Identification of the relevant amino acids driving allele-specific immunity is an important step in the design of malaria vaccines that target polymorphic antigens. The characterization of the AMA1 protein crystal structure [9
] has allowed identification of immunologically important amino acid residues in this malaria vaccine antigen, using both in vitro [5
] and in vivo approaches [7
]. In particular, molecular epidemiological tools and approaches have the potential to inform the choice of antigenic variants to include in multivalent, broadly efficacious vaccines [1
], and these tools can be used to assess the impact of malaria vaccines on the frequency of alleles observed in field trials [14
The finding that a monovalent AMA1 vaccine was efficacious only against homologous alleles in a human vaccine trial is consistent with in vitro studies [4
] and animal studies [2
] suggesting that AMA1 generates allele-specific immune responses. The observed patterns of natural acquisition of allele-specific immunity to AMA1 are consistent with a gradual accumulation of protective responses to a repertoire of AMA1 variants by people repeatedly exposed to malaria over years [23
In contrast to a bivalent AMA1 vaccine that showed no overall efficacy nor any suggestion of allele-specific efficacy [15
], the FMP2.1/AS02A
vaccine was highly efficacious against clinical malaria with vaccine-type AMA1 c1L. The mean antibody responses to FMP2.1/AS02A
vaccine were much higher and more sustained as compared to antibody responses to the bivalent AMA1 vaccine, suggesting that the latter vaccine failed simply because it was not immunogenic enough. In secondary analyses, the FMP2.1/AS02A
vaccine was shown to have an efficacy of about 20% against all clinical malaria episodes [14
], with varying statistical significance. This marginal overall efficacy may represent the vaccine having an effect exclusively directed against vaccine-type and closely related alleles with respect to the cluster of highly polymorphic amino acids located in domain 1 of AMA1.
The allele-specific efficacy of the FMP2.1/AS02A vaccine was confirmed by an analysis of the incidence of the vaccine and nonvaccine c1L haplotypes in clinical episodes before and after vaccination, which revealed a decreased incidence of the 3D7 AMA1 c1L haplotype in the malaria vaccine group as compared to the rabies group following vaccination. With 3D7 representing 13.5% of c1L haplotypes, an overall efficacy of approximately 20% against clinical malaria episodes suggests that there may be other AMA1 amino acid positions outside of c1L that are targeted by protective antibodies. While it is possible that FMP2.1/AS02A would offer protective efficacy against clinical malaria caused by parasites with c1L that matches 3D7 at position 197 but is not identical to 3D7 at other positions within c1L, we found no such haplotypes among the 600 samples sequenced in this study.
The finding that this malaria vaccine was efficacious only against clinical malaria with AMA1 alleles homologous to the vaccine allele in c1L points to the need for a multiple-allele vaccine. Although a multivalent vaccine including all unique AMA1 alleles would be practically impossible, by using molecular epidemiological approaches it may be possible to group sequences that are closely related and/or to identify amino acids that play a fundamental role in allele-specific efficacy. Representative alleles that cover a large proportion of the population may be selected and used as vaccine antigens, as has been done for a successful multivalent pneumococcal vaccine [24
]. Focusing on representative alleles may reduce the number of alleles to a number that can feasibly be managed by vaccine developers. A multivalent vaccine that comprises 10 of the most prevalent alleles may cover >70% of alleles identified in the study area (Supplementary Figure 2
). In corroboration of this finding, immunological analyses have been used to assess the allele specificity of the immune response by characterizing antibody responses against variants of AMA1 [13
]. These analyses revealed that epitopes within AMA1 of the 3D7 strain of P. falciparum
are representative of both D10 and S35 epitopes (which are identical in domain 1 but differ in domains 2 and 3), suggesting that 3D7 may be used to achieve cross-protection against both D10 and S35 strains in a multivalent malaria vaccine.
Even though a barrier blocking clinical episodes with vaccine-type alleles (a sieve effect) [25
] was observed at several amino acid positions in domains 1 and 3, the strongest barrier induced by the malaria vaccine was seen at amino acid position 197, which is located in c1L of domain 1. Alleles defined on the basis of this position showed vaccine efficacy identical to that for the whole c1L, suggesting that this position may be the most critical amino acid in antibody binding. Therefore, instead of considering all polymorphic positions of AMA1 to define haplotypes, position 197 alone might be used to define which alleles to include in a vaccine. This would reduce the number of alleles required to design a multivalent AMA1-based vaccine to only 6, which would represent 100% coverage of all alleles, where “alleles” is defined on the basis of AMA1 position 197.
The latest findings that a mixed-antigen formulation may produce the same immunological response as sequential infections with the same antigens [26
] further support the feasibility of developing a multivalent AMA1 vaccine, either as a stand-alone blood-stage malaria vaccine or, more likely, as a component of a multistage, multiple antigen vaccine [27
], with higher efficacy than that achieved by the most advanced malaria vaccine, RTS,S/AS01, which is currently being assessed in a large phase III trial in Africa [28