A diversity-covering approach to immunization with Plasmodium falciparum apical membrane antigen 1 induces broader allelic recognition and growth inhibition responses in rabbits

Abstract

Plasmodium falciparum apical membrane antigen 1 (PfAMA1), a candidate malaria vaccine, is polymorphic. This polymorphism is believed to be generated predominantly under immune selection pressure and, as a result, may compromise attempts at vaccination. Alignment of 355 PfAMA1 sequences shows that around 10% of the 622 amino acid residues can vary between alleles and that linkages between polymorphic residues occur. Using this analysis, we have designed three diversity-covering (DiCo) PfAMA1 sequences that take account of these linkages and, when taken together, on average incorporate 97% of amino acid variability observed. For each of the three DiCo sequences, a synthetic gene was constructed and used to transform the methylotrophic yeast Pichia pastoris, allowing recombinant expression. All three DiCo proteins were reactive with the reduction-sensitive monoclonal antibody 4G2, suggesting the DiCo sequences had conformations similar to those of naturally occurring PfAMA1. Rabbits were immunized with FVO strain PfAMA1 or with the DiCo proteins either individually or as a mixture. Antibody titers and the ability to inhibit parasite growth in vitro were determined. Animals immunized with the DiCo mix performed similarly to animals immunized with FVO AMA1 when measured against FCR3 strain parasites but outperformed animals immunized with FVO AMA1 when assessed against other strains. The levels of growth inhibition (approximately 70%) induced by the mix of three DiCo proteins were comparable for FVO, 3D7, and HB3, suggesting that a considerable degree of diversity in AMA1 is adequately covered. This suggests that vaccines based upon the DiCo mix approach provide a broader functional immunity than immunization with a single allele.

FIG. 1.

Alignment of aa 100 to 545 of DiCo1, DiCo2, DiCo3, and the yeast-expressed HB3 AMA1, 3D7 AMA1, and FVO AMA1, as well as the consensus sequence (Cons) that was derived from the alignment of the 355 PfAMA1 sequences obtained from the GenBank database. Residues that were changed in order to prevent N glycosylation in P. pastoris (residues 162, 288, 373, 422, 423, and 499) are underlined.

FIG. 2.

SDS-PAGE and Western blot analyses of purified DiCo proteins. (A) SDS-PAGE analysis. Lane 1, benchmark marker; lane 2, DiCo1; lane 3, DiCo2; lane 4, DiCo3; lane 5, DiCo mix. (B) Western blot analysis with 4G2 as the primary antibody. Lane 1, DiCo1; lane 2, DiCo2; lane 3, DiCo3; lane 4, DiCo mix. Samples were not reduced.

FIG. 3.

IgG antibody levels to various AMA1 variants. The box at the top of each panel indicates the ELISA coating antigen, and the antigen(s) used for immunization is listed on the x axis. The box indicates medians and 25 and 75 percentiles. For each treatment group, open circles (○) represent rabbit 1, filled circles (•) rabbit 2, open squares (□) rabbit 3, filled squares (▪) rabbit 4, and open diamonds (⋄) rabbit 5.

FIG. 4.

Growth-inhibiting titers versus IgG concentration in individual rabbits according to vaccine antigen and test strain. The top line of each panel indicates the test strain used for the GIA, and the second line indicates the antigen(s) used for immunization. For each treatment group, open circles (○) represent rabbit 1, filled circles (•) rabbit 2, open squares (□) rabbit 3, filled squares (▪) rabbit 4, and open diamonds (⋄) rabbit 5.

FIG. 5.

Growth-inhibiting titers versus IgG concentration in groups of rabbits according to vaccine antigen and test strain. The top line of each panel indicates the test strain used for the GIA, and the symbols indicate the antigen(s) used for immunization. For each treatment group, open circles (○) represent DiCo mix, filled circles (•) DiCo1, open squares (□) DiCo2, filled squares (▪) DiCo3, and open diamonds (⋄) FVO AMA1.