Structural basis of antigenic escape of a malaria vaccine candidate

Abstract

Antibodies against the malaria vaccine candidate apical membrane antigen-1 (AMA-1) can inhibit invasion of merozoites into RBC, but antigenic diversity can compromise vaccine efficacy. We hypothesize that polymorphic sites located within inhibitory epitopes function as antigenic escape residues (AER). By using an in vitro model of antigenic escape, the inhibitory contribution of 24 polymorphic sites of the 3D7 AMA-1 vaccine was determined. An AER cluster of 13 polymorphisms, located within domain 1, had the highest inhibitory contribution. Within this AER cluster, antibodies primarily targeted five polymorphic residues situated on an alpha-helical loop. A second important AER cluster was localized to domain 2. Domain 3 polymorphisms enhanced the inhibitory contribution of the domain 2 AER cluster. Importantly, the AER clusters could be split, such that chimeras containing domain 1 of FVO and domain 2 + 3 of 3D7 generated antisera that showed similarly high level inhibition of the two vaccine strains. Antibodies to this chimeric protein also inhibited unrelated strains of the parasite. Interstrain AER chimeras can be a way to incorporate inhibitory epitopes of two AMA-1 strains into a single protein. The AER clusters map in close proximity to conserved structural elements: the hydrophobic trough and the C-terminal proteolytic processing site. This finding led us to hypothesize that a conserved structural basis of antigenic escape from anti-AMA-1 exists. Genotyping high-impact AER may be useful for classifying AMA-1 strains into inhibition groups and to detect allelic effects of an AMA-1 vaccine in the field.

Fig. 1.

Clusters of AMA-1 polymorphic sites. (Left) The table shows the 24 polymorphic differences between 3D7 and FVO AMA-1. Dark lines separate domain boundaries. (Right) Polymorphic face of P. falciparum AMA-1 (15, 16, 28). Residues selectively switched from FVO to 3D7 type are shown as solid amino acids. Polymorphisms were switched domain-wise: D1 (red plus blue plus orange plus purple plus sky blue), D2 (yellow), or D3 (wheat). Polymorphic clusters within domain 1 were also switched: C1 (red plus blue), C2 (orange), C1-L (red), C3 (purple), and C1+C2 (red plus blue plus orange). The C-terminal proteolytic cleavage site of AMA-1 (Pro; brown) (23) and the side-chains of the hydrophobic trough residues (green) (15) are also shown. Domain 1 residues not included in the cluster chimeras are colored sky blue. Figures were made by using PyMOL software (www.pymol.org).

Fig. 2.

Purity and reactivity of AMA-1 chimeras. (A) Chimeric AMA-1 proteins analyzed for purity by nonreduced SDS/PAGE stained with Coomassie blue. (B) Western blot with mAb 4G2dc1. Anti-3D7 and anti-FVO AMA-1 rabbit sera pools (1:10,000 dilution) were mixed with an equal volume of 0.5 mg/ml of FVO or 3D7 AMA-1 proteins, respectively. The cross-reactive antibodies were blocked, and the free strain-specific anti-3D7 (C) and anti-FVO (D) antibodies were used in a Western blot. Densitometric analysis of Fig. 2C blot is shown in SI Fig. 9. Two batches of C1+C2 were loaded on these gels.

Fig. 3.

Five serial dilutions (x axis) of the 3D7, the FVO, or the domain chimeras were compared for their ability to reverse invasion inhibition (y axis), mediated by 7% anti-3D7 AMA-1 serum pool against the 3D7 parasite target. P. berghei AMA-1 (PbAMA) was used as the negative control antigen.

Fig. 4.

Five serial dilutions (x axis) of the 3D7, FVO AMA-1, or the domain 1 cluster chimeras were compared for their ability to reverse invasion inhibition (y axis), mediated by 7% anti-3D7 AMA-1 serum pool against the 3D7 parasite target. P. berghei AMA-1 (PbAMA) was used as the negative control antigen.

Fig. 5.

Anti-3D7 AMA-1 serum pool (7% concentration)-mediated inhibition was reversed by a 1.75 μM concentration of 3D7, FVO, or the chimeric AMA-1 proteins. The percent invasion was calculated as (% parasitemia in test well/% parasitemia in adjuvant control well). RE (y axis) was determined by subtracting % invasion in the presence of FVO AMA-1 antigen from % invasion in the presence of the test antigen and then adjusting RE for 3D7 AMA-1 to 100% and FVO AMA-1 to 0% (not plotted). This calculation allowed the reversal of inhibition due to cross-reactive antibodies to be canceled out. Mean RE ± SD of three independent experiments is shown. RE/residue (dots) = mean RE/number of residues displayed by a particular chimera.

Fig. 6.

Left y axis (bars), concentration of IgG required for 50% invasion inhibition (ED₅₀) was determined against 3D7 and FVO target parasites. The mean ED₅₀ (± SD) was plotted from three separate GIA experiments (SI Fig. 12). Right y axis (circles), ELISA units (ng/ml IgG that resulted in OD₄₁₅ = 1.0) determined by using the homologous 3D7 or FVO AMA-1 coat antigens.

Fig. 7.

Individual rabbit sera from each vaccine group were tested in a GIA by using multiple serum dilutions against 3D7, FVO, CAMP, 7G8, HB3, DD2, and M24 strains (SI Fig. 14). The mean invasion (+SD) of three rabbits at 20% serum dilution was plotted.