WO2006019427A2 - Compositions et methodes de prophylaxie, de traitement et de detection d'infections paludeennes - Google Patents

Compositions et methodes de prophylaxie, de traitement et de detection d'infections paludeennes

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Publication number
WO2006019427A2
WO2006019427A2 PCT/US2005/012248 US2005012248W WO2006019427A2 WO 2006019427 A2 WO2006019427 A2 WO 2006019427A2 US 2005012248 W US2005012248 W US 2005012248W WO 2006019427 A2 WO2006019427 A2 WO 2006019427A2
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WIPO (PCT)
Prior art keywords
epitope
seq
amino acid
acid residues
comprises amino
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PCT/US2005/012248
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English (en)
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WO2006019427A3 (fr
Inventor
Altaf A. Lal
Lihua X. Xiao
Charles W. Todd
Paul K. Schnake
Zhiyong Zhou
Ya-Ping Shi
Robert M. Wohlhueter
Venkatachalam Udhayakumar
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The Governement Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services, Centers For Disease Control And Prevention
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Application filed by The Governement Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services, Centers For Disease Control And Prevention filed Critical The Governement Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services, Centers For Disease Control And Prevention
Publication of WO2006019427A2 publication Critical patent/WO2006019427A2/fr
Publication of WO2006019427A3 publication Critical patent/WO2006019427A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/002—Protozoa antigens
    • A61K39/015—Hemosporidia antigens, e.g. Plasmodium antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • 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.
  • 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.
  • P. malaria protozoal Plasmodium
  • P. vivax a species of the protozoal Plasmodium
  • P. malariae a 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%.
  • 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.
  • the parasite 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.
  • 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.
  • 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.
  • Recombinant polynucleotides such as recombinant DNA, cDNA and RNA sequences
  • 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.
  • 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.
  • the pharmaceutical compositions further include an immunostimulant such as an adjuvant.
  • methods 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.
  • FIG. 1 is a schematic map of the recombinant chimeric polypeptide FALVAC- 1A-His showing locations of various epitopes and spacers.
  • 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 ⁇ g/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.
  • 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.
  • 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.
  • FIG. 5 shows reverse-phase HPLC tracings of FALVAC- 1 A (Lot F 134A) in native (top) and reduced (bottom) states, respectively.
  • samples were acidified and injected on a C ]8 reverse phase column and eluted between 5%-90% buffer B (0.1%TFA/80% acetonitrile/H 2 0) over 35 minutes.
  • the reduced sample was incubated at 37 0 C for 30 minutes with 2M urea plus 10OmM DTT prior to acidification and injection.
  • FIG. 6 is two graphs ((A) and (B)) showing the time courses of rabbit antibody responses to FALVAC-I A-His.
  • 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.
  • 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 ( ⁇ ).
  • 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 ⁇ g 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 ⁇ g FALVAC-IA-His (diagonally hatched bars), or 0.1 ⁇ g FALVAC-IA-His (wavy line bars).
  • FIG. 10 is a bar graph showing IL-4 ELISPOT results from C57BL/6mice immunized (s.c.) on days 0 and 14 with 10 ⁇ g 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 ⁇ g FAL VAC-I A-His (diagonally hatched bars), or 0.1 ⁇ g FALVAC-IA-His (wavy line bars).
  • 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 AlPO 4 alone; or (vi) 200 ug AlPO 4 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.
  • 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 ( ⁇ ); (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 AlPO 4 alone (0); or (vi) 200 ug AlPO 4 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.
  • 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.
  • 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.
  • 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.
  • SEQ ID NO: 3 is an amino acid sequence of a representative P. falciparum sporozoite stage p592 epitope.
  • SEQ ID NO: 4 is an amino acid sequence of a representative P. falciparum sporozoite stage p593 epitope.
  • SEQ ID NO: 5 is an amino acid sequence of a representative P. falciparum sporozoite stage p594 epitope.
  • SEQ ID NO: 6 is an amino acid sequence of a representative P. falciparum blood stage p597 epitope.
  • SEQ ID NO: 7 is an amino acid sequence of a representative P. falciparum blood stage p598 epitope.
  • SEQ ID NO: 8 is an amino acid sequence of a representative P. falciparum liver stage p595 epitope.
  • SEQ ID NO: 9 is an amino acid sequence of a representative P. falciparum sporozoite stage p519 epitope.
  • SEQ ID NO: 10 is an amino acid sequence of a representative P. falciparum blood stage p600 epitope.
  • SEQ ID NO: 11 is an amino acid sequence of a representative P. falciparum blood stage p601 epitope.
  • SEQ ID NO: 12 is an amino acid sequence of a representative P. falciparum blood stage p545 epitope.
  • SEQ ID NO: 13 is an amino acid sequence of a representative P. falciparum liver stage p596 epitope.
  • SEQ ID NO: 14 is an amino acid sequence of a representative P. falciparum blood stage p606 epitope.
  • SEQ ID NO: 15 is an amino acid sequence of a representative P. falciparum blood stage p543 epitope.
  • SEQ ID NO: 16 is an amino acid sequence of a representative P. falciparum blood stage p544 epitope.
  • SEQ ID NO: 17 is an amino acid sequence of a representative P. falciparum blood stage p546 epitope.
  • SEQ ID NO: 18 is an amino acid sequence of a representative P. falciparum blood stage p603 epitope.
  • SEQ ID NO: 19 is an amino acid sequence of a representative P. falciparum blood stage p602 epitope.
  • SEQ ID NO: 20 is an amino acid sequence of a representative P. falciparum blood stage p607 epitope.
  • SEQ ID NO: 21 is an amino acid sequence of a representative P. falciparum blood stage p604 epitope.
  • SEQ ID NO: 22 is an amino acid sequence of a representative P. falciparum blood stage p605 epitope.
  • SEQ ID NO: 23 is an amino acid sequence of a representative P. falciparum blood stage p599 epitope.
  • SEQ ID NO: 24 is a FALVAC-IA forward primer.
  • SEQ ID NO : 25 is a FALVAC- 1 A reverse primer.
  • 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).
  • GPGPG SEQ ID NO: 26
  • PGPG amino acid sequences of exemplar reverse-turn spacers
  • 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.
  • 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.
  • polypeptides including fusion proteins
  • polynucleotides as described herein are isolated.
  • 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.
  • 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).
  • 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.
  • the chimeric polypeptide may further include one or more other blood stage sporozoite, and/or liver stage epitopes.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the chimeric polypeptide includes at least 6 spacers, such as at least 8, at least 10 or at least 12 spacers separating adjacent epitopes.
  • 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.
  • 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.”
  • compositions are also disclosed.
  • 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.
  • 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.
  • a method for stimulating an immune response in a subject 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.
  • a method for detecting a malarial infection in a subject 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
  • 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.
  • 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.
  • an adjuvant may increase the titer of antibodies induced in an animal by the immunogenic agent.
  • an adjuvant may alter the particular epitopic sequences that are specifically bound by antibodies induced in an animal.
  • 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.
  • Amino acid 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.
  • 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).
  • 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.
  • 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).
  • 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
  • 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).
  • 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.
  • Antigenicity refers to the ability to specifically bind to the antigen binding site of an antibody or T-cell receptor for the antigen.
  • Cellular 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • epitopes typically have a maximum sequence of about 100 amino acids, for example, about 50, 25 or 18 amino acids in length.
  • 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.
  • 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.
  • alternative sequences that are functional variants of a specifically disclosed P. falciparum epitope sequence also are contemplated (as described in additional detail below).
  • 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.
  • a multi-stage, multivalent chimeric polypeptide including P. falciparum epitopes is processed to yield the component epitopes for presentation by antigen-presenting cells.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Humoral immunity refers to immunity that can be transferred with immune serum from one animal to another.
  • humoral immunity refers to immunity resulting from the introduction of specific antibodies and/or stimulation of the production of specific antibodies.
  • Immune 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).
  • 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.
  • 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.
  • 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").
  • an immune response is a T cell response, such as a CD4+ response or a CD8+ response.
  • the response is a B cell response, and results in the production of specific antibodies to the immunogenic agent.
  • 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.
  • 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.
  • IPTG Isopropyl thiogalactoside
  • 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.
  • 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.
  • Mimetic refers to a molecule (such as an organic chemical compound) that mimics the activity of another molecule.
  • Multistage refers to more than one stage in the life cycle of an organism.
  • 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.
  • Multivalent refers to a molecule comprising more than one epitope.
  • 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.
  • 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.
  • 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.
  • 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).
  • Passive immunity refers to immunity acquired by the introduction by immune system components into an animal rather than by stimulation
  • 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).
  • 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.
  • 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 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.”
  • 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).
  • Prophylaxis or treating 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.
  • 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.
  • purified does not require absolute purity; rather, it is intended as a relative term.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • spacer amino acid sequence is two or more amino acids that serve to link two peptide subsequences in a single polypeptide sequence.
  • 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.
  • GPGPG an exemplar reverse turn-inducing sequence
  • 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.”
  • 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.
  • two compounds are said to "specifically bind" when the binding constant for complex formation between the components exceeds about 10 4 L/mol, for example, exceeds about 10 6 L/mol, exceeds about 10 8 L/mol, or exceeds about 10 10 L/mol.
  • the binding constant for two components may be determined using methods that are well known in the art.
  • 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.
  • stage 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.
  • stage 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.
  • stages of the life cycle of a malaria parasite include the sexual stage, the blood stage, the liver stage and the sporozoite stage.
  • Subject A living multi-cellular vertebrate ("animal”), and a category that includes both human and veterinary subjects, including human and non-human mammals.
  • 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.
  • NCBI Basic Local Alignment Search Tool (BLASTTM) (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.
  • 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 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.
  • 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.
  • 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.
  • a vector is a viral vector.
  • Viral vectors include, but are not limited to, retroviral and adenoviral vectors.
  • the singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise.
  • the word “or” is intended to include “and” unless the context clearly indicates otherwise.
  • “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.
  • 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.
  • 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.
  • epitopes from all life stages are included in a disclosed chimeric polypeptide.
  • 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.
  • 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 include: p519 NANPNANPNANP (SEQ ID NO: 9) and functional variants thereof.
  • 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
  • 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.
  • 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).
  • 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
  • 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
  • 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.
  • 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
  • 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.
  • 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
  • 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.
  • 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
  • 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
  • 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
  • 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
  • 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
  • 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.
  • chimeric polypeptides include the p545, p546, p594, p606 and p607 epitopes in any order.
  • 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).
  • sequenceofthechimericpolypeptide may be included in the sequenceofthechimericpolypeptide,and suchsequencesmay(butneednot)possessfurtherimmunogenic orantigenicproperties.
  • 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 ⁇ 519, 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 ⁇ 604 epitope, or a ⁇ 605 epitope are possible in multiple combinations of order of epitopes chosen from the group of a p519 epitope, a
  • all of these optional epitopes are included in the chimeric polypeptide.
  • 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.
  • chimeric polypeptides with one or more epitopes in addition to the p545, p546, p594, p606, and ⁇ 607 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; p5
  • spacer amino acid sequences and/or additional peptide sequences may be present between epitopes of any disclosed chimeric polypeptide.
  • 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 ⁇ 594, p545, p606, ⁇ 546, or p607 epitopes).
  • 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.
  • reverse-turn sequences which permit a chimeric polypeptide chain to make a fold or bend.
  • GPGPG SEQ ID NO: 26
  • 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).
  • GPG GPG
  • PGP PGP
  • PGPG SEQ ID NO: 27
  • GGPGG SEQ ID NO: 28
  • APAPA SEQ ID NO: 29 GGG
  • GGGGGG SEQ ID NO: 30
  • APGPA SEQ ID NO: 31
  • spacers such as, KG or other glycine-containing spacers that do not also include structurally rigid prolines
  • spacers 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).
  • 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.
  • the chimeric polypeptides are fusion proteins of the chimeric polypeptide with a fusion partner.
  • chimeric polypeptides with one or more epitopes in addition to the p545, p546, p594, p606, and p607 epitopes and one 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, p594, spacer, p606, p607, spacer, p602; p545, spacer, p546, spacer, p594, spacer, p606, p607, spacer,
  • 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.
  • a chimeric polypeptide may be conjugated to an immunoglobulin Fc region (see also the description of fusion proteins below).
  • 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.
  • 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).
  • 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
  • variants 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 (or epitope) from which it is derived.
  • variants include, without limitation, sequence variants of, peptide derivatives of, or fusion proteins including a disclosed chimeric polypeptide (or, as applicable, an epitope thereof).
  • a variant of a chimeric polypeptide 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).
  • 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.
  • homologs and variants 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.
  • 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.
  • 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.
  • 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.
  • G Glycine
  • A Alanine
  • V Valine
  • V Valine
  • L Leucine
  • I Isoleucine
  • Aromatic Phenylalanine (F), Tyrosine (Y), Tryptophan (W);
  • Acidic Aspartic acid (D), Glutamic acid (E), Asparagine (N), Glutamine (Q).
  • 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.
  • a conservative variant of an epitope is also a functional variant of the epitope.
  • 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.
  • 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.
  • one conservative substitution is included in one or more epitopic peptide subsequences of a chimeric polypeptide.
  • two conservative substitutions or less are included in an individual antigenic peptide subsequence or an entire chimeric polypeptide.
  • 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.
  • 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.
  • 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.
  • 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.
  • a given epitope binds an antibody
  • 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).
  • 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).
  • 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).
  • a soluble major histocompatibility complex molecule such as a Class II HLA-DR or -DQ molecule
  • 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.
  • the peptide/class II complexes are transferred to wells coated with anti-DR Mab L234 antibody (15 ⁇ g/ml) or anti-DQ Mab HB144 (3.5 ⁇ g/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.
  • an optimal concentration of the biotinylated indicator peptide (0.1 ⁇ M-5 ⁇ M) is incubated with ten-fold dilutions (0.01 . ⁇ M-100 ⁇ M) 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-( ⁇ O.D. in presence of competitor peptide / ⁇ O.D.
  • the concentration of a competitor peptide required to inhibit 50% of binding of the biotinylated indicator peptide is determined and a peptide with an IC 50 of less than 100 ⁇ M (such as an IC 50 of less than 50 ⁇ M, less than 25 ⁇ M or less than 10 ⁇ M) is considered a functional variant of the epitope.
  • 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.
  • carboxylic acid groups of the protein 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 NR 1 R 2 wherein R] and R 2 are each independently H or Ci-Cj ⁇ alkyl, or combined to form a heterocyclic ring, such as a 5- or 6- membered ring.
  • Amino groups of the peptide 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 C 1 -C 16 alkyl or dialkyl amino or further converted to an amide.
  • a pharmaceutically acceptable acid addition salt such as the HCl, HBr, acetic, benzoic, toluene sulfonic, maleic, tartaric and other organic salts
  • Hydroxyl groups of the peptide side chains may be converted to CpCig alkoxy or to a C 1 -C 16 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 r Ci 6 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 C 2 -C 4 alkylenes.
  • Thiols can be protected with any one of a number of well-recognized protecting groups, such as acetamide groups.
  • protecting groups such as acetamide groups.
  • 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.
  • 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).
  • a mimetic mimics the binding of a malaria epitope or malaria protein to an antibody.
  • 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.
  • Fusion proteins may generally be prepared using standard techniques, including chemical conjugation.
  • 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.
  • 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.
  • 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.
  • 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.
  • stop codons that end translation and transcription termination signals may be present 3' to the DNA sequence encoding the second polypeptide.
  • 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).
  • an immunological fusion partner is derived from protein D, a surface protein of the gram-negative bacterium Haemophilus influenza B (WO 91/18926).
  • 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.
  • 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.
  • 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.
  • a variant of a chimeric polypeptide 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.
  • assays for determining antibody binding and T-cell reactivity are well known in the art.
  • 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.
  • 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, 125 I-labeled (or other detectable) Protein A.
  • variant chimeric polypeptide or epitope
  • 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.
  • Chimeric polypeptides 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 0 C to 65 0 C, in a solution of 5xSSC overnight; and washing twice at 65 0 C for 20 minutes with each of 2x, 0.5x and 0.2x SSC containing 0.1% SDS.
  • 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 0 C to 72 0 C. 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.
  • PCR polymerase chain reaction
  • 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.
  • a suitable RNA polymerase promoter such as T7 or SP6
  • 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).
  • antigen-presenting cells such as dendritic cells
  • 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 are typically 22-30 nucleotides in length, but may be longer in some embodiments.
  • 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.
  • Nucleotide sequences as described herein may be joined to a variety of other nucleotide sequences using established recombinant DNA techniques.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • lipid-based systems including oil-in- water emulsions, micelles, mixed micelles, and liposomes (i.e. an artificial membrane vesicle).
  • liposomes i.e. an artificial membrane vesicle
  • compositions described herein may be used in methods for stimulating an immune response in a subject against malarial infections, such as P. falciparum infections.
  • 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.
  • 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).
  • 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.
  • the disclosed antibodies and/or chimeric polypeptides may be used in assays to detect the presence of malarial infection in a subject.
  • 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).
  • 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).
  • immune response-modifying agents such as the polypeptides and polynucleotides disclosed herein.
  • the methods of prophylaxis or treatment comprise stimulation of an immune response.
  • 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.
  • agents with established immune reactivity such as autologous effector cells or antibodies
  • 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.
  • 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.
  • cytokines such as IL-2
  • 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.
  • antigen-presenting cells such as dendritic, macrophage, monocyte, fibroblast and/or B cells
  • antigen-presenting cells may be pulsed with immunoreactive polypeptides or transfected with one or more polynucleotides using standard techniques.
  • 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).
  • 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.
  • compositions including pharmaceutical compositions
  • administration such as intracutaneous, intramuscular, intravenous or subcutaneous injection
  • intranasally such as by aspiration
  • 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.
  • the compositions may be administered by injection (such as intracutaneous, intramuscular, intravenous or subcutaneous injection), intranasally (such as by aspiration) or orally.
  • injection such as intracutaneous, intramuscular, intravenous or subcutaneous injection
  • intranasally such as by aspiration
  • 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.
  • the amount of each present in a dose ranges, for a 4.5 kg infant, from about 200 ng/kg to about 200 ⁇ g/kg (such as, from about 3 ⁇ g/kg to about 55 ⁇ g/kg), or, for an 80 kg adult, from about 12.5 ng/kg to about 12.5 ⁇ g/kg (such as, from about 185 ng/kg to about 3 ⁇ g/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.
  • an appropriate dosage and treatment regimen provides the active composition(s) in an amount sufficient to provide a therapeutic and/or prophylactic benefit.
  • 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 ⁇ g to 1 mg of the disclosed immunogenic polypeptides, for example, 10 ⁇ g to 800 ⁇ g of the polypeptide, such as from 15 ⁇ g to 250 ⁇ g.
  • "booster" doses may comprise from 1 ⁇ g to 1 mg of the disclosed immunogenic polypeptides, for example, from 10 ⁇ g to 800 ⁇ g, such as from 15 ⁇ g to 250 ⁇ g.
  • an effective amount of a composition administered to a 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 ⁇ g of a chimeric polypeptide is administered for an initial immunization, and approximately 5-500 ⁇ g is administered for a subsequent booster immunization. In another example, approximately 5-500 ⁇ g of a chimeric polypeptide is mixed together with approximately 20-2000 ⁇ g of an adjuvant, such as aluminum phosphate, and administered.
  • an adjuvant such as aluminum phosphate
  • a chimeric polypeptide is mixed with about 40-1000 ⁇ g of an adjuvant and is administered, or about 15-100 ⁇ g of a chimeric polypeptide is mixed with about 60-400 ⁇ g of an adjuvant and is administered.
  • 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.
  • a biological sample for example, blood, sera, sputum urine and/or biopsies obtained from the subject
  • binding agents such as antibodies against one or more malarial epitopes.
  • the disclosed chimeric polypeptides can be used in assays to indicate the presence or absence of a malarial infection since they contain malarial epitopes.
  • specific binding agents such as serum antibodies can be detected by their binding to the chimeric polypeptides.
  • 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.
  • 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.
  • 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.
  • 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.
  • the solid support may be a test well in a microliter plate or a nitrocellulose or other suitable membrane.
  • 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).
  • 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.
  • 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 ⁇ g, such as from about 100 ng to about 1 ⁇ g, is sufficient to immobilize an adequate amount of chimeric polypeptide.
  • a plastic microtiter plate such as polystyrene or polyvinylchloride
  • 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.
  • 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.
  • 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).
  • 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.
  • a detection reagent preferably a second antibody capable of binding to a different site on the polypeptide
  • 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.
  • PBS phosphate-buffered saline
  • an appropriate contact time is a period of time that is sufficient to detect the presence of polypeptide within a sample obtained from an individual with malaria.
  • 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.
  • an incubation time of about 30 minutes is generally sufficient.
  • 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.
  • 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.
  • 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.
  • 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.
  • a sample generating a signal that is three standard deviations above the predetermined cut-off value is considered positive for the infection.
  • 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.
  • 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.
  • 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.
  • a sample generating a signal that is higher than the cut-off value determined by this method is considered positive for a malarial infection.
  • the assay is performed in a flow-through or strip test format, wherein the chimeric polypeptide is immobilized on a membrane, such as nitrocellulose.
  • a membrane such as nitrocellulose.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ g such as from about 50 ng to about 500 ng. Such tests can typically be performed with a very small amount of biological sample.
  • 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.
  • 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.
  • 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 ⁇ g/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.
  • CD4+ T cells activation may be detected by evaluating proliferation of the T cells.
  • 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.
  • 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.
  • 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.
  • one or more immunogenic portions of other malarial proteins may be present, either incorporated into a fusion protein or as a separate compound.
  • a pharmaceutical composition may comprise DNA encoding one or more of the disclosed polypeptides, such that the polypeptide(s) is (are) generated in situ.
  • 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.
  • 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.
  • a viral expression system e.g., vaccinia or other pox virus, retrovirus, or adenovirus
  • vaccinia or other pox virus e.g., vaccinia or other pox virus, retrovirus, or adenovirus
  • 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.
  • 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.
  • any suitable pharmaceutically acceptable carrier known to those of ordinary skill in the art may be employed in a disclosed pharmaceutical composition.
  • 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.
  • 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.
  • 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.
  • 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
  • Suitable biodegradable microspheres are disclosed, for example, in U.S. Pat. Nos. 4,897,268 and 5,075,109.
  • 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.
  • 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.
  • buffers e.g., neutral buffered saline or phosphate buffered saline
  • carbohydrates e.g., glucose, mannose, sucrose or dextrans
  • mannitol e.glycine
  • additional proteins polypeptides or amino acids
  • additional proteins e.glycine
  • antioxidants e.g., antioxidants, chelating agents such as EDTA or glutathione
  • immunostimulants such as adj
  • 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.
  • any of a variety of immunostimulants may be employed in the pharmaceutical compositions.
  • 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-de ⁇ ved 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.
  • Cytokines such as GM- CSF
  • 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- ⁇ ., IL-2 and IL-12
  • Th2-type cytokines such as IL-4, IL-5, IL-6 and IL-10
  • a subject may support an immune response that includes ThI- and Th2-type responses.
  • Thl-type cytokines 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Still other adjuvants include polymers and co-polymers.
  • copolymers such as polyoxyethylene-polyoxypropylene copolymers and block co-polymers may be used.
  • a particular example of a polymeric adjuvant is polymer P 1005.
  • 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 ⁇ g to 800 ⁇ g, such as from 50 ⁇ g to 500 ⁇ g.
  • 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.
  • 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.
  • a suitable carrier or excipient 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.
  • 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.
  • APCs antigen presenting cells
  • APCs 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.
  • 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.
  • secreted vesicles antigen-loaded dendritic cells called exosomes
  • exosomes antigen-loaded dendritic cells
  • 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.
  • dendritic cells may be differentiated ex vivo by adding a combination of cytokines such as GM-CSF, IL-4, IL-13 and/or TNF- ⁇ to cultures of monocytes harvested from peripheral blood.
  • 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- ⁇ , CD40 ligand, LPS, flt3 ligand and/or other compound(s) that induce differentiation, maturation and proliferation of dendritic cells.
  • 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).
  • 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).
  • 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.
  • 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 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).
  • polypeptides or fusion proteins may be covalently conjugated to an immunological partner that provides T cell help (e.g., a carrier molecule).
  • an immunological partner that provides T cell help e.g., a carrier molecule.
  • a dendritic cell may be loaded with a non-conjugated immunological partner, separately or in the presence of the desired composition.
  • 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.
  • formulations may be stored as suspensions, solutions or as emulsions in oily or aqueous vehicles.
  • a pharmaceutical composition may be stored in a freeze-dried condition requiring only the addition of a sterile liquid carrier immediately prior to use.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • two compounds are said to "specifically bind" when the binding constant for complex formation exceeds about 10 4 L/mol, for example, exceeds about 10 6 L/mol, exceeds about 10 s L/mol, or exceeds about 10 10 L/mol.
  • the binding constant may be determined using methods well known in the art.
  • 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.
  • a malarial infection such as a P. falciparum infection
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • a carrier protein for example, in a fusion protein construct
  • 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
  • an adjuvant such as ISA-720, QS-21, TiterMax Gold, non-ionic copolymer in a water-in-oil emulsion, or aluminum phosphate
  • 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.
  • 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.
  • 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.
  • Monoclonal antibodies may be isolated from the supernatants of growing hybridoma colonies.
  • 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.
  • 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.
  • Isolated antibodies 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.
  • 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
  • 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.
  • 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).
  • a variety of routes may be used for administration of binding agents and conjugates made therefrom.
  • 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.
  • 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.
  • 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.
  • 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.
  • T cells may be derived from related or unrelated humans, non-human mammals, cell lines or cultures.
  • 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.
  • a polypeptide or polynucleotide is present within a delivery vehicle, such as a microsphere, to facilitate the generation of specific T cells.
  • 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.
  • 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 ⁇ g/ml, for example, between 200 ng/ml-25 ⁇ g/ml) for 3-7 days that results in at least a two fold increase in proliferation of the T cells indicates specificity.
  • a peptide or polypeptide for example, at 100 ng/ml-100 ⁇ g/ml, for example, between 200 ng/ml-25 ⁇ g/ml
  • 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.
  • 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
  • kits for use within any of the diagnostic methods described above typically comprise two or more components necessary for performing a diagnostic assay.
  • Components may be compounds, reagents, containers and/or equipment.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a number of spacer amino acid sequences were added between certain epitopic peptides.
  • 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.
  • 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.
  • 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.
  • 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.
  • a chimeric polypeptide 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.
  • 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.
  • spacers such as KAA and KG spacers, may optionally be placed between one or more pairs of epitopes along the sequence.
  • KAA spacers may be placed after the CTL epitopes as in FALVAC-IA to aid in their presentation to the immune system.
  • GPGPG spacers SEQ ID NO: 26
  • epitopes between the spacers are possible while still providing a compact structure.
  • CSP circumsporozoite protein
  • LSA- liver stage antigen- 1
  • MSP- merozoite surface protein- 1
  • MSP-2 merozoite surface protein-2
  • AMA-I apical membrane antigen- 1
  • EBA-17 5 erythrocyte binding antigen- 17 5
  • 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.
  • 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.
  • 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.
  • one or more GPGPG spacers may be replaced by other short peptide sequences (for example, 3- 6 residues in length) that permit, or promote, reverse turns.
  • 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.
  • 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.
  • 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.
  • 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
  • the epitopes may be separated with appropriate spacer sequences, and additional sequences may be added to aid in expression or isolation.
  • GPGPG spacers SEQ ID NO: 26
  • immunogenic and/or non-immunogenic protein partners may be added to provide fusion proteins comprising the chimeric polypeptides.
  • poly-Arginine or poly-Histidine sequences, FLAG tags or other sequences known for such purposes may be added.
  • 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.
  • 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.
  • 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 SinglesTM Kit (Novagen, Madison, WI).
  • 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).
  • 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 ⁇ m 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.
  • 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.
  • 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.
  • 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.
  • 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
  • HCP E. coli host cell protein
  • N.D. Not determined
  • the theoretical molecular weight (MW) of FALVAC-I A-His is 42,661 daltons.
  • 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.
  • this Example demonstrates that an exemplar multivalent, multistage malarial vaccine, FALVAC-IA-His, consistently can be expressed and purified.
  • the molecule reproducibly folded to assume an energetically stable, defined molecular conformation that was stable when frozen and thawed for use.
  • this Example illustrates the feasibility of constructing chimeric polypeptide antigens including selected antigenic determinants and spacer determinants that provide an energetically stable conformation.
  • 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 ⁇ g 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.
  • 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 l ⁇ g/ml FALVAC-IA or 10 ⁇ g/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 4 0 C. 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 H 3 PO 4 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.
  • 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.
  • 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).
  • NIIMALVAC-I multivalent, multistage P falciparum vaccine
  • the physico-chemical properties of NIIMALVAC-I and FALVAC-IA for example, their aqueous solubility and pi values, are distinctly different (see Table 4).
  • Tins Example illustrates that FALVAC-lA-induced rabbit antibodies react with native parasite antigens in both the sporozoite and blood stages.
  • IFA immunofluorescence assay
  • the slides were incubated at 37°C, 100% relative humidity for 30 minutes, then washed 3 times by aspiration and reapplication of 20 ⁇ l PBS. Following the final aspiration, 10 ⁇ l 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.
  • 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.
  • ISI sporozoite invasion
  • falciparum CSP protein was used as positive control at 200 ⁇ g/ml, with 3 wells containing complete medium as negative control. Fifty ⁇ l 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 ⁇ g/ml) as primary antibody and peroxidase labeled goat anti-mouse IgG (KPL) as the secondary antibody.
  • NFSl (10 ⁇ g/ml) as primary antibody
  • KPL peroxidase labeled goat anti-mouse IgG
  • 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.
  • ADCI antibody dependent cellular inhibition
  • 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 37 0 C in a mixed gas containing 5% O 2 , 5% CO 2 , and 90% N 2 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.
  • HE vital dye hydroethidine
  • dilutions of purified antibodies from immunized and control rabbits are added to micro-cultures of parasitized human type-0 erythrocytes (100 ⁇ L of 1.5% (v/v) erythrocytes with 1% parasitemia).
  • the cultures are incubated for 18 hours at 37 0 C in 90% N 2 , 5% O 2 , 5% CO 2 , washed, incubated with fresh medium for another 8 hours and then radiolabeled with 14 C-isoleucine and 3 H-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).
  • 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.
  • IFN-G Interferon gamma
  • IL-4 Interleukin-4
  • 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.
  • 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).
  • 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.
  • ELISPOT enzyme-linked immunospot assay
  • RPMI medium RPMI- 1640, 10% heat inactivated fetal bovine serum (FBS), 50 ⁇ g/ml gentamicin, 0.1 mM nonessential amino acids, 0.1 mM MEM vitamins, 1% L-glutamine and 2 mM ⁇ -mercaptoethanol
  • 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 10 6 cells/ml in complete RPMI medium. The blocking medium in the assay plates was removed and 10 5 mouse mononuclear cells (100 ul of 10 6 cells/ml in complete RPMI medium) were added to appropriate wells.
  • hypotonic shock Lysis Buffer, eBiosciences, San Diego, CA
  • the blocking medium in the assay plates was removed and 10 5 mouse mononuclear cells (100 ul of 10 6 cells/ml in complete RPMI medium) were added to appropriate wells.
  • any IFN-G or IL-4 secreted by activated cells bound to its respective antibody on the membrane at the bottom of the well.
  • the plates were washed twice with 200 ⁇ l distilled water to lyse and remove the cells, then four times with PBS-0.01% Tween 20 (P-T20).
  • the spots were developed by adding 100 ul of substrate solution (AEC Chromogen/substrate, BDTM ELISPOT AEC Substrate Set, BD Biosciences Pharmingen) for 15 minutes at room temperature.
  • substrate solution AEC Chromogen/substrate, BDTM ELISPOT AEC Substrate Set, BD Biosciences Pharmingen
  • 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/10 6 spleen cells.
  • 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.
  • 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.
  • ConA Concanavalin A
  • FALVAC-IA the immunizing antigen
  • FIG. 10 shows the day 23 IL-4 ELISPOT data for C57BL/6 mice.
  • 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.
  • Interferon gamma is produced by T h l 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.
  • cytokines such as interleukin-2, IFN-G, lymphotoxin and tumor necrosis factor- beta.
  • IL-4 is secreted by T h 2 lymphocytes that recognize antigens presented by B lymphocytes.
  • T h 2 lymphocytes produce cytokines such as interleukins 2, 4, 5, 10, and 13, which promote antibody production.
  • 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.
  • 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.
  • 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).
  • 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 -70 0 C 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).
  • 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.).
  • FALVAC-IA One hundred (100) ul FALVAC-IA was added to give 1.0 and 0.1 ug/ml final concentration in the well.
  • 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.
  • FALVAC-lA-immunized monkeys 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.
  • 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.
  • FALVAC-IA 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.
  • 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.
  • 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.
  • PBMCs also are examined for proliferative responses to FALVAC-IA.
  • SI stimulation index

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Abstract

L'invention concerne des polypeptides immunogènes chimériques. Lesdits polypeptides immunogènes chimériques comprennent des épitopes peptidiques dérivés de différents stades du cycle biologique du parasite paludéen, et, en particulier, du parasite Plasmodium falciparum. Dans certains modes de réalisation, des séquences d'acides aminés d'espacement sont insérées entre des épitopes. Les polypeptides immunogènes chimériques selon l'invention peuvent être utilisés pour stimuler une réponse immunitaire dirigée contre le parasite P. falciparum chez un sujet.
PCT/US2005/012248 2004-04-14 2005-04-11 Compositions et methodes de prophylaxie, de traitement et de detection d'infections paludeennes WO2006019427A2 (fr)

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EP2249857A2 (fr) * 2008-01-18 2010-11-17 Aeras Global TB Vaccine Foundation Compositions de vaccin contre la malaria et constituants qui déclenchent une immunité à médiation cellulaire
WO2014028644A1 (fr) * 2012-08-15 2014-02-20 Cyvax, Inc. Procédés et compositions pour la prévention d'un état

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2249857A2 (fr) * 2008-01-18 2010-11-17 Aeras Global TB Vaccine Foundation Compositions de vaccin contre la malaria et constituants qui déclenchent une immunité à médiation cellulaire
JP2011509672A (ja) * 2008-01-18 2011-03-31 アエラス グローバル ティービー ワクチン ファウンデーション マラリアワクチン組成物および細胞媒介免疫性を惹起する成分
EP2249857A4 (fr) * 2008-01-18 2012-02-29 Aeras Global Tb Vaccine Foundation Compositions de vaccin contre la malaria et constituants qui déclenchent une immunité à médiation cellulaire
US8728492B2 (en) 2008-01-18 2014-05-20 Aeras Global Tb Vaccine Foundation Malaria vaccine compositions and constituents which elicit cell mediated immunity
WO2014028644A1 (fr) * 2012-08-15 2014-02-20 Cyvax, Inc. Procédés et compositions pour la prévention d'un état

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