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Title:  Malaria immunogenic composition

United States Patent:  6,660,498

Issued:  December 9, 2003

Inventors:  Hui; George S. N. (Honolulu, HI); Pang; Lap-Yin (Kwai Chung, HK); Ho; Walter K. K. (Taipo, HK)

Assignee:  University of Hawaii (Honolulu, HI); The Chinese University of Hong Kong (Hong Kong, HK); Queen Emma Foundation (Honolulu, HI)

Appl. No.:  710000

Filed:  November 10, 2000

Abstract

We utilized the silkworm (Bombyx mori)/baculovirus system to produce recombinant Major Merozoite Surface Protein 1 (MSP142) because of the low cost and potential high yield of this expression system. The MSP142 (3D7 sequence) was cloned into the baculovirus, BmNPV with the melittin signal sequence. The recombinant virus, BmNPV-Sp42 was used to infect silkworms for the expression of MSP142 (Sp42). One recombinant clone expressed high level of Sp42 with an estimated 0.5 mg of antigen produced within a single worm. The Sp42 was recognized by monoclonal and polyclonal antibodies specific for parasite MSP1 in direct binding and competitive binding ELISAs, suggesting that Sp42 possesses antigenic determinants similar to parasite MSP142. Immunogenicity studies were performed in rabbits. Sp42 induced high titers of antibodies crossreactive with MSP1. Specificity analyses showed that anti-Sp42 antibodies reacted primarily against conserved determinants on MSP1-19. Our results showed that the silkworm expression system can produce recombinant MSP142 that are antigenically and immunogenically comparable to other recombinant MSP1 antigens expressed in other eukaryotic systems. The low cost ad high level of protein expression makes it an attractive alternative for the development of a human malaria vaccine.

SUMMARY OF THE INVENTION

In order to meet these needs, the present invention is directed to the use of a baculovirus expression system to produce a recombinant C terminal 42 kDa fragment of the Major Merozoite Surface Protein (MSP142) for use in a malaria vaccine. The expression system of the invention is silkworms infected with recombinant nuclear polyhedrosis virus (NPV). The silkworms are preferably Bombyx mori silkworms (BmNPV). The malaria vaccine finds use in treating and preventing malaria including malaria resulting from the four species of the protozoal parasites of the genus Plasmodium that infect humans: P. falciparum P, vivax, P. malariae and P. ovale.

The present invention is further directed to a method of producing a malaria vaccine, comprising: (a) expressing an immunogenic fragment of MSP 142 in a baculovirus expression system; (b) purifying the immunogenic fragment; and (c) formulating the immunogenic fragment in a malaria vaccine. In the method, the immunogenic fragment may include all or a portion of the MSP 142 protein. The MSP 142 protein may include a hexa-histidine tail.

In the method of purifying a malaria vaccine of the invention, the immunogenic fragment may be purified by chromatography or electrophoresis. The chromatography purification method may be ion exchange chromotogaphy, metal chelate affinity chromatography; molecular weight sieving, high pressure liquid chromatography, affinity chromatography or antibody affinity chromatography. The electrophoresis procedure may be agarose, acrylamide or isoelectric focusing electrophoresis.

In the method of producing a malaria vaccine of the invention the vaccine may include an adjuvant. The adjuvant may be selected from the group consisting of aluminum phosphate, aluminum hydroxide, saponin, Quil A, muramyl dipeptide, monophosphoryl lipid A muramyl tripeptide, cytokines, diphteriatoxoid, exotoxin A, granulocyte-macrophage colony stimulating factor and phospholipid conjugates. The adjuvant may further be selected from the group consisting of Adjumer.TM.; PCPP salt; polyphophazene; polyidi(carboxylatophenoxyl)phosphazene; Adju-Phos; Aluminum phosphate gel; .beta.-glucan; glucan; Gamma inulin/alum composite adjuvant; aluminum hydroxide gel; alum; N,N-dioctadecyl-N1, N1 -bis(2-hydroexyethyl) propanediamine; N-(2-Deoxy-2-L-leucylamino-.beta.-D-glucopyranosyl)-N-octadecyldodecanoyla mide; Calcitriol; 25-dihydroxyvitamin D3; 1,25-di(OH)2 D3 ; 1,25-DHCC; 1.alpha.,25-dihdroxycholecalciferol; 9,10-seco(5Z,7E)-5,7,10(19)-cholestatriene-1.alpha.,3.beta.,25-triol; Block Copolymer P1205; Cytokine-containing Liposomes; Cytokine-containing Dehydration Rehydration Vesicles; Dimethyl dioctadecylammonium bromide; demethyl distearylammonium bromide; Dehydroepiandrosterone; 5-androsten-3.beta.-o1-17-one; dehydroisoandrosterone; androstenolone; prasterone; transdehydroandrosterone; Dimyristoyl phosphatidyl choline; sn-3-phosphatidyl choline-1,2-dimyristoyl; 1,2-dimyristoyl-sn-3-phosphatidyl choline; Dimyristoyl pposphatidylglcerol; sn-3-phosphatidyl glycerol-1,2-dimyristoyl, sodium salt; 1,2-dimyritoyl-sn-3-phosphatidyl glycerol; Deoxycholic Acid Sodium Salt; Gamma Inulin; Interleukin-1.beta.; IL-10; IL-1; human Interleukin 1.beta. mature polypeptide Interferon-.gamma.; Immunoliposomes Containing Antibodies to Costimulatory Molecules; ImmTher.TM.; N-acetylglucosaminyl-N-acetyhnuramyl-L-Ala-D-isoGlu-L-Ala-Glycerol dipalmitate; Imiquimod; 1-(2-methypropyl)-IH-imidazo[4,5-c]quinolin-4-amine; GMDP; N-acetylglucosaminyl-(.beta.1-4)-N-acetylmuramyl-L-alanyl-D-isoglutamine; Gerbu Adjuvant; Interleukin-2; IL-2; T-cell growth factor; aldesleukin (des-alanyl-1, serine-125 human interleukin 2); Proleukin.RTM.; Teceleukin.RTM.; Interleukin-7; IL-7; Interleukin-12; IL-12; natural killer cell stimulatory factor (NKSF); cytotoxic lymphocyte maturation factor (CLMF); ISCOM(s).TM.; Immune stimulating complexes; Iscoprep 7.0.3..TM.; Liposomes; Liposomes (L) containing protein or Th-Cell and/or B-cell peptides, or microbes with or without co-entrapped interleukin-2, BisHOP or DOTMA; Loxoribine; 7-allyl-8-oxoguanosine; LT-OA or LT Oral Adjuvant; E. coli labile enteroxtoxin protoxin; MONTANIDE ISA 720; metabolizable oil adjuvant; MPL.TM.; 3-Q-dsacyl-4'-monophosphoryl lipid A; 3D-MLA; MF59; MTP-PE; N-acetyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1,2-dipalmitoyl-sn-glcero-3 -(hydroxy-phosphoryloxy)) ethylamide, mono sodium salt; MTP-PE Liposomes; MTP-PE Antigen presenting liposomes; Murametide; Nac-Mur-L-Ala-D-Gln-OCH3; Murapalmitine; Nac-Mur-L-Thr-D-isoGln-sn-glycerol dipalmitoyl; D-Murapalmitine; Nac-Mur-D-Ala-D-isoGln-sn-glcerol dipalmitoyl; NAGO; Neuraminidase-galactose oxidase; Non-Ionic Surfactant Vesicles; NISV.; Pleuran; PLGA, PGA, and PLA; Homo-and co-polymers of lactic and glycolic acid; Lactide/glycolide polymers; poly-lactic-co-glycolide; Pluronic L121; Poloxamer 401; PMMA; Polymethyl methacrylate; PODDS.TM.; Proteinoid microspheres; Poly rA; Poly rU; Poly-adenylic acid-poly-uridylic acid complex; Polysorbate 80; Tween 80; Sorbitan mono-9-octadecenoate poly(oxy-1,2-ethanediyl) derivatives; Protein Cochleates; QS-21; Stimulon.TM. QS-21 Adjuvant; Rehydragel HPA; High Protein Adsobency Aluminum Hydroxide Gel; alum; Rehydragel LV; low viscosity alluminum hydroxide gel; alum; S-28463; 4-Amino-otec, -dimethyl-2-ethoxmethyl-1H-imidazo[4,5-c]quinoline-1-ethanol; SAF-1; Scalvo peptide; IL-1.beta. 163-171 peptide; Span 85; Arlacel 85, sorbitan trioleate; Specol; Marcol 52 (mineral oil, paraffins, and cycloparaffins, chain length 13-22 C atoms) Span 85 (emulsifier, sorbitan trioleate) Tween 85 (emulsfier, polyoxyethylene-20-trioleate); Squalane; Spinacane; Robane.RTM.; 2,6,10,15,19,23-hexamethyltetracosane; Squalene; Spinacene; Supraene; 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22 tetracosahexaene; Stearyl Tyrosine; Octadecyl tyrosine hydrochloride; Theramide.TM.; N-acetylglucosaminyl-N-acetylinuramyl-L-Ala-D-isoGlu-L-Ala-dipalmitoxy propylmadie (DTP-DPP); Threonyl-MDP; Termurtide.TM.; [thr1 ]-MDP ; N-acetyl muramyl-L-threonyl-D-isoglutamine; Ty Particles; and Ty-VLPs, (Virus Like Particles)

In the method of purifying a malaria vaccine of the invention the vaccine may include cholera toxin. The cholera toxin may be cholera toxin subunit A or cholera toxin subunit B.

The present invention is also directed to a method of immunizing a patient to malaria, comprising (a) preparing a malaria vaccine by expressing an immunogenic fragment of MSP142 in a baculovirus expression system; (b) purifying the immunogenic fragment; (c) formulating the immunogenic fragment in a malaria vaccine; and (d) administering the vaccine to said patient.

In the method of of immunizing a patient to malaria, the immunogenic fragment may be purified by chromatography or electrophoresis. The chromatography purification method may be ion exchange chromotogaphy, molecular weight sieving, high pressure liquid chromatography, affinity chromatography or antibody affinity chromatography. The electrophoresis procedure may be selected from agarose, acrylamide and isoelectric focusing electrophoresis.

In the method of immunizing a patient to malaria, the vaccine may include an adjuvant. In the method of immunizing a patient to malaria, the vaccine may include cholera toxin. The cholera toxin may be cholera toxin subunit A or cholera toxin subunit B.

The present invention is also directed to a malaria vaccine produced by the method of: (a) expressing an immunogenic fragment of MSP142 in a bacculovirus expression system; (b) purifying the immunogenic fragment; and (c) formulating the immunogenic fragment in a malaria vaccine.

The vaccine may further include an adjuvant. The vaccine may include cholera toxin. The cholera toxin may be cholera toxin subunit A or cholera toxin subunit B.

The present invention is further directed to a method of treating a patient with malaria, compromising: (a)expressing an immunogenic fragment of MSP142 in a baculovirus expression system; purifying the immunogenic fragment; (c) formulating the immunogenic fragment in a malaria vaccine; and (d) administering the malaria vaccine to the patient.

In the method of treating a patient with a malaria vaccine the immunogenic fragment may be purified by chromatography or electrophoresis. The chromatography purification method may be ion exchange chromotogaphy, molecular weight sieving, high pressure liquid chromatography, affinity chromatography and antibody affinity chromatography. The electrophoresis procedure may be selected from agarose, acrylamide and isoelectric focusing electrophoresis.

In the method of treating a patient with a malaria vaccine of the invention the vaccine may include an adjuvant. In the method of treating a patient with a malaria vaccine of the invention the vaccine may include cholera toxin. The cholera toxin may be cholera toxin subunit A or cholera toxin subunit B.

The invention is further directed to a method of purifying an immunogenic fragment of MSP142, comprising: expressing the immunogenic fragment in a bacculovirus expression system wherein the baculovirus expression system is Bombyx mori silkworms infected with nuclear polyhedrosis virus and purifying the immunogenic fragment by chromatography or electrophoresis.

In the method purifying an immunogenic fragment of MSP142, the chromatography purification method may be selected from ion exchange chromotogaphy, molecular weight sieving, high pressure liquid chromatography, affinity chromatography and antibody affinity chromotography. and the electrophoresis procedure may be agarose, acrylamide or isoelectric focusing electrophoresis.

The present ivention is further directed to an isolated and purified immunogenic fragment of MSP142 purified by the methods of the invention.

The present invention is further directed to a silkworm capable of expressing recombinant MSP 142. The silkworm may be Bombyx mori silkworm infected with recombinant nuclear polyhedrosis virus.

DETAILED DESCRIPTION OF THE INVENTION

DEFINITIONS

To ensure a complete understanding of the invention, the following definitions are provided:

Malaria: Malaria is any group of diseases usually intermittent or remittent characterized by attacks of chills fever, and sweating caused by a parsitic protozoan which is transferred to the human bloodstream by a mosquito.

Major Merozoite Surface Protein 1 (MSP1): Major merozoite surface protein 1 (MSP1) is a surface protein of Plasmodium falciparum which is a parasite which causes malaria. The C-terminal 42 kDa fragment of Plasmodium falciparum MSP142 is also known as PfMSP-142. Pf MSP-142 ; MSP1-42 and PfMSP1-42 are synonymous. The MSP142 is a protein of approximately 195 kDa in size. The N-terminal region of PfMSP1-42 comprises of non-conserved (or dimorphic) amino acid sequences, and the C-terminal region of PfMSP142 comprises conserved sequences. The C-terminal conserved region of PfMSP142 (a 19 kDa fragment), is also known as MSP1-19. Limited variant-specific sequences are found at the C-terminal region of PSP1-42 (or MSP1-19) and these include the sequences EKNG, ETSR, and QKNG.

Malaria Vaccine: A malaria vaccine is a preparation used as a preventive innoculation against malaria or as a treatment for malaria including the malaria caused by the four species of the protozoal parasites of the genus Plasmodium that infect man, P. falciparum P. vivax, P. malariae and P. ovale The malaria vaccine of the present invention includes purified MSP142 or antigenic fragments thereof isolated from silkworms infected with a baculovirus vector containing all or part of the MSP142 sequence. The vaccine may further include adjuvants and/or various carriers as further described below.

Polypeptide: Polypeptide means any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation).

Substantially Pure Polypeptide: Substantially pure polypeptide means a MSP142 polypeptide which has been separated from components which naturally accompany it. Typically, the polypeptide is substantially pure when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight MSP142 polypeptide. A substantially pure MSP142 polypeptide may be obtained, for example, by extraction from a natural source (e.g., a plasmodium) by expression of a recombinant nucleic acid encoding a MSP142 polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

A protein is substantially free of naturally associated components when it is separated from those contaminants which accompany it in its natural state. Thus, a protein which is chemically synthesized or produced in a cellular system different from the cell from which it naturally originates will be substantially free from its naturally associated components. Accordingly, substantially pure polypeptides include, without limitation, those derived from eukaryotic organisms but synthesized in E. coli or other prokaryotes, or those derived from a eukaryotic cell. which does not normally synthesize such a protein, or those derived from a eukaryotic cell engineered to overexpress such a protein.

Immunogenic Fragment: Immunogenic fragments are peptide fragments of a least 5-8 amino acids of a protein such as MSP 142 wherein the fragaments are capable of inducing an immune response in a mammal such as a human. The treated mammal mounts an immune response resulting in the production of antibodies against the MSP 142 immunogenic fragments of which circulate in the mammal's blood stream

Recombinant Protein: A recombinant protein is a protein expressed in a non-native organism or cell. In the context of the present invention, recombinant MSP 142 is MSP142 expressed in a silkworm cell. Herein recombinant MSP1-42, recombinant MSP142 and Sp42 are synonymous.

Substantially Pure DNA: Substantially pure DNA means DNA that is free of the genes which, in the naturally-occurring genome of the organism from which the DNA of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or which exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.

Positioned for Expression: Positioned for expression means that the DNA molecule is positioned adjacent to a DNA sequence which directs transcription and translation of the sequence (i.e., facilitates the production of, e.g., a recombinant MSP142 polypeptide or RNA molecule).

Taking into account these definitions, the present invention is directed to a vaccine comprising purified MSP142. The MSP142 vaccine of the invention is produced by expressing MSP 142 in a baculovirus expression system in silkworms, purifying the MSP142 protein and preparing a vaccine containing the purified MSP142

1. Baculovirus Expression System

Baculoviruses are a diverse group of closed-circular double-stranded DNA viruses of the family Baculoviridae, which are found mostly in insects. The baculo portion of the name refers to the rod-shaped capsids of the virus particles. The Baculoviridae can be divided into two sub-families: the Eubaculovirinae (occluded baculoviruses) and the Nudibaculovirinae (non-occluded baculoviruses). The Eubaculovirinae produce crystalline proteinaceous structures called occlusion bodies, which are absent in the Nudibaculovirinae. The Eubaculovirinae subfamily is made up of two genera: granulosis viruses (GVs) and nuclear polyhedrosis viruses (NPVs). The virus used in the present invention, the Bombyx mori Nuclear Polyhedrosis Virus (BmNPV), is a NPV.

There are a number of advantages in using the Bombyx mori Nuclear Polyhedrosis Virus (BmNPV) baculovirus as an expression vector. Firstly, being eukaryotic origin, the insect cells can correctly fold and modify the expressed foreign proteins with their biological activities retained. Secondly, the double-stranded DNA genome of the virus can be easily manipulated by general molecular biology techniques and the rod-shaped viral capsid is rather "flexible" in accommodating large DNA insert. Thirdly, the recombinant baculoviruses cannot survive in the environment owing to the lack of protection by occlusion bodies, thus posing less biohazards. Fourth, in contrast to the Autographa californica Nuclear Polyhedrosis Virus (AcNPV) expression system which is limited to cell culture and is expensive, the BmNPV system used in the present invention can be adopted to produce recombinant proteins in silkworm larvae in large quantity and can be produced at a fraction of the cost of using cell culture. Fifth, BmNPV has a narrow host range and is therefore biologically safer to use. Because of these advantages, the BmNPV baculovirus expression system of the present invention is an ideal system for the expression of MSP-142 protein.

2. Cloning and Expression of MSP 142.

Insect cells are the preferred hosts for the baculovirus vectors of the invention. In the present invention, the preferred host is silkworm Bombyx mori cells.

The large size of the BmNPV genomic DNA (.about.130 kbp) makes it sensitive to minor mechanical damage such as shearing during pipetting. Because of this, direct insertion of PfMSP-142 DNA into the viral genome by conventional cloning techniques is not possible. In order to insert the PfMSP-142 DNA into BmNPV, an indirect cloning method was employed as detailed in the Examples below. The PCR product of PfMSP-142 was first cloned into a BmNPV-based transfer vector, pBM030, to generate the recombinant pBM030-PfMSP-142 transfer vector. Recombinant BmNPVs were then generated by cotransfecting BmN cells with the purified BmNPV genomic DNA and the recombinant transfer vector. By homologous recombination, the cloned PfMSP-142 DNA sequence was inserted into the viral genome. A total of three recombinant BmNPVs were produced by this method as described in the Examples below. Each of the recombinant BmNPVs carries a different form of PfMSP-142, namely a secretory form (the sp42 construct) with a honeybee melittin signal peptide, an intracellular form with a hexa-histidine tag fused to the N-terminal (the hp42 construct), and the original unmodified intracellular form (the p42 construct).

3. Purification of MSP 142

Methods for purifying recombinant MSP 142 are well known and can be generally divided into chromatographic methods, for example, ion exchange chromatography, molecular weight sieving, high pressure liquid chromatography, affinity chromatography, antibody affinity chromatography and electrophoretic methods, e.g., electrophoresis on agarose or acrylamide gels and isoelectric focusing. Any of these methods can be adapted to purify MSP 142.

A preferred method of purifying MSP142 is immuno affinity chromatography. In immunoaffinity chromatography, an antibody to MSP 142 is immobilized on a chromatographic substrate, a mixture containing MSP 142 is applied to the substrate under conditions allowing the antibody to bind MSP 142, the unbound material is removed by washing, and the bound MSP 142 is eluted using, for example, high or low pH, protein denaturants or chaotropes.

For example, MSP 142 may be purified by affinity chromatography using one or a combination of immobilized antibodies such as those described below covalently bound to agarose beads or bound non-covalently via a goat-anti mouse IgM antibody to Staphylococcus aureaus protein G beads. MSP 142 isolation can also be achieved, for example, by incubating cell extracts with anti-MSP 142 antibodies, described below, attached to a solid phase, such as chemical conjugation to agarose beads.

4. Vaccine Preparation

To prepare vaccines, purified MSP 142 or immunogenic fragments of MSP 142 are formulated and packaged using methods and materials known to those skilled in the art of vaccines, examples of which are described below. As used herein, an immunogenic fragment of a protein is a protein fragment of at least five to eight amino acids that elicits an immune response in an animal or individual.

a. Adjuvants

Adjuvants may, optionally, be employed and are preferred in some embodiments. Vaccines can be combined with an adjuvant, in an amount effective to enhance the immune response. Various adjuvants which find use in the invention include those described at http://www.niaid.nih.gov/aidsvaccine/pdf/compendium.pdf which is hereby incorporated by reference. In addition, a common adjuvant widely used in humans is alum, that is, aluminum phosphate or aluminum hydroxide. Saponin and its purified component Quil A, Freund's complete adjuvant and other adjuvants used in research and veterinary applications have toxicities which limit their potential use in human vaccines. Chemically defined preparations such as muramyl dipeptide, monophosphoryl lipid A, and phospholipid conjugates such as those described by Goodman-Snitkoff, et al., J. Immunol. 147: 410-415 (1991) which is hereby incorporated by reference, can also be used.

Further adjuvants include Adjumer.TM.; PCPP salt; polyphophazene; polyidi(carboxylatophenoxy)lphosphazene; Adju-Phos; Aluminum phosphate gel; .beta.-glucan; glucan; Gamma inulin/alum composite adjuvant; aluminum hydroxide gel; alum; N,N-dioctadecyl-N1,N1 -bis(2-hydroexyethyl) propanediamine; N-(2-Deoxy-2-L-leucylamino-.beta.-D-glucopyranosyl)-N-octadecyldodecanoyla mide; Calcitriol; 25-dihydroxyvitamin D3; 1,25-di(OH)2 D3 ; 1,25-DHCC; 1.alpha., 25-dihdroxycholecalciferol; 9,10-seco(5Z,7E)-5,7,10(19)-cholestatriene-1.alpha.,3.beta.,25-triol; Block Copolymer P1205; Cytokine-containing Liposomes; Cytokine-containing Dehydration Rehydration Vesicles; Dimethyl dioctadecylammonium bromide; demethyl distearylammonium bromide; Dehydroepiandrosterone; 5-androsten-3.beta.-o1-17-one; dehydroisoandrosterone; androstenolone; prasterone; transdehydroandrosterone; Dimyristoyl phosphatidyl choline; sn-3-phosphatidyl choline-1,2-dimyristoyl; 1,2-dimyristoyl-sn-3-phosphatidyl choline; Dimyristoyl pposphatidylglcerol; sn-3-phosphatidyl glycerol-1,2-dimyristoyl, sodium salt; 1,2-dimyritoyl-sn-3-phosphatidyl glycerol; Deoxycholic Acid Sodium Salt; Gamma Inulin; Interleukin-1.beta.; IL-10; IL-1; human Interleukin 1.beta. mature polypeptide Interferon-.gamma.; Immunoliposomes Containing Antibodies to Costimulatory Molecules; ImmTher.TM.; N-acetylglucosaminyl-N-acetyhnuramyl-L-Ala-D-isoGlu-L-Ala-Glycerol dipalmitate; Imiquimod; 1-(2-methypropyl)-1H-imidazo[4,5-c]quinolin-4-amine; GMDP; N-acetylglucosaminyl-(.beta.1-4)-N-acetylmuramyl-L-alanyl-D-isoglutamine; Gerbu Adjuvant; Interleukin-2; IL-2; T-cell growth factor; aldesleukin (des-alanyl-1, serine-125 human interleukin 2); Proleukin.RTM.; Teceleukin.RTM.; Interleukin-7; IL-7; Interleukin-12; IL-12; natural killer cell stimulatory factor (NKSF); cytotoxic lymphocyte maturation factor (CLMF); ISCOM(s).TM.; Immune stimulating complexes; Iscoprep 7.0.3..TM.; Liposomes; Liposomes (L) containing protein or Th-Cell and/or B-cell peptides, or microbes with or without co-entrapped interleukin-2, BisHOP or DOTMA; Loxoribine; 7-allyl-8-oxoguanosine; LT-OA or LT Oral Adjuvant; E. coli labile enteroxtoxin protoxin; MONTANIDE ISA 720; metabolizable oil adjuvant; MPL.TM.; 3-Q-dsacyl-4'-monophosphoryl lipid A; 3D-MLA; MF59; MTP-PE; N-acetyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1,2-dipalmitoyl-sn-glcero-3 -(hydroxy-phosphoryloxy)) ethylamide, mono sodium salt; MTP-PE Liposomes; MTP-PE Antigen presenting liposomes; Murametide; Nac-Mur-L-Ala-D-Gln-OCH3; Murapalmitine; Nac-Mur-L-Thr-D-isoGln-sn-glycerol dipalmitoyl; D-Murapalmitine; Nac-Mur-D-Ala-D-isoGln-sn-glcerol dipalmitoyl; NAGO; Neuraminidase-galactose oxidase; Non-Ionic Surfactant Vesicles; NISV.; Pleuran; PLGA, PGA, and PLA; Homo-and co-polymers of lactic and glycolic acid; Lactide/glycolide polymers; poly-lactic-co-glycolide; Pluronic L121; Poloxamer 401; PMMA; Polymethyl methacrylate; PODDS.TM.; Proteinoid microspheres; Poly rA; Poly rU; Poly-adenylic acid-poly-uridylic acid complex; Polysorbate 80; Tween 80; Sorbitan mono-9-octadecenoate poly(oxy-1,2-ethanediyl) derivatives; Protein Cochleates; QS-21; Stimulon.TM. QS-21 Adjuvant; Rehydragel HPA; High Protein Adsobency Aluminum Hydroxide Gel; alum; Rehydragel LV; low viscosity alluminum hydroxide gel; alum; S-28463; 4-Amino-otec,-dimethyl-2-ethoxmethyl-1H-imidazo[4,5-c]quinoline-1-ethanol; SAF-1; Scalvo peptide; IL-1.beta. 163-171 peptide; Span 85; Arlacel 85, sorbitan trioleate; Specol; Marcol 52 (mineral oil, paraffins, and cycloparaffins, chain length 13-22 C atoms) Span 85 (emulsifier, sorbitan trioleate) Tween 85 (emulsfier, polyoxyethylene-20-trioleate); Squalane; Spinacane; Robane.RTM.; 2,6,10,15,19,23-hexamethyltetracosane; Squalene; Spinacene; Supraene; 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22 tetracosahexaene; Stearyl Tyrosine; Octadecyl tyrosine hydrochloride; Theramide.TM.; N-acetylglucosaminyl-N-acetylinuramyl-L-Ala-D-isoGlu-L-Ala-dipalmitoxy propylmadie (DTP-DPP); Threonyl-MDP; Termurtide.TM.; [thr1 ]-MDP;N-acetyl muramyl-L-threonyl-D-isoglutamine; Ty Particles; Ty-VLPs, (Virus Like Particles)

For oral administration, it is known that an admixture of trace amounts of cholera toxin (CT), either cholera toxin subunit A, cholera toxin subunit B, or both, and a second antigen stimulate a mucosal immunity to the co-administered antigen. Furthermore, there is a dramatic humoral immune response to the second antigen instead of the immune tolerance that is elicited by oral delivery of the antigen alone. Thus, mucosally delivered CT functions as a powerful immunostimulant or adjuvant of both mucosal and humoral immunity. It is therefore preferred to enhance immunogenicity of the orally administered antigen by including CT in the vaccine.

For parenteral administration, adjuvants include muramyl dipeptides, muramyl tripeptide, cytokines, diphtheria toxoid, and exotoxin A. Commercially available adjuvants include QS-21 from Cambridge Biosciences, Worcester, Mass., and monophosphoryl lipid A (MPLA) from Ribi Immunochem.

A group of growth factors termed colony stimulating factors which support survival, clorial expansion, and differentiation of hematopoietic progenitor cells are also useful as adjuvants. Granulocyte-macrophage colony stimulating factor (GM-CSF) belongs to this group and induces partially committed progenitor cells to divide and differentiate in the granulocyte-macrophage pathways.

The commercially available GM-CSF from the Immunex Corporation is provided as a sterile, white, preservative-free, lyophilized powder and is intended for intravenous infusion following reconstitution with 1 ml sterile water for injection, USP and is know as LEUKINE. The pH of the reconstituted, isotonic solution is 7.4+0.3. When used as an adjuvant, LEUKINE may be reconstituted with sterile water or MSP142 with the vaccine preparation of this invention. If reconstituted with water then LEUKINE is administered by intramuscular injection at the same site as immunization with the MSP142 vaccine or is first mixed with the MSP142 vaccine preparation. The vaccine/GM-CSF mixture obtained by either reconstituting the GM-CSF with the MSP142 vaccine preparation directly or by mixing the water reconstituted GM-CSF with the MSP142 vaccine is then administered by intramuscular, subcutaneous or intradermal injection.

b. Carriers

Numerous carriers for administration of MSP 142 vaccine compounds are known. These include, but are not limited to, simple liquid carriers, and polymeric and lipid compositions. Simple liquid carriers, such as water or a buffered saline, can be used, either alone or in combination with other carriers.

The carrier may also be a polymeric delayed-release system. Synthetic polymers are particularly useful in the formulation of a vaccine to effect the controlled release of antigens. An example of this is described by Kreuter, Microcapsules and Nanoparticles in Medicine and Pharmacology, pages 125-148 (M. Donbrow, ed., CRC Press) which is incorporated herein by reference. The use of other particles have demonstrated that the adjuvant effect of these polymers depends on particle size and hydrophobicity.

Microencapsulation has been applied to the injection of microencapsulated pharmaceuticals to give a controlled release. A number of factors contribute to the selection of a particular polymer for microencapsulation. The reproducibility of polymer synthesis and the microencapsulation process, the cost of the microencapsulation materials and process, the toxicological profile, the requirements for variable release kinetics and the physicochemical compatibility of the polymer and the antigens are all factors that must be considered. Examples of useful polymers are polycarbonates, polyesters, polyurethanes, polyorthoesters, and polyamides, particularly those that are biodegradable.

A frequent choice of a carrier for pharmaceuticals and more recently for antigens is poly (d,1-lactide-co-glyco-lide) (PLGA). This is a biodegradable polyester that has a long history of medical use in erodible sutures, bone plates and other temporary prostheses, where it has exhibited no toxicity. A wide variety of pharmaceuticals including peptides and antigens have been formulated into PLGA microcapsules. A body of data has accumulated on the adaptation of PLGA for the controlled release of antigen, for example, as reviewed by Eldridge et al., Current Topics in Microbiology and Immunology 146: 59-66 (1989) which is hereby incorporated by reference. The PLGA microencapsulation process uses a phase separation of a water-in-oil emulsion. In this process, the MSP 142 vaccine is prepared as an aqueous solution and the PLGA is dissolved in a suitable organic solvent such as methylene chloride and ethyl acetate. These two immiscible solutions are co-emulsified by high-speed stirring. A non-solvent for the polymer is then added, causing precipitation of the polymer around the aqueous droplets to form embryonic microcapsules. The microcapsules are collected, and stabilized with one of an assortment of agents (polyvinyl alcohol (PVA), gelatin, alginates, polyvinylpyrrolidone (PVP), methyl cellulose) and the solvent removed by either drying in vacuo or solvent extraction.

Proteosomes, combinations of protein and liposomes, can also be used as carriers for combination vaccines, using the MSP 142 as the protein component. The procedures and materials for the use of proteosomes are as described in Lowell et al., Science 240: 800 (1988); Lowell, in New Generation Vaccines (Woodrow and Levine, eds., Marcel Dekker, N.Y., 1990), Ch. 12, pages 141-160; and Orr et al., Infect. Immun. 61: 2390 (1993) which are hereby incorporated by reference.

It will be understood by those skilled in the art that the immunogenic MSP 142 vaccine composition can contain other physiologically acceptable ingredients such as water, saline or a mineral oil such as Drakeol.TM., Markol.TM., and squalene, to form an emulsion, or in combination with aqueous buffers, or encapsulated within a capsule or enteric coating to protect the protein from degradation while passing through the stomach.

5. Vaccine Administration

In a preferred embodiment, the MSP 142 vaccine is packaged in a single dosage for immunization by parenteral, that is, intramuscular, intradermal or subcutaneous, administration; or nasopharyngeal, that is, intranasal, administration. The effective dosage is determined using standard techniques, such as antibody titer. The antigen may be lyophilized for resuspension at the time of administration or in solution. If administered with adjuvant, the adjuvant may be administered in combination with or in the vicinity of the MSP 142 vaccine.

Immunity is measured using assays to detect and quantitate antibodies that bind to the MSP 142. Cellular immunity is measured using assays that measure specific T-cell responses such as delayed type hypersensitivity (DTH) and lymphocyte proliferation. The dosage is determined by the antigen loading and by standard techniques for determining dosage and schedules for administration for each antigen, based on titer of antibody elicited by the antigen administration. As used herein, a dose effective to elicit an immune response is considered to be one that causes antibody titer to increase compared to untreated animals or individuals, using any of the known methods of titering antibodies.

Circulating antibodies to recombinant MSP 142 are detected by enzyme immunoassay using recombinant MSP 142 as antigen. Such assays are described in. detail in the Examples below.

Claim 1 of 10 Claims

We claim:

1. A method of producing a malaria immunogenic composition, comprising:

(a) expressing an immunogenic fragment of MSP 142 in a baculovirus expression system, wherein said expression system comprises one or more silkworms infected with a nuclear polyhedrosis virus, and wherein said silkworms comprise hemolymph;

(b) collecting said hemolymph from said one or more silkworms and combining said hemolymph with a reducing agent;

(c) purifying said immunogenic fragment from said hemolymph; and

(d) formulating said immunogenic fragment in a malaria immunogenic composition.



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