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,
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
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
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
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
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
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
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