Identification of antigenically active tryptic fragments of apical membrane antigen-1 (AMA1) of Plasmodium chabaudi malaria: strategies for assembly of immunologically active peptides
Introduction
Malaria continues to be one of the major health problems in many tropical countries with potentially one-third of the world’s population at risk from infection. In some countries, the incidence of disease and mortality is increasing; important contributing factors being the appearance of insecticide-resistant mosquito vectors and drug-resistant parasites. Because of this the development of an effective vaccine for malaria is a priority.
Apical membrane antigen-1 (AMA1) is one of several merozoite surface antigens rated highly as potential vaccine candidates against malaria. This protein can be detected in blood-stage parasites just prior to schizont rupture. A mature form of AMA1 can be detected in the rhoptries of merozoites and about the time of schizogony a smaller N-terminally processed form spreads circumferentially around the merozoite surface [1]. Although AMA1 is expressed only for a narrow time interval around the time of schizogony it is a target of the protective host immune response. Furthermore, both native and recombinant forms of the antigen have been able to induce various levels of protection against challenge from Plasmodium parasites in various animal models [2], [3], [4], [5], [6]. The vaccine potential of the Plasmodium falciparum AMA1 ectodomain is currently in the early stages of clinical assessment.
The primary structure of AMA1, found in various Plasmodium species, contains 16 conserved cysteine residues [7], [8], [9] which form eight intra-molecular disulphide bonds [10]. The protective immune response against AMA1 is largely directed toward disulphide bond-dependent conformational epitopes as the reduced and alkylated antigen gives poor protection against parasite challenge in vaccine trials [5] and is poorly recognised by antibodies from the serum of hyperimmune individuals who live in malaria endemic regions [11]. Using antibodies from hyperimmune mice we were interested in identifying and then characterising the vaccine potential of disulphide-linked peptide fragments from trypsin-digested recombinant P. chabaudi adami AMA1.
An antigenically active fragment was identified and found to represent part of a loop region, closed by a disulphide bond, of a putative sub-domain of AMA1. Two synthetic peptide-based constructs representing this antigenically active tryptic fragment were assembled. One of the constructs was made using two individually synthesised peptide chains linked through cysteine residues to yield a peptide heterodimer. The second construct was synthesised as a linear 45 amino acid residue peptide which was then folded by in vitro oxidation of the two cysteine residues to yield a closed loop. In this way, both constructs were assembled to incorporate a disulphide bond of the same assignment as that present within the native sequence. The two peptide constructs were examined for immunogenic activity and vaccine potential by inoculation into mice.
Section snippets
Peptide synthesis
Chemicals used were of analytical grade or better. Fmoc amino acids, N,N′-dimethylformamide (DMF), piperidine, trifluoracetic acid (TFA), O-benzotriazole-N,N,N′,N′-tetra-methyl-uronium-hexafluorophosphate (HBTU), 1-hydroxybenzotriazole (HOBt) and diisopropylethylamine (DIPEA) were obtained from Auspep Pty. Ltd. (Melbourne, Australia). Phenol and triisopropylsilane (TIPS) were from Aldrich (Milwaulke, WI) and trinitrobenzylsulphonic acid (TNBSA) and 2,2′-dithiodipyridine (DTDP) from Fluka
Fractionation of AMA1 tryptic fragments
Trypsin digestion of AMA1 and subsequent separation by reversed-phase HPLC yields a well-resolved tryptic fingerprint map [10]. Screening of individual fractions by ELISA indicated that two fractions (56 and 58) contained antigenic activity. One of these contained a peptide complex containing several disulphide-linked peptides, which could not be clearly defined and was therefore not investigated further. Mass spectral and Edman degradation analyses of the second fraction, however, revealed two
Discussion
The use of synthetic peptides as vaccine candidates has attracted a great deal of interest over the last two decades (for reviews, see [15], [16], [17]) but no peptide-based vaccine is yet in widespread use. Some of the reasons for the, as yet, unrealised potential of peptide-based vaccines centre around the poor immunogenicity exhibited by many peptide immunogens. This poor immunogenicity may be due to (i) a lack of conformational similarity between the peptide and the sequence within the
Acknowledgements
This work was supported by a grant from the Cooperative Research Centre for Vaccine Technology and a grant from the National Health and Medical Research Council of Australia. We would like to express our appreciation to Mary Macris who was always willing to carry out the mass spectral analyses for us.
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Present address: Department of Biochemistry, La Trobe University, Boondoora 3086, Australia.