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. 2002 Dec;70(12):6948-60.
doi: 10.1128/IAI.70.12.6948-6960.2002.

In vitro studies with recombinant Plasmodium falciparum apical membrane antigen 1 (AMA1): production and activity of an AMA1 vaccine and generation of a multiallelic response

Michael C Kennedy  1 , Jin Wang, Yanling Zhang, Aaron P Miles, Farideh Chitsaz, Allan Saul, Carole A Long, Louis H Miller, Anthony W Stowers
Affiliations

In vitro studies with recombinant Plasmodium falciparum apical membrane antigen 1 (AMA1): production and activity of an AMA1 vaccine and generation of a multiallelic response

Michael C Kennedy et al. Infect Immun. 2002 Dec.

Abstract

Apical membrane antigen 1 (AMA1) is regarded as a leading malaria blood-stage vaccine candidate. While the overall structure of AMA1 is conserved in Plasmodium spp., numerous AMA1 allelic variants of P. falciparum have been described. The effect of AMA1 allelic diversity on the ability of a recombinant AMA1 vaccine to protect against human infection by different P. falciparum strains is unknown. We characterize two allelic forms of AMA1 that were both produced in Pichia pastoris at a sufficient economy of scale to be usable for clinical vaccine studies. Both proteins were used to immunize rabbits, singly and in combination, in order to evaluate their immunogenicity and the ability of elicited antibodies to block the growth of different P. falciparum clones. Both antigens, when used alone, elicited high homologous anti-AMA1 titers, with reduced strain cross-reactivity. Similarly, sera from rabbits immunized with a single antigen were capable of blocking the growth of homologous parasite strains at levels theoretically sufficient to clear parasite infections. However, heterologous inhibition was significantly reduced, providing experimental evidence that AMA1 allelic diversity is a result of immune pressure. Encouragingly, rabbits immunized with a combination of both antigens exhibited titers and levels of parasite inhibition as good as those of the single-antigen-immunized rabbits for each of the homologous parasite lines, and consequently exhibited a broadening of allelic diversity coverage.

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Figures

FIG. 1.
FIG. 1.
Alignment of native and synthetic AMA1 amino acid sequences. The GenBank sequences for P. falciparum FVO AMA1 (AJ277646 and U84348) and P. falciparum F3D7 AMA1 (U65407) are aligned with the sequences used for recombinant protein production in this study (SynFVO and Syn3D7). The three mutations unique to U84348 among all P. falciparum AMA1's, and so not used in the synthetic FVO sequence, are indicated by a solid background. The single glycosylation site mutated on the basis of alignment with allelic variations in other P. falciparum AMA1 sequences is shown as a boxed “K.” Another amino acid in the SynFVO sequence was mutated to T (boxed), because a T always accompanies a K in the previous position (both in other P. falciparum sequences and in the AMA1 sequences of other Plasmodium species). Mutations indicated by shading were chosen by alignment with sequences of other Plasmodium species, because all P. falciparum AMA1 sequences were conserved in this region. The first three of these were from other primate malaria species (Plasmodium vivax, Plasmodium cynomolgi, and/or Plasmodium fragile), and the last was from P. chabaudi. Lowercase letters in the synthetic recombinant sequences represent vector-derived sequences. The signal peptide, transmembrane, and cytoplasmic domains present in the native sequences, but not used in the recombinant proteins, are underlined.
FIG. 2.
FIG. 2.
Purity and integrity of PpAMA1's. SDS-PAGE on 4-to-20% gradient Tris-glycine polyacrylamide gels was performed on serial fourfold dilutions of purified PpAMA1 FVO (A and B) and PpAMA1 3D7 (C and D). Quantities loaded were, from left to right, 12.5, 3.1, 0.8, and 0.2 μg. Electrophoresis was performed either under nonreducing conditions (A and C) or with 5% (vol/vol) β-mercaptoethanol (B and D). Scanning laser densitometry showed yields of 96.9% (nonreduced) or 81.9% (reduced) full-length PpAMA1 FVO and 97.6% (nonreduced) or 48.4% (reduced) full-length PpAMA1 3D7. Single asterisks indicate bands whose N-terminal sequence was identified by Edman degradation as Y1VQNYWEHPYQKSDVYHPIN (SynFVO) or Y1VQNYWEHPYQNSDVYRPIN (Syn3D7); double asterisks indicate bands whose N-terminal sequence was S355AFLPTGAFKADRYKSH.
FIG. 3.
FIG. 3.
Mean growth inhibition levels of rabbit sera raised against recombinant PpAMA1's. Groups of five rabbits each were immunized with 50 μg of either PpAMA1 FVO (solid symbols) or PpAMA1 3D7 (open symbols). Each rabbit serum was assayed individually for growth inhibition against three parasite lines (FVO [circles], 3D7 [inverted triangles], or HB3 [squares]), and the mean percent inhibition of invasion of RBCs for each group of five rabbits is shown along the y axis. GIAs for each serum were conducted at three (with HB3 parasites) or four concentrations, as shown on the x axis. Lines represent regression of homologous results (i.e., PpAMA FVO versus FVO parasites and PpAMA 3D7 versus 3D7 parasites) by use of a hyperbolic equation.
FIG. 4.
FIG. 4.
Homologous and heterologous growth inhibition of P. falciparum by sera raised against two alleles of AMA1. Individual sera from each of four rabbits in each of the three immunization groups (PpAMA1 FVO, PpAMA 3D7, or a combination of both) were tested at three different serum concentrations (25, 12.5, and 6.25%) for their abilities to inhibit the growth of five different strains of P. falciparum.
FIG. 5.
FIG. 5.
Homologous and heterologous GIAs comparing rabbit groups receiving single versus combined immunogens. Rabbits from each of the three immunization groups were compared at the highest serum concentration (25%) for their abilities to inhibit the growth of five different P. falciparum strains. Horizontal lines show the mean value for each group. Groups were compared for statistical significance by an unpaired t test.
FIG. 6.
FIG. 6.
Effects of antigenic differences on growth-inhibitory activity. Rabbits were immunized with either PpAMA1 FVO (blue), PpAMA1 3D7 (red), or a combination of both (green). Sera from immunized rabbits were used in GIAs against the M24, HB3, D10, FVO, and 3D7 P. falciparum clones. The number of amino acid differences between an immunizing AMA1 allele (FVO, 3D7, or a combination of both) and the parasite clone tested for inhibition is plotted on the x axis, and the mean level of inhibition obtained against that parasite is plotted on the y axis. For the combined immunization, only those amino acids present in neither immunizing allele are taken into account. The dotted line is the result of a linear regression of all data points.
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