Paths to a malaria vaccine illuminated by parasite genomics

Abstract

More human death and disease is caused by malaria parasites than by all other eukaryotic pathogens combined. As early as the sequencing of the first human genome, malaria parasite genomics was prioritized to fuel the discovery of vaccine candidate antigens. This stimulated increased research on malaria, generating new understanding of the cellular and molecular mechanisms of infection and immunity. This review of recent developments illustrates how new approaches in parasite genomics, and increasingly large amounts of data from population studies, are helping to identify antigens that are promising lead targets. Although these results have been encouraging, effective discovery and characterization need to be coupled with more innovation and funding to translate findings into newly designed vaccine products for clinical trials.

Figure 1

Haploid Plasmodium falciparum genome containing 14 chromosomes. The whole genome of 23 Mb contains ∼5500 protein-coding genes. Subtelomeric regions shaded orange are highly divergent in sequence and gene content among species; the core genome shaded blue has a high level of synteny among Plasmodium species, although some genes are species-specific. Loci encoding the 15 antigens incorporated in reported clinical vaccine trials are shown. Only six of these have clear orthologs in all Plasmodium species (shown in bold font). The full names of each of these antigens are given in Box 2. A region of chromosome 10 is enlarged on the bottom right of the figure to show a cluster of antigen-encoding genes including several that are highly polymorphic (most are members of the msp3-like gene family flanked by unrelated antigen genes at both ends of the cluster).

Figure 2

30-Year timeline of P. falciparum vaccine antigen sequence description and first clinical trial publication for each antigen. Dates of first description of gene sequences are shown by arrows above the timeline for each of the 15 P. falciparum antigens with sequences incorporated into vaccines reported in published clinical trials (arrows below the timeline indicate the first trial in each case). The color shading indicates the parasite life-cycle stage at which each of the antigens is mainly expressed: pre-erythrocytic (blue), asexual blood stage (red), sexual and zygote transmission stage (green). The nature of the vaccine constructs varied extensively, based on synthetic peptides, recombinant proteins, virus-like particles, or viral vectored systems. In some cases only a short sequence representing part of an antigen was included in the vaccine, combined with sequences from other antigens, whereas in other cases most of a primary sequence of a single antigen was incorporated. For several of the antigens, other vaccine constructs have also been designed and tested at later dates that are not shown on the scheme. The date of publication of the first parasite genome sequence in 2002 is marked by a broken arrow above the dateline.

Figure 3

Example of variation in expression of a malaria parasite antigen. The P. falciparum MSPDBL2 protein is encoded by an msp3-like gene expressed on merozoites contained in mature blood-stage schizonts. The expression is variable among individual parasites because most schizonts are negative whereas a minority are positive. The figure shows example data from one parasite clone (HB3) (reproduced with modification from [66]). (A) A single microscopy field showing fixed parasite-infected erythrocytes. The left panel shows immunofluorescence of schizonts that react with an anti-MSPDBL2 antibody; the right panel shows DAPI (4′,6-diamidino-2-phenylindole)-stained parasite DNA in the cells (schizonts having multiple blue clustered nuclei). (B) Proportions of mature schizonts positive for MSPDBL2 vary among different subclones of the HB3 parasite. The eight subclones shown were cultured separately for several weeks, and then analyzed by microscopy counting of hundreds of mature schizonts from each to determine the percentage positive (with 95% confidence intervals).

Figure 4

Genome-wide scanning for malaria parasite genes potentially under balancing selection. P. falciparum population genomic data from clinical isolates sampled from two different West African populations (Guinea and The Gambia) are plotted and compared. Each point represents a gene with at least three SNPs in each population (3316 genes in total included) on a scatterplot of Tajima's D values for the polymorphic site frequency spectra in Guinea (n = 100 isolates) and Gambia (n = 52 isolates) (reproduced with modification from [84]). This shows negatively skewed frequency distributions for most genes in both populations (with genome-wide average values of less than −1.0), indicating more rare alleles than expected under a neutral equilibrium, and probably reflects demographic growth of P. falciparum populations historically. Against this background, a minority of genes have positive Tajima's D values, indicating those at which allele frequencies are more balanced than expected under neutrality. Enlarged symbols are shown for the genes with values above 1.0 in both populations that are most likely to be under balancing selection. These include several known antigens that are labeled for illustration, together with other genes that have not been previously studied.

Figure 5

Genome-wide scan of gene polymorphism in P. falciparum versus divergence with the chimpanzee parasite P. reichenowi. Each point represents comparative data for two different indices (HKA ratio and MK skew) for a gene, with sequence data from five P. falciparum laboratory lines compared with a single P. reichenowi genome (reproduced with modification from reference [10]). Several known antigen genes are labeled for illustration. The HKA ratio for each gene (on the x axis) is the average pairwise nucleotide polymorphism in P. falciparum (π) divided by the average interspecies nucleotide divergence (K), with a broken line shown at an arbitrary ratio of 0.15 to visually indicate genes with highest relative levels of polymorphism on the right of the plot. The MK skew for each gene (on the y axis) is the log₂-transformed odds ratio from the 2 × 2 table of numbers of nonsynonymous and synonymous polymorphisms within P. falciparum over the numbers of nonsynonymous and synonymous interspecies fixed differences. An MK skew value of zero represents an odds ratio of 1.0 reflecting where the intraspecific and interspecific ratios do not differ. Positive MK skew values indicate ratios for genes reflecting an excess of nonsynonymous polymorphism or deficit of nonsynonymous fixed differences compared with neutral expectations, marking in red those that are significant with Fisher's exact tests. Three of these genes that also have high HKA ratios are shown in bold outline (including AMA1). Some genes are not plotted here as they have infinite MK skew values (although significant in a number of cases by Fisher's exact test), mostly due to having no synonymous polymorphisms. Abbreviations: HKA, Hudson–Kretman–Aguade test; MK, McDonald–Kreitman test.

Figure I

Parasites are depicted here as oval-shaped cells expressing a single antigen type on the surface, encoded by a gene at a particular chromosomal locus marked by a similar shape which is enlarged for visibility. Transcriptionally active loci are indicated with an asterisk. For absolute schematic simplicity, no cellular details (e.g., nucleus) are shown. (A) Allelic polymorphism of a single locus with fixed expression (as for many malaria vaccine candidates such as CSP, TRAP, MSP1, and AMA1). (B) Variation due to alternative expression of different loci with minimal allelic polymorphism (as for malaria parasite antigens including some of the EBA and Rh ligands using alternative erythrocyte receptors). (C) Variable expression of highly polymorphic loci contributing to a very extensive potential repertoire of diversity (as for parasite genes encoding PfEMP1, and for some of the CLAG/RhopH1 and MSP3-like proteins).