Parasite Immunology, 1996:18: 159–161

Brief communication

Can software be used to predict antigenic regions in Plasmodiumfalciparum peptides?

MARIA DE FA TIMA FERREIRA-DA-CRUZ1, SALVATORE GIOVANNI-DE-SIMONE2,3,DALMA MARIA BANIC 1,4, MARILENE CANTO-CAVALHEIRO1, DANIEL CAMUS4 &CLAUDIO TADEU DANIEL-RIBEIRO 1

1Department of Immunology, World Health Organization Collaborating Center for Research and Training in the Immunology of Parasitic Diseases.Instituto Oswaldo Cruz-Fiocruz Av. Brasil 4365, Cep 21045.900. Rio de Janeiro, Brazil2Department of Biochemistry and Molecular Biology, Instituto Oswaldo Cruz3Universidade Federal Fluminense, Niteroi, Rio de Janeiro4Unite de Biologie et de Biochimie Parasitaires et Fongiques, INSERM U42, Villeneuve d’Ascq, France

SUMMARY

We compared the antigenicity of p126Plasmodium falci-parumpeptides with predicted antigenic regions identifiedusing the methods described by Garnieret al. (1978) andChou & Fasman (1974). For this purpose nine differentP.falciparum peptides were synthesized in accordance withthe deduced amino acid sequence of the p126 gene, andtheir reactivity was tested using an enzyme linked immuno-sorbent assay against sera from individuals with a naturalmalaria infection. Both predictive methods gave similarantigenic-index scores, however, a comparison of thesepredictive results with data obtained by ELISA showedthat the probability of a correct prediction was onlyaround 45% for both cases. Thus, in our view computersoftwares could not be used in isolation for screeningpurposes, and other parameters must also be taken intoaccount when using such software to assess antigenicity.

Keywords p126 antigen,P. falciparum, peptides, malaria

INTRODUCTION

The use of synthetic peptides that correspond to immuno-dominant regions onP. falciparumantigens constitutes apromising strategy for malaria vaccine development. How-ever, the complexity of protein structures and the immensedifficulties involved in defining and synthesizing proteinantigenic sites, along with the high cost of peptide synthesisand the time-consuming nature of peptide-antigenicity test-ing techniques, remain major hurdles in this endeavor. Ourlaboratories have been working with malaria transmissionand immunity, and in this context our research has focusedon the humoral immune response againstP. falciparumantigens (Banicet al. 1994, Ferreira-da-Cruzet al. 1995).One such antigen is p126, a molecule that has been shown toinduce protective immunity in monkeys (Delplaceet al.1988, Knappet al. 1992). With a view to bypassing theproblems mentioned above we compared the antigenicity ofp126 P. falciparum peptides with predicted antigenicregions identified using the methods described by Garnieret al. 1978, Chou & Fasman 1974).

MATERIALS AND METHODS

Four peptides (13 amino acids in length) included on thenaturally processed fragments (pII and pVIII at the 50 kDapolypeptide; pI and pIX at the 73 kDa polypeptide) of thep126 protein (Debrabantet al. 1992) were commerciallyobtained from Ne´osystem (France). The other five peptides(pIII–pVII) were of a similar length and were preparedmanually using the 9-fluorenylmethoxycarbonyl (Fmoc)-method on Wang resin (Van Regen Mortelet al. 1988).The reactivity of the peptides was tested using an enzymelinked immunosorbent assay (ELISA) against a panel of

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Correspondence: M. de Fatima Ferreira-da-CruzReceived: 21 June 1994Accepted for publication: 19 October 1995

positive crudeP. falciparumantigen sera from 84 indivi-duals with a natural malaria infection. The hydrophilicityprofile was based on the Kyte-Doolittle hydropathy scale(Kyte & Doolittle 1982) using 7 size window. A graphicalrepresentation of the predicted antigenic determinants on thep126 P. falciparum molecule was generated using theprograms PEPPLOT and PLOTSTRUCTURE (Modrow &Wolf 1986) on a VAX-computer. Antigenic indexes werecalculated using the method described by Jameson & Wolf(1988).

RESULTS AND COMMENTS

The positivity of the ELISA ranged from 24% to 63%, and thebest reactivity was observed with peptides pIV (63%) and pIII(60%). No reactivity was observed with peptides pI, pII, pVIIIand pIX. Analysis of the hydropathic profile of p126 proteinshowed that the sequences corresponding to peptides pII,

pIII, pIV, pV and pVI (Table 1) exhibited a similar medianlevel of hydrophilicity, while peptides pI, pVIII and pIXdisplayed only discrete hydrophilicity. Peptide pVI showeda high level of hydrophobicity. The secondary structureprediction was based on the methods of Chou & Fasman(1974) and Garnieret al. (1978). Each of these two methods,when used on its own, gives a large number of�-pleated anda helical structures for the p126 antigen, especially themethod of Chou & Fasman. Computer predictions of regionsof immunogenicity, surface probability, flexibility andhydrophilicity are superimposed on the peptide sequences(Figure 1). There appears to be a good correlation betweenthe serologically determined immunogenicity of the mole-cular regions corresponding to the sequences of peptidespIII, pIV and pV and the predicted score on antigenic indexand the three other parameters mentioned above. Similarly,the absence of any reactivity with peptide pI, at the extremeC terminus, coincides with a region of predicted lowimmunogenicity and loop formation. In contrast, however,the predicted-antigenic index score for peptides pVI andpVII (both with positive ELISA reactions) was zero, irre-spective of the presence (pVI) or absence (pVII) of an�-helix structure. Positive-predicted antigenic sites werefound for pII and pIX, but these peptides did not reactwith any of the human sera analysed, in spite of the fact thatpIX gave the highest score of all on the antigenic predictiveindex. According to the computer-predicted antigenic index,no antigenic site was expected in pVI, an extremely hydro-phobic peptide. However, when tested by ELISA, thispeptide gave 56% of positive results. This positivity wassimilar to that (60%) observed with the hydrophilic peptidepIII, which was predicted to have 5 possible antigenic sites.

M.de F.Ferreira-da-Cruzet al. Parasite Immunology

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Table 1 Sequence of synthetic peptides from the p126 protein

Peptide Amino acid sequence Residue

pI EDIVSNFKEIKAE 370–382pII TEDDDEDDYTEYK 383–395pIII SWIFASKYHLETI 588–600pIV VGENCPKVEDHWM 660–672pV KGYTAYESERFHD 693–705pVI MDAFVKIIKTDVM 707–719pVII FKVDMYGPTHCHF 791–802pVIII DTAGSGQSGKESN 879–890pIX KLDTNNECYFCYV 977–989

Figure 1 Comparison between the predicted regions of the p126 molecule. The computer predictions of flexibility, surface probability andhydrophilicity were generated as described in Material and Methods. The position of each synthetic peptides is shown as dashed vertical lines.

Similarly, pII, which gave maximum hydrophilicity, was notfound to be antigenic in our assays. Thus, linear analysis ofpeptide sequences in relation to the hydrophilicity indexdoes not seem to constitute an absolute criterion for asses-sing antigenicity. A more detailed discussion of this subjecthas been provided by Atassi (1984).

In conclusion, we found that both predictive methods(Chou & Fasman, 1974 & Garnieret al. 1978) gave similarantigenic-index scores, corroborating previous studies(Geysenet al. 1987). However, a comparison of thesepredictive results with data obtained by ELISA showedthat the probability of a correct prediction was onlyaround 45% for both methods. Thus, in our view computersoftwares can not be used in isolation for screening pur-poses, and other parameters will have to be included in suchsoftware before it can be usefully employed to assessantigenicity.

ACKNOWLEDGEMENTS

This work received financial support from the Science andTechnology for Development Program of the Commissionof the European Communities and from Brazilian NationalResearch Council (CNPq). Maria de Fa´tima Ferreira-da-Cruz is recipient of a CNPq fellowship (Pesq 2C 302325/84-0 NV).

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Volume 18, Number 3, March 1996 Antigenic predictions forP. falciparumpeptides

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