Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1996-10-07
2002-07-09
Minnifield, Nita (Department: 1645)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C424S184100, C424S268100, C424S272100, C424S130100, C424S278100, C424S192100, C435S252300, C435S173300, C435S069100, C435S069300, C435S342000, C435S252330, C435S235100, C435S325000, C536S023100, C536S023700, C530S350000, C530S300000
Reexamination Certificate
active
06417341
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to a non-naturally occurring immunogenic polypeptide derived from a merozoite antigen of Plasmodium species, and to methods of production thereof. The invention also relates to the use of this immunogenic polypeptide in a subunit vaccine against malaria.
BACKGROUND OF THE INVENTION
Apical membrane antigen 1 (AMA-1) of Plasmodium species (previously also referred to as RMA-1) is a merozoite protein that is considered a leading candidate for inclusion in a malaria vaccine (see International Patent Publication No. WO 89/07645). AMA-1 is found associated with the merozoite apical organelles but subsequently it relocates to the merozoite surface prior to merozoite invasion of the erythrocyte. Early studies with PK66, the homologue of AMA-1 expressed in the simian parasite
Plasmodium knowlesi
, showed that merozoite invasion could be inhibited with a monoclonal antibody to AMA-1
1,2
and that immunization with the purified parasite antigen provided partial protection of rhesus monkeys against infection with
P. knowles
3
.
The AMA-1 gene sequence has been determined for a number of different isolates of
P. falciparum
4,5
and for several other Plasmodium species
6-8
. The deduced amino acid sequence is typical of a type I integral membrane protein with an NH
2
-terminal signal sequence and a presumed transmembrane domain towards the COOH-terminus. A comparison of the various AMA-1 sequences shows this to be a relatively conserved protein which contrasts with MSA-1 and MSA-2, two other well characterised merozoite surface antigens. In the large NH
2
-terminal, presumably ectodomain, there are 16 cysteine residues that are conserved in all AMA-1 sequences. This indication that folding of the ectodomain is stabilised by intramolecular disulphide bonds is supported by the observation that the mobility of AMA-1 on SDS-PAGE varies depending on whether or not the sample buffer contains a reducing agent.
Because AMA-1 is relatively conserved in the genus it has been possible to identify clones corresponding to the homologue in other species of Plasmodium. Thus, AMA-1 of the simian malaria,
P. fragile
7
and the murine malaria
P. chabaudi adami
6
are available for studies of vaccine efficacy using appropriate host-parasite combinations. Because of the presumed disulphide bonded structure, the AMA-1s of both
P. fragile
and
P. chabaudi
were expressed in insect cells using recombinant baculovirus to produce antigen for use in preclinical vaccine trials. The
P. fragile
AMA-1 expressed in this way was isolated by lentil lectin chromatography and then anion- and cation-exchange chromatography. Saimiri monkeys immunized with the semi-purified molecule were partially protected against challenge with
P. fragile
. In this trial the degree of protection among immunized monkeys was positively correlated with the titre of antibodies induced by immunization. All surviving monkeys were drug-treated to eliminate persisting parasitaemias and re-challenged with
P. falciparum
. Three of four control monkeys developed transient, low parasitaemias whereas no
P. falciparum
parasitaemias were detected in the five immunized animals that were re-challenged. Thus, immunization with
P. fragile
AMA-1 provides protection against the homologous parasite and also may provide some cross-immunity against
P. falciparum
over and above that provided by a prior infection with
P. fragile.
Subsequently, the protective efficacy of AMA-1 was studied in mice using the
P. chabaudi
homologue of AMA-1. Initially, the full-length
P. chabaudi
AMA-1 was expressed in insect cells using recombinant baculovirus and a vaccine trial was carried out using a lysate of insect cells infected with the recombinant baculovirus (In preliminary experiments cell lysates induced antibody responses that approached those induced by the semi-purified
P. fragile
antigen). C3H/He mice immunized with insect cell lysates containing the
P. chabaudi
AMA-1 were protected against challenge with
P. chabaudi
. None of the immunized mice died and peak parasitaemias were very much lower than those in mice that were immunized with lysates of insect cells infected with non-recombinant baculovirus.
Although it may be possible to base a vaccine against
P. falciparum
infections of humans on baculovirus-expressed full-length
P. falciparum
AMA-1, there are several disadvantages to using this approach for a human vaccine. First, it is more expensive to use a eukaryotic expression system than it is to use
E. coli
as the host cell. Second, the antigen is glycosylated in insect cells and this introduces microheterogeneity into the protein which could make it difficult to manufacture reproducible batches of purified material. Third, AMA-1 is a type I integral membrane protein and therefore the full-length molecule would be difficult to work with in aqueous buffers without added detergent. In view of the above disadvantages, the present inventors have examined the possibility of producing AMA-1 in
E. coli
in a form soluble in aqueous buffers and capable of inducing a protective immune response.
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, parti
Anders Robin Fredric
Crewther Pauline Elizabeth
Hodder Anthony Neil
Leet Mary Shu Mai
Pye David
Minnifield Nita
Saramane Pty. Ltd.
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