Internet for Pharmaceutical and Biotech Communities
| Newsletter | Advertising |
 
 
 

  

Pharm/Biotech
Resources

Outsourcing Guide

Cont. Education

Software/Reports

Training Courses

Web Seminars

Jobs

Buyer's Guide

Home Page

Pharm Patents /
Licensing

Pharm News

Federal Register

Pharm Stocks

FDA Links

FDA Warning Letters

FDA Doc/cGMP

Pharm/Biotech Events

Consultants

Advertiser Info

Newsletter Subscription

Web Links

Suggestions

Site Map
 

 
   

 

  Pharmaceutical Patents  

 

Title:  Recombinant P. falciparum merozoite protein-1.sub.42 vaccine
United States Patent: 
7,563,883
Issued: 
July 21, 2009

Inventors:
 Angov; Evelina (Bethesda, MD), Lyon; Jeffrey A. (Silver Spring, MD), Darko; Christian Asare (Silver Spring, MD), Cohen; Joe D. (Brussels, BE)
Assignee: 
The United States of America as represented by the Secretary of the Army (Washington, DC)
Appl. No.:
 11/889,578
Filed: 
August 14, 2007


 

Patheon


Abstract

In this application is described the expression and purification of a recombinant Plasmodium falciparum (FVO) MSP-1.sub.42. The method of the present invention produces a highly purified protein that retains folding and disulfide bridging of the native molecule. The recombinant MSP-1.sub.42 is useful as a diagnostic reagent, for use in antibody production, and as a vaccine.

Description of the Invention

SUMMARY OF THE INVENTION

The present invention satisfies the needs discussed above. The present invention provides a vaccine grade E. coli expressed recombinant MSP-1.sub.42 (FVO) that is properly folded. Characterization of the MSP-1.sub.42 by immunoblotting with mAbs specific for conformation-dependent epitopes show that the protein contains properly formed epitopes on the highly structured MSP-1.sub.19 portion. The purified protein was immunogenic in mice, rabbits and in Aotus monkeys. Rabbit anti-sera raised against the protein induced antibodies that inhibited P. falciparum growth in vitro. Aotus monkeys were protected against an experimental erythrocytic stage P. falciparum FVO strain challenge.

Therefore, a major aim of the present invention resides in the production of large amounts of MSP-1.sub.42 that maintain conformational epitopes critical to epitope formation in pure form (>95% pure) for diagnostic, prophylactic and therapeutic purposes.

This may not seem complicated but, as with most strategies for protein purification, proved to be difficult and unpredictable. E. coli was chosen as a host, even though it had gone out of favor, for two reasons: (1) E. coli was known to produce high level of recombinant proteins and (2) recombinant proteins produced in E. coli are not glycosylated, which is consistent with the capabilities of malaria parasites. Several hurdles had to be overcome to achieve the desired expression level as soluble cytoplasmic form that can be sufficiently purified from host cell proteins without sacrificing proper folding of the protein. Problems with E. coli endotoxin levels and the presence of non-MSP-1.sub.42 contaminants had to be resolved.

The P. falciparum FVO genomic DNA extending from amino acid 1349 to amino acid 1713 of the full length P. falciparum FVO MSP-1 (Miller, et al, 1993, Mol. Biochem. Parasitol., 59, 1-14) was amplified using primers containing restriction sites compatible with cloning into the expression construct pET(AT)PfMSP-1.sub.42(3D7) (FIG. 1, see Original Patent), which contains an MSP1.sub.42 sequence from a P. falciparum 3D7 allele. The 3D7 allele was removed and the FVO allele was inserted into the expression vector using appropriate restriction sites. The sequenced clones were found to be identical in this region to Genbank Accession number, L20092. This construct was found to express inadequate amounts of the protein for use as a vaccine. In order to increase the expression of the protein, a single synonymous codon change at amino acid position #158, from ATC to ATA. (codon numbering starts from the ATG, initiation codon of the transcript) was introduced by PCR. The expressed protein is identical to the native amino acid for P. falciparum (FVO) MSP-1.sub.42. The substitution was theorized to improve proper translation and folding of the protein, and hence its expression because it harmonized codon usage rates for this codon between E. coli and F. falciparum. Indeed, expression of the protein improved sufficiently to allow the use of the protein for a vaccine. The sequence of the final expressed FVO fragment is set forth in SEQ ID NO:1, with the sequence of the expressed protein in SEQ ID NO:2. Additional improvements to protein expression were achieved by further harmonizing codon frequencies between E. coli and P. falciparum. SEQ ID NO:3 sets forth the sequence of the FVO MSP1-42 gene with harmonized 5' 100 nucleotides, N-mod. SEQ ID NO:4 sets forth the complete MSP1.sub.42 harmonized throughout the complete gene.

The E. coli expressed MSP-1.sub.42 fragment is comprised from amino acid 1349 (Ala) to amino acid 1713 (Ser) from the full length P. falciparum FVO MSP-1 [Miller, 1993, supra]. The MSP-1.sub.42 DNA used to prepare this clone was produced by PCR amplification of P. falciparum FVO genomic DNA; and the fragment was subcloned into the expression vector, pET(AT)PfMSP-1.sub.42 (3D7), that was previously described for the expression of the MSP-1.sub.42 3D7 allele, (Angov et. al. (2003) Molec. Biochem. Parasitol; in press) (FIG. 1A, see Original Patent). The final product contains 17 non-MSP-1.sub.42 amino acids that include the hexa-histidine tag for Ni.sup.+2 chelating chromatography at the N-terminus (FIG. 1B, see Original Patent). Soluble expression of MSP-1.sub.42 was induced by addition of 0.1 mM IPTG. MSP-1.sub.42 was purified under GMP conditions using a two-step chromatographic method; that included a Ni.sup.+2-NTA Sepharose affinity resin followed by a Q-Sepharose ion exchanger. The purified bulk was concentrated and buffer exchanged using ultrafiltration. The final protein was purified to greater than 95% of MSP-1. Vaccination of rabbits with the purified MSP-1.sub.42 elicited neutralizing antibodies.

Therefore, it is an object of the present invention to provide a recombinant P. falciparum FVO MSP-1.sub.42 for use in diagnostic assays and for production of antibodies.

It is another object of the present invention to provide compositions comprising purified recombinant P. falciparum FVO MSP-1.sub.42.

It is yet another object of the present invention to provide novel vector constructs for recombinantly expressing P. falciparum MSP-1.sub.42, as well as host cells transformed with said vector.

It is also an object of the present invention to provide a method for producing and purifying recombinant P. falciparum FVO MSP-1.sub.42 protein comprising:

growing a host cell containing a vector expressing P. falciparum FVO MSP-1.sub.42 proteins in a suitable culture medium,

causing expression of said vector sequence as defined above under suitable conditions for production of soluble protein and,

lysing said transformed host cells and recovering said MSP-1.sub.42 protein such that it retains its native folding and is essentially free of host toxins.

It is also an object of the present invention to provide diagnostic and immunogenic uses of the recombinant P. falciparum FVO MSP-1.sub.42 protein of the present invention, as well as to provide kits for diagnostic use for example in malaria screening and confirmatory antibody tests.

It is also an object of the present invention to provide monoclonal or polyclonal antibodies, more particularly human monoclonal antibodies or mouse monoclonal antibodies which are humanized which react specifically with MSP-1.sub.42 epitopes, either comprised in peptides or conformational epitopes comprised in recombinant proteins.

It is also an object of the present invention to provide possible uses of anti-MSP-1.sub.42 monoclonal antibodies for malaria antigen detection or for therapy of chronic malaria infection.

It is yet another object of the present invention to provide a malaria vaccine comprising MSP-1.sub.42 of the present invention, in an amount effective to elicit an immune response in an animal against P. falciparum; and a pharmaceutically acceptable diluent, carrier, or excipient.

It is another object of the present invention to provide a method for eliciting in a subject an immune response against malaria, the method comprising administering to a subject a composition comprising MSP-1.sub.42 of the present invention. In one aspect of the invention, the DNA vaccine is delivered along with an adjuvant, for example ADJUVANT B.

It is another object of the present invention to provide a method for preventing malaria infection in an animal comprising administering to the animal the MSP-1.sub.42 of the present invention.

The vaccine according to the present invention is inherently safe, is not painful to administer, and should not result in adverse side effects to the vaccinated individual.

DETAILED DESCRIPTION

The P. falciparum antigen of the present invention can be made by any recombinant method that provides the epitope of interest. For example, recombinant expression in E. coli is a preferred method to provide non-glycosylated antigens in `native` conformation. This is most desirable because natural P. falciparum antigens are not glycosylated. Proteins secreted from mammalian cells may contain modifications including galactose or sialic acids that may be undesirable for certain diagnostic or vaccine applications. However, it may also be possible and sufficient for certain applications, as it is known for proteins, to express the antigen in other recombinant hosts such as baculovirus and yeast or higher eukaryotes, as long as glycosylation is inhibited.

The proteins according to the present invention may be secreted or expressed within compartments of the cell. Preferably, however, the proteins of the present invention are expressed within the cell and are released upon lysing the cells.

It is also understood that the isolates used in the examples section of the present invention were not intended to limit the scope of the invention and that an equivalent sequence from a P. falciparum isolate from another allele, e.g. 3D7, or CAMP, can be used to produce a recombinant MSP-1.sub.42 protein using the methods described in the present application. Other new strains of Plasmodium may be a suitable source of MSP-1.sub.42 sequence for the practice of the present invention.

The MSP-1.sub.42 protein of the present invention is expressed as part of a recombinant vector. The present invention relates more particularly to the MSP-1.sub.42 nucleic acid sequence in recombinant nucleic acid vector pET(AT)FVO as represented in SEQ ID NO:1, 3 or 4, or parts thereof. The MSP-1.sub.42 (FVO) insert DNA was PCR amplified from P. falciparum (FVO) strain genomic DNA and ligated into the pET(AT)PfMSP-1.sub.42(3D7) (modified from vector pET32a from Novagen (Madison, Wis.)). This plasmid comprises, in sequence, a T7 promoter, optionally a lac operator, a ribosome-binding site, restriction sites to allow insertion of the structural gene and a T7 terminator sequence. Other vectors described include pET(K)FVO.B "initiation complex" (FIG. 1D, see Original Patent) harmonized and pET(K)FVO.C [Full gene harmonized](FIG. 1E, see Original Patent) wherein the ampicillin and tetracycline antibiotic resistance genes have been replaced with a kanamycin resistance gene. Examples of other plasmids which contain the T7 inducible promoter include the expression plasmids pET-17b, pET-11a, pET-24a-d(+), and pEt-9a, all from Novagen (Madison, Wis.); see the Novagen catalogue.

The present invention also contemplates host cells transformed with a recombinant vector as defined above. In a preferred embodiment, E. coli strain BL21(DE3) (F-ompT hsdSB(rB-mB-) gal dcm (DE3)) is employed. The above plasmids may be transformed into this strain or other strains of E. coli having the following characteristics: a T7 RNA polymerase gene, Lon, ompT protease mutants or any other E. coli with a protease deficiency such as E. coli B834 DE3, Origami DE3. Preferably, the host includes BL21 (DE3) and any of its precursors. Other host cells such as insect cells can be used taking into account that other cells may result in lower levels of expression.

Eukaryotic hosts include lower and higher eukaryotic hosts as described in the definitions section. Lower eukaryotic hosts include yeast cells well known in the art. Higher eukaryotic hosts mainly include mammalian cell lines known in the art and include many immortalized cell lines available from the ATCC, inluding HeLa cells, Chinese hamster ovary (CHO) cells, Baby hamster kidney (BHK) cells, PK15, RK13 and a number of other cell lines. MSP-1.sub.42 expressed in these cells will be glycosylated unless the cells have been altered such that glycosylation of the recombinant protein is not possible. It is expected that when producing MSP-1.sub.42 in a eukaryotic expression system, extensive investigation into methods for expressing, isolating, purifying, and characterizing the protein would be required as eukaryotic cells post-translationally modify this protein and this would alter protein structure and immunogenicity.

Methods for introducing vectors into cells are known in the art. Please see e.g., Maniatis, Fitsch and Sambrook, Molecular Cloning; A Laboratory Manual (1982) or DNA Cloning, Volumes I and II (D. N. Glover ed. 1985) for general cloning methods. Host cells provided by this invention include E. coli containing pET(AT)FVO.A [single pause site], E. coli containing pET(K)FVO.B [initiation complex harmonized] and E. coli containing pET(K)FVO.C[Full gene harmonized].

A preferred method for isolating or purifying MSP-1.sub.42 as defined above is further characterized as comprising at least the following steps:

(i) growing a host cell as defined above transformed with a recombinant vector expressing MSP-1.sub.42 proteins in a suitable culture medium,

(ii) causing expression of said vector sequence as defined above under suitable conditions for production of a soluble protein,

(iii) lysing said transformed host cells and recovering said MSP-1.sub.42 protein such that it retains its native conformation and is essentially pure.

Once the host has been transformed with the vector, the transformed cells are grown in culture in the presence of the desired antibiotic. For FDA regulatory purposes, it is preferable to use tetracycline or kanamycin. When cells reach optimal biomass density, in this case about 0.4-0.6 OD.sub.600 in small culture flasks or 5-7 OD.sub.600 in bulk fermentors, the cells are induced to produce the recombinant protein. The inventors have found after trial and error that for expression of a soluble MSP-1.sub.42, it was necessary to cool the culture to a range of about 10.degree. C.-20.degree. C., more preferably about 15.degree. C.-28.degree. C., most preferably about 24 to 26.degree. C. prior to induction. The concentration of inducer, i.e. IPTG, added affects the maximal protein synthesis. It was found that a concentration of 0.1 mM IPTG was best, however, a range of 0.05 to 0.5 mM would be sufficient to produce 80-100% of maximal.

The cells were then collected and lysed to release the recombinant protein. Preferably, lysis should occur at a paste to buffer ratio of 1:3 w/v to reduce viscosity and volume of sample loaded on Ni-NTA column. Preferably, lysis is in the presence of imidazole that reduces non-specific binding of E. coli proteins to Ni resin, and benzonase that is able to digest E. coli nucleic acids. Lysis is preferably at a temperature of about 0.degree. C.-24.degree. C., more preferably about 5-15.degree. C. in order to retain native folding of the MSP-1.sub.42 protein and to reduce proteolysis. A high salt concentration of about 0.5-1.0 M is preferable during extraction procedures. Salts used include NaCl or other monovalent ions.

Preferably, the E. coli endotoxin is separated and removed from the recombinant protein. This can be done several ways. For MSP-1.sub.42, endotoxin was removed by applying to a Ni.sup.+2-NTA column. The removal of endotoxin depended on washing in high salt, about 0.5 to about 1.5 M, preferably about 1000 mM NaCl at a flow rate of about 20 to about 35 ml/min, preferably about 30 ml/min. The cell paste to resin ratio can be about 5:1 to about 20:1 w/v, preferably about 12:1 w/v. The recombinant protein can be eluted by addition of high concentration of imidazole, 500 to 1500 mM, preferably 1000 mM at pH 8.0, in a phosphate buffer of about 5-25 mM, more preferably about 10 mM sodium phosphate buffer.

At this point the recombinant protein is about 50% pure. If further purity is required, ion-exchange chromatography can be utilized. The column is preferably with an ionic character such that a pH to enhance protein binding. Reducing the buffer pH to 7.2 and increasing the salt to 250 mM elutes the protein from the resin. Under these conditions, the endotoxin and nucleic acid remain bound to the resin and are therefore removed from the protein.

The present invention further relates to a composition comprising at least one of the following:

MSP-1.sub.42 alone (SEQ ID NO: 1, 3, and 4) spanning amino acids to 1349-1713 of MSP-1,

MSP-1.sub.42 plus 17 amino acids at N-terminal in final expression vector construct pET(AT)FVO.A, the final expressed product referred to as FMP003. The peptide sequence contains additional nonMSP1 amino acids, i.e. MAHHHHHHPGGSGSGTM (SEQ ID NO:6 containing His6Tag and nonMSP1 nucleotide linker sequence.

The present invention also relates to a composition-comprising peptides or polypeptides as described above, for in vitro detection of malaria antibodies present in a biological sample.

The present invention also relates to a composition comprising at least one of the following MSP-1.sub.42 conformational epitopes:

epitope recognized by monoclonal antibodies 12.10, 12.8, 7.5, 2.2, 1E1 (Blackman et al., 1990, supra; Conway et al., 1991, Parasitology 103, 1-6; McBride et al., 1982, Science 217, 254-257; Mackay et al., 1985, Embo J. 4, 3823-3829).

epitope recognized by monoclonal antibody 5.2 (Chang et al., 1988, Exp. Parasitol. 67, 1-11).

The present invention also relates to an MSP-1.sub.42 specific antibody raised upon immunizing an animal with a peptide or protein composition, with said antibody being specifically reactive with any of the polypeptides or peptides as defined above, and with said antibody being preferably a monoclonal antibody.

The present invention also relates to an MSP-1.sub.42 specific antibody screened from a variable chain library in plasmids or phages or from a population of human B-cells by means of a process known in the art, with said antibody being reactive with any of the polypeptides or peptides as defined above, and with said antibody being preferably a monoclonal antibody.

The MSP-1.sub.42 specific monoclonal antibodies of the invention can be produced by any hybridoma liable to be formed according to classical methods from splenic or lymph node cells of an animal, particularly from a mouse or rat, immunized against the Plasmodium polypeptides or peptides according to the invention, as defined above on the one hand, and of cells of a myeloma cell line on the other hand, and to be selected by the ability of the hybridoma to produce the monoclonal antibodies recognizing the polypeptides which has been initially used for the immunization of the animals.

The antibodies involved in the invention can be labelled by an appropriate label of the enzymatic, fluorescent, or radioactive type.

The monoclonal antibodies according to this preferred embodiment of the invention may be humanized versions of mouse monoclonal antibodies made by means of recombinant DNA technology, departing from parts of mouse and/or human genomic DNA sequences coding for H and L chains from cDNA or genomic clones coding for H and L chains.

Alternatively the monoclonal antibodies according to this preferred embodiment of the invention may be human monoclonal antibodies. These antibodies according to the present embodiment of the invention can also be derived from human peripheral blood lymphocytes of patients infected with malaria, or vaccinated against malaria. Such human monoclonal antibodies are prepared, for instance, by means of human peripheral blood lymphocytes (PBL) repopulation of severe combined immune deficiency (SCID) mice, or by means of transgenic mice in which human immunoglobulin genes have been used to replace the mouse genes.

The invention also relates to the use of the proteins or peptides of the invention, for the selection of recombinant antibodies by the process of repertoire cloning.

Antibodies directed to peptides or single or specific proteins derived from a certain strain may be used as a medicament, more particularly for incorporation into an immunoassay for the detection of Plasmodium strains for detecting the presence of MSP-1.sub.42 antigens, or antigens containing MSP-1.sub.42 epitopes, for prognosing/monitoring of malaria disease, or as therapeutic agents.

Alternatively, the present invention also relates to the use of ant of the above-specified MSP-1.sub.42 monoclonal antibodies for the preparation of an immunoassay kit for detecting the presence of MSP-1.sub.42 antigen or antigens containing MSP-1.sub.42 epitopes in a biological sample, for the preparation of a kit for prognosing/monitoring of malaria disease or for the preparation of a malaria medicament.

The present invention also relates to a method for in vitro diagnosis or detection of malaria antigen present in a biological sample, comprising at least the following steps:

(i) contacting said biological sample with any of the MSP-1.sub.42 specific monoclonal antibodies as defined above, preferably in an immobilized form under appropriate conditions which allow the formation of an immune complex,

(ii) removing unbound components,

(iii) incubating the immune complexes formed with heterologous antibodies, which specifically bind to the antibodies present in the sample to be analyzed, with said heterologous antibodies conjugated to a detectable label under appropriate conditions,

(iv) detecting the presence of said immune complexes visually or mechanically (e.g. by means of densitometry, fluorimetry, colorimetry).

The present invention also relates to a kit for in vitro diagnosis of a malaria antigen present in a biological sample, comprising:

at least one monoclonal antibody as defined above, with said antibody being preferentially immobilized on a solid substrate,

a buffer or components necessary for producing the buffer enabling binding reaction between these antibodies and the malaria antigens present in the biological sample, and

a means for detecting the immune complexes formed in the preceding binding reaction.

The kit can possibly also include an automated scanning and interpretation device for inferring the malaria antigens present in the sample from the observed binding pattern.

Monoclonal antibodies according to the present invention are suitable both as therapeutic and prophylactic agents for treating or preventing malaria infection in susceptible malaria-infected subjects. Subjects include rodents such as mice or guinea pigs, monkeys, and other mammals, including humans.

In general, this will comprise administering a therapeutically or prophylactically effective amount of one or more monoclonal antibodies of the present invention to a susceptible subject or one exhibiting malaria infection. Any active form of the antibody can be administered, including Fab and F(ab').sub.2 fragments. Antibodies of the present invention can be produced in any system, including insect cells, baculovirus expression systems, chickens, rabbits, goats, cows, or plants such as tomato, potato, banana or strawberry. Methods for the production of antibodies in these systems are known to a person with ordinary skill in the art. Preferably, the antibodies used are compatible with the recipient species such that the immune response to the MAbs does not result in clearance of the MAbs before parasite can be controlled, and the induced immune response to the MAbs in the subject does not induce "serum sickness" in the subject. Preferably, the MAbs administered exhibit some secondary functions such as binding to Fc receptors of the subject.

Treatment of individuals having malaria infection may comprise the administration of a therapeutically effective amount of MSP-1.sub.42 antibodies of the present invention. The antibodies can be provided in a kit as described below. The antibodies can be used or administered as a mixture, for example in equal amounts, or individually, provided in sequence, or administered all at once. In providing a patient with antibodies, or fragments thereof, capable of binding to MSP-1.sub.42, or an antibody capable of protecting against malaria in a recipient patient, the dosage of administered agent will vary depending upon such factors as the patient's age, weight, height, sex, general medical condition, previous medical history, etc.

In general, it is desirable to provide the recipient with a dosage of antibody which is in the range of from about 1 pg/kg-100 pg/kg, 100 pg/kg-500 pg/kg, 500 pg/kg-1 ng/kg, 1 ng/kg-100 ng/kg, 100 ng/kg-500 ng/kg, 500 ng/kg-1 ug/kg, 1 ug/kg-100 ug/kg, 100 ug/kg-500 ug/kg, 500 ug/kg-1 mg/kg, 1 mg/kg-50 mg/kg, 50 mg/kg-100 mg/kg, 100 mg/kg-500 mg/kg, 500 mg/kg-1 g/kg, 1 g/kg-5 g/kg, 5 g/kg-10 g/kg (body weight of recipient), although a lower or higher dosage may be administered.

In a similar approach, another prophylactic use of the monoclonal antibodies of the present invention is the active immunization of a patient using an anti-idiotypic antibody raised against one of the present monoclonal antibodies. Immunization with an anti-idiotype that mimics the structure of the epitope could elicit an active anti-MSP-1.sub.42 response (Linthicum, D. E. and Farid, N. R., Anti-Idiotypes, Receptors, and Molecular Mimicry (1988), pp 1-5 and 285-300).

Likewise, active immunization can be induced by administering one or more antigenic and/or immunogenic epitopes as a component of a subunit vaccine. Vaccination could be performed orally or parenterally in amounts sufficient to enable the recipient to generate protective antibodies against this biologically functional region, prophylactically or therapeutically. The host can be actively immunized with the antigenic/immunogenic peptide in pure form, a fragment of the peptide, or a modified form of the peptide. One or more amino acids, not corresponding to the original protein sequence can be added to the amino or carboxyl terminus of the original peptide, or truncated form of peptide. Such extra amino acids are useful for coupling the peptide to another peptide, to a large carrier protein, or to a support. Amino acids that are useful for these purposes include: tyrosine, lysine, glutamic acid, aspartic acid, cysteine and derivatives thereof. Alternative protein modification techniques may be used e.g., NH.sub.2-acetylation or COOH-terminal amidation, to provide additional means for coupling or fusing the peptide to another protein or peptide molecule or to a support.

The antibodies capable of protecting against malaria are intended to be provided to recipient subjects in an amount sufficient to effect a reduction in the malaria infection symptoms. An amount is said to be sufficient to "effect" the reduction of infection symptoms if the dosage, route of administration, etc. of the agent are sufficient to influence such a response. Responses to antibody administration can be measured by analysis of subject's vital signs.

The present invention more particularly relates to a composition comprising at least one of the above-specified peptides or a recombinant MSP-1.sub.42 protein composition as defined above, for use as a vaccine for immunizing a mammal, preferably humans, against malaria, comprising administering a sufficient amount of the composition possibly accompanied by pharmaceutically acceptable adjuvant(s), to produce an immune response.

Immunogenic compositions can be prepared according to methods known in the art. The present compositions comprise an immunogenic amount of one or more recombinant MSP-1.sub.42 protein or peptides as defined above, usually combined with a pharmaceutically acceptable carrier, preferably further comprising an adjuvant.

The proteins of the present invention, preferably purified MSP-1.sub.42 from one of P. falciparum, e.g. FVO and 3D7, are expected to provide a particularly useful vaccine antigen, since the antigen is able to induce invasion inhibitory antibodies as well as high titer antibodies that react with schizont-infected-erythrocytes.

Pharmaceutically acceptable carriers include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers; and inactive virus particles. Such carriers are well known to those of ordinary skill in the art.

Preferred adjuvants to enhance effectiveness of the composition include, but are not limited to: montanide, aluminum hydroxide (alum), N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP) as found in U.S. Pat. No. 4,606,918, N-acetyl-normuramyl-L-alanyl-D-isoglutamine(nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl-s- n-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE) and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate, and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion. Any of the 3 components MPL, TDM or CWS may also be used alone or combined 2 by 2. Additionally, adjuvants such as Stimulon (Cambridge Bioscience, Worcester, Mass.) or SAF-1 (Syntex) may be used. Further, Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA) may be used for non-human applications and research purposes.

The adjuvant used in the examples below, Adjuvant B, is described in U.S. Pat. No. 6,146,632, with the formulation 10.68 mg squalene, 11.86 mg tocopherol, 4.85 mg Tween 80, 50 ug 3D-MPL, and 50 ug QS21 and consisting of an oil-in water emulsion comprising the squalene and alpha-tocopherol, the emulsion being in admixture with the QS21 and 3-DPML;

All documents cited herein are hereby incorporated by reference thereto.

The immunogenic compositions typically will contain pharmaceutically acceptable vehicles, such as water, saline, glycerol, ethanol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, preservatives, and the like, may be included in such vehicles.

Typically, the immunogenic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. The preparation also may be emulsified or encapsulated in liposomes for enhanced adjuvant effect. The MSP-1.sub.42 proteins of the invention may also be incorporated into Immune Stimulating Complexes together with saponins, for example QuilA (ISCOMS).

Immunogenic compositions used as vaccines comprise a `sufficient amount` or `an immunologically effective amount` of the proteins of the present invention, as well as any other of the above-mentioned components, as needed. `Immunologically effective amount`, means that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment, as defined above. This amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated (e.g. nonhuman primate, primate, etc.), the capacity of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, the strain of malaria infection, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials. Usually, the amount will vary from 0.01 to 1000 ug/dose, more particularly from about 1.0 to 100 ug/dose most preferably from about 10 to 50 ug/dose.

The proteins may also serve as vaccine carriers to present homologous (e.g. other malaria antigens, such as EBA-175 or AMA-1) or heterologous (non-malaria) antigens. In this use, the proteins of the invention provide an immunogenic carrier capable of stimulating an immune response to other antigens. The antigen may be conjugated either by conventional chemical methods, or may be cloned into the gene encoding MSP-1.sub.42 fused to the 5' end or the 3' end of the MSP-1.sub.42 gene. The vaccine may be administered in conjunction with other immunoregulatory agents.

The compounds of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby these materials, or their functional derivatives, are combined in admixture with a pharmaceutically acceptable carrier vehicle. Suitable vehicles and their formulation, inclusive of other human proteins, e.g., human serum albumin, are described, for example, in Remington's Pharmaceutical Sciences (16th ed., Osol, A. ed., Mack Easton Pa. (1980)). In order to form a pharmaceutically acceptable composition suitable for effective administration, such compositions will contain an effective amount of the above-described compounds together with a suitable amount of carrier vehicle.

Additional pharmaceutical methods may be employed to control the duration of action. Control release preparations may be achieved through the use of polymers to complex or absorb the compounds. The controlled delivery may be exercised by selecting appropriate macromolecules (for example polyesters, polyamino acids, polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, or protamine sulfate) and the concentration of macromolecules as well as the method of incorporation in order to control release. Another possible method to control the duration of action by controlled release preparations is to incorporate the compounds of the present invention into particles of a polymeric material such as polyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylene vinylacetate copolymers. Alternatively, instead of incorporating these agents into polymeric particles, it is possible to entrap these materials in microcapsules prepared, for example, interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly(methylmethacylate)-microcapsules, respectively, or in colloidal drug delivery systems, for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences (1980).

Administration of the compounds, whether antibodies or vaccines, disclosed herein may be carried out by any suitable means, including parenteral injection (such as intraperitoneal, subcutaneous, or intramuscular injection), in ovo injection of birds, orally, or by topical application of the antibodies (typically carried in a pharmaceutical formulation) to an airway surface. Topical application of the antibodies to an airway surface can be carried out by intranasal administration (e.g., by use of dropper, swab, or inhaler which deposits a pharmaceutical formulation intranasally). Topical application of the antibodies to an airway surface can also be carried out by inhalation administration, such as by creating respirable particles of a pharmaceutical formulation (including both solid particles and liquid particles) containing the antibodies as an aerosol suspension, and then causing the subject to inhale the respirable particles. Methods and apparatus for administering respirable particles of pharmaceutical formulations are well known, and any conventional technique can be employed. Oral administration may be in the form of an ingestable liquid or solid formulation.

The treatment may be given in a single dose schedule, or preferably a multiple dose schedule in which a primary course of treatment may be with 1-10 separate doses, followed by other doses given at subsequent time intervals required to maintain and or reinforce the response, for example, at 1-4 months for a second dose, and if needed, a subsequent dose(s) after several months. Examples of suitable treatment schedules include: (i) 0, 1 month and 6 months, (ii) 0, 7 days and 1 month, (iii) 0 and 1 month, (iv) 0 and 6 months, or other schedules sufficient to elicit the desired responses expected to reduce disease symptoms, or reduce severity of disease.

The present invention also provides kits that are useful for carrying out the present invention. The present kits comprise a first container means containing the above-described antibodies. The kit also comprises other container means containing solutions necessary or convenient for carrying out the invention. The container means can be made of glass, plastic or foil and can be a vial, bottle, pouch, tube, bag, etc. The kit may also contain written information, such as procedures for carrying out the present invention or analytical information, such as the amount of reagent contained in the first container means. The container means may be in another container means, e.g. a box or a bag, along with the written information.

The present invention also relates to a method for in vitro diagnosis of malaria antibodies present in a biological sample, comprising at least the following steps

(i) contacting said biological-sample with a composition comprising any of the MSP-1.sub.42 peptides as defined above, preferably in an immobilized form under appropriate conditions which allow the formation of an immune complex, wherein said peptide or protein can be a biotinylated peptide or protein which is covalently bound to a solid substrate by means of streptavidin or avidin complexes,

(ii) removing unbound components,

(iii) incubating the immune complexes formed with heterologous antibodies, with said heterologous antibodies having conjugated to a detectable label under appropriate conditions,

(iv) detecting the presence of said immune complexes visually or mechanically (e.g. by means of densitometry, fluorimetry, colorimetry).

The present invention also relates to a kit for determining the presence of malaria antibodies, in a biological sample, comprising:

at least one peptide or protein composition as defined above, possibly in combination with other polypeptides or peptides from Plasmodium or other types of malaria parasite, with said peptides or proteins being preferentially immobilized on a solid support, more preferably on different microwells of the same ELISA plate, and even more preferentially on one and the same membrane strip,

a buffer or components necessary for producing the buffer enabling binding reaction between these polypeptides or peptides and the antibodies against malaria present in the biological sample,

means for detecting the immune complexes formed in the preceding binding reaction,

possibly also including an automated scanning and interpretation device for inferring the malaria parasite present in the sample from the observed binding pattern.

The immunoassay methods according to the present invention utilize MSP-1.sub.42 domains that maintain linear (in case of peptides) and conformational epitopes (proteins) recognized by antibodies in the sera from individuals infected with a malaria parasite. The MSP-1.sub.42 antigens of the present invention may be employed in virtually any assay format that employs a known antigen to detect antibodies. A common feature of all of these assays is that the antigen is contacted with the body component suspected of containing malaria antibodies under conditions that permit the antigen to bind to any such antibody present in the component. Such conditions will typically be physiologic temperature, pH and ionic strength using an excess of antigen. The incubation of the antigen with the specimen is followed by detection of immune complexes comprised of the antigen.

Design of the immunoassays is subject to a great deal of variation, and many formats are known in the art. Protocols may, for example, use solid supports, or immunoprecipitation. Most assays involve the use of labeled antibody or polypeptide; the labels may be, for example, enzymatic, fluorescent, chemiluminescent, radioactive, or dye molecules. Assays which amplify the signals from the immune complex are also known; examples of which are assays which utilize biotin and avidin or streptavidin, and enzyme-labeled and mediated immunoassays, such as ELISA assays.

The immunoassay may be, without limitation, in a heterogeneous or in a homogeneous format, and of a standard or competitive type. In a heterogeneous format, the polypeptide is typically bound to a solid matrix or support to facilitate separation of the sample from the polypeptide after incubation. Examples of solid supports that can be used are nitrocellulose (e.g., in membrane or microtiter well form), polyvinyl chloride (e.g., in sheets or microtiter wells), polystyrene latex (e.g., in beads or microtiter plates, polyvinylidine fluoride (known as Immunolon..TM..), diazotized paper, nylon membranes, activated beads, and Protein A beads. For example, Dynatech Immunolon..TM..1 or Immunlon..TM.. 2 microtiter plates or 0.25 inch polystyrene beads (Precision Plastic Ball) can be used in the heterogeneous format. The solid support containing the antigenic polypeptides is typically washed after separating it from the test sample, and prior to detection of bound antibodies. Both standard and competitive formats are known in the art.

In a homogeneous format, the test sample is incubated with the combination of antigens in solution. For example, it may be under conditions that will precipitate any antigen-antibody complexes that are formed. Both standard and competitive formats for these assays are known in the art.

In a standard format, the amount of malaria antibodies in the antibody-antigen complexes is directly monitored. This may be accomplished by determining whether labeled anti-xenogeneic (e.g. anti-human) antibodies which recognize an epitope on anti-malaria antibodies will bind due to complex formation. In a competitive format, the amount of malaria antibodies in the sample is deduced by monitoring the competitive effect on the binding of a known amount of labeled-antibody (or other competing ligand) in the complex.

Complexes formed comprising anti-malaria antibody (or in the case of competitive assays, the amount of competing antibody) are detected by any of a number of known techniques, depending on the format. For example, unlabeled malaria antibodies in the complex may be detected using a conjugate of anti-xenogeneic Ig complexed with a label (e.g. an enzyme label).

In an immunoprecipitation or agglutination assay format the reaction between the malaria antigens and the antibody forms a network that precipitates from the solution or suspension and forms a visible layer or film of precipitate. If no anti-malaria antibody is present in the test specimen, no visible precipitate is formed.

There currently exist three specific types of particle agglutination (PA) assays. These assays are used for the detection of antibodies to various antigens when coated to a support. One type of this assay is the hemagglutination assay using red blood cells (RBCs) that are sensitized by passively adsorbing antigen (or antibody) to the RBC. The addition of specific antigen antibodies present in the body component, if any, causes the RBCs coated with the purified antigen to agglutinate.

To eliminate potential non-specific reactions in the hemagglutination assay, two artificial carriers may be used instead of RBC in the PA. The most common of these are latex particles. However, gelatin particles may also be used. The assays utilizing either of these carriers are based on passive agglutination of the particles coated with purified antigens.

The MSP-1.sub.42 proteins, peptides, or antigens of the present invention will typically be packaged in the form of a kit for use in these immunoassays. The kit will normally contain in separate containers the MSP-1.sub.42 antigen, control antibody formulations (positive and/or negative), labeled antibody when the assay format requires the same and signal generating reagents (e.g. enzyme substrate) if the label does not generate a signal directly. The MSP-1.sub.42 antigen may be already bound to a solid matrix or separate with reagents for binding it to the matrix. Instructions (e.g. written, tape, CD-ROM, etc.) for carrying out the assay usually will be included in the kit.

Immunoassays that utilize the MSP-1.sub.42 antigen are useful in screening blood for the preparation of a supply from which potentially infective malaria parasite is lacking. The method for the preparation of the blood supply comprises the following steps. Reacting a body component, preferably blood or a blood component, from the individual donating blood with MSP-1.sub.42 proteins of the present invention to allow an immunological reaction between malaria antibodies, if any, and the MSP-1.sub.42 antigen. Detecting whether anti-malaria antibody--MSP-1.sub.42 antigen complexes are formed as a result of the reacting. Blood contributed to the blood supply is from donors that do not exhibit antibodies to the native MSP-1 antigens.

The present invention further contemplates the use of MSP-1.sub.42 proteins, or parts thereof as defined above, for in vitro monitoring malaria infection or prognosing the response to treatment (for instance with chloroquine, mefloquine, Malarone) of patients suffering from malaria infection comprising:

incubating a biological sample from a patient with malaria infection with an MSP-1.sub.42 protein or a suitable part thereof under conditions allowing the formation of an immunological complex,

removing unbound components,

calculating the anti-MSP-1.sub.42 titers present in said sample (for example at the start of and/or during the course of therapy),

monitoring the natural course of malaria infection, or prognosing the response to treatment of said patient on the basis of the amount anti-MSP-1.sub.42 titers found in said sample at the start of treatment and/or during the course of treatment.

Patients who show a decrease of 2, 3, 4, 5, 7, 10, 15, or preferably more than 20 times of the initial anti-MSP-1.sub.42 titers could be concluded to be long-term, sustained responders to malaria therapy.

It is to be understood that smaller fragments of the above-mentioned peptides also fall within the scope of the present invention. Said smaller fragments can be easily prepared by chemical synthesis and can be tested for their ability to be used in an assay as detailed above.

The present invention also relates to a kit for monitoring malaria infection or prognosing the response to treatment (for instance to medication) of patients suffering from malaria infection comprising:

at least one MSP-1.sub.42 peptide as defined above,

a buffer or components necessary for producing the buffer enabling the binding reaction between these proteins or peptides and the anti-MSP-1.sub.42 antibodies present in a biological sample,

means for detecting the immune complexes formed in the preceding binding reaction,

possibly also an automated scanning and interpretation device for inferring a decrease of anti-MSP-1.sub.42 titers during the progression of treatment.

The present invention also relates to a serotyping assay for detecting one or more serological types or alleles of malaria parasite present in a biological sample, more particularly for detecting antibodies of the different types or alleles of malaria parasites to be detected combined in one assay format, comprising at least the following steps:

(i) contacting the biological sample to be analyzed for the presence of malaria antibodies of one or more serological types, with at least one of the MSP-1.sub.42 compositions as defined above, preferentially in an immobilized form under appropriate conditions which allow the formation of an immune complex,

(ii) removing unbound components,

(iii) incubating the immune complexes formed with heterologous antibodies, with said heterologous antibodies-being conjugated to a detectable label under appropriate conditions,

(iv) detecting the presence of said immune complexes visually or mechanically (e.g. by means of densitometry, fluorometry, calorimetry) and inferring the presence of one or more malaria serological types present from the observed binding pattern.

It is to be understood that the compositions of proteins or peptides used in this method are recombinantly expressed type-specific or allele-specific proteins or type-specific peptides.

The present invention further relates to a kit for serotyping one or more serological types or alleles of malaria parasite present in a biological sample, more particularly for detecting the antibodies to these serological types of malaria parasites comprising:

at least one MSP-1.sub.42 protein or MSP-1.sub.42 peptide, as defined above,

a buffer or components necessary for producing the buffer enabling the binding reaction between these proteins or peptides and the anti-MSP-1 antibodies present in a biological sample,

means for detecting the immune complexes formed in the preceding binding reaction,

possibly also an automated scanning and interpretation device for detecting the presence of one or more serological types present from the observed binding pattern.

The present invention also relates to the use of a peptide or protein composition as defined above, for immobilization on a solid support and incorporation into a reversed phase hybridization assay, preferably for immobilization as parallel lines onto a solid support such as a membrane strip, for determining the presence or the genotype of malaria parasite according to a method as defined above. Combination with other type-specific or allele-specific antigens from other malaria parasites also lies within the scope of the present invention.
 

Claim 1 of 4 Claims

1. An isolated recombinant vector comprising a DNA sequence encoding MSP-1.sub.42 wherein said DNA sequence is SEQ ID NO:4.

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.
 

 

     
[ Outsourcing Guide ] [ Cont. Education ] [ Software/Reports ] [ Training Courses ]
[ Web Seminars ] [ Jobs ] [ Consultants ] [ Buyer's Guide ] [ Advertiser Info ]

[ Home ] [ Pharm Patents / Licensing ] [ Pharm News ] [ Federal Register ]
[ Pharm Stocks ] [ FDA Links ] [ FDA Warning Letters ] [ FDA Doc/cGMP ]
[ Pharm/Biotech Events ] [ Newsletter Subscription ] [ Web Links ] [ Suggestions ]
[ Site Map ]