The allele frequencies in endemic populations appear to be under balancing selection [12], and antibodies against the sequences have been associated with protection from malaria [11], [12], [14] and [19]. Allele-specific growth inhibition has been reported with an antibody-dependant cellular inhibition HKI-272 concentration (ADCI) assay [13], although antibodies alone are not inhibitory except for a report of activity with one monoclonal antibody [20]. Previously, we demonstrated how an epitope mapping approach could be used to characterize
the complex antigenic polymorphism seen in the K1-like block 2 repeat sequences, and employed this in the design of a single synthetic sequence termed the K1 Super Repeat (K1SR) [15]. Immunization of mice with this K1SR antigen elicited a broad check details antibody repertoire against P. falciparum isolates bearing diverse K1-like allelic types. Here we present the design and characterization of a polyvalent hybrid protein incorporating the K1SR sequence together with K1-like flanking block 2 sequences, T helper cell epitope sequences near the junction of blocks 1 and 2, and MAD20-like
and R033-like block 2 allele sequences. To investigate the immunogenic contributions of each module that made up the final construct, five other sub-component constructs were designed and tested for comparative immunogenicity. Antibody responses were largely dependent on the presence of the T helper cell epitopes, and showed expected combinations of allele specificity. Antibodies to the full polyvalent hybrid Bay 11-7085 protein raised in both mice and rabbits displayed a broad repertoire with serological coverage against isolates of all allelic types. Six
recombinant antigens were constructed, five of which were designed as comparative reagents (antigens 1–5, Fig. 1A and Supplementary Fig. 1) to validate the final candidate immunogen (+)T-K1SR-R033-Wellcome (antigen 6, Fig. 1A and Supplementary Fig. 1). The DNA sequence encoding each antigen was generated using a modular construction, with each module separated by restriction enzyme sites (Supplementary Fig. 1). For constructs incorporating the K1-like 3D7 module (antigens 1 and 3, Fig. 1A), PCR products were amplified from 3D7 parasite genomic DNA using the primer pair KTPfK1F1BamH1 (5′-GGGGATCCGTAACACATGAAAGTTAT-3′) and KTPfR1Sac1M1 (5′-GGGAGCTCGCTTGCATCAGCTGGAGG-3′). This module also included the sequence for a conserved T-cell epitope within MSP1 block 1 (T1, amino acid position 20–39: VTHESYQELVKKLEALEDAV) and a polymorphic T-cell epitope (T2, amino acid position 44–63: GLFHKEKMILNEEEITTKGA) [21], spanning the junction of blocks 1 and 2. The R033-type block 2 module was amplified from R033 parasite genomic DNA using the primer pairs KTPfR033F1Sac1M2 (5′-GGGAGCTCAAGGATGGAGCAAATACT-3′) and KTPfR033R1Kpn1M2 (5′-GGGGTACCACTTGAATCATCTGAAGG-3′).