Immunological and functional properties of a non-repeat region in Plasmodium falciparum antigen Pf155/RESA

Sammanfattning: Plasmodium falciparum malaria is one of the most prevalent of all the great insect-borne diseases causing high morbidity and mortality in humans. As conventional anti-malarial treatment is becoming increasingly inefficient, alternative approaches to combat the parasite, like vaccine development, are of high priority. Among other antigens, Pf155/RESA has been taken into consideration for inclusion in a vaccine against the asexual blood stages of P. falciparum. Repeat sequences of this antigen induce neutralizing antibodies and are potentially involved in protective immune responses. However, the genetic restriction in the immune responses to these epitopes has made it of importance to define additional regions in this antigen which may be suitable for inclusion in a vaccine. The present studies were aimed at investigating potential immunodominant B- and T-cell epitopes in the non-repeat regions of Pf155/RESA suitable for inclusion in subunit vaccine immunogens.In order to define immunodominant B- and T-cell epitopes, short overlapping peptides were synthesized, corresponding to three different non-repeat sequences of Pf155/RESA. Sera collected from malaria endemic countries recognized pepetides representing N-terminal sequences of the antigen. Based on high antibody reactivity in ELISA, two peptides were selected for affinity purification of antibodies from Liberian sera. The purified antibodies were shown to be efficient in parasite growth inhibition in vitro. Furthermore, the peptides had the ability to stimulate peripheral blood mononuclear cells from P. falciparum-immune donors to proliferate and to secrete IL-4 and/or IFN-g. Although the frequency of responders was high, the magnitude of the responses was generally low.Antibodies were raised in rabbits against synthetic peptides corresponding to different non-repeat sequences in Pf155/RESA. Although all antisera reacted strongly with the corresponding peptide, they reacted only weakly with full-length Pf155/RESA. Antibodies to some of the non-repeat sequences inhibited merozoite invasion in vitro, notably, also the invasion of parasites deficient in Pf155/RESA, indicating the presence of an antigen highly homologous to Pf155/RESA.Upon infection with P. falciparum, erythrocytes expose cryptic determinants in band 3, which are thought to mediate cytoadherence to endothelial cells. The N-terminal non-repeat region of Pf155/RESA contains a hexapeptide motif which is highly homologous to the cytoadherence related sequences of erythrocyte band 3. Synthetic peptides containing this hexapeptide motif inhibited the binding of P. falciparum-infected erythrocytes to melanoma cells in vitro. Antibodies raised against the largely overlapping sequences displayed highly different specificity patterns. Antibodies to the cytoadherence related motif of Pf155/RESA as well as antibodies raised against the peptides corresponding to the band 3 motif inhibited cytoadherence but not parasite growth. In contrast antibodies to sequences adjacent to the Pf155/RESA cytoadherence motif inhibited parasite growth in vitro but had no effect on cytoadherence.In summary, although the target antigens for the anti-parasitic activities displayed by the antibodies in these studies are unclear, sequences in non-repeat regions of Pf155/RESA may induce antibodies with capacity to inhibit parasite growth in vitro or to block the cytoadherence of P. falciparum infected erythrocytes to endothelial cells. The combination of non-repeat sequences with repeat sequences of Pf155/RESA in a subunit vaccine may provide immunogens with an improved capacity to induce parasite neutralizing antibody responses.