Our analysis of the diversity and dynamics of the P. falciparum
AMA-1 protein within infected individuals in Mali provides direct evidence from human infections that agrees with the findings of a recent in vitro study (13
) that showed that residues within the c1L cluster of domain I had the greatest effect on antigenic escape. Our results also corroborate the findings of an examination of the structure of AMA-1 in complex with an inhibitory monoclonal antibody that demonstrated that mutations at positions 197, 200, 201, 204, and 225 (all of which are located in c1L or c1) abrogated binding of this antibody (28
). These combined findings suggest that residues within domain I c1L may be useful for categorizing parasites into groups for inclusion in a polyvalent AMA-1 vaccine and for monitoring vaccine-induced selection in clinical trials of AMA-1–based vaccines. Measures of vaccine-induced selection and allele-specific antibody responses in efficacy trials of AMA-1 vaccines will be required to confirm the importance of these residues because other factors, including immune responses to other antigens, could influence the dynamics of AMA-1 diversity. We plan to do such analyses on samples from phase 2 trials of both monovalent (6
) and bivalent (5
) AMA-1 vaccines in Mali. Further immunological investigations are also warranted to more comprehensively map the putative epitopes responsible for the associations observed in this study.
The inability to detect effects of AMA-1 type on risk of clinical illness after 6 weeks is consistent with the short-lived antibody responses reported in immunological studies (15
) and might be interpreted as evidence that protective AMA-1 antibody responses are too transient for AMA-1 vaccines to offer clinically meaningful protection. B cell memory may persist after antibody concentrations wane, however, and vaccine-induced immunity to AMA-1 may persist longer than naturally acquired humoral immunity: In a recent phase 1 study of an AMA-1–based vaccine in children, anti-AMA-1 antibody titers increased by at least 100-fold after immunization and remained high throughout the 1-year follow-up period (29
). Indeed, a malaria vaccine must perform better than naturally acquired immunity, which confers only temporary protection against disease, but in doing so, vaccine-induced immunity may impose even greater selection pressure on the protein than does natural immunity. The strength, duration, and allele specificity of vaccine-induced immune responses, as well as their effect on the distribution of AMA-1 alleles in vaccinated individuals, are being evaluated in a phase 2 trial of the same vaccine that showed prolonged, high-antibody responses in the phase 1 trial (29
The clusters of polymorphisms were defined on the basis of their proximity to one another in the crystal structure (13
); however, it is possible that there were immunologically important polymorphisms outside of clusters c1 and c1L whose effects were masked because of their being grouped with less important residues. For example, based on the random forest analysis, amino acid changes at positions 172 and 175 were good predictors of the development of symptoms, and these findings are supported by results from growth-inhibitory assays that indicate that residue 175 (along with other residues) has a significant effect on erythrocyte invasion (14
The prevalence of AMA-1 alleles differed among parasites from Africa, Asia, and South America. These results suggest that vaccine efficacy might also vary by geographic location, depending on the strain(s) targeted by the vaccine. For example, a vaccine based on the FVO strain might have higher efficacy in Asia than in West Africa, and a vaccine based on the 7G8 strain might have the highest efficacy in South America but low efficacy in Asia or Africa. Some malaria vaccines have shown varying efficacy by geography (30
); however, parasite genotyping was not done in these trials to determine whether geographic differences in allele prevalences contributed to the variable efficacy. The geographical differences in AMA-1 haplotype prevalences highlight the need to measure vaccine antigen allele prevalences in malaria endemic areas before testing and distribution of vaccines.
Examination of additional published AMA-1 sequences did not show evidence of a maximum amount of existing AMA-1 diversity. Analysis of 63% more AMA-1 ectodomain sequences resulted in a 57% increase in the number of observed AMA-1 ectodomain haplotypes. Likewise, a 122% increase in the number of domain I c1L sequences resulted in a 92% increase in the number of observed domain I c1L haplotypes. This seemingly limitless amount of diversity may pose a major obstacle for development of an effective polyvalent AMA-1–based vaccine.
A bivalent AMA-1 vaccine recently failed to demonstrate efficacy in Mali (5
), although it is not yet known whether this resulted from a relatively poorly immunogenic formulation and/or inadequate cross-protection between the two vaccine strains and the diverse parasites in nature. An ongoing efficacy trial of a more highly immunogenic formulation of a monovalent AMA-1 vaccine will provide evidence of whether and to what extent vaccine-induced immunity is cross-protective or allele-restricted. Even a polyvalent recombinant subunit vaccine may not be feasible if 10 to 20 haplotypes are required to cover most of the diversity in a single geographic location. Nevertheless, it may be possible to select or engineer vaccine antigens that are more cross-protective. A recent study aligned published AMA-1 sequences to determine the location and extent of polymorphism in the protein and to identify linkage between specific residues, and this information was used to design three artificial AMA-1 constructs that share conserved amino acids while representing the greatest number of polymorphisms (32
). Data from molecular epidemiological studies and/or analyses of protein structure can be used to identify subsets of residues (13
) that may be particularly important to take into consideration in engineering such artificial antigens, ensuring that the most important residues for protection are represented.
It may be desirable to avoid polymorphism altogether by engineering vaccine constructs that boost the immune response to protective epitopes in conserved regions of AMA-1 and other proteins. At least one epitope that inhibits parasite invasion has been identified in the domain II loop on the nonpolymorphic face of AMA-1 (34
). If subunit vaccines based on conserved epitopes can divert the immune response away from highly polymorphic regions, they may be able to induce strain-transcending immunity. Alternatively, genomic and proteomic approaches are being used to identify new vaccine targets that are not immunodominant and likely to be more conserved than the current highly immunodominant and polymorphic candidates (35
). Live attenuated whole-organism vaccines may also hold promise for circumventing the extreme polymorphism in individual antigens exemplified by this study (38