Induction of Protective Immune Responses by Immunization with Linear Multiepitope Peptides Based on Conserved Sequences fromPlasmodium falciparumAntigens

Abstract

A cysteine-containing peptide motif, EWSPCSVTCG, is found highly conserved in the circumsporozoite protein (CSP) and the thrombospondin-related anonymous protein (TRAP) of all thePlasmodium species analyzed so far and has been shown to be crucially involved in the sporozoite invasion of hepatocytes. We have recently shown that peptide sequences containing this motif, and also the antibodies raised against the motif, inhibit the merozoite invasion of erythrocytes. However, during natural infection, and upon immunization with recombinant CSP, this motif represents a cryptic epitope. Here we present the results of immunization studies with two linear multiepitopic constructs, a 60-residue (P60) and a 32-residue (P32) peptide, containing the conserved motif sequence. Both the peptides per se generated high levels of specific antibodies in BALB/c mice. P32 was found to be genetically restricted toH-2d and H-2b haplotypes of mice, whereas P60 was found to be immunogenic in five different strains of mice. The antibody response was predominantly targeted to the otherwise cryptic, conserved motif sequence in P60. Anti-P60 antibodies specifically stained the asexual blood stages ofPlasmodium falciparum and Plasmodium yoelii in an immunofluorescence assay, recognized a 60- to 65-kDa parasite protein in an immunoblot assay, and blocked P. falciparummerozoite invasion of erythrocytes in a dose-dependent manner. Immunization with P60 also induced significant levels of the cytokines interleukin-2 (IL-2), IL-4, and gamma interferon in BALB/c mice. Moreover, >60% of mice immunized with P60 survived a heterologous challenge infection with a lethal strain of P. yoelii. These results indicate that appropriate medium-sized synthetic peptides might prove useful in generating specific immune responses to an otherwise cryptic but critical and putatively protective epitope in an antigen and could form part of a multicomponent malaria vaccine.

Plasmodium vivax Pv12 B-cell epitopes and HLA-DRβ1*-dependent T-cell epitopes in vitro antigenicity

“…These results indicated a specific T-cell response for Pv 12 epitopes and ascertained these epitopes’ antigenicity when recognised by immune system memory cells. They correlated proliferation and cytokine production, as seen in other studies with Plasmodium antigens [ 37 , 70 , 71 ].…”

Immune responses to multiple antigen peptides containing T and B epitopes from Plasmodium falciparum circumsporozoite protein of Brazilian individuals naturally exposed to malaria

“…MAP constructs with coupled T and B epitopes permit normal processing and antigen presentation, and lead to cooperative cell interactions that will amplify the humoral response against the antigen (8,15–17). In addition, it has been shown that a lysine core contributes significantly to immunogenicity levels (6–8).…”

DNA prime–protein boost immunization in monkeys: efficacy of a novel construct containing functional domains ofPlasmodium cynomolgiCS and TRAP

“…Antisera obtained from vaccinated animals after three DNA immunizations and after protein booster did contain high levels of antibodies, which reacted in ELISA with a synthetic peptide PcP60. This peptide is based on the RII and downstream sequences of PcCS and is homologous to the PfP60 peptide reported earlier [9]. Our earlier studies have shown that the anti‐RII antibodies inhibit sporozoite invasion and can also protect mice from heterologous challenge [8,9].…”

“…TRAP, like CS, is involved in invasion of hepatocytes by sporozoite in a RII‐dependent manner [7]. Studies by different groups including our own have suggested that antibodies and cellular immune responses against peptides based on the sequences around the RII of CS and TRAP of Plasmodium falciparum inhibit sporozoite invasion of HepG2 cells [8,9], and protect immunized mice against lethal Plasmodium berghei challenge [8,10]. Both TRAP and CS also contain a number of T cell epitopes, and cytotoxic T lymphocyte (CTL) responses specific to these proteins have been detected in volunteers immunized with attenuated sporozoites and in persons living in endemic areas [11,12].…”