Debate

Candidate antigens for an asexual blood-stage vaccine

Few who were actively engaged in malaria vaccine research 20 years ago (including myself) would have imagined that, in 2005, there would still be a prediction of a 10–20-year horizon before vaccines become part of malaria-control strategies. Why is it still proving so challenging to produce effective vaccines?

The 1980s were a period of great activity in the field of parasitology that generated a vast amount of research published in ∼20 specialist and several other learned journals. There was, however, no vehicle for disseminating the exciting discoveries that were being made in an immediate, easily readable and accessible form suitable for teachers, students and others interested in aspects of the subject that were broader than their own particular specialty. To fill this perceived gap, Elsevier decided in 1985 that it would be worthwhile publishing Parasitology Today to complement its other successful Trends journals. In this article, I review the breadth of coverage of topics published during the first year of Parasitology Today, most of which continue to be of interest today.

A solid-phase affinity assay was set up for the determination of the affinity of the interaction between the human monoclonal antibody (mAb) 33G2 and peptides corresponding to repeated sequences in three blood stage antigens of the malaria parasite Plasmodium falciparum. The epitope of this mAb is of interest due to the parasite blocking capacity of the mAb. Previous studies with PEPSCAN have defined the minimal epitope for the mAb as the pentapeptide VTEEI, a sequence frequently found in antigen Pf332. In the previous study, epitopes responsible for the cross-reactivity of the mAb with antigens Pf155/RESA and Pf11.1 were also identified. In the affinity assay described herein, the mAb was coated on a solid phase and binding of a labelled peptide was displaced by homologous or heterologous peptides. The affinity of peptides corresponding to Pf332 increased with increasing length, and the highest affinity was displayed by a dimer (23 amino acids) of a Pf332 repeat (K = 1.9 × 10⁸ M⁻¹). Peptide length did not influence the binding of peptides corresponding to the Pf155/RESA and Pf11.1 repeats, which had lower affinities comparable to that of the shortest Pf332 octapeptide (K = 2.2 × 10⁴ M⁻¹). Only peptides containing binding sites as defined by PEPSCAN analysis showed a measurable binding. When using peptides as inhibitors in peptide ELISA, binding correlated with the affinity of the peptides, but only the high affinity peptides were inhibitory. In contrast, a poor correlation was found when peptides were used directly for coating in ELISA. Although the results of the affinity determinations were in concordance with the PEPSCAN analysis regarding the specificity of the mAb, they indicate that the optimal binding site may be more complex than revealed by PEPSCAN. This may have implications for selecting peptide sequences comprising the mAb 33G2 epitope for the construction of vaccine immunogens.

Population effects of malaria vaccination programs will depend on a complex interaction of the stage specificity of the vaccine, its duration of effectiveness, whether it is responsive to natural boosting, the strategy implemented, the proportion vaccinated and the pre-existing endemic conditions. In this article, Elizabeth Halloran and Claudio Struchiner review models of malaria transmission that incorporate aspects of immunity relevant to studying the effects of stage-specific malaria vaccination programs. They discuss the difference in the assumptions and applicability of the models and compare their predictions. Experience with malaria has demonstrated the difficulty in eliminating transmission, so emphasis needs to be on the new host-parasite balance that will be induced by the vaccination program. Although Halloran and Struchiner advise caution in interpreting the results of such models, they conclude that quantitative and theoretical analysis will be important in planning and evaluating interventions with malaria vaccines.

The human monoclonal antibody 33G2 has earlier been shown to inhibit merozoite reinvasion of red blood cells in Plasmodium falciparum cultures in vitro and to inhibit cytoadherence of infected red blood cells to melanoma cells in vitro. 33G2 cross-reacts with a family of P. falciparum antigens, Ag332, Pf11.1 and Pf155/RESA, sharing a common feature of repeated sequences consisting of regularly spaced pairs of glutamic acid. Peptides corresponding to residues 2–19 of the known amino acid sequence of Ag332 have been shown earlier to have the highest inhibitory capacity of antibody binding to infected red blood cells. Using the PEPSCAN method, overlapping hepta-, hexa-, penta- and tetrapeptides corresponding to residues 1–19 of the known sequence of Ag332 were synthesized. Antibody fine specificity was examined by synthesizing an octapeptide (residues 1–8) and all possible single amino acid substitutions. The monoclonal antibody was shown to react with a linear 5-amino acid-long sequence corresponding to Ag332 residues 3–7: VTEEI. These amino acids were irreplaceable or only partially replaceable in the replacement set analysis. Furthermore, epitope analogs corresponding to sequences contained within the Pf11.1 repeats and overlapping heptapeptides corresponding to Pf155/RESA repeats were synthesized. Reactivity to epitope analogs and Pf155/RESA peptides provided information which may explain antibody cross-reactivity. The defined epitope of this monoclonal antibody is of interest as a potential B cell epitope for the development of a malaria subunit vaccine.

For selection of immunogens capable of inducing high levels of antibodies reactive with the Plasmodium falciparum antigen Pf155/RESA, rabbits were immunized with synthetic peptides corresponding to sequences based on the repeat subunits EENVEHDA and (EENV)₂ from the C-terminus of this antigen. The antibodies obtained were analyzed with regard to binding to synthetic peptides in ELISA and to reactivity with parasite antigens by immunofluorescence or immunoblotting. All antisera reacted with both the peptides EENVEHDA and (EENV)₂ as well as with Pf155/RESA. Antibody fractions specific for each of the two peptides were prepared by affinity chromatography on insolubilized peptides. Strong reactivity with antigens in the membrane of erythrocytes infected with early stages of the parasite as well as reactivity with Pf155/RESA in immunoblotting correlated with reactivity of antibody with (EENV)₂. Antibody preparations reactive with EENVEHDA and depleted of (EENV)₂ reactivity showed only a weak reactivity with Pf155/RESA but reacted also with P. falciparum polypeptides of 250, 210, and 88 kDa. In immunofluorescence, these antibodies stained mainly the intraerythrocytic parasite. Both EENVEHDA- and (EENV)₂specific antibodies inhibited merozoite reinvasion in P. falciparum in vitro cultures, the latter antibodies being the most efficient. This study defines the specificity and crossreactivity with other P. falciparum antigens of antibodies to the C-terminal repeats of Pf155/RESA.