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 plasmodium falciparum merozoite surface proteins 4 and 5 and their use
United States Patent: 
8,026,354
Issued: 
September 27, 2011

Inventors: 
Longacre; Shirley (Paris, FR), Polson; Hannah (Paris, FR), Perraut; Ronald (ointe a Pitre Cedex, FR), Nato; Faridabano (Antony, FR)
Assignee: 
Institut Pasteur (Paris, FR), Centre Nationale de la Recherche Scientifique (Paris, FR)
Appl. No.: 
11/603,310
Filed: 
November 22, 2006


 

Training Courses -- Pharm/Biotech/etc.


Abstract

Accordingly, the invention provides constructs in which the nucleic acids encoding Plasmodium falciparum MSP4 and MSP5, and the resulting polypeptides, have been modified. More particularly, this invention provides constructs encoding recombinant MSP4 and MSP5 polypeptides, which are expressed as soluble, secreted polypeptides in a baculovirus-insect cell expression system. It was surprisingly found that the recombinant polypeptides contain an EGF-like domain at the C-terminus that is properly folded in the polypeptide.

Description of the Invention

SUMMARY OF THE INVENTION

This invention aids in fulfilling these needs in the art. The invention provides a purified or recombinant nucleic acid molecule comprising a DNA sequence of SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, or 8 and a purified or recombinant nucleic acid molecule encoding the amino acid sequence of SEQ ID NOS: 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22. The invention also encompasses purified nucleic acid molecules complementary to these sequences.

The invention also encompasses purified polypeptides encoded by the purified or recombinant nucleic acid molecules comprising a DNA sequence of SEQ ID NOS: 1-8, as predicted by the sequence.

The invention includes purified double-stranded nucleic acid molecules comprising the DNA sequence of SEQ ID NOS: 1-8, and purified double-stranded nucleic acid molecules encoding the amino acid sequence of SEQ ID NOS: 9-22. Both single-stranded and double-stranded RNA and DNA nucleic acid molecules are encompassed by the invention. These molecules can be used to detect both single-stranded and double-stranded RNA and DNA variants encoding polypeptides encompassed by the invention. A double-stranded DNA probe allows the detection of nucleic acid molecules equivalent to either strand of the nucleic acid molecule.

Purified nucleic acid molecules that hybridize to a denatured, double-stranded DNA comprising the DNA sequence of SEQ ID NOS: 1-8, and encoding the amino acid sequence of SEQ ID NOS: 9-22 under conditions of moderate stringency in 50% formamide and 6.times.SSC, at 42.degree. C. with washing conditions of 60.degree. C., 0.5.times.SSC, 0.1% SDS are encompassed by the invention.

The invention further encompasses purified nucleic acid molecules derived by in vitro mutagenesis from SEQ ID NOS: 1-8. In vitro mutagenesis includes numerous techniques known in the art including, but not limited to, site-directed mutagenesis, random mutagenesis, and in vitro nucleic acid synthesis.

The nucleic acid molecules of the invention, which include DNA and RNA, are referred to herein as "recombinant MSP4 and MSP5 nucleic acids" or "recombinant MSP4 and MSP5 DNA", and the amino acids encoded by these molecules are referred to herein as "recombinant MSP4 and MSP5 polypeptides" or "the polypeptides of the invention".

The invention also encompasses purified nucleic acid molecules degenerate from SEQ ID NOS: 1-8 as a result of the genetic code, purified nucleic acid molecules, which are allelic variants of recombinant MSP4 and MSP5 nucleic acids, or a species homolog of recombinant MSP4 and MSP5 nucleic acids. The invention also encompasses recombinant vectors that direct the expression of these nucleic acid molecules and host cells transformed, transfected or infected with these vectors.

Purified polyclonal or monoclonal antibodies that bind to recombinant MSP4 or MSP5 polypeptides are encompassed by the invention and are referred to herein as "the antibodies of the invention".

The invention further encompasses methods for the production of recombinant MSP4 and MSP5 polypeptides, including culturing a host cell under conditions promoting expression, and recovering the polypeptide from the culture medium or cellular pellets. Especially, the expression of recombinant MSP4 and MSP5 polypeptides in baculovirus insect expression systems is encompassed by the invention.

This invention also provides labelled recombinant MSP4 and MSP5 polypeptides. Preferably, the labelled polypeptides are in purified form. It is also preferred that the unlabelled or labelled polypeptide is capable of being immunologically recognized by human body fluid containing antibodies to malaria. The polypeptides can be labelled, for example, with an immunoassay label selected from the group consisting of radioactive, enzymatic, fluorescent, chemiluminescent labels, and chromophores.

Immunological complexes between the recombinant MSP4 or MSP5 polypeptides of the invention and antibodies recognizing those are also provided. The immunological complexes can be labelled with an immunoassay label selected from the group consisting of radioactive, enzymatic, fluorescent, chemiluminescent labels, and chromophores.

Furthermore, this invention provides an in vitro method for detecting MSP4 and/or MSP5 polypeptides of a Plasmodium parasite. The method comprises providing a composition comprising a biological material suspected of containing MSP4 and/or MSP5 polypeptides of a Plasmodium parasite (malaria parasite), and assaying for the presence of MSP4 and/or MSP5 polypeptides of a Plasmodium parasite. The MSP4 and MSP5 polypeptides of a Plasmodium parasite are typically assayed by electrophoresis or by immunoassay with the antibodies of the invention. This method can be used for the detection of Plasmodium parasites in a biological sample, and in a preferred embodiment for detection of Plasmodium falciparum and Plasmodium vivax parasites.

This invention also provides an in vitro diagnostic method for the detection of the presence or absence of antibodies, which bind to an antigen comprising the recombinant or purified MSP4 or MSP5 polypeptides of the invention or mixtures thereof. The method comprises contacting the antigen with a biological fluid for a time and under conditions sufficient for the antigen and antibodies in the biological fluid to form an antigen-antibody complex, and then detecting the formation of the complex. The detection step can further comprise measuring the formation of the antigen-antibody complex. The formation of the antigen-antibody complex is preferably measured by immunoassay based on Western blot technique, ELISA (enzyme linked immunosorbent assay), indirect immunofluorescent assay, or immunoprecipitation assay. This method can be used for the detection of an immunological response to a Plasmodium parasite in a biological fluid coming from an animal or a human patient malaria infected. In a preferred embodiment this method can be used for the detection of an immunological response to a Plasmodium falciparum infection or a Plasmodium vivax infection.

A diagnostic kit for the detection of the presence or absence of antibodies, which bind to the recombinant MSP4 or MSP5 polypeptides of the invention or mixtures thereof, contains antigen comprising the recombinant MSP4 and/or MSP5 polypeptides, or mixtures thereof, and means for detecting the formation of immune complexes between the antigen and antibodies. The antigen and the means are present in an amount sufficient to perform the detection.

A diagnostic kit for the detection of the presence or absence of MSP4 and/or MSP5 polypeptides of Plasmodium parasite, contains the antibodies of the invention, and means for detecting the formation of immune complexes between an antigen and the antibodies. The antibodies and the means are present in an amount sufficient to perform the detection.

This invention also provides an immunogenic composition comprising a recombinant MSP4 or MSP5 polypeptide of the invention or a mixture thereof in an amount sufficient to induce an immunogenic or protective response in vivo, in association with a pharmaceutically acceptable immunostimulator therefore. A vaccine composition of the invention comprises a sufficient amount of the recombinant MSP4 and/or MSP5 polypeptide and a pharmaceutically acceptable immunostimulator therefore to induce neutralizing antibodies.

The MSP4 and MSP5 polypeptides of the invention are thus useful as a portion of a diagnostic composition for detecting the presence of antibodies to antigenic proteins associated with malaria.

In addition, the recombinant MSP4 and MSP5 polypeptides can be used to raise antibodies for detecting the presence of antigenic proteins associated with malaria.

The polypeptides of the invention can be also employed to raise neutralizing antibodies that either inactivate the parasite, reduce the viability of the parasite in vivo, or inhibit or prevent parasite replication. The ability to elicit parasite-neutralizing antibodies is especially important when the polypeptides of the invention are used in immunizing or vaccinating compositions.

Following is a vaccine, which includes (A) the natural signal sequence (B) a C-terminal His tag and (C) the acid repeat region that resembles that of the P. falciparum antigen most strongly correlated with protective antibody responses in the field (MSP4p20) -- see Original Patent.

DETAILED DESCRIPTION OF THE INVENTION

Two recently identified merozoite surface antigens, Plasmodium falciparum merozoite surface proteins 4 and 5 (PfMSP4 and PfMSP5), are promising protein constituents of a potential multi-component anti-malaria vaccine. The msp4 and msp5 genes both code for 272 residue proteins, each with a single C-terminal EGF-like domain and GPI attachment motif [10,11] and are located in tandem on chromosome 2, just upstream of msp2. Membrane association at the merozoite surface has been demonstrated for both proteins, and human immune sera have been shown to react with recombinant MSP4 expressed in Escherichia coli [12,13].

In 3 murine species of Plasmodium, P. yoelii, P. chabaudi, and P. berghei, there is only a single gene at the MSP4 and MSP5 locus (MSP4/5), which shows some degree of homology to each [14-16]. This gene is denoted MSP4/5 and has been used to investigate protective immunity in the P. yoelii lethal challenge model [17].

MSP4/5 has been shown to confer protection using a variety of immunization strategies, and efficacy is maximized when delivered in conjunction with MSP1p19 [17-20]. In addition, there appears to be no strain specificity in immune responses induced by the murine MSP4/5 protein [21]. In P. falciparum, msp4 and msp5 each have a single intron at homologous locations [22].

Plasmodium falciparum merozoite surface protein 4 (PfMSP4) protein sequence includes a secretory signal sequence, a C-terminal EGF-like domain, and GPI-attachment signal [10].

The Pfmsp4 gene sequence is 960 bp in length, includes one intron of 144 bp and encodes 272 amino acids residues. The Pfmsp5 gene sequence is 955 bp in length, includes one intron of 136 bp and encodes 272 amino acids residues. As for PfMSP4, the PfMSP5 protein sequence consists of a secretory signal sequence, a C-terminal EGF-like domain, and a GPI-attachment signal [11,22]. Downstream of this cluster of MSP genes is the highly conserved adenylosuccinate lysase (ASL) gene, which has proved to be a useful handle to facilitate the identification of this locus in other species (FIG. 1 (see Original Patent)).

Much current data support the notion that PfMSP4 and PfMSP5 are good vaccine candidates. Nevertheless, several published findings show that recombinant analogs of PfMSP4 produced in two different expression systems (E. coli and yeast) differ in antigenicity and induce conformationally-independent responses against the EGF-like domain of the protein, a phenomenon not seen with human immune sera [13]. If recognition of conformational epitopes in this region of the protein is important for protection, as is the case with MSP1-19, it is imperative to generate a product that faithfully reproduces all epitopes.

Accordingly, the invention provides constructs in which the nucleic acids encoding Plasmodium falciparum MSP4 and MSP5, and the resulting polypeptides, have been modified to achieve optimal expression in insect cells. More particularly, this invention provides constructs encoding recombinant MSP4 polypeptides, which are expressed as soluble, secreted polypeptides in a baculovirus-insect cell expression system. The recombinant polypeptides contain an EGF-like domain at the C-terminus that appears to be properly folded. This is indicated by a marked reduction (50-60%) in polyclonal rabbit sera recognition of MSP4 when the protein is irreversibly reduced on ELISA plates (see FIG. 12 (see Original Patent))

One of the recombinant MSP4 polypeptides of the invention is a MSP4 exo-antigen (minus C-terminal hydrophobic residues of the GPI attachment site, thus allowing protein secretion) with a deletion of 30 amino acids from the polymorphic region near the N-terminus. This polypeptide of the invention is referred to as MSP4p30.

Another one of the recombinant MSP4 polypeptides of the invention is a MSP4 exo-antigen (minus C-terminal hydrophobic residues of the GPI attachment site, thus allowing protein secretion) without a deletion of 30 amino acids from a polymorphic region near the N-terminus. This polypeptide of the invention is referred to as MSP4p40 and is the full-length gene product.

Another recombinant MSP4 polypeptide of the invention is a 20 kDa polypeptide corresponding approximately to the C-terminal half of MSP4, starting around the sequence KSPKE (SEQ ID NO: 43) motif and including the EGF domain. Upstream supplementary amino acid residues could be included, in particular residues of the MSP4p40 sequence localized upstream the KSPKE (SEQ ID NO: 43) motif. This recombinant polypeptide of the invention is referred to as MSP4p20.

In addition, this invention provides recombinant MSP5 polypeptides including p35 and p45 forms that are produced simultaneously. Both contain a post-translational modification, likely to involve the covalent attachment of a fatty acid residues (myristoylation) that can boost immunogenicity. These recombinant polypeptides of the invention are referred to as MSP5p45 and MSP5p35.

Nevertheless, the recombinant MSP4 and MSP5 polypeptides of the invention can be also expressed as C-terminal GPI anchored entities using either their native GPI signal sequences or that from another GPI anchored protein sequence signaling for GPI modification. Such GPI modified entities would be expected to substantially enhance immunogenicity of the recombinant MSP4 and MSP5 polypeptides in the absence of any adjuvants of immunity.

The recombinant polypeptides of the invention are described in greater detail with reference to their corresponding SEQ ID NOS. as follows -- see Original Patent.

PvMSP4p20 (sequence of proposed vaccine construct based on Plasmodium vivax sequence of MSP4 as given in "alignment of amino acid sequences of PfMSP4 and PvMSP4) SEQ ID NO: 30

These polypeptides are individually and collectively referred to herein as "the recombinant MSP4 and MSP5 polypeptides" of the invention. Similarly, the nucleic acids encoding these polypeptides are referred to as "the recombinant MSP4 and MSP5 nucleic acids" of the invention.

The implications for this invention are widespread. This discovery of the recombinant MSP4 and MSP5 polypeptides enables construction of expression vectors comprising nucleic acid sequences encoding recombinant MSP4 and MSP5 polypeptides of the invention; host cells transfected or transformed with the expression vectors; biologically active recombinant MSP4 and MSP5 polypeptides and recombinant MSP4 and MSP5 polypeptides as isolated or purified proteins; antibodies immunoreactive with recombinant MSP4 and MSP5 polypeptides, diagnostic use of the recombinant MSP4 and MSP5 polypeptides and antibodies directed against the recombinant MSP4 and MSP5 polypeptides in detection of Plasmodium parasite and malaria infection and vaccine use of the recombinant MSP4 and MSP5 polypeptides to protect against Plasmodium infection.

As used herein, the term "recombinant MSP4 and MSP5 polypeptides" also refers to a genus of polypeptides that further encompasses proteins having the amino acid sequence of SEQ ID NOS: 9-22, as well as those proteins and polypeptides having a high degree of similarity (at least 90% homology) with such amino acid sequences and which proteins and polypeptides are immunoreactive. In addition, recombinant MSP4 and MSP5 polypeptides refers to the gene products of the nucleotides of SEQ ID NOS: 1-8.

The term "purified" as used herein, means that the recombinant MSP4 and MSP5 polypeptides are essentially free of association with other proteins or polypeptides, for example, as a purification product of recombinant host cell culture or as a purified product from a non-recombinant source. The term "substantially purified" as used herein, refers to a mixture that contains recombinant MSP4 and MSP5 polypeptides and is essentially free of association with other proteins or polypeptides, but for the presence of known proteins that can be removed using a specific antibody, and which substantially purified recombinant MSP4 and MSP5 polypeptides can be used as antigens.

A recombinant MSP4 and MSP5 polypeptide "variant" as referred to herein means a polypeptide substantially homologous to recombinant MSP4 and MSP5 polypeptides, but which has an amino acid sequence different from that of recombinant MSP4 and MSP5 polypeptides because of one or more deletions, insertions, or substitutions. The variant amino acid sequence preferably is at least 80% identical to a recombinant MSP4 and MSP5 polypeptide amino acid sequence, most preferably at least 90% identical. The percent identity can be determined, for example by comparing sequence information using the GAP computer program, version 6.0 described by Devereux et al. (Nucl. Acids Res. 12:387, 1984) and available from the University of Wisconsin Genetics Computer Group (UWGCG). The GAP program utilizes the alignment method of Needleman and Wunsch (J. Mol. Biol. 48:443, 1970), as revised by Smith and Waterman (Adv. Appl. Math 2:482, 1981). The preferred default parameters for the GAP program include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the weighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res. 14:6745, 1986, as described by Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358, 1979; (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.

Variants can comprise conservatively substituted sequences, meaning that a given amino acid residue is replaced by a residue having similar physiochemical characteristics. Examples of conservative substitutions include substitution of one aliphatic residue for another, such as Ile, Val, Leu, or Ala for one another, or substitutions of one polar residue for another, such as between Lys and Arg; Glu and Asp; or Gln and Asn. Other such conservative substitutions, for example, substitutions of entire regions having similar hydrophobicity characteristics, are well known. Examples of variants of the MSP4 and MSP5 polypeptides of the invention are proteins that result from alternate mRNA splicing events or from proteolytic cleavage of the recombinant MSP4 and MSP5 polypeptides. Variations attributable to proteolysis include, for example, differences in the termini upon expression in different types of host cells, due to proteolytic removal of one or more terminal amino acids from the recombinant MSP4 and MSP5 polypeptides. Variations attributable to frame shifting include, for example, differences in the termini upon expression in different types of host cells.

As stated above, the invention provides isolated and purified, or homogeneous, recombinant MSP4 and MSP5 polypeptides. Variants and derivatives of recombinant MSP4 and MSP5 polypeptides that can be used as antigens can be obtained by mutations of nucleotide sequences coding for recombinant MSP4 and MSP5 polypeptides. Alterations of the amino acid sequence can be accomplished by any of a number of conventional methods. Mutations can be introduced at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analogue having the desired amino acid insertion, substitution, or deletion.

Alternatively, oligonucleotide-directed, site-specific mutagenesis procedures can be employed to provide an altered gene wherein predetermined codons can be altered by substitution, deletion, or insertion. Exemplary methods of making the alterations set forth above are disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); Kunkel (Proc. Natl. Acad. Sci. USA 82:488, 1985); Kunkel et al. (Methods in Enzymol. 154:367, 1987); and U.S. Pat. Nos. 4,518,584 and 4,737,462, all of which are incorporated by reference.

Within an aspect of the invention, recombinant MSP4 and MSP5 polypeptides can be utilized to prepare antibodies that specifically bind to recombinant MSP4 and MSP5 polypeptides. The term "antibodies" is meant to include polyclonal antibodies, monoclonal antibodies, fragments thereof, such as F(ab')2 and Fab fragments, as well as any recombinantly produced binding partners. Antibodies are defined to be specifically binding if they bind recombinant MSP4 and MSP5 polypeptides with a Ka of greater than or equal to about 107 M-1. Affinities of binding partners or antibodies can be readily determined using conventional techniques, for example, those described by Scatchard et al., Ann. N.Y. Acad. Sci., 51:660 (1949). Polyclonal antibodies can be readily generated from a variety of sources, for example, horses, cows, goats, sheep, dogs, chickens, rabbits, mice, or rats, using procedures that are well known in the art.

The invention further encompasses isolated fragments and oligonucleotides derived from the nucleotide sequences of SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, and 8. The invention also encompasses polypeptides encoded by these fragments and oligonucleotides.

Nucleic acid sequences within the scope of the invention include isolated DNA and RNA sequences that hybridize to the native recombinant MSP4 and MSP5 nucleic acids disclosed herein under conditions of moderate or severe stringency, and which encode recombinant MSP4 and MSP5 polypeptides. As used herein, conditions of moderate stringency, as known to those having ordinary skill in the art, and as defined by Sambrook et al. Molecular Cloning: A Laboratory Manual, 2 ed. Vol. 1, pp. 1.101-104, Cold Spring Harbor Laboratory Press, (1989), include use of a prewashing solution for the nitrocellulose filters 5.times.SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridization conditions of 50% formamide, 6.times.SSC at 42.degree. C. (or other similar hybridization solution, such as Stark's solution, in 50% formamide at 42.degree. C.), and washing conditions of about 60.degree. C., 0.5.times.SSC, 0.1% SDS. Conditions of high stringency are defined as hybridization conditions as above, and with washing at 68.degree. C., 0.2.times.SSC, 0.1% SDS. The skilled artisan will recognize that the temperature and wash solution salt concentration can be adjusted as necessary according to factors such as the length of the probe.

Due to the known degeneracy of the genetic code, wherein more than one codon can encode the same amino acid, a DNA sequence can vary from that shown in SEQ ID NOS: 1-8, and still encode a recombinant MSP4 and MSP5 polypeptide having the amino acid sequence of SEQ ID NOS: 9-22. Such variant DNA sequences can result from silent mutations (e.g., occurring during PCR amplification), or can be the product of deliberate mutagenesis of a native sequence.

The invention thus provides equivalent isolated DNA sequences, encoding recombinant MSP4 and MSP5 polypeptides, selected from: (a); (a) DNA comprising the nucleotide sequence of SEQ ID NOS: 1-8 (b) DNA capable of hybridization to a DNA of (a) under conditions of moderate stringency and which encode recombinant MSP4 and MSP5 polypeptides; and (c) DNA which is degenerate as a result of the genetic code to a DNA defined in (a), or (b) and which encodes recombinant MSP4 and MSP5 polypeptides. The polypeptides encoded by such DNA equivalent sequences are encompassed by the invention.

DNA that is equivalent to the DNA sequence of SEQ ID NOS: 1-8 will hybridize under moderately stringent conditions to the DNA sequence that encode polypeptides comprising amino acid sequences of SEQ ID NOS: 9-22. Examples of recombinant MSP4 and MSP5 polypeptides encoded by such DNA, include, but are not limited to, recombinant MSP4 and MSP5 polypeptide fragments and recombinant MSP4 and MSP5 polypeptides comprising inactivated N-glycosylation site(s), inactivated protease processing site(s), or conservative amino acid substitution(s), as described above. The polypeptides encoded by DNA derived from other species of Plasmodium, wherein the DNA will hybridize to the complement of the DNA of SEQ ID NOS: 1-8 are also encompassed.

Recombinant expression vectors containing a nucleic acid sequence encoding recombinant MSP4 and MSP5 polypeptides can be prepared using well known methods. The expression vectors include a recombinant MSP4 and MSP5 DNA sequence operably linked to suitable transcriptional or translational regulatory nucleotide sequences, such as those derived from a mammalian, microbial, viral, or insect gene. Examples of regulatory sequences include transcriptional promoters, operators, or enhancers, an mRNA ribosomal binding site, and appropriate sequences which control transcription and translation initiation and termination. Nucleotide sequences are "operably linked" when the regulatory sequence functionally relates to the recombinant MSP4 and MSP5 DNA sequence. Thus, a promoter nucleotide sequence is operably linked to a recombinant MSP4 and MSP5 DNA sequence if the promoter nucleotide sequence controls the transcription of the recombinant MSP4 and MSP5 DNA sequence. The ability to replicate in the desired host cells, usually conferred by an origin of replication, and a selection gene by which transformants are identified can additionally be incorporated into the expression vector.

In addition, sequences encoding appropriate signal peptides that are not naturally associated with recombinant MSP4 and MSP5 polypeptides can be incorporated into expression vectors. For example, a DNA sequence for a signal peptide (secretory leader) can be fused in-frame to the recombinant MSP4 and MSP5 nucleotide sequences so that the recombinant MSP4 and MSP5 polypeptides are initially translated as a fusion protein comprising the signal peptide. A signal peptide that is functional in the intended host cells enhances extra-cellular secretion of the recombinant MSP4 and MSP5 polypeptides. The signal peptide can be cleaved from the recombinant MSP4 and MSP5 polypeptides upon secretion of recombinant MSP4 and MSP5 polypeptides from the cell.

Expression vectors for use in prokaryotic host cells generally comprise one or more phenotypic selectable marker genes. A phenotypic selectable marker gene is, for example, a gene encoding a protein that confers antibiotic resistance or that supplies an autotrophic requirement. Examples of useful expression vectors for prokaryotic host cells include those derived from commercially available plasmids. Commercially available vectors include those that are specifically designed for the expression of proteins. These include pMAL-p2 and pMAL-c2 vectors, which are used for the expression of proteins fused to maltose binding protein (New England Biolabs, Beverly, Mass., USA).

Promoter sequences commonly used for recombinant prokaryotic host cell expression vectors include .beta.-lactamase (penicillinase), lactose promoter system (Chang et al., Nature 275:615, 1978; and Goeddel et al., Nature 281:544, 1979), tryptophan (trp) promoter system (Goeddel et al., Nucl. Acids Res. 8:4057, 1980; and EP-A-36776), and tac promoter (Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, p. 412, 1982).

Suitable host cells for expression of recombinant MSP4 and MSP5 polypeptides include prokaryotes, yeast or higher eukaryotic cells. Insect cells are preferred. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are described, for example, in Pouwels et al. Cloning Vectors: A Laboratory Manual, Elsevier, N.Y., (1985). Cell-free translation systems could also be employed to produce recombinant MSP4 and MSP5 polypeptides using RNAs derived from DNA constructs disclosed herein.

It will be understood that the present invention is intended to encompass the previously described proteins or polypeptides in isolated or purified form, whether obtained using the techniques described herein or other methods. In a preferred embodiment of this invention, the recombinant MSP4 and MSP5 polypeptides are substantially free of human tissue and human tissue components, nucleic acids, extraneous proteins and lipids, and adventitious micro-organisms, such as bacteria and viruses. It will also be understood that the invention encompasses equivalent proteins having substantially the same biological and immunogenic properties.

Depending on the use to be made of the recombinant MSP4 and MSP5 polypeptides of the invention, it may be desirable to label them. Examples of suitable labels are radioactive labels, enzymatic labels, fluorescent labels, chemiluminescent labels, and chromophores. The methods for labelling proteins of the invention do not differ in essence from those widely used for labelling immunoglobulin. The need to label may be avoided by using labelled antibody to the antigen of the invention or anti-immunoglobulin to the antibodies to the antigen as an indirect marker.

Once the recombinant MSP4 and MSP5 polypeptides of the invention have been obtained, they have been used to produce polyclonal and monoclonal antibodies reactive therewith. Thus, a protein or polypeptide of the invention can be used to immunize an animal host by techniques known in the art. Such techniques usually involve inoculation, but they may involve other modes of administration. A sufficient amount of the polypeptide is administered to create an immunogenic response in the animal host. Any host that produces antibodies to the antigen of the invention can be used. Once the animal has been immunized and sufficient time has passed for it to begin producing antibodies to the antigen, polyclonal antibodies can be recovered. The general method comprises removing blood from the animal and separating the serum from the blood. The serum, which contains antibodies to the antigen, can be used as an antiserum to the antigen. Alternatively, the antibodies can be recovered from the serum. Affinity purification is a preferred technique for recovering purified polyclonal antibodies to the antigen from the serum.

Monoclonal antibodies to the antigens of the invention can also be prepared. One method for producing monoclonal antibodies reactive with the antigens comprises the steps of immunizing a host with the antigen; recovering antibody producing cells from the spleen of the host; fusing the antibody producing cells with myeloma cells deficient in the enzyme hypoxanthine-guanine phosphoribosyl transferase to form hybridomas; selecting at least one of the hybridomas by growth in a medium comprising hypoxanthine, aminopterin, and thymidine; identifying at least one of the hybridomas that produces an antibody to the antigen; culturing the identified hybridoma to produce antibody in a recoverable quantity; and recovering the antibodies produced by the cultured hybridoma.

These polyclonal or monoclonal antibodies can be used in a variety of applications. Among these is the neutralization of corresponding proteins. They can also be used to detect Plasmodium parasite antigens in biological preparations or in purifying corresponding proteins, glycoproteins, or mixtures thereof, for example, when used in an affinity chromatographic column.

The recombinant MSP4 and MSP5 polypeptides can be used as antigens to detect the presence of antibodies specific for malaria parasite in biological samples and to evaluate the levels of such antibodies in those biological samples, which constitute a signal for current or previous infection. Such biological samples of course include human tissue and human cells, as well as biological fluids, such as human body fluids, including human sera. When used as a reagent in an immunoassay for determining the presence or concentration of the antibodies to malaria, the antigens of the present invention provide an assay that is convenient, rapid, sensitive, and specific.

More particularly, the antigens of the invention can be employed for the detection of malaria by means of immunoassays that are well known for use in detecting or quantifying humoral components in fluids. Thus, antigen-antibody interactions can be directly observed or determined by secondary reactions, such as precipitation or agglutination. In addition, immunoelectrophoresis techniques can also be employed. For example, the classic combination of electrophoresis in agar followed by reaction with anti-serum can be utilized, as well as two-dimensional electrophoresis, rocket electrophoresis, and immunolabeling of polyacrylamide gel patterns (Western Blot or immunoblotting). Other immunoassays in which the antigens of the present invention can be employed include, but are not limited to, radioimmunoassay, competitive immunoprecipitation assay, enzyme immunoassay, and immunofluorescence assay. It will be understood that turbidimetric, colorimetric, and nephelometric techniques can also be employed. An immunoassay based on Western Blot technique is preferred.

Immunoassays can be carried out by immobilizing one of the immunoreagents, either an antigen of the invention or an antibody of the invention to the antigen, on a carrier surface while retaining immunoreactivity of the reagent. The reciprocal immunoreagent can be unlabeled or labelled in such a manner that immunoreactivity is also retained. These techniques are especially suitable for use in enzyme immunoassays, such as enzyme linked immunosorbent assay (ELISA) and competitive inhibition enzyme immunoassay (CIEIA).

When either the antigen of the invention or antibody to the antigen is attached to a solid support, the support is usually a glass or plastic material. Plastic materials moulded in the form of plates, tubes, beads, or disks are preferred. Examples of suitable plastic materials are polystyrene and polyvinyl chloride. If the immunoreagent does not readily bind to the solid support, a carrier material can be interposed between the reagent and the support. Examples of suitable carrier materials are proteins, such as bovine serum albumin, or chemical reagents, such as gluteraldehyde or urea. Coating of the solid phase can be carried out using conventional techniques.

The invention provides immunogenic recombinant MSP4 and MSP5 polypeptides, and more particularly, protective polypeptides for use in the preparation of vaccine compositions against malaria. These polypeptides can thus be employed as vaccines by administering the polypeptides to a mammal susceptible to malaria infection. Conventional modes of administration can be employed. For example, administration can be carried out by oral, sublingual, respiratory, or parenteral routes. Intradermal, subcutaneous, intramuscular, and intravenous routes of administration are preferred when the vaccine is administered parenterally.

The major purpose of the immune response in a malaria-infected mammal is to inactivate the malaria parasites and to facilitate malaria parasite killing and clearance of parasite infected red blood cells. The B-cell arm of the immune response has the major responsibility for inactivating blood-stage malaria parasites. The principal manner in which this is achieved is by neutralization of infectivity (inhibition of erythrocyte invasion) and antibody dependent cellular cytotoxicity (ADCC). The target antigen must be conserved to be effective against re-infection with other parasite strains, and capable of inducing T helper cell activity (CD4+) to generate a long-lived memory response. T cell mediated mechanism for destruction of parasite infected cells is provided by cytotoxic (CD8+) T lymphocytes (CTL) that could recognize recombinant MSP4 and MSP5 antigens expressed in combination with Class I histocompatibility antigens at the surface of hepatic cells in the preerythrocytic phase of infection.

Following is a vaccine, which includes (A) the natural signal sequence (B) a C-terminal His tag and (C) the acid repeat region that resembles that of the P. falciparum antigen most strongly correlated with protective antibody responses in the field (MSP4p20).

TABLE-US-00004 [SEQ ID NO: 30] MKVAYFLSVLDLLIIFSLYFDGRRSAFAGIAACIRHGRILGEGGE/QNST PGSGGQTGDHSAEAENGDYNEQGDDHGDDHGDDHGDDHGDEQDGEDYDDA EDDDLYELSEVDENANLCLDNNGGCGDDKICENLGKGIVKCLCKPGYKLV GTECVEHHHHHH

The ability of the recombinant MSP4 and MSP5 polypeptides and vaccines of the invention to induce protective levels of neutralizing antibody (i.e., antibodies elicited during a humoral response to the antigen, which directly blocks the ability of the pathogen to infect red blood cells) in a host can be enhanced by emulsification with an adjuvant (immunostimulator), incorporating in a liposome, coupling to a suitable carrier, or by combinations of these techniques. For example, the recombinant MSP4 and MSP5 polypeptides of the invention can be administered with a conventional adjuvant, such as aluminium phosphate and aluminium hydroxide gel, in an amount sufficient to potentiate humoral or cell-mediated immune responses in the host.

The immunization schedule will depend upon several factors, such as the susceptibility of the host to infection and the age of the host. A single dose of the vaccine of the invention can be administered to the host or a primary course of immunization can be followed in which several doses at intervals of time are administered. Subsequent doses used as boosters can be administered as needed following the primary course.

The recombinant MSP4 and MSP5 proteins, polypeptides, and vaccines of the invention can be administered to the host in an amount sufficient to induce immune responses that prevent or inhibit parasite infection and replication in vivo so as to reduce the parasite burden in the host and diminish clinical symptoms. An immunogenic response can be obtained by administering the polypeptides of the invention to the host in amounts ranging from 10 to 500 micrograms per dose, preferably about 50 to 100 micrograms per dose. The proteins and vaccines of the invention can be administered together with a physiologically acceptable carrier. For example, a diluent, such as water or a saline solution, can be employed.

Another aspect of the invention provides a method of DNA vaccination. The method also includes administering any combination of the nucleic acids encoding recombinant MSP4 and MSP5 polypeptides, the proteins and polypeptides per se, with or without carrier molecules, to an individual. In embodiments, the individual is an animal, and is preferably a mammal. More preferably, the mammal is selected from the group consisting of a human, a dog, a cat, a bovine, a pig, and a horse. In an especially preferred embodiment, the mammal is a human.

Those of skill in the art are cognizant of the concept, application, and effectiveness of nucleic acid vaccines (e.g., DNA vaccines) and nucleic acid vaccine technology as well as protein and polypeptide based technologies. The nucleic acid based technology allows the administration of nucleic acids encoding recombinant MSP4 and MSP5 polypeptides, naked or encapsulated, directly to tissues and cells without the need for production of encoded proteins prior to administration. The technology is based on the ability of these nucleic acids to be taken up by cells of the recipient organism and expressed to produce an immunogenic determinant to which the recipient's immune system responds. Typically, the expressed antigens are displayed on the surface of cells that have taken up and expressed the nucleic acids, but expression and export of the encoded antigens into the circulatory system of the recipient individual is also within the scope of the present invention. Such nucleic acid vaccine technology includes, but is not limited to, delivery of naked DNA and RNA and delivery of expression vectors encoding recombinant MSP4 and MSP5 polypeptides. Although the technology is termed "vaccine", it is equally applicable to immunogenic compositions that do not result in a protective response. Such non-protection inducing compositions and methods are encompassed within the present invention.

Although it is within the present invention to deliver nucleic acids encoding recombinant MSP4 and MSP5 polypeptides and carrier molecules as naked nucleic acid, the present invention also encompasses delivery of nucleic acids as part of larger or more complex compositions. Included among these delivery systems are viruses, virus-like particles, or bacteria containing the nucleic acid encoding recombinant MSP4 and MSP5 polypeptides. Also, complexes of the invention's nucleic acids and carrier molecules with cell permeabilizing compounds, such as liposomes, are included within the scope of the invention. Other compounds, such as molecular vectors (EP 696,191, Samain et al.) and delivery systems for nucleic acid vaccines are known to the skilled artisan and exemplified in, for example, WO 93 06223 and WO 90 11092, U.S. Pat. No. 5,580,859, and U.S. Pat. No. 5,589,466 (Vical's patents), which are incorporated by reference herein, and can be made and used without undue or excessive experimentation.
 

Claim 1 of 11 Claims

1. A purified nucleic acid molecule comprising SEQ ID NO: 7.
 

____________________________________________
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 ]