptide subsequence or an entire chimeric polypeptide. Conservative substitutions provide conservative variants and or functional variants of individual peptides, chimeric polypeptides and proteins.

[0164] A functional variant of a chimeric polypeptide substantially retains the function, such as antigenicity or immunogenicity (such as determined in one or more ELISA assays and/or a T-cell reactivity assay), of the chimeric polypeptide from which it is derived. As applied to a disclosed chimeric polypeptide, the term "functional variant" includes, without limitation, a different chimeric polypeptide having the same epitopes as an unaltered chimeric polypeptide but which retains a function or property (such as immunogenicity) of the unaltered chimeric polypeptide, but where the epitopes are in different positions (such as different orders) along the chimeric polypeptide from the C-terminus to the N-terminus. Functional variants also include chimeric polypeptides that differ from an unaltered chimeric polypeptide in the number and position of any spacers present between individual subsequences and the presence or absence of additional sequences, such as affinity sequences. Functional variants can have sequence alterations in any one or more epitopes, one or more spacers or otherwise, including, but are not limited to, conservative substitutions, deletions, mutations, frameshifts, and insertions. Thus, in one example, functional variants of an epitope include peptides of varying lengths, such as between 6 and 100 amino acids long (for example, peptides of between 6 and 50 amino acids in length, between 8 and 25 amino acids in length or between 8 and 18 amino acids in length) that retain a function or property of the unaltered epitope.

[0165] In one embodiment, a given epitope binds an antibody, and a functional variant is a peptide that binds the same antibody (detected, for example, in an ELISA assay; see, for example, Munesingh et al., Eur. J. Immunol, 12:3015, 1991). Thus a functional variants include peptides which have the substantially the same binding specificity as an unaltered epitope, and may be used as a reagent in place of the epitope (such as in a diagnostic assay or vaccine).

[0166] In an alternative embodiment, a functional variant of an epitope is a peptide that specifically binds a soluble major histocompatibility complex molecule (such as a Class II HLA-DR or -DQ molecule) as measured in a competitive ELISA assay against unaltered epitope (see, for example, U.S. Patent No. 6,669,945 and Hammer et al., J. Exp. Med., 180:2353, 1994). Briefly, purified DR or DQ molecules are added to each well of a 96 well plate along with biotinylated indicator peptide (such as a biotinylated, but otherwise unaltered epitope) in citrate-phosphate buffer containing 2% n-octyl-glucoside, PMSF, EDTA and protease inhibitors. A binding buffer at pH 7 is used for most DQ and DR assays, with the exception of the DRB 1*0701 binding buffer which is pH 5. Following incubation overnight at room temperature (RT) the peptide/class II complexes are transferred to wells coated with anti-DR Mab L234 antibody (15 μg/ml) or anti-DQ Mab HB144 (3.5 μg/ml). Following a two-hour incubation, the wells were washed with PBS+1% Tween, and the capture of the biotinylated peptide/class II molecule complexes is revealed by addition of alkaline phosphatase-labeled streptavidin and substrate, p-nitrophenylphosphate (Kierkegaard and Perry, Gaithersburg, Md.). Optical densities are determined in a Titertek MC Multiscan ELISA reader (Flow Labs) using a 405 nm filter. For the peptide competition assays, an optimal concentration of the biotinylated indicator peptide (0.1 μM-5 μM) is incubated with ten-fold dilutions (0.01 .μM-100 μM) of the unlabelled competitor peptides. The ability of the unlabelled competitor peptide to compete with biotinylated indicator peptide for binding to the class II molecule is revealed by measuring optical density (O.D.). Inhibition is calculated as a percentage using the formula: 100x[l-(Δ O.D. in presence of competitor peptide /Δ O.D. in absence of competitor)]. The concentration of a competitor peptide required to inhibit 50% of binding of the biotinylated indicator peptide (IC₅₀) is determined and a peptide with an IC₅₀ of less than 100 μM (such as an IC₅₀ of less than 50 μM, less than 25 μM or less than 10 μM) is considered a functional variant of the epitope.

[0167] Also contemplated herein are chimeric polypeptides or one or more epitopes thereof (such as, p592, p593, p594, p597, p598, p595, p519, p600, p601, p545, p596, p606, p543, p544, p546, p603, p602, p607, p604, p605, or p599), having one or more amino acids or one or more peptides sequences that have been modified by a variety of chemical techniques to produce derivatives having essentially the same activity as the unmodified peptides, and optionally having other desirable properties. For example, carboxylic acid groups of the protein, whether carboxyl-terminal or side chain, maybe provided in the form of a salt of a pharmaceutically acceptable cation or esterified to form a Q-Cie ester, or converted to an amide of formula NR₁R₂ wherein R] and R₂ are each independently H or Ci-Cjβ alkyl, or combined to form a heterocyclic ring, such as a 5- or 6- membered ring. Amino groups of the peptide, whether amino- terminal or side chain, may be in the form of a pharmaceutically acceptable acid addition salt, such as the HCl, HBr, acetic, benzoic, toluene sulfonic, maleic, tartaric and other organic salts, or may be modified to C₁-C₁₆ alkyl or dialkyl amino or further converted to an amide.

[0168] Hydroxyl groups of the peptide side chains may be converted to CpCig alkoxy or to a C₁-C₁₆ ester using well-recognized techniques. Phenyl and phenolic rings of the peptide side chains may be substituted with one or more halogen atoms, such as fluorine, chlorine, bromine or iodine, or with C_rCi₆ alkyl, Ci-Cig alkoxy, carboxylic acids and esters thereof, or amides of such carboxylic acids. Methylene groups of the peptide side chains can be extended to homologous C₂-C₄ alkylenes. Thiols can be protected with any one of a number of well-recognized protecting groups, such as acetamide groups. Those of ordinary skill in the art will also recognize methods for introducing cyclic structures into the peptides of this invention to select and provide conformational constraints to the structure that result in enhanced stability.

[0169] Peptidomimetic and organomimetic embodiments are also within the scope of the present invention, whereby the three-dimensional arrangement of the chemical constituents of such peptido- and organomimetics mimic the three-dimensional arrangement of the peptide backbone and component amino acid side chains, resulting in such peptido- and organomimetics of the proteins of this invention having measurable or enhanced ability to bind an antibody. For computer modeling applications, a pharmacophore is an idealized, three-dimensional definition of the structural requirements for biological activity. Peptido- and organomimetics can be designed to fit each pharmacophore with current computer modeling software (using computer assisted drag design or CADD). See Walters, "Computer-Assisted Modeling of Drugs", in Klegerman & Groves (eds.), Pharmaceutical Biotechnology, Interpharm Press, pp.165-174, 1993, and Munson (ed.), Principles of Pharmacology, Ch. 102, 1995, for descriptions of techniques used in CADD. Also included within the scope of the invention are mimetics prepared using such techniques. In one embodiment, a mimetic mimics the binding of a malaria epitope or malaria protein to an antibody.

[0170] Alternatively, a chimeric polypeptide according to the disclosure may be a fusion protein that comprises two or more immunogenic chimeric polypeptide as described herein, or that comprises at least one immunogenic chimeric polypeptide as described herein and an unrelated sequence, such as a soluble protein, for example, a soluble P. falciparum protein. A fusion partner also may, for example, assist in providing T helper epitopes (an immunological fusion partner), preferably T helper epitopes recognized by humans, or may assist in expressing the protein (an expression enhancer) at higher yields than the native recombinant peptide or polypeptide. Certain fusion partners are both immunological and expression enhancing fusion partners. Other fusion partners may be selected so as to increase the solubility of the protein or to enable the protein to be targeted to desired intracellular compartments. Still further fusion partners include affinity tags, which facilitate purification of the protein.

[0171] Fusion proteins may generally be prepared using standard techniques, including chemical conjugation. Alternatively, a fusion protein may be expressed as a recombinant protein, allowing the production of increased levels, relative to a non-fused protein, in an expression system. Briefly, DNA sequences encoding the polypeptide components may be assembled separately, and ligated into an appropriate expression vector. The 3' end of the DNA sequence encoding one polypeptide component is ligated, with or without a sequence coding a peptide linker, to the 5 ' end of a DNA sequence encoding the second polypeptide component so that the reading frames of the sequences are in phase. This permits translation into a single fusion protein that retains the biological activity of both component polypeptides.

[0172] A peptide spacer also may be employed to separate first and second polypeptide components of a fusion protein by a distance sufficient to assist folding into secondary and tertiary structures. Peptide linker sequence may be incorporated into the fusion protein using standard techniques well known in the art. Suitable peptide linker sequences may be chosen based on the following factors: (1) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the first and second peptides or polypeptides; and/or (3) the lack of hydrophobic or charged residues that might react with epitopes. Peptide linker sequences may contain Pro, GIy, Asn and Ser residues. Near neutral amino acids, such as Thr and Ala may also be used in the linker sequence. Amino acid sequences which may be usefully employed as linkers include those disclosed in Maratea et ah, Gene 40:39-46, 1985; Murphy et at, Proc. Natl. Acad. Sci. USA 83:8258-8262, 1986; and in U.S. Pat. Nos. 4,935,233 and 4,751,180. The spacer may generally be from 1 to about 50 amino acids in length.

[0173] Ligated DNA sequences are operably linked to suitable transcriptional or translational regulatory elements. The regulatory elements responsible for expression of DNA may be located 5' to the DNA sequence encoding the first polypeptides. Similarly, stop codons that end translation and transcription termination signals may be present 3' to the DNA sequence encoding the second polypeptide. [0174] Fusion proteins are also provided comprising a disclosed immunogenic chimeric polypeptide together with an unrelated immunogenic protein. The immunogenic protein may be capable of eliciting a recall response. Examples of such proteins include tetanus, tuberculosis and hepatitis proteins (see, for example, Stoute et al. New Engl. J. Med., 336:86-91, 1997).

[0175] Within some embodiments, an immunological fusion partner is derived from protein D, a surface protein of the gram-negative bacterium Haemophilus influenza B (WO 91/18926). For example a protein D derivative may comprise approximately the first third of the protein (e.g., the first N-terminal 100-110 amino acids), and a protein D derivative may be lipidated. Within other embodiments, the first 109 residues of a Lipoprotein D fusion partner may be included on the N-terminus to provide the polypeptide with additional exogenous T-cell epitopes and to increase the expression level in E coli (thus functioning as an expression enhancer). The lipid tail assists optimal presentation of the antigen to antigen presenting cells. Other fusion partners include the non-structural protein from influenza virus, NSl (hemagglutinin). Typically, the N-terminal 81 amino acids are used, although different fragments that include T-helper epitopes may be used.

[0176] In another embodiment, the immunological fusion partner is the protein known as LYTA, or a portion thereof (such as a C-terminal portion). LYTA is derived from Streptococcus pneumoniae, which synthesizes an N-acetyl-L-alanine amidase known as amidase LYTA (encoded by the LytA gene; Gene 43:265-292, 1986). LYTA is an autolysin that specifically degrades certain bonds in the peptidoglycan backbone. The C-terminal domain of the LYTA protein is responsible for the affinity to the choline or to some choline analogues such as DEAE. This property has been exploited for the development of E. coli C-LYTA expressing plasmids useful for expression of fusion proteins. Purification of hybrid proteins containing the C-LYTA fragment at the amino terminus has been described (see, for example, Biotechnology 10:795-798, 1992). A repeat portion of LYTA may be incorporated into a fusion protein. A repeat portion is found in the C-terminal region starting at residue 178, for example, a repeat portion may incorporate residues 188-305 of LYTA.

[0177] As mentioned previously, a variant of a chimeric polypeptide (or of an epitope) substantially retains a function, such as antigenicity or immunogenicity (such as determined in one or more ELISA assays and/or a T-cell reactivity assay), of the chimeric polypeptide (or epitope) from which it is derived. Immunogenicity of a chimeric polypeptide, its component epitopes, or variants or derivatives of a disclosed chimeric polypeptide or its epitope can be confirmed according to the methods described, for example, in the Examples. In addition, assays for determining antibody binding and T-cell reactivity are well known in the art. For example, screens for antigenicity or immunogenicity may generally be performed using well known methods such as those described in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988, or in Paul, Fundamental Immunology, 3rd ed., 243-247 (Raven Press, 1993) and references cited therein. In particular assays, a peptide may be immobilized on a solid support and contacted with subject sera to allow binding of antibodies within the sera to the immobilized polypeptide. Unbound sera may then be removed and bound antibodies detected using, for example, ¹²⁵I-labeled (or other detectable) Protein A. [0178] The ability of a variant chimeric polypeptide (or epitope) to react with antigen-specific antisera may be unchanged relative to the reference chimera (or epitope), or may be enhanced or diminished by less than 30%, for example, less than 20%, such as less than 10%, relative to the reference sequence.

[0179] Chimeric polypeptides (including variants thereof) may be prepared using any of a variety of well known techniques, including, for example, the techniques described in the Examples. Recombinant polypeptides encoded by DNA sequences as described below may be readily prepared from the DNA sequences using any of a variety of known expression vectors. Expression may be achieved in any appropriate host cell that has been transformed or transfected with an expression vector containing a DNA molecule that encodes a recombinant polypeptide. Suitable host cells include prokaryotes, yeast, higher eukaryotic and plant cells. For example, suitable host cells include E. coli, yeast, insect cells, and mammalian cell lines such as COS or CHO. Supernatants from suitable host/vector systems which secrete recombinant polypeptide into culture media may be first concentrated using a commercially available filter. Following concentration, the concentrate may be applied to a suitable purification matrix such as an affinity matrix or an ion exchange resin. One or more reverse phase HPLC steps also can be employed to further purify a recombinant polypeptide.

[0180] A particular example of a suitable expression system is a baculovirus expression system employing the Sf21 insect cell line, which is described in Chatterjee et ah, Gene, 171:209-213, 1996. Additional examples include pET expression systems (Novagen, Madison, WI) and Topo expression systems (Invitrogen, San Diego CA).

[0181] Polypeptides having fewer than about 100 amino acids, and generally fewer than about 50 amino acids, may also be generated by synthetic means, using techniques well known to those of ordinary skill in the art. For example, such polypeptides may be synthesized using any of the commercially available solid-phase techniques, such as the Merrifield solid-phase synthesis method, where amino acids are sequentially added to a growing amino acid chain (see, for example, Merrifield, J. Am Chem. Soc. 85:2149-2146, 1963). Equipment for automated synthesis of polypeptides is commercially available from suppliers such as Perkin Elmer/Applied BioSystems Division (Foster City, CA), and may be operated according to the manufacturer's instructions.

IV. Polynucleotides

[0182] Disclosed polynucleotides include DNA or RNA sequences that encode all or a portion of the disclosed chimeric polypeptides, sequences that are complementary to such polynucleotide sequences (such as cDNA), and variants, such as conservative variants, of such polynucleotide sequences. Polynucleotides complementary to any such sequences are also encompassed by the disclosure. Polynucleotides may be single stranded (coding or antisense), double stranded or triplexed, and may be DNA (genomic, cDNA or synthetic) or RNA molecules. RNA molecules include HnRNA molecules, which contain introns and correspond to a DNA molecule in a one-to-one manner, and mRNA molecules, which do not contain introns. Additional coding or non-coding sequences may, but need not, be present in a polynucleotide, and a polynucleotide may, but need not, be linked to other molecules and/or support materials. [0183] Polynucleotide variants may contain one or more substitutions, additions, deletions and/or insertions such that the immunogenicity of the encoded peptide or polypeptide is not diminished, relative to the particularly disclosed peptides and polypeptides. The effect on the immunogenicity of the encoded polypeptide may generally be assessed as described herein. Variants typically exhibit at least about 70% identity, more typically at least about 80% identity and most typically at least about 90% identity to a polynucleotide sequence that encodes a disclosed chimeric polypeptide, or a fragment or fusion protein thereof. The term "variants" also encompasses homologous genes of xenogenic origin.

[0184] Two polynucleotide (or polypeptide sequences) are said to be "identical" if the sequence of nucleotides (or amino acids) in the two sequences is the same when aligned for maximum correspondence as described below. Comparisons between two sequences are typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. A "comparison window" as used herein, refers to a segment of at least about 20 contiguous positions, usually 30 to about 75, 40 to about 50, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.

[0185] Optimal alignment of sequences for comparison may be conducted using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, Wis.), using default parameters. This program embodies several alignment schemes described in Dayhoff, M. O., "A model of evolutionary change in proteins—Matrices for detecting distant relationships," in Atlas of Protein Sequence and Structure, Dayhoff, M. O. (ed.), National Biomedical Research Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358, 1978; Hein, "Unified Approach to Alignment and Phylogenes," Methods in Enzymology, vol. 183, Academic Press, Inc., San Diego, CA, pp. 626-645, 1990; Higgins and Sharp, CABIOS, 5:151-153 (1989); Myers and Muller, CABIOS 4:11-17 (1988); Robinson, Comb. Theor., 11:105 (1971); Santou and Nes, MoI. Biol. Evol, 4:406-425, 1987; Sneath and Sokal, Numerical Taxonomy-the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA, 1973; and Wilbur and Lipman, Proc. Natl. Acad., Sd. USA, 80:726-730, 1983.

[0186] The "percentage of sequence identity" may, for example, be determined by comparing two optimally aligned sequences over a window of comparison of at least 20 positions, wherein the portion of the polynucleotide (or polypeptide) sequence in the comparison window may comprise additions or deletions (i.e. gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12 percent, as compared to the reference sequences (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid bases (or amino acids) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e. the window size) and multiplying the results by 100 to yield the percentage of sequence identity.

[0187] Variants may also, or alternatively, be substantially homologous to a native gene, or a portion or complement thereof. Such polynucleotide variants are capable of hybridizing under moderately stringent conditions to a DNA sequence encoding a disclosed peptide or polypeptide (or a complementary sequence). Suitable moderately stringent conditions include pre- washing in a solution of 5xSSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50 ⁰C to 65 ⁰C, in a solution of 5xSSC overnight; and washing twice at 65 ⁰C for 20 minutes with each of 2x, 0.5x and 0.2x SSC containing 0.1% SDS.

[0188] It will be appreciated that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode the disclosed peptides, polypeptides, fusion proteins and variants (such as conservative variants) thereof. Thus, polynucleotides that vary due to differences in codon usage are specifically contemplated.

[0189] Polynucleotides and variants thereof may be prepared using any of a variety of techniques. There are numerous amplification techniques for obtaining a full length coding sequences from partial cDNA sequences. Within such techniques, amplification is generally performed via polymerase chain reaction (PCR). Any of a variety of commercially available kits may be used to perform the amplification step. Primers may be designed using, for example, software well known in the art. Typically, primers may be 22-30 nucleotides in length, have a GC content of at least 50% and anneal to the target sequence at temperatures of about 68 ⁰C to 72 ⁰C. The amplified fragments, such as overlapping fragments, may then be assembled into a contiguous sequence. Other methods employing amplification may also be employed to obtain a full length cDNA sequence.

[0190] Polynucleotides and variants thereof, and primers for use in amplification, may be prepared by any method known in the art, including chemical synthesis by, for example, solid phase phosphoramidite chemical synthesis. Modifications in a polynucleotide sequence may also be introduced using standard mutagenesis techniques, such as oligonucleotide-directed site-specific mutagenesis (see Adelman et al., DNA, 2:183, 1983). Alternatively, RNA molecules may be generated by in vitro or in vivo transcription of DNA sequences, provided that the DNA is incorporated into a vector with a suitable RNA polymerase promoter (such as T7 or SP6). Certain portions may be used to prepare an encoded polypeptide. Alternatively, in order to immunize the subject the nucleic acid sequences may be administered such that the encoded polypeptide is generated in vivo {e.g., by transfecting antigen-presenting cells, such as dendritic cells, with a cDNA construct encoding a disclosed peptide or polypeptide, and administering the transfected cells to the subject).

[0191] A portion of a coding sequence, or of a complementary sequence, may also be designed as a probe or primer to detect a P. falciparum infection. Probes and may be labeled with a variety of reporter groups, such as radionuclides and enzymes, and are typically at least 10 nucleotides in length, more typically at least 20 nucleotides in length and still more typically at least 30 nucleotides in length. Primers, as noted above, are typically 22-30 nucleotides in length, but may be longer in some embodiments.

[0192] Any polynucleotide may be further modified to increase stability in vivo. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3 '-ends; the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages in the backbone; and/or the inclusion of nontraditional bases such as inosine, queosine and wybutosine, as well as acetyl- methyl-, thio- and other modified forms of adenine, cytidine, guanine, thymine and uridine.

[0193] Nucleotide sequences as described herein may be joined to a variety of other nucleotide sequences using established recombinant DNA techniques. For example, a polynucleotide may be cloned into any of a variety of cloning vectors, including plasmids, phagemids, lambda phage derivatives and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors and sequencing vectors. In general, a vector will contain an origin of replication functional in at least one organism, convenient restriction endonuclease sites and one or more selectable markers. Other, additional coding and non-coding elements may be added depending upon the use and the organism or cell in which expression is desired.

[0194] Within certain embodiments, polynucleotides may be formulated so as to permit entry into a cell, for example, a mammalian cell, and expression therein. Such formulations are particularly useful for prophylactic and therapeutic purposes, as described below. It will be appreciated that there are many ways to achieve expression of a polynucleotide in a target cell, and any suitable method may be employed. For example, a polynucleotide may be incorporated into a viral vector such as, but not limited to, adenovirus, adeno-associated virus, retrovirus, or vaccinia or other pox virus (such as an avian pox virus). The polynucleotides may also be administered as naked plasmid vectors. Techniques for incorporating DNA into such vectors are well known. A retroviral vector may additionally transfer or incorporate a gene for a selectable marker (to aid in the identification or selection of transduced cells) and/or a targeting moiety, such as a gene that encodes a ligand for a receptor on a specific target cell, to render the vector target specific. Targeting may also be accomplished using an antibody as is known in the art.

[0195] Other formulations for therapeutic purposes include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in- water emulsions, micelles, mixed micelles, and liposomes (i.e. an artificial membrane vesicle). The preparation and use of such systems is well known.

V. Prophylaxis and/or Treatment of Malarial Infections

[0196] The compositions described herein may be used in methods for stimulating an immune response in a subject against malarial infections, such as P. falciparum infections. Within such methods, compositions such as pharmaceutical compositions are typically administered to a subject. A "subject" refers to any animal, but more typically is a human. A subject may or may not be afflicted with a malarial infection. Accordingly, the compositions may be used to inhibit the later development of a malarial infection (prophylaxis) in an uninfected subject or to treat a subject afflicted with a malarial infection (treatment). Although it is desirable to administer an immunogenic agent 1-3 months prior to an initial infection or possible exposure (for example, prior to travel to an area in which malaria is endemic) the compositions may be administered after initial infection to ameliorate disease progression, or after initial infection, to treat the disease. A malarial infection may be diagnosed using any generally accepted criteria, including the presence of malaria symptoms or on the basis of a screening test for the presence of the malarial parasite. For example, the disclosed antibodies and/or chimeric polypeptides may be used in assays to detect the presence of malarial infection in a subject. Alternatively, microscopic examination of a blood smear for the presence parasites, for example, parasitized erythrocytes, merosoites, or gametocytes can reveal a malarial infection in a subject (see, for example, Example 11).

[0197] Within certain embodiments, stimulation of an immune response refers to active immunotherapy where in vivo stimulation of the endogenous host immune system to react against malarial infections occurs upon administration of immune response-modifying agents (such as the polypeptides and polynucleotides disclosed herein). In these embodiments, the methods of prophylaxis or treatment comprise stimulation of an immune response.

[0198] Within other embodiments, immunotherapy may be passive immunotherapy, in which treatment involves the delivery of agents with established immune reactivity (such as autologous effector cells or antibodies) that can directly or indirectly mediate anti-malarial effects and does not necessarily depend on an intact host immune system. Examples of effector cells include T cells as discussed above, T lymphocytes (such as CD8+ cytotoxic T lymphocytes and CD4+ T-helper tumor-infiltrating lymphocytes), killer cells (such as Natural Killer cells and lymphokine-activated killer cells), B cells and antigen- presenting cells (such as dendritic cells and macrophages) expressing a peptide or polypeptide provided herein. T cell receptors and antibody receptors specific for the peptides and polypeptides disclosed herein may be cloned, expressed and transferred into other vectors or effector cells for adoptive immunotherapy. The polypeptides (or immunogenic portions thereof) provided herein may also be used to generate antibodies or anti-idiotypic antibodies (as described, for instance, in the Examples and U.S. Pat. No. 4,918,164) for passive immunotherapy.

[0199] Effector cells, such as autologous effector cells, may generally be obtained in sufficient quantities for adoptive immunotherapy by growth in vitro, as described herein. Culture conditions for expanding single antigen-specific effector cells to several billion in number with retention of antigen recognition in vivo are well known. Such in vitro culture conditions typically use intermittent stimulation with antigen, often in the presence of cytokines (such as IL-2) and non-dividing feeder cells. As noted above, immunogenic peptides and polypeptides as provided herein may be used to rapidly expand antigen- specific T cell cultures in order to generate a sufficient number of cells for immunotherapy. In particular, antigen-presenting cells, such as dendritic, macrophage, monocyte, fibroblast and/or B cells, may be pulsed with immunoreactive polypeptides or transfected with one or more polynucleotides using standard techniques. For example, antigen-presenting cells can be transfected with a polynucleotide having a promoter appropriate for increasing expression in a recombinant virus or other expression system. Cultured effector cells for use in therapy advantageously are able to grow and distribute widely, and to survive long term in vivo. Cultured effector cells can be induced to grow in vivo and to survive long term in substantial numbers by repeated stimulation with antigen supplemented with IL-2 (see, for example, Cheever et al., Immunological Reviews 157:177, 1997).

[0200] Alternatively, a vector expressing a disclosed chimeric polypeptide or fusion protein may be introduced into antigen presenting cells taken from a subject and clonally propagated ex vivo for transplant back into the same subject. Transfected cells may be reintroduced into the subject using any means known, for example, in sterile form by intravenous or intraperitoneal administration.

[0201] Routes and frequency of administration of the disclosed compositions, as well as dosage, will vary from individual to individual, and may be readily established using standard techniques. In general, the compositions (including pharmaceutical compositions) may be administered by injection (such as intracutaneous, intramuscular, intravenous or subcutaneous injection), intranasally (such as by aspiration) or orally. For example, between 1 and 10 doses may be administered over a 52 week period. In one embodiment, 6 doses are administered, at intervals of 1 month, and booster vaccinations may be given periodically thereafter. Alternate protocols may be appropriate for individual subjects. A suitable dose is an amount of a composition that, when administered to a subject, is capable of promoting an anti-malarial immune response, that is at least 10-50% above the basal (untreated) level. Such response can be monitored by measuring the anti-malarial antibodies in a subject or by vaccine-dependent generation of cytolytic effector cells capable of killing the malaria parasite (or one of its life stages) in vitro. Such compositions also may be capable of causing an immune response that leads to an improved clinical outcome (such as less frequent infections, or complete or partial or longer disease-free survival) in treated subjects as compared to non-treated subjects. In general, for pharmaceutical compositions comprising one or more polypeptides, the amount of each present in a dose ranges, for a 4.5 kg infant, from about 200 ng/kg to about 200 μg/kg (such as, from about 3 μg/kg to about 55 μg/kg), or, for an 80 kg adult, from about 12.5 ng/kg to about 12.5 μg/kg (such as, from about 185 ng/kg to about 3 μg/kg). Suitable doses can be readily adjusted for subjects of different body weights by those of ordinary skill in the art. Suitable doses can be administered in a variety of volumes (depending upon the concentration of the agent) and most likely will vary with the size of the subject, but will typically range from about 0.1 mL to about 5 mL, for example, from about 0.5 mL to 1.0 mL.

[0202] In general, an appropriate dosage and treatment regimen provides the active composition(s) in an amount sufficient to provide a therapeutic and/or prophylactic benefit. Such a response can be monitored by establishing an improved clinical outcome in treated subjects as compared to non-treated subjects. Increases in preexisting immune responses to a malarial infection generally correlate with an improved clinical outcome. Such immune responses may generally be evaluated using standard proliferation, cytotoxicity or cytokine assays, which may be performed using samples obtained from a subject before and after treatment. Nonetheless, an immune response may be stimulated in a subject with 1 to 5 dosages. An initial dose may comprise from 1 μg to 1 mg of the disclosed immunogenic polypeptides, for example, 10 μg to 800 μg of the polypeptide, such as from 15 μg to 250 μg. Subsequent, "booster" doses may comprise from 1 μg to 1 mg of the disclosed immunogenic polypeptides, for example, from 10 μg to 800 μg, such as from 15 μg to 250 μg.

[0203] In other embodiments, an effective amount of a composition administered to a subject (such as, a human subject) will vary depending upon a number of factors associated with that subject, including whether the subject previously has been exposed to P. falciparum. An effective amount of a composition can be determined by varying the dosage of the product and measuring the resulting cellular and humoral immune and/or therapeutic responses. For example, about 5-500 μg of a chimeric polypeptide is administered for an initial immunization, and approximately 5-500 μg is administered for a subsequent booster immunization. In another example, approximately 5-500 μg of a chimeric polypeptide is mixed together with approximately 20-2000 μg of an adjuvant, such as aluminum phosphate, and administered. In yet other examples, about 10-250 μg of a chimeric polypeptide is mixed with about 40-1000 μg of an adjuvant and is administered, or about 15-100 μg of a chimeric polypeptide is mixed with about 60-400 μg of an adjuvant and is administered.

VI. Detecting Malarial Infections

[0204] In general, a malarial infection may be detected in a subject based on analysis of a biological sample (for example, blood, sera, sputum urine and/or biopsies obtained from the subject) for the presence of binding agents such as antibodies against one or more malarial epitopes. In other words, the disclosed chimeric polypeptides can be used in assays to indicate the presence or absence of a malarial infection since they contain malarial epitopes. Thus, specific binding agents such as serum antibodies can be detected by their binding to the chimeric polypeptides.

[0205] There are a variety of assay formats known for using a binding agent to detect its binding partner in a sample (see, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988). In general, the presence or absence of a malarial infection in a subject may be determined by (a) contacting a biological sample obtained from a subject with a chimeric polypeptide; (b) detecting in the sample a level of the binding agent that binds to the chimeric polypeptide; and (c) comparing the level of binding agent with a predetermined cut-off value.

[0206] In one embodiment, the assay involves the use of a chimeric polypeptide immobilized on a solid support to bind to malaria antibodies in the sample. The bound antibodies may then be detected using a detection reagent that contains a reporter group and specifically binds to the binding agent/chimeric polypeptide complex. Such detection reagents may comprise, for example, a binding agent that specifically binds to the polypeptide or an antibody or other agent that specifically binds to the binding agent, such as an antiimmunoglobulin, protein G, protein A or a lectin. [0207] The solid support may be any material known to which the disclosed chimeric polypeptides, combinations of such chimeric polypeptides, fusion proteins comprising the disclosed chimeric polypeptides, or polynucleotides coding for the same may be attached. For example, the solid support may be a test well in a microliter plate or a nitrocellulose or other suitable membrane. Alternatively, the support may be a bead or disc, such as glass, fiberglass, latex or a plastic material such as polystyrene or polyvinylchloride. The support may also be a magnetic particle or a fiber optic sensor, such as those disclosed, for example, in U.S. Pat. No. 5,359,681. The chimeric polypeptide may be immobilized on the solid support using a variety of techniques that are described in the patent and scientific literature (see, for example U.S. Pat. No. 4,948,836). In the context of the disclosure, the term "immobilization" refers to both noncovalent association, such as adsorption, and covalent attachment (which may be a direct linkage between the agent and functional groups on the support or may be a linkage by way of a cross- linking agent). Adsorption may be achieved by contacting the chimeric polypeptide, in a suitable buffer, with the solid support for a suitable amount of time. The contact time varies with temperature, but is typically between about 1 hour and about 1 day. In general, contacting a well of a plastic microtiter plate (such as polystyrene or polyvinylchloride) with an amount of chimeric polypeptide ranging from about 10 ng to about 10 μg, such as from about 100 ng to about 1 μg, is sufficient to immobilize an adequate amount of chimeric polypeptide.

[0208] Covalent attachment of binding agent to a solid support may generally be achieved by reacting the support with a bifunctional reagent that will react with both the support and a functional group, such as a hydroxyl or amino group, on the chimeric polypeptide. For example, the chimeric polypeptide may be covalently attached to supports having an appropriate polymer coating using benzoquinone or by condensation of an aldehyde group on the support with an amine and an active hydrogen on the binding partner (see, for example, the Pierce Immunotechnology Catalog and Handbook, 1991, at A12-A13).

[0209] In certain embodiments, the assay is a two-antibody sandwich assay. This assay may be performed by first contacting an antibody that has been immobilized on a solid support (for example, in a well of a microtiter plate) with the sample such that polypeptides within the sample are allowed to bind to the immobilized antibody. Unbound sample is then removed from the immobilized polypeptide-antibody complexes and a detection reagent (preferably a second antibody capable of binding to a different site on the polypeptide) containing a reporter group is added. The amount of detection reagent that remains bound to the solid support is then determined using a method appropriate for the specific reporter group.

[0210] More specifically, once the antibody is immobilized on the support as described above, the remaining protein binding sites on the support are typically blocked. Any suitable blocking agent known to those of ordinary skill in the art, such as bovine serum albumin or a nonionic surfactant such as Tween 20 (Sigma Chemical Co., St. Louis, Mo.). The immobilized antibody is then incubated with the sample, and polypeptide is allowed to bind to the antibody. The sample may be diluted with a suitable diluent, such as phosphate-buffered saline (PBS) prior to incubation. In general, an appropriate contact time (i.e., incubation time) is a period of time that is sufficient to detect the presence of polypeptide within a sample obtained from an individual with malaria. Typically, the contact time is sufficient to achieve a level of binding that is at least about 95% of that achieved at equilibrium between bound and unbound polypeptide. The time necessary to achieve equilibrium may be readily determined by assaying the level of binding that occurs over a period of time. At room temperature, an incubation time of about 30 minutes is generally sufficient.

[0211 ] Unbound sample may then be removed by washing the solid support with an appropriate buffer, such as PBS containing 0.01-0.10% Tween 20. The second antibody, which contains a reporter group, may then be added to the solid support. Reporter groups include fluorescent, radiographic and enzymatic reporter groups.

[0212] The detection reagent is then incubated with the immobilized antibody-chimeric polypeptide complex for an amount of time sufficient to detect the bound antibody. An appropriate amount of time may generally be determined by assaying the level of binding that occurs over a period of time. Unbound detection reagent is then removed and bound detection reagent is detected using the reporter group. The method employed for detecting the reporter group depends upon the nature of the reporter group. For radioactive groups, scintillation counting or autoradiographic methods are generally appropriate. Spectroscopic methods may be used to detect dyes, luminescent groups and fluorescent groups. Biotin may be detected using avidin, coupled to a different reporter group (commonly a radioactive or fluorescent group or an enzyme). Enzyme reporter groups may generally be detected by the addition of substrate (generally for a specific period of time), followed by spectroscopic or other analysis (such as chromatographic analysis) of the reaction products.

[0213] To determine the presence or absence of a malarial infection the signal detected from the reporter group that remains bound to the solid support is generally compared to a signal that corresponds to a predetermined cut-off value. In one embodiment, the cut-off value for the detection of a malarial infection is the average mean signal obtained when the immobilized antibody is incubated with samples from subjects without a malarial infection. In general, a sample generating a signal that is three standard deviations above the predetermined cut-off value is considered positive for the infection. In an alternate embodiment, the cut-off value is determined using a Receiver Operator Curve, according to the method of Sackett et al, Clinical Epidemiology: A Basic Science for Clinical Medicine, Little Brown and Co., 1985, p. 106-7. Briefly, in this embodiment, the cut-off value maybe determined from a plot of pairs of true positive rates (that is sensitivity) and false positive rates (100%-specificity) that correspond to each possible cut-off value for the diagnostic test result. The cut-off value on the plot that is the closest to the upper left-hand corner (that is the value that encloses the largest area) is the most accurate cut-off value, and a sample generating a signal that is higher than the cut-off value determined by this method may be considered positive. Alternatively, the cut-off value may be shifted to the left along the plot, to minimize the false positive rate, or to the right, to minimize the false negative rate. In general, a sample generating a signal that is higher than the cut-off value determined by this method is considered positive for a malarial infection.

[0214] In a related embodiment, the assay is performed in a flow-through or strip test format, wherein the chimeric polypeptide is immobilized on a membrane, such as nitrocellulose. In the flow- through test, antibodies within the sample bind to the immobilized chimeric polypeptide as the sample passes through the membrane. A second, labeled binding agent then binds to the binding agent-chimeric polypeptide complex as a solution containing the second binding agent flows through the membrane. The detection of bound second binding agent may then be performed as described above. In the strip test format, one end of the membrane to which chimeric polypeptide is bound is immersed in a solution containing the sample. The sample migrates along the membrane through a region containing the second binding agent and to the area of the immobilized chimeric polypeptide. The concentration of second binding agent at the area of immobilized chimeric polypeptide indicates the presence of a malarial infection. Typically, the concentration of second binding agent at that site generates a pattern, such as a line, that can be read visually. The absence of such a pattern indicates a negative result. In general, the amount of chimeric polypeptide immobilized on the membrane is selected to generate a visually discernible pattern when the biological sample contains a level of antibody that would be sufficient to generate a positive signal in the two-antibody sandwich assay, in the format discussed above. The amount of chimeric polypeptide immobilized on the membrane ranges from about 25 ng to about 1 μg such as from about 50 ng to about 500 ng. Such tests can typically be performed with a very small amount of biological sample.

[0215] Of course, numerous other assay protocols exist that are suitable for use with the disclosed polypeptides and fusion proteins. The descriptions above are only exemplary. For example, it will be apparent that the above protocols may be readily modified to use the disclosed polypeptides to detect antibodies that bind to such polypeptides in a biological sample. The detection of such malarial protein specific antibodies may correlate with the presence of a malarial infection.

[0216] A malarial infection may also be detected based on the presence of T cells that specifically react with disclosed chimeric polypeptides (and/or fusion proteins including the same) in a biological sample. Within certain methods, a biological sample comprising CD4+ and/or CD8+ T cells isolated from a subject is incubated with a disclosed chimeric polypeptide, fusion protein including a disclosed chimeric polypeptide, a polynucleotide encoding such a polypeptide or fusion protein and/or an APC that expresses at least an immunogenic portion of a disclosed chimeric polypeptide or fusion protein including a disclosed chimeric polypeptide. The presence or absence of specific activation of the T cells is then detected. Suitable biological samples include, but are not limited to, isolated T cells. For example, T cells may be isolated from a subject by routine techniques (such as by Ficoll/Hypaque density gradient centrifugation of peripheral blood lymphocytes). T cells may be incubated in vitro for 2-9 days (typically 4 days) at 37° C with polypeptide (for example, at a concentration of 5-25 μg/ml). It may be desirable to incubate another aliquot of a T cell sample in the absence of the polypeptide to serve as a control. For CD4+ T cells, activation may be detected by evaluating proliferation of the T cells. For CD8+ T cells, activation may be detected by evaluating cytolytic activity. A level of proliferation that is at least two fold greater and/or a level of cytolytic activity that is at least 20% greater than in disease-free subjects indicates the presence of a malarial infection in the subject.

VII. Pharmaceutical Compositions

[0217] In some embodiments, disclosed immunogenic chimeric polypeptides (including fusion proteins), polynucleotides, and/or binding agents are incorporated into pharmaceutical compositions (such as immunogenic compositions or vaccines). Pharmaceutical compositions comprise one or more such immunogenic chimeric polypeptides (including fusion proteins), polynucleotides, and/or binding agents and a physiologically acceptable carrier (also referred to as a pharmaceutically acceptable carrier). Pharmaceutical compositions also may comprise an immunostimulant. An immunostimulant is any substance that enhances or potentiates an immune response to an exogenous antigen. Examples of immunostimulants include adjuvants, biodegradable microspheres (such as polylactic galactide microspheres) and liposomes (see, for example, U.S. Pat. No. 4,235,877). Vaccine preparation is generally described, for example, in M. F. Powell and M. J. Newman, eds., Vaccine Design: The subunit and adjuvant approach, Plenum Press, NY, 1995. Pharmaceutical compositions within the scope of the disclosure also may contain other compounds, which may be either biologically active or inactive. For example, one or more immunogenic portions of other malarial proteins may be present, either incorporated into a fusion protein or as a separate compound.

[0218] A pharmaceutical composition may comprise DNA encoding one or more of the disclosed polypeptides, such that the polypeptide(s) is (are) generated in situ. As noted above, the DNA may be present within any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid expression systems, bacteria and viral expression systems. Numerous gene delivery techniques are well known in the art, including those described by Rolland, CHt. Rev. Therap. Drug Carrier Systems 15:143-198, 1998, and references cited therein. Appropriate nucleic acid expression systems contain DNA sequences for expression in the subject (such as a suitable promoter and terminating signal). Bacterial delivery systems involve the administration of a bacterium (such as Bacillus-Calmette-Guerrin) that expresses the polypeptide on its cell surface and/or secretes it. In one embodiment, the DNA is introduced using a viral expression system (e.g., vaccinia or other pox virus, retrovirus, or adenovirus), which may involve the use of a non-pathogenic (defective), replication competent virus. Suitable systems are disclosed, for example, in Fisher-Hoch et ah, Proc. Natl. Acad. ScL USA, 86:317-321, 1989; Flexner et ah, Ann. NY. Acad. ScL, 569:86-103, 1989; Flexner et ah, Vaccine, 8:17-21, 1990; U.S. Pat. Nos. 4,603,112, 4,777,127, 4,769,330, and 5,017,487; PCT publication nos. WO 89/01973 and WO 91/02805; Berkner, Biotechniques, 6:616-627, 1988; Rosenfeld et ah, Science, 252:431-434, 1991; Kolls et ah, Proc. Natl. Acad. ScL USA, 91:215-219, 1994; Kass-Eisler et ah, Proc. Natl. Acad. ScL USA, 90:11498- 11502, 1993; Guzhian et ah, Circulation, 88:2838-2848, 1993; and Guzman et ah, Cir. Res., 73: 1202-1207, 1993. Techniques for incorporating DNA into such expression systerhs are known. DNA also may also be incorporated as "naked DNA," as described, for example, in Ulmer et ah, Science, 259:1745-1749, 1993 and Cohen, Science, 259:1691-1692, 1993. Uptake of naked DNA may be increased by coating the DNA onto biodegradable beads, which are more efficiently transported into cells.

[0219] Any suitable pharmaceutically acceptable carrier known to those of ordinary skill in the art may be employed in a disclosed pharmaceutical composition. For example, Martin, Remington 's Pharmaceutical Sciences, 15th Edition, Mack Publishing Co., Easton, PA, 1975, describes compositions and formulations suitable for pharmaceutical delivery of the described polypeptides and nucleic acids. In general, the nature of the pharmaceutically acceptablecarrier will depend on the particular mode of administration being employed. Pharmaceutical compositions may be formulated for any appropriate manner of administration, including for example, oral (including buccal or sublingual), nasal, rectal, aerosol, topical, intravenous, intraperitoneal, intradermal, subcutaneous or intramuscular administration. For parenteral administration, such as subcutaneous injection, the pharmaceutically acceptable carrier may comprise water, saline, alcohol, a fat, a wax or a buffer. Other exemplar parenteral formulations comprise pharmaceutically and physiologically acceptable fluids such as physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like. For oral administration, any of the above carriers or a solid carrier, such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, and magnesium carbonate, may be employed. Biodegradable microspheres (e.g., polylactate polyglycolate) may also be employed as carriers for the pharmaceutical compositions. Suitable biodegradable microspheres are disclosed, for example, in U.S. Pat. Nos. 4,897,268 and 5,075,109. For solid compositions (for example, powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. Pharmaceutically acceptable carriers can also optionally contain 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.

[0220] The disclosed pharmaceutical compositions may also comprise buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, and additional proteins, polypeptides or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, and immunostimulants (such as adjuvants, for example, aluminum phosphate, aluminum hydroxide, or aluminum hydroxyphosphate) and/or preservatives. Alternatively, compositions of the present invention may be formulated as a lyophilizate, or stored at temperatures from about 4⁰C to -100⁰C. Compositions may also be encapsulated within liposomes using well known technology. Furthermore, the compositions may be sterilized, for example, by filtration, radiation and/or heat. A bacteriostatic agent such as phenoxy-ethanol or thimerosal also may be added to the composition.

[0221 ] Any of a variety of immunostimulants may be employed in the pharmaceutical compositions. For example, an adjuvant may be included. Some adjuvants contain a substance designed to protect the antigenic peptide or polypeptide from rapid catabolism, for example, aluminum hydroxide or mineral oil, and a stimulator of immune responses, such as lipid A, Bordetella pertussis- or Mycobacterium tuberculosis-deήved proteins. Suitable adjuvants are commercially available as, for example, Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ.), TiterMax Gold (TiterMax, Norcross, GA), ISA-720 (Seppic, France) ASO-2 (SmithKlineGlaxo, Rixensart, Belgium); aluminum salts such as aluminum hydroxide gel (alum), aluminum hydroxyphosphate, or aluminum phosphate; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and saponins such as quil A and QS-21 (Antigenics, Framingham, MA). Cytokines, such as GM- CSF or interleukin-2, -7, or -12, also may be used as adjuvants.

[0222] The adjuvant composition may be designed to induce an immune response predominantly of the ThI type. High levels of Thl-type cytokines (e.g., IFN-G, TNF-α., IL-2 and IL-12) tend to favor the induction of cell mediated immune responses to an administered antigen. In contrast, high levels of Th2-type cytokines (such as IL-4, IL-5, IL-6 and IL-10) tend to favor the induction of humoral immune responses. Following administration of a pharmaceutical composition as provided herein, a subject may support an immune response that includes ThI- and Th2-type responses. However, within certain embodiments, in which a response is predominantly ThI -type, the level of Thl-type cytokines will increase to a greater extent than the level of Th2-type cytokines. The levels of these cytokines may be readily assessed using standard assays. For a review of the families of cytokines, see Mosmann and Coffman, Ann. Rev. Immunol. 7:145-173, 1989.

[0223] Adjuvants for use in eliciting a predominantly Thl-type response include, for example, a combination of monophosphoryl lipid A, such as 3-de-O-acylated monophosphoryl lipid A (3D-MPL), together with an aluminum salt. MPL adjuvants are available from Corixa (Seattle, WA; see also U.S. Pat. Nos. 4,436,727; 4,877,611; 4,866,034 and 4,912,094). CpG-containing oligonucleotides (in which the CpG dinucleotide is unmethylated) also induce a predominantly ThI response. Such oligonucleotides are well known and are described, for example, in PCT publications WO 96/02555 and WO 99/33488. Imrnunostimulatory DNA sequences are also described, for example, by Sato et ah, Science 273:352, 1996. Another adjuvant is a saponin such as QS21 (Antigenics, Framingham, Mass.), which may be used alone or in combination with other adjuvants. For example, an enhanced system involves the combination of a monophosphoryl lipid A and saponin derivative, such as the combination of QS21 and 3D-MPL as described in WO 94/00153, or a less reactogenic composition where the QS21 is quenched with cholesterol, as described in WO 96/33739. Other formulations comprise an oil-in-water emulsion and tocopherol. An adjuvant formulation involving QS21, 3D-MPL and tocopherol in an oil-in-water emulsion is described in WO 95/17210.

[0224] Still further adjuvants include Montanide ISA 720 (Seppic, France), SAF (Chiron, California, United States), ISCOMS (CSL), MF-59 (Chiron), the ASO-2 series of adjuvants (SmithKlineGlaxo, Rixensart, Belgium), Detox (Corixa, Seattle, WA), RC-529 (Corixa, Seattle, WA), Aminoalkyl glucosaminide 4-phosphates (AGPs), copolymer adjuvants, CpG oligonucleotide motifs and combinations of CpG oligonucleotide motifs, bacterial extracts (such as mycobacterial extracts), detoxified endotoxins, and membrane lipids. Combinations of two or more adjuvants are also possible.

[0225] Still other adjuvants include polymers and co-polymers. For example, copolymers such as polyoxyethylene-polyoxypropylene copolymers and block co-polymers may be used. A particular example of a polymeric adjuvant is polymer P 1005.

[0226] Adjuvants are utilized in an adjuvant amount, which can vary with the adjuvant, mammal and immunogen. Typical amounts of non-emulsion adjuvants can vary from about 1 ng to about 500 mg per administration, for example, from 10 μg to 800 μg, such as from 50 μg to 500 μg. For emulsion adjuvants (oil-in-water and water-in-oil emulsions) the amount of the oil phase can vary from about 0.1% to about 70%, for example between about 0.5% and 5% oil in an oil-in-water emulsion and between about 30% and 70% oil in a water-in-oil emulsion. In one particular embodiment, a final formulation of adjuvant with antigen is a water-in-oil emulsion of approximately 70% (w/w) oil phase and approximately 30% (w/w) aqueous phase. Those of ordinary skill in the art will appreciate appropriate concentrations of adjuvants, and such amounts can be readily determined. [0227] Any pharmaceutical composition provided herein may be prepared using well known methods that result in a combination of antigen, immunostimulant and a suitable carrier or excipient. Such compositions may be administered as part of a sustained release formulation (such as a capsule, sponge or gel comprising polysaccharides) that provides a slow release of the composition following administration. Such formulations may generally be prepared using well known technology (see, for example, Coombes et al., Vaccine 14:1429-1438, 1996) and administered by, for example, subcutaneous implantation at the desired target site. Sustained-release formulations may contain a polypeptide, polynucleotide or antibody dispersed in a carrier matrix and/or contained within a reservoir surrounded by a rate controlling membrane.

[0228] Carriers for use with the disclosed compositions are biocompatible, and may also be biodegradable, and the formulation also may provide a relatively constant level of active component release. Suitable carriers include microparticles of poly(lactide-co-glycolide), as well as polyacrylate, latex, starch, cellulose and dextran. Other delayed-release carriers include supramolecular biovectors, which comprise a non-liquid hydrophilic core (e.g., a cross-linked polysaccharide or oligosaccharide) and, optionally, an external layer comprising an amphophilic compound, such as a phospholipid (see, for example, U.S. Pat. No. 5,151,254 and PCT publication nos. WO 94/20078, WO/94/23701 and WO 96/06638). The amount of active compound contained within a sustained release formulation depends upon the site of implantation, the rate and expected duration of release and the nature of the condition to be treated or prevented.

[0229] Any of a variety of delivery vehicles may be employed with the disclosed pharmaceutical compositions to facilitate production of an antigen-specific immune response to the P. falciparum parasite in one or more of its life stages. Delivery vehicles include antigen presenting cells (APCs), such as dendritic cells, macrophages, B cells, monocytes and other cells that may be engineered to be efficient APCs. Such cells may, but need not, be genetically modified to increase the capacity for presenting the antigen, to improve activation and/or maintenance of the T cell response, to have anti¬ malarial effects and/or to be immunologically compatible with the receiver {i.e., matched HLA haplotype). APCs may generally be isolated from any of a variety of biological fluids and organs, including tumor and peritumoral tissues, and may be autologous, allogeneic, syngeneic or xenogeneic cells.

[0230] Certain embodiments employ dendritic cells or progenitors thereof as antigen- presenting cells. Dendritic cells are highly potent APCs (see, for example, Banchereau and Steinman, Nature 392:245-251, 1998). In general, dendritic cells may be identified based on their typical shape (stellate in situ, with marked cytoplasmic processes, or dendrites, visible in vitro), their ability to take up, process and present antigens with high efficiency, and their ability to activate naive T cell responses. Dendritic cells may, of course, be engineered to express specific cell-surface receptors or ligands that are not commonly found on dendritic cells in vivo or ex vivo, and such modified dendritic cells are contemplated. As an alternative to dendritic cells, secreted vesicles antigen-loaded dendritic cells (called exosomes) may be used (see Zitvogel et ah, Nature Med. 4:594-600, 1998).

[0231 ] Dendritic cells and progenitors may be obtained from peripheral blood, bone marrow, tumor-infiltrating cells, peritumoral tissues-infiltrating cells, lymph nodes, spleen, skin, umbilical cord blood or any other suitable tissue or fluid. For example, dendritic cells may be differentiated ex vivo by adding a combination of cytokines such as GM-CSF, IL-4, IL-13 and/or TNF-α to cultures of monocytes harvested from peripheral blood. Alternatively, CD34 positive cells harvested from peripheral blood, umbilical cord blood or bone marrow may be differentiated into dendritic cells by adding to the culture medium combinations of GM-CSF, IL-3, TNF-α, CD40 ligand, LPS, flt3 ligand and/or other compound(s) that induce differentiation, maturation and proliferation of dendritic cells.

[0232] Dendritic cells are conveniently categorized as either "immature" or "mature" cells, which allows a simple way to discriminate between two well characterized phenotypes. However, this nomenclature should not be construed to exclude all possible intermediate stages of differentiation. Immature dendritic cells are characterized as APCs with a high capacity for antigen uptake and processing, which correlates with the high expression of Fey receptor and mannose receptor. The mature phenotype is typically characterized by a lower expression of these markers, but a high expression of cell surface molecules responsible for T cell activation such as class I and class II MHC, adhesion molecules (e.g., CD54 and CDl 1) and costimulatory molecules (e.g., CD40, CD80, CD86 and 4-1BB).

[0233] APCs may generally be transfected with a polynucleotide encoding a disclosed chimeric polypeptide or fusion protein (or immunogenic portion or variant thereof) such that the chimeric polypeptide, fusion protein or an immunogenic portion thereof, is expressed on the cell surface. Such transfection may take place ex vivo, and a composition comprising such transfected cells may then be used for therapeutic purposes, as described herein. Alternatively, a gene delivery vehicle that targets a dendritic or other antigen presenting cell may be administered to a subject, resulting in transfection that occurs in vivo. In vivo and ex vivo transfection of dendritic cells, for example, may generally be performed using any methods known in the art, such as those described in PCT publication WO 97/24447, or the gene gun approach described by Mahvi et al., Immunology and Cell Biology 75:456-460, 1997. Antigen loading of dendritic cells may be achieved by incubating dendritic cells or progenitor cells with one or more of the disclosed chimeric polypeptides or fusion proteins, DNA (naked or within a plasmid vector) or RNA coding for the same, or with antigen-expressing recombinant bacteria or viruses (e.g., vaccinia, fowlpox, adenovirus or lentivirus vectors). Prior to loading, polypeptides or fusion proteins may be covalently conjugated to an immunological partner that provides T cell help (e.g., a carrier molecule). Alternatively, a dendritic cell may be loaded with a non-conjugated immunological partner, separately or in the presence of the desired composition.

[0234] Pharmaceutical compositions may be presented in unit-dose or multi-dose containers, such as sealed ampoules or vials. Such containers are typically hermetically sealed to preserve sterility of the formulation until use. In general, formulations may be stored as suspensions, solutions or as emulsions in oily or aqueous vehicles. Alternatively, a pharmaceutical composition may be stored in a freeze-dried condition requiring only the addition of a sterile liquid carrier immediately prior to use.

VIII. Binding Agents

[0235] Disclosed binding agents include antibodies, complementary nucleic acid sequences, antigen-presenting cells and immune system cells. Disclosed antibodies are immune system proteins, or antigen-binding (epitope-binding) fragments thereof, that are capable of binding any of the disclosed chimeric polypeptide sequences, corresponding nucleic acid sequences coding the chimeric polypeptides and fusion proteins comprising the chimeric polypeptides. Antibodies also can be combinations of antibodies that bind to particular portions of the chimeric polypeptides, for example, the portions corresponding to SEQ ID NOs: 5, 12, 14, 17 and 20 (or sequences with 90%, 95% or 98% identity thereto). Alternatively, antibodies can be a combination of antibodies that binds to at least 75%, 80%, 90% or 95% of all of the immunogenic portions (epitopes) of a chimeric polypeptide. For example, antibodies can be a combination of antibodies that binds to at least 14, 15, 16, 17, 18, 19, or 20 of the 21 epitopic peptide sequences that are included in the chimeric polypeptide sequence set forth as SEQ ED NO 2, or residues 1- 374 thereof.

[0236] Antigen presenting cells include dendritic cells, macrophages, monocytes, fibroblasts and B-cells that express a disclosed immunogenic polypeptide, a fusion protein comprising such an immunogenic polypeptide, or a polynucleotide coding for the same. Immune system cells, such as T cells, include T cells that are specific for the disclosed chimeric polypeptides, polynucleotides coding for the disclosed chimeric polypeptides and fusion proteins comprising the disclosed chimeric polypeptides.

[0237] For example, disclosed antibodies include combinations of antibodies that bind the epitopic sequences set forth as SEQ ID NOs: 5, 12, 14, 17 and 20, or a combination of epitopic sequences having at least 90%, 95% or 98% identity to SEQ ID NOs: 5, 12, 14, 17, and 20.

[0238] Disclosed antibodies and antigen-binding fragments thereof specifically bind to the disclosed chimeric polypeptides, polynucleotides coding the chimeric polypeptides, fusion proteins including the disclosed chimeric polypeptides, or at least some portion (such as 75%, 80%, 90% or 95%) of the immunogenic peptide sequences. These antibodies are therefore capable of identifying each of these epitopes. As used herein, an antibody, or antigen-binding fragment thereof, is said to "specifically bind" if it reacts at a detectable level (within, for example, an ELISA) with the disclosed compositions, and does not react detectably with unrelated peptides, polypeptides, nucleic acids and proteins under similar conditions. As used herein, "binding" refers to a noncovalent association between two separate molecules such that a complex is formed. The ability to bind may be evaluated by, for example, determining a binding constant for the formation of the complex. The binding constant is the value obtained when the concentration of the complex is divided by the product of the component concentrations. In general, two compounds are said to "specifically bind" when the binding constant for complex formation exceeds about 10⁴ L/mol, for example, exceeds about 10⁶ L/mol, exceeds about 10^s L/mol, or exceeds about 10¹⁰ L/mol. The binding constant may be determined using methods well known in the art.

[0239] Binding agents are also believed to be further capable of differentiating between subjects with and without a malarial infection, such as a P. falciparum infection, using the representative assays provided herein. Typically, antibodies or other binding agents that bind to disclosed peptides, polypeptides, polynucleotides and fusion proteins will generate a signal indicating the presence of an infection in at least about 20% (such as at least about 30%, 40% or 50%) of subjects with the disease, and will generate a negative signal indicating the absence of the disease in at least about 80% (such as at least about 90%, 95% or 98%) of individuals without the infection. To determine whether a binding agent satisfies this requirement, biological samples (such as blood, sera, sputum urine and/or biopsies) from subjects with and without an infection (as determined using standard clinical tests) may be assayed as described herein for the presence of the naturally occurring molecules that bind to the binding agent. It will be apparent that a statistically significant number of samples with and without the disease should be assayed. Although it is beneficial that each binding agent satisfy the above criteria; however, is should also be recognized that binding agents may be used in combination to improve sensitivity.

[0240] Any agent that satisfies the above requirements may be a binding agent. For example, a binding agent may be a ribosome, with or without a peptide component, a nucleic acid molecule (such as an RNA molecule) or a polypeptide. In particular embodiments, a binding agent is an antibody or an antigen- binding fragment thereof. Antibodies may be prepared by any of a variety of techniques known to those of ordinary skill in the art (see, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988). In general, antibodies can be produced by cell culture techniques, including the generation of monoclonal antibodies as described herein, or via transfection of antibody genes into suitable bacterial or mammalian cell hosts, in order to allow for the production of recombinant antibodies. In one technique, an immunogenic composition comprising an immunogenic chimeric polypeptide is initially injected into any of a wide variety of mammals (e.g., mice, rats, rabbits, sheep or goats). In this step, the chimeric polypeptide may serve as the immunogen without modification. Alternatively, particularly for relatively short polypeptides, a superior immune response may be elicited if the polypeptide is joined to a carrier protein (for example, in a fusion protein construct) such as bovine serum albumin or keyhole limpet hemocyanin. The immunogenic polypeptide in combination with an adjuvant (such as ISA-720, QS-21, TiterMax Gold, non-ionic copolymer in a water-in-oil emulsion, or aluminum phosphate) is injected into the animal host, preferably according to a predetermined schedule incorporating one or more booster immunizations, and the animals are bled periodically. Polyclonal antibodies specific for the immunogenic polypeptide may then be purified from such antisera by, for example, affinity chromatography using the polypeptide coupled to a suitable solid support.

[0241] Monoclonal antibodies specific for an immunogenic polypeptide or an immunogenic portion thereof may be prepared, for example, using the technique of Kohler and Milstein, Eur. J. Immunol., 6:511-519, 1976. Briefly, these methods involve the preparation of immortal cell lines capable of producing antibodies having the desired specificity (i.e., reactivity with the polypeptide of interest). Such cell lines may be produced, for example, from spleen cells obtained from an animal immunized as described above. The spleen cells are then immortalized by, for example, fusion with a myeloma cell fusion partner, preferably one that is syngeneic with the immunized animal. A variety of cell fusion techniques may be employed. For example, the spleen cells and myeloma cells may be combined with a nonionic detergent for a few minutes and then plated at low density on a selective medium that supports the growth of hybrid cells, but not myeloma cells. A particular selection technique uses HAT (hypoxanthine, arninopterin, thymidine) selection. After a sufficient time, usually about 1 to 2 weeks, colonies of hybrids are observed. Single colonies are selected and their culture supernatants tested for binding activity against the peptide or polypeptide. Hybridomas having high reactivity and specificity are typically selected.

[0242] Monoclonal antibodies may be isolated from the supernatants of growing hybridoma colonies. In addition, various techniques may be employed to enhance the yield, such as injection of the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host, such as a mouse. Monoclonal antibodies may then be harvested from the ascites fluid or the blood. Contaminants may be removed from the antibodies by conventional techniques, such as chromatography, gel filtration, precipitation, and extraction. The disclosed peptides and polypeptides may be used in the purification process in, for example, in an affinity chromatography step.

[0243] In certain embodiments, antigen-binding fragments of antibodies are prepared. Such fragments include Fab fragments, which may be prepared using standard techniques. Briefly, immunoglobulins may be purified from rabbit serum by affinity chromatography on Protein A bead columns (see, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988) and digested using papain to yield Fab and Fc fragments. The Fab and Fc fragments may be separated by affinity chromatography on protein A bead columns.

[0244] Isolated antibodies (and other binding agents) may be coupled to one or more therapeutic agents to further assist prophylaxis or treatment of malarial infections. Suitable therapeutic agents may be selected from any class of anti-malarial agent. Examples of suitable anti-malarial agents include quinoline compounds (such as chloroquin, diiodohdroxyquin, primacrine, quinine, quinacrine or pyronaradine), pyrimidine derivatives (such as trimethoprim or pyramethamine), aryl biguanides (such as chlorguanide, chlorpyroguanii), natural products (such as artemisinin) or other anti-protozoan drugs (such as atovaquone, atebrin, proguanil, malarone, maloprim, fansidar, mefloquine, doxycycline, sulphadoxine, N-l-(5,6-Dimethoxy-4-pyrimidynyl)-sulafanilamide and halfantrin). Additional information about such anti-malarial drugs and their dosages is found in Muson, Principles of Pharmacology, pp. 1440-1452, 1994.

[0245] It may be desirable to couple more than one agent to an antibody. In one embodiment, multiple molecules of an agent are coupled to one antibody molecule. In another embodiment, more than one type of agent may be coupled to one antibody. Regardless of the particular embodiment, antibody conjugates with more than one agent may be prepared in a variety of ways. For example, more than one agent may be coupled directly to an antibody molecule, or linkers which provide multiple sites for attachment can be used. Alternatively, a carrier can be used.

[0246] A carrier may bear the agents in a variety of ways, including covalent bonding either directly or via a linker group. Suitable carriers include proteins such as albumins (see U.S. Pat. No. 4,507,234), peptides and polysaccharides such as aminodextran (see U.S. Pat. No. 4,699,784). A carrier may also bear an agent by noncovalent bonding or by encapsulation, such as within a liposome vesicle (see, for example, U.S. Pat. Nos. 4,429,008 and 4,873,088).

[0247] A variety of routes may be used for administration of binding agents and conjugates made therefrom. Typically, administration will be intravenous, intramuscular, or subcutaneous. It will be evident that the precise dose of the binding agent/conjugate will vary depending upon the binding agent used, the antigen density and the rate of clearance of the binding agent/conjugate.

[0248] In addition to antibodies, disclosed binding agents include T cells specific for a disclosed chimeric polypeptide, and in particular specific for individual epitopes of the chimeric polypeptide that provoke a T-cell reaction. Such T-cells can be used for the ready indentification of functional variants of the chimeric polypeptides. Such cells may generally be prepared in vitro or ex vivo, using standard procedures. For example, T cells may be isolated from bone marrow, peripheral blood, or a fraction of bone marrow or peripheral blood of a subject, using a commercially available cell separation system, such as the Isolex System, available fromNexell Therapeutics, Inc. Irvine, Calif, (see also U.S. Pat. Nos. 5,240,856 and 5,215,926, and PCT publications WO 89/06280; WO 91/16116 and WO 92/07243). Alternatively, T cells may be derived from related or unrelated humans, non-human mammals, cell lines or cultures.

[0249] T cells may be stimulated with a disclosed chimeric polypeptide (such as a T-cell epitope containing chimeric polypeptide), a polynucleotide encoding the chimeric polypeptide and/or an antigen presenting cell (APC) that expresses the chimeric polypeptide. Stimulation is performed under conditions and for a time sufficient to permit the generation of T cells that are specific for the polypeptide, or an immunogenic portion thereof. For example, a polypeptide or polynucleotide is present within a delivery vehicle, such as a microsphere, to facilitate the generation of specific T cells.

[0250] T cells are considered to be specific for a chimeric polypeptide (such as a T-cell epitope containing chimeric polypeptide) if the T cells specifically proliferate, secrete cytokines or kill target cells either coated with the polypeptide or expressing a gene encoding the polypeptide. T cell specificity may be evaluated using any of a variety of standard techniques. For example, a chromium release assay or proliferation assay may be used, and a stimulation index of more than a two fold increase in lysis and/or proliferation, compared to negative controls, indicates T cell specificity. Such assays may be performed, for example, as described in Chen et al., Cancer Res. 54:1065-1070, 1994. Alternatively, detection of the proliferation of T cells may be accomplished by a variety of known techniques. For example, T cell proliferation can be detected by measuring an increased rate of DNA synthesis (such as by pulse-labeling cultures of T cells with tritiated thymidine and measuring the amount of tritiated thymidine incorporated into DNA), where contact with a peptide or polypeptide (for example, at 100 ng/ml-100 μg/ml, for example, between 200 ng/ml-25 μg/ml) for 3-7 days that results in at least a two fold increase in proliferation of the T cells indicates specificity. Specificity may also be evaluated by activation of T cells as measured using standard cytokine assays in which at least a two fold increase in the level of cytokine release (e.g., TNF or IFN-G) is indicative of specific T cell activation (see, for example, Coligan et al., Current Protocols in Immunology, Greene, ed., Vol. 1, Wiley Interscience, 1998). T cells activated in response to a disclosed polypeptide, polynucleotide or polypeptide-expressing APC may be CD4+ and/or CD8+. Specific T cells may be expanded using standard techniques. For example, the T cells are derived from either a subject or a related, or unrelated, donor and are administered to the subject following stimulation and expansion.

[0251 ] For therapeutic purposes, CD4+ or CD8+ T cells that proliferate in response to a disclosed polypeptide, polynucleotide or APC can be expanded in number either in vitro or in vivo. Proliferation of such T cells in vitro may be accomplished in a variety of ways. For example, the T cells can be re-exposed to a disclosed polypeptide with or without the addition of T cell growth factors, such as interleukin-2, and/or stimulator cells that synthesize the polypeptide or an immunogenic portion of the polypeptide. Alternatively, one or more T cells' that proliferate in the presence of the polypeptide can be expanded in number by cloning. Methods for cloning cells include limiting dilution. IX. Diagnostic Kits

[0252] Also disclosed are kits for use within any of the diagnostic methods described above. Such kits typically comprise two or more components necessary for performing a diagnostic assay. Components may be compounds, reagents, containers and/or equipment. For example, one container within a kit may contain a chimeric polypeptide that specifically binds to a malarial antibody. Such chimeric polypeptide may be provided attached to a support material, as described above. One or more additional containers may enclose elements, such as reagents or buffers, to be used in the assay. Such kits also may contain a detection reagent as described above that contains a reporter group suitable for direct or indirect detection of antibody binding to the chimeric polypeptide. Instructions for performing the assay also may be included in the kit. In some examples, kit components will be enclosed in a single packaging unit, such as a box or other container, which packaging unit may have compartments into which one or more components of the kit can be placed.

EXAMPLES

[0253] The following examples demonstrate (among other things) the utility of using a multi-epitope approach to design conformationally stable molecules that are immunogenic and can induce antibodies that react with intact pathogen to mediate immunological effector functions. These examples are provided to illustrate certain particular features and/or embodiments, but should not be construed to limit the invention to the particular features or embodiments described.

Example 1 Design of the Recombinant Immunogenic Polypeptide FALVAC-IA

[0254] Table 1 shows the epitopic peptide sequences and spacer amino acid sequences of a particular embodiment of the disclosed chimeric polypeptides. This embodiment is referred to in this and the following examples as "FALVAC-IA." The complete sequence of the chimeric FALVAC-IA polypeptide may be read from top to bottom in the column of amino acid sequences listed in Table 1, and concludes with an optional polyhistidine affinity tag at the C-terminus. In some instances, the embodiment of FALVAC-I A that contains a poly-His tag may be referred to as FALVAC-I A-His. FALVAC-IA without the optional poly-His tag corresponds to amino acid residues 1-374 of SEQ ID NO: 2. Another embodiment of FALVAC-IA corresponds to amino acid residues 2-374 (or residues 2-380) of SEQ ID NO: 2 because, as explained in more detail below, the initial methionine of FALVAC-IA can be clipped off during processing, which is very unlikely to substantially effect its P. faciparum-spedfic immunogenicity. Still another embodiment of FALVAC-IA includes a mixture of polypeptides having residues 1-374 of SEQ ID NO: 2 and polypeptides having residues 2-374 of SEQ ID NO: 2, or a mixture of polypeptides having residues 1 -380 of SEQ ID NO: 2 and polypeptides having residues 2-380 of SEQ ID NO: 2.

[0255] A combination of 21 immunogenic peptides (12 B-cell epitopes, 5 T-proliferation epitopes, 2 CTL epitopes, 1 T-helper epitope and 1 CTL + T-helper epitope) was selected for expression as a single chimeric polypeptide. The combination of epitopes covers a range of the natural human and P. falciparum genetic diversity, and includes epitopes from the sporozoite, liver and blood stages of the life cycle of the P. falciparum parasite. In particular, epitopic peptide sequences from immunogenic proteins associated with the sporozite stage (CSP), the liver stage (LSA-I), and the blood stage (MSP-I, MSP-2, AMA-I, EBA-175, and RAP-I) were included. The epitopes of the CSP, LSA-I, MSP-I, AMA-I, and RAP-I proteins were identified through vaccine-related field studies of naturally acquired protective immunity against malaria. The epitopes for the MSP-2 and EBA-175 were identified as conferring protection against invasion of erythrocytes by the parasite.

[0256] In addition to the immunogenic P. falciparum peptides, a number of spacer amino acid sequences were added between certain epitopic peptides. For example, a total of five GPGPG spacer sequences (SEQ ID NO: 26), which tend to induce 180° reverse turns in a polypeptide structure, were added between epitopic peptides to aid folding of the resulting polypeptide chain into a more stable, compact form. For example, GPGPG spacers (SEQ ID NO: 26) were added at regular intervals along the length of the chimeric polypeptide. Five KG spacers also were added to further increase the flexibility of certain regions of the recombinant polypeptide and further aid in folding. These were added throughout the chimeric polypeptide. KAA spacers also were added immediately after the carboxy termini of two CTL epitopes (p595 and p596) to aid in their processing and presentation in the Th-I pathway. A Met-Ala dipeptide was added to the N-terminus to provide the translation start signal (Met) and for cloning purposes (Ala). An affinity tag of six histidines was optionally added to the carboxy terminus to facilitate purification of the polypeptide.

[0257] The arrangement of epitopes and spacers within FALVAC-IA was designed to be compatible with an energetically stable protein, using the INSIGHT suite and SEQFOLD module (Accelrys, San Diego, CA). Several variations of spacer placements were threaded within the molecule onto known protein folds in the SCOP (structural characterization of proteins) database. The final arrangement of epitopes and spacers, as described above, proved to have internal energies comparable to natural proteins of similar size (for example PDB entries ldxi or lado) upon homology modeling to the best SCOP matches and energy minimalization of the models. That energetically feasible conformations are available to FALVAC-IA was borne out (at least) by its apparent stability to thermal denaturation as evidenced by circular dichroism (see Example 3 below).

[0258] FIG. 1 is a schematic diagram showing the epitope and spacer sequence for the chimeric polypeptide FALVAC-IA. FALVAC-IA is expected to have a compact structure in solution because of the GPGPG spacers (SEQ ID NO: 26) in its sequence, and this also is reflected in the schematic diagram of FIG. 1. The life stage of the malarial parasite that is the source of each of the epitopic peptides also is shown in FIG. 1.

[0259] It should be understood that as another aspect of the disclosure, functional and conservative variants of the FALVAC-IA polypeptide are provided. Such alternative chimeric polypeptides include those where the individual peptide sequences that comprise the FALVAC-IA polypeptide are redistributed or re-ordered along the sequence. In addition, the distribution and number of the spacers along the sequence may be altered to provide functional variants. Other embodiments include chimeric polypeptides comprising conservative variants of the particular epitopic peptides listed in Table 1.

[0260] In a particular alternative embodiment of the disclosed chimeric polypeptides, the relatively even distribution of the GPGPG spacers (SEQ ID NO: 26) in FALVAC-IA is retained to provide a functional variant having a compact structure. For example, in one embodiment, a chimeric polypeptide is provided that comprises the same epitopes as FALVAC-IA, in the same order, but with 6 GPGPG spacers (SEQ ID NO: 26) located evenly along the sequence. For example, a first GPGPG spacer (SEQ ID NO: 26) is situated after the first 3 epitopes, and the remainder is individually situated after every subsequent group of 3 epitopes along the sequence of the chimeric polypeptide. Other spacers, such as KAA and KG spacers, may optionally be placed between one or more pairs of epitopes along the sequence. In particular, KAA spacers may be placed after the CTL epitopes as in FALVAC-IA to aid in their presentation to the immune system. Of course, other alternative embodiments with fewer or greater numbers of GPGPG spacers (SEQ ID NO: 26) and alternative numbers of epitopes between the spacers are possible while still providing a compact structure.

Abbreviations: CSP, circumsporozoite protein; LSA- 1, liver stage antigen- 1; MSP- 1, merozoite surface protein- 1 ; MSP-2, merozoite surface protein-2; AMA-I, apical membrane antigen- 1; EBA-17₅, erythrocyte binding antigen- 17₅; RAP-I, rhoptry associated protein- 1.

The entire sequence of this exemplar recombinant FALVAC-IA chimeric polypeptide runs continuously down the column labeled "Amino Acid Sequence." The first two residues, "MA," are non-plasmodial and are present to provide a start signal for expression (Met) and for cloning purposes (Ala). The first residue (Met) can be later cleaved from the expressed chimeric polypeptide without substantially affecting the immunogenicity of the chimeric polypeptide. Similarly, the poly-His tag is a non-plasmodial sequence, which facilitates purification of the FALVAC-IA chimera. It is believed that the poly-His tag (or other tag predominantly used for purification purposes) also can be removed without substantially affecting the P.falciparum-specific immunogenicity of the chimera.

[0262] Additional functional variants of FALVAC-IA are possible through alteration of the spacer sequences used between certain of the 21 epitopic peptides in the sequence. For example, one or more GPGPG spacers (SEQ ID NO: 26) may be replaced by other short peptide sequences (for example, 3- 6 residues in length) that permit, or promote, reverse turns. For example, other peptide sequences rich in proline and glycine may be used to promote compact folding of the chimeric polypeptide and to rninimize formation of junctional epitopes that may interfere with stimulus of an immune response by individual epitopes. In addition, the KG spacers of FALVAC-IA may be replaced with short peptide sequences (for example, 2-4 residues in length) that comprise one or more small amino acids (such as G and A) and promote the flexibility of the peptide. Furthermore, the KAA spacers may be replaced with other short peptide spacers that promote in vivo epitope processing. Spacers that promote epitope processing include peptide sequences containing amino acids, such as K, N, C, G, and/or A.

[0263] Particular alternative linear arrangements of the epitopes listed in Table 1 that can provide functional variants include the following combinations of 21 epitopes; p593 p594 p597 p598 p595 p519 p600 p601 p545 p596 p606 p543 p544 p546 p603 p602 p607 p604 p605 p599 p592; p592 p594 p597 p598 p595 p600 p545 p596 p543 p544 p546 p602 p607 p603 p604 p605 p599 p593 p519 p601 p606; p592 p594 p597 p598 p595 p600 p545 p596 p543 p544 p546 p602 p607 p603 p604 p605 p599 p593 p519 p601p606; and p594 p595 p600 p545 p596 p543 p592 p607 p603 p605 p599 p593 p519 p601p606 p604 p597 p598 p544 ρ546 p602. The epitopes may be separated with appropriate spacer sequences, and additional sequences may be added to aid in expression or isolation. For example, GPGPG spacers (SEQ ID NO: 26) may be added. Also, immunogenic and/or non-immunogenic protein partners may be added to provide fusion proteins comprising the chimeric polypeptides. Furthermore, to facilitate purification of a chimeric polypeptide, poly-Arginine or poly-Histidine sequences, FLAG tags or other sequences known for such purposes may be added.

[0264] We had previously designed a multi-stage, multi-valent P. faciparum vaccine without taking into account the energy costs associated with particular conformations of that antigen (see NIIMALVAC-I as described, e.g., in U.S. Pat. No. 6,828,416, and Shi et al, Proc. Natl. Acad. Sci USA, 96:1615-1620, 1999). In addition to its ability to assume an energy-stable conformation, FALVAC-IA has several additional advantages over NIIMALVAC-I. For example, FALVAC-IA omits 3 epitopes found in NIIMALVAC-I: A thrombospondin related protein (TRAP) determinant that bears homology to a human gene, the tetanus toxoid epitope that did not induce T-cell responses, and the gametocyte epitope from Pf27 that did not induce any transmission-blocking antibodies. Moreover, unlike NIIMALVAC-I, FALVAC-IA includes (i) 2 epitopes from EBA- 175 (peptides pF2-8 and pF2-16) that react with conformational antibodies specific for the EBA-RII domain (Ockenhouse et al., MoI. Biochem. Parasitol., 113:9-21, 2001); (ii) a RAP-I epitope, which has been modified from LTPLEELY to KNTLTPLEELYPT, based on the information that monoclonal antibodies recognizing this epitope inhibit the growth of P. falciparum (Harnyuttanakorn et al, MoI. Biochem. Parasitol. 55:177-186, 1992); and (iii) an EBA-175 epitope, which was modified fromNEREDERTLTKYEDEDIVLK to TLTKEYEDIVLKSHMNRESDD based on the information that sera against this peptide block blood stage parasite growth in vifro (Jakobsen et al., Infect. Immunity, 66:4203-4207, 1998).

Example 2 Expression and Purification of FALVAC-I A-His

[0265] The amino acid sequence of FALVAC-I A-His (SEQ ID NO: 2) was reverse translated and codon-optimized for E. coli to yield a corresponding DNA coding sequence using the program Backtranslation (Entelechon BmbH, Gegensburg, Germany). One such DNA sequence is given as SEQ ID NO: 1. Degenerate variants of this DNA sequence also are specifically contemplated, as are DNA sequences that encode conservative and functional variants of the chimeric polypeptide sequence.

[0266] Twenty-four overlapping oligonucleotides (50-114 nucleotides in length) coding for the sense and anti-sense strands of the DNA coding for FALVAC-I A-His, were chemically synthesized at the Core Facility at the Centers for Disease Control and Prevention (Atlanta, GA) and their sequences were verified. The gene was then assembled using the polymerase chain reaction (PCR). The twenty-four overlapping single-strand oligonucleotide primers were assembled into the DNA sequence coding for FALVAC- lA-His by repeated annealing and extension steps. Generation of a larger DNA sequence by PCR amplification of overlapping oligonucleotides is a technique common in the art and is described generally in Withers-Martinez et ah, "PCR-base gene synthesis as an efficient approach for expression of the A-T-rich malaria genome," Protein Eng., 12:1113-1120, 1990. The nucleic acid sequence was amplified using two flanking primers (SEQ ID NO: 24, 5'-TCC ATG GCG AAA CCG AAA CAC AAG AAG CTG AAG-3'; SEQ ID NO: 25, 5'-TGC GGC CGC TCA TTA GTG GTG GTG GTG GTG GTG TCC CAG ATC ATC TTT ATA TTT CGC CAG CAC CTT TTC G-3') containing the restriction enzyme sites Nco I and Not I, and a sequence encoding 6 histidines. The artificial gene was cloned into pGEM-T easy vector system (Promega) for sequence verification. The cloned gene was then subcloned into the pET24d vector (Novagen, EMD Biosciences, San Diego, CA) containing the Kanamycin resistance gene, which was used to transform E. coli BLR(DE)3 cells with the Singles™ Kit (Novagen, Madison, WI).

[0267] Starter cultures were grown up overnight at 37⁰C in shaker flasks and used to inoculate 4 x 200 ml cultures of LB broth containing 30 μg/ml Kanamycin with about 1.67 x 10⁷ cells/ml. These cultures were incubated at 37⁰C until ODβoo ⁼ 0.6-1.0, at which time FAL VAC-I A-His expression was induced by addition of IPTG (BioVectra, Prince Edward island, Canada) to ImM. Following incubation for a further 3 hours, during which time the product accumulated in inclusion bodies, the cultures were harvested by centrifugation at 4°C and the cell pellets stored at -70⁰C for subsequent purification of the expressed protein.

[0268] A three-step procedure of centrifugation, affinity chromatography and ion-exchange chromatography was used to purify FALVAC-I A-His from bacterial cultures. The E. coli cells were lysed in BugBuster buffer plus benzonase (Novagen) and EDTA-free protease inhibitors (Roche Diagnostics, Indianapolis, IN) at pH 8.0. The crude inclusion body preparation was pelleted by centrifugation (15,000 x g for 15 minutes), and treated with BugBuster buffer plus lysozyme (United States Biomedical Corporation, Cleveland, OH). Then, the inclusion bodies were washed 4 times with 1:10 BugBuster buffer in 20 mM Tris, 150 mM NaCl pH 8.0 (all routine laboratory chemicals from Sigma, St Louis, MO). This washing procedure advantageously decreased contamination by host cell proteins. The washed inclusion bodies were solubilized in 100 mM sodium phosphate, 10 mM Tris, 8 M urea pH 8.0, clarified by centrifugation (43,000 x g for 25 minutes), and the FALVAC-1A-His adsorbed onto Talon Cobalt affinity resin (BD Bioscience, Palo Alto, CA) via the carboxy terminal 6-His moiety. The affinity column was washed with the sodium phosphate/tris/urea buffer at pH 7.0, and eluted with the sodium phosphate/tris/urea buffer at pH 5.9. The eluted FALVAC-I A-His was dialyzed against 50 mM Tris, 8 M urea, 10 mM EDTA pH 9.0, clarified by 0.2 μm filtration (Acrodisc, Pall Life Sciences, Ann Arbor, MI) and then passed over Q FF Sepharose (Amersham Biosciences, Piscataway, NJ) equilibrated in the same buffer to remove high and low molecular weight contaminating bands. The first unbound, flow through peak of FALVAC-IA-His was collected. The solubilized FALVAC-I A-His was then refolded by dialysis using stepwise removal of urea (6M, 4M, 2M and OM urea) in 10 mM sodium phosphate, 500 mM NaCl pH 7.2. Finally, the product was concentrated by ultrafiltration (Centriprep, Millipore Corporation, Bedford, MA) to 300-800 μg/ml, sterile filtered (0.2 μm Acrodisc), and stored at -70⁰C. Example 3 Characterization of FALVAC-I A-His Polypeptide

[0269] This Example shows FALVAC-I A-His is reproducibly produced and isolated from bacterial cultures in substantial amounts. Moreover, the recombinant polypeptide assumes a consistent and energetically stable tertiary structure. The production of a stable multivalent polypeptide with a defined conformation represents a significant step forward in the development of defined vaccine antigens.

A. Methods

[0270] The purity of FALVAC-I A-His produced as described in Example 2 was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), running approximately 3 μg/lane on precast 4 ~ 15% gradient bis-Tris gels (Bio-Rad, Hercules, CA) in a Mini-PROTEAN II cell (Bio-Rad). Prestained molecular weight markers (Bio-Rad) were also run. The gels were stained with Gelcode Blue Stain Reagent (Piece, Rockford, IL), destained, scanned on a laser densitometer (LABSCAN, Amersham Pharmacia Biotech, Piscataway, NJ) and the data analyzed with ImageMaster ID Elite software version 4.20 (Amersham Pharmacia Biotech) to determine the percent purity. The concentrations of E. coli host cell protein and endotoxin were determined by immuno-assay (E. coli HCP ELISA, Cygnus Technologies, Inc., Southport, NC and QCL-1000^® Chromogenic LAL Endpoint Assay, Cambrex - Biotherapeutic Products, Walkersville, MD, respectively) according to the manufacturer's instructions. FALVAC-IA-His concentration was measured by the Bradford protein assay (Bio-Rad) against bovine serum albumin standards.

[0271] Full length and tryptically digested samples were analyzed by MALDI-TOF MS on a Bruker Reflex IV (Broker Daltonics, Billerica, MA) equipped with delayed extraction and a nitrogen laser. Samples were mixed with matrix (sinapinic acid (SA) or α-Cyano^-hydroxycinnamic acid (HCCA) 10 mg/ml in 60% acetonitrile/1% acetic acid/15% methanol) and spotted onto a stainless steel target and allowed to dry. Full length samples were mixed with SA and spectra were collected in positive linear mode. Digested samples were mixed with HCCA and spectra were collected in positive reflector mode.

[0272] HPLC analyses were run on an Ultra Plus II HPLC system (MicroTech Scientific, Vista, CA). Buffer A was 0.1% TFA (Applied Biosystems)/H₂O, buffer B was 0.1%TFA/80% acetonitrile (JTBaker)/H₂O. Aliquots of each batch of purified FALVAC-IA were acidified and injected on a 1 x 150mm Zorbax 300A C₁₈ Reverse phase HPLC column (MicroTech Scientifc). Peaks were eluted between 5% - 90% buffer B over 35 minutes. Analyses with reduced samples were done in the same manner except that they were incubated at 37°C for 30 minutes with 2M urea plus 10OmM DTT prior to acidification and injection.

[0273] Circular dichroism spectra were obtained on a Jasco J-810 spectropolarimeter (Jasco, Inc., Easton, MD) equipped with a temperature programmable sample holder. Buffer background (10 mM sodium phosphate, 500 mM NaCl pH 7.2) was subtracted from FALVAC-I A-His spectra captured in that buffer. Chemical denaturation was accomplished by addition of 4 M guanidinium HCl and thermal stability was monitored by taking spectra in five-degree intervals between 10° and 95°C. For estimation of secondary structure contributions we used the CDSSTR program within the "CDPro" suite of Sreerama and Woody (Anal. Biochem., 287:252-260, 2000). B. FAL VA C-I A-His Characterization

[0274] The final yield of FALVAC-I A-His expressed and purified as described in Example 2 was approximately 10 mg/liter of culture (see Table 2). The purification process was reproducible and gave product lots with similar characteristics. As shown in Table 2 and FIG. 2, the product was >90% pure by gel chromatography with low endotoxin and E. coli host cell contamination.

[0275] Table 2. Purity and Analysis of Different F ALVAC-I A-His Lots

From 800 ml culture.

² Abbreviations: HCP, E. coli host cell protein; N.D., Not determined

[0276] The theoretical molecular weight (MW) of FALVAC-I A-His is 42,661 daltons. A MW determination by MALDI-TOF mass spectrometry showed that the actual MW of FALVAC-I A-His was 42,530 daltons, which is 131 daltons less than the theoretical value (see FIG. 3). It is commonly known that E. coli frequently clips the N-terminal Met (MW=131) following protein synthesis (Hirel et al., Proc Natl Acad Sd USA, 86:8247-8251, 1989). Consistently, Edman degradation sequencing of 6 FALVAC-1A-His lots (F071A, F072A, F134A to F137A) gave the N terminal sequence AKPKHKK (residues 2-8 of SEQ ID NO: 2). In each case, the sequence lacked the start methionine (refer, e.g., to Table 1), which indicated that this amino acid had been clipped by E. coli at some point during the expression and/or isolation process. With the N-terminal methionine removed, the theoretical arid measured molecular weights of FALVAC- 1 A-His concurred. Additional studies using peptide mapping and positive immunoreactivity by epitope- specific sera with FALVAC-I A-His were consistent with the actual amino acid sequence and composition of purified protein FALVAC-IA-His being identical to the desired theoretical sequence.

[0277] Unlike native proteins, an artificial recombinant molecule does not have a structural conformation that has been refined by evolution. Accordingly, an artificial polypeptide can assume numerous structural conformations in solution, many or even most of which conformations may not be stable. As one result, hydrophobic residues of artificial polypeptides can be exposed, which is likely to cause aggregation of the artificial polypeptide in aqueous solution when, for example, hydrophobic residues on adjacent molecules interact so as to avoid the polar solvent.

[0278] Increased understanding of the molecular interactions between amino acids and the ability to design computer programs to model these interactions have been (and continue to be) developed. For example, Dahiyat and Mayo designed a 28-amino acid zinc finger motif that folded correctly (Dahiyat and Mayo, Science, 278:82-87, 1997). Similarly, Harbury et al. successfully designed a set of artificial right handed coiled-coil bundles (Harbury et al, Science, 282:1462-1467, 1998), and Kuhlman et al. designed a 93-amino acid α/β protein displaying a new globular protein fold (Kuhlman et al., Science, 302:1364-1368, 2003). As described herein, FALVAC-1A-His included spacer groups that were believed to enhance stability of the polypeptide by aiding folding to a more compact form (GPGPG; SEQ ID NO: 26) and allowing for flexibility (KG).

[0279] The molecular conformation of FALVAC- 1A-His in solution following refolding was determined by circular dichroism (CD) studies in the far UV. As shown in FIG. 4, refolded FALVAC-I A-His had a defined molecular conformation. Deconvolution of the component plots indicated that the secondary structure of FALVAC-I A-His consisted of approximately 20% α-helix, 27% β-sheet, 22% turns and 30% random coil. This structure could be disrupted by the addition of 4 M guanidine, a known chaotropic agent (see FIG. 4). On the other hand, CD spectra of FALVAC-I A-His at temperatures from 10°C to 85°C were superimposable, which demonstrates that the FALVAC-IA-His structure was thermally stable. Thermal stability of FALVAC-I A-His structure may indicate the presence of intramolecular disulfide bonds. Each of 6 FALVAC-IA-His lots (F071A, F072A, F134A to F137A) yielded the same CD results showing that each lot had the same secondary structural components when produced as described in Example 2.

[0280] Six FALVAC-IA-His lots (F071A, F072A, F134A to F137A) were also examined by RP-HPLC in the native and reduced state. As shown for two representative examples in FIG. 5, a shoulder in the native state disappeared following reduction. These observations are also consistent with the presence of disulfide bonds in the molecule.

[0281] The stability of FALVAC-I A-His was further demonstrated by measuring polypeptide degradation as a function of temperature and time. Six-hundred (600) ul aliquots of FALVAC-IA lot F136A at 500 ug/ml in 0.5M NaCl/PBS in 1.0 Marsh polypropylene tubes were placed at -7O⁰C, 4°C and 37°C. Samples were assayed by SDS-PAGE at time 0, 4, and 8 weeks. FALVAC-IA-His was stable at - 7O⁰C and was only somewhat degraded at 4°C after 8 weeks. Such long-term degradation at 4°C may be due to residual protease activity in the preparation.

[0282] In summary, this Example demonstrates that an exemplar multivalent, multistage malarial vaccine, FALVAC-IA-His, consistently can be expressed and purified. Advantageously, the molecule reproducibly folded to assume an energetically stable, defined molecular conformation that was stable when frozen and thawed for use. Taken together, this Example illustrates the feasibility of constructing chimeric polypeptide antigens including selected antigenic determinants and spacer determinants that provide an energetically stable conformation.

Example 4 Immunogenicity of the FALVAC-IA Chimeric Polypeptide in Rabbits

[0283] This Example demonstrates that FALVAC-IA (produced as described in Example 2) induces antibody responses in rabbits in vivo. Rabbit antibodies produced by immunization with FALVAC-IA recognize the whole molecule and its individual peptide epitopes.

[0284] New Zealand white rabbits (Myrtle's Rabbitry, Thompson's Station, TN) were kept in AAALAC-accredited facilities at the CDC with rabbit chow and water ad lib. They were immunized intramuscularly on days 0, 28, 56 and 176 with 50 μg FALVAC-IA (lot F007A) formulated with the following 4 adjuvants: (i) Squalene/Span 80/CRL-1005 copolymer water-in-oil emulsion (Squalene and Span 80, Sigma, St Louis, MO.; CRL-1005 was a gift from Dr. Robert Hunter, SynthRx, Houston, TX.); (ii) Montanide ISA-720 (a gift from Seppic, Inc. Fairfϊeld, NJ.); (iii) 50 μg QS-21 (a gift from Antigenics, Inc. Lexington, MA.); and (iv) aluminum phosphate (Adju-Phos, HCI Biosector, Frederikssund, Denmark). Aluminum phosphate (pi = 5) was selected because at physiological pH it has a negative charge, whereas FALVAC-IA (pi = 8.3) is positively charged and its binding to the adjuvant exceeds 95%. The rabbits were bled every 2 weeks and exsanguinated at the final bleed. The sera were separated by centrifugation and stored at -7O⁰C.

[0285] Serum antibody titers against FALVAC-IA and its individual antigenic peptides were determined by ELISA. Immunlon 2HB plates (Thermo Electron, Franklin, MA) were coated overnight at 4°C with either lμg/ml FALVAC-IA or 10 μg/ml peptide in phosphate buffered saline (PBS), blocked with 5% nonfat dried milk in PBS + 0.05% Tween 20 (diluent) and washed with PBS + 0.05% Tween (PBS-T). Sera were diluted two fold in diluent from 1: 800 to 1:1,638,400 for anti-FALVAC-lA, 1:100 to 1:204,800 for anti-peptide, 100 ul were added per well and the plates incubated overnight at 4⁰C. Known positive and negative sera were included on each plate as controls. The plates were washed between additions with PBS-T. The assay was developed by the addition of 1:5000 HRP Goat anti-rabbit IgG (Southern Biotech, Birmingham, AL) for 1 hour at room temperature followed by TMB Peroxidase Substrate (KPL, Inc., Gaithersburg, MD.) for 5 minutes at room temperature. The reactions were stopped with IM H₃PO₄ and read at 450 nm on a SpectraMax (Molecular Devices, Sunnyvale CA). Titers were determined using an optical density of 0.1 as the cutoff.

[0286] As shown in FIGs. 6A and 6B, FALVAC-IA was immunogenic and induced high titer antibodies. As expected, the water-in-oil emulsion adjuvants, Squalene/Span/Copolymer and Montanide ISA-720, induced the highest and longest lasting antibody titers. However, the adjuvants with greater potential for human use, QS-21 and aluminum phosphate induced high titers, especially after boosting (see, FIG. 6).

[0287] Antibody titers against each of the component FALVAC-IA epitopes were also determined by ELISA. As shown in Table 3, there was a range of activity against the different epitopes, although the pattern was similar between rabbits and adjuvants.

[0288] Table 3. Rabbit Anti-FALVAC-lA Peptide ELISA Titers of Final Bleed sera.

In CSP, the first epitope, CTL+T, KPKDELDYENDIEKKICKMEKCS (residues 23-4₅ of SEQ ID NO 2) runs continuously into the second T epitope, SVFNVVNS (residues 46-₅3 of SEQ ID NO 2) Antibody responses were determined jointly against the peptide, KPKDELDYENDIEKKICKMEKCSSVFNVVNS (residues 23-₅3 of SEQ ID NO 2)

² Titers marked with a dash were <100

³ Rabbit No

[0289] As expected, rabbits with higher ELISA titers to the whole molecule had higher titers to the component epitopes. Antibody responses were seen against peptides with sequences identical to B cell epitopes from CSP, MSP-I, AMA-I and RAP-I Responses to B cell epitopes from MSP-2 and EBA-175 were generally lower or absent The immunogemcity of the MSP-2 and EBA-175 epitopes in the respective parent proteins may have been conformationally determined and such conformation may not be retained in FALVAC-IA Alternatively the MSP-2 and EBA-175 epitopes may not have been correctly displayed in the synthetic antigen and/or not properly processed/presented to the immune system in an immunogenic form The lower response to these particular epitopes does not affect the overall importance and usefulness of FALVAC-IA because MSP-2 and EBA-175 represent a minor part of the FALVAC-IA molecule as a whole and, as discussed above, the intact molecule induces a strong immune response.

[0290] FALVAC-IA was compared to the multivalent, multistage P falciparum vaccine (referred to in those references as NIIMALVAC-I) described, for example, in U.S Pat. No. 6,828,416 (see also, Shi et al, Proc Natl Acad Sci £/5/4, 96:1615-1620, 1999). The physico-chemical properties of NIIMALVAC-I and FALVAC-IA, for example, their aqueous solubility and pi values, are distinctly different (see Table 4).

[0291 ] Table 4. Compaπson of Selected Physical Properties of NIIMALVAC-I and FALVAC-IA-His

[0292] In addition, the FALVAC-IA anti-peptide ELISA data (see Table 3) were compared with historical data from rabbits comparably immunized with NIIMALVAC-I (Shi et al , Proc Natl Acad Sa USA, 96:1615-1620, 1999). Considering the 18 epitopes that are common to NIIMAL VAC-I and FALVAC-IA, only 11 of those epitopes from NIIMALVAC-I stimulated an antibody response in at least one animal compared to 14 such epitopes in FALVAC-IA. The epitopes that did not stimulate an antibody response to NIIMALVAC-I were p592, p598, p543 and p599. As indicated in Table 3, these peptides stimulated responses in 5, 4, 4, and 4 of the 8 FALVAC-I A-immunized rabbits, respectively. These findings were unexpected and demonstrate that, in addition to having certain distinct epitopes and useful spacer determinants that NIIMALVAC-I does not, FALVAC-IA induces a better immune response than does NIIMALVAC-I to several epitopes the polypeptides have in common.

Example 5 Anti-FALVAC-IA-His Antibodies Bind to Sporozoite and Blood Stage Parasites

[0293] Tins Example illustrates that FALVAC-lA-induced rabbit antibodies react with native parasite antigens in both the sporozoite and blood stages.

[0294] Twelve-well immunofluorescence assay (IFA) slides with the wells coated with either P. falciparum sporozoites or blood stages were removed from freezer storage and allowed to reach room temperature. The test rabbit sera were diluted two fold from 1 :20 to 1 : 10240 (10 steps) in phosphate buffered saline (PBS). Appropriate dilutions of known positive control sera were also prepared. Ten μl of each specimen dilution were added to 10 wells respectively, starting from the highest dilution. Negative control (PBS) and positive control samples were added to the remaining 2 wells on each slide, respectively. The slides were incubated at 37°C, 100% relative humidity for 30 minutes, then washed 3 times by aspiration and reapplication of 20 μl PBS. Following the final aspiration, 10 μl of conjugate solution (1/50 dilution of FITC-labeled goat anti-rabbit IgG in PBS + 0.01% Evan Blue dye) were added to each well and the slides incubated for a further 30 minutes as above. The slides were again washed 3 times with PBS, aspirated, and mounted under a cover slip in buffered glycerol, pH 9.0. The slides were viewed by a fluorescence microscope at 2Ox magnification and scored for fluorescence positivity by 2 independent observers on a scale of: 4+, 3+, 2+, 1+, +/-, Negative. The titer was determined from the last well with 1+ score.

[0296] Antibody reactivity in the immunofluorescence assays was adjuvant dependent, generally following the pattern of reactivity seen in ELISA assays (see Example 4). This Example and the foregoing examples have shown (among other things) that anti-F ALVAC-I A antibodies react with isolated FALVAC-IA polypeptide, its isolated immunogenic peptides and intact P. falciparum parasites. Examples provided below shows that these antibodies also have anti-parasitic activity.

Example 6 FALVAC-I A-His Rabbit Antisera Inhibits Sporozoite Invasion of Hepatocytes

[0297] This Example demonstrates that anti-F AL V AC-I A-His antibodies produced as described in Example 4 inhibited P. falciparum sporozoite invasion of liver cells in vitro.

[0298] Inhibition of sporozoite invasion (ISI) assays are known in the art (see, e.g., Hollingdale et ah, J. Immunol, 132:909-913, 1984). Briefly, on day 1, 50,000 HepG2-A16 cells/well were seeded on 8 well glass Chamber Slides (Lab-Tek No. 177402, Nalgene Nunc International, Rochester, NY) previously coated with ECL Cell Attachment matrix (Upstate USA, Inc. Charlottesville, VA). On day 4, 5 million sporozoites were obtained from 220 infected Anopheles stephensi mosquitoes by microcentrifugation and a DEAE cellulose column (DE52 Whatman Inc, Clifton, NJ), and diluted in complete medium (Eagle's Minimum Essential Medium supplemented with 10% fetal Bovine serum, BSA, insulin, and penicillin- streptomycin) to a concentration of 400,000 sporozoites/ml. The sera were diluted 1:50 in complete medium and 50 μl were added to triplicate HepG2-A16 wells after removing the complete medium. NFSl, a monoclonal antibody against P. falciparum CSP protein, was used as positive control at 200 μg/ml, with 3 wells containing complete medium as negative control. Fifty μl of the sporozoite suspension (20,000 sporozoites/well) were added to all wells. The chamber slides were incubated for 3 hours at 37°C, washed with PBS, fixed in cold methanol for 10 minutes and washed with PBS. The chamber slides were immunostained using NFSl (10 μg/ml) as primary antibody and peroxidase labeled goat anti-mouse IgG (KPL) as the secondary antibody. Color was developed using a DAB reagent kit (KPL, Inc.) and the slides were mounted in Permount (Fisher Scientific, Fairlawn, NJ) under 24 x 60 mm cover glasses. The number of sporozoites that had invaded the HepG2-A16 cells/well was counted by phase contrast microscopy and the percent inhibition calculated relative to the negative controls.

[0299] As shown in Table 6, two sera from Rabbits 3123 and 333 immunized with the copolymer emulsion formulation had >90% ISI activity, indicating that the sporozoite-specific CSP epitopes in FALVAC-IA (see, e.g., Table 1) were capable of stimulating antibody responses with functional activity against sporozoites in vitro. In general, ISI activity was associated with the highest IFA titers to sporozoites (see Example 5). The NFSl positive control routinely gave >90% ISI.

[0300] Table. 6. Inhibition of S orozoite Invasion

Example 7 FALVAC-I A-His Antibody-Dependent Cellular Inhibition (ADCI) of Blood-Stage Parasites

[0301] This Example illustrates that FALVAC- lA-His-elicited antibodies have significant inhibitory effects on in vitro growth of blood-stage parasites in the presence of monocytes.

[0302] IgG fractions were purified from each final titer rabbit serum by adsorbing 3 ml serum with 20 ml immobilized Protein A (PIERCE, Rockford, IL) on the AKTA prime liquid chromatography system (Amersham Pharmacia Biotech, Piscataway, NJ) and eluting the bound antibody following the manufacturer's instructions. The purified IgG fractions were dialyzed in RPMI 1640 medium, concentrated to approximately 4 mg/ml, sterile filtered (0.22 um Acrodisc, Pall), and stored at -20°C.

[0303] Antibody dependent cellular inhibition (ADCI) assays were performed using described methods (see, for example, Bouharoun-Tayoun et al., J. Exp.Med., 182:409-418; 1995; Shi et al., Am. J. Trop. Med. Hyg., 60:135-141, 1999). Briefly, peripheral blood mononuclear cells (PBMN) were isolated from 5 normal human blood donors. Each cell donor was used 4 to 8 times. Purified antibodies were added at at 75 ug/ml final concentration into FC27 strain blood-stage parasite cultures (0.3% parasitemia with 60% schizonts, and 1% hematocrit), along with 80,000 rhIFN-G (100 ng/ml)-activated human monocytes. The cell cultures were incubated at 37⁰C in a mixed gas containing 5% O₂ , 5% CO₂, and 90% N₂ for 72 hours, with medium and antibody replacement every 24 hours. Parasites were stained with the vital dye hydroethidine (HE) and parasitemias were determined by a flow cytometry-based parasite enumerating procedure using FACScan.

[0304] Only assays where the positive control (purified IgG from a serum pool of clinically immune Kenyan adults) gave greater than 10% inhibition were used to estimate ADCI. Differences between experimental groups and the positive control were evaluated by Student's t test. As shown in FIG. 7 (and despite the inherent variability of the ADCI assay), 7 of the 8 rabbit sera had ADCI activities that were comparable to a positive pool of human immune sera. Only serum from rabbit R3123 had significantly less (p <0.05; indicated by "*") ADCI activity than the positive control. Human studies have linked ADCI activity to functional immunity (Bouharoun-Tayoun et al., J. Exp. Med., 182:409-418, 1995; Oeuvray et al, Infect. Immun., 68:2617-20, 2000). Thus, the foregoing ADCI results support the belief that FALVAC-IA can provide functional immunity in humans.

[0305] This and the prior Examples demonstrate that FALVAC-IA was immunogenic and effective against P. falciparum in vitro. The reactivities of native P. falciparum epitopes are determined by both their sequences and their conformations within the proteins in which the epitopes are found. Because anti-FALVAC-1 A antibodies reacted with intact parasites and were able to mediate in vitro effector activity, it is believed that many of the epitopes in the FALVAC-IA polypeptide retained or replicated sufficient of their original, native conformation to induce these antibodies. Example 8 Growth Inhibition Assays (GIA) Using Purified Antibodies Specific for FALVAC-IA

[0306] Purified IgG fractions from the rabbit sera were tested in the Growth Inhibition Assay (GIA) by the MVI GIA Reference Center, National Institutes of Allergy and Infectious Diseases, Rockville, MD., using their standardized method (Carole Long, personal communication). Exemplar growth inhibition assays have been described (see, for example, Chula et al., J. Infect. Dis., 144:270-278, 1981 and Vernes et ah, Am. F. Trop. Med. Hyg., 33:197-203, 1984). In some examples, dilutions of purified antibodies from immunized and control rabbits are added to micro-cultures of parasitized human type-0 erythrocytes (100 μL of 1.5% (v/v) erythrocytes with 1% parasitemia). The cultures are incubated for 18 hours at 37⁰C in 90% N₂, 5% O₂, 5% CO₂, washed, incubated with fresh medium for another 8 hours and then radiolabeled with ¹⁴C-isoleucine and ³H-hypoxanthine for 16 hours. Inhibition of parasite growth is determined either by a decreased incorporation of radioactivity (relative to control) or low parasitemias determined microscopically (relative to control).

[0307] No GIA activity was detected using antibodies isolated from FALVAC-IA immunized rabbits. Nonetheless, it is known from the IFA data (see Table 5) that the rabbit sera do react with the blood stages of P. falciparum. The lack of reactivity of purified rabbit IgG fractions in this Example might relate to the pecularities of the GIA assay. Moreover, the significance of GIA assay data is unclear because presently there is no known in vivo correlate to in vitro GIA results.

Example 9 Immunogenicity of FALVAC-IA in a Mouse Model System

[0308] This Example demonstrates that in response to FALVAC-IA immunization mice produce anti-F AL VAC-I A antibodies and T cells that secrete Interferon gamma (IFN-G) and Interleukin-4 (IL-4) upon antigen re-stimulation.

A. Mouse Immunization

[0309] Four strains of mice (outbred ICR and 3 inbred strains, C57BL/6 (H-2^b), B10.BR (H-2^k) and B10.D2 (H-2^d)) in groups of 8-10 were immunized with 3 x 10 ug doses of FALVAC-IA alone or with FALVAC-IA formulated with: (i) Squalene/Span/polymer water-in-oil emulsion; (ii) Montanide ISA-720; (iii) QS-21 ; or (iv) aluminum phosphate. Mice were immunized by subcutaneous injection on days 0, 14 and 28, and blood was collected on days 21, 42, 63 and 147.

[0310] FIG. 8 shows the reactivities of sera collected from C57BL/6 mice as a function of time after the first immunization. Mouse serum reactivity to immobilized FALVAC-IA was measured by ELISA (see, e.g., Example 4). As seen with rabbit FALVAC-IA immunization, the type of adjuvant used had an effect on the amplitude of mouse antibody responses. The responses of the other 3 mouse strains were similar to that shown for C57BL/6 mice. These results demonstrate that FALVAC-IA can stimulate antibodies in 4 strains of mice with differing H-2 regions and consequent immune response characteristics.

B. Cellular Responses

[0311] The same four mouse strains described above were immunized (4 mice/group) with 10 μg FALVAC-IA by subcutaneous injection on each of days 0 and 14. The FALVAC-IA antigen was formulated with (i) Squalene/Span/polymer water-in-oil emulsion; (ii) Montanide ISA-720; (iii) QS-21; or (iv) aluminum phosphate. One group received PBS alone, i.e. a no antigen control.

[0312] The enzyme-linked immunospot assay (ELISPOT) was used to detect

FALVAC-I A-specifϊc mouse IFN-G and IL-4 producing cells. On the day prior to spleen cell harvest, the wells of Immunospot™ M200 96-well plates (BD Biosciences Pharmingen, San Diego, CA, USA) were coated with either purified anti-mouse IFN-G or anti-mouse IL-4 (BD Biosciences Pharmingen) at 5 μg/ml final concentration in PBS, pH 7.2 and incubated overnight at 4⁰C. The following day, the plates were washed once with complete RPMI medium (RPMI- 1640, 10% heat inactivated fetal bovine serum (FBS), 50 μg/ml gentamicin, 0.1 mM nonessential amino acids, 0.1 mM MEM vitamins, 1% L-glutamine and 2 mM β-mercaptoethanol) and blocked with the same medium for 2 hours at room temperature.

[0313] Mouse spleen cells were harvested on days 21 (2 mice/group) and 23 (2 mice/group) to assay for ELISPOT responses. Spleen cells from 2 mice per group were pooled prior to assay. The red blood cells in the spleen cells were lysed by hypotonic shock (Lysis Buffer, eBiosciences, San Diego, CA), then the resultant mononuclear cell preparation was washed, counted and adjusted to 1 x 10⁶ cells/ml in complete RPMI medium. The blocking medium in the assay plates was removed and 10⁵ mouse mononuclear cells (100 ul of 10⁶ cells/ml in complete RPMI medium) were added to appropriate wells. One hundred (100) ul of FALVAC-IA were added to appropriate wells to give 0.5 ug/ml final concentration. Concanavalin A (ConA; Sigma, St Louis, MO.) at 0.25 ug/ml final concentration was used as a positive control, and both bovine serum albumin (BSA; Sigma, 0.25 μg/ml final concentration) and complete RPMI medium alone were used in other wells as negative controls. The plates and cells were incubated for 20-24 hours (for IFN-G) or 40-48 hours (IL-4) at 37⁰C in 5% CO₂/95% air, 100% relative humidity. During this incubation, any IFN-G or IL-4 secreted by activated cells bound to its respective antibody on the membrane at the bottom of the well. After incubation the plates were washed twice with 200 μl distilled water to lyse and remove the cells, then four times with PBS-0.01% Tween 20 (P-T20).

[0314] One hundred (100) μl of detection antibody (biotinylated anti-mouse IFN-G or anti-mouse IL-4; BD Biosciences Pharmingen,) at 2 μg/ml in dilution buffer (10% FBS in PBS) were added to all wells and the plates incubated at room temperatures for 2 hours. After three washes with P-T20, 100 μl of 1:100 dilution of streptavidin-HRP concentrate (BD Biosciences Pharmingen,) in dilution buffer were added, and the plates incubated at room temperature for 1 hour. Following four washes with P-T20 and two washes with PBS, the spots were developed by adding 100 ul of substrate solution (AEC Chromogen/substrate, BD™ ELISPOT AEC Substrate Set, BD Biosciences Pharmingen) for 15 minutes at room temperature. The substrate reactions were stopped by washing the wells with distilled water. The plates were air dried and the colored spots corresponding to the number of IFN-G or IL-4 secreting cells were enumerated using the ELISPOT Reader System ELR02 (AID Autoimmun Diagnostika GmbH, StraBberg, Germany), designed for automated evaluation of ELISPOT plates. The results (discussed below) are presented as number of ELISPOTS/10⁶ spleen cells.

[0315] FIG. 9 shows the day 23 IFN-G ELISPOT data for C57BL/6 mice. Spleen cells from each of the 4 groups that were immunized with FALVAC-IA and adjuvant were unresponsive to the medium control and the BSA specificity control. However, spleen cells from these same groups responded by producing IFN-G-secreting cells to the positive control, Concanavalin A (ConA), and the immunizing antigen, FALVAC-IA. Mice that had been injected with just PBS and no antigen only responded to the positive control, and not to the negative control (BSA) or the test antigen (FALVAC-IA). Similar responses were demonstrated for the other 3 mouse strains.

[0316] In addition, IL-4-secreting cells were demonstrated by ELISPOT in these same animals. FIG. 10 shows the day 23 IL-4 ELISPOT data for C57BL/6 mice. In a similar manner to the IFN-G data, the IL-4 data demonstrate the specific induction of IL-4-secreting cells in all groups of mice that were immunized with FALVAC-IA plus adjuvant. IL-4 ELISPOTs were also detected in all 4 mouse strains.

[0317] Interferon gamma is produced by T_hl lymphocytes. These lymphocytes recognize antigens presented by macrophages and function primarily to activate and to promote cell-mediated immunity by producing cytokines such as interleukin-2, IFN-G, lymphotoxin and tumor necrosis factor- beta. These cytokines enable T8-lymphocytes to proliferate and to differentiate into CTL capable of destroying infected host cells, for example liver cells infected with P. falciparum. These cytokines also promote the replication of T4-lymphocytes and stimulate the production of opsonizing and complement- activating antibodies that improve the efficiency of phagocytosis. On the other hand, IL-4 is secreted by T_h2 lymphocytes that recognize antigens presented by B lymphocytes. These T_h2 lymphocytes produce cytokines such as interleukins 2, 4, 5, 10, and 13, which promote antibody production. Collectively these cytokines (i) enable activated B lymphocytes to proliferate, synthesize and secrete antibodies, (ii) promote the differentiation of B lymphocytes into antibody-secreting plasma cells, and (iii) enable antibody producing cells to switch the class of antibodies being produced.

[0318] This Example illustrates, among other things, that FALVAC-IA is capable of inducing T cells that secrete IFN-G and IL-4 upon re-stimulation in vitro in 4 strains of mice with differing genetic backgrounds, and in combination with different adjuvants. The demonstration of IFN-G and IL-4 secreting cells in mice following immunization with FALVAC-IA indicates that this molecule has the potential to induce the full spectrum of cellular and antibody-mediated immune responses that would be required to provide immune effector functions against all stages of the malaria parasites.

Example 10 Immunogenicity and Protective Efficacy of FALVAC-IA in a Monkey Model System

A. Aotus Immunization

[0319] Six Aotus monkeys per group were immunized by intramuscular injection with one of the below-described antigens on each of days 0, 28, and 84. Antigens were formulated as follows: (i) Montanide ISA-720 alone; (ii) Montanide ISA 720 plus 50 ug FALVAC-IA; (iii) 50 ug QS-21 alone; (iv) 50 ug QS-21 plus 50 ug FALVAC-IA; (v) 200 ug Al (as AlPO₄) alone; or (vi) 200 ug Al (as AlPO₄) plus 50 ug FALVAC-IA. There were no vaccine-attributable adverse events associated with the immunizations. The monkeys were bled on day 98.

B. Antibody Characterization

[0320] FALVAC-IA was immunogenic in Aotus monkeys and induced high titers of antibodies specific for isolated FALVAC-IA (as determined by ELISA), and the sporozoite and blood-stage parasites (as determined by immunoflurorescence assay) (see FIG. 11). An ELISA using Day 98 Aotus sera and isolated immunogenic FALVAC-IA peptides showed that the intensity of the responses to the individual epitopes by Aotus monkeys (see Table 7) followed a similar pattern to that observed for rabbits (see Example 4 and Table 3).

Number of responding Aotus monkeys (n = 6) with ELISA titers >200.

² Geometric mean titers of responding animals.

³ In CSP, the first epitope, CTL+T, KPKDELDYENDIEKKICKMEKCS (residues 23-45 of SEQ ID NO: 2) runs continuously into the second T epitope, SVFNWNS (residues 46-53 of SEQ ID NO: 2). Antibody responses were determined jointly against the peptide, KPKDELD YENDIEKKICKMEKCSSVFNWNS (residues 23-53 of SEQ ID NO: 2).

[0322] Like the rabbit, there was a strong Aotus anti-FALVAC-1 A antibody response to B cell epitopes from CSP, MSP-I, AMA-I and RAP-I. However, Aotus monkeys had a generally ln^'gher antibody response to MSP-2 and EBA-175 B cell epitopes than did rabbits. Also in contrast to the rabbits, the type of adjuvant used (e.g., ISA-720, QS-21 and AlPO₄) had much less impact on the magnitude of Aotus anti-peptide responses. This similarity was also seen in the IFA studies, where the Aotus responses with the 3 different adjuvants were much more comparable than noted in the rabbits (see FIG. 11).

C. Cellular Responses.

[0323] Peripheral blood mononuclear (PBMN) cells were isolated from FALVAC-lA-immunized Aotus monkeys on days 0, 28, 56, 98 and 168, and were cultured with FALVAC-IA to determine lymphocyte proliferation by tritiated thymidine incorporation. Peripheral blood samples (approximately 4 mis) were collected aseptically in heparinized tubes. These tubes were centrifuged at 1,000 rpm and the clear plasma remaining on top of the cells was collected and stored at -70⁰C for serological studies. The cells were then diluted 1:2 with RPMI 1640 medium (GIBCO-BRL, Grand Island, NY). PBMN were isolated on Ficoll-Hypaque (Fico/Lite-LymphoH, Atlanta Biologicals, Atlanta, GA) by centrifugation. The diluted Aotus blood was layered on top of 4 ml Ficoll-Hypaque in a 15-ml tube and centrifuged at 1,500 rpm for 30 minutes at room temperature. The PBMN at the interface were collected, washed three times in RPMI 1640 medium (GIBCO-BRL, Grand Island, NY) supplemented with 5% fetal bovine serum, and resuspended in culture medium (RPMI 1640 supplemented with 5% fetal bovine serum, 2 mM glutamine, and 50 ug/ml gentamicin per ml). Viable PBMN counts were made under a phase contrast microscope by the trypan blue dye exclusion test. A total of 200,000 PBMN in 100 ul of culture medium were added to each well of a flat-bottomed 96-well plate (Costar, Cambridge, MA.). One hundred (100) ul FALVAC-IA was added to give 1.0 and 0.1 ug/ml final concentration in the well. Phytohemagglutinin (PHA; Sigma Chemical Co., St. Louis, Mo.) at 10 ug/ml final concentration was used as a positive control and culture medium as a negative control in all the experiments. Each condition was tested in triplicate wells. Cultures were incubated at 37°C in a 5% CO₂/95% air atmosphere for 5 days and then labeled with 1 mCi of [³H]thymidine (specific activity, 2 Ci [74.0 GBq] per mmol) (DuPont, NEN Research Products, Boston, Mass.) overnight. Cultures were harvested with a Skatron harvester, and the radioactivity was determined in a liquid scintillation counter. The geometric mean counts per minute (cpm) for each set of triplicateplicate wells were calculated. The experimental/control (E/C) values were determined as the geometric mean cpm of FALVAC-IA- or PHA- stimulated cultures divided by the geometric mean cpm of the negative control cultures.

[0324] All three FALVAC-I A/adjuvant formulations stimulated the production of lymphocytes that responded by proliferation in vitro to FALVAC-IA (see FIG. 12). The FIG. 12 data are presented as group mean E/C values. Aluminum phosphate induced the highest responses; QS-21 and ISA-720 were lower that AlPO₄, but comparable to each other. The maximum response was at day 56 for AlPO₄ and QS-21. The ISA-720 response persisted longer. These data indicate that FALVAC-IA can produce a cellular immune response in a primate.

[0325] The showing in this Example of both antibody and cellular responses in Aotus monkeys is consistent with and extends the data obtained from mice (see Example 9). These results further demonstrate that FALVAC-IA is capable of inducing the full spectrum of cellular and antibody-mediated immune responses that would be required to provide immune effector functions against all stages of the malaria parasites.

D. Immune Protection

[0326] The Aotus nancymai model was used to evaluate protection against challenge with P. falciparum-infected erythrocytes following immunization with FALVAC-IA. It should be noted that this model only tests protection against P. falciparum blood stages and not against the pre-erythrocytic sporozoite and liver stages. FALVAC-IA was designed to induce immunity to all three of these stages.

[0327] FALVAC-lA-immunized monkeys (see Section A, above) were challenged on day 98 with 10,000 P. falciparum FVO strain ring-infected erythrocytes. Parasitemia in infected animals was monitored by daily examination of thick and thin blood smears. Smears were stained with Giemsa stain, examined microscopically, and parasite counts were recorded per microliter of blood. Animals were treated when parasitemia >200,000/ul. Following the initial challenge, animals were rested and re-challenged on day 183 with 50,000 P. falciparum strain FC/H4 ring-infected erythrocytes.

[0328] There were no statistically significant differences in parasitemia metrics between the FALVAC-immunized groups and their respective adjuvant-only controls following either the primary or secondary challenge. It is possible that a different FALVAC-IA immunization protocol will provide Aotus monkeys protection against P. falciparum challenge. In addition, unlike humans and other hosts, A. nancymai only supports P. falciparum blood stages but not its pre-erythrocytic life stages. Thus, in other biological systems (such as humans) that are immunized with FALVAC-IA and naturally infected by mosquito bite, the full spectrum of FALVAC-I A-induced responses will be brought to bear against all parasite stages with a likely different and improved outcome.

[0329] In summary, Examples 4, 5, and 7-11 demonstrate that FALVAC-IA was immunogenic in 3 mammalian species having a variety of genetic backgrounds. Such immune responses were both antibody and cell mediated. The majority of the component epitopes were immunogenic and immune reactivity was noted throughout the molecule. Moreover, anti-F AL VAC-I A antibodies had in vitro anti¬ parasitic activity.

Example 11

In Vitro Determination of Immune Reactivity to FALVAC-IA in Individuals Naturally Exposed to Malaria

[0330] Sera from individuals naturally exposed to P. falciparum malaria are tested for reactivity to FALVAC-IA in vitro by ELISA. It is also determined whether FALVAC-IA can stimulate peripheral blood mononuclear cells from individuals naturally exposed to malaria to produce cellular immune response in vitro, for example, as measured by interferon-gamma and IL-4 production.

Example 12

Detailed Determination of Immune Reactivity to FALVAC-IA in Individuals Naturally Exposed to Malaria

[0331] Lymphocyte proliferation, cytokine, and antibody responses to FALVAC-IA are tested in non-immune children and clinically immune adults from a malaria endemic area. Finger prick samples of heparinized blood are used. The serum samples are used in determining the antibody response against the vaccine antigen and/or peptides in the vaccine antigen using an 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, FALVAC-IA. The T-cell proliferation is measured quantitatively and the cell culture supernatant is used for measuring cytokine levels using published techniques (LaI et al., Infect. Immun., 64:1054-1059,1996; Coligan et ah, Current Protocols in Immunology, 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., 1996). [0332] PBMCs also are examined for proliferative responses to FALVAC-IA. The individuals with a stimulation index (SI) value greater than 2.5 are considered positive responders.

[0333] It should be understood that the foregoing relates only to particular embodiments and that numerous modifications or alterations may be made without departing from the true scope and spirit of the invention as defined in the following claims.

Claims

1. A chimeric immunogenic polypeptide, comprising: a p545 epitope; a p546 epitope; a p594 epitope; a p606 epitope; and a p607 epitope.

2. The chimeric immunogenic polypeptide of claim 1 further comprising one or more of the following epitopes: a p519 epitope; a p543 epitope; a p544 epitope; a p592 epitope; a p593 epitope; a p595 epitope; a ρ596 epitope; a p597 epitope; a ρ598 epitope; a p599 epitope; a p600 epitope; a p601 epitope; a p602 epitope; a p603 epitope; a p604 epitope; and a p605 epitope.

3. The chimeric immunogenic polypeptide of claim 1, further comprising a combination of the following epitopes: a p519 epitope; a p543 epitope; a ρ544 epitope; a p592 epitope; a p593 epitope; a p595 epitope; a p596 epitope; a p597 epitope; a p598 epitope; a p599 epitope; a p600 epitope; a p601 epitope; a p602 epitope; a p603 epitope; a p604 epitope; and a p605 epitope.

4. The chimeric immunogenic polypeptide of claim 1, further comprising a spacer separating at least some adjacent epitopes.

5. The chimeric immunogenic polypeptide of claim 4, further comprising spacers separating multiple adjacent epitopes.

6. The chimeric immunogenic polypeptide of claim 5, wherein the spacers comprise two or more different spacers.

7. The chimeric immunogenic polypeptide of claim 4, wherein the one or more spacers are independently GPGPG, GPG, PGP, PGPG, GGPGG, APAPA, GGG, GGGG, APGPA, KG, GAA, G, RAA, CAA, NAA or KAA spacers.

8. The chimeric polypeptide of claim 7, wherein the one or more spacers are independently GPGPG, KG or KAA.

9. The chimeric immunogenic polypeptide of claim 4, wherein the spacer improves the immunogenicity of the chimeric polypeptide compared to the chimeric polypeptide without the spacer.

10. The chimeric immunogenic polypeptide of claim 1, wherein the p545 epitope comprises amino acid residues 163-175 of SEQ ID NO: 2 or a functional variant thereof, the p546 epitope comprises amino acid residues 228-248 of SEQ ID NO: 2 or a functional variant thereof, the p594 epitope comprises amino acid residues 46-53 of SEQ ID NO: 2 or a functional variant thereof, the p606 epitope comprises amino acid residues 182-206 of SEQ ID NO: 2 or a functional variant thereof, and the p607 epitope comprises amino acid residues 294-313 of SEQ ID NO: 2 or a functional variant thereof.

11. The chimeric immunogenic polypeptide of claim 2, wherein the p519 epitope comprises amino acid residues 110-121 of SEQ ID NO: 2 or a functional variant thereof, the p543 epitope comprises amino acid residues 207-214 of SEQ ID NO: 2 or a functional variant thereof, the ρ544 epitope comprises amino acid residues 220-227 of SEQ ID NO: 2 or a functional variant thereof, the p592 epitope comprises amino acid residues 3-18 of SEQ ID NO: 2 or a functional variant thereof, the p593 epitope comprises amino acid residues 23-45 of SEQ ID NO: 2 or a functional variant thereof, the p595 epitope comprises amino acid residues 98-106 of SEQ ED NO: 2 or a functional variant thereof, the p596 epitope comprises amino acid residues 176-183 of SEQ ID NO: 2 or a functional variant thereof, the p597 epitope comprises amino acid residues 56-73 of SEQ ID NO: 2 or a functional variant thereof, the p598 epitope comprises amino acid residues 74-92 of SEQ ID NO: 2 or a functional variant thereof, the p599 epitope comprises amino acid residues 358-374 of SEQ ID NO: 2 or a functional variant thereof, the p600 epitope comprises amino acid residues 122-139 of SEQ ID NO: 2 or a functional variant thereof, the p601 epitope comprises amino acid residues 145-162 of SEQ ID NO: 2 or a functional variant thereof, the p602 epitope comprises amino acid residues 273-291 of SEQ ID NO: 2 or a functional variant thereof, the p603 epitope comprises amino acid residues 249-270 of SEQ ID NO: 2 or a functional variant thereof, the p604 epitope comprises amino acid residues 319-336 of SEQ ID NO: 2 or a functional variant thereof, and the p605 epitope comprises amino acid residues 339-355 of SEQ ID NO: 2 or a functional variant thereof.

12. The chimeric immunogenic polypeptide of claim 10, wherein the p545 epitope comprises amino acid residues 163-175 of SEQ ID NO: 2, the p546 epitope comprises amino acid residues 228-248 of SEQ ID NO: 2, the p594 epitope comprises amino acid residues 46-53 of SEQ ID NO: 2, the p606 epitope comprises amino acid residues 182-206 of SEQ ID NO: 2, and the p607 epitope comprises amino acid residues 294-313 of SEQ ID NO: 2.

13. The chimeric immunogenic polypeptide of claim 11 , wherein the p519 epitope comprises amino acid residues 110-121 of SEQ ID NO: 2, the p543 epitope comprises amino acid residues 207-214 of SEQ ID NO: 2, the p544 epitope comprises amino acid residues 220-227 of SEQ ID NO: 2, the p592 epitope comprises amino acid residues 3-18 of SEQ ID NO: 2, the p593 epitope comprises amino acid residues 23-45 of SEQ ID NO: 2, the p595 epitope comprises amino acid residues 98-106 of SEQ ID NO: 2, the p596 epitope comprises amino acid residues 176-183 of SEQ ID NO: 2, the p597 epitope comprises amino acid residues 56-73 of SEQ ID NO: 2, the p598 epitope comprises amino acid residues 74-92 of SEQ ID NO: 2, the p599 epitope comprises amino acid residues 358-374 of SEQ ID NO: 2, the p600 epitope comprises amino acid residues 122-139 of SEQ ID NO: 2, the p601 epitope comprises amino acid residues 145-162 of SEQ ID NO: 2, the p602 epitope comprises amino acid residues 273-291 of SEQ ID NO: 2, the p603 epitope comprises amino acid residues 249-270 of SEQ ID NO: 2, the ρ604 epitope comprises amino acid residues 319-336 of SEQ ID NO: 2, and the p605 epitope comprises amino acid residues 339-355 of SEQ ID NO: 2.

14. The chimeric immunogenic polypeptide of claim 1 comprising in the following order p- 592-spacer-p593-p594-spacer-p597-p598-spacer-p595-spacer-p519-p600-spacer-p601-p545-p596-spacer- p606-p543-spacer-p544-ρ546-p603-spacer-p602-spacer-p607-spacer-p604-spacer-p605-spacer-p599.

15. The chimeric immunogenic polypeptide of claim 14 comprising p-592-GPGPG-p593- P594-KG-P597-P598-GPGPG-P595-KAA-P519-P600-GPGPG-P601-P545-P596-KAA-P606-P543-GPGPG- p544_p546-p603-KG-p602-KG-p607-GPGPG-p604-KG-p605-KG-p599.

16. The chimeric immunogenic polypeptide of claim 15, comprising amino acid residues 3- 374 of SEQ ID NO: 2.

17. The chimeric immunogenic polypeptide of claim 16, comprising amino acid residues 1- 374 of SEQ ID NO: 2.

18. The chimeric immunogenic polypeptide of claim 17, comprising the amino acid sequence of SEQ ID NO: 2.

19. The chimeric immunogenic polypeptide of claim 1 further comprising an affinity or targeting sequence.

20. The chimeric immunogenic polypeptide of claim 19, wherein the affinity sequence is a C- terminal poly-His affinity tag.

21. A polynucleotide coding for the chimeric immunogenic polypeptide of claim 1.

22. An antibody that binds at least a portion of the chimeric polypeptide of claim 1.

23. A combination of antibodies that binds to at least 75% of the epitopic peptide sequences of the chimeric polypeptide of claim 1.

24. A cell expressing the chimeric immunogenic polypeptide of claim 1.

25. The cell of claim 24, wherein the cell is a T-cell, a T-lymphocyte, a killer cell, a B cell or an antigen-presenting cell.

26. A pharmaceutical composition, comprising: a chimeric immunogenic polypeptide according to claim 1 ; and a pharmaceutically acceptable carrier.

27. The pharmaceutical composition of claim 26 further comprising an immunostimulant.

28. The pharmaceutical composition of claim 27, wherein the immunostimulant is an adjuvant.

29. The pharmaceutical composition of claim 26, comprising the chimeric immunogenic polypeptide set forth as amino acid residues 3-374 of SEQ ID NO: 2, or a functional variant thereof.

30. The pharmaceutical composition of claim 26, comprising the chimeric immunogenic polypeptide set forth as SEQ ID NO: 2 or residues 3-374 thereof.

31. A pharmaceutical composition, comprising: a binding agent that specifically binds a chimeric immunogenic polypeptide according to claim 1 ; and a pharmaceutically acceptable carrier.

32. The pharmaceutical composition of claim 31, wherein the binding agent specifically binds to a chimeric polypeptide as set forth as amino acid residues 3-374 of SEQ ID NO: 2.

33. The pharmaceutical composition of claim 31 , wherein the binding agent comprises an antibody.

34. The pharmaceutical composition of claim 33, wherein the binding agent comprises a combination of antibodies that bind to at least 75% of epitopic peptide sequences in the chimeric immunogenic polypeptide.

35. The pharmaceutical composition of claim 31 , wherein the binding agent is an effector cell.

36. The pharmaceutical composition of claim 35, wherein the effector cell is a T-cell, T- lymphocyte, a killer cell, a B cell or an antigen-presenting cell.

37. A method for stimulating an immune response in a subject comprising administering to the subject an effective amount of a pharmaceutical composition according to claim 26.

38. A method for treating a subject having a malarial infection comprising administering to the subject an effective amount of the pharmaceutical composition according to claim 31.

39. A method for detecting a malarial infection in a subject, comprising: contacting a biological sample obtained from a subject with an immobilized chimeric polypeptide according to claim 1; and quantitating antibody binding to the immobilized chimeric polypeptide, wherein binding of at least a predetermined quantity of antibody to the immobilized chimeric polypeptide indicates the malarial infection in the subject.