B- and T-cell responses in congenic mice to repeat sequences of the malaria antigen Pf332: effects of the number of repeats

Abstract

The Plasmodium falciparum antigen Pf332 comprises degenerated 11-amino-acid repeats with regularly spaced pairs of glutamic acid. Epitopes formed by such repeats are recognized by polyclonal and monoclonal antibodies that interfere with the life cycle of the blood stages of the malaria parasite. In order to study the immunogenicity of one such Pf332 repeat sequence (SVTEEIAEEDK), fusion proteins containing ZZ (two IgG binding domains of staphylococcal protein A) and dimers, trimers or tetramers of the malarial sequence were injected into mice. To analyse possible major histocompatibility complex class II restrictions of the immune response, mice of different H-2 haplotypes were used. A significant antibody response was elicited by administration of all the three fusion proteins in mice expressing the I-A k allele (B10.BR, B10.A(2R) and B10.A(4R)) whereas B10 and C57BL/6 (H-2 b) mice were low responders. In comparison, B10.D2 (H-2 d) mice were low responders to fusion proteins with 2 or 3 repeats but responded well to the protein containing 4 repeats. Lymph node cells from B10.BR (H-2 k) mice, primed in vivo with ZZ-fusion proteins containing either 2 or 4 repeats, proliferated in vitro in response to repeat sequences fused to ZZ or to an unrelated fusion partner, as well as to a synthetic peptide containing less than two repeats. In contrast, a response of lymph node cells from B10.D2 (H-2 d) mice was only obtained when a fusion protein containing 4 repeats was used both for in vivo priming and in vitro restimulation. Antibodies from all responding mice reacted with the native parasite protein in immunofluorescence. However, when measured by binding to a panel of synthetic peptides, the fine specificities of the antibodies were found to depend on the number of Pf332 repeats used for immunization. Thus, the number of repeat sequences influences both B- and T-cell responses. Since immunodominant repeat regions are a common feature for several malarial antigens involved in immune protection, the present findings have implications for the design of proper immunogens to be incorporated in malaria vaccines.