1 Department of Biochemistry, La Trobe University, Bundoora, 3083, Victoria, 2 Cooperative Research Centre for Diagnostics, 3 Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria and 4 Cooperative Research Centre for Vaccine Technology, Australia
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Abstract |
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Keywords: combined phage display/epitope mapping/malaria antigen/Plasmodium falciparum
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Introduction |
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Furthermore, evidence was presented to show that the region of MABEL that shared homology with AMA1 was the region that bound to erythrocytes when expressed on COS cells, reinforcing the suggestion that AMA1 is involved in the invasion process (Noe and Adams, 1998). AMA1 is a type I integral membrane protein and has 16 cysteine residues in the ectodomain that are spatially conserved in all Plasmodium spp. studied (Peterson et al., 1990
; Cheng and Saul, 1994
; Dutta et al., 1995
). The cysteine connectivities within AMA1 suggest that the ectodomain of the protein can be divided into four domains (pro-domain, domain-1, domain-2 and domain-3) restricted by intramolecular disulfide bonds (Hodder et al., 1996
). The AMA1 protein of P.falciparum differs slightly from AMA1 of other species in that the prosequence contains a 55-residue insert absent from AMA1 molecules from other species (Peterson et al., 1990
). A proteolytic event removes a small fragment from the N-terminus of AMA1, reducing the initial polypeptide from 80 to 62 kDa, at about the time the protein migrates onto the surface of the merozoite (Narum and Thomas, 1994
). The functional consequences of this processing event, as well as the position of the precise cleavage sites within AMA1, are unknown and further studies with defined reagents are required to elucidate the mechanisms involved.
Antibodies that bind to AMA1 and inhibit the invasion of merozoites have been described (Deans et al., 1982; Kocken et al., 1998
) and immunization of mice and monkeys with either native or recombinant AMA1 induces a protective immune response against their respective plasmodial infections (Deans et al., 1988
; Collins et al., 1994
; Crewther et al., 1996
; Anders et al., 1997
). These observations are further supported by adoptive transfer experiments (Anders et al., 1997
). Additional immunization studies indicate that the correct disulfide conformation is required for protective antibody responses (Anders et al., 1997
).
We are in the process of developing a panel of monoclonal antibodies in order to study the function of P.falciparum AMA1 and its potential role as a mediator of erythrocyte invasion. To this end we have generated two MAbs which recognize both recombinant refolded P.falciparum AMA1 and the native molecule expressed in parasites. Preliminary studies indicated that one of these Mabs only binds to non-reduced AMA1 whereas the other recognizes both reduced and non-reduced forms of the protein. Thus both MAbs recognize different surface features of AMA1. In order to characterize these MAbs and determine the precise nature of the epitope on AMA1, we used the approach of phage display. Since the first reports that foreign peptides can be displayed on the surface of filamentous phage (Smith, 1985), the display of proteins, protein domains and peptides on bacteriophage has proved to be a powerful approach to elucidate the molecular nature of many different proteinprotein interactions (Wilson and Finlay, 1998
). Phage displayed antibody fragments have been selected against a variety of antigens including AMA1 (Fu et al., 1997
) and cancer antigens (Cai and Garen, 1996
), growth factor mimotopes have been generated (Wrighton et al., 1996
) and receptorligand interactions have been elucidated using this technology (Kiewitz and Wolfes, 1997
). Phage display technology relies on the fusion of a DNA sequence encoding a protein or polypeptide with the gene encoding a structural protein present on bacteriophage. When the phage protein gpIII is expressed it is fused with the protein or peptide sequence of interest, which is then incorporated into a phage particle as part of the natural phage assembly process. The result is a phage displaying the fusion protein on its surface and containing within the phage particle the genetic material encoding for the displayed peptide or protein (Smith and Scott, 1993
).
In this paper, we describe the display of defined domains and fragments of AMA1 in the context of bacteriophage M13 and show that a library comprising random fragments of the molecule is an efficient method of epitope mapping antibodies specific for linear and conformationally constrained epitopes of AMA1. We also demonstrate that the isolation of mimotopes from random peptide libraries displayed on phage, specific for one of the antibodies, can allow the fine mapping of a linear epitope to within a few residues. In addition to identifying epitopes, these fragment libraries will have great potential in delineating functional domains of AMA1 which may be involved in erythrocyte invasion.
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Materials and methods |
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Monoclonal antibodies to P.falciparum AMA1 3D7 strain were generated by standard procedures (Harlow and Lane, 1988). Briefly, mice were immunized with recombinant, refolded P.falciparum AMA1, spleens excised and B cells isolated. Fusions were carried out and individual clones isolated. The ability of secreted antibodies to react with AMA1 was assayed in an ELISA format, immunoblotting of native AMA1 from cultured parasites and immunofluorescence assay of 3D7 schizonts and merozoites. Two antibodies were selected from the panel generated: 5G8, recognizing a reduction insensitive epitope, and 1F9, which reacted with AMA1 only in the non-reduced state.
Oligonucleotide primers were generated in order to amplify the entire ectodomain and individual subdomains as described by Hodder et al. (Hodder et al., 1996). PCR reactions were carried out as previously described (Deans et al., 1984
) and the products restricted with appropriate enzymes and ligated into the correspondingly restricted phagemid vector pHENH6. This phagemid vector contains a copy of the M13 bacteriophage geneIII and a multicloning site between the periplasmic signal sequence and the functional geneIII protein (gpIII) (a kind gift from Peter Waterhouse, CSIRO Plant Industry, Canberra, Australia). Ligation of dsDNA into the multicloning site allows expression of the polypeptide of interest with the geneIII product and subsequent display of the fusion protein on M13 bacteriophage after phage rescue. The ligation product was transformed into Escherichia coli TG1 cells (Stratagene) by electroporation and the resulting clones were assayed for the presence of the AMA1 domains by PCR using autologous primers. Clones containing appropriate AMA1 fragments were cultured in 10 ml of 2TY broth containing 50 µg/ml ampicillin to an optical density (OD) of 0.6 at 600 nm and 1x1011 PFU M13 helper phage was added and incubated for 30 min at 37°C. This culture was then transferred to 200 ml of `Super Broth' containing 70 µg/ml kanamycin and incubated at 30°C, shaking at 200 r.p.m. for 16 h. Phage were isolated by PEG precipitation as previously described (Adda et al., 1999
) and phage titres established.
Generation and affinity panning of phage displayed AMA1 gene library
The AMA1 ectodomain PCR product was digested with DNase I for various times and the digestion products were separated by agarose electrophoresis. The time point which showed the broadest size distribution was chosen, the DNA was purified and ragged ends blunted with the aid of Vent polymerase. pHENH6 phagemid vector was restricted with PstI and similarly blunted with Vent polymerase. The blunt products of the randomly fragmented AMA1 ectodomain were then ligated into the treated pHENH6 vector and the ligation products were transformed into E.coli TG-1 cells by electroporation. The diversity of the gene library was estimated to be in the order of 5x108 individual clones by limiting dilution of the transformed bacterial cells. The essential random distribution of fragments in the library was assayed by PCR and sequencing of 12 individual clones. Coverage appeared to be random and clones were isolated with fusions in both directions and in a random set of frames. The library was displayed on M13 bacteriophage and the phage isolated by PEG precipitation in the same manner as the defined fragments. Affinity panning of the phage library was carried out as previously described (Adda et al., 1999). Briefly, 1.0 µg of MAb in 100 µl of PBS was added to each of 10 wells in a 96-well ELISA plate. The plate was washed x2 in PBS0.2% Tween 20 and 1x 1012 phage particles in 100 µl of PBS were added. The plate was incubated at 20°C for 1 h and the non-adherent phage particles were washed away by washing x2 in PBSTween 20. Adherent phage were eluted by the addition of 0.2 M glycine, pH 2.2. The eluate was immediately added to 10 ml of log phase (OD 0.6) E.coli TG-1 cells and incubated at 37°C for 1 h to allow the eluted phage to infect the E.coli. Ampicillin was added to 50 µg/ml and the culture was incubated for a further 1 h at 37°C, then 5x1011 M13 helper phage were added and the culture incubated for a further 1 h at 37°C. The culture was added to 200 ml of SB containing ampicillin (50 µg/ml) and kanamycin (70 µg/ml) and incubated at 30°C, shaking at 200 r.p.m. for 16 h. Phage were harvested as previously described and another round of panning was carried out with the freshly isolated phage. Four rounds of panning were carried out on each MAb. Phage titrations were carried out after each round of panning to ensure that equal numbers of phage particles were added to each round of panning. Individual clones were selected after every round of panning, their inserts amplified by PCR and sequenced in order to establish the identity of the fragments of AMA1 binding to each MAb.
Panning a random peptide library on MAb5G8
A random peptide library was also panned on the 5G8 MAb. This library was a kind gift from Professor George Smith (University of Missouri, Columbia, MO). This library is in fd-tet phage background and requires no phage rescue and produces phage constitutively. An E.coli K91 culture containing the 15mer peptide library was propagated overnight in 200 ml of 2xTY at 37°C and phage were harvested in the same manner as previously described. Phage panning was carried out as described above and by Adda et al. (Adda et al., 1999). Isolated clones were amplified by PCR and the products sequenced in order to establish the identity of the peptide fused to the gpIII protein. The specificity of the interaction between these peptides and MAb5G8 was further demonstrated by ELISA and immunoblotting of the phage preparations.
Construction of phage displaying defined mutations within the AYP motif
The following oligonucleotides were designed to introduce mutations in the amino acid sequence of the tripeptide motif:
AYP:AGTTTCGGCCCCAGCGGCCCCGTCTATTGGATATGCGTGTTG
AYA:AGTTTCGGCCCCAGCGGCCCCGTCTATTGCATATGCGTGTTG
AAA:AGTTTCGGCCCCAGCGGCCCCGTCTATTGCTGCTGCGTGTTG
AAP:AGTTTCGGCCCCAGCGGCCCCGTCTATTGGTGCTGCGTGTTG
The SfiI restriction sites are shown in bold. Oligonucleotides were synthesized and used as the 3' primers in a polymerase chain reaction with AMA1 DNA as a template. The common 5' primer corresponding to the YQQEDSG amino acid sequence of AMA1 was TCACTCGGCCGACGGGGCCTACCAACAAGAAGATTCAGGAG. Products of PCR were extracted from 15% polyacrylamide gels, purified using a Qiaex II kit (Qiagen) and cleaved with SfiI restriction endonuclease. The cleaved products were ligated at 16°C overnight at a 3:1 molar ratio of insert to fUSE5 phage vector (Smith and Scott, 1993). Following purification with Qiaex II the ligated products were transformed into MC1061 E.coli cells (Stratagene) using a Bio-Rad Gene Pulser. Clones from each ligation were sequenced using primers within the vector sequence flanking the insert site (CCTCATAGTTAGCGTAACG) and DNA with the correct sequences subsequently re-transformed into K91 E.coli cells (a kind gift from G.Smith, University of Missouri). Phage were produced and used as described above.
Immunoblotting of phage displayed AMA1 fragments
2x1012 phage particles were separated by SDSPAGE and transferred on to an Immobilon PVDF membrane. The membrane was blocked in 10% skimmed milk and the primary antibody (either monoclonal or rabbit anti-AMA1 polyclonal serum) was added for 1 h. The membrane was washed for 1 h in PBS containing 0.2% Tween 20 with three changes. Secondary antibody was then added (either HRP-conjugated goat anti-rabbit or rabbit anti-mouse), incubated for 30 min and the washing procedure was repeated. The blots were visualized by enhanced chemiluminescence.
ELISA of phage displayed AMA1 fragments
Anti-AMA1 antibodies (50 µl) were added to wells of a Maxisorp (Nunc) ELISA plate at a concentration of 5 µg/ml for the MAbs or 1/1000 dilution for the antiserum and allowed to absorb for 1 h at 37°C. The plates were then blocked with 10% skimmed milkPBS for 1 h at 37°C and subsequently washed in PBS0.2%Tween 20 three times. 1x1012 phage particles (or dilutions thereof) were added to the wells and incubated at 20°C for 1 h, followed by washing and addition of HRP-conjugated anti-M13 MAb at the appropriate dilution and incubation at 20°C for 30 min. The plates were then washed four times and H2O2 substrate and OPD colour reagent (Sigma) were added according to the manufacturer's instructions. The reactions were stopped by the addition of 0.5 M H2SO4 and the plates were read spectrophotometrically. Results were displayed graphically. All reactions were carried out in duplicate. In some instances peptide competition assays were carried out. In this case dilutions of peptide were added to known amounts of phage prior to the addition of phage to the plate. The assays were then carried out in the same manner as described.
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Results |
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Determination of the cysteine connectivities allowed the assignment of four putative domains, pro-sequence, domain-1, domain-2 and domain-3, in the AMA1 ectodomain that is exposed on the exterior surface of the merozoite (Hodder et al., 1996). We designed specific oligonucleotides to amplify fragments corresponding to these domains with an additional two fragments, one comprising the whole ectodomain and the other containing the pro-sequence plus domain-1 (Figure 1A
). These fragments were cloned into the phagemid expression vector pHENH6, transformed into E.coli and phage expressing the corresponding protein fragments were harvested.
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A gene fragment corresponding to the ectodomain of AMA1 was amplified and digested with DNase I for various times. Conditions that yielded fragments between 50 and 400 bp were used to produce a pool of AMA1 ectodomain gene fragments, (Figure 2A), which were then cloned into pHENH6 to give a library size of ~108 independent clones. Several clones picked at random and sequenced revealed an essentially random distribution in orientation and sequence (Figure 2B
), thus confirming that our library was representative of AMA1 ectodomain sequence. It should be noted that, as expected, most of the clones were out of frame; however, the size of the library is such that there should be adequate coverage of the AMA1 for practical purposes.
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In an effort to define more precisely the epitope of MAb5G8, we panned a library of phage displaying a 15-residue random peptide library. Four rounds of panning resulted in significant enrichment of phage that bound specifically to MAb5G8 as assessed by ELISA, whereas no binding was seen with pooled phage from any of the four rounds of panning when MAb5G8 was replaced with BSA (Figure 5A). When the proteins of selected phage clones were separated by SDSPAGE and blotted on to nylon membranes, MAb5G8 recognized the peptide fused to gpIII (Figure 5B
, clones 1, 2, 3); however, there was no binding of MAb5G8 to phage displaying a peptide that was isolated by panning on an irrelevant protein (Figure 5B
, clone C). MAb5G8 also recognized bacterially expressed AMA1 under the same conditions (Figure 5B
, lane A). Selected clones after four rounds of panning (Figure 5D
, clones 1, 2, 3, 4) were shown to bind to MAb 5G8-coated ELISA plates, in a dose-dependent manner, whereas a clone containing a peptide isolated by panning the same library on an unrelated protein (Figure 5D
, control) was unable to bind MAb5G8 even at 1011 CFU/ml. Evidence that these phage clones were binding specifically to the antigen binding site of MAb5G8 was obtained by competing the interaction between MAb5G8 and the phage displaying the binding peptide with recombinant AMA1. Incorporation of recombinant AMA1 in an ELISA assay consisting of phage displaying a binding peptide and MAb5G8 resulted in a 3-fold reduction of binding of two individual phage clones isolated from the fourth round of panning, to immobilized MAb5G8 (Figure 5C
), although this inhibition of phage binding to MAb5G8 by AMA1 was not complete at the concentrations examined.
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Confirmation that the AYP motif is critical for the binding of MAb5G8 to AMA1 was achieved by examining the binding of phage displaying the 19-residue AMA1 sequence with mutations introduced into the motif. As can be seen in Figure 7, the three mutants with mutations in either the tyrosine or the proline or both result in a dramatic reduction in binding compared with phage displaying the 19-residue peptide with the intact AYP motif. Hence these two residues contribute a significant amount of energy to the binding of this epitope to MAb5G8.
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MAb1F9 recognizes refolded recombinant AMA1 only in its non-reduced form, hence the epitope for this MAb on AMA1 is dependent on the correct folding of the molecule and its stabilization by disulphide bonds. Western blot analysis of the defined AMA1 fragments expressed on phage using the MAb1F9 showed that the MAb recognized the whole AMA1 ectodomain and also fragments containing pro-sequence plus domain-1 (Pro+1) and domain-1 (Dom-1) alone. MAb1F9 does not bind to domain-2 (Dom-2) or domain-3 (Dom-3) fragments (Figure 8A and B). When the AMA1 random fragment library was panned on MAb1F9 two in-frame clones were isolated that localized to domain-1 (Figure 8B
). The larger of the fragments (1.2.1) spanned the two loops defined by cysteines 2 and 3 and cysteines 4 and 5. The smaller fragment (1.4.1) was contained within fragment 1.2.1 and consisted of 26 residues N-terminal to cysteine 2 and terminated immediately after cysteine 3. It has been shown that cysteines 2 and 3 are connected in AMA1 forming a loop within domain-1 which is itself delineated by the connectivity between cysteines 1 and 6 (Hodder et al., 1996
). Interestingly, no fragments were observed to contain odd numbers of cysteine residues, presumably reflecting a combination of the dependence of this epitope on disulphide and the intolerance of the bacterial system for lone unpaired cysteines (Kay et al., 1993
; Zhong et al., 1994
). Confirmation that these fragments were recognized by MAb1F9 was obtained by Western blotting of phage containing these fragments with MAb1F9 (Figure 8B
). Furthermore, this binding was significantly reduced when the phage were electrophoresed under reducing conditions (data not shown).
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Discussion |
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AMA1 represents a severe test of the limits of the phage display approach since the molecule has a complex folded structure stabilized by eight intramolecular disulphide bonds (Hodder et al., 1996). The spacing of 16 cysteine residues is absolutely conserved in all Plasmodium spp. so far examined (Peterson et al., 1989
; Dutta et al., 1995
; Marshall et al., 1995
). The eight disulphide bonds partition the AMA1 ectodomain into three putative domains plus a prodomain and presumably stabilized the tertiary structure (Hodder et al., 1996
). It was difficult to predict whether AMA1 fragments expressed on phage would be presented in a form resembling the native structure and whether the appropriate disulphide connections would be made. As a first step, we expressed defined AMA1 fragments corresponding to the putative domains on phage. Interestingly, the complete AMA1 ectodomain was found to be expressed on phage despite its relatively large size (Mr
60 kDa). The phagemid system used in this study probably facilitates the expression of large protein fragments on phage since there will be an average of less than one copy of the fusion protein per phage particle (Lasters et al., 1997
). The additional four copies of wild-type gpIII protein promote attachment and entry of the phage into a host bacterial cell. Phage expressing the AMA1 ectodomain were recognized by monoclonal antibodies MAb5G8, MAb1F9 and rabbit antiserum raised against AMA1 by ELISA and by Western blotting. Furthermore, MAb1F9 recognizes a reduction sensitive epitope which is present on these phage. Hence we conclude that at least a proportion of phage are expressing AMA1 ectodomain in a correctly folded form resembling the native conformation AMA1 conformation with respect to the 1F9 antibody.
The presence of two faster migrating MAb1F9 reactive bands in these preparations (Figure 8B, lane Pro+1+2+3), suggests that there was some proteolytic cleavage of the ectodomain from the phage framework either during phage assembly or, more likely, after phage preparation. Although different in molecular weights, the pattern of cleavage products is similar to the proteolytic cleavage products of AMA1 in parasites as described by Crewther et al. (Crewther et al., 1990
). It is tempting to speculate that the processing events that occur in AMA1 around the time of merozoite invasion of erythrocytes are reproduced in this in vitro phage expression system, presumably via a bacterial protease. The importance of this proteolysis in parasites is unknown and this phage system may aid in defining the molecular events involved.
In addition to the AMA1 ectodomain, smaller fragments of AMA1 have been expressed on phage with intact conformational epitopes. Thus the MAb1F9 epitope, which is reduction sensitive, is retained on domain-1 expressed on phage (Figures 1 and 8). On Western blots, however, the smear surrounding the band corresponding to domain-3, which contains closely spaced cysteine residues in a cysteine knot-like motif (Figure 1
), suggests that there may be some misfolding of this domain, possibly leading to incorrect cysteine pairing. Thus the favourable oxidizing environment in the periplasm promotes disulphide bond formation and facilitates the expression of functional proteins (Skerra and Pluckthun, 1988
; Bardwell, 1994
).
Examination of clones from the random fragment library revealed that most were random in size, orientation and sequence. It was possible to enrich from this library several clones that had AMA1 sequences in frame with the gpIII protein of the phage, by panning on MAb5G8 and MAb1F9. The two antibodies enriched a different sub-set of clones originating from distinct domains of AMA1. Several rounds of panning were sufficient to locate the regions on AMA1 that contain the epitopes for these two MAbs.
The epitope of MAb 5G8 was mapped to a 19-residue linear sequence within the AMA1 pro-sequence. The MAb5G8 fragment lies within a 55 amino acid sequence that is found only in P.falciparum AMA1 and not AMA1 from other species of malaria (Peterson et al., 1989). Consistent with this, MAb5G8 did not bind to AMA1 from P.chabaudi. Thus, as has been demonstrated in other systems (Zhong et al., 1994
; Wang and Yu, 1998), phage display of random fragments is a convenient approach for epitope mapping for malaria antigens.
Phage display libraries expressing random peptides have been used for defining binding sites for antibodies and other proteins in a wide variety of systems including malaria (Kay et al., 1993; Wrighton et al., 1996
; Adda et al., 1999
; Koivunen et al., 1999
). Four rounds of panning of MAb5G8 on a 15-residue random peptide library produced a single binding peptide which contained within the sequence a three residue motif AYP which was found within the 19 residues common to all the enriched fragments from the random fragment library. Further experiments demonstrated that of the two overlapping peptides that span the 19-residue fragment only those containing AYP were able to interact with MAb5G8. AYP is critical for MAb5G8 binding, but does not appear to be sufficient in itself since the small peptide consisting of the 12 natural residues on AMA1 that include AYP binds to MAb5G8 with 100-fold greater apparent affinity than the mimotope. Thus amino acid sequences flanking clearly affect interaction with MAb5G8. A search of malaria proteins in the WHO malaria database identified 12 proteins with AYP in their primary sequence. However, MAb 5G8 is specific for AMA1 and this is consistent with the epitope consisting of AYP in the context of appropriate flanking residues and accessible on the surface of the folded protein. Although the flanking residues may affect the overall binding affinity of the epitope to MAb5G8, mutation of either the tyrosine or the proline residue or both to alanine results in almost complete abolition of binding (Figure 7
). Hence these two residues must confer the bulk of the binding energy of this epitope to the antibody.
MAb1F9, which only recognizes AMA1 in the oxidized form, was used to pan the random fragment library. The epitope was contained within a 57-residue region that comprised cysteines 2 and 3 along with 26 residues N-terminal to cysteine 2. Cysteines 2 and 3 form one of the three intramolecular disulphide bonds in domain-1 of the AMA1 ectodomain as described by Hodder et al. (Hodder et al., 1996). No smaller fragments binding to MAb1F9 which contrasted with the fragments isolated by panning on MAb5G8. Hence the MAb1F9 epitope most likely comprises two or more clusters of amino acids brought into close contact and stabilized by the disulphide bond involving cysteines 2 and 3. Further fine mapping of the MAb1F9 epitope could use a strategy described by Jespers and colleagues (Jespers et al., 1997
), where a library of mutants is created on phage and by negative selection the residues that contribute to binding can be identified.
The use of the phage display for the identification of the minimal epitopes may allow the synthesis of small peptides or fragments rather than large native proteins which can be difficult to produce in a recombinant form. Immunization with these peptides or fragments may focus the immune response to regions of antigens that are known to generate a protective antibody response. Such an approach could have important benefits if immunization with AMA1, like MSP-1, generates antibodies which bind close to the epitope of protective antibodies and block the protective action of serum antibodies (Guevara Patino et al., 1997).
AMA1 can be considered to be a prototype of other P.falciparum cysteine-rich proteins whose structure and function are governed by intramolecular disulphide bonds. The induction of protective immune responses by MSP119 and AMA1, the two leading P.falciparum asexual stage vaccine candidates, is dependent on conformational epitopes stabilized by disulphide bonds. Other malaria proteins with important functions in development of the malaria parasite, e.g. var, eba, also have cysteine-rich domains and clearly phage display is a potentially useful approach for analysing structurefunction relationships in these proteins.
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Notes |
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Acknowledgments |
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References |
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Received January 1, 2001; revised June 8, 2001; accepted June 18, 2001.