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Title:  Malaria merozoite antigen subunit vaccine

United States Patent:  6,417,341

Inventors:  Anders; Robin Fredric (North Melbourne, AU); Crewther; Pauline Elizabeth (North Carlton, AU); Leet; Mary Shu Mai (Flemington, AU); Hodder; Anthony Neil (Ocean Grove, AU); Pye; David (Bullengarook, AU)

Assignee:  Saramane Pty., Ltd. (Victoria, AU)

Appl. No.:  687387

Filed:  October 7, 1996

PCT Filed:  February 3, 1995

PCT NO:  PCT/AU95/00049

371 Date:  October 7, 1996

102(e) Date:  October 7, 1996

PCT PUB.NO.:  WO95/21192

PCT PUB. Date:  August 10, 1995

Abstract

An immunogenic polypeptide for use in inducing an immune response against Plasmodium infection comprises an amino acid sequence corresponding to a non-full length fragment of the apical membrane antigen 1 (AMA-1) of Plasmodium species which does not include a transmembrane domain thereof, and which is stabilised by folding thereof. Production of the immunogenic polypeptide by expression of a recombinant DNA molecule in a host cell, and methods and compositions using the immunogenic polypeptide are disclosed.

SUMMARY OF THE INVENTION

According to the present invention there is provided a non-naturally occurring immunogenic polypeptide comprising an amino acid sequence corresponding to a non-full length fragment of the apical membrane antigen 1 (AMA-1) of Plasmodium species, said polypeptide not including an amino acid sequence corresponding to the transmembrane domain of AMA-1 and being stabilised by folding.

Folding of the polypeptide is preferably achieved by generation of intramolecular disulphide bonds which stabilise the polypeptide in a conformation required for inducing a protective immune response.

The amino acid sequence of the immunogenic polypeptide corresponding to a non-full length fragment of AMA-1, may be a fragment corresponding to the mature ectodomain of the antigen not including the transmembrane domain thereof, or a portion thereof.

Preferably, the AMA-1 fragment is a fragment of P. falciparum AMA-1, however the present invention also extends to AMA-1 fragments of other Plasmodium species, including P. vivax, P. fragile and P. chabaudi adami

Preferably also, the AMA-1 fragment is a fragment which contains at least two of the conserved cysteine residues of the AMA-1 molecule. It will be appreciated, however, that the fragment may contain from two to all of the sixteen conserved cysteine residues of AMA-1.

The immunogenic polypeptide in accordance with this invention is preferably produced by recombinant DNA technology.

Accordingly, in another aspect the present invention provides a recombinant DNA molecule comprising a nucleotide sequence which encodes a polypeptide comprising an amino acid sequence corresponding to a non-full length fragment of the apical membrane antigen 1 (AMA-1) of Plasmodium species, said polypeptide not including an amino acid sequence corresponding to the transmembrane domain of AMA-1.

The recombinant DNA molecule may also comprise an expression control sequence operatively linked to the nucleotide sequence as described above.

The present invention also extends to a recombinant DNA cloning vector containing a recombinant DNA molecule as broadly described above, as well as to a host cell such as E. coli containing such a recombinant DNA molecule or recombinant DNA cloning vector. Such a host cell, of course, provides means for the production of the immunogenic polypeptide of the present invention using techniques which are well known to persons skilled in this art.

As an alternative to the prokaryotic host cell, the recombinant DNA molecule of this invention may be cloned using a eukaryotic host cell such as yeast, with folding and disulphide bond formation being achieved by passage of the expressed polypeptide through the secretory pathway of the cells.

It has been found that a stabilised conformation is required to enable the AMA-1 fragment to induce a protective response, and accordingly where the immunogenic fragment is produced as a recombinant product, particularly in a prokaryotic expression system, refolding of the product with disulphide bond formation is required to produce an efficacious immunogenic polypeptide.

In work leading to the present invention, the present inventors have discovered a fragment of AMA-1 corresponding to the mature ectodomain which, when expressed in E. coli, purified by Ni-chelate chromatography, refolded by dilution in the presence of reduced and oxidized glutathione, concentrated by anion-exchange chromatography, and injected formulated with an appropriate adjuvant, induces an immune response which protects against malaria.

Accordingly, the present invention extends to a vaccine composition, or composition for inducing an immune response in a host, comprising an immunogenic polypeptide as broadly described above, and a pharmaceutically acceptable carrier or diluent. Such a vaccine composition preferably also comprises an adjuvant.

The formulation of such vaccine compositions is well known to persons skilled in this field. Suitable pharmaceutically acceptable carriers and/or diluents include any and all conventional solvents, dispersion media, fillers, solid carriers, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art, and it is described, by way of example, in Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Company, Pennsylvania, USA. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the vaccine compositions of the present invention is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

The present invention also provides a method for actively immunizing a host against Plasmodium infection, which comprises administering to the host an effective amount of a vaccine composition as described above.

The active component is administered in prophylactically effective amounts. A prophylactically effective amount means that amount necessary at least partly to attain the desired effect, that is to induce an immune response against Plasmodium infection. Such amounts will depend, of course, on individual patient parameters including age, physical condition, size, weight and concurrent treatment. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

Claim 1 of 10 Claims

What is claimed is:

1. A DNA expression vector comprising a polynucleotide encoding a polypeptide consisting of AMA-1B or fragments thereof from plasmodium species P. falciparum or P. vivax, and

wherein said AMA-1B polypeptide contains 16 conserved cysteine residues and said fragments thereof are comprised of from 2 to 16 conserved cysteine residues of a mature ectodomain of apical membrane antigen (AMA-1).
 


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